WO2018140041A1

WO2018140041A1 - Nucleic acid complex including a frame and a metallic nanoparticle

Info

Publication number: WO2018140041A1
Application number: PCT/US2017/015418
Authority: WO
Inventors: Aleksandr Polyakov; Fausto D'APUZZO; Anita Rogacs; Raghuvir N. SENGUPTA
Original assignee: Hewlett-Packard Development Company, L.P.
Priority date: 2017-01-27
Filing date: 2017-01-27
Publication date: 2018-08-02

Abstract

According to an example, a nucleic acid complex may include a frame including nucleic acid staples and a nucleic acid scaffold; and a metallic nanoparticle, in which the metallic nanoparticle is attached to the frame at ultiple points along a face of the frame.

Description

NUCLEIC ACID COMPLEX INCLUDING A FRAME AND A METALLIC

NANOPARTICLE

BACKGROUND

[0001] Surface enhanced Raman scattering or surface enhanced Raman spectroscopy (SERS) is a surface-sensitive technique that enhances Raman scattering by molecules absorbed on rough metal surfaces or by nanostructures. The enhancement factor may be used to detect single molecules. Raman spectroscopy has many possible applications, but application and

commercialization of Raman spectroscopy devices is still limited because Raman spectroscopy has weak signal intensity and low reproducibility.

BRIEF DESCRIPTION OF THE DRAWING

[0002] Features of the present disclosure are illustrated by way of example and not limited in the following figure(s), in which like numerals indicate like elements, in which:

[0003] Fig. 1 is an isometric view illustrating an example frame;

[0004] Fig. 2 is an isometric view illustrating an example nucleic acid complex;

[0005] Fig 3 is an isometric view illustrating an example detection device;

[0006] Fig 4 is a flowchart of an example method of making a detection device; and

[0007] Fig. 5 is a schematic drawing of an example system for performing surface enhanced Raman spectroscopy using a nucleic acid complex. DETAILED DESCRIPTION

[0008] For simplicity and illustrative purposes, the present disclosure is described by referring mainly to an example thereof. In the following description, numerous specific details are set forth in order to provide a thorough

understanding of the present disclosure. It will be readily apparent however, that the present disclosure may be practiced without limitation to these specific details. In other instances, some methods and structures have not been described in detail so as not to unnecessarily obscure the present disclosure. As used herein, the terms "a" and "an" are intended to denote at least one of a particular element, the term "includes" means includes but not limited to, the term "including" means including but not limited to, and the term "based on" means based at least in part on.

[0009] Raman scattering refers to a phenomenon in which, when light passes through a certain medium, a portion of the light undergoes a frequency shift and progresses in a different direction. The term "SERS" (surface enhanced Raman spectroscopy or surface enhanced Raman scattering) refers to a phenomenon in which, when a molecule is located around a metal nanostructure, the intensity of Raman scattering of the molecule is significantly increased.

Disclosed herein is a nucleic acid complex 5 that may be used in a detection device in which, for example, surface enhanced Raman spectroscopy, may be performed on a target molecule of interest. As shown in Figs. 2 and 3, the nucleic acid complex 5 may include a frame 7 including a nucleic acid staple and a nucleic acid scaffold; and a metallic nanoparticle 40, in which the metallic nanoparticle 40 is attached to the frame 7 at multiple points along a face 20 of the frame 7. In an aspect, there is disclosed a self-assembled sensor architecture based on nucleic acid/metal nanoparticles complexes that may effectively control and/or define a position of the metallic nanoparticles relative to a target analyte and to each other. This architecture may leverage the nanometer addressability of nucleic acid nanostructures in order to maximize the Raman signal intensity and reproducibility of the assemblies. [0010] As illustrated in Fig. 1 , the frame 7 may include a geometric shape with an open interior. The frame 7 may have a uniform width 10 along a first plane and a face 20 of the frame 7 along a second plane. The geometric shape may include a circle, triangle, square, rhombus, pentagon, hexagon, octagon, decagon, parallelogram, or the like. In an aspect, the frame 7 may have any shape so long as a width 10 of the frame 7 is substantially uniform to control a distance between metallic nanoparticles 40 attached to opposite faces of the frame 7.

[0011 ] The geometric shape of the frame 7 may be formed from the nucleic acid staple and the nucleic acid scaffold. Each of the nucleic acid staple and the nucleic acid scaffold may independently include an oligonucleotide. Each oligonucleotide may include a hybridization region in which two single stranded nucleic acids may hybridize to provide a double stranded region. The double stranded nucleic acid may be formed through hybridization of an oligonucleotide with itself, for example, a single oligonucleotide chain may fold over onto itself at a hybridization region. In another aspect, the double stranded nucleic acid may be formed through hybridization with another oligonucleotide, for example, a nucleic acid staple may hybridize with a nucleic acid scaffold at their respective hybridization regions. In an aspect, each of the nucleic acid staple and the nucleic acid scaffold may independently include a hybridization region. In another aspect, each of the nucleic acid staple and the nucleic acid scaffold may independently include multiple hybridization regions, and may hybridize to multiple other oligonucleotides.

[0012] The frame 7 may include nucleic acids including, but not limited to, deoxyribonucleic acids (DNAs), ribonucleic acids (RNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs), nucleic acid-like structures, a combination thereof, and an analogue thereof. The frame 7 may include nucleotide analogues having bonding properties similar to or better than those of a naturally occurring nucleotide. The frame 7 may include a nucleic acid analogue having a synthetic backbone. The synthetic backbone analogue may include phosphodiester, phosphorothioate, phosphorodithioate, methyl phosphonate, phosphoramidate, alkyl phosphotriester, sulfamate, 3'-thioacetal, methylene(methylimino), 3'-N-carbamate, morpholino carbamate, PNAs, modified phosphodiester, or modified methylphosphonate bonds. The nucleic acid staple and/or the nucleic acid scaffold (e.g. , DNA) used for preparing the frame 7 may each independently be naturally occurring, modified, or synthetic nucleic acid.

[0013] In an aspect, the nucleic acid scaffold may be a long (about 8 kb) viral DNA strand that may be folded under during hybridization with a set of shorter (about < 100 b) sequences of a nucleic acid staple. The nucleic acid staples sequences may be obtained with the aid of open-source DNA origami software, such as caDNAno. In an aspect, a p8064 viral DNA scaffold may be folded into a ring-shaped nucleic acid complex 5 by hybridization with about 250 nucleic acid staples. In addition, other geometric shapes for the frame 7 of the nucleic acid complex 5 may be obtained by leveraging other DNA engineering strategies. For example, longitudinal stress and torque may be locally generated by introducing intentional defects in the double helix. Additionally, curved and twisted segments may be implemented into the design of the frame 7.

[0014] The frame 7 may be self-assembled. For example, the frame 7 may be spontaneously formed by covalent or non-covalent bonds between atoms, or other types of molecular interactions. The oligonucleotides of the nucleic acid staple and the nucleic acid scaffold may include a sequence or region that is complementary to and, thus, able to hybridize with, the sequences of other oligonucleotides of the nucleic acid staple and the nucleic acid scaffold. The nucleic acids may, therefore, be self-assembled through hybridization between complementary sequences.

[0015] The nucleic acid scaffold and the nucleic acid staples may be selected so that they form a frame 7 with single digit nm precision at a width 10 of the frame 7. The ability to form a frame 7 with this degree of precision may enable the accurate positioning of the metallic nanoparticles 40 to the frame 7 to form a nucleic acid complex 5 for optical excitation, i.e., optical excitation is greatest in a gap between the metallic nanoparticles 40, which in an aspect may be a width 10 of the frame 7 along a first plane, as shown in Fig. 2.

[0016] As illustrated in Fig. 3, which depicts a detection device 50, the nucleic acid complex 5 may further include a linker 30 which, in turn, may include a moiety capable of reversibly or irreversibly coupling the linker 30 to a selected target molecule (analyte); and (b) a moiety or moieties capable of reversibly or irreversibly coupling the linker to the frame 7. Examples of a linker 30 include, but are not limited to, oligonucleotide, avidin, biotin, antibody, aptamers, synthetic high-affinity ligands (SHALS). As shown in Fig. 3, the linker 30 may span an interior of the frame 7. The linker 30 may be immobilized on the frame 7 through a nucleic acid handle that may be synthesized to base-pair with a section of the frame 7. The position of the binding sites for the linker 30 may be controlled down to the fundamental base-pair pixel by harnessing the sub-nanometer

addressability of a nucleic acid sequence.

[0017] In an aspect, the linker 30 may be positioned within the frame 7 to maximize a signal, such as a Raman signal. The linker 30 may be positioned in a region where the highest electric field intensity may occur. The linker 30 may also be positioned in a region where possible sources of background signals are not located.

[0018] The frame 7 may be exposed to a sample containing an analyte. The sample may be blood, urine, a mucous membrane, saliva, a body fluid, a tissue, a biopsy material, a combination thereof, or the like. The analyte may be organic molecules, inorganic molecules, biomolecules, or the like. Non-limiting examples of the analyte include amino acids, peptides, polypeptides, proteins, carbohydrates, fatty acid, lipids, nucleotides, oligonucleotides, polynucleotides, glycoproteins, such as prostate specific antigen (PSA), proteoglycans, lipoproteins, lipopolysaccharides, drugs, drug metabolites, small organic molecules, inorganic molecules and natural or synthetic polymers. Non-limiting examples of the analyte also may include glucose, free fatty acids, lactic acid, C-reactive protein and anti-inflammatory mediators, such as cytokines, eicosanoids, or leukotrienes. The biomolecules may be a protein, a nucleic acid, a sugar, or a combination thereof. The biomolecules may be pathogens.

[0019] The analyte may be immobilized on the frame 7 by including a linker 30, such as an aptamer sequence. The linker 30 domain, which may bind specifically to the analyte of interest, may be composed of DNA, RNA, or both. In addition, linker 30, such as an aptamer sequence, may include commercially available chemical modifications, such as 2'-methoxy substitutions, to confer greater stability against nuclease degradation, and 5'- and 3'-thiol modifiers, to conjugate the linker 30 to reporter molecules.

[0020] The nucleic acid complex 5 may be detectibly labeled. The label may include a fluorescent molecule, a radioactive isotope, an enzyme, an antibody, other detectable moiety, non-fluorescent organic molecule, inorganic nanoparticles, or other detectably moiety. The label may facilitate the detection and measurement of an analyte by a sensor 104 when nanoparticles 40 are attached to the detection device 50.

[0021] In an aspect, a width 10 of the frame 7 along a first plane may control the distance between metallic nanoparticles 40. By controlling the distance between metallic nanoparticles 40, a user may overcome sensor-to sensor signal variation in a detection device 50. The nucleic acid complex 5 may be designed to control the placement of the metallic nanoparticles 40, for example, on a scale below 10 nm, thereby resulting in a minimal variation in the amplitude of a signal from nominally identical detection devices 50. The disclosed nucleic acid complex 5 may be used in a detection device 50 with controlled spatial placement of metallic nanoparticles 40 that form high field enhancement gaps with high reproducibility.

[0022] The metallic nanoparticles 40 may be attached to the frame 7 at multiple points along a face 20 of the frame 7 in which the face 20 of the frame 7 lies along a second plane. In an aspect, the nucleic acid staple of the frame 7 may be modified with a functional group that may form a covalent bond with the metallic nanoparticle 40. The nucleic acid staple with the functional group may be positioned within the nucleic acid complex 5 with high spatial precision through complementing a specific sequence on the nucleic acid complex 5. In an aspect, the functional group may be a sulfur-containing group including a thiol group or a sulfhydryl group. The functional group may be an alcohol and/or phenol derivative, and may be a compound having a formula of RSH containing sulfur in an oxygen site, in which R may be substituted or unsubstituted alkyl, cyclic or heterocyclic aromatic group, or aliphatic group. Also, the functional group may be thiol ester or dithiol ester having a formula of RSR' or RSSR', respectively, in which R and R' may be independently substituted or unsubstituted alkyl, cyclic or heterocyclic aromatic group, or aliphatic group. Further, the functional group may be an amino group (— NH₂) or carboxyl group.

[0023] In an aspect, the frame 7 may be ring-shaped including about 12 nucleic acid staples in which each nucleic acid staple may be modified with a thiol functional group. Half of the nucleic acid staples may protrude for about 1 nm on each face 20, along a second plane, of the ring frame 7 so that each face may attach a metallic nanoparticle 40 with 6 thiol-containing groups.

[0024] The metallic nanoparticles 40 may be optically active materials. The metallic nanoparticles 40 may include, but are not limited to, gold (Au), silver (Ag), copper (Cu), sodium (Na), aluminum (Al), chromium (Cr), platinum (Pt), ruthenium (Ru), palladium (Pd), iron (Fe), cobalt (Co), nickel (Ni), and a combination thereof. Also, metal ions or metal ion chelates may be used. The metallic nanoparticles 40 may be chemically reduced, coated or functionalized with other metallic or non-metallic layers, or subjected to laser ablation.

[0025] The detection device 50, as illustrated in Fig. 3, may include a nucleic acid complex 5 including a frame 7 including nucleic acid staples and a nucleic acid scaffold. The nucleic acid complex 5 may also include a metallic nanoparticle 40 attached to the frame 7 at multiple points along a face 20 of the frame 7. The detection device 50 may also include a linker 30. The linker 30 may span an interior of the frame 7 of the nucleic acid complex 5. The linker 30 may include any segment that may bind an analyte.

[0026] The detection device 50 may be used in various detection techniques, such as Raman spectroscopy, surface plasmon resonance (SPR), mid infrared (MIR) spectroscopy, surface enhanced infrared absorption (SEIRA), surface enhanced Raman spectroscopy (SERS), and the like.

[0027] With reference now to Fig. 4, there is shown a flow diagram of an example method 60 of making a detection device 50. In the method 60, a nucleic acid scaffold and a nucleic acid staple may be hybridized to produce a frame 7 (block 62). In addition, at block 64, a linker 30 may be attached to the frame 7 so that the linker 30 spans an interior of the frame 7. At block 66, an analyte may be bound to the linker 30 and at block 68, a metallic nanoparticle 40 may be attached at multiple points along a face 20 of the frame 7. In an aspect, by binding an analyte to the linker 30 an issue relating to non-specific adsorption of the analyte to the metallic nanoparticle 40 may be avoided. In another aspect, after the analyte is bound to the linker 30 the method may further include a wash step or multiple wash steps, before attaching the metallic nanoparticle 40 to the frame 7.

[0028] The metallic nanoparticle 40 may be positioned on either side of the linker 30. In an aspect, the linker 30 with the attached analyte may be positioned between metallic nanoparticles 40.

[0029] The metallic nanoparticles 40 may be attached to the frame 7 at multiple points of attachment in order to increase the binding strength of the metallic nanoparticle 40 to the frame 7. In an aspect, the metallic nanoparticle 40 is attached at two points, three points, four points, five points, six points, or seven points, etc., to the frame 7.

[0030] To enable the aggregation of a stable solution of colloidal metallic nanoparticles 40, depending on the metallic nanoparticle size, concentration and charge, the colloid stability may be decreased by a small addition of salt to incrementally screen the negative charge of the metallic nanoparticle 40 allowing them to get close enough to each other and to the negatively charged nucleic acid complex 5 to allow the assembly through the functional group binding, such as a thiol group.

[0031] In order to obtain a high yield of dimers, i.e., two metallic nanoparticles 40 attach to parallel faces 20 of the frame 7 of the nucleic acid complex 5, the relative concentration of metallic nanoparticles 40, frame 7, and salt may be finely tuned. In an aspect, with a ring shaped frame 7 the salt concentration may be below 1 .4 mM (MgCI₂) and a concentration ratio of frame 7:metallic nanoparticles 40, may be about 1 :2. The status of the aggregation of the metallic nanoparticles 40 to the frame 7 may be monitored in real-time for fine tuning by Dynamic Light Scattering measurements or by depositing the metallic nanoparticles 40/frame 7 assemblies on a conductive substrate and performing scanning electron microscope imaging.

[0032] There is also disclosed a kit including a first container including a nucleic acid staple, a second container including a nucleic acid scaffold, a third container including a linker 30, and a fourth container including a metallic nanoparticle 40. A user may combine the nucleic acid staple and the nucleic acid scaffold to form a frame 7. The frame 7 may be combined with the linker 30. The frame 7 with the linker 30 may be exposed to a sample containing an analyte so that the analyte may bind to the linker 30. The metallic nanoparticle 40 may be attached to the frame 7 using functional groups.

[0033] An example SERS system 100 is illustrated schematically in Fig. 5. The system 100 may include a detection device 50 and may be used to perform Raman spectroscopy. The SERS system 100 may include the detection device 50, an excitation source 102, and a sensor 104. The SERS system 100 may include various optical components positioned between the excitation source 102 and the detection device 50, and various optical components between the detection device 50 and the sensor 104. In addition, an analyte 106 may have been attached to the linker 30 as discussed above.

[0034] The excitation source 102 may include any suitable source for emitting radiation at a desired wavelength, and may emit a tunable wavelength of radiation. For example, commercially available semiconductor lasers, helium-neon lasers, carbon dioxide lasers, light emitting diodes, incandescent lamps, and many other known radiation-emitting sources may be used as the excitation source 102. The wavelengths that are emitted by the excitation source 102 may be any suitable wavelength for analyzing the analyte using SERS.

[0035] The sensor 104 may receive and detect the Raman scattered photons and may include a monochromator (or any other suitable device for determining the wavelength of the Raman scattered photons) and a device such as, for example, a photomultipiier for determining the quantity of Raman scattered photons (intensity).

[0036] To perform SERS using the system 100, a user may provide a detection device 50 that may be irradiated with excitation radiation or light from the source 102. Raman scattered photons scattered by the analyte 106 bound to the nucleic acid complex 5 may be detected by the sensor 104.

[0037] EXAMPLES

[0038] Example 1 - A long (8 kb) viral DNA sequence (nucleic acid scaffold) and about 250 short (less than about 6kb) nucleic acid staples were mixed together in 1 :10 molar ratio. A quantity of 1 pico-mole of scaffold and 10 pmol of staples were added to 50 microliters of T.E buffer (T.E. = 40 mM Tris, 2 mM EDTA and 18 mM MgCI₂, with a pH = 8.1 , adjusted with HCI). The solution was annealed in a PCR tube by heating to 95° C and slowly cooling (rate of 0.1 ° C/min) to room temperature over the course of several hours in a BioRad thermo-cycler.

[0039] The frame was purified from the extra oligonucleotides by running 12 microliters of the annealing product, mixed with a loading dye (30% glycerol) in a 2% agarose gel (stained with Ethidium Bromide) at 70 V for 3 hours. The products were imaged under ultraviolet illumination. The slow migrating band corresponding to the frame was removed from the gel with a razor blade and extracted with a Freeze n' Squeeze kit (BioRad) and stored at 4° C until further use.

[0040] Example 2 - The linker 30 can be mixed with the nucleic acid staples and the nucleic acid scaffold following conditions described above to generate frame 7. If an aptamer sequence is to be included, an additional sequence containing an aptamer domain, specific to the analyte of interest, as well as a hybridization domain that is complementary to the linker 30, will be included. Following agarose gel purification, this complex can be exposed to various concentrations of the analyte of interest. The free analyte can be removed from the mixture via Amicon centrifugal filter units with molecular weight cutoffs that exceed the molecular weight of the analyte but not that for the frame 7:aptamer:analyte complex. [0041] Example 3 - Thiol functional groups usually come in the form of a di-sulfide group for stability reasons and were "activated" when ready for binding to the metallic (gold) nanoparticle. Activation was achieved by suspending the nucleic acid complex in 50 microliters of the relevant buffer (e.g., tri-acetate EDTA (TAE) or tri-borate EDTA (TBE) with pH = 5.2). 10 microliters of 1 .0 N dithiothreitol (DTT) was added, gently vortexed, and incubated at room temperature for about 15 minutes. (The 1 .0 N DTT was prepared by dissolving 1 .545 g of DTT in 20 ml of 0.01 M sodium acetate (pH 5.2).) The excess DTT was removed from the mixture by extracting with ethyl acetate 3 times, using 50 microliter per extraction. After vortexing and centrifuging, the upper layer was removed.

[0042] Example 4 - At this point the metallic nanoparticles were mixed together with the "activated" oligonucleotide structure in a frame:nanoparticle molar ratio of 1 :2 and the salt concentration was adjusted to 1 mM MgCI₂. Metallic nanoparticles can be purchased or synthesized, in this example they were purchased in solution from Sigma Aldrich. The mixture was incubated for 1 h at room temperature.

[0043] Although described specifically throughout the entirety of the instant disclosure, representative examples of the present disclosure have utility over a wide range of applications, and the above discussion is not intended and should not be construed to be limiting, but is offered as an illustrative discussion of aspects of the disclosure.

[0044] What has been described and illustrated herein is an example of the disclosure along with some of its variations. The terms, descriptions and figures used herein are set forth by way of illustration only and are not meant as limitations. Many variations are possible within the spirit and scope of the disclosure, which is intended to be defined by the following claims - and their equivalents - in which all terms are meant in their broadest reasonable sense unless otherwise indicated.

Claims

What is claimed is:

1 . A nucleic acid complex, comprising:

a frame including a nucleic acid staple and a nucleic acid scaffold; and a metallic nanoparticle,

wherein the metallic nanoparticle is attached to the frame at multiple points along a face of the frame.

2. The nucleic acid complex of claim 1 , wherein the frame includes a closed geometric shape with an open interior, the frame having a uniform width along a first plane, and the face of the frame along a second plane.

3. The nucleic acid complex of claim 2, wherein the closed geometric shape includes one of a circle, a triangle, a square, a rhombus, a pentagon, a hexagon, an octagon, a decagon, and a parallelogram

4. The nucleic acid complex of claim 1 , wherein two metallic nanoparticles are attached to the frame and a width of the frame defines a distance between the two metallic nanoparticles.

5. The nucleic acid complex of claim 1 , wherein the nucleic acid staple is modified with a functional group to form a covalent bond with the metallic nanoparticle.

6. The nucleic acid complex of claim 5, wherein the functional group includes one of a thiol group, an alcohol-containing group, a thiol ester group, and an amino group.

7. The nucleic acid complex of claim 1 , wherein the nucleic acid scaffold includes a single-strand nucleic acid.

8. The nucleic acid complex of claim 1 , wherein the nucleic acid staple and the nucleic acid scaffold self-assemble into the frame.

9. The nucleic acid complex of claim 1 .wherein the metallic nanoparticle is gold, silver, copper, sodium, aluminum, chromium, platinum, ruthenium, palladium, iron, cobalt, nickel, or a combination thereof.

10. The nucleic acid complex of claim 1 , further comprising a linker that spans the interior of the frame.

1 1 . A detection device, comprising:

a nucleic acid complex including a frame including nucleic acid staples and a nucleic acid scaffold; and a metallic nanoparticle, wherein the metallic nanoparticle is attached to the frame at multiple points along a face of the frame; and

a linker.

12. The detection device of claim 1 1 , wherein the linker includes a segment that binds with an analyte.

13. A method of making a detection device, comprising:

hybridizing a nucleic acid scaffold and a nucleic acid staple to produce a frame;

attaching a linker to the frame so that the linker spans an interior of the frame;

binding an analyte to the linker; and

attaching a metallic nanoparticle at multiple points along a face of the frame.

14. The method of claim 13, wherein the metallic nanoparticle is positioned on either side of the linker.

15. The method of claim 13, wherein the multiple points of attachment increase the binding strength of the metallic nanoparticle to the frame.