WO2018185735A1

WO2018185735A1 - Anti-microbial combination

Info

Publication number: WO2018185735A1
Application number: PCT/IB2018/052437
Authority: WO
Inventors: Darren John Day; Jennifer Pamela SOUNDY
Original assignee: Victoria Link Limited
Priority date: 2017-04-07
Filing date: 2018-04-09
Publication date: 2018-10-11

Abstract

The present invention relates to anti-microbial agents (AMA) having bacteriostatic and/or bactericidal properties, compositions and combinations comprising an AMA, and the use of such compositions, combinations and/or AMAs for use in inhibiting the growth and/or proliferation of microorganisms, for use in the manufacture of medicaments for treating microbial infection, and/or for use for treating microbial infection.

Description

ANTI-MICROBIAL COMBINATION

FIELD OF THE INVENTION

This invention relates generally to anti-microbial agents (AMA) having bacteriostatic and/or bactericidal properties, compositions and combinations comprising an AMA, and the use of such compositions, combinations and/or AMAs to inhibit the growth and/or proliferation of microorganisms, in the manufacture of medicaments for treating microbial infection, and/or for treating microbial infection.

BACKGROUND OF THE INVENTION

According to the World Health Organization (WHO) Fact sheet No 194 (April 2015) anti-microbial resistance threatens the effective prevention and treatment of an ever- increasing range of infections caused by bacteria, parasites, viruses and fungi. The WHO considers anti-microbial resistance to be an increasingly serious threat to global public health and calls on all government sectors and society in general to act, noting that anti-microbial resistance is present in all parts of the world, and that new resistance mechanisms emerge and spread globally. For example, there are high proportions of antibiotic resistance in bacteria that cause common infections (e.g. urinary tract infections, pneumonia, bloodstream infections) in all regions of the world, with a high percentage of hospital-acquired infections caused by highly resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA) or multidrug- resistant Gram-negative bacteria.

For example, Pseudomonas aeruginosa is a clinically important, opportunistic, gram- negative bacterium that is responsible for a wide variety of severe hospital-acquired infections. It largely effects immune-compromised patients, particularly those with cystic fibrosis, as well as causing infections in burn wounds, and forming biofilms on implanted devices such as urinary catheters and heart stents. P. aeruginosa is intrinsically resistant to many anti-microbial agents due to the low permeability of its outer membrane, and upregulation of multidrug efflux pump systems [1]. It can also acquire resistance mechanisms through horizontal gene transfer and chromosomal mutations. This multidrug resistance is further amplified by the ability of the bacteria to form biofilms. Biofilms are a matrix of excreted exopolysaccharides, proteins and DNA that is formed over the colonised bacterial population which protects the bacteria inside from antibiotics [2]. These biofilm bacteria have adaptive changes in gene expression and shift to a metabolically less active state, making the infections harder to clear and are responsible for the recalcitrance of disease.

In view of the economic and health reasons as discussed above, the continued use of currently available small molecule antibiotics has significant limitations and poses a threat to the global public health. There is a need for new anti-microbial agents that do not have the cost and/or health and/or environmental issues that are emerging with the overuse of antibiotics.

It is an object of the invention to provide at least one anti-microbial agent and/or at least one anti-microbial combination and/or a composition comprising at least anti- microbial agent or anti-microbial combination and/or to provide methods of using such an agent and/or combination to inhibit the growth and/or proliferation of

microorganims and/or in the the manufacture of medicaments for treating microbial infections and/or in methods of treating microbial infections, and/or to at least provide the public with a useful choice. SUMMARY OF THE INVENTION

Anti-microbial combination

In a first aspect, the invention relates to an anti-microbial combination comprising a polynucleotide comprising a targeting component and an anti-microbial component, wherein the targeting component specifically binds a target molecule or cell, and wherein the anti-microbial component comprises a polynucleotide in association with an anti-microbial agent.

In another aspect the invention relates to a combination of formula I: TC-AMC wherein TC = a targeting component that specifically binds to a target molecule or cell, and

AMC = an anti-microbial component comprising i) a polynucleotide that forms a metal nanocluster with at least three metal atoms, or a polynucleotide that chelates at least one metal ion, or both, and ii) an anti-microbial agent.

In another aspect the invention relates to a combination of formula II: AptPA-AMC wherein

AptPA = is an aptamer or functional fragment thereof that specifically binds a Pseudomonas aeruginosa cell, and

AMC= an anti-microbial component comprising i) a polynucleotide comprising a nucleic acid sequence that is 5'-(CxN_y)_z-3' wherein C = cytosine; N= any combination of adenosine (A), guanosine (G), thymidine (T), analogues of C, A, G and T, and modified deoxynucleosides; x = an integer from 2 to 7; y = an integer from 0 to 7; and z = any integer, and ii) an anti-microbial agent comprising at least three atoms of silver or

copper metal, or at least three silver or copper ions associated with the polynucleotide in i).

In another aspect the invention relates to a combination of formula Ila :

AptPA-AMC wherein

AMC= an anti-microbial component comprising i) a polynucleotide comprising a nucleic acid sequence that is 5'-(CxN_y)_z-3' wherein C = cytosine; N= any combination of adenosine (A), guanosine (G), thymidine (T), analogues of C, A, G and T, and modified deoxynucleosides; x = an integer from 2 to 7; y = an integer from 0 to 7; and z = any integer, and ii) an anti-microbial agent comprising at least three atoms of silver and/or copper metal, and/or at least three silver or copper ions associated with the polynucleotide in i).

In another aspect the invention relates to a method of making an anti-microbial combination comprising : a) synthesizing a polynucleotide comprising a targeting component comprising a polynucleotide that specifically binds to a target molecule or cell, and an anti- microbial component comprising a nucleic acid sequence that forms a metal nanocluster with at least three metal atoms, or is a nucleic acid sequence that chelates at least one metal ion, or both, and b) associating the polynucleotide synthesized in a) with at least one antimicrobial agent to make the anti-microbial combination. In another aspect the invention relates to an anti-microbial combination as described herein made by a method as described herein.

In another aspect the invention relates to an anti-microbial component (AMC) comprising i) a polynucleotide that forms a metal nanocluster with at least three metal atoms, or a polynucleotide structure that chelates at least one metal ion, or both, and ii) an anti-microbial agent.

In another aspect the invention relates to a composition comprising an anti-microbial combination, a targeting component, an anti-microbial component, a combination of formula I, or a combination of formula II or Ila as described herein and a carrier, diluent or excipient.

In another aspect the invention relates to the use of an anti-microbial combination, a targeting component, an anti-microbial component, a combination of formula I, a combination of formula II or Ila, or a composition as described herein, in the manufacture of a medicament for treating microbial infection. In another aspect the invention relates to a method of treating a microbial infection comprising administering an anti-microbial combination, an anti-microbial component, a combination of formula I, a combination of formula II or Ila, or a composition as described herein to a subject in need thereof. In another aspect the invention relates to an anti-microbial combination, an antimicrobial component, a combination of formula I, a combination of formula II or Ila, or a composition as described herein for use in treating, or when used to treat, microbial infection.

In another aspect the invention relates to the use of an anti-microbial component as described herein to inhibit the growth and/or proliferation of at least one bacterial species, wherein the anti-microbial component comprises a polynucleotide in association with an anti-microbial agent, wherein the polynucleotide comprises an imotif structure and the anti-microbial agent is at least one metal ion.

In another aspect the invention relates to the use of an anti-microbial component as described herein in the manufacture of a medicament for treating microbial infection, wherein the anti-microbial component comprises a polynucleotide in association with an anti-microbial agent, wherein the polynucleotide comprises an imotif structure and the anti-microbial agent is at least one metal ion.

In another aspect the invention relates to a method of inhibiting the growth and/or proliferation of at least one bacterial species comprising contacting the at least one bacterial species with an anti-microbial component as described herein, wherein the anti-microbial component comprises a polynucleotide in association with an antimicrobial agent, wherein the polynucleotide comprises an imotif structure and the anti-microbial agent is at least one metal ion. In another aspect the invention relates to a method of treating a microbial infection comprising administering an anti-microbial component to a subject in need thereof, wherein the anti-microbial component comprises a polynucleotide in association with an anti-microbial agent, wherein the polynucleotide comprises an imotif structure and the anti-microbial agent is at least one metal ion. In another aspect the invention relates to an anti-microbial component for use in inhibiting the growth and/or proliferation of at least one bacterial species treating, or when used to treat, microbial infection, wherein the anti-microbial component comprises a polynucleotide in association with an anti-microbial agent, wherein the polynucleotide comprises an imotif structure and the anti-microbial agent is at least one metal ion.

In another aspect the invention relates to a polynucleotide selected from the group consisting of SEQ ID NO: 32, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42 and SEQ ID NO: 43.

In another aspect the invention relates to the use of an anti-microbial component to inhibit the growth and/or proliferation of at least one bacterial species, wherein the anti-microbial component consists essentially of a polynucleotide structure in association with an anti-microbial agent, wherein the polynucleotide structure comprises an i-motif structure, wherein the anti-microbial agent is at least one metal ion, preferably at least one Ag⁺, wherein the imotif structure comprises a single nucleic acid strand that forms the i- motif structure or at least two, preferably four, nucleic acid strands that form the imotif structure, wherein the nucleic acid strand or strands that form the i-motif structure comprise a nucleic acid sequence comprising at least one, preferably at least two, preferably four poly C tracts (pCts), wherein the bacterial species is selected from the group consisting of Pseudomonas aeruginosa, Salmonella enterica serovar typhi, Klebsiella pneumoniae, Enterobacter cloacae, Listeria innocua, Escherichia coli, Acinetobacter baumannii, Staphylococcus aureus, and Staphylococcus epididymis.

While various embodiments of certain aspects of the invention are set out above, the invention is not limited thereto. Additional embodiments of the aspects of the invention set out above are further described in the Detailed Description and set out in the claims of the application.

Other aspects and embodiments of the invention may become apparent from the following description which is given by way of example only and with reference to the accompanying drawings. It is intended that reference to a range of numbers disclosed herein (for example, 1 to 10) also incorporates reference to all rational numbers within that range (for example, 1, 1.1, 2, 3, 3.9, 4, 5, 6, 6.5, 7, 8, 9 and 10) and also any range of rational numbers within that range (for example, 2 to 8, 1.5 to 5.5 and 3.1 to 4.7) and, therefore, all sub-ranges of all ranges expressly disclosed herein are hereby expressly disclosed. These are only examples of what is specifically intended and all possible combinations of numerical values between the lowest value and the highest value enumerated are to be considered to be expressly stated in this application in a similar manner.

BRIEF DESCRIPTION OF THE DRAWINGS The invention will now be described by way of example only and with reference to the drawings in which :

Figure 1. Tuneable fluorescence of DNA scaffolded silver nanoclusters. The sequence of the DNA scaffold for AgNC synthesis plays a role in the emitted fluorescence of the clusters. Displayed in this figure is red, orange, yellow and green fluorescence (from left to right) of AgNCs.

Figure 2. Comparison of binding affinity of JN27 and JN27.SH. Increasing

concentrations of FAM labelled aptamer (left to right -10 nM, 50 nM, 100 nM, 250 nM) JN27 (black, solid line) and JN27.SH (grey, solid line) were incubated with P.

aeruginosa PA692. Cells were washed, re-suspended in buffer and analysed by flow cytometry to look for an increase in median fluorescence compared to bacteria with no aptamer (black dotted), and a difference between the two aptamers. Increased binding of the FAM labelled aptamers results in a right shift in the histograms.

Figure 3. Aptamer sequences and their dissociation constants (Kd).

Figure 4. Predicted aptamer secondary structures. Figure 5. Specificity of aptamer binding to Pseudomonas aeruginosa species compared with other bacterial species. FAM-labelled aptamer (250 nM) was incubated with P. aeruginosa strains (ΤΆ692 and PAOl) or other bacterial species (Salmonella enterica, Klebsiella pneumoniae, Enterobacter cloacae, Listeria innocua and

Escherichia coli). Cells were washed, re-suspended in phosphate buffer and analysed by flow cytometry to measure the relative fluorescence intensity (arbitrary

fluorescence units, AFU) due to bound aptamer. Figure 6. The effect of aptamers on the growth rate of P. aeruginosa PA692. 1 μΜ of each aptamer was incubated with 10⁵ CFU of bacteria and the growth rate monitored by measurement of OD₆₀₀ every 30 min for 16 hours. No significant differences in growth rate were seen between aptamer treated and untreated cells. Figure 7. Fluorescence spectra of aptamer-AgNCs. Excitation (ex) and emission (em) spectra are as indicated.

Figure 8. Thermal stability of JN21-NC fluorescence. The emission spectrum of aptamer in MES buffer pH6.5 was recorded at 10°C and the relative fluorescence intensity set to 100%. The temperature was then raised in 5°C increments and the emission spectrum recorded and the relative intensity determined (grey bars). The experiment was repeated with fresh aptamer except after each temperature jump the sample was cooled to 10°C and the fluorescence intensity determined. The recovered fluorescence expressed as a percentage of the initial fluorescence is shown by the black bars. Figure 9. CD spectra of JN 21 split and JN 17 3'. Aptamer alone, aptamer-AgNC, and aptamer-Ag⁺- Circular dichroism (CD) spectra of aptamers JN21 split and JN 17 3'. Spectra of 1 μΜ solutions in a 2 mm pathlength cuvette were recorded for the aptamer alone (black solid line), aptamer-AgNC (black dashed line), and aptamer to which a 6-fold molar excess of silver nitrate was added to form a complex of aptamer- chelated silver ions (aptamer-Ag⁺) (grey solid line).

Figure 10. Fluorescence spectra of aptamer-AgNC after addition of Ag⁺. The fluorescence emission (solid line) and excitation (dashed line) spectrum of JN 17 was recorded (black) prior to the addition of a 6-molar excess of silver nitrate (grey). Chelation of extra silver shifts the emission from the red into the green part of the visible spectrum.

Figure 11. Antibacterial activity of Aptamer-AgNCs. Growth assays showing antibacterial activity of Aptamer-AgNCs. An exponentially growing culture of P.

aeruginosa PA692 was diluted such that 2xl0⁵ cfu were present in 150 μΙ_ of growth media, then aptamers were added to a final concentration of between 10 μΜ and 78 nM as indicated in the legend. Growth was monitored by measuring the increase in OD₆₀₀ at 5 minute intervals for 20 hrs. Figure 12. Bacterial titre (cfu) as a percentage of starting inoculum after 20 hr exposure to 10 μΜ aptamer-AgNC. Bacterial titre (cfu) as a percentage of starting inoculum after 20 hr exposure to 10 μΜ aptamer-AgNC. P. aeruginosa was culture and treated with aptamer as described in Figure 11. After 20hrs growth, the number of viable cells was enumerated by plating onto agar and culturing overnight. The number of cfu present as a percentage of the initial inoculum size is presented.

Figure 13. Antibacterial activity of Aptamer chelated Ag⁺.

Growth assays showing antibacterial activity of Aptamer-Ag⁺. An exponentially growing culture of P. aeruginosa PA692 was diluted such that 2xl0⁵ cfu were present in 150 μΙ of growth media then aptamer with chelated silver ions was prepared and added to a final concentration of between 10 μΜ and 78 nM as indicated in the legend. Growth was monitored by measuring the increase in OD₆₀₀ at 5 minute intervals for 20 hrs.

Figure 14. Bacterial titre (cfu) as a percentage of starting inoculum after 20 hr exposure to 2.5 μΜ aptamer- Ag⁺- P. aeruginosa was cultured and treated with aptamer as described in Figure 13. After 20hrs growth, the number of viable cells was enumerated by plating onto agar and culturing overnight. The number of cfu present as a percentage of the initial inoculum size is presented.

Figure 15. Bacterial growth on agar plates after exposure to aptamer-Ag⁺. Bacterial titre (cfu) agar plates with no dilution after exposure to 2.5 μΜ aptamer-Ag⁺ for 20 hours. Bacteria were cultured as described in Figure 13 and were treated with 2.5 μΜ aptamer-Ag⁺ for 20 hrs. Undiluted samples (100 μΙ) were plated directly and grown overnight. The JN27 5' Ag⁺ and t21lpl7 3'Ag⁺ treated cultures had less than 3 colonies present compared with the untreated control that was completely overgrown forming a bacterial lawn. Individual colonies can be seen on the JN 17 3' Ag⁺ and JN21 split Ag⁺ treated plates.

Figure 16. Growth assays showing synergy between aptamer-AgNC and ciprofloxacin action. P. aeruginosa was cultured and treated with aptamer as described in Figure 11 and treated with either no aptamer (control) or 1 μΜ or 5 μΜ aptamer-NC, in the presence or absence of 0.5 μg/ml ciprofloxacin. Growth was monitored by measuring the increase in OD₆₀₀ at 5 minute intervals for 20 hrs.

Treatment with both aptamer-NC and ciprofloxacin inhibited growth to a greater extent than the sum of the individual treatments. Figure 17. The antibacterial activity of aptamer-Ag⁺ against different strains of P. aeruginosa. Growth curves showing antibacterial activity of 2.5 μΜ aptamer- Ag⁺ against different strains of P. aeruginosa. Different laboratory strains and clinical isolates of P. aeruginosa were cultured as described in Figure 13 and were treated with 2.5 μΜ of aptamer-Ag⁺. Untreated control (squares) and treatment with the equivalent amount of silver nitrate (2.5 μΜ, triangles) are presented for comparison. Growth was monitored by measuring the increase in OD₆₀₀ at 5 minute intervals for 20 hrs.

Figure 18. Kaplan Meier survival analysis of Galleria mellonella larvae infected with P. aeruginosa then treated with aptamer-AgNC or aptamer- Ag⁺.

Galleria mellonella survival curves for larvae treated with aptamer-AgNC and aptamer- Ag⁺. Larvae were infected with approximately 20 cfu of P. aeruginosa then treated with 10 μΙ of the indicated concentration of aptamer-AgNC or aptamer- Ag⁺ 30 min later. Survival was monitored at 1 hr intervals. Figure 19. Propidium iodide (PI) uptake following treatment with aptamer- AgNC or aptamer-Ag⁺. PI staining histograms after treatment with 5 μΜ aptamer- AgNC and aptamer-Ag⁺. P. aeruginosa was treated with 5 μΜ of aptamer-AgNC or aptamer-Ag⁺ then stained with propidium iodide and analysed by flow cytometry to identify depolarized or dead cells. Depolarization of death causes an increasing right shift in the histogram.

Figure 20. Time course for killing of P. aeruginosa with aptamer-Ag⁺. The table shows the number of cfu obtained after plating an inoculum containing 2xl0⁵ cfu following treatment with 1 μΜ JN21 split Ag⁺, after 1 and 3 hrs of treatment.

Figure 21. The seventh SELEX round shows enhanced binding to P.

aeruginosa. Binding of the seventh SELEX round (dark grey) was assessed relative to the starting library (light grey) to P. aeruginosa (A) and E. coli (B). The fluorescence of unstained cells is indicated in black. The seventh SELEX round shows increased binding to P. aeruginosa (right shift in the histogram) relative to the starting library, but both the starting library and the seventh SELEX round bind equally poorly to E. coli.

Figure 22. Aptamer dissociation constants (Kd) Figure 23. Specificity of aptamer binding to Pseudomonas aeruginosa species compared with other bacterial species. FAM-labelled aptamer (250 nM) was incubated with P. aeruginosa strains (ΤΆ692 and PAOl) or other bacterial species (Salmonella enterica, Klebsiella pneumoniae, Enterobacter cloacae, Listeria innocua and Escherichia coli). Cells were washed, re-suspended in PBS and analysed by flow cytometry to measure the relative fluorescence intensity (arbitary fluorescence units, AFU) due to bound aptamer. All aptamer candidates preferentially bound the P.

aeruginosa strains.

Figure 24. Antibacterial activity of aptamer chelated Ag⁺. LB was inoculated with 2xl0⁵ CFU of P. aeruginosa strain PA692 and treated with aptamer-Ag⁺s from 10 μΜ and 78 nM as indicated in the legend. Growth was monitored by measuring the increase in OD₆₀₀ at 5 minute intervals for 20 hrs.

Figure 25. Position of the AgNC poly C sequence effects antimicrobial activity.

LB was inoculated with P. aeruginosa strain PA692 and either untreated (black solid), or treated with 2.5 μΜ of JN21-AgNC in which the AgNC forming sequence was either 5' (dark grey solid), 3' (light grey solid), 5' with T7 spacer (black dashed), 3' with T7 spacer (dark grey dashed), or split (light grey dashed).

Figure 26. Antibacterial activity of aptamer-NCs. LB was inoculated with 2xl0⁵ CFU of P. aeruginosa strain PA692 and treated with aptamer-AgNCs from 10 μΜ and 78 nM as indicated in the legend. Growth was monitored by measuring the increase in OD₆₀₀ at 5 minute intervals for 20 hrs.

Figure 27. Circular dichroism spectra showing i-motif formation upon titration with Ag⁺. Spectra show the formation of a trough at about260 nm upon additon of excess silver ions as illustrated for the graph labelled Full iMotif. The indicated spectral changes are characteristic of i-motif formation.

Figure 28. Aptamers target delivery of AgNCs. P. aeruginosa strain PA692 was treated with 2.5 μΜ AgNC (NC, dark grey dashed) or 2.5 μΜ aptamer-AgNC and growth compared to no treatment control (black solid). JN27 5'-AgNC (dark grey solid), JN21 split-AgNC (light grey solid), St21Lpl7 split-AgNC (black dashed). The untargeted NC has some antimicrobial activity but conjugation to the aptamer sequences enhances the antibacterial activity resulting in a shift to the right in the graphs. Figure 29. Agar plates showing remaining PA692 after 20 hours treatment with 0, 5 or 10 μΜ aptamer-AgNC The control plate (Ctrl) that was not treated with aptamer-NC is an overgrown lawn of bacteria. For JN27 AgNC and St21Lpl7 AgNC individual colonies are visble with 5 μΜ treatment and no colonies with 10 μΜ treatement. Fo JN21 AgNC the plate shows an overgrown lawn at 5 μΜ treatment and individual colonies at 10 μΜ treatment.

Figure 30. Agar plates showing remaining PA692 after 20 hours treatment with 0, 2.5, 5 or 10 μΜ aptamer-Ag⁺. The control plate (Ctrl) that was not treated with aptamer-AgiM is an overgrown lawn of bacteria. For JN27 AgiM, JN21 AgiM and St21Lpl7 AgiM a few colonies are visible at 2.5 μΜ treatment and no colonies at 5 μΜ or 10 μΜ treatment.

Figure 31. Growth assays showing synergy between aptamer-AgNC and ciprofloxacin action. P. aeruginosa was cultured and treated with either no aptamer (control), 1 μΜ or 5 μΜ aptamer-AgNC, in the presence or absence of 0.5 μg/ml ciprofloxacin. Growth was monitored by measuring the increase in OD₆₀₀ at 5 minute intervals for 20 hrs. Treatment with aptamer-AgNC and ciprofloxacin together inhibited growth to a greater extent than the sum of the individual treatments.

Figure 32. The antibacterial activity of aptamer-Ag⁺ against different strains of P. aeruginosa. Different laboratory strains and clinical isolates of P. aeruginosa were cultured as described in Figure 17 and were treated with 2.5 μΜ of aptamer-Ag⁺. Untreated control (black solid) and treatment with the equivalent amount of silver nitrate (2.5 μΜ, light grey dashed) are presented for comparison. Growth was monitored by measuring the increase in OD₆₀₀ at 5 minute intervals for 20 hrs. All aptamer-Ag⁺ conjugates prevented growth while the same concentration of silver nitrate slowed growth but was much less effective than the aptamer-Ag⁺.

Figure 33. Time-to-kill assay for aptamer-AgNCs. P. aeruginosa strain PA692 was resuspended in MES buffer and treated with 5 μΜ or 10 μΜ aptamer-AgNC and the number of viable CFU remaining at each time point determined by agar plating. The aptamer-AgNC cause rapid killing of the bacteria. Figure 34. Time-to-kill assay for aptamer-Ag⁺. P. aeruginosa strain PA692 was resuspended in MES buffer and treated with 1 μΜ or 2.5 μΜ aptamer-Ag⁺ and the number of viable CFU remaining at each time point determined by agar plating. The aptamer-Ag⁺ cause rapid killing of the bacteria. Figure 35. Comparison between dose dependent antimicrobial effects of aptamer-AgNC and aptamer-Ag⁺. The fold increase in lag phase compared to control growth was compared for each concentration of treatment with aptamer-AgNC or aptamer-Ag⁺, as determined by the growth assays in figure 24 and 26. Figure 36. Aptamer-AgNCs depolarise P. aeruginosa cells after 10 min and 1 hr of treatment. P. aeruginosa strain PA692 was treated with either, 1 μg/mL ciprofloxacin, 5 or 10 μΜ aptamer-AgNC, or not at all (no treatment) for 10 min and 1 hr. Cells were then stained with 1.5 μg/mL propidium iodide (PI) and the fraction of high PI fluorescing (permeable cells) cells recorded. The positive control are cells killed with isopropanol.

Figure 37. Aptamer-Ag⁺s depolarise P. aeruginosa cells after 10 min and 1 hr of treatment. P. aeruginosa strain PA692 was treated with no treatment, or a 2-fold dilution series of aptamer-Ag⁺ from 5 μΜ to 0.078 μΜ, for 10 min or 1 hr then stained with propidium iodide (PI, 1.5 μg/mL) and the high PI fluorescing (permeable cells) population recorded.

Figure 38. Kaplan Meier survival analysis of Galleria mellonella larvae infected with P. aeruginosa then treated with aptamer-AgNC or aptamer- Ag⁺.

Larvae were infected with approximately 10 CFU of P. aeruginosa PAOl . Panel A+B show larvae infected with PAOl as above and then treated with 10 μΜ of each aptamer-AgNC and the survival tracked for 24 hours as two independent experiments.

Figure 39. Dose response graphs of PAOl and isogenic MexB efflux pump mutant after treatment with ciprofloxacin or aptamer-AgNCs. Graphs show growth of the bacteria at each treatment concentration as a percentage of growth compared to no treatment control. Panel A shows the difference in IC50 between P. aeruginosa strain PAOl and MexB when treated with ciprofloxacin. Panels B-D show no significant difference in IC50 between PAOl and MexB treated with aptamer-AgNCs.

Figure 40. Dose response graphs of PAOl and isogenic MexB efflux pump mutant after treatment with ciprofloxacin or aptamer-Ag⁺s. Graphs show growth of the bacteria at each treatment concentration as a percentage of growth compared to no treatment control. Panel A shows the difference in IC50 between P. aeruginosa strain PAOl and MexB when treated with ciprofloxacin. Panels B-D show no significant difference in IC50 between PAOl and MexB treated with aptamer-Ag⁺s. Figure 41. Changes to an immature biofilm after treatment with aptamer-Ag⁺ or ciprofloxacin. Biofilms were grown for 20 hours from PA692 and treated with 5 μΜ aptamer-Ag⁺ or ciprofloxacin once for 6 hours (panels A+B) or twice for 6 then 17 hours (panels C+D). Panels A+C show the growth of planktonic cells shed from the biofilm during antimicrobial challenge, panels B+D show the reduction in biofilm biomass after treatments.

Figure 42. Changes to a mature biofilm after treatment with aptamer-Ag⁺ or ciprofloxacin. Biofilms were grown for 44 hours from P. aeruginosa strain PA692 and treated with 5 μΜ aptamer-Ag⁺ or ciprofloxacin once for 6 hours. Panel A shows planktonic growth of cells shed from the biofilm during treatment. Panel B shows the reduction in biofilm biomass after treatment.

Figure 43. Antibacterial activity of i-motif2 (SEQ ID NO:38) against various bacterial strains. Growth assays showing antibacterial activity of i-motif2. An exponentially growing culture of each bacterial strain was diluted such that 2xl0⁵ cfu were present in 150 μΙ of growth media then Ag⁺ stabilised i-motif2 was prepared and added to a final concentration of between 2.5 μΜ and 0.62 μΜ as indicated in the legend. Growth was monitored by measuring the increase in OD₆₀₀ at 5 minute intervals for 17 hrs.

Figure 44. Antibacterial activity of a series of i-motif sequences against P. aeruginosa strain PA692. Growth assays showing antibacterial activity of various i- motif sequences. An exponentially growing culture of PA692 was diluted such that 2xl0⁵ cfu were present in 150 μΙ of growth media then Ag⁺ stabilised I-motifs were prepared and added to a final concentration of between 5 μΜ and 1.25 μΜ as indicated in the legend . Growth was monitored by measuring the increase in OD₆₀₀ at 5 minute intervals for 17 hrs.

Figure 45. Antibacterial activity of a series of i-motif sequences against P. aeruginosa strain PAOl. Growth assays showing antibacterial activity of various i- motif sequences. An exponentially growing culture of PA01 was diluted such that 2xl0⁵ cfu were present in 150 μΙ of growth media then Ag⁺ stabilised I-motifs were prepared and added to a final concentration of between 5 μΜ and 1.25 μΜ as indicated in the legend . Growth was monitored by measuring the increase in OD₆₀₀ at 5 minute intervals for 17 hrs. Figure 46. Circular dichroism spectra showing Ag⁺ stabilised i-motif formation upon addition of Ag⁺ of various predicted i-motif forming

sequences. Spectra show the formation of a trough at about 260 nm and a peak at 290 nm upon addition of excess silver ions. The indicated bathochromic shift in spectral properties are characteristic of i-motif formation.

Figure 47. Antibacterial activity of a series of i-motif sequences against S. aureus. Growth assays showing antibacterial activity of various i-motif sequences. An exponentially growing culture of S. aureus was diluted such that 2xl0⁵ cfu were present in 150 μΙ of growth media then Ag⁺ stabilised i-motifs were prepared and added to a final concentration of between 5 μΜ and 1.25 μΜ as indicated in the legend. Growth was monitored by measuring the increase in OD₆₀₀ at 5 minute intervals for 17 hrs.

Figure 48. Antibacterial activity of I-motif2 (SEQ ID NO: 38) against various bacterial strains and synergy with co-treatment with 2 mM EDTA. Growth assays showing antibacterial activity of i-motif2 and EDTA. Exponentially growing cultures of bacteria were diluted such that 2xl0⁵ cfu were present in 150 μΙ of growth media then Ag⁺ stabilised i-motif2 was prepared and added to a final concentration of 1.25 μΜ with or without 2 mM EDTA as indicated in the legend. Growth was monitored by measuring the increase in OD₆₀₀ at 5 minute intervals for 17 hrs. Figure 49. Antibacterial activity of half i-motif 1 (SEQ ID NO: 39) against S. aureus from 0.078 μΜ to 1.25 μΜ and synergy with co-treatment with a range of concentrations of EDTA. Growth assays showing antibacterial activity of half i-motifl and EDTA. Exponentially growing cultures of S. aureus were diluted such that 2xl0⁵ cfu were present in 150 μΙ of growth media then Ag⁺ stabilised half i-motifl was prepared and added to a final concentration range of between 1.25 μΜ and 0.078 μΜ, with 0.1, 0.15, 0.2, 0.5, or 0.75 mM EDTA as indicated in the legend. Growth was monitored by measuring the increase in OD₆₀₀ at 5 minute intervals for 17 hrs.

DETAILED DESCRIPTION OF THE INVENTION

Definitions The following definitions are presented to better define the present invention and as a guide for those of ordinary skill in the art in the practice of the present invention. Unless otherwise specified, all technical and scientific terms used herein are to be understood as having the same meanings as is understood by one of ordinary skill in the relevant art to which this disclosure pertains.

Examples of definitions of common terms in microbiology, molecular biology and biochemistry can be found in Methods for General and Molecular Microbiology, 3rd Edition, C. A. Reddy, et al. (eds.), ASM Press, (2008); Encyclopedia of Microbiology, 2nd ed., Joshua Lederburg, (ed .), Academic Press, (2000); Microbiology By Cliffs Notes, I. Edward Alcamo, Wiley, (1996); Dictionary of Microbiology and Molecular Biology, Singleton et al. (2d ed.) (1994); Biology of Microorganisms 11th ed., Brock et al., Pearson Prentice Hall, (2006); Genes IX, Benjamin Lewin, Jones & Bartlett

Publishing, (2007); The Encyclopedia of Molecular Biology, Kendrew et al. (eds.), Blackwell Science Ltd., (1994); and Molecular Biology and Biotechnology: a

Comprehensive Desk Reference, Robert A. Meyers (ed.), VCH Publishers, Inc., (1995).

It is also believed that practice of the present invention can be performed using standard microbiological, molecular biology and biochemistry protocols and procedures as known in the art, and as described, for example in Ellington, A. D., and Szostak, J.

W. (1990) In vitro selection of RNA molecules that bind specific ligands, Nature 346,

818-822; Fang, X., and Tan, W. (2010) Aptamers generated from cell-SELEX for molecular medicine: a chemical biology approach, Accounts of chemical research 43, 48-57, and other commonly available reference materials relevant in the art to which this disclosure pertains, and which are all incorporated by reference herein in their entireties.

The term "fragment" as used herein is used interchangeably with the term "functional fragment" and means the same thing. The term "a functional fragment thereof" when used in reference to a polynucleotide as described herein refers to that portion of the polynucleotide that is required for the activity of that polynucleotide, wherever that activity may be, and which could not reasonable be expected to have occurred by random chance. In the case of a polynucleotide that specifically binds a target molecule or cell, "a functional fragment thereof" means that portion of the polynucleotide that is required for specific binding.

The term "a targeting component" as used herein with reference to a polynucleotide or functional fragment thereof means a polynucleotide or functional fragment thereof that specifically binds a target molecule or cell. In one embodiment a targeting component of an anti-microbial combination as described herein comprises, consists essentially of, or consists of an aptamer or functional fragment thereof.

The term "specifically binds" as used herein with reference to a "targeting component" of a polynucleotide as described herein means that the polynucleotide binds a given target molecule cell with a "high binding affinity". In an embodiment where the targeting component comprises, consists essentially of, or consists of an aptamer or functional fragment thereof, "a high binding affinity" means that on average, the binding affinity of an aptamer that binds to a given Gram negative bacterial species is at least 10 μΜ, preferably at least 5 μΜ, preferably at least 2 μΜ, preferably at least 0.5 μΜ.

A skilled person in the art using suitable conditions as described herein and known in the art can employ a "targeting component" as described herein to specifically bind to a target molecule or cell to distinguish that target molecule or cell from amongst a population of non-target molecules or cells. The basis of this determination is the high binding affinity that a "targeting component", and particularly an aptamer or functional fragment thereof as described herein, has for a target molecule or cell as compared to a non-target molecule or cell.

The term "aptamer" (including a "functional fragment thereof") means to a

polynucleotide or functional fragment thereof that binds, preferably that specifically binds, a target molecule or cell without the formation of canonical Watson-Crick complementary base pairs between the nucleotide residues in the aptamer and the residues of a target molecule to which the aptamer binds. Although internal Watson- Crick base pairing within the aptamer may be present, specific binding between an aptamer and a target molecule or cell is based on the three dimensional structure of the aptamer and target, and is selected for in the design and evolution of the aptamer.

The term "specifically binds" when used in particular reference to an aptamer or functional fragment of an aptamer as described herein means that the primary basis for the aptamer or functional fragment thereof binding to the target molecule or cell does not involve the formation of complementary nucleotide base pairs between the aptamer and the target molecule or cell.

A person of skill in the art recognizes that it is well-known in the art that the nucleotide sequence of an aptamer may include base pairs that are not required for specific binding of the aptamer to a given target, and that smaller fragments of an aptamer, even fragments having below 50% sequence identity may still be capable of effectively binding to a target (Alsager, Omar A., et al. "Ultrasensitive Colorimetric Detection of 173-Estradiol : The Effect of Shortening DNA Aptamer Sequences." Analytical chemistry 87.8 (2015) : 4201-4209).

The terms "in association", "associated with", "associating the" (and other

grammatical variations of these terms) as used herein with reference to an "antimicrobial component" or "a polynucleotide" that is "in association" or "associated with" an anti-microbial agent or with reference to "associating the" "anti-microbial component" or "polynucleotide" with an "anti-microbial agent" means that the antimicrobial agent is connected to the "anti-microbial component" or "polynucleotide" by at least one chemical bond.

The term "at least one chemical bond" as used herein means at least one covalent, coordination, or ionic bond. The phrase "that associates, under the appropriate conditions" and similar

grammatical constructions as may be used herein refers to the conditions that are appropriate as known in the art for forming a silver nanocluster comprising at least 3 atoms of AgO with a polynucleotide scaffold. Determination and utilization of such conditions is considered to be within the skill of those in the art. The terms "anti-microbial agent" and "additional anti-microbial agent" as used herein refer to any agent that has microbiocidal and/or microbiostatic activity against a microorganism or microscopic lifecycle stage of an organism. Preferably an "antimicrobial agent" and/or an "additional anti-microbial agent" have microbiocidal, preferably bactericidal activity. Included are agents that have activity against viruses, bacteria, protists and fungi. Preferred anti-microbial agents used in the context of the present invention are anti-bacterial agents having bactericidal and/or bacteriostatic properties. An anti-microbial agent can be an organic or inorganic.

In the context of the present disclosure, "inhibiting the growth and/or proliferation" (and grammatical variations thereof) of at least one bacterial species refers to a measureable reduction in the number of bacteria present, and/or in the duration of the bacterial presence or infection. In some embodiments, "inhibiting the growth and/or proliferation" of a bacterial species is determined by comparative assay of the optical density at 600_nm over time, of a bacterial control culture vs. a bacterial culture treated with an antibacterial combination or composition as described herein.

The term "conjugate" and "conjugated" as used herein mean bound by at least one covalent bond. For example, a "conjugate" will comprise at least two components joined together by at least one covalent bond.

The terms "complex" and "complexed" as used herein means bound by at least one non-covalent chemical bond that may be a coordination bond or an ionic bond. For example, a "complex" will comprise at least two components joined together by at least one coordination bond or by at least one ionic bond or that are physically associated by other molecular interactions, such as but not limited to pi pi stacking.

The term "polynucleotide scaffold" as used herein refers to a polynucleotide sequence that links a targeting component as described herein to an antimicrobial agent as described herein. In some embodiments a polynucleotide scaffold may be designed to minimize steric conflicts between the targeting component, or at least a portion of a targeting component, and an anti-microbial agent. The polynucleotide scaffold also functions to stabilize nanociusters and /or retain bound metal ions. The polynucleotide may enhance the specificity of the targeting component. In some embodiments the polynucleotide scaffold, or at least a portion of the polynucleotide scaffold, interacts and/or associates with the targeting component and contributes to the binding of a target cell or molecule by the targeting component. In some embodiments a polynucleotide scaffold may form secondary structures either alone or in combination with other nucleic acid residues in an anti-microbial combination as described herein. For example, the nucleic acid residues in the polynucleotide scaffold may interact with the nucleic acid residues in a targeting component. In some embodiments the polynucleotide scaffold forms an i-motif, or at least a portion of an i-motif.

The term "i-motif" as used herein refers to a polynucleotide sequence that is cytosine rich whose core is arranged as two parallel duplexes that are inter-chelated in an anti- parallel manner. An "i-motif" can be formed by one strand, or two strands or four strands spatially arranged to form the "i-motif" core. The "i-motif" structure may be stabilised by acidic pH or bound metal ions such as silver or copper. Polynucleotides adopting the "i-motif" structure can be identified on the basis of their spectroscopic properties, in particular their circular dichroism spectra, enhance fluorescence emission upon binding the dye neutral red, and UV difference spectra following thermal denaturation or metal ion binding. The spectral properties of i-motif forming polynucleotides are well reported in the literature [23], [24], [25].

The term "i-motif" is used interchangeably herein with the term "i-motif structure" and means the same thing. In particular, for the purposes of this specification, the terms "imotif", "i-motif", "i-motif structure" and grammatical variations thereof mean a polynucleotide or polynucleotides that is/ar silver stabilised to form an i-motif (also termed a "silver stabilized i-motif" herein) in which the polnucleotide(s) exist in free solution at neutral pH in a configuration that is not a fully folded i-motif. The addition of Ag⁺ alters the conformation of the polynucleotide(s) stabilizing the

polynucleotide(s) to form a silver stabilised n i-motif. In some embodiments where the i-motif is formed from a single polynucleotide (i.e., a single nucleic acid strand) the addition of Ag⁺ bends the polynucleotide into the fully folded i-motif.

The term "silver nanocluster" as used herein means a collection of 3 to 50 silver atoms (Ag) that is formed by the reduction of silver ions and stabilized by interaction with a polynucleotide scaffold. In some embodiments the polynucleotide scaffold is a DNA scaffold.

The term "silver ion" as used herein means Ag⁺. The term "silver metal" as used herein means Ag.

The term "aptamer-Ag⁺" as used herein means aptamers that form i-motifs that remain stably associated with Ag⁺.

The term "statistically significant" as used herein refers to the likelihood that a result or relationship is caused by something other than random chance. A result may be found to be statistically significant using statistical hypothesis testing as known and used in the art. Statistical hypothesis testing provides a "P-value" as known in the art, which represents the probability that the measured result is due to random chance alone. It is believed to be generally accepted in the art that levels of significance of 5% (0.05) (e.g., the observed result occurs by chance less than 5% of the time) or lower are considered to be strongly statistically significant, and P values between 0.2 and 0.05 are considered to be weakly significant. As used herein, the terms "treat", "treating" and "treatment" refer to therapeutic measures which reduce, alleviate, ameliorate, manage, prevent, restrain, stop or reverse microbial infection including the symptoms associated with or related to microbial infection. The subject may show observable or measurable (statistically significant) decrease in one or more of the symptoms associated with or related microbial infection as known to those skilled in the art, as indicating improvement.

The term "effective amount" as used herein means an amount effective to protect against, delay, reduce, stabilise, improve or treat microbial infection as known in the art, and/or as described herein. In particular, an "effective amount" of a targeting component, an anti-microbial component and/or an anti-microbial combination as described is an amount that is sufficient to achieve at least a lessening of the symptoms associated with a microbial infection that is being or is to be treated or that is sufficient to achieve a reduction in microbial growth, or that is sufficient to increase in microbial susceptibility to other therapeutic agents or natural immune clearance.

In some embodiments, an effective amount is an amount sufficient to achieve a statistically different result as compared to an untreated control.

A "formulation agent" as used herein refers to any compound or material that facilitates or optimizes the production, handling, storage, transport, application and/or persistence of a targeting component, an anti-microbial component, an anti-microbial combination, and or a composition of, or for use in the invention, but not limited thereto.

"Subject" as used herein is an animal, preferably a vertebrate animal or an

invertebrate animal. Preferably the vertebrate animal is a mammal. Preferably the mammal includes human and non-human mammals including but not limited to cats, dogs, horses, cows, sheep, deer, mice, rats, primates (including gorillas, rhesus monkeys and chimpanzees), pigs, possums and other domestic farm or zoo animals, but not limited thereto. Preferably, the mammal is human. The term "about" when used in connection with a referenced numeric indication means the referenced numeric indication plus or minus up to 10% of that referenced numeric indication. For example, "about 100" means from 90 to 110 and "about six" means from 5.4 to 6.6.

The term "comprising" and grammatical variations thereof as used in this specification and claims means "consisting at least in part of". When interpreting statements in this specification, and claims which include the term "comprising", it is to be understood that other features that are additional to the features prefaced by this term in each statement or claim may also be present. Related terms such as "comprise" and "comprised" are to be interpreted in similar manner.

The term "consisting essentially of" and grammatical variations thereof as used herein means the specified materials or steps and those that do not materially affect the basic and novel characteristic(s) of the claimed invention.

The term "consisting of" and grammatical variations thereof as used herein means the specified materials or steps of the claimed invention, excluding any element, step, or ingredient not specified in the claim.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates generally to the inventors unexpected identification of anti-microbial combinations that comprise at least one polynucleotide component in association with at least one anti-microbial agent. In particular the inventors have identified that a polynucleotide that specifically binds a molecule or cell can be combined with a polynucleotide that associates with an anti-microbial agent into an anti-microbial combination to deliver the anti-microbial agent directly to the target molecule or cell.

In some embodiments of the anti-microbial combination, a polynucleotide that specifically binds a molecule or cell is termed a "targeting component" as described herein. In some embodiments of the anti-microbial combination, a polynucleotide that associates with an anti-microbial agent is termed an "anti-microbial component" as described herein.

In one non-limiting example, an anti-microbial combination comprises a targeting component that is an aptamer or functional fragment thereof that specifically binds Pseudomonas aeruginosa and an anti-microbial component comprising a nucleic acid sequence encoding a polynucleotide scaffold that associates, under the appropriate conditions, with sufficient silver ions to form a DNA scaffolded silver nanocluster upon reduction The silver in the nanocluster in the anti-microbial combination is

predominantly in the form of silver metal (Ag). In some embodiments the nanocluster may comprise associated silver ions. In another non-limiting example, an anti-microbial combination comprises a targeting component that is an aptamer or functional fragment thereof that specifically binds Pseudomonas aeruginosa and an anti-microbial component that is a polynucleotide scaffold that forms, or that forms part of, an i-motif where the i-motif associates, under the appropriate conditions, with at least one silver (Ag⁺) or copper (Cu⁺, Cu⁺⁺) ion.

An important aspect of the inventors work is their unexpected determination that a targeting component as described herein, particularly an aptamer or functional fragment thereof that specifically binds to a pathogenic bacterial cell, can potentiate the activity and/or efficacy of other anti-microbial agents. Surprisingly, the inventors have identified that the anti-microbial components, anti-microbial combinations and compositions described herein have unexpectedly high efficacy for killing bacteria, particularly Pseudomonas spp. bacteria.

To the best of the inventors' knowledge, particular targeting components, antimicrobial components, anti-microbial combinations, and compositions comprising such as described herein are unknown in the art. In particular, the inventor's believe they are the first to employ the following combinations, at least:

• a targeting component comprising a polynucleotide that specifically targets a molecule or cell, and

• an anti-microbial component comprising o a nucleic acid sequence encoding a polynucleotide scaffold that

associates, under the appropriate conditions, with sufficient silver ions (Ag⁺) to form, upon reduction, a silver nanocluster comprising at least 3 Ag, or o a nucleic acid sequence encoding a polynucleotide scaffold that forms, or that forms part of, an i-motif, that associates under the appropriate conditions with silver ions, as an anti-microbial, particularly as an anti-bacterial combination.

The inventors further believe that they are the first to identify that polynucleotide that forms, or that forms part of an i-motif, when complexed with at least one silver or copper ion, is effective therapeutically when used alone as an anti-microbial, particularly an anti-bacterial agent. The inventors have also identified that an antimicrobial component that is an i-motif-Ag⁺ combination (also termed an "imotif alone") herein can be used to inhibit the growth and/or proliferation of

microorganisms, particularly bacteria, and therapeutically to treat microbial, particularly bacterial infection. Additionally, the inventors disclose herein how combining such an anti-microbial component with a targeting component as described herein provides an effective anti-microbial combination.

Aptamers

Disclosed herein is the selection and characterisation of DNA aptamers which bind with a high affinity and specificity to live biofilm derived P. aeruginosa cells. These aptamers or a functional fragment thereof may be used in or as a targeting

component as described herein to deliver anti-microbial components as described herein to the site of microbial, and particularly of Pseudomonas infection. In some embodiments, the anti-microbial components themselves, as described herein, may be used alone in therapeutic applications that do not require specific targeting, for example, topical applications, but not limited thereto.

The inventor's further believe that they are the first to provide a nti -Pseudomonas spp. aptamers that bind to live Pseudomonas spp. cells. A person of skill in the art will appreciate that the targeting component of an anti-microbial combination as described herein may be an aptamer or a functional fragment thereof as described herein that specifically binds target bacteria, particularly Pseudomonas spp. bacteria, and more particularly Pseudomonas aeruginosa.

Aptamers show potential as new therapeutics against pathogenic bacteria. They have been shown to have bacteriostatic effects against Salmonella [3], and inhibitory properties against Mycobacterium [4]. Aptamers are short, single stranded DNA or RNA oligonucleotides that can be selected to a wide variety of microbial targets including viruses [5] and bacteria [6, 7]. Aptamers are made by a Systematic

Evolution of Ligands by Exponential Enrichment (SELEX) approach [8, 9], and this process can be modified to select aptamers to whole bacteria. Aptamers are relatively non-immunogenic and non-toxic due to their small size, and can be rapidly chemically synthesised and modified, making them an interesting candidate for new drug development. Aptamers form stable secondary structures which allows them to bind to their target with high affinity and specificity, and potentially cause a therapeutic effect. The aptamers described herein are made by chemical synthesis. However, a person of skill in the art recognizes that aptamers as described herein may be made by other means as known in the art, for example, by enzymatic synthesis in vitro or in vivo expression in a permissive cell, but not limited thereto. What is important is that based on the disclosure herein by the inventors of the primary nucleotide sequences of the aptamers as described herein, a skilled worker can make any of the aptamers as described herein as known in the art, including any aptamer or a functional fragment thereof having one or more modifications to that aptamer as described herein as would be appropriate in the context of the invention and/or for use in the present invention.

Based in part on the long felt need for new anti-microbial agents which will overcome at least some of the limitations of currently used antibiotics, the inventors provide, among other things, "aptabiotics". Aptabiotics comprise bacterial species-specific polynucleotide aptamers or a functional fragment thereof, that specifically bind to bacteria, particularly Gram-negative bacteria, and that are complexed with antimicrobial components as described herein. In some embodiments the anti-microbial components comprise polynucleotide scaffolds comprising silver nanoclusters (AgNC) . In some embodiments the anti-microbial components comprise polynucleotide scaffolds that form, or that form part of, an i-motif. In one embodiment an aptabiotic specifically binds live whole bacterial cells. In one embodiment the live whole bacterial cells are Pseudomonas spp. cells, preferably P. aeruginosa cells.

The inventors have unexpectedly found that aptabiotics can be used to deliver AgNCs and i-motif/ Ag⁺ complexes to specifically targeted living bacteria to render such otherwise multi-drug resistant bacteria, susceptible to antibiotic action. Aptabiotics, targeting components and/or anti-microbial components as described herein may have both intrinsic anti-microbial activity and synergistic activity with existing

fluoroquinolone antibiotics that are used clinically to treat pathogenic bacterial infections, particularly Pseudomonas spp. infections, particularly P. aeruginosa infections [10]. Without wishing to be bound by theory the inventors believe that the therapeutic, anti-microbial use of aptabiotics, targeting components and/or antimicrobial components provides a generic approach for targeted anti-microbial therapy applicable to many pathogenic microorganisms, particularly bacteria. In particular, the inventors believe that the aptabiotics disclosed herein have synergistic activity generally with existing antibiotics that are used clinically, particularly with

Pseudomonas spp. infections, particularly P. aeruginosa infections. The anti-microbial activity of silver ions and silver nanoparticles is well reported [11- 13]. Silver ions are also able to synergistically increase the efficacy of many antibiotics [14], however, the medicinal use of silver has been limited as it has not been possible to restrict its toxicity to just bacterial cells at the infection site. Aptabiotics are a novel therapeutic that specifically deliver a toxic dose of silver to target cell. Additionally, aptabiotics augment the activity of existing antibiotics, while the circumventing resistance mediated by multi-drug resistance efflux pumps, as neither the silver metal nor silver ions are extruded by theses pumps.

The mechanism of silver metal toxicity is still controversial but it is generally accepted that the major mode of action for silver ions is reaction with cellular thiols, particularly glutathione, to deplete antioxidant defences [11, 12]. It is also generally accepted that silver nanoparticles and silver ions increase membrane permeability, react with cellular thiols, disrupt electron transport by the respiratory chain and bind to DNA.

Polynucleotide scaffolded AgNCs are small clusters of silver atoms stabilised by polynucleotides, with a well-defined, small (3-50) number of silver atoms, that exhibit bright fluorescence. Disclosed herein are AgNCs (Fig. 1) with tuneable fluorescence properties that depend on the sequence of the associated polynucleotide scaffolding.

Recent work has also demonstrated that silver ions can be associated with

polynucleotides to form high order secondary structures termed i-motifs. Such structures may be formed by polydeoxynucleotide sequences rich in cytosine and are four stranded nucleic acid secondary structures comprising two parallel duplexes hydrogen bonded in an antiparallel orientation by intercalated cytosine=-cytosine base pairs [21, 28, 29, 30]. I-motif DNA structures have been used for various

nanotechnological applications, however the inventor's believe they are the first to recognize and employ, in any therapeutic embodiment, the anti-microbial, and particularly anti-bacterial properties of i-motif polynucleotides, particularly i-motif DNAs, associated with silver or copper ions. An anti-microbial combination comprising an aptamer or functional fragment thereof as described herein can specifically bind a target molecule or cell, particularly a live target bacterial cell (including Pseudomonas aeruginosa) and deliver silver nanoclusters or silver ions that disrupt membrane integrity and cellular activities. The anti-microbial combination described herein also inhibits growth of bacteria in liquid culture, and biofilm formation, and synergizes with antibiotics, particularly floroquinone antibiotics such as Ciprofloxacin, to enhance antibiotic efficacy. Although silver nanoparticles (colloidal silver) are used in wound dressing and fabrics, and silver nano-coatings are used in medical implants, to the best of the inventor's knowledge, neither the targeted delivery of silver metal (Ag) in the form of nanoclusters or nanoparticles, nor the targeted delivery of silver ions (Ag⁺) complexed to i-motif polynucleotide scaffolds, to bacteria, using aptamers has been previously reported.

The aptabiotics described herein are novel anti-microbial agents when used directly to inhibit the growth of a targeted microbial pathogen, (for example, P. aeruginosa but not limited thereto). Additionally and importantly, the aptabiotics as described herein may restore sensitivity to antibiotics of a targeted pathogen where that pathogen was previously resistant to those antibiotics) (for example, where the targeted pathogen is P.

aeruginosa, but not limited thereto).

ANTI-MICROBIAL COMBINATIONS In a first aspect the invention relates to an anti-microbial combination comprising a polynucleotide comprising a targeting component and an anti-microbial component, wherein the targeting component specifically binds a target molecule or cell, and wherein the anti-microbial component comprises a polynucleotide in association with an anti-microbial agent. Targeting component

In one embodiment the targeting component comprises a polynucleotide or functional fragment thereof that specifically binds the target molecule or cell. In one

embodiment the targeting component consists essentially of a polynucleotide or functional fragment thereof that specifically binds the target molecule or cell. In one embodiment the targeting component consists of a polynucleotide or functional fragment thereof that specifically binds the target molecule or cell.

In some embodiments the targeting component comprises a polynucleotide or functional fragment thereof that is a single stranded nucleic acid and may be either single stranded RNA or DNA. Preferably the polynucleotide or functional fragment thereof is DNA. In some embodiments, the targeting component comprises a polynucleotide or functional fragment thereof that comprises a ribonucleotide, deoxyribonucleotide, or other type of nucleic acid, or two or more different types of nucleic acids. In some embodiments the targeting component comprises a polynucleotide or functional fragment thereof that comprises one or more modified bases, sugars, polyethylene glycol spacers or backbone modifications. In some embodiments, the targeting component comprises a polynucleotide or functional fragment thereof that comprises one or more 2' sugar modifications, such as a 2'-0- alkyl (e.g., 2'-0-methyl or 2'-0- methoxyethyl) or a 2'-fluoro modification, but not limited thereto. In one embodiment the targeting component comprises a polynucleotide or functional fragment thereof that comprises a nucleic acid sequence that specifically binds the target molecule or cell. In one embodiment the nucleic acid sequence is an

oligonucleotide. In one embodiment the oligonucleotide is selected from the group consisting of an antisense oligonucleotides, shRNAs, siRNAs and aptamers. In one embodiment the targeting component comprises an aptamer or a functional fragment thereof. In one embodiment the targeting component consists essentially of an aptamer or a functional fragment thereof. In one embodiment the targeting component consists of an aptamer or a functional fragment thereof.

In some embodiments, the polynucleotide, aptamer or functional fragment thereof is about 120, about 115, about 110, about 105, about 100, about 95, about 90, about 85, about 80, about 75, about 70, about 65, about 60, about 55, about 50, about 45, about 40, about 35, about 30, about 25, about 20, about 15, or about 10 nucleotides in length.

In some embodiments the polynucleotide, aptamer or functional fragment thereof is 120, 110, 100, 90, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, or 10 nucleotides in length.

In some embodiments, the polynucleotide, aptamer or functional fragment thereof is less than 120, less than 115, less than 110, less than 105, less than 100, less than 95, less than 90, less than 85, less than 80, less than 75, less than 70, less than 65, less than 60, less than 55, less than 50, less than 45, less than 40, less than 35, less than 30, less than 25, less than 20, less than 15, or less than 10 nucleotides in length In some embodiments the polynucleotide, aptamer or functional fragment thereof is about 110 to about 120, about 100 to about 110, about 90 to about 100, about 80 to about 90, about 70 to 80, about 60 to 70, about 50 to 60, about 40 to 50, about 30 to 40, about 20 to 30, or about 10 to 20 nucleotides in length. In some embodiments the polynucleotide, aptamer or functional fragment thereof is 110 to 120, 100 to 110, 90 to 100, 80 to 90, 70 to 80, 60 to 70, 50 to 60, 40 to 50, 30 to 40, 20 to 30, or 10 to 20 nucleotides in length.

In some embodiments the polynucleotide, aptamer or functional fragment thereof is about 105 to about 115, about 95 to about 105, about 85 to about 95, about 75 to 85, about 65 to 75, about 55 to 65, about 45 to 55, about 35 to 45, about 25 to 35, or about 15 to 25 nucleotides in length.

In some embodiments the polynucleotide, aptamer or functional fragment thereof is 105 to 115, 95 to 105, 85 to 95, 75 to 85, 65 to 75, 55 to 65, 45 to 55, 35 to 45, 25 to 35, or 15 to 25 nucleotides in length. In some embodiments the polynucleotide, aptamer or functional fragment thereof is about 90 to about 80 or about 88 to about 82 or about 86 to about 84, or about 86, or about 85 or about 84 nucleotides in length, preferably 90 to 80 or 88 to 82 or 86 to 84, or 86, or 85 or 84 nucleotides in length.

In some embodiments, the polynucleotide, aptamer or functional fragment thereof is about 40 to about 50 or about 42 to about 48 or about 44 to about 46, or about 43 or about 45 nucleotides in length, preferably 40 to 50 or 42 to 48 or 44 to 46 or 43 or 45 nucleotides in length.

In some embodiments, the polynucleotide, aptamer or functional fragment thereof is about 30 to about 40 or about 32 to about 38 or about 34 to about 36, or about 32 or about 35 or about 36 or about 38 nucleotides in length, preferably 30 to 40 or 32 to 38 or 34 to 36, or 32 or 35 or 36 or 38 nucleotides in length.

In some embodiments the polynucleotide, aptamer or functional fragment thereof is about 20 to about 30, or about 22 to about 28 or about 24 to about 26 or about 24 or about 25 or about 26 nucleotides in length, preferably 20 to 30, or 22 to 28 or 24 to 26 or 24 or 25 or 26 nucleotides in length. A person of skill in the art will appreciate that the polynucleotide, aptamer or functional fragment thereof may be any length polynucleotide, aptamer or functional fragment thereof that falls within the size parameters set out herein. By way of non- limiting example a polynucleotide, aptamer or functional fragment thereof may be about 86, about 85, about 76, about 61, about 54, about 43, about 38, about 32, or about 27 nucleotides in length or may be 84, 78, 70, 58, 43, 35, or 26 nucleotides in length. What is important is that the polynucleotide, aptamer or functional fragment thereof specifically binds the target molecule or cell.

In one embodiment the aptamer or functional fragment thereof comprises a polynucleotide having a least 70% nucleic acid sequence identity to a polynucleotide selected from the group consisting of SEQ ID NO: 1 to 30. Preferably the aptamer or a functional fragment thereof comprises a polynucleotide having at least 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% nucleic acid sequence identity to a polynucleotide selected from the group consisting of SEQ ID NOs: 1 to 30. In one embodiment the aptamer or functional fragment thereof comprises a polynucleotide selected from the group consisting of SEQ ID NO: 1 to 30.

In one embodiment the aptamer or functional fragment thereof consists essentially of a polynucleotide having a least 70% nucleic acid sequence identity to a polynucleotide selected from the group consisting of SEQ ID NO: 1 to 30. Preferably the aptamer or a functional fragment thereof consists essentially of a polynucleotide having at least 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% nucleic acid sequence identity to a polynucleotide selected from the group consisting of SEQ ID NOs: 1 to 30. In one embodiment the aptamer or functional fragment thereof consists essentially of a polynucleotide selected from the group consisting of SEQ ID NO: 1 to 30.

In one embodiment the aptamer or functional fragment thereof consists of a polynucleotide having a least 70% nucleic acid sequence identity to a polynucleotide selected from the group consisting of SEQ ID NO: 1 to 30. Preferably the aptamer or a functional fragment thereof consists of a polynucleotide having at least 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% nucleic acid sequence identity to a polynucleotide selected from the group consisting of SEQ ID NOs: 1 to 30. In one embodiment the aptamer or functional fragment thereof consists of a polynucleotide selected from the group consisting of SEQ ID NO: 1 to 30.

In one embodiment the aptamer is selected from the group consisting of JN27 (SEQ ID NO: 1); JN08 (SEQ ID NO: 2); JN27.SH (SEQ ID NO: 3); JN27 5' NC (SEQ ID NO: 4); JN27 3' NC (SEQ ID NO: 5);JN27 split NC (SEQ ID NO: 6); JN08.SH (SEQ ID NO: 7); JN08 5' NC (SEQ ID NO: 8); JN08 3' NC (SEQ ID NO: 9); JN08 split NC (SEQ ID NO: 10); JN21.SH (SEQ ID NO: 11); JN21 5' NC (SEQ ID NO: 12); JN21 3' NC (SEQ ID NO: 13); JN21 split NC (SEQ ID NO: 14); JN 17.SH (SEQ ID NO: 15); JN 17 5' SH (SEQ ID NO: 16); JN 17 3' SH (SEQ ID NO: 17); JN 17 split NC (SEQ ID NO: 18); St21Lpl7 (SEQ ID NO: 19); St21Lpl7 5' NC (SEQ ID NO: 20); St21Lpl7 3' NC (SEQ ID NO:

21); St21Lpl7 split NC (SEQ ID NO: 22); Stl7Lp21 (SEQ ID NO: 23); Stl7Lp21 5' NC (SEQ ID NO: 24); Stl7Lp21 3' NC (SEQ ID NO: 25); Stl7Lp21 split NC (SEQ ID NO: 26); St08Lpl7 (SEQ ID NO: 27); St08Lpl7 5' NC (SEQ ID NO: 28); St08Lpl7 3' NC (SEQ ID NO: 29) and St08Lpl7 split NC (SEQ ID NO: 30). In some embodiments the aptamer is a functional fragment of any one of JN27 (SEQ ID NO: 1); JN08 (SEQ ID NO: 2); JN27.SH (SEQ ID NO: 3); JN27 5' NC (SEQ ID NO: 4); JN27 3' NC (SEQ ID NO: 5);JN27 split NC (SEQ ID NO: 6); JN08.SH (SEQ ID NO: 7); JN08 5' NC (SEQ ID NO: 8); JN08 3' NC (SEQ ID NO: 9); JN08 split NC (SEQ ID NO: 10); JN21.SH (SEQ ID NO: 11); JN21 5' NC (SEQ ID NO: 12); JN21 3' NC (SEQ ID NO: 13); JN21 split NC (SEQ ID NO: 14); JN 17.SH (SEQ ID NO: 15); JN 17 5' SH (SEQ ID NO: 16); JN 17 3' SH (SEQ ID NO: 17); JN 17 split NC (SEQ ID NO: 18);

St21Lpl7 (SEQ ID NO: 19); St21Lpl7 5' NC (SEQ ID NO: 20); St21Lpl7 3' NC (SEQ ID NO: 21); St21Lpl7 split NC (SEQ ID NO: 22); Stl7Lp21 (SEQ ID NO: 23);

Stl7Lp21 5' NC (SEQ ID NO: 24); Stl7Lp21 3' NC (SEQ ID NO: 25); Stl7Lp21 split NC (SEQ ID NO: 26); St08Lpl7 (SEQ ID NO: 27); St08Lpl7 5' NC (SEQ ID NO: 28); St08Lpl7 3' NC (SEQ ID NO: 29) or St08Lpl7 split NC (SEQ ID NO: 30).

JN 17 (SEQ ID NO: 1); JN 17-SH (SEQ ID NO: 2) ; JN21 (SEQ ID NO: 3); JN21-SH (SEQ ID NO: 4); JN27 (SEQ ID NO: 5) and JN27-SH (SEQ ID NO: 6).

In one embodiment, the aptamer is about 86 nucleotides in length and comprises SEQ ID NO: 1 or a functional fragment thereof. In one embodiment, the aptamer is about 86 nucleotides in length and consists essentially of SEQ ID NO: 1 or a functional fragment thereof. In one embodiment, the aptamer is about 86 nucleotides in length and consists of SEQ ID NO: 1 or a functional fragment thereof. In one embodiment, the aptamer is 86 nucleotides in length and comprises SEQ ID NO: 1 or a functional fragment thereof. In one embodiment, the aptamer is 86 nucleotides in length and consists essentially of SEQ ID NO: 1 or a functional fragment thereof. In one embodiment, the aptamer is 86 nucleotides in length and consists of SEQ ID NO: 1 or a functional fragment thereof.

In one embodiment, the aptamer is about 85 nucleotides in length and comprises SEQ ID NO: 2 or a functional fragment thereof. In one embodiment, the aptamer is about 85 nucleotides in length and consists essentially of SEQ ID NO: 2 or a functional fragment thereof. In one embodiment, the aptamer is about 85 nucleotides in length and consists of SEQ ID NO: 2 or a functional fragment thereof.

In one embodiment, the aptamer is 85 nucleotides in length and comprises SEQ ID NO: 2 or a functional fragment thereof. In one embodiment, the aptamer is 85 nucleotides in length and consists essentially of SEQ ID NO: 2 or a functional fragment thereof. In one embodiment, the aptamer is 85 nucleotides in length and consists of SEQ ID NO: 2 or a functional fragment thereof.

In one embodiment, the aptamer is about 43 nucleotides in length and comprises SEQ ID NO: 3 or a functional fragment thereof. In one embodiment, the aptamer is about 43 nucleotides in length and consists essentially of SEQ ID NO: 3 or a functional fragment thereof. In one embodiment, the aptamer is about 43 nucleotides in length and consists of SEQ ID NO: 3 or a functional fragment thereof.

In one embodiment, the aptamer is 43 nucleotides in length and comprises SEQ ID NO: 3 or a functional fragment thereof. In one embodiment, the aptamer is 43 nucleotides in length and consists essentially of SEQ ID NO: 3 or a functional fragment thereof. In one embodiment, the aptamer is 43 nucleotides in length and consists of SEQ ID NO: 3 or a functional fragment thereof.

In one embodiment, the aptamer is about 26 nucleotides in length and comprises SEQ ID NO: 7 or a functional fragment thereof. In one embodiment, the aptamer is about 26 nucleotides in length and consists essentially of SEQ ID NO: 7 or a functional fragment thereof. In one embodiment, the aptamer is about 26 nucleotides in length and consists of SEQ ID NO: 7 or a functional fragment thereof.

In one embodiment, the aptamer is 26 nucleotides in length and comprises SEQ ID NO: 7 or a functional fragment thereof. In one embodiment, the aptamer is 26 nucleotides in length and consists essentially of SEQ ID NO: 7 or a functional fragment thereof. In one embodiment, the aptamer is 26 nucleotides in length and consists of SEQ ID NO: 7 or a functional fragment thereof.

In one embodiment, the aptamer is about 45 nucleotides in length and comprises SEQ ID NO: 11 or a functional fragment thereof. In one embodiment, the aptamer is about 45 nucleotides in length and consists essentially of SEQ ID NO: 11 or a functional fragment thereof. In one embodiment, the aptamer is about 45 nucleotides in length and consists of SEQ ID NO: 11 or a functional fragment thereof.

In one embodiment, the aptamer is 45 nucleotides in length and comprises SEQ ID NO: 11 or a functional fragment thereof. In one embodiment, the aptamer is 45 nucleotides in length and consists essentially of SEQ ID NO: 11 or a functional fragment thereof. In one embodiment, the aptamer is 45 nucleotides in length and consists of SEQ ID NO: 11 or a functional fragment thereof.

In one embodiment, the aptamer is about 36 nucleotides in length and comprises SEQ ID NO: 15 or a functional fragment thereof. In one embodiment, the aptamer is about 36 nucleotides in length and consists essentially of SEQ ID NO: 15 or a functional fragment thereof. In one embodiment, the aptamer is about 36 nucleotides in length and consists of SEQ ID NO: 15 or a functional fragment thereof.

In one embodiment, the aptamer is 36 nucleotides in length and comprises SEQ ID NO: 15 or a functional fragment thereof. In one embodiment, the aptamer is 36 nucleotides in length and consists essentially of SEQ ID NO: 15 or a functional fragment thereof. In one embodiment, the aptamer is 36 nucleotides in length and consists of SEQ ID NO: 15 or a functional fragment thereof.

In one embodiment, the aptamer is about 38 nucleotides in length and comprises SEQ ID NO: 19 or a functional fragment thereof. In one embodiment, the aptamer is about 38 nucleotides in length and consists essentially of SEQ ID NO: 19 or a functional fragment thereof. In one embodiment, the aptamer is about 38 nucleotides in length and consists of SEQ ID NO: 19 or a functional fragment thereof.

In one embodiment, the aptamer is 38 nucleotides in length and comprises SEQ ID NO: 19 or a functional fragment thereof. In one embodiment, the aptamer is 38 nucleotides in length and consists essentially of SEQ ID NO: 19 or a functional fragment thereof. In one embodiment, the aptamer is 38 nucleotides in length and consists of SEQ ID NO: 19 or a functional fragment thereof.

In one embodiment, the aptamer is about 35 nucleotides in length and comprises SEQ ID NO: 23 or a functional fragment thereof. In one embodiment, the aptamer is about 35 nucleotides in length and consists essentially of SEQ ID NO: 23 or a functional fragment thereof. In one embodiment, the aptamer is about 35 nucleotides in length and consists of SEQ ID NO: 23 or a functional fragment thereof.

In one embodiment, the aptamer is 35 nucleotides in length and comprises SEQ ID NO: 23 or a functional fragment thereof. In one embodiment, the aptamer is 35 nucleotides in length and consists essentially of SEQ ID NO: 23 or a functional fragment thereof. In one embodiment, the aptamer is 35 nucleotides in length and consists of SEQ ID NO: 23 or a functional fragment thereof.

In one embodiment, the aptamer is about 32 nucleotides in length and comprises SEQ ID NO: 27 or a functional fragment thereof. In one embodiment, the aptamer is about 32 nucleotides in length and consists essentially of SEQ ID NO: 27 or a functional fragment thereof. In one embodiment, the aptamer is about 32 nucleotides in length and consists of SEQ ID NO: 27 or a functional fragment thereof.

In one embodiment, the aptamer is 32 nucleotides in length and comprises SEQ ID NO: 27 or a functional fragment thereof. In one embodiment, the aptamer is 32 nucleotides in length and consists essentially of SEQ ID NO: 27 or a functional fragment thereof. In one embodiment, the aptamer is 32 nucleotides in length and consists of SEQ ID NO: 27 or a functional fragment thereof.

A polynucleotide, aptamer or functional fragment thereof of the invention may have intrinsic anti-microbial activity, particularly bacteriostatic or bactericidal activity. However, the inventors have also found that the intrinsic activity of the

polynucleotide, aptamer or functional fragment thereof may be potentiated by combining the polynucleotide, aptamer or functional fragment thereof with an antimicrobial component as described herein to form an anti-microbial combination of the invention. Specific binding

In one embodiment the targeting component specifically binds the target molecule or cell with a binding affinity of Kd = about 10 μΜ, preferably about 5 μΜ, preferably about 1 μΜ, preferably about 0.5 μΜ. In one embodiment the binding affinity is Kd = 10 μΜ, preferably 5 μΜ, preferably 1 μΜ, preferably 0.5 μΜ. In one embodiment the binding affinity is Kd = less than 10 μΜ, preferably less than 5 μΜ, preferably less than 1 μΜ, preferably less than 0.5 μΜ. Target molecule or cell

In one embodiment the target molecule is a molecule on a cell. In one embodiment the cell is a microorganism. In one embodiment the microorganism is selected from the group consisting of fungi, viruses, bacteria, and protists. In one embodiment the target microorganism is a living microorganism. In one embodiment the living microorganism is comprised in a biofilm. In one embodiment the living

microorganism is a bacteria, preferably a Gram negative bacteria, preferably

Pseudomonas spp., preferably Pseudomonas aeruginosa.

In one embodiment the target cell is a microbial cell selected from the group consisting of eukaryotic cells and prokaryotic cells. In one embodiment the target cell is a prokaryotic cell, preferably a bacterial cell, preferably a Gram negative bacterial cell, preferably a Pseudomonas spp. cell, preferably a Pseudomonas aeruginosa cell.

Polynucleotide scaffolds and anti-microbial agents

In one embodiment the polynucleotide in association with the anti-microbial agent is complexed to the anti-microbial agent by at least one chemical bond. In one embodiment the chemical bond is a coordination bond or an ionic bond.

Polynucleotide scaffold

In one embodiment the polynucleotide in association with the anti-microbial agent is, or comprises a nucleic acid sequence that encodes a polynucleotide scaffold. In one embodiment the polynucleotide in association with the anti-microbial agent, is or consists essentially of a nucleic acid sequence that encodes a polynucleotide scaffold. In one embodiment the polynucleotide in association with the anti-microbial agent consists of a nucleic acid sequence that encodes a polynucleotide scaffold. In one embodiment the polynucleotide scaffold, or at least a portion of the polynucleotide scaffold, comprises a nucleic acid sequence that associates and/or interacts with a targeting component as described herein, and contributes to the binding of a target cell or molecule by the targeting component. In one embodiment the targeting component is an aptamer or a functional fragment thereof. In one embodiment the polynucleotide scaffold comprises a polynucleotide that is 5'- (CxN_y)z-3' wherein C = cytosine; N= any combination of adenosine (A), guanosine (G), thymidine (T), analogues of C, A, G and T, and modified deoxynucleosides; x = an integer from 2 to 7; y = an integer from 0 to 7; and z = an integer from 1 to 8. In one embodiment x is an integer that is 3, 4, 5, 6 or 7 and y is an integer that is 2, 3, 4 or 5. In one embodiment the polynucleotide scaffold comprises at least one additional nucleoside residue either 5' or 3' or both, of the nucleic acid sequence that associates with the ant-microbial agent.

In one embodiment the polynucleotide scaffold comprises a polynucleotide that is 5'- (CxN_y)z-3' wherein C = cytosine; N= any combination of adenosine (A), guanosine (G), thymidine (T), analogues of C, A, G and T, and modified deoxynucleosides; x = an integer from 2 to 7; y = an integer from 0 to 7; and z = an integer from 1 to at least

100, preferably at least 1000, preferably at least 10,000, preferably at least 100,000.

In one embodiment x is an integer that is 3, 4, 5, 6 or 7 and y is an integer that is 2, 3, 4 or 5. In one embodiment the polynucleotide scaffold comprises at least one additional nucleoside residue either 5' or 3' or both, of the nucleic acid sequence that associates with the ant-microbial agent.

In one embodiment the polynucleotide scaffold, or at least part of the polynucleotide scaffold is conjugated to the 5' end of the targeting component. In one embodiment the polynucleotide scaffold, or at least part of the polynucleotide scaffold is conjugated to the 3' end of the targeting component. In one embodiment a polynucleotide scaffold, or at least part of a polynucleotide scaffold is conjugated to the 5' end of the targeting component and a polynucleotide scaffold, or at least part of a polynucleotide scaffold, is conjugated to the 3' end of the targeting component. In one embodiment the polynucleotide scaffold is complexed with at least one atom of silver (Ag). In one embodiment the at least one atom of Ag is complexed with the polynucleotide scaffold by at least one coordination bond. In one embodiment the polynucleotide scaffold is complexed with a silver nanocluster (AgNC). In one embodiment the AgNC is complexed with the polynucleotide scaffold by at least one coordination bond.

In one embodiment a silver nanocluster comprises at least 3 silver atoms (Ag). In one embodiment a silver nanocluster comprises about 3 to 50 silver atoms, preferably 3 to 50 silver atoms. In some embodiments, a silver nanocluster comprises about 3 to about 12 atoms, preferably 3 to about 12 atoms, preferably about 3 to 12 atoms, preferably 3 to at least 12 atoms, preferably at least 3 to 12 atoms, preferably 3 to 12 atoms.

In one embodiment the polynucleotide scaffold comprises at least 4 nucleotides. In one embodiment the polynucleotide scaffold comprises at least 4 and less than 30 nucleotides. In one embodiment the polynucleotide scaffold comprises 4 to 30 nucleotides, preferably 4 to 29, preferably 4 to 28, preferably 4 to 27, preferably 4 to

26, preferably 4 to 25 nucleotides.

In one embodiment (C_xN_y) in the polynucleotide scaffold is at least 4 nucleotides. In one embodiment (C_xN_y) in the polynucleotide scaffold comprises at least 4 and less than 30 nucleotides. In one embodiment (C_xN_y) in the polynucleotide scaffold comprises 4 to 30 nucleotides, preferably 4 to 29, preferably 4 to 28, preferably 4 to

27, preferably 4 to 26, preferably 4 to 25 nucleotides.

In one embodiment the polynucleotide scaffold consists essentially of at least 4 nucleotides. In one embodiment the polynucleotide scaffold consists essentially of at least 4 and less than 30 nucleotides. In one embodiment the polynucleotide scaffold consists essentially of 4 to 30 nucleotides, preferably 4 to 29, preferably 4 to 28, preferably 4 to 27, preferably 4 to 26, preferably 4 to 25 nucleotides.

In one embodiment (C_xN_y) in the polynucleotide scaffold consists essentially of at least 4 and less than 30 nucleotides. In one embodiment (CxN_y) in the polynucleotide scaffold consists essentially of 4 to 30 nucleotides, preferably 4 to 29, preferably 4 to

28, preferably 4 to 27, preferably 4 to 26, preferably 4 to 25 nucleotides.

In one embodiment (CxN_y) in the polynucleotide scaffold consists of 4 and less than 30 nucleotides. In one embodiment (C_xN_y) in the polynucleotide scaffold consists of 4 to 30 nucleotides, preferably 4 to 29, preferably 4 to 28, preferably 4 to 27, preferably 4 to 26, preferably 4 to 25 nucleotides.

In one embodiment the DNA scaffold consists of at least 4 nucleotides. In one embodiment the DNA scaffold consists of at least 4 and less than 30 nucleotides. In one embodiment the DNA scaffold consists of 4 to 30 nucleotides, preferably 4 to 29, preferably 4 to 28, preferably 4 to 27, preferably 4 to 26, preferably 4 to 25 nucleotides.

In one embodiment the polynucleotide comprises about 10 nucleotides, preferably consists essentially of about 10 nucleotides, preferably consists of about 10 nucleotides. In one embodiment the polynucleotide comprises 10 nucleotides, preferably consists essentially of 10 nucleotides, preferably consists of 10 nucleotides.

In one embodiment the polynucleotide scaffold comprises SEQ ID NO: 31 or a functional fragment thereof. In one embodiment the polynucleotide scaffold consists essentially of SEQ ID NO: 31 or a functional fragment thereof. In one embodiment the polynucleotide scaffold consists of SEQ ID NO: 31 or a functional fragment thereof. i- motif

Under acidic conditions C-rich sequences can fold to form i-motifs that are four stranded structures comprising two parallel duplexes hydrogen bonded in an antiparallel orientation by interchelated cytosine to hemiprotonated cytosine base pairs [29], in which the strands may originate from a single or multiple molecules [30, 34]. i-motifs can also form at neutral pH particularly when cytosine to cytosine base pairing is stabilized by Ag⁺ ions [21]. Indeed, the reduction of Ag⁺ stabilized i-motif to a fluorescent nanocluster has been employed as the basis of a sensitive assay for the detection of Ag⁺ [35].

Without wishing to be bound by theory, the inventors believe that the C-rich regions within certain aptamer sequences are non-suited to form intra-strand i-motifs as they do not contain a suitable loop region to allow i-motif folding. However, the inventors have determined that modified aptamers containing a C-rich tract on both the 5' and 3' end of the DNA sequence can form an intra-strand i-motif by coming together with the addition of Ag⁺. Again without wishing to be bound by theory, the inventors believe that the polynucleotide scaffolds described herein, alone or combined with aptamers as described herein, will form dimer or tetramer inter-strand i-motifs stabilized by Ag⁺, with two or four separate sequences coming together. The CD spectra presented in example 3 are consistent with the inventor's

determination that the C-rich regions added to the aptamers of the invention as described herein formed i-motifs. The formation of imotif structures is demonstrated by the spectra observed in Figure 27, which shows the spectral shifts expected when these structures form as described herein, with the addition of silver. The CD spectra of an aptamer-i-motif with a classical i-motif sequence is presented for comparison (Fig . 27). The inventors believe that i-motif structures can be formed from polynucleotide sequences in many different ways, such as by varying the C-tract region nucleotides and length, as well as the nucleotides and lengths composing the bend in the i-motif structure. Without wishing to be bound by theory, the inventors believe that quite unexpectedly, and based solely on their work as described herein, any sequence that forms an i-motif at neutral and physiological pH due to the stabilising binding of Ag⁺ within the i-motif structure will have antimicrobial activity, particularly bacteriostatic and/or bactericidal activity.

The inventors have also determined that the formation of a silver stabilised i-motif enhances the antimicrobial activity of silver ions in an anti-microbial component as described herein, particularly as compared with silver ions alone. While the exact mechanism is unknown, the inventors believe that the imotif structure formed by a polynucleotide as described herein acts to facilitate the uptake of an anti-microbial component as described herein, by the bacteria. This increased uptake results in enhanced inhibition of the growth and/or proliferation of both Gram-positive and Gram-negative bacterial species, particularly a) Gram-negative bacterial species, preferably Pseudomonas spp., preferably P. aeruginosa; Salmonella spp., preferably S. enterica; Klebsiella spp., preferably K. pneumoniae; Enterobacter spp., preferably E. cloacae; Acinetobacter spp., preferably A. baumannii and Escherichia coli and b) Gram-positive bacterial species, preferably Listeria spp., preferably L. innocua; and/or Staphylococcus spp., preferably S. aureus or S. epididymis. Again without wishing to be bound by theory the inventors believe that based solely on their work as described herein, a skilled worker will appreciate that any DNA sequence able to form a silver stabilised i-motif is reasonably expected to have antimicrobial, particularly bacteriostatic and/or bactericidal activity.

Circular dichroism is a simple spectroscopy method for detecting the formation of i- motif structures in DNA and is the most accepted method for detecting i-motif formation in the literature. Other possible methods for i-motif detection include the interchelation of dyes and thermal difference spectroscopy. It was reasoned that the C-rich regions conjugated to the aptamers were not suited to form intra-strand i-motifs as they do not contain a suitable turn region to allow the strand to fold back on itself and form the i-motif structure and do not contain the four poly C regions required. However, without wishing to be bound by theory, the inventors believe there is the possibility that the aptamers containing a C-rich tract on both the 5' and 3' end of the DNA sequence. The stem region of the aptamer brings the 5' and 3' poly C tracts into juxtaposition being functionally equivalent to a turn region in a full iMotif. This 'split' half i-motif can form an intra-strand full i-motif upon the addition of Ag⁺ by dimerising, the resulting structure being a full iMotif formed by two strands containing two poly C regions separated by alternative turn structure. The possibility of dimer and tetramer inter-strand i-motifs stabilised by Ag⁺ are also possible with two or four separate C-rich sequences coming together. The ability of forming inter-strand i-motifs by dimerization was confirmed by the CD spectra of a half i-motif sequence (SEQ ID NO: 39), producing a spectra of a classic full i-motif upon the addition of silver by titration. If they were not able to dimerise to form i- motifs the CD spectra would have shown characteristics of a hairpin DNA structure, rather than the i-motif observed.

Quite unexpectedly, the inventors have determined that polynucleotide structures that form i-motifs by themselves are useful, when combined with an anti-microbial agent, as an anti-microbial therapeutic, either targeted to a particular cell or molecule with an aptamer or untargeted. Without wishing to be bound by theory, the inventors believe that chelation of the Ag⁺ into the i-motif makes the silver stabilized i-motif more suited for both topical applications and for use in internal medicine as the amount of free Ag⁺ is reduced, i-motif anti-microbial therapeutics as described herein are envisaged as useful for topical treatment of infections such as otitis externa, burn wounds and ulcers as well as treatment of infections in the cystic fibrosis lung, or in urinary tract.

The data provided herein demonstrates that i-motifs alone exhibit strong antimicrobial activity against a wide range of different bacterial species, supporting the utility of i- motifs as useful in broad spectrum antibiotic therapies. In some embodiments i- motifs may be provided in the form of a solution, emulsion, bound to carrier nanoparticles or ai hydrogel for topical application or for use as a wash or lavage. By way of non-limiting example, a solution comprising an anti-microbial agent that was an i-motif alone as described herein could be formulated at concentration of from 10 μΜ to 0.0001 μΜ active agent, depending on the application and addition of additives. The action of i-motifs alone can be enhanced by treating simultaneously with EDTA, a common wash in topical treatments before application of a drug. The inventors have unexpectedly found that the action of Ag⁺ stabilised i-motifs is strongly synergistic when imotifs alone are combined with EDTA for co-treatment. This synergistic action is seen against a large number of diverse Gram positive and negative bacterial species. Accordingly, in one aspect the invention relates to an anti-microbial component (AMC) comprising i) a polynucleotide that forms a metal nanocluster with at least three metal atoms, or a polynucleotide structure that chelates at least one metal ion, or both, and ii) an anti-microbial agent.

In another aspect the invention relates to the use of an anti-microbial component comprising i) a polynucleotide that forms a metal nanocluster with at least three metal atoms, or a polynucleotide structure that chelates at least one metal ion, or both, and ii) an anti-microbial agent to inhibit the growth and/or proliferation of at least one bacterial species.

In one embodiment i) is a polynucleotide structure that chelates at least one metal ion.

In one embodiment, the anti-microbial component comprises the polynucleotide structure in association with the anti-microbial agent, wherein the polynucleotide structure comprises an i-motif structure and the anti-microbial agent is at least one metal ion. In one embodiment the antimicrobial component consists essentially of, preferably consists of, the polynucleotide structure and the at least one metal ion.

In one embodiment the polynucleotide structure comprises, consists essentially of, or consists of a single nucleic acid strand that forms the i-motif structure.

In one embodiment the polynucleotide structure comprises, consists essentially of, or consists of at least two, preferably four, nucleic acid strands that form the imotif structure.

In one embodiment the imotif structure is formed under suitable conditions. In one embodiment the suitable conditions are conditions in which the nucleic acid strand or strands that form the imotif structure exist in free solution at neutral pH in a configuration that is not a full formed i-motif, wherein the addition of the at least one metal ion stabilizes the nucleic acid strand or strands to form the i-motif structure. In one embodiment the at least one metal ion is Cu²⁺ or Ag⁺, preferably Ag⁺.

In one embodiment the nucleic acid strand or strands that form the imotif structure comprise a nucleic acid sequence comprising at least one, preferably at least two, preferably four poly C tracts (pCts).

In one embodiment the nucleic acid strand or strands that form the imotif structure comprise a nucleic acid sequence comprising at least one pair of pCts separated by a turn region (TR), and have the formula (pCt-TR-pCt)n, wherein n is an integer from 1 to at least 10, preferably to at least 100, preferably to at least 1000, preferably to at least 10,000, preferably to at least 100,000.

In one embodiment the poly-C tracts are selected from the group consisiting of CCC, where m = ten

consecutive C nucleotide residues or more.

In one embodiment the turn regions compise only A and/or T nucleotide residues. In one embodiment the turn regions are selected from the group consisting of A, T, AA, TT, AT, TA, AAT, ATA, TAA, ATT, TTA, TTT, AAAA, AAAT, AATA, ATAA, TAAA, AATT, ATTA, TTAA, ATTT, TTTA, and ΤΠΤ.

In one embodiment the turn region is a targeting component as described herein. In one embodiment the turn region is an aptamer as described herein.

In one embodiment the poly-C tracts or turn regions or both are variant poly-C tracts or variant turn regions, wherein a variant poly-C tract or variant turn region differs from a poly-C tract or turn region by the inclusion of either a single G residue, or a doublet GG residue pair. Non-limiting examples of such nucleic acid strands having variant poly-C tracts and/or variant turn regions are provided in SEQ ID NO: 38-SEQ ID NO: 43.

In one embodiment the polynucleotide structure is formed from a nucleic acid strand comprising a concatamer of nucleic acid sequences having alternating pCts and TRs. In one embodiment the polynucleotide structure is formed from four separate nucleic acid strands each strand comprising at least one, preferably one, pCt.

In one embodiment the i-motif structure consists essentially of, preferably consists of, a single nucleic acid strand, or of at least two separate nucleic acid strands, or of four separate nucleic acid strands.

In one embodiment the polynucleotide or polynucleotide structure comprises a nucleic acid sequence that is 5'-(CxN_y)_z-3' wherein C = cytosine; N = any combination of adenosine (A), guanosine (G), thymidine (T), analogues of C, A, G and T, and modified deoxynucleosides; x = an integer from 2 to 7; y = an integer from 0 to 7; and z = an integer from 1 to 2, preferably from 1 to at least 10, preferably to at least 100, preferably to at least 1000, preferably to at least 10,000, preferably to at least 100,000.

In one embodiment x is an integer that is 3, 4, 5, 6 or 7 and y is an integer that is 2, 3, 4 or 5. In one embodiment the nucleic acid sequence that forms the i-motif, or at least part of the i-motif, comprises at least one additional nucleoside residue 5' or 3' of the nucleic acid sequence that forms the i-motif, or that forms part of the i-motif.

In one embodiment the polynucleotide structure comprises, preferably consists essentially of, preferably consists of, a nucleic acid selected from the group consisting of SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42 and SEQ ID NO: 43.

In one embodiment the at least one bacterial species is a Gram-negative bacterial species.

In one embodiment the Gram-negative bacterial species is a mammalian pathogen, preferably a human, horse or dog pathogen.

In one embodiment the Gram-negative bacterial species is selected from the group consisting of Pseudomonas spp. belonging to the P. aeruginosa group, P. chlolraphis group, P. fluorescens group, P. pertucinogena group, P. putida group, P. stutzeri group, or the P. syringae group, preferably wherein the Pseudomonas spp. is P. aeruginosa, P. florescens, P. putida, P. stutzeri, P. tabaci, P. angulata, P. phaseolicola, P. pisi, P. glycinea, P. solanacearum, P. caryophylli, P. cepacia, P. marginalis, P. savastonoi, P. marginata or P. syringae; Salmonella spp., preferably S. enterica; Klebsiella spp., preferably K. pneumoniae; Enterobacter spp., preferably E. cloacae; Acinetobacter spp., preferably A. baumannii and Escherichia coll.

In one embodiment the least one bacterial species is a Gram-positive bacterial species.

In one embodiment the Gram-positive bacterial species is a mammalian pathogen, preferably a human, horse or dog pathogen.

In one embodiment the Gram-positive bacterial species is a Listeria spp., preferably L. innocua; and/or a Staphylococcus spp., preferably S. aureus or S. epididymis. In another aspect the invention relates to the use of an anti-microbial component to inhibit the growth and/or proliferation of at least one bacterial species, wherein the anti-microbial component consists essentially of a polynucleotide structure in association with an anti-microbial agent, wherein the polynucleotide structure comprises an i-motif structure, wherein the anti-microbial agent is at least one metal ion, preferably at least one Ag⁺, wherein the imotif structure comprises a single nucleic acid strand that forms the i- motif structure or at least two, preferably four, nucleic acid strands that form the imotif structure, wherein the nucleic acid strand or strands that form the i-motif structure comprise a nucleic acid sequence comprising at least one poly C tract (pCts), wherein the bacterial species is selected from the group consisting of Pseudomonas aeruginosa, Salmonella enterica serovar typhi, Klebsiella pneumoniae, Enterobacter cloacae, Listeria innocua, Escherichia coli, Acinetobacter baumannii, Staphylococcus aureus, and Staphylococcus epididymis. In one embodiment the imotif structure comprises at least two, preferably four, separate nucleic acid strands that form the imotif structure.

The person of skill in the art appreciates that additionally, all of the embodiments set out herein that relate to the first aspect or any other aspect of the invention, including the anti-microbial component, and the use of the anti-microbial component to inhibit the growth and/or proliferation of at least one bacterial species, are also specifically contemplated herein as part of this aspect of the invention that relates to the use of an anti-microbial component to inhibit the growth and/or proliferation of at least one bacterial species, wherein the anti-microbial component consists essentially of a polynucleotide structure in association with an anti-microbial agent, and are specifically contemplated herein.

In another aspect the invention relates to the use of an anti-microbial component comprising i) a polynucleotide that forms a metal nanocluster with at least three metal atoms, or a polynucleotide structure that chelates at least one metal ion, or both, and an anti-microbial agent in the manufacture of a medicament for treating microbial infection, wherein the antimicrobial component comprises a polynucleotide in association with an anti-microbial agent, wherein the polynucleotide comprises an imotif structure and the anti-microbial agent is at least one metal ion.

The person of skill in the art appreciates that additionally, all of the embodiments set out herein that relate to the first aspect or any other aspect of the invention, including the anti-microbial component, and the use of the anti-microbial component to inhibit the growth and/or proliferation of at least one bacterial species, are also specifically contemplated herein as part of this aspect of the invention that relates to the use of an anti-microbial component in the manufacture of a medicament for treating microbial infection, and are specifically contemplated herein.

In another aspect the invention relates to a method of inhibiting the growth and/or proliferation of at least one bacterial species comprising contacting the at least one bacterial species with an anti-microbial component comprising i) a polynucleotide that forms a metal nanocluster with at least three metal atoms, or a polynucleotide structure that chelates at least one metal ion, or both, and ii) an anti-microbial agent, wherein the anti-microbial component comprises a polynucleotide in association with an anti-microbial agent, wherein the polynucleotide comprises an i-motif structure and the anti-microbial agent is at least one metal ion.

The person of skill in the art appreciates that additionally, all of the embodiments set out herein that relate to the first aspect or any other aspect of the invention, including the anti-microbial component, and the use of the anti-microbial component to inhibit the growth and/or proliferation of at least one bacterial species, are also specifically contemplated herein as part of this aspect of the invention that relates to the method of inhibiting the growth and/or proliferation of at least one bacterial species comprising contacting the at least one bacterial species with an anti-microbial component, and are specifically contemplated herein.

In another aspect the invention relates to a method of treating a microbial infection comprising administering an anti-microbial component comprising i) a polynucleotide that forms a metal nanocluster with at least three metal atoms, or a polynucleotide that chelates at least one metal ion, or both, and an anti-microbial agent to a subject in need thereof, wherein the anti-microbial component comprises a polynucleotide in association with an anti-microbial agent, wherein the polynucleotide comprises an imotif structure and the anti-microbial agent is at least one metal ion.

The person of skill in the art appreciates that additionally, all of the embodiments set out herein that relate to the first aspect or any other aspect of the invention, including the anti-microbial component, and the use of the anti-microbial component to inhibit the growth and/or proliferation of at least one bacterial species, are also specifically contemplated herein as part of this aspect of the invention that relates to the method of treating a microbial infection comprising administering an anti-microbial component, and are specifically contemplated herein.

In another aspect the invention relates to an anti-microbial component comprising i) a polynucleotide that forms a metal nanocluster with at least three metal atoms, or a polynucleotide structure that chelates at least one metal ion, or both, and ii) an anti-microbial agent for use in inhibiting the growth and/or proliferation of at least one bacterial species treating, or when used to treat, microbial infection, wherein the polynucleotide in i) comprises an i-motif structure and the anti-microbial agent in ii) is at least one metal ion.

The person of skill in the art appreciates that additionally, all of the embodiments set out herein that relate to the first aspect or any other aspect of the invention, including the anti-microbial component, and the use of the anti-microbial component to inhibit the growth and/or proliferation of at least one bacterial species, are also specifically contemplated herein as part of this aspect of the invention that relates to the antimicrobial component for use in inhibiting the growth and/or proliferation of at least one bacterial species treating, or when used to treat, microbial infection, and are specifically contemplated herein.

The following additional embodiments are further contemplated as specific

embodiments of any of the aspects set out herein, including aspects that relate to and/or encompass imotif structures and aspects that relate to and/or encompass antimicrobial combinations of formulas I, II and Ila, and including uses and methods of the anti-microbial components and anti-microbial combinations disclosed herein.

In one embodiment a polynucleotide structure as described herein either forms, or forms part of an "i-motif". i-motifs are four stranded nucleic acid secondary structures comprising two parallel duplexes hydrogen bonded in an antiparallel orientation by intercalated cytosine=-cytosine base pairs, i-motifs can be formed by

polydeoxynucleotide sequences rich in cytosine.

In one embodiment the polynucleotide in association with the anti-microbial agent comprises a polynucleotide scaffold. In one embodiment the polynucleotide scaffold comprises a polynucleotide that forms, or that forms part of, an i-motif. In one embodiment the polynucleotide forms, or forms part of, an i-motif that binds at least one metal ion. In one embodiment the at least one metal ion is selected from the group consisting of silver ions and copper ions, preferably silver ions. In one embodiment the polynucleotide that chelates at least one metal ion a nucleic acid sequence that forms, or that forms part of, an i-motif. In one embodiment the polynucleotide forms, or forms part of, an i-motif that binds at least one metal ion. In one embodiment the at least one metal ion is selected from the group consisting of silver ions and copper ions, preferably silver ions.

In one embodiment the polynucleotide that forms an i-motif, or that forms part of an i-motif comprises a polynucleotide that is 5'-(CxN_y)_z-3' wherein C = cytosine; N = any combination of adenosine (A), guanosine (G), thymidine (T), analogues of C, A, G and T, and modified deoxynucleosides; x = an integer from 2 to 7; y = an integer from 0 to 7; and z = an integer from 1 to 8.

In one embodiment the polynucleotide that forms an i-motif, or that forms part of an i-motif comprises a polynucleotide that is 5'-(CxN_y)_z-3' wherein C = cytosine; N = any combination of adenosine (A), guanosine (G), thymidine (T), analogues of C, A, G and T, and modified deoxynucleosides; x = an integer from 2 to 7; y = an integer from 0 to 7; and z = an integer from 1 to at least 100, preferably at least 1000, preferably at least 10,000, preferably at least 100,000.

In one embodiment a polynucleotide that forms an i-motif comprises a nucleic acid sequence having at least two repeating poly C tracts separating a turn region with the i-motif being formed by concatermerisation of the nucleic acid having the alternating poly C tracts separating the turn region.

In one embodiment the polynucleotide that forms an i-motif, or that forms part of an i-motif is conjugated to the 5' end of a targeting component. In one embodiment the polynucleotide that forms an i-motif, or that forms part of an i-motif is conjugated to the 3' end of a targeting component. In one embodiment a polynucleotide that forms an i-motif, or that forms part of an i-motif is conjugated to the 5' end of a targeting component and a polynucleotide that forms an i-motif, or that forms part of an i-motif is conjugated to the 3' end of a targeting component. In one embodiment the polynucleotide that forms an i-motif, or that forms part of an i-motif comprises about 24 nucleotides, preferably consists essentially of about 24 nucleotides, preferably consists of about 24 nucleotides. In one embodiment the polynucleotide that forms an i-motif, or that forms part of an i-motif comprises 24 nucleotides, preferably consists essentially of 24 nucleotides, preferably consists of 24 nucleotides.

In one embodiment the polynucleotide that forms an i-motif, or that forms part of an i-motif comprises a nucleic acid selected from the group consisting of SEQ ID NO: 32, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 41 and SEQ ID NO: 43, or a functional fragment thereof. In one embodiment the

polynucleotide that forms an i-motif, or that forms part of an i-motif consists essentially of a nucleic acid selected from the group consisting of SEQ ID NO: 32, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 41 and SEQ ID NO: 43, or a functional fragment thereof. In one embodiment the polynucleotide that forms an i-motif, or that forms part of an i-motif consists of a nucleic acid selected from the group consisting of SEQ ID NO: 32, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 41 and SEQ ID NO: 43, or a functional fragment thereof.

In one embodiment the anti-microbial agent is selected from the group consisting of anti-viral agents, anti-bacterial agents, anti-protist agents and anti-fungal agents. Preferably the anti-microbial agent is an anti-bacterial agent.

In one embodiment the anti-microbial agent is an organic or inorganic anti-microbial agent. In one embodiment the organic anti-microbial agent is an antibiotic.

Preferably the antibiotic is selected from the group consisting of penicillins,

cephalosporins, tetracyclins, macrolides, glycopeptides, quinolones and

oxazolidinones.

In one embodiment the inorganic anti-microbial agent is selected from the group consisting of metals and small molecules. In one embodiment the metal is a noble metal. In one embodiment the metal is elemental metal or is a metal ion. In one embodiment the elemental metal is silver (Ag) or copper (Cu), preferably silver.

In one embodiment the elemental metal is Ag. In one embodiment the Ag is selected from the group consisting of at least one atom of Ag, a complex of at least 2 atoms of Ag, an Ag nanocluster, and a Ag nanoparticle. Preferably the Ag is comprised in an Ag nanocluster (AgNC). Preferably the Ag nanocluster comprises about 3 to 50 Ag atoms, preferably 3 to 50 atoms of Ag. Preferably an Ag nanocluster comprises about 3 to about 12 atoms, preferably 3 to about 12 atoms, preferably about 3 to 12 atoms, preferably 3 to at least 12 atoms, preferably at least 3 to 12 atoms, preferably 3 to 12 atoms of Ag.

In one embodiment the metal ion is Ag⁺. In one embodiment the anti-microbial agent is Ag, a nanocluster of Ag, a nanoparticle of Ag, or Ag⁺.

In one embodiment the anti-microbial agent is Cu, a nanocluster of Cu, a nanoparticle of Cu, Cu⁺ or Cu⁺⁺. In another aspect the invention relates to a combination of formula I:

TC-AMC wherein

TC = a targeting component that specifically binds to a target molecule or cell, and AMC= an anti-microbial component comprising i) a polynucleotide that forms a metal nanocluster with at least three metal atoms, or is a polynucleotide that chelates at least one metal ion, or both, and ii) an anti-microbial agent. The person of skill in the art appreciates that additionally, all of the embodiments set out herein that relate to the first aspect or any other aspect of the invention, including but not limited to embodiments relating to targeting components, anti-microbial components, and anti-microbial agents, and uses and methods related to or encompassing such targeting and/or anti-microbial components and/or agents are also specifically contemplated herein as part of this aspect of the invention that relates to the combination of formula I, and are specifically contemplated herein.

In another aspect the invention relates to a combination of formula II:

AptPA-AMC wherein AptPA = is an aptamer or functional fragment thereof that specifically binds a

Pseudomonas aeruginosa cell, and

AMC= an anti-microbial component comprising i) a polynucleotide comprising a nucleic acid sequence that is 5'-(CxN_y)_z-3' wherein C = cytosine; N= any combination of adenosine (A), guanosine (G), thymidine (T), analogues of C, A, G and T, and modified deoxynucleosides; x = an integer from 2 to 7; y = an integer from 0 to 7; and z = any integer, and ii) an anti-microbial agent comprising at least three atoms of silver or coper metal, or at least three silver or copper ions associated with the polynucleotide in i).

The person of skill in the art appreciates that additionally, all of the embodiments set out herein that relate to the first aspect or any other aspect of the invention, including but not limited to embodiments relating to targeting components, anti-microbial components and anti-microbial agents, and uses and methods related to or

encompassing such targeting and/or anti-microbial components and/or agents are also specifically contemplated herein as part of this aspect of the invention that relates to the combination of formula II, and are specifically contemplated herein.

In another aspect the invention relates to a combination of formula Ila :

AptPA-AMC wherein

AMC= an anti-microbial component comprising i) a polynucleotide comprising a nucleic acid sequence that is 5'-(CxN_y)_z-3' wherein C = cytosine; N= any combination of adenosine (A), guanosine (G), thymidine (T), analogues of C, A, G and T, and modified deoxynucleosides; x = an integer from 2 to 7; y = an integer from 0 to 7; and z = any integer, and an anti-microbial agent comprising at least three atoms of silver and/or copper metal, and/or at least three silver or copper ions associated with the polynucleotide in i). The person of skill in the art appreciates that additionally, all of the embodiments set out herein that relate to the first aspect or any other aspect of the invention, including but not limited to embodiments relating to targeting components, anti-microbial components and anti-microbial agents, and uses and methods related to or

encompassing such targeting and/or anti-microbial components and/or agents are also specifically contemplated herein as part of this aspect of the invention that relates to the combination of formula Ila, and are specifically contemplated herein.

In one embodiment the polynucleotide in i) in formula II and/or formula Ila comprises a nucleic acid sequence having at least 50% nucleic acid sequence identity with SEQ ID NO: 31 and SEQ ID NO: 32 or with any one of SEQ ID NO: 38 to SEQ ID NO: 43. Preferably the polynucleotide in i) comprises a nucleic acid sequence having at least 55%, preferably at least 60%, preferably at least 65%, preferably at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% nucleic acid sequence identity with SEQ ID NO: 31 or SEQ ID NO: 32 or with any one of SEQ ID NO: 38 to SEQ ID NO: 43. In one embodiment the polynucleotide in i) consists essentially of SEQ ID NO: 31 or SEQ ID NO: 32 or with any one of SEQ ID NO: 38 to SEQ ID NO: 43. In one embodiment the polynucleotide in i) consists of SEQ ID NO: 31 or SEQ ID NO: 32 or with any one of SEQ ID NO: 38 to SEQ ID NO: 43.

In additional various embodiments of the combination of formula II and/or formula Ila, AptPA is an aptamer that specifically binds a Pseudomonas aeruginosa cell as described herein for the first, or any other aspect of the invention. Additionally, in various embodiments of the combination of formula II and/or formula Ila, the anti- microbial component, polynucleotide in i), and the anti-microbial agent are as described herein for the first or any other aspect of the invention.

In another aspect the invention relates to a method of making an anti-microbial combination comprising : a) synthesizing a polynucleotide comprising a targeting component comprising a polynucleotide that specifically binds to a target molecule or cell, and an antimicrobial component comprising a nucleic acid sequence that forms a metal nanocluster with at least three metal atoms, or is a nucleic acid sequence that chelates at least one metal ion, or both, and b) associating the polynucleotide synthesized in a) with at least one antimicrobial agent to make the anti-microbial combination.

In one embodiment the method comprises an initial step of designing the targeting component comprising making a polynucleotide that specifically binds to a target molecule or cell. In one embodiment making the polynucleotide comprises evolving the polynucleotide using SELEX. In one embodiment making the polynucleotide comprises amplifying at nucleic acid template using SEQ ID NO: 33 and SEQ ID NO: 34 as amplification primers.

In one embodiment the method comprises an initial step of designing the anti- microbial component. In one embodiment designing the anti-microbial component comprises designing a nucleic acid sequence that forms a metal nanocluster with at least three metal atoms, or is a nucleic acid sequence that chelates at least one metal ion, or both. In one embodiment the metal nanocluster is a silver or copper nanocluster. In one embodiment the metal ion is a silver or copper ion. In one embodiment the method comprises the step of confirming that the targeting component specifically binds to a target molecule or cell.

In one embodiment associating the polynucleotide synthesized in a) comprises binding at least 3 silver or copper atoms to the polynucleotide. In one embodiment binding comprises binding at least 3 silver or copper ions to the polynucleotide to form a complex, and then contacting the complex with at least one reducing agent to form a silver (AgNC) or copper (CuNC) nanocluster.

The person of skill in the art appreciates that additionally, all of the embodiments set out herein that relate to the first aspect or any other aspect of the invention, including but not limited to embodiments relating to targeting components, anti-microbial components and anti-microbial agents, and uses and methods related to or encompassing such targeting and/or anti-microbial components and/or agents are also specifically contemplated herein as part of this aspect of the invention that relates to a method of making an anti-microbial combination, and are specifically contemplated herein. In another aspect the invention relates to a composition comprising an anti-microbial combination, a targeting component, an anti-microbial component, a combination of formula I, or a combination of formula II or Ila as described herein for any aspect of the invention and a carrier, diluent or excipient.

In one embodiment the composition is a pharmaceutical composition. In one embodiment the composition or pharmaceutical composition comprises an effective amount of the anti-microbial combination, targeting component, anti-microbial component, combination of formula I, or combination of formula II or Ila. In one embodiment the effective amount is a therapeutically effective amount.

In one embodiment the effective amount is an amount of the anti-microbial combination, targeting component, anti-microbial component, combination of formula I, or combination of formula II or Ila that kills at least 50%, preferably at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.9%, at least 99.99%, at least 99.995%, or preferably at least 99.999%of a target microorganism, preferably a target bacteria, as described herein for any aspect of the invention, when contacted to the target microorganism. In this embodiment, the target microorganism comprises a starting population of cells (i.e., the number of cells before treatment begins) of at least 1.0 x 10⁵ cells.

In one embodiment killing of at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.9%, at least 99.99%, at least 99.995%, or preferably at least 99.999% of the target

microorganism, preferably the target bacteria, occurs in less than about 4 hours after the target microorganism is contacted . Preferably killing occurs in less than 4 hours.

In one embodiment killing occurs in less than about 3 hours, preferably less than about 2 hours, preferably less than about 1 hour. In one embodiment killing occurs in less than 3 hours, preferably less than 2 hours, preferably less than 1 hour. In one embodiment the concentration of the anti-microbial combination, targeting component, anti-microbial component, combination of formula I, or combination of formula II or Ila in the composition or pharmaceutical composition is from about 500 nM to about 10 μΜ, or from about 500 nM to 10 μΜ, or from 500 nM to about 10 μΜ, or from 500 nM to 10 μΜ. In one embodiment the concentration is about 1 μΜ to about 5 μΜ, or is about 1 μΜ to 5 μΜ, or is 1 μΜ to about 5 μΜ or is 1 u μΜ to 5 μΜ. In one embodiment where the antimicrobial agent is an AgNC or Ag/CuNC as described herein, preferably an AgNC, the concentration is less than 5 μΜ, about 5 μΜ or is 5 μΜ. In one embodiment where the antimicrobial agent is Ag⁺, Cu⁺ or Cu⁺⁺ as described herein, the concentration is less than 1 μΜ, is about 1 μΜ, or is 1 μΜ.

In one embodiment the concentration of the combination of formula I or II or Ila or of the anti-microbial component comprising an i-motif and silver ions shows therapeutic activity against a target cell with an IC50 of less than 10 μΜ, preferably less than 7.5 μΜ, preferably less than 5 μΜ, preferably less than 2.5 μΜ, preferably less than 1 μΜ. In one embodiment the concentration of the combination of formula I or II or Ila shows therapeutic activity against a target cell with an IC50 of about 7.5 μΜ, preferably about 5 μΜ, preferably about 2.5 μΜ, preferably about 1 μΜ. In one embodiment the carrier, diluent or excipient is a buffer. In one embodiment the buffer is a zwitterionic buffer. In one embodiment the zwitterionic buffer is selected from the group consisting of MES, MOPS, HEPES and TRIS, preferably MES. In one embodiment the buffer is an inorganic buffer. In one embodiment the inorganic buffer is selected from the group consisting of citrate, acetate, phosphate and cacodylate. Buffers with low concentrations of chloride ions are preferred to prevent precipitation of AgCI. In one embodiment the buffer maintains the composition or pharmaceutical composition in a pH range of about 6 to about 8 or of about 6 to 8 or of 6 to about 8 or of 6 to 8, preferably about 6.5 to about 7.5 or about 6.5 to 7.5 or 6.5 to about 7.5 or 6.5 to 7.5, preferably about pH 6.5, 7 or 7.5, preferably at pH 6.5± .2, 7± .2 or 7.5± .2, preferably at pH 6.5, 7 or 7.5, preferably at pH 6.5.

In one embodiment the composition or pharmaceutical composition comprises pharmaceutically acceptable carriers, proteins, small peptides, salts, excipients, thickeners, diluents, buffers, preservatives, surface active agents, neutral or cationic lipids, lipid complexes, liposomes, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers and the like in addition to an anti-microbial combination, a targeting component, an anti-microbial component, a combination of formula I, or a combination of formula II or Ila as described herein. Such

compositions and formulations can be used as described herein.

A pharmaceutically acceptable carrier may be liquid or solid and is selected as known in the art, in view of a planned manner of administration. A pharmaceutically acceptable carrier provides for the desired bulk, consistency, or other

pharmaceutically desirable property of a pharmaceutical composition that is to be used or delivered in a particular context. A pharmaceutically acceptable carrier typically includes binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone (PVP) or hydroxypropyl

methylcellulose, and the like, fillers such as lactose or other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.); disintegrates (e.g., starch, sodium starch glycolate, etc.); or wetting agents (e.g., sodium lauryl sulphate, etc.). In one non-limiting example, the carrier is formulated to allow application of a composition or pharmaceutical composition as described herein by aerosol directly to the lungs.

Penetration enhancers may be included in pharmaceutical compositions in order to enhance the delivery of a useful agent. Examples of penetration enhancers include fatty acids, bile salts, chelating agents, surfactants and non-surfactants, but are not limited thereto. Single penetration enhancers may be used alone or in combination with any other penetration enhancer disclosed herein.

Examples of fatty acids (and derivatives thereof) useful as penetration enhancers include, but are not limited to, oleic acid, lauric acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid and physiologically acceptable salts thereof.

Suitable chelating agents are well known and disclosed in the art and will include a number of different agents that may be selected by the skilled worker, the skilled worker being aware that the selected agent should not introduce chloride ions into the composition. Such agents include, but are not limited to, disodium

ethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g., sodium salicylate, 5- methoxysalicylate and homovanilate) and N-acyl derivatives of collagen.

Likewise, numerous surfactants are well known and disclosed in the art. Examples include, but are not limited to, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether. Examples of non-surfactants include, but are not limited to, unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanone derivatives and non-steroidal antiinflammatory agents such as diclofenac sodium, indomethacin and phenylbutazone The anti-microbial combination, a targeting component, an anti-microbial component, a combination of formula I, or a combination of formula II as described herein can be formulated in pharmaceutical compositions that contain additional functional or therapeutic components. Such other components can be considered adjunct components as may be conventionally found in pharmaceutical compositions, at their art-established usage levels. Examples of such components include compatible pharmaceutically-active materials such as local anaesthetics or anti-inflammatory agents. Additional materials useful in physically formulating various dosage forms of a pharmaceutical composition may also be included, such as dyes, flavouring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers.

A person skilled in the art will be able to formulate a anti-microbial combination, a targeting component, an anti-microbial component, a combination of formula I, or a combination of formula II or Ila as described herein as a composition, preferably a pharmaceutical composition, by determining an appropriate mode of administration of the anti-microbial combination, targeting component, anti-microbial component, combination of formula I, or combination of formula II or Ila with reference to the literature and as described herein, and then formulating the composition for such mode with reference to the literature and as described herein. By way of non-limiting example, a formulation of the composition for topical application would be preferred for the treatment and prevention of certain microbial infections of the skin or mucosa, a formulation of the composition for systemic application would be preferred for the treatment of systemic microbial infections or localized internal microbial infections, and aerosol delivery to the lungs, such as when treating cystic fibrosis, but not limited thereto. In one embodiment the pharmaceutical composition is formulated for administration, or is in a form for administration, to a subject in need thereof. In one embodiment administration is selected from the group consisting of is topical, intranasal, epidermal, transdermal, oral or parenteral. In one embodiment parenteral administration is selected from the group consisting of direct application, systemic, subcutaneous, intraperitoneal or intramuscular injection, intravenous drip or infusion, inhalation, insufflation or intrathecal or intraventricular administration. In one embodiment administration is by aerosol delivery.

In one embodiment the anti-microbial combination, targeting component, antimicrobial component, combination of formula I, or combination of formula II or Ila as described herein is formulated for, or is in a form for, parenteral administration in any appropriate solution, including sterile aqueous solutions which may also contain buffers, diluents and other suitable additives

In one embodiment the anti-microbial combination, targeting component, anti- microbial component, combination of formula I, or combination of formula II or Ila as described herein is formulated for, or is in a form for oral administration in powders or granules, aqueous or non-aqueous suspensions or solutions, capsules, pills, lozenges or tablets. Thickeners, flavouring agents, diluents, emulsifiers, dispersing aids or binders may be desirable. In one embodiment the anti-microbial combination, targeting component, antimicrobial component, combination of formula I, or combination of formula II or Ila as described herein is formulated for, or is in a form for topical, aerosol, or direct administration in transdermal patches, subdermal implants, ointments, lotions, creams, gels, drops, pastes, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.

In one embodiment, the direct administration is direct application or local application. In one embodiment direct or local application comprises application of the antimicrobial combination, targeting component, anti-microbial component, combination of formula I or combination of formula II or Ila as described herein in combination with a delivery reagent or additional anti-microbial agent, but not limited thereto.

A person skilled in the art will be able to choose the appropriate mode of

administration of an anti-microbial combination, a targeting component, an antimicrobial component, a combination of formula I, or a combination of formula II or Ila as described herein with reference to the literature and as described herein. By way of non-limiting example, a systemic application would be preferred for the treatment and prevention of certain microbial infections whereas a local application would be preferred for the treatment of others, but not limited thereto.

In one embodiment the composition or pharmaceutical composition comprises at least one formulation agent. In one embodiment, the composition comprises at least two formulation agents. In one embodiment the composition or pharmaceutical composition further comprises an additional anti-microbial agent. In one embodiment the composition or pharmaceutical composition comprises a combination of one or more additional anti-microbial agents and one or more formulation agents. In some embodiments the composition or pharmaceutical composition is formulated as pre-prepared composition or in a concentrated form. In one embodiment the additional anti-microbial agent is selected from the group consisting of anti-viral agents, anti-bacterial agents, anti-protist agents and antifungal agents.

In one embodiment an additional anti-microbial agent is an agent that is capable of contributing to the control (e.g., treatment and/or prevention) of microbial infection, particularly bacterial infection, preferably infection by Pseudomonas spp. bacteria, more preferably infection by P. aeruginosa, but not limited thereto.

Suitable additional anti-microbial agents for use in the present invention may be capable of controlling Pseudomonas spp., particularly P. aeruginosa directly, or may be capable of potentiating the effect of any one or all of SEQ ID NO: 1 to 30 and 38 to 43 for controlling Pseudomonas spp., particularly P. aeruginosa. Additional antimicrobial agents may be included directly in the composition of or useful in the invention, or may be administered separately, either simultaneously or sequentially as appropriate according to a use or method of the invention.

In one embodiment the additional anti-microbial agent is an organic or inorganic anti- microbial agent. In one embodiment the organic anti-microbial agent is an

antibacterial agent. In one embodiment the antibacterial agent is an antibiotic. In one embodiment the antibiotic is selected from the group consisting of penicillins, cephalosporins, tetracyclins, macrolides, glycopeptides, quinolones and

oxazolidinones. In one embodiment the inorganic additional anti-microbial agent is selected from the group consisting of metals and small molecules. In one embodiment the metal is a noble metal. In one embodiment the metal is elemental metal or is a metal ion. In one embodiment the elemental metal or metal ion is the elemental form of, or an ion of, silver (Ag), mercury (Hg), platinum (Pt), gold (Au), copper (Cu) or arsenic (As). The person of skill in the art appreciates that additionally, all of the embodiments set out herein that relate to the first aspect or any other aspect of the invention, including but not limited to embodiments relating to targeting components, anti-microbial components and anti-microbial agents, and uses and methods related to or encompassing such targeting and/or anti-microbial components and/or agents are also specifically contemplated herein as part of this aspect of the invention that relates to a composition comprising an anti-microbial combination, a targeting component, an anti-microbial component, a combination of formula I, or a combination of formula II or Ila, and are specifically contemplated herein.

In another aspect the invention relates to the use of an anti-microbial combination, a targeting component, an anti-microbial component, a combination of formula I, a combination of formula II or Ila, or a composition as described herein for the first or any other aspect of the invention, in the manufacture of a medicament for treating microbial infection. In one embodiment the medicament comprises an effective amount of the anti-microbial combination, targeting component, anti-microbial component, combination of formula I, combination of formula II or Ila, or

composition. In one embodiment the effective amount is a therapeutically effective amount.

In one embodiment the medicament comprises at least one additional anti-microbial agent. In one embodiment the at least one additional anti-microbial agent is an antibiotic. In one embodiment the medicament comprises an effective amount of the additional anti-microbial agent. In one embodiment the effective amount of the at least one additional anti-microbial agent is a therapeutically effective amount.

In one embodiment the medicament consists essentially of an effective amount of the anti-microbial combination, targeting component, anti-microbial component, combination of formula I, or combination of formula II or Ila and an additional antimicrobial agent. In one embodiment the effective amount of the additional anti- microbial agent is a therapeutically effective amount.

In one embodiment the medicament is formulated for administration, or is in a form for administration, to a subject in need thereof.

In one embodiment the medicament is in a form for, or is formulated for topical, intranasal, epidermal, transdermal, oral or parenteral administration. In one embodiment parenteral administration is selected from the group consisting of direct application, systemic, subcutaneous, intraperitoneal or intramuscular injection, intravenous drip or infusion, inhalation, insufflation or intrathecal or intraventricular administration. In one embodiment the medicament is in a form for, or is formulated for, parenteral administration in any appropriate solution, preferably in a sterile aqueous solution which may also contain buffers, diluents and other suitable additives.

In one embodiment the medicament formulated for, or is in a form for oral

administration selected from the group consisting of a powder, a granule, an aqueous suspension, an aqueous solution, a non-aqueous suspension, a non-aqueous solution, a capsule, a pill, a lozenge, and a tablet.

When administered orally, the addition of one or more of the following may be desirable: thickeners, flavouring agents, diluents, emulsifiers, dispersing aids or binders.

In one embodiment the medicament is formulated for, or is in a form for topical or direct administration selected from the group consisting of transdermal patches, subdermal implants, ointments, lotions, creams, gels, drops, pastes, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be used as required or desired in this embodiment.

In one embodiment, the direct administration is direct application or local application. In one embodiment direct or local application comprises application of the medicament in combination with a delivery reagent or additional anti-microbial agent. A person skilled in the art will be able to choose the appropriate mode of

administration of the medicament with reference to the literature and as described herein. By way of non-limiting example, a systemic application would be preferred for the treatment and prevention of certain microbial infections whereas a local application would be preferred for the treatment of others, but not limited thereto. In one embodiment the medicament is for, is formulated for, or is in a form for administration separately, simultaneously or sequentially with an additional antimicrobial agent.

By way of non-limiting example, one additional anti-microbial agent that may be included in the composition of, or for use in the invention, is an antibiotic that is, or is suspected of being effective against a target cell, particularly a target bacterial cell. In one embodiment the target bacterial cell is a Gram negative bacterial cell. The inventors have determined that various combinations of a targeting component, anti-microbial component, combination of formula I, combination of formula II or Ila, or composition as described herein and an antibiotic, particularly ciprofloxin, have an interworking relationship and provide an unexpected synergistic bactericidal effect when used together as disclosed herein.

In one embodiment the medicament comprises an anti-microbial combination, targeting component, anti-microbial component, combination of formula I,

combination of formula II or Ila, or composition as described herein and an antibiotic, wherein the medicament is for, is formulated for, or is in a form for separate, simultaneous or sequential administration to a subject.

combination of formula II or Ila, or composition as described herein and an antibiotic, wherein the medicament is for, is formulated for, or is in a form for administration to a subject that has shown a non-response or reduced response to treatment with the antibiotic alone.

The person of skill in the art appreciates that additionally, all of the embodiments set out herein that relate to the first aspect or any other aspect of the invention, including but not limited to embodiments relating to targeting components, anti-microbial components and anti-microbial agents, and uses and methods related to or encompassing such targeting and/or anti-microbial components and/or agents are also specifically contemplated herein as part of this aspect of the invention that relates to the use of an anti-microbial combination, a targeting component, an anti-microbial component, a combination of formula I, a combination of formula II or Ila, or a composition as described herein, in the manufacture of a medicament for treating microbial infection, and are specifically contemplated herein.

In another aspect the invention relates to the use of an anti-microbial combination, targeting component, anti-microbial component, combination of formula I,

combination of formula II or Ila, or composition as described herein and an antibiotic in the manufacture of a medicament for treating a microbial infection.

In another aspect the invention relates to the use of an anti-microbial component as described herein in the manufacture of a medicament for treating microbial infection, wherein the anti-microbial component comprises a polynucleotide in association with an anti-microbial agent, wherein the polynucleotide comprises an i-motif structure and the anti-microbial agent is at least one metal ion.

encompassing such targeting and/or anti-microbial components and/or agents are also specifically contemplated herein as part of this aspect of the invention that relates to the use of an anti-microbial component as described herein in the manufacture of a medicament for treating microbial infection, and are specifically contemplated herein.

In another aspect, the invention relates to use of an anti-microbial component as described herein to inhibit the growth and/or proliferation of at least one bacterial species, wherein the anti-microbial component comprises a polynucleotide in association with an anti-microbial agent, wherein the polynucleotide comprises an i- motif structure and the anti-microbial agent is at least one metal ion.

In one embodiment the anti-microbial component inhibits the growth and/or proliferation of at least one bacterial species. In one embodiment the antibacterial component is bacteriostatic or bactericidal or both for the at least one bacterial species. In one embodiment the at least one bacterial species is a Gram-negative bacterial species.

In one embodiment the Gram-negative bacterial species is selected from the group consisting of Pseudomonas spp. belonging to the P. aeruginosa group, P. chlolraphis group, P. fluorescens group, P. pertucinogena group, P. putida group, P. stutzeri group, or the P. syringae group, preferably wherein the Pseudomonas spp. is P. aeruginosa, P. florescens, P. putida, P. stutzeri, P. tabaci, P. angulata, P. phaseolicola, P. pisi, P. glycinea, P. solanacearum, P. caryophylli, P. cepacia, P. marginalis, P. savastonoi, P. marginata or P. syringae- Salmonella spp., preferably S. enterica; Klebsiella spp., preferably K. pneumoniae; Enterobacter spp., preferably E. cloacae; Acinetobacter spp., preferably A. baumannii and Escherichia coll.

In one embodiment the Gram-negative bacterial species is selected from the group consisting of Xanthomonas spp., preferably X. phaseoli, X. oryzae, X. runi, X. juglandis, X. campestris or X. vascularum; Erwinia spp., preferably E. amylovora, E. tracheiphila, E. stewartii or E. carotovora; Corynebacterium spp., preferably C.

insidiosum, C. michiganese or C. facians; Streptomyces spp., preferably S. scabies or S. ipomoeae; Agrobacterium spp., preferably A. tumefaciens, A. rubi or A. rhizogenes; Mycoplasma spp. and Sprioplasma spp..

In one embodiment the at least one bacterial species is a Gram-positive bacterial species.

In one embodiment the Gram-positive bacterial species is a Listeria spp., preferably L. innocua; and/or a Staphylococcus spp., preferably S. aureus or S. epididymis. In one embodiment an anti-microbial component as described herein is in the form of, or is formulated as a disinfectant.

The formulation of an anti-microbial component as described herein as a disinfectant is believed to be within the skill of those in the art in view of the present disclosure and common general knowledge. In some embodiments, the combination is formulated as, or is in the form of, a composition comprising an anti-microbial component as described herein and a carrier, diluent or excipient.

In one embodiment the composition consists essentially of the anti-microbial component. In one embodiment the carrier, diluent or excipient is a buffer. In one embodiment the buffer is a zwitterionic buffer. In one embodiment the zwitterionic buffer is selected from the group consisting of MES, MOPS, HEPES and TRIS, preferably MES or MOPS. In one embodiment the buffer is an inorganic buffer. In one embodiment the inorganic buffer is selected from the group consisting of citrate, acetate, phosphate, carbonate and cacodylate. Buffers with low concentrations of chloride ions are preferred to prevent precipitation of AgCI. In one embodiment the buffer maintains the composition in a pH range of about 6 to about 8 or of about 6 to 8 or of 6 to about 8 or of 6 to 8, preferably about 6.5 to about 7.5 or about 6.5 to 7.5 or 6.5 to about 7.5 or 6.5 to 7.5, preferably about pH 6.5, 7 or 7.5, preferably at pH 6.5± .2, 7± .2 or 7.5± .2, preferably at pH 6.5, 7 or 7.5, preferably at pH 6.5. The formulation of an anti-microbial component as a composition in the form of a solid, liquid, paste, gel, particle, nanoparticle, emulsion, cream, ointment, lotion, liniment, solution, suspension, stick, block, pill, lozenge, powder, slurry, mist or vapour for use to inhibit the growth and/or proliferation of at least one Gram-negative bacterial species, or to treat a Gram-negative bacterial infection, disease and/or condition as described herein is believed to be within the skill of those in the art as described herein and in light of common general knowledge.

In one embodiment the anti-microbial component is formulated as a composition, pharmaceutical or cosmetic composition that comprises acceptable carriers, particularly pharmaceutically acceptable or cosmetically acceptable carriers, proteins, small peptides, salts, excipients, thickeners, diluents, buffers, preservatives, surface active agents, neutral or cationic lipids, lipid complexes, liposomes, penetration enhancers, carrier compounds and/or other carriers in addition to the anti-microbial component. A person skilled in the art will be able to formulate an anti-microbial component as described herein as a composition, particularly a pharmaceutical or cosmetic composition, by determining an appropriate mode of use, application and/or administration of the composition with reference to the literature and as described herein, and then formulating the composition for such mode with reference to the literature and as described herein. By way of non-limiting example, a formulation of the composition as a pharmaceutical composition for topical application would be preferred for inhibiting the growth and/or proliferation of certain Gram-negative bacteria, or for the treatment and prevention of certain Gram-negative bacterial infections, diseases and/or conditions of the skin or mucosa that are caused by and/or associated with at least one Gram-negative bacterial species. In another non-limiting embodiment, a formulation of the composition as a pharmaceutical composition for systemic application would be preferred for the treatment of systemic or localized internal bacterial infections, diseases and/or conditions of the skin or mucosa that are caused by and/or associated with at least one Gram-negative bacterial species. In some embodiments, an anti-microbial component or pharmaceutical composition as contemplated herein may be formulated according to conventional pharmaceutical practice and may be: Semisolid formulations: Gels, pastes, mixtures. Liquid formulations: Solutions, suspensions, drenches, emulsions. The person of skill in the art appreciates that additionally, all of the embodiments set out above that relate to the first or any other aspect of the invention also relate to this aspect of the invention that is the use of an anti-microbial component as described herein to inhibit the growth and/or proliferation of at least one bacterial species, wherein the anti-microbial component comprises a polynucleotide in association with an anti-microbial agent, wherein the polynucleotide comprises an i motif structure and the anti-microbial agent is at least one metal ion, and are specifically contemplated herein.

In another aspect the invention relates to a method of inhibiting the growth and/or proliferation of at least one bacterial species comprising contacting the at least one bacterial species with an anti-microbial component as described herein, wherein the anti-microbial component comprises a polynucleotide in association with an antimicrobial agent, wherein the polynucleotide comprises an imotif structure and the anti-microbial agent is at least one metal ion. In one embodiment contacting comprises contacting an object or part thereof that comprises the at least one bacterial species.

In one embodiment contacting comprises contacting a surface in and/or on the object or part thereof.

In one embodiment contacting is for a sufficient time to allow the combination or composition to inhibit the growth and/or proliferation of the at least one Gram- negative and/or Gram-positive bacterial species on and/or in the object.

In one embodiment contacting comprises directly or indirectly applying the

combination or composition to the object or part thereof. In one embodiment applying is directly applying. In one embodiment applying is indirectly applying.

In one embodiment applying comprises applying the anti-microbial component to the object or part thereof at least two times.

In one embodiment applying is applying as a coating or partial coating. In some embodiments, applying comprises applying the combination or composition at least lx, or 2x, or 3x, or 4x, or 5x, or 6x, or 7x, or 8x, or 9x, preferably lOx, or more. In some embodiments applying is least lx per day (lx/d), at least 2x/d, at least 3x/d, at least 4x/day, at least 5x/day, at least 6x/day, at least 7x/day, at least 8x/day, at least 9x/day, at least lOx/day.

In one embodiment the object is an animal or part thereof, or plant or part thereof.

In one embodiment the animal is a mammal.

In one embodiment the mammal is selected from the group consisting of canines, felines, bovines, ovines, equines, cervines, caprines, porcines, lagomorphs, rodents, camelids and hominids.

In one embodiment the mammal is selected from the group consisting of cats, dogs, rats, stoats, ferrets, possums, guinea pigs, mice, hamsters, zebra, elephants, lions, tigers, cheetah, monkeys, apes, macaques, tarsiers, lemurs, giraffes, prairie dogs, meerkats, bears, otters, tapiers, cows, horses, pigs, sheep, goats, deer, minks, hippopotami and humans.

In one embodiment the animal is a bird selected from the group consisting of chickens, ducks, pheasants, pigeons, ostriches, turkeys and geese.

In one embodiment the part of the animal is the hair, skin or hide, preferably human, cow, deer, sheep or horse hair, skin or hide. In one embodiment the part of the plant is selected from the group consisting of roots, shoots, stalks, stems, trunks, branches, leaves, buds, flowers, and seeds.

In one embodiment contacting is to an animal or part thereof, and the at least one bacterial species is a Gram-negative bacterial species.

In one embodiment the Gram-negative bacterial species is selected from the group consisting of Pseudomonas spp. belonging to the P. aeruginosa group, P. chlolraphis group, P. fluorescens group, P. pertucinogena group, P. putida group, P. stutzeri group, or the P. syringae group, preferably wherein the Pseudomonas spp. is P.

aeruginosa, P. florescens, P. putida, P. stutzeri, P. tabaci, P. angulata, P. phaseolicola, P. pisi, P. glycinea, P. solanacearum, P. caryophylli, P. cepacia, P. marginalis, P.

savastonoi, P. marginata or P. syringae- Salmonella spp., preferably S. enterica; Klebsiella spp., preferably K. pneumoniae; Enterobacter spp., preferably E. cloacae; Acinetobacter spp., preferably A. baumannii and Escherichia coll.

In one embodiment contacting is to an animal or part thereof, and the at least one bacterial species is a Gram-negative bacterial species. In one embodiment the Gram-positive bacterial species is a Listeria spp., preferably L. innocua; and/or a Staphylococcus spp., preferably S. aureus or S. epididymis.

In some embodiments inhibiting the growth and/or proliferation of the at least one bacterial species comprises inhibiting or reducing a bacterial infection, disease and or condition caused by or associated with the bacterial species. In some embodiments the bacterial infection, disease or condition is a bacterial infection, disease or condition of cystic fibrosis, neutropenia, HIV/AIDS, urinary tract infections, community-acquired pneumonia, ventilator-associated pneumonia, endocarditis, meningitis, ocular infections, ear infections, including but not limited to, perichondritis of the ear otitis, externa, malignant otitis externa, and skin and softissue infections, including but not limited to, burns, necrotising fascilitis, ecthyma gangrenosum, green nail syndrome, foot infections, puncture wounds and

osteomyelitis, and hot tub folliculitis.

In one embodiment, the combination or composition is formulated as a coating, or is in the form of, a coating or a partial coating. In one embodiment the anti-microbial component is formulated as, or is in a form of, a disinfectant.

In one embodiment the object is in inanimate article, material or substance, or part thereof. In one embodiment the object is an object on which bacterial species are known or suspected of being present and/or growing. In one embodiment the object is used in food processing, hygiene, medicine, dentistry or any other industry where contamination by bacterial species poses a health risk and/or is desired to be prevented and/or reduced. In some embodiments the object is selected from the group consisting of medical devices, surgical devices, surgical instruments, surgical implants, stents, catheters, dental devices, dental instruments dental prostheses, dental implants, contact lenses, bandages, wound dressings, and food processing equipment. The person of skill in the art appreciates that additionally, all of the embodiments set out above that relate to the first or any other aspect of the invention also relate to this method aspect of the invention that is a method of inhibiting the growth and/or proliferation of at least one bacterial species comprising contacting the at least one bacterial species with an anti-microbial component as described herein, wherein the anti-microbial component comprises a polynucleotide in association with an antimicrobial agent, wherein the polynucleotide comprises an imotif structure and the anti-microbial agent is at least one metal ion.

In another aspect the invention relates to a method of treating a microbial infection comprising administering an anti-microbial combination, a targeting component, an anti-microbial component, a combination of formula I, a combination of formula II or Ila, or a composition as described herein to a subject in need thereof.

In another aspect the invention relates to a method of treating a microbial infection comprising administering an anti-microbial component to a subject in need thereof, wherein the anti-microbial component comprises a polynucleotide in association with an anti-microbial agent, wherein the polynucleotide comprises an imotif structure and the anti-microbial agent is at least one metal ion.

In one embodiment of the method of treating aspects, administration is local or systemic administration. In one embodiment, administration is topical, intranasal, epidermal, and transdermal, oral or parenteral. In one embodiment oral

administration comprises aerosol delivery to the lungs. In one embodiment, parenteral administration is selected from the group consisting of direct application, systemic, subcutaneous, intraperitoneal or intramuscular injection, intravenous drip or infusion, inhalation, insufflation or intrathecal or intraventricular administration. In one embodiment of the method of treating aspects, administration is transient administration. In one embodiment transient administration comprises administration of an anti-microbial combination, a targeting component, an anti-microbial

component, a combination of formula I, a combination of formula II or Ila, or a composition as described herein for a sufficient period of time to provide a treatment or achieve a therapeutic result without the presence of the agent being harmful or causing significant deleterious effects to the subject. Administration can be rapid (e.g., by injection), or can occur over a period of time (e.g., by slow infusion or

administration of slow release formulations). In one embodiment of the method of treating aspects administration comprises administering a composition or pharmaceutical composition as described herein, wherein the composition or pharmaceutical composition comprises about 100 nM to about 10 μΜ, or from about 100 nM to 10 μΜ, or from 100 nM to about 10 μΜ, or from 100 nM to 10 μΜ of the anti-microbial combination, targeting component, antimicrobial component, combination of formula I, or combination of formula II or Ila. In one embodiment the concentration is about 1 μΜ to about 5 μΜ, or is about 1 μΜ to 5 μΜ, or is 1 μΜ to about 5 μΜ or is 1 u μΜ to 5 μΜ. In one embodiment where the antimicrobial agent is an AgNC as described herein, the concentration is less than 5 μΜ, about 5 μΜ or is 5 μΜ. In one embodiment of the method of treating aspects where the antimicrobial agent is Ag⁺ as described herein, the concentration is less than 1 μΜ, is about 1 μΜ, or is 1 μΜ.

In one embodiment of the method of treating aspects administration comprises administering about 100 nM to about 10 μΜ, or from about 100 nM to 10 μΜ, or from 100 nM to about 10 μΜ, or from 100 nM to 10 μΜ of the anti-microbial combination, targeting component, anti-microbial component, combination of formula I, or combination of formula II or Ila. In one embodiment administration comprises administering about 1 μΜ to about 5 μΜ, or is about 1 μΜ to 5 μΜ, or is 1 μΜ to about 5 μΜ or is 1 u μΜ to 5 μΜ. In one embodiment where the antimicrobial agent is an AgNC as described herein, administration comprises administering less than 5 μΜ, about 5 μΜ or is 5 μΜ. In one embodiment where the antimicrobial agent is Ag⁺ as described herein, administration comprises administering less than 1 μΜ, is about 1 μΜ, or is 1 μΜ .

In one embodiment of the method of treating aspects administration comprises administering a sufficient amount of an anti-microbial combination, a targeting component, an anti-microbial component, a combination of formula I, or a

combination of formula II or Ila to achieve a concentration of about 100 nM to about 10 μΜ, or from about 100 nM to 10 μΜ, or from 100 nM to about 10 μΜ, or from 100 nM to 10 μΜ of the anti-microbial combination, targeting component, anti-microbial component, combination of formula I, or combination of formula II or Ila at the site of microbial infection. In one embodiment the concentration is about 1 μΜ to about 5 μΜ, or is about 1 μΜ to 5 μΜ, or is 1 μΜ to about 5 μΜ or is 1 u μΜ to 5 μΜ. In one embodiment where the antimicrobial agent is an AgNC as described herein, the concentration is less than 5 μΜ, about 5 μΜ or is 5 μΜ. In one embodiment where the antimicrobial agent is Ag⁺ as described herein, the concentration is less than 1 μΜ, is about 1 μΜ, or is 1 μΜ.

In one embodiment of the method of treating aspects a sufficient amount comprises about 100 nM to about 10 μΜ, or from about 100 nM to 10 μΜ, or from 100 nM to about 10 μΜ, or from 100 nM to 10 μΜ of the anti-microbial combination, targeting component, anti-microbial component, combination of formula I, or combination of formula II or Ila. In one embodiment a sufficient amount is about 1 μΜ to about 5 μΜ, or is about 1 μΜ to 5 μΜ, or is 1 μΜ to about 5 μΜ or is 1 u μΜ to 5 μΜ. In one embodiment where the antimicrobial agent is an AgNC as described herein, a sufficient amount is less than 5 μΜ, about 5 μΜ or is 5 μΜ. In one embodiment where the antimicrobial agent is Ag⁺ as described herein, a sufficient amount is less than 1 μΜ, is about 1 μΜ, or is 1 μΜ.

In one embodiment of the method of treating aspects administration comprises administering a composition or pharmaceutical composition as described herein, wherein the composition or pharmaceutical composition comprises about 1 nM to about 50 μΜ, or from about InM to 50 μΜ, or from 1 nM to about 50 μΜ, or from 1 nM to 50 μΜ of the anti-microbial combination, targeting component, anti-microbial component, combination of formula I, or combination of formula II or Ila. In one embodiment the concentration is about 1 μΜ to about 10 μΜ, or is about 1 μΜ to 10 μΜ, or is 1 μΜ to about 10 μΜ or is 1 μΜ to 10 μΜ. In one embodiment where the antimicrobial agent is an AgNC as described herein, the concentration is less than 20 μΜ, about 20 μΜ or is 20 μΜ. In one embodiment of the method of treating aspects where the antimicrobial agent is Ag⁺ as described herein, the concentration is less than 10 μΜ, is about 10 μΜ, or is 10 μΜ. In one embodiment of the method of treating aspects administration comprises administering about 1 nM to about 50 μΜ, or from about 1 nM to 50 μΜ, or from 1 nM to about 50 μΜ, or from 1 nM to 50 μΜ of the anti-microbial combination, targeting component, anti-microbial component, combination of formula I, or combination of formula II or Ila. In one embodiment administration comprises administering about 1 μΜ to about 10 μΜ, or is about 1 μΜ to 10 μΜ, or is 1 μΜ to about 10 μΜ or is 1 μΜ to 10 μΜ. In one embodiment where the antimicrobial agent is an AgNC as described herein, administration comprises administering less than 20 μΜ, about 20 μΜ or is 20 μΜ. In one embodiment where the antimicrobial agent is Ag⁺ as described herein, administration comprises administering less than 10 μΜ, is about 10 μΜ, or is 10 μΜ. In one embodiment of the method of treating aspects administration comprises administering a sufficient amount of an anti-microbial combination, a targeting component, an anti-microbial component, a combination of formula I, or a

combination of formula II or Ila to achieve a concentration of about 1 nM to about 10 μΜ, or from about 1 nM to 50 μΜ, or from 1 nM to about 50 μΜ, or from 1 nM to 50 μΜ of the anti-microbial combination, targeting component, anti-microbial component, combination of formula I, or combination of formula II or Ila at the site of microbial infection. In one embodiment the concentration is about 1 μΜ to about 10 μΜ, or is about 1 μΜ to 10 μΜ, or is 1 μΜ to about 10 μΜ or is 1 u μΜ to 10 μΜ. In one embodiment where the antimicrobial agent is an AgNC as described herein, the concentration is less than 10 μΜ, about 10 μΜ or is 10 μΜ. In one embodiment where the antimicrobial agent is Ag⁺ as described herein, the concentration is less than 10 μΜ, is about 10 μΜ, or is 10 μΜ.

In one embodiment of the method of treating aspects a sufficient amount comprises about 1 nM to about 50 μΜ, or from about 1 nM to 50 μΜ, or from 1 nM to about 50 μΜ, or from 1 nM to 50 μΜ of the anti-microbial combination, targeting component, anti-microbial component, combination of formula I, or combination of formula II or Ila . In one embodiment a sufficient amount is about 1 μΜ to about 10 μΜ, or is about 1 μΜ to 10 μΜ, or is 1 μΜ to about 10 μΜ or is 1 u μΜ to 10 μΜ. In one embodiment where the antimicrobial agent is an AgNC as described herein, a sufficient amount is less than 10 μΜ, about 10 μΜ or is 10 μΜ . In one embodiment where the

antimicrobial agent is Ag⁺ as described herein, a sufficient amount is less than 10 μΜ, is about 10 μΜ, or is 10 μΜ.

An anti-microbial combination, a targeting component, anti-microbial component, combination of formula I, or combination of formula II or Ila, as described herein may be usefully employed in the methods described when formulated as bioequivalent compounds, including pharmaceutically acceptable salts and prodrugs. Such formulation encompasses any pharmaceutically acceptable salts, esters, or salts of such esters, or any other compound which, upon administration to an animal including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Pharmaceutically acceptable salts retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto. An anti-microbial combination, targeting component, anti-microbial component, combination of formula I, or combination of formula II or Ila, as described herein can also be formulated as a prodrug or in prodrug form as known in the art. A prodrug is a therapeutic agent that is prepared in an inactive form that is converted to an active form (i.e., drug) within the body or cells of a subject by the action of endogenous enzymes or other chemicals and/or conditions.

The formulation of compositions, pharmaceutical compositions and medicaments as described herein, and their subsequent administration following a method of treating a microbial infection as described herein is believed to be within the skill of those in the art. A particular and effective dosage regime will be dependent on severity of the infection to be treated and on the responsiveness of the treated subject to the course of treatment. An effective treatment may last from several hours to several days to several months, or until an acceptable therapeutic outcome is effected or assured or until an acceptable reduction of the infection is observed. An optimal dosing schedule (s) may be calculated from drug accumulation as measured in the body of a treated subject. It is believed to be within the skill of persons in the art to be able to easily determine optimum and/or suitable dosages, dosage formulations and dosage regimes. Of course, the optimum dosages may vary depending on the relative potency of a given anti-microbial combination, targeting component, anti-microbial component, combination of formula I, combination of formula II or Ila, or composition as described herein, but will be estimable from an EC50s found to be effective in suitable cells in vitro and in an appropriate in vivo animal model. In general, dosage is from 0.001 g to 99 g per kg of body weight, and may be given once or more daily, weekly, monthly or yearly, but not limited thereto. In one embodiment of the method of treating aspects, the anti-microbial combination, targeting component, anti-microbial component, combination of formula I,

combination of formula II or Ila, or composition as described herein, is administered in conjunction with the administration of an additional anti-microbial agent and/or an additional therapeutic agent. In one embodiment the additional anti-microbial agent is as described herein.

In one embodiment of the method of treating aspects the additional therapeutic agent is any appropriate therapeutic agent used to treat or prevent any symptom, side effect or other consequence of treatment, either as a result of the use of the anti-microbial combination, targeting component, anti-microbial component, combination of formula I, combination of formula II or Ila, or composition as described herein, in the methods described herein, or for any other reason related to the desired treatment. In a preferred embodiment, the additional therapeutic agent is active against microbial infection and/or is used to treat a symptom, side-effect or cause of a microbial infection.

Where the anti-microbial combination, targeting component, anti-microbial component, combination of formula I, combination of formula II or Ila, or composition as described herein, is formulated with an additional anti-microbial agent and/or an additional therapeutic agent, then the dosing of the anti-microbial combination, targeting component, anti-microbial component, combination of formula I,

combination of formula II, or composition as described herein, and the additional therapeutic agent can be separate, simultaneous or concurrent, as is appropriate. Repetition rates for dosing can be based on measured residence times and

concentrations of a given agent in the cells, fluids or tissues of the subject that is being or that is to be treated.

Maintenance therapy may be desirable in successfully treated patient in order to prevent the recurrence of the infection, wherein the anti-microbial combination, targeting component, anti-microbial component, combination of formula I,

combination of formula II or Ila, or composition as described herein, is administered in maintenance doses, ranging from 0.001 g to 99 g per kg of body weight, once or more daily, to once every 5 years.

The person of skill in the art appreciates that additionally, all of the embodiments set out above that relate to the first or any other aspect of the invention also apply equally to this aspect of the invention that is a method of treating a microbial infection comprising administering an anti-microbial component to a subject in need thereof, wherein the anti-microbial component comprises a polynucleotide in association with an anti-microbial agent, wherein the polynucleotide comprises an i motif structure and the anti-microbial agent is at least one metal ion.

In another aspect the invention relates to an anti-microbial combination, a targeting component, an anti-microbial component, a combination of formula I, a combination of formula II or Ila, or a composition as described herein for use in treating, or when used to treat, microbial infection. In another aspect the invention relates to an anti-microbial component for use in inhibiting the growth and/or proliferation of at least one bacterial species treating, or when used to treat, microbial infection, wherein the anti-microbial component comprises a polynucleotide in association with an anti-microbial agent, wherein the polynucleotide comprises an i-motif structure and the anti-microbial agent is at least one metal ion.

In another aspect the invention relates to a polynucleotide selected from the group consisting of SEQ ID NO: 32, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42 and SEQ ID NO: 43. In one embodiment the polynucleotide is selected from the group consisting of SEQ ID NO: 32, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42 and SEQ ID NO: 43, and optionally SEQ ID NO: 31.

In one embodiment the polynucleotide is single stranded. In one embodiment the polynucleotide is comprised in a polynucleotide structure that forms an i-motif. In one embodiment the i-motif comprises at least one of SEQ ID NO: 32, SEQ ID NO: 38,

SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42 and SEQ ID NO: 43.

In one embodiment the i-motif comprises, consists essentially of, or consists of at least one of SEQ ID NO: 32, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42 and SEQ ID NO: 43. In one embodiment the i-motif comprises, consists essentially of, or consists of SEQ ID NO: 31.

The person of skill in the art appreciates that additionally, all of the embodiments set out above that relate to the first or any other aspect of the invention apply equally to these aspects of the invention that relate to an anti-microbial component as described herein for use in inhibiting the growth and/or proliferation of at least one bacterial species treating, or when used to treat, microbial infection, wherein the anti-microbial component comprises a polynucleotide in association with an anti-microbial agent, wherein the polynucleotide comprises an iimotif structure and the anti-microbial agent is at least one metal ion. Various aspects of the invention will now be illustrated in non-limiting ways by reference to the following examples. EXAMPLES

EXAMPLE 1:

Selection and characterisation of DNA aptamers selected to live biofilm derived P. aeruginosa cells. General Methodologies

Bacterial culture

The bacterial strain used for SELEX selection was Pseudomonas aeruginosa 692 (ATCC 14502) obtained from New Zealand Culture Collection (Porirua, NZ).

Other bacterial strains used in the following assays were: Pseudomonas aeruginosa PAOl (ATCC 15692), Salmonella enterica serovar typhi (ATCC 19430), Klebsiella pneumoniae (ATCC 13883), Enterobacter cloacae (ATCC 13047), Listeria innocua (ATCC 33090) and Escherichia coli.

Additional P. aeruginosa strains used in various assays as described herein were ARL1079, ARL1204, ARL1205, ARL1236, and PA918. These strains are clinical isolates gifted to the inventors by a colleague (D. Day - Personal communication).

All bacterial strains were cultured in standard Luria Broth (LB) medium at 37°C for 16 h with aeration. After growth, the cells were diluted 1 : 100 in fresh LB and grown for 4 hours to allow them to reach exponential phase.

Biofilm growth The selection strain was grown as a biofilm in a microfluidic flow cell with an 800 μηη channel depth (Ibidi, Germany). The flow cell was inoculated with 200 μL of exponentially growing culture diluted to an OD₆₀₀ = 0.1. Cells were left to adhere for 2 hours prior to commencing flow of growth media (0.5x LB broth) at a flow rate of 1 mL/hr driven by a syringe pump (KD Scientific). Cultures were grown for 42 hours at ambient temperature (22°C) prior to washing loosely adhered planktonic cells by increasing the flow rate to 5 mL/hr for 1 hour. The biofilm was collected by fluidic agitation and suspended in 1 mL 0.85% NaCI. Biofilm cells were washed three times in 0.85% NaCI with vortexing to dissociate and remove excess alginate and

exopolysaccharides characteristic of biofilms. Cells were then washed by centrifugation and re-suspended in wash buffer consisting of phosphate buffered saline (PBS) containing 0.05% Tween 20 prior to washing with SELEX binding buffer (wash buffer, containing 1% BSA) . The cells were re-suspended in binding buffer to a density of ~10⁶ CFU/mL in preparation for SELEX selection

SELEX Aptamers were selected against live Pseudomonas aeruginosa cells (P. aeruginosa 692 -ATCC 14502) grown as a biofilm using a modified whole-bacterium SELEX [6, 15] protocol. The random ssDNA library (Integrated DNA technologies, USA) consisted of a random 45 nucleotide site flanked by two constant primer regions,

ATGAGAGCGTCGGTGTGGTA (SEQ ID NO: 33)-N₄5-TACTTCCGCACCCTCCTACA (SEQ ID NO: 34). Before cell-SELEX the ssDNA library was denatured by heating at 95°C for 5 min then snap cooled on ice for 10 min. For the first round of selection 1 nmol at 1 μΜ of ssDNA was incubated with bacteria in 1 mL binding buffer for 30 min at room temperature (22°C) with gentle agitation. Aptamer bound cells were recovered by centrifugation (3,100xg, 10 min) and washed twice with 1 mL wash buffer to remove unbound oligonucleotides. Washing time and volumes were increased in subsequent rounds to increase the stringency of selection. Bound oligonucleotides were recovered by re-suspending cells in 100 μL of TE buffer (10 mM Tris-HCI pH 8.0 containing 1 mM EDTA) and heating to 95°C for 5 min then centrifugation at 14,000xg for 15 min. The supernatant that contained the eluted aptamer sequences was kept and used for PCR amplification to generate the next library.

Library amplification was monitored by real time PCR using a CFX-Connect Real Time PCR Detection System (BioRad, USA) with SYBR green chemistry to determine the optimal number of amplification cycles to prevent the formation of non-specific amplification products [6]. Amplification was performed using SensiMix SYBR & Fluorescein mix (Bioline, UK), with 100 nM forward primer (5'-

ATGAGAGCGTCGGTGTGGTA-3') (SEQ ID NO: 35) and 100 nM reverse primer (5'- TGTAGG AGGGTGCGGAAGTA- 3 ') (SEQ ID NO: 36) (IDT, USA) in a total volume of 100 μL. A thermal cycle consisting of an initial enzyme activation step of 95°C for 10 min, then 95°C for 10 sec and 65°C for 15 sec for the optimised number cycles was used for amplification and the quality of the PCR product was confirmed by agarose gel electrophoresis. The single stranded DNA library was regenerated by asymmetric PCR [16, 17]. The asymmetric PCR was performed using 50 μL of the dsDNA PCR product as template in a final volume of 100 μL. Amplification was achieved using KAPA2G HotStart DNA polymerase (10 U) (KAPABiosystems, USA) with the supplied KAPA2G buffer A, and contained 0.2 mM dNTPS, 1 μΜ FAM labelled forward primer (5'-FAM- ATG AG AGC GTC GGTGTG GTA- 3 ') (SEQ ID NO: 37) and 50 nM reverse primer. The PCR was run for 20 cycles using the same cycling conditions. The ssDNA PCR product was desalted using an Illustra™ NAP™-5 column (GE Healthcare, UK) in to wash buffer and stored at -20°C until the next round of selection. ssDNA concentration was determined by 3% agar electrophoresis and densitometry analysis against known concentration standards using ImageJ [18].

At round 4, 5 and 6 of SELEX a negative selection was performed against

exponentially growing PA692 to bias the selection process for outer membrane markers present in biofilm and stationary phase cells. A culture of 3xl0⁶ CFU/mL exponentially growing PA692 was incubated with 100 nM ssDNA library from the previous round for 30 min at room temperature then centrifuged (3,100xg, 10 min) to pellet cells. The supernatant was removed and used for the next round of positive selection. A total of 7 rounds of SELEX were performed. Cloning and sequencing of aptamers

After the 7^th round of selection the evolved aptamer pool was PCR amplified using unmodified primers and purified using the QIAEX II Gel Extraction Kit (Qiagen, Germany). Cloning was performed using the TOPO-TA cloning kit (Thermo Fisher Scientific, USA) following the manufacturer's instructions. White colonies were picked and grown individually overnight at 37°C, before being analysed for the correct sized insert by PCR using the aptamer specific primers. Plasmid from clones containing the desired insert were isolated using the GeneJET plasmid miniprep kit (Thermo Fisher Scientific, USA) following the supplied protocol and were sequenced by Macrogen Inc. (Korea) using the M 13R-pUC primer, and the aptamer sequences were extracted using the Geneious software (Biomatters, NZ). Aptamer candidates were synthesised (IDT, USA) both FAM labelled and unlabelled.

Flow cytometry

Flow cytometry was used to assess binding of the aptamer libraries and the aptamer candidates. Cells were grown in LB to an OD6oo=0.1, washed once in flow buffer (FB) (lx PBS containing 0.1% BSA), then re-suspended in 200 μΙ_ of FB. A BD FACSCanto II flow cytometer (BD Biosciences, USA) was used to identify bacteria cells based upon forward and side scatter profiles. Aptamer binding was performed in a volume of 300 μΙ_ in FB containing 10 μΙ_ of cells suspension and the desired concentration of library or aptamer. The samples were incubated at room temperature for 15 min with aptamer, collected by centrifugation (13,000xg, 2 min) then re-suspended in 300 μΙ_ PBS for flow analysis. At least 10,000 events in the gated bacterial population were collected and data analysed using Flowing Software 2.5.1 (Turku Centre for

Biotechnology, Finland).

Metabolic activity

To determine whether aptamer binding induced changes in metabolic activity incorporation of 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) was used. CTC is oxidised to an insoluble fluorescent precipitate by metabolically active cells [19]. Cells were incubated with aptamer for 15 min then 5 mM CTC was added and incubated for a further 15 min before being analysed by flow cytometry. Changes in cell

permeability were determined by staining with propidium iodide (2 μg/mL, 2 min) and flow cytometry analysis.

Measurement of binding affinity The dissociation constants were determined by flow cytometry by incubating a set number of bacterial cells with a range of FAM labelled aptamer concentrations from 1 nM to 250 nM . Samples were incubated for 15 minutes at room temperature, collected by centrifugation (13,000xg, 2 min) then re-suspended in 300 μΙ_ PBS for flow analysis. Binding of aptamers to E. coli cells was used as a measure of non-specific binding and calculation of Kds was done on GraphPad Prism 5 using a one site binding minus non-specific binding model.

Aptamer effect on bacterial growth

Growth assays were performed using an Enspire 2300 Multilabel plate reader (Perkin Elmer, USA) to follow the growth of the bacteria by OD₆₀₀ measurement in a 96-well plate. 10⁵ CFU of exponentially growing bacteria were washed and re-suspended in LB and added to each well with 1 μΜ of unlabelled aptamer and made up to a total volume of 200 μL with LB. Wells with LB only and bacteria only were used as controls for contamination and normal growth respectively. Each condition was done in triplicate per plate. OD₆₀₀ readings were taken every 30 minutes for betweenl6 and 20 hours while the plate was incubated in the machine at 37°C. Triplicate data was averaged and blank subtracted and plotted against time. Results

Aptamer selection

Aptamers were selected to P. aeruginosa bacteria grown as a biofilm. Whole-cell SELEX was undertaken for seven cycles with three counter-selection steps to exponentially growing P. aeruginosa to bias the selection towards biofilm associated epitopes. Flow cytometric analysis of fluorescently labelled aptamer pools was carried out to assess the binding affinity of each library. The seventh library showed binding enrichment to target cells (Fig. 21) as well as potential metabolic effects and was cloned in to E. coli. 27 different clones were identified by sequencing. Screening of aptamer candidates

Single-stranded aptamer candidates were prepared from the plasmids by asymmetric PCR using a fluorescently labelled primer to allow analysis by flow cytometry, and changes in metabolic activity and membrane permeability were determined by staining with CTC and PI respectively. The aptamer candidates were ranked according to binding affinity and their ability to alter metabolic activity or membrane permeability. Two candidates, JN08 and JN27, were chosen to be chemically synthesised and investigated further.

The secondary structures of the aptamers were determined using mfold [20] and the secondary structure of JN27 indicated a single stem-loop as being the only significant stable structure, while JN08 contained two putative stem-loops. The stem-loop of JN27 (SEQ ID NO: 1) was hypothesised as being important for binding so a truncated version comprising just the stem-loop (JN27-SH, SEQ ID NO: 3) was synthesised and its binding compared to the full length aptamers. The truncated version showed increased binding when compared to the original length aptamer which confirmed the importance of the stem loop structure (Fig. 2).

Having identified a putative stem-loop structure as being important for binding, mfold was used to predict the secondary structures of the top binding candidates from the initial screen. Several of the tighter binders, including JN08 (SEQ ID NO: 2) had similar stem-loop structures that we reasoned may also be important for binding. Three additional stem-loop structures were synthesised both fluorescently and non- fluorescently labelled and evaluated for binding. The truncated aptamer candidates JN 17.SH (SEQ ID NO: 15), JN21.SH (SEQ ID NO: 11) and JN08.SH (SEQ ID NO: 7) all showed tight binding to P. aeruginosa.

To further investigate the stem loop motifs, in silico mating was done to mix and match stems and loops of identified aptamers and produce three new aptamer candidates for binding testing - St21Lpl7 (SEQ ID NO: 19), Stl7Lp21 (SEQ ID NO: 23) and St08Lpl7 (SEQ ID NO: 27). All aptamers and secondary structures are identified in figures 3 and 4.

Binding affinity of individual aptamers

The Kds were determined for all identified aptamers and are displayed in figure 3 and figure 22. JN27-SH displayed a lower Kd than JN27. Without wishing to be bound by theory, the inventors believe that this result supports their hypothesis that the stem- loop motif is important for binding. All Kds are in the low nanomolar range, 10-55 nM .

Determination of species specificity

To determine whether the aptamers were selective for P. aeruginosa, the identified aptamers were evaluated for binding to other bacterial species (Salmonella enterica serovar typhi, Klebsiella pneumoniae, Enterobacter cloacae, Listeria innocua, and Escherichia coli) in comparison with P. aeruginosa PAOl and PA692 by flow cytometry. All aptamers were selective for P. aeruginosa with a very small amount of cross- reactivity with Salmonella and to a lesser extent Enterobacter (Fig. 5, Fig. 23, Table 1).

Table 1. Specificity of aptamer binding to P. aeruginosa and other bacterial strains (values indicate relative binding intensity l=highest).

Determination of intrinsic antibacterial effects

Each of the aptamers were tested for possible antibacterial effects, either

bacteriostatic or bactericidal. These effects were determined by changes in PI and CTC staining of the bacterial population, indicative of metabolic changes, after a 15 min incubation with 1 μΜ aptamer. Preliminary studies showed a change with the enzymatically made aptamers, however the pure, chemically synthesised aptamers did not show any changes in metabolism upon incubation with aptamer. Growth assays were also used to look for an increase in lag phase and time to killing for the aptamer treatments compared to control (Fig. 6). These results were also negative, showing that these aptamers have no intrinsic bacteriostatic and/or bactericidal activity, even though they are highly specific for P. aeruginosa.

Conclusion

The specific nature of the aptamer binding to Pseudomonas opens doors to potential uses in diagnostics, such as a biosensor for detection of the pathogen in water in hospitals. Pseudomonas forming biofilms inside taps and on equipment is a problem, especially in neonatal wards where an opportunistic infection is usually fatal. These aptamers could be used for the early detection of bacterial contamination. Aptamers are also currently being explored as alternative therapies. The aptamer sequences described herein can be used as targeted therapeutic aptamer conjugates to deliver drugs or other small molecules specifically to the bacteria, increasing the effective concentration at the infection site. This is a problem faced by common antibiotic treatments as the effective concentration is often too low and induces resistance mechanisms. P. aeruginosa is an important bacterial pathogen that needs combatting. As described herein, the inventors have identified a series of aptamers that bind with high affinity and specificity to P. aeruginosa. These aptamers can be used diagnostically and/or therapeutically by utilising the aptamers as a targeting component as described herein to conjugate to antibiotics or other small molecules for specific delivery to target cells or molecules. Example 2:

Generation of Pseudomonas aeruginosa specific DNA aptamers for the targeted delivery of antibacterial silver nanoclusters and i-motif stabilised Ag⁺

General Methodologies Aptamer-silver nanocluster (AgNC) and aptamer-Ag⁺ synthesis

Oligonucleotide DNA templates for nanocluster generation were synthesised by Integrated DNA Technologies (IDT, USA) and were prepared in TE buffer (10 mM Tris- HCI pH 8.0 containing 1 mM EDTA) to a final concentration of 250 μΜ and stored at - 20°C before use. Sequences used were obtained from our previously reported

Pseudomonas aeruginosa specific aptamers. Nanoclusters were prepared in 10 mM MES buffer (pH 6.5) containing 12.5 μΜ oligonucleotide template and 75 μΜ AgNO₃. The mixture was incubated in the dark at 4°C for 1 hour prior to the slow addition of 75 μΜ NaBH₄ while the reaction mixture was vigorously mixed by vortexing for 30 seconds. The reaction mixture was then transferred to 4°C and kept in the dark.

Fluorescent nanoclusters formed over the next 1-2 hours and were left to mature overnight prior to use. Excess salts and reagents were removed by concentration through an Amicon Ultra-0.5 mL centrifugal filter with a 3000 molecular weight cut-off (Merck Millipore, Germany) to a final volume of approximately 10-15 μΙ_, followed by dilution with milliQ H2O to 100 μΙ_. The sample was then concentrated again to a volume of 10-15 μΙ_ and diluted to a final working volume of 150 μΙ_.

DNA chelated Ag⁺ was generated in a similar manner, they were prepared in 10 mM MES buffer (pH 6.5) containing 12.5 μΜ oligonucleotide template and 75 μΜ AgNO₃. However there was no reduction of the silver ions by NaBH₄. The silver ions remained bound to the DNA without reduction to silver molecules. Excess salts and reagents were then removed in the same manner with a centrifugal filter as AgNCs, and the final working volume resuspended to 150 μΙ_.

Physical characterisation

Reaction yield

The yield of recovered aptamer was determined by UV-visible spectroscopy. The concentration of aptamer-AgNC or aptamer-Ag⁺ was determined using the extinction coefficient at 260 nm of the native oligonucleotide. All reference to a concentration of aptabiotic used herein refers to this method of concentration determination.

Fluorescence spectroscopy

Due to the fluorescence properties of silver nanoclusters an excitation and emission spectra could be obtained for each aptamer-AgNCs. A Cary Eclipse fluorescence spectrophotometer (Agilent Technologies, USA) was used to find the excitation and emission wavelengths using a slow scan rate in a 10 mm pathlength quartz cuvette at room temperature.

Circular dichroism spectroscopy For spectroscopic studies the aptamers or aptamer-AgNCs were suspended in MES buffer (pH = 6.5) at a concentration of 10 μΜ. Circular dichroism (CD) spectra were acquired from 350 to 200 nm at a scan rate of 60 nm/min in a 2 mm path length quartz cell at room temperature.

Aptamer-AgNC and aptamer-Ag⁺ biological characterisation Bacterial culture

The bacterial strains used for aptamer-AgNC and aptamer-Ag⁺ characterisation were P. aeruginosa 692 (ATCC 14502) and PAOl (ATCC 15692) obtained from New Zealand Culture Collection (Porirua, NZ).

Other P. aeruginosa strains used in the following assays were: P. aeruginosa 918 (ATCC 27853), and P. aeruginosa clinical isolates - ARL1024 (catheter, urine), ARL1205 (left thigh), ARL1079 (tracheal aspirate), ARL1236 (blood).

The bacteria were cultured in standard LB medium at 37°C for 16 h with aeration. After growth, the cells were diluted 1 : 100 in fresh LB and grown for 3 hours to allow them to reach exponential phase before use. Bacterial growth assays

Growth assays were performed in 96 well microtitre plate format. Growth was monitored by measuring absorbance at 600 nm. Wells containing LB growth media were inoculated with 10⁵ CFU of exponentially growing bacteria and treated with aptabiotic or other test substrates. OD₆₀₀ readings were taken every 5 minutes for at least 16 hours while the plate was incubated at 37°C. Resulting data was plotted against time to give the bacterial growth curves.

Another assay was performed to look at time to killing of the aptamer-AgNC and aptamer-Ag⁺. A starting inoculum of 2xl0⁵ cfu of PA692 in LB was incubated with 1 μΜ of aptamer-Ag⁺. After 1 and 3 hours the number of bacteria remaining was enumerate using a standard agar plating protocol. These numbers were compared to the number of cfu that went in to the experiment as a control.

Metabolic effects measured by flow cytometry

Bacteria was incubated with 5 μΜ aptamer-AgNC or aptamer-Ag⁺ for 30 mins before addition of 2 μg/mL propidium iodide (PI) to look for changes in cell permeability. The cells were incubated for 2 minutes with PI then changes in cell permeability were determined by flow cytometry analysis.

In vivo model of infection - Galleria mellonella

Wax moth larvae (Galleria mellonella) were used as in vivo models of infection to test bactericidal and/or bacteriostatic effects of the aptamer-AgNCs and aptamer-Ag⁺ by intrahaemocoelic injection. Fifth instar larvae (130-200 mg in weight) were obtained from Biosuppliers (NZ) and starved for 24 hours before the experiment. A bacterial culture of PAOl was grown as previously described and diluted in lxPBS to a final concentration of 10³ cfu/mL to get 10 cfu per larvae (in a 10 μL injection). Larvae were chilled on ice before injection to slow movement and then 10 μL of prepared bacterial suspension was injected into the right hind proleg. Aptamer-AgNCs or aptamer-Ag⁺ were then administered as a second 10 μL injection in to the left hind proleg at a concentration of 10, 5 or 2.5 μΜ. Larvae injected with PBS only were used as a control for injection related mortality. Injected larvae were then incubated at 37°C and checked for death every hour from 12 hours to 24 hours post injection.

Death was characterised as the production of melanin blackening the larval skin and no response to stimulus.

Results

Physical characterisation of the silver nanoclusters DNA scaffolded silver nanoclusters have many unique properties, one of these is fluorescence which allows characterisation of the nanoclusters by measuring their excitation and emission spectra. Our Aptamer-AgNC candidates produced emission spectra in the orange to red region of the visible spectrum (Fig. 7). The differences in fluorescent properties could relate to how many atoms of silver are in each cluster and also their association with the surrounding DNA sequence. Fluorescence spectroscopy was also used to measure the stability of the NCs by heating and cooling the same sample to find the temperature at which the NCs became unstable, indicated by a loss of fluorescence, and also whether the NCs would reform when cooled back down, indicated by a return of fluorescence. It was found that the fluorescence intensity started to decrease from 40°C upwards with an almost complete loss of fluorescence occurring at 90°C. However, this decrease in fluorescence above 40°C was recovered to a significant degree by cooling the NC back to 10°C, as seen in figure 8.

Circular dichroism spectroscopy was used to determine whether the formation of the silver nanoclusters changes the conformation of the DNA secondary structure. Both JN 17 and JN21 NCs had similar CD spectra to their native oligo before cluster synthesis. This indicates that the formation of the NC on the aptamer sequence does not change the structure significantly and that it appears that the aptamer maintains its secondary structure which is important for binding. Interestingly there is a significant change in the CD spectra of JN 17 and JN21 when Ag⁺ is added to the sample in the form of AgNO₃ and left to bind for an hour (Fig. 9). The spectra obtained is characteristic of the formation of an i-motif according to relevant literature [21]. I- motifs are characterised as an upwards peak at 290 nm and a downwards peak at 260 nm, also as the concentration of Ag⁺ increases the spectra shifts down and to the right. I-motifs can form in cytosine rich oligonucleotides where strings of C's fold in a certain way. The NC sequences attached to the aptamer sequences are not long enough to form intra-strand i-motifs. However, without wishing to be bound by theory, the inventors hypothesize the aptamers are coming together as dimers or tetramers to form inter-strand i-motifs with each other in the presence of Ag⁺.

Aptamers JN 17, JN21, JN27 and st21lpl7 identified as forming i-motifs in the presence of Ag⁺ were tested for antibacterial activity, as the Ag⁺ remained associated with the DNA after removing the excess silver from the sample (Figs 13, Fig. 24).

A study by Lee et al. [22] showed that the addition of Ag⁺ to prepared and purified silver nanoclusters changed the fluorescence from red to green due to the Ag⁺ inducing a conformational change. They utilised this phenomenon as a method to detect free silver in solution. To determine whether the same spectral shift occurred with the aptamer-AgNCs we describe, a 12 fold molar excess of Ag⁺ was added to the aptamer-AgNCs and the change in fluorescence with time observed. After 30 minutes a green emission was detectable with an excitation wavelength at 502 nm and emission wavelength at 572 nm that was not there previous present prior to the addition Ag⁺. The red emission spectra of the nanocluster remained although diminished in intensity relative to that prior to the addition of Ag⁺ (Fig. 10).

Biological characterisation

The main aim of this study was to test the antibacterial properties of aptamer-AgNCs and aptamer-Ag⁺. In previous research we conducted it was shown that the aptamers themselves had no effect on the growth of the bacteria so we hypothesised that the targeting ability of the aptamer to P. aeruginosa combined with the silver could make an effective antimicrobial.

Growth curves were used to determine antimicrobial effects by following the OD₆₀₀ of the samples for 16-20 hours, with a measurement taken every 5 minutes. After seeing initial positive results we wanted to determine whether the placement of the nanocluster sequence had an effect on the antibacterial activity. A series of aptamer-AgNCs were tested using the JN21 and JN 17 aptamers. The NC sequence was placed on the 5' or 3' side, or both, as well as with or without a poly T5 linker between the NC sequence and the aptamer. We hypothesised that the linker would allow the NC to form away from the aptamer sequence and make it more effective. Surprisingly it was the opposite that was seen. The aptamer-AgNCs with no linker had a stronger bactericidal effect then those with the linker, irrespective of being on the 5' or 3' side (Fig. 25). Without wishing to be bound by theory, the inventors believe that this result is consistent with the CD spectral analysis shown in figure 9, and supports the hypothesis that the nanoclusters do not significantly alter the aptamer secondary structure. The JN21 aptamer was also more effective as the split NC sequence on both the 5' and 3' side, and this, along with JN 17 split NC, JN27 5'-NC and St21Lpl7 split- NC were our top candidates for further investigation.

All four showed bacteriostatic effects (Fig. 11, Fig. 26). Concentrations were based on the concentration of the DNA, assuming that each synthesis is the same when following the same standard protocol. After 20 hours CFU counts were performed on the concentration that had complete inhibition of growth as determined from the measured optical density (OD₆₀₀) . The number of CFU formed after 24 hr growth was determined and compared to the starting inoculum size. At 5 μΜ all aptamer-AgNCs had no growth after 16 hours, and the number of CFU for 10 μΜ showed that JN21 had 99.9985% killing compared to the untreated control, while JN 17 had 99.995 % (Fig. 12). This indicates that at 10 μΜ concentration both aptamer-AgNCs (JN 17 and JN21) are bactericidal. Dose response curves show that the EC50 for JN21 and JN 17 AgNCs are 2.439 μΜ and 2.18 μΜ respectively.

Growth curves were also used to assess the antibacterial activity of aptamer-Ag⁺. Without wishing to be bound by theory, the inventors theorized that because silver toxicity is thought to be mediated by silver ions, aptamers chelated with Ag⁺ would be more effective than the nanoclusters that contain metallic Ag. The aptamer-Ag⁺ had strong antibacterial activity (Fig. 13, Fig. 24). All four aptamer-Ag⁺ tested showed no growth at 2.5 μΜ after 20 hours of culture. Enumeration of viable CFU was performed for the 2.5 μΜ treatment after 20 hours to determine the extent of killing. JN27 showed 99.996% killing, St21lpl7 99.995% killing, JN21 99.7% killing and JN 17 93.5% (Fig. 14 and 15). From dose response curves the EC50 for JN27, St21Lpl7, JN21 and JN 17 Ag⁺ are 0.7887 μΜ, 1.015 μΜ, 1.029 μΜ and 1.179 μΜ respectively.

When P. aeruginosa cultures were co-treated with both aptamer-AgNC (1 μΜ and 5 μΜ) and ciprofloxacin (at V2 MIC concentration, 0.5 μg/mL), synergy was observed such that the combined treatment was more effective than the effect seen by either the aptamer-AgNC or ciprofloxacin alone (Fig. 16, Fig 31.) and determined by the time required for the culture to grow to an OD600 of 0.2. This indicates the use of silver NCs to re-sensitise resistant bacteria to common antibiotics synergistically enhance antibiotic therapy.

The aptamer-Ag⁺ compositions were also able to kill a range of different strains of P. aeruginosa other than the test strains PA692 and PA01. A range of strains, as indicated in Fig. 17, were treated with 2.5 μΜ aptamer-Ag⁺ and the growth monitored by OD600 measurement as previously described. All P. aeruginosa strains were highly susceptible to aptamer-Ag⁺ treatment, compared with the equivalent concentration of AgNOs (Fig. 17, Fig 32). The efficacy of the aptamer-AgNC and aptamer-Ag⁺ was then tested in vivo using Galleria mellonella larvae which is an established invertebrate (insect) model for infection. The P. aeruginosa strain PAOl was used as the infecting bacteria as it is highly virulent being capable of killing larvae at low doses. Larvae were injected with the bacteria followed by a second injection containing the treatment after 15 minutes. 10 μΜ of the JN27 aptamer-AgNC was effective at increasing the median survival time by 3 hours, Log-rank (Mantel-Cox) testing showed a statistically significant difference (P value=0.0013) (Fig. 18). A comparison between larvae treated with lower dose of 5 μΜ JN21 aptamer-AgNC or JN21 aptamer-Ag⁺ showed an increase of 4.5 hours to the median survival time for those treated with the aptamer-Ag⁺ (Fig. 18). Log-rank testing showed a significant difference (P value=0.0023) between the survival curves for the control and the JN21 Ag⁺ treatment, and no significant difference between the control and the NC treatment at 5 μΜ.

Changes in metabolic activity were also tested. Bacteria were incubated with 5 μΜ aptamer-AgNC and aptamer-Ag⁺ for 30 mins before addition of 2 μg/mL propidium iodide (PI) for 2 mins to look for changes in cell permeability by flow cytometry analysis. Bacterial populations showed an increase in PI uptake after 30 min treatment indicating that the aptabiotic is permeabilising the bacterial membrane (Fig. 19). This is seen as a right shift in PI fluorescence from gate H2 to gate H3, with the fraction visible in H3 gate increasing from 30% in the control to over 70% for all treatment groups. This increase in PI uptake was also seen after only a 5 min treatment, showing that the action of the aptabiotic is rapid.

To determine the time required to achieve killing of an inoculum containing 1 xlO⁵ cells, enumeration of cfu after treatment with 1 μΜ of JN21split Ag⁺ was undertaken. After 1 hour of treatment only 31 cells of the starting 2xl0⁵ inoculum remained and after 3 hours only 2 cells remained (Fig. 20).

Example 3:

Continued testing of aptamer-AgNCs and aptamer-Ag⁺ Method Circular dichroism studies were performed as in example 2. Growth assays were performed as in example 2. Galleria mellonella were performed as in example 2. Time to kill assays

To determine the time required for the aptamer-AgNC and aptamer-Ag⁺ constructs to kill P. aeruginosa an inoculum of 2xl0⁵ CFU of exponentially growing PA692 was incubated with 5 and 10 μΜ aptamer-AgNC, or 1 and 2.5 μΜ aptamer-Ag⁺ in 10 mM MES buffer. After 10 min, 1 and 3 hrs, an aliquot of the culture was diluted to reduce the concentration of aptabiotic to allow enumeration of viable bacteria by plating on agar. Metabolic effects measured by flow cytometry

Flow cytometry was performed using a BD FACSCanto II flow cytometer (BD

Biosciences, USA). Cells were grown in LB to an OD₆₀₀ of 0.1 then adjusted to give a final concentration of 5xl0⁶ CFU/mL. The culture was washed once in LB before being re-suspended in LB containing 0.1% BSA. LB was used to prevent de-energisation due to nutrient deprivation as occurs when cells are re-suspended in phosphate buffer. A 10 μL aliquot of cells (5xl0⁴ CFU) was incubated with 5 and 10 μΜ aptamer-AgNC, or a range of aptamer-Ag⁺ concentrations (5 μΜ to 0.078 μΜ) each in a total volume of 50 μL for 10 min or 1 hr before addition of 1.5 μg/mL propidium iodide (PI) and 1.5 mM SYT09. Cells were identified by their forward and side scatter properties and by staining with 1.5 mM SYT09. At least 10,000 events in the gated bacterial population were collected and the data analysed using Flowing Software 2.5.1 (Turku Centre for Biotechnology, Finland).

Results

Circular dichroism studies of i-motif formation All three aptamer-AgNCs had similar CD spectra compared to the respective native oligo prior to AgNC synthesis. The native oligonucleotides showed a peak at approximately 280 nm and a trough around 240 nm. The formation of AgNC did not cause a significant shift in the wavelengths indicating no conformational changes to the aptamer upon AgNC formation. The addition of a 6-fold molar excess of AgNO₃, however, induced a bathochromic shift in the spectra with the trough shifting to approximately between 250-265 nm, and the peak shifting down and across to approximately 290 nm. These changes induced by addition of Ag⁺ are consistent with the formation of a folded structure with spectral characteristics similar to an i-motif as has been previously reported [21, 28, 29, 30]. The formation of the i-motif structure was further investigated by titrating a range of molar excess concentrations of AgNO₃ with 10 μΜ of the native oligonucleotide (which has the NC poly C sequence but no NCs generated), and the CD spectra was obtained. Figure 27 shows the incremental bathochromic shift of the spectra from native oligo to a spectra indicative of an i-motif conformation with the increase in Ag⁺. A classic full i- motif sequence was also used to demonstrate a characteristic Ag⁺ stabilised i-motif spectra, and a half i-motif to show that they can come together as dimer to give a characteristic i-motif CD spectra.

I-motifs can form in cytosine rich oligonucleotides where strings of cytosines fold in a certain way. The NC sequences attached to the aptamer sequences are not long enough to form intra-strand i-motifs. However, without wishing to be bound by theory, the inventors hypothesize the aptamers are coming together as dimers or tetramers to form inter-strand i-motifs with each other in the presence of Ag⁺.

Aptamers JN21 split, JN27 5' and st21lpl7 split identified as forming i-motifs in the presence of Ag⁺ were tested for antibacterial activity, as the Ag⁺ remained stably associated with the DNA after removing the excess silver from the sample (Fig. 13 and Fig. 24). The term "aptamer-Ag⁺" as used herein means aptamers that form i-motifs that remain stably associated with Ag⁺.

Aptamer targeting enhances antimicrobial action

To determine whether the AgNCs show antimicrobial activity and whether aptamer targeting would improve their efficacy, P. aeruginosa cultures were treated with either AgNC alone (untargeted) or aptamer-AgNC. Figure 28 shows the growth of P.

aeruginosa without treatment (black solid), when treated with 2.5 μΜ AgNC (dark grey dashed), or when treated with 2.5 μΜ of the same nanocluster forming sequence conjugated to either aptamer JN27 (dark grey solid), JN21 (light grey solid) or St21Lpl7 (black dashed). Treatment with the untargeted AgNC results in a significant increase in the time required for the culture to achieve the same density as the untreated control (arrowed line). The growth delay is greater for all of the aptamer conjugated AgNCs relative to the untargeted AgNC indicating enhanced killing due to aptamer targeting.

Dose dependent killing of P. aeruginosa by aptamer-AgNCs and aptamer-Ag⁺s

Figure 26 shows the effect that a 2-fold dilution series in the range 10 μΜ to 78 nM of different aptamer-AgNCs has on the growth of PA692 (three independent experiments were performed and similar results were obtained for each). Concentrations were based on the concentration of the DNA, assuming that each synthesis is the same when following the same standard protocol. In the absence of treatment (black solid) cultures grew rapidly and achieved an optical density greater than 0.1 by

approximately 300 minutes. Increasing amounts of aptamer-AgNC caused a

proportional increase in the apparent lag time in a dose-dependent manner, with no growth being detected for the 5 μΜ or 10 μΜ treatment by the end of the experiment (960 min). At the final time point of each experiment the 5 and 10 μΜ wells which showed no growth (no turbidity) were spread on to LB agar plates and cultured for 24 hours to allow for enumeration of any viable bacteria at these treatment

concentrations which had not had time to reach a detectable threshold by experiment completion, or to determine whether no growth was due to complete sterilisation of the starting inoculum. As expected the control plate was completely overgrown as a dense lawn that produces large amounts of the coloured pigments pyocyanin and pyoverdine (Figure 29). While there was no detectable growth for 5 and 10 μΜ in the plate assay, a fraction of the initial inoculums were still viable for the 5 μΜ treatments, with JN21 split-AgNC treatment producing a lawn comparable to control. The 10 μΜ treatments showed complete sterilisation of the culture for JN27 5' and St21Lpl7 split- AgNC and very few remaining CFU for JN21 split-AgNC.

Growth curves were also used to assess the antibacterial activity of aptamer-Ag⁺. The aptamer-Ag⁺ had strong antibacterial activity (Fig. 24). All aptamer-Ag⁺ tested showed no growth at 2.5 μΜ after 20 hours of culture. Enumeration of viable CFU was performed for the 2.5 μΜ treatment after 20 hours to determine the extent of killing. JN27 5' showed 99.996% killing, St21lpl7 3' 99.995% killing and JN21 split 99.7% killing (Fig. 14). To determine whether no growth in the clear wells at higher concentrations was due to the length of the experiment or complete sterilisation, the whole wells were spread on to agar plates. In figure 30 it can be seen that at 2.5 μΜ there are still viable cells remaining, as was evident from the CFU counts in the previous experiment. At 5 μΜ and 10 μΜ, however, complete sterilisation of the cultures was seen for all aptamer-Ag⁺ tested - JN27 5'-Ag⁺, JN21 split-Ag⁺ and St21Lpl7 split-Ag⁺. Aptamer-AgNC and aptamer-Ag⁺ conjugates rapidly kill P. aeruginosa

To determine the time required to achieve killing, an inoculum containing 2xl0⁵ CFU was treated with 5 or 10 μΜ of JN27 5', JN21 split or St21Lpl7 split-AgNC, or 1 μΜ and 2.5 μΜ of the same aptamers made as aptamer-Ag⁺ rather than reduced to AgNCs. After 10 min, 1 hr and 3 hrs, traces of aptamer-AgNC/Ag⁺ were removed by dilution then plated for enumeration of CFU. For aptamer-AgNCs 5 μΜ treatment the onset of killing was shown to be rapid for JN27 and JN21 as after 10 min the population treated with JN27 showed 24 ±7.9% death and JN21 46 ± 12.7% death, whereas St21Lpl7 was only 9.2 ± 4.6%. After 1 hr these apt-AgNCs had killed 79.5 ±5.3, 80.7 ±3.3%, and 47.6 ±6.2% respectively. At 3 hrs they had killed 90.8 ±3.14%, 87.9 ±0.49%, and 56.6 ± 13.2% of the starting population respectively (Figure 33, Table 2). For the 10 μΜ treatment it was expected that the killing effect would be more rapid and pronounced due to the higher concentration but the data shows the opposite effect. At 10 min they showed 10.9 ±5.4% for JN27, 15.8 ±8.2% for JN21 and 17.6 ±9.1% for St21Lpl7. After 1 hr it was 61 ±5%, 61.8 ± 4.2% and 66.1 ±9.4% respectively, and at 3 hrs this had slightly increased to 73.3 ±8.2%, 71 ±8% and 66.4 ± 10.3% respectively (Figure 33, table 2).

For aptamer-Ag⁺ 1 μΜ treatment resulted in approximately 50% bacterial death for JN27 and JN21 (49.4 ±8.7% and 50.7 ± 10.2%) after 10 min, with 23.2 ±7.3% for St21Lpl7. After 1 hr these aptamer-Ag⁺s had killed 83.1 ±4.7%, 80.1 ±4.4%, and 89.5 ±7.7% respectively. At 3 hrs they had killed 98.4 ±0.3%, 98.1 ±0.5%, and 99.8 ±0.1% of the starting population respectively (Figure 34, table 2). A very similar trend was seen with the 2.5 μΜ treatment of JN27, JN21 and St21lpl7-Ag⁺ with 55.3 ±9.4%, 53.2 ± 14% and 49 ± 12.1% respectively at 10 min. At 1 hour they had killed 88.7 ±2.1%, 86.4 ±3.7% and 91.2 ±8.6% respectively, and at 3 hours they had killed 98.3 ±0.8%, 98.5 ±0.8% and 99.9 ±0.07% respectively (Fig. 34, table 2). These results show the rapid killing effect of the aptamer-AgNCs and aptamer-Ag⁺, as well as the increase in antimicrobial effect of the aptamer-Ag⁺ compared to aptamer-AgNCs.

Table 2. Percentage of bacterial population dead at each time point for the time-to-kill assay with aptamer-AgNCs and aptamer-Ag⁺s.

This comparison in antimicrobial effect between aptamer-AgNC and aptamer-Ag⁺ is also shown in figure 35, when the increase in lag phase of the PA692 culture caused by each concentration of treatment is compared between the two types of aptamer- silver conjugate. Aptamer-Ag⁺ has a stronger antimicrobial effect for all three aptamers tested.

Aptamer-AgNC and aptamer-Ag⁺ conjugates depolarise P. aeruginosa cells

The metabolic effects of aptamer-AgNC and aptamer-Ag⁺ treatment were explored using flow cytometry to look at the permeabilisation of the cells using the viability stain propidium iodide. One mode of silver toxicity is through disruption of membrane integrity and disruption of electron transport leading to a decreased proton motive force (pmf) for ATP synthesis and other energy requiring processes. The vital dye PI is excluded from healthy bacterial cells because of its low membrane permeability and extrusion by pmf energised efflux pumps; dead, damaged or de-energised bacteria show an increase in PI uptake that can be readily measured by flow cytometry.

Exponentially grown P. aeruginosa when washed and stained with PI can be fractionated into two populations, those that are highly impermeable and show high levels of PI extrusion, and those that have increased permeability.

Similar results were seen at both the 10 min and 1 hr time points for aptamer-AgNC showing that action of the apt-AgNC has a rapid onset. Three independent

experiments were performed and after a 10 min treatment with aptamer-AgNC the bacterial population shifted from 9.35 ±0.25% of the control in the high PI population to 54.34 ±4.9%, 52.58 ± 1.2% and 36.1 ±0.89% respectively for treatment with JN21 split, JN27 5' and St21Lpl7 split-AgNCs, as shown in Figure 36 with no change in ciprofloxacin treatment compared to control being seen. After a 1 hr treatment the bacterial population shifted from 9.61 ±0.12% of the control in the high PI population to 50.09 ±5.7%, 41.14 ±8.7% and 30.7 ±6.3% respectively for treatment with JN21 split, JN27 5' and St21Lpl7 split AgNCs, as shown in Figure 36. For aptamer-Ag⁺ testing a range of concentrations from 0.0078 μΜ to 5 μΜ were tested for depolarisation of the bacteria (Fig. 37). For all aptamers tested after 10 min the most depolarisation was seen for those treated with 1.25 μΜ and after 1 hour the most depolarisation occurred with 0.63 μΜ (n=2). Aptamer-AgNC and aptamer-Ag⁺ conjugates enhance survival in an

invertebrate model of bacterial infection

Figure 38A and 38B shows larvae injected with PAOl and then treated with three different aptamer-AgNCs at 10 μΜ to look for an increase in median survival time compared to untreated control. Two independent experiments were performed and similar results obtained for each. Each treatment was given as a second injection 20 min following infection with 10 CFU of PA01. All larvae for the untreated control were dead by 20 hours (black solid). Treatment with JN21 split-AgNC (black dashed) and st211 p 17 split-AgNC (grey dashed) increased the median survival time to 19 hours, compared to 18 hours for control, and JN27 5'-AgNC (grey solid) increased median survival to 20 hours.

Aptamer-AgNC and aptamer-Ag⁺ conjugates are not substrates for efflux pumps

P. aeruginosa has high resistance to many antibiotics through expression of multidrug resistance pumps of the RND class [31, 32, 33]. These broad specificity pumps are able to extrude antimicrobials such that the intracellular concentration does not achieve a sufficient therapeutic dose. We examined the sensitivity of P. aeruginosa PA01 and an isogenic efflux pump MexB knockout mutant, to the antibiotic

ciprofloxacin and to each aptamer-AgNC and aptamer-Ag⁺ (Figure 39 and 40, table 3). Panel A shows a dose response plot for PA01 (black) and MexB (red) treatment with ciprofloxacin for 16 hrs, and panel B-D treatment with each aptamer-AgNC for 16 hrs. The efflux mutant is highly sensitive to ciprofloxacin with the IC50 being 0.01781 μg/mL, compared to 0.2507 μg/mL for wild type PA01, whereas the aptamer-AgNCs and aptamer-Ag⁺s had similar sensitivity in the MexB mutant and wild type strain. These data are consistent with the aptabiotic being a poor efflux pump substrate. It was considered that the rapid killing action of the aptabiotic may not have allowed time for it to be effluxed, however if this was the case we would expect to see less of an antimicrobial effect for sub IC50 concentrations between the wildtype and the mexB knockout, however the antimicrobial effect was observed to be the same. Table 3. The IC50 of PA01 and the isogenic efflux mutant MexB when treated with ciprofloxacin, aptamer-AgNC and aptamer-Ag⁺.

Aptamer-AgNC and aptamer-Ag⁺ conjugates cause dissociation of established biofilms

Aptamer-AgNCs and aptamer-Ag⁺s were tested as a treatment (5 μΜ) against both immature (20 hour) and mature (44 hour) biofilms and their action compared to that of the common fluoroquinolone antibiotic ciprofloxacin at lxMIC or 0.5xMIC for planktonic PA692. Immature biofilms were treated either once for 6 hours, or twice with the second treatment lasting 17 hours. This was to see if the replacement of the aptabiotic with fresh aptabiotic to keep the concentration high would increase the killing effect as it is hypothesised that the aptabiotic degrades during treatment. For the one and two treatments of 20 hour biofilms with aptamer-Ag⁺ it was found that all treatments significantly decreased the biofilm biomass as determined by crystal violet staining (Fig. 41 B). With just one treatment the aptamer-Ag⁺s were more effective at decreasing the biomass than both concentrations of ciprofloxacin, but after two treatments they were about equal to the higher concentration of ciprofloxacin (Fig. 41 D). It was also found that all treatments could limit or inhibit the growth of planktonic cells shed from the biofilm during antimicrobial challenge (Fig. 41A and C). Treatment of a mature biofilm showed that the aptamer-Ag⁺s were more effective than both concentrations of ciprofloxacin at dissociating the biomass (Fig. 42 B) and inhibiting planktonic growth from dispersed cells (panel A) as shown in Figure 42. There was a statistically significant difference between groups for both the decrease in biomass (n=6) and inhibition of planktonic growth (n=6) as determined by one-way ANOVA (F(5,30)=41.7, p= .0001) and (F(5,30)=263, p= .0001) respectively. Dunnet's post hoc test revealed that these changes compared to control growth were statistically significant for all treatments. These results show that aptamer-Ag⁺s could have potential use as biofilm dissociating agents. Aptamer-AgNCs were also tested for antimicrobial biofilm dissociating effects at 5 μΜ. They had a smaller antimicrobial effect then the aptamer-Ag⁺s, and were similar in ability to the two concentrations of ciprofloxacin tested.

Example 4: Broad spectrum utility of Ag⁺ i-motifs Methods

Growth assays were performed as in example 2.

Bacterial strains tested - P. aeruginosa 692 (ATCC 14502), P. aeruginosa PAOl (ATCC 15692), Salmonella enterica serovar typhi (ATCC 19430), Klebsiella pneumoniae (ATCC 13883), Enterobacter cloacae (ATCC 13047), Listeria innocua (ATCC 33090), Escherichia coli DH5a, Staphylococcus aureus (ATCC 25923), and Staphylococcus epididymis (ATCC 14990).

Circular dichroism spectra were performed as in example 3, except a 6-fold molar excess of Ag⁺ was added rather than a titration performed. A 12-fold molar excess was required in once instance, as indicated, to form the i-motif.

Results

Figure 43 shows that the Ag⁺ stabilised i-motif (SEQ ID NO 38) has bactericidal or bacteriostatic activity against both Gram-negative bacteria and Gram-positive bacteria. Complete inhibition of growth was achieved at a concentration of 2.5 μΜ for P. aeruginosa, E. coli, and K. pneumoniae, (Fig 43A, B, D, F) while the same concentration suppressed growth of S. aureus, L. innocua, E. cloacae, S . epidermidis, S. enterica (Fig 43C, E, G H I). Growth inhibition was observed at all concentrations of the Ag⁺ stabilised i-motif tested. Table 4 records the time required for the culture to attain an OD600 of 0.1 as a measure of the extent of growth inhibition relative to the untreated control. Similar results would be expected if other Gram-positive and Gram- negative were tested for susceptibility. Table 4. The time taken (minutes) for different bacterial species treated with a range of concentrations (μΜ) of i-motif2 (SEQ ID NO:38) to reach a threshold value of 0.1 OD600- Dashes indicate the threshold was not met during the 1015 minute duration of the experiment.

To demonstrate that the antimicrobial activity is an inherent property of Ag⁺ stabilised i-motifs due to the presence of silver ions, a selection of silver i-motif forming sequences were evaluated. Sequences were selected from a set comprising poly-C tracts and turns, such that the i-motif is formed by concatermerisation of alternating poly-C and turns. The poly-C tracts were selected from a set that contained the sequences: CCC, CCCC, CCCCC, CCCCCC, CCCCCCC, CCCCCCCC, CCCCCCCCC, and the turns from the set: A, T,

AA, TT, AT, TA, AAT, ATA, TAA, ATT, TTA, TTT, AAAA, AAAT, AATA, ATAA, TAAA, AATT, ATTA, TTAA, ATTT, TTTA, M M . Sequences that varied from the possible combinations derived from the set of pol-C tracts and turns was allowed by inclusion of G or GG in either one or more of the turn or poly-C tracts. The extent of concatermerisation (the number of poly-C/turns used) was restricted to 6 for the purposes of demonstration only. The sequences experimentally evaluated were, SEQ ID NO 31, SEQ ID NO 32,

SEQ ID NO 38, SEQ ID NO 39, SEQ ID NO 40, SEQ ID NO 41, SEQ ID 42, SEQ ID 43 and are presented as an indicative set of possible Ag⁺ stabilised i-motifs. This set of sequences were all confirmed to form Ag⁺ stabilised I-motif structures by circular dichroism spectroscopy (figure 46).

Figure 44 and Figure 45 show that all the Ag⁺ i-motif sequences tested inhibited the growth of P. aeruginosa strains PA692 and PAOl respectively, but that the i-motif forming sequences differ in their antimicrobial activity. Table 5 shows that all seven of the different Ag⁺ stabilised i-motifs tested were either bacteriostatic or bactericidal for P. aeruginosa strain PAOl at the concentrations tested . Table 5 records the time required for the cultures to obtain an OD₆₀₀ of 0.1. No growth was observed after 1015 minutes in culture for any of the Ag⁺ i-motifs at 5 μΜ concentration. Table 5. The time taken (minutes) for P. aeruginosa strain PAOl treated with a range of concentrations of different i-motif structures to reach a threshold value of O.l OD600- Dashes indicate the threshold value was not met during the time 1015 period and no growth occurred.

Table 6 shows that all seven of the different Ag⁺ stabilised i-motifs tested were either bacteriostatic or bactericidal for P. aeruginosa strain PA692 at the concentrations tested . Table 6 records the time required for the cultures to obtain an OD600 of 0.1. No growth was observed after 1015 minutes in culture for any of the Ag⁺ i-motifs at 2.5 and 5 μΜ concentrations.

Table 6. Time taken (minutes) for P. aeruginosa strain 692 treated with a range of concentrations of different i-motif structures to reach a threshold value of 0.1 OD600 in a maximum time period of 1015 minutes. Dashes indicate the threshold was not met during the time period.

Figure 47 shows that all the Ag⁺ i-motif sequences tested had an inhibitory effect on the growth of Staphylococcus aureus, and that the i-motif forming sequences differ in their antimicrobial activity.

Table 7 shows that all seven of the different Ag⁺ stabilised i-motifs tested were either bacteriostatic or bactericidal for S. aureus at the concentrations tested. Table 7 records the time required for the cultures to obtain an OD600 of 0.1. Table 7. Time taken (minutes) for S. aureus treated with a range of

concentrations of different i-motif structures to reach a threshold value of 0.1 OD600 in a maximum time period of 1015 minutes. Dashes indicate the threshold was not met during the time period.

It is well reported in the literature that EDTA co-treatment with antibiotics can improve the efficacy of anti-pseudomonal therapy, as EDTA is thought to increase the permeability of the bacterial cell wall by chelation of Mg²⁺ ions (Chauhan et al. 2012). Table 8 shows that co-treatment with a low concentration of EDTA (2 mM) significantly enhance the antimicrobial action of silver stabilised i-motif against a variety of different Gram-negative and Gram-positive bacteria, specifically Klebsiella, Salmonella and Enterobacter. Although for some bacterial strains treatment with EDTA alone caused an inhibitory effect so potential synergy can't be seen at this concentration of EDTA.

Table 8. Time taken (minutes) for different bacterial species treated with 1.25 μΜ of i-motif2 (SEQ ID NO:38), with or without 2 mM EDTA co- treatment, to reach a threshold value of 0.1 OD600 . Dashes indicate the threshold value was not met during the time period (1015 minutes) of the experiment and no growth occurred . Bacteria were treated with 2 mM EDTA alone also as a control.

Growth curves for the data presented in Table 8 are shown in Figure 48.

Surprisingly the Gram-positive organism S .aureus also showed strong anti-microbial synergy between EDTA treatment and half i-motifl (SEQ ID NO: 39). The half i-motifl was used in the concentration range 0.078 μΜ to 1.25 μΜ, with EDTA in the range 0.1 mM to 0.75 mM. Figure 49 shows growth curves for the various combinations of half i- motifl and EDTA, and demonstrates that EDTA enhances the antibacterial effect of half i-motifl above that of EDTA treatment alone, or half i-motif treatment alone. Table 9. Time taken (minutes) for S. aureus treated with 0.078 to 1.25 μΜ of half i-motifl (SEQ ID NO: 39), with or without 0.1 to 0.75 mM EDTA co- treatment, to reach a threshold value of 0.1 OD600 . Dashes indicate the threshold value was not met during the time period (1015 minutes) of the experiment and no growth occurred . Bacteria were treated with EDTA alone also as a control.

Appendix A:

Nucleic Acid Sequences

SEQ ID NO: 1

JN27 - 5'-ATG AGA GCG TCG GTG TGG TAA CTA GTC TGA TTT CTA TTT CCT TTA ATT AGT CTG CAC ACA TTG CAT TGT AGG AGG GTG CGG AAG TA-3'

SEQ ID NO: 2

JN08 - 5'-ATG AGA GCG TCG GTG TGG TAA CTT AAT CCT CTA GTT TTA ATT CTA GAT ACG GCG CAT GCA CTC TAT GTA GGA GGG TGC GGA AGT A-3'

SEQ ID NO: 3 JN27.SH - 5'-TAA TTA GTC TGC ACA CAT TGC ATT GTA GGA GGG TGC GGA AGT A-3' SEQ ID NO: 4

JN27 5' NC - 5'- TCC CCC CCG TCT GCA CAC ATT GCA TTG TAG GAG GGT GCG GAA G-3'

SEQ ID NO: 5 JN27 3' NC - 5'- G TCT GCA CAC ATT GCA TTG TAG GAG GGT GCG GAA TCC CCC CCT G-3'

SEQ ID NO: 6

JN27 split NC - 5'- TCC CCC GTC TGC ACA CAT TGC ATT GTA GGA GGG TGC GGA AGC CCC T-3' SEQ ID NO: 7

JN08.SH - 5'-ATG CAC TCT ATG TAG GAG GGT GCG GA-3' SEQ ID NO: 8

JN08 5' NC- 5'-TCC CCC CCT ATG CAC TCT ATG TAG GAG GGT GCG GA-3' SEQ ID NO: 9 JN08 3' NC - 5'-ATG CAC TCT ATG TAG GAG GGT GCG GAT CCC CCC C-3' SEQ ID NO: 10

JN08 split NC - 5'-TCC CCC ATG CAC TCT ATG TAG GAG GGT GCG GAC CCC T-3' SEQ ID NO: 11 JN21.SH - 5'-AGA GCG TCG GTG TGG TAA CTG TTC AGG AGG ATG ACA TTG TCG CCT-

3'

SEQ ID NO: 12

JN21 5' NC - 5'-TCC CCC CCT AGA GCG TCG GTG TGG TAA CTG TTC AGG AGG ATG ACA TTG TCG CCT-3' SEQ ID NO: 13

JN21 3' NC - 5'-AGA GCG TCG GTG TGG TAA CTG TTC AGG AGG ATG ACA TTG TCG CCT CCC CCC CT-3'

SEQ ID NO: 14

JN21 split NC - 5'- TCC CCC AGC GTC GGT GTG GTA ACT GTT CAG GAG GAT GAC ATT GTC GCC CCT-3'

SEQ ID NO: 15

JN 17.SH - 5'-ATC CTC CTA TCT ATC GGT AGT TGT AGG AGG GTG CGG-3' SEQ ID NO: 16

JN 17 5' NC - 5'-TCC CCC CCT ATC CTC CTA TCT ATC GGT AGT TGT AGG AGG GTG CGG-3'

SEQ ID NO: 17

JN 17 3' NC - 5'-ATC CTC CTA TCT ATC GGT AGT TGT AGG AGG GTG CGG TCC CCC CCT-3'

SEQ ID NO: 18 JN 17 split NC - 5'- TCC CCC ATC CTC CTA TCT ATC GGT AGT TGT AGG AGG GTG CCC CT-3'

SEQ ID NO: 19

St21Lpl7 - 5'- AAG CGT CGG TGT TCT ATC GGT AGT TGA CAC CGA CGC CT-3' SEQ ID NO: 20

St21Lpl7 5' NC - 5'- TCC CCC CCT AAG CGT CGG TGT TCT ATC GGT AGT TGA CAC CGA CGC CT-3'

SEQ ID NO: 21

St21Lpl7 3' NC - 5'- AAG CGT CGG TGT TCT ATC GGT AGT TGA CAC CGA CGC CTT CCC CCC CT-3'

SEQ ID NO: 22

St21Lpl7 split NC- 5'- TCC CCC AAG CGT CGG TGT TCT ATC GGT AGT TGA CAC CGA CGC CTC CCC T-3'

SEQ ID NO: 23 Stl7Lp21 - 5'-ATC CTC CTA GGT AAC TGT TCA GGA TGT AGG AGG GT-3' SEQ ID NO: 24

Stl7Lp21 5' NC - 5'-TCC CCC CCT ATC CTC CTA GGT AAC TGT TCA GGA TGT AGG AGG GT-3'

SEQ ID NO: 25 Stl7Lp21 3' NC - 5'- ATC CTC CTA GGT AAC TGT TCA GGA TGT AGG AGG GTC CCC CCC T-3'

SEQ ID NO: 26

Stl7Lp21 split NC - 5'-TCC CCC ATC CTC CTA GGT AAC TGT TCA GGA TGT AGG AGG GTC CCC T-3' SEQ ID NO: 27 St08Lpl7 - 5'-ATG CAC TCT TCT ATC GGT AGT TGA GGG TGC GG-3' SEQ ID NO: 28

St08Lpl7 5' NC - 5'-TCC CCC CCT ATG CAC TCT TCT ATC GGT AGT TGA GGG TGC GG-

3' SEQ ID NO: 29

St08L.pl 7 3' NC - 5'-ATG CAC TCT TCT ATC GGT AGT TGA GGG TGC GGT CCC CCC CT-

3'

SEQ ID NO: 30

St08Lpl7 split NC - 5'-TCC CCC ATG CAC TCT TCT ATC GGT AGT TGA GGG TGC GGT CCC CT-3'

SEQ ID NO: 31

NC sequence - 5'-TTCCCCCCCT-3' SEQ ID NO: 32 i-motif sequence - 5'-TCC CCT TCC CCT TCC CCT TCC CCT- 3' SEQ ID NO: 33

Forward primer flanking region - 5'-ATG AGA GCG TCG GTG TGG TA-3' SEQ ID NO: 34

Reverse primer flanking region - 5'-TAC TTC CGC ACC CTC CTA CA-3' SEQ ID NO: 35 Forward primer for PCR - 5'- ATG AGA GCG TCG GTG TGG TA-3' SEQ ID NO: 36

Reverse primer for PCR - 5'- TGT AGG AGG GTG CGG AAG TA-3' SEQ ID NO: 37 FAM labelled forward primer for PCR - 5'-FAM-ATG AGA GCG TCG GTG TGG TA-3'

SEQ ID NO: 38

I-motif2 - 5'- TCC CCT AAC CCC TAA CCC CTA ACC CCT- 3' SEQ ID NO: 39 Half i-motifl - 5'-TCC CCT AAC CCC T-3' SEQ ID NO: 40

I-motif3 - 5'- TCC CCC TTT CCC CCT TTC CCC CTT TCC CCC T - 3' SEQ ID NO: 41

I-motif4 - 5'- ACC CAA CCC AAC CCA ACC CG - 3' SEQ ID NO: 42

Half i-motif2 - 5'- TCC CCC TTT CCC CCT - 3' SEQ ID NO: 43

Half i-motif3 - 5'- GCC CCC CAT ACC CCC CG - 3'

Although the invention has been described by way of example and with reference to particular embodiments, it is to be understood that modifications and/or

improvements may be made without departing from the scope of the invention.

In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognise that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

In this specification where reference has been made to patent specifications, other external documents, or other sources of information, this is generally for the purpose of providing a context for discussing the features of the invention. Unless specifically stated otherwise, reference to such external documents is not to be construed as an admission that such documents, or such sources of information, in any jurisdiction, are prior art, or form part of the common general knowledge in the art. All headings and sub-headings are used herein for convenience only and should not be construed as limiting the invention in any way. Any combination of the above- described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. Unless otherwise stated, all exact values provided herein are representative of corresponding approximate values (e. g., all exact exemplary values provided with respect to a particular factor or measurement can be considered to also provide a corresponding approximate measurement, modified by "about," where appropriate).

All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise indicated. No language in the specification should be construed as indicating any element is essential to the practice of the invention unless as much is explicitly stated. The citation and incorporation of patent documents herein is done for convenience only and does not reflect any view of the validity, patentability and/or enforceability of such patent documents, The description herein of any aspect or embodiment of the invention using terms such as reference to an element or elements is intended to provide support for a similar aspect or embodiment of the invention that "consists of," "consists essentially of" or "substantially comprises" that particular element or elements, unless otherwise stated or clearly contradicted by context (e. g. , a composition described herein as comprising a particular element should be understood as also describing a composition consisting of that element, unless otherwise stated or clearly contradicted by context). This invention includes all modifications and equivalents of the subject matter recited in the aspects or claims presented herein to the maximum extent permitted by applicable law. All publications and patent applications cited in this specification are herein incorporated by reference in their entireties as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to a person of skill the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

INDUSTRIAL APPLICATION

The anti-microbial combinations, targeting components, anti-microbial components and anti-microbial combinations disclosed herein all have industrial application as bacteriostatic and/or bactericidal agents, can be formulated into conjugates, complexes and compositions having bacteriostatic and/or bactericidal activity, and can be used in both the manufacture of medicaments for, and the direct treatment of, microbial infection.

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Claims

WHAT WE CLAIM IS:

1. Use of an anti-microbial component comprising a polynucleotide structure that chelates at least one metal ion and an anti-microbial agent to inhibit the growth and/or proliferation of at least one bacterial species, wherein the polynucleotide structure is in association with the anti-microbial agent, wherein the polynucleotide comprises an i-motif structure and the anti-microbial agent is at least one metal ion.

2. The use of claim 1, wherein the antimicrobial agent consists essentially, preferably consists of, the at least one polynucleotide and the at least one metal ion.

3. The use of claim 1 or claim 2, wherein the polynucleotide structure comprises, consists essentially of, or consists of a single, preferably two, preferably four nucleic acid strands, that form the i-motif structure.

4. The use of claim 3, wherein the i-motif structure is formed under suitable conditions, wherein the suitable conditions are conditions in which the nucleic acid strand or strands that form the imotif structure exist in free solution at neutral pH in a configuration that is not a full formed i-motif, wherein the addition of the at least one metal ion stabilizes the nucleic acid strand or strands to form the i-motif structure.

5. The use of any one of claims 1 to 4, wherein the at least one metal ion is Ag⁺.

6. The use of any one of claims 3 to 5, the nucleic acid strand or strands that form the imotif structure comprise a nucleic acid sequence comprising at least one, preferably at least two, preferably four poly C tracts (pCts).

7. The use of claim 6, the nucleic acid strand or strands that form the imotif structure comprise a nucleic acid sequence comprising at least one pair of pCts separated by a turn region (TR), and have the formula (pCt-TR-pCt)n, wherein n is an integer from 1 to at least 10, preferably to at least 100, preferably to at least 1000, preferably to at least 10,000, preferably to at least 100,000.

8. The use of any one of claims 1 to 7, wherein the polynucleotide structure comprises a nucleic acid sequence that is 5'-(CxN_y)_z-3' wherein C = cytosine; N= any combination of adenosine (A), guanosine (G), thymidine (T), analogues of C, A, G and T, and modified deoxynucleosides; x = an integer from 2 to 7; y = an integer from 0 to 7; and z = an integer from 1 to at least 10, preferably at least 100, preferably to at least 1000, preferably to at least 10,000, preferably to at least 100,000.

9. The use of claim 8, wherein x is an integer that is 3, 4, 5, 6 or 7 and y is an integer that is 2, 3, 4 or 5.

10. The use of any one of claims 1 to 9, wherein the polynucleotide structure comprises, preferably consists essentially of, preferably consists of, a nucleic acid selected from the group consisting of SEQ ID NO: 32, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42 and SEQ ID NO: 43.

11. The use of any one of claims 1 to 10, wherein the at least one bacterial species is a Gram-negative bacterial species, preferably wherein the Gram-negative bacterial species is selected from the group consisting of Pseudomonas spp. belonging to the P. aeruginosa group, P. chlolraphis group, P. fluorescens group, P. pertucinogena group, P. putida group, P. stutzeri group, or the P. syringae group, preferably wherein the Pseudomonas spp. is P. aeruginosa, P. florescens, P. putida, P. stutzeri, P. tabaci, P. angulata, P. phaseolicola, P. pisi, P. glycinea, P. solanacearum, P. caryophylli, P. cepacia, P. marginalis, P. savastonoi, P. marginata or P. syringae- Salmonella spp., preferably S. enterica; Klebsiella spp., preferably K. pneumoniae; Enterobacter spp., preferably E. cloacae; Acinetobacter spp., preferably A. baumannii and Escherichia coli.

12. The use of any one of claims 1 to 11, wherein the at least one bacterial species is a Gram-positive bacterial species, preferably a Listeria spp., preferably L. innocua; and/or a Staphylococcus spp., preferably S. aureus or S. epididymis.

13. Use of an anti-microbial component to inhibit the growth and/or proliferation of at least one bacterial species, wherein the anti-microbial component consists essentially of a polynucleotide structure in association with an anti-microbial agent, wherein the polynucleotide structure comprises an i-motif structure, wherein the anti-microbial agent is at least one metal ion, preferably at least one Ag⁺, wherein the imotif structure comprises a single nucleic acid strand that forms the i- motif structure or at least two, preferably four, nucleic acid strands that form the imotif structure, wherein the nucleic acid strand or strands that form the i-motif structure comprise a nucleic acid sequence comprising at least one poly C tract (pCts), and wherein the bacterial species is selected from the group consisting of Pseudomonas aeruginosa, Salmonella enterica serovar typhi, Klebsiella pneumoniae, Enterobacter cloacae, Listeria innocua, Escherichia coli, Acinetobacter baumannii, Staphylococcus aureus, and Staphylococcus epididymis.

14. The use of claim 13 wherein the polynucleotide selected from the group consisting of SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42 and SEQ ID NO: 43.

15. A polynucleotide selected from the group consisting of SEQ ID NO: 32, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42 and SEQ ID NO: 43.

16. A method of treating a microbial infection comprising administering an antimicrobial component to a subject in need thereof, wherein the anti-microbial component comprises a polynucleotide in association with an anti-microbial agent, wherein the polynucleotide comprises an imotif structure and the anti-microbial agent is at least one metal ion.

17. The method of claim 16, wherein the antimicrobial agent consists essentially, preferably consists of, the at least one polynucleotide and the at least one metal ion.

18. The method of claim 16 or claim 17, wherein the at least one metal ion is Ag⁺.

19. The method of any one of claims 16 to 18 wherein the i-motif structure is formed under suitable conditions, wherein the suitable conditions are conditions in which the nucleic acid strand or strands that form the imotif structure exist in free solution at neutral pH in a configuration that is not a full formed i-motif, wherein the addition of the at least one metal ion stabilizes the nucleic acid strand or strands to form the i- motif structure.

20. The method of claim 19, wherein the nucleic acid strand or strands that form the imotif structure comprise a nucleic acid sequence comprising at least one, preferably at least two, preferably four poly C tracts (pCts).

21. The method of claim 19 or claim 20, wherein the i-motif consists essentially of, preferably consists of, a single nucleic acid strand, or of at least two separate nucleic acid strands, or of four separate nucleic acid strands.

22. The method of any one of claims 16 to 21, wherein the i-motif structure comprises a nucleic acid sequence that is 5'-(CxN_y)_z-3' wherein C = cytosine; N = any combination of adenosine (A), guanosine (G), thymidine (T), analogues of C, A, G and T, and modified deoxynucleosides; x = an integer from 2 to 7; y = an integer from 0 to 7; and z = an integer from 1 to at least 10, preferably at least 100, preferably to at least 1000, preferably to at least 10,000, preferably to at least 100,000.

23. The method of claim 22, wherein x is an integer that is 3, 4, 5, 6 or 7 and y is an integer that is 2, 3, 4 or 5.

24. The method of any one of claims 16 to 23, wherein the polynucleotide

comprises, preferably consists essentially of, preferably consists of, a nucleic acid selected from the group consisting of SEQ ID NO : 31, SEQ ID NO: 32, SEQ ID NO : 38, SEQ ID NO: 39, SEQ ID NO : 40, SEQ ID NO : 41, SEQ ID NO : 42 and SEQ ID NO: 43.

25. The method of any one of claims 16 to 24, wherein the at least one bacterial species is a Gram-negative bacterial species.

26. The method of any one of claims 16 to 25, wherein the Gram-negative bacterial species is selected from the group consisting of Pseudomonas spp. belonging to the P. aeruginosa group, P. chlolraphis group, P. fluorescens group, P. pertucinogena group, P. putida group, P. stutzeri group, and the P. syringae group, preferably P. aeruginosa, P. florescens, P. putida, P. stutzeri, P. tabaci, P. angulata, P. phaseolicola, P. pisi, P. glycinea, P. solanacearum, P. caryophylli, P. cepacia, P. marginalis, P. savastonoi, P. marginata or P. syringae.

27. An anti-microbial combination comprising a polynucleotide comprising a targeting component and an anti-microbial component, wherein the targeting component specifically binds a target molecule or cell, and wherein the anti-microbial component comprises a polynucleotide in association with an anti-microbial agent.

28. The anti-microbial combination of claim 27, wherein the targeting component comprises a polynucleotide or functional fragment thereof that specifically binds the target molecule or cell.

29. The anti-microbial combination of claim 28, wherein the polynucleotide or functional fragment thereof comprises an aptamer.

30. The anti-microbial combination of any one of claims 27 to 29 wherein the targeting component comprises any one of SEQ ID NO: 1 to SEQ ID NO : 30.

31. The anti-microbial combination of claim 29 or claim 30, wherein the aptamer or functional fragment thereof comprises any one of SEQ ID NO: 1, 2, 3, 7, 11, 15, 19, 23, or 27.

32. The anti-microbial combination of any one of claims 27 to 31, wherein the targeting component specifically binds the target molecule or cell with a binding affinity of Kd = about 10 μΜ, preferably about 5 μΜ, preferably about 1 μΜ, preferably about 0.5 μΜ.

33. The anti-microbial combination of any one of claims 27 to 32, wherein the target cell is a microorganism, preferably a bacterium, preferably a Gram-negative bacterium, preferably a Pseudomonas spp. bacterium, preferably P. aeruginosa.

34. The anti-microbial combination of any one of claims 27 to 33, wherein the polynucleotide in association with the anti-microbial agent is, or comprises a nucleic acid sequence that encodes a polynucleotide scaffold.

35. The anti-microbial combination of claim 34, wherein the polynucleotide scaffold comprises a polynucleotide that is 5'-(CxN_y)_z-3' wherein C = cytosine; N = any combination of adenosine (A), guanosine (G), thymidine (T), analogues of C, A, G and T, and modified deoxynucleosides; x = an integer from 2 to 7; y = an integer from 0 to 7; and z = an integer from 1 to 8, preferably wherein x is an integer that is 3, 4, 5, 6 or 7 and y is an integer that is 2, 3, 4 or 5.

36. The anti-microbial combination of claim 34 or 35, wherein the polynucleotide scaffold, or at least part of the polynucleotide scaffold is conjugated to the 5' end of the targeting component or is conjugated to the 3' end of the targeting component, or wherein the polynucleotide scaffold, or at least part of the polynucleotide scaffold, is conjugated to the 5' end of the targeting component and the polynucleotide scaffold, or at least part of the polynucleotide scaffold, is conjugated to the 3' end of the targeting component.

37. The anti-microbial combination of any one of claims 27 to 36, wherein the AMC comprises SEQ ID NO: 1, 2, 4-6, 8-10, 12-14, 16-18, 20-22, 24-26 or 28-30.

38. The anti-microbial combination of any one of claims 27 to 37, wherein the polynucleotide in association with an anti-microbial agent is complexed to the antimicrobial agent by at least one chemical bond, preferably at least one coordination bond or at least one ionic bond.

39. The anti-microbial combination of any one of claims 27 to 38, wherein the antimicrobial agent comprises a silver nanocluster (AgNC) or a silver ion (Ag+).

40. A combination of formula I:

TC-AMC wherein

TC = a targeting component that specifically binds to a target molecule or cell, and

AMC an anti-microbial component comprising i) a polynucleotide that forms a metal nanocluster with at least three metal atoms, or is a polynucleotide that chelates at least one metal ion, or both, and an anti-microbial agent.

41. A combination of formula II:

AptPA-AMC wherein

42. A method of making an anti-microbial combination comprising : a) synthesizing a polynucleotide comprising a targeting component comprising a polynucleotide that specifically binds to a target molecule or cell, and an antimicrobial component comprising a nucleic acid sequence that forms a metal nanocluster with at least three metal atoms, or is a nucleic acid sequence that chelates at least one metal ion, or both, and b) associating the polynucleotide synthesized in a) with at least one antimicrobial agent to make the anti-microbial combination.

43. An anti-microbial combination made by the method of claim 42.

44. A composition comprising a polynucleotide as defined in claim 15 or an antimicrobial combination targeting component, or anti-microbial component as defined in claim 27, or a combination as defined in claim 40 or claim 41, and a carrier, diluent or excipient.

45. Use of a polynucleotide as defined in claim 15, an anti-microbial combination, a targeting component, or anti-microbial component as defined in claim 27, or a combination as defined in claim 40 or claim 41 in the manufacture of a medicament for treating microbial infection.

46. A method of treating a microbial infection comprising administering a

polynucleotide as defined in claim 15, an anti-microbial combination, a targeting component, or anti-microbial component as defined in claim 27, a combination as defined in claim 40 or claim 41, or a composition as defined in claim 42 to a subject in need thereof.

47. A polynucleotide as defined in claim 15, an anti-microbial combination, a targeting component, or anti-microbial component as defined in claim 27, a combination as defined in claim 40 or claim 41, or a composition as defined in claim 42 for use in treating, or when used to treat, microbial infection.

48. Use of an anti-microbial component in the manufacture of a medicament for treating microbial infection, wherein the anti-microbial component comprises a polynucleotide in association with an anti-microbial agent, wherein the polynucleotide comprises an i-motif structure and the anti-microbial agent is at least one metal ion.

49. A method of inhibiting the growth and/or proliferation of at least one bacterial species treating a microbial infection comprising administering an anti-microbial component to a subject in need thereof, wherein the anti-microbial component comprises a polynucleotide in association with an anti-microbial agent, wherein the polynucleotide comprises an i-motif structure and the anti-microbial agent is at least one metal ion.

50. An anti-microbial component for use in inhibiting the growth and/or proliferation of at least one bacterial species treating, or when used to treat, microbial infection, wherein the anti-microbial component comprises a polynucleotide in association with an anti-microbial agent, wherein the polynucleotide comprises an i-motif structure and the anti-microbial agent is at least one metal ion.

51. An anti-microbial component for use in treating, or when used to treat, microbial infection, wherein the anti-microbial component comprises a polynucleotide in association with an anti-microbial agent, wherein the polynucleotide comprises an i- motif structure and the anti-microbial agent is at least one metal ion.