Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/10—Transferases (2.)
  • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
  • C12N9/1241—Nucleotidyltransferases (2.7.7)
  • C12N9/1252—DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/96—Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
  • C12Y207/07—Nucleotidyltransferases (2.7.7)
  • C12Y207/07007—DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase

Definitions

• the present invention provides a new activatable DNA polymerase comprising a DNA polymerase protein coupled to a nucleic acid, uses thereof and methods of DNA synthesis or diagnosis as well as a new apparatus for carrying out the methods of DNA synthesis.
• DNA polymerases are arguably one of the most successful applications of enzymes to biotechnology with applications in molecular biology, genome sequencing, sensing and diagnostics among others [1 ], Yet, a common practical problem of these assays is that significant polymerase or exonuclease activities (e.g.,3 1 to 5' exonuclease activity) of the DNA polymerase during sample preparation can lead to loss of product yield and to lowered sensitivity and specificity [2], Polymerase activity can elongate missprimed events, including primer dimers and unspecific off target binding events, resulting in by-products that can compete with the desired amplicon. 3' terminal primer degradation by 3' to 5' exonuclease activity can enhance off-target binding also inducing misspriming. Furthermore, exonucleolytic degradation of primers and template decrease the overall product yield [2-3],
• the unavoidable heating step makes this type of strategy non-viable for mesophilic DNA polymerases, such as the Phi29 DNA polymerase (Phi29 pol), which is used for whole-genome amplification and sensing applications [1 a, 6].
• Phi29 DNA polymerase Phi29 DNA polymerase
• the most common hot-start strategies employ specific aptamers or antibodies to selectively block the DNA polymerases.
• the development of such aptamers and antibodies requires time-consuming screening of libraries and laborious optimization steps and each aptamer or antibody is specific for a certain polymerase [2e, 4, 7],
• Taq polymerase An interesting alternative to hot-start polymerases was the development of a light- activated Taq polymerase, but only partial recovery of the enzymatic activity was shown and the approach was specific for the Taq polymerase (Taq pol) [8], Overall, these problems call for strategies of broader application and easier implementation.
• the present invention provides a new activatable DNA polymerase which fulfills these needs.
• the present invention provides an activatable DNA polymerase comprising a DNA polymerase protein unit coupled to a nucleic acid unit, preferably wherein the nucleic acid unit has a length which is sufficient to block activity of the DNA polymerase protein unit, optionally further comprising at least one linker unit between the polymerase protein unit and the nucleic acid unit, wherein the nucleic acid unit is coupled (a) by a covalent chemical bond or by streptavidin-biotin; and/or (b) by its terminal nucleotide.
• the present invention provides a method of synthesizing a nucleic acid comprising: a) providing a sample comprising at least one nucleic acid template, components for nucleic acid synthesis and the activatable DNA polymerase of the invention, b) activating the activatable DNA polymerase in the sample of step a), thereby generating an activated DNA polymerase, and c) subjecting the sample comprising the activated DNA polymerase of step b) to reaction conditions that allow synthesis of a nucleic acid complementary to the at least one nucleic acid template.
• the present invention provides a method of determining the presence or absence of a single stranded nucleic acid of interest comprising: a) providing a sample to be tested for comprising a single stranded nucleic acid of interest, b) adding the activatable DNA polymerase according to the invention to the sample of step a), wherein at least a part of the nucleic acid unit is single stranded and complementary to the single stranded nucleic acid of interest, c) subjecting the sample with the activatable DNA polymerase of step b) to reaction conditions which allow hybridization, preferably hybridization between the single stranded part of the nucleic acid unit of the activatable DNA polymerase unit and the single stranded nucleic acid of interest, thereby activating the activatable DNA polymerase of the invention, and d) determining activity of the activatable DNA polymerase of step c), wherein activity of the activatable DNA polymerase indicates the presence of the nucleic acid of
• the invention provides the use of a nucleic acid for reversibly inhibiting a DNA polymerase wherein the nucleic acid is coupled to the DNA polymerase (a) by a covalent chemical bond or by streptavidin-biotin; and/or (b) by its terminal nucleotide.
• the nucleic acid has a length which is sufficient to block the activity of the DNA polymerase, more preferably wherein the nucleic acid is at least partially single stranded as described herein.
• the invention provides the use of the activatable DNA polymerase of the invention in an in vitro nucleic acid synthesis process, preferably wherein the activatable DNA polymerase comprises a cleavable site wherein upon cleavage at least a part of the nucleic acid unit is removed and preferably wherein the cleavable site is a photocleavable site.
• the cleavage of the photocleavable site is by irradiation with light, preferably wherein the photocleavable site is an o-nitrobenzyl based photocleavable site and/or the light has a wavelength between 300 nm to 400 nm, preferably between 310 nm to 370 nm, more preferably between 315 nm to 365 nm, most preferably at about 315 or at about 365 nm.
• the invention provides the use of the activatable DNA polymerase of the invention for in vitro diagnosis, such as diagnosis of a disease, a medical condition and/or detection of pathogens in a sample, such as from a human or an animal (e.g., mammal or bird), food, drink, soil or water (e.g., a drinking water, a waste water or a hydrologic sample).
• a sample such as from a human or an animal (e.g., mammal or bird), food, drink, soil or water (e.g., a drinking water, a waste water or a hydrologic sample).
• Figure 1 Scheme showing the mechanism of Light-Start DNA polymerases.
• the sequence at the bottom of the Figure represents an example of the nucleic acid unit (SEQ ID NO: 1 ).
• the asterisk (*) denotes a photocleavable unit.
• Figure 2 Light-activation of Phi29 DNA pol.
• Phi29 pol-PC_oligoScr left gel
• Phi29 pol_oligo2 constructs right gel
• a light pulse of 365 nm 120 s was applied (lane 2). All activities assays were performed with 20 nM enzyme for 2 h at 30 °C.
• Figure Caption. Phi29 pol is represented in dark and Phi29 pol-PC_Oligo in light grey.
• Figure 3 Polymerase and nuclease activity of different polymerases can be blocked, a) 3' to 5' exo activity of Phi29 pol-PC_oligo. Exonuclease activity of the unmodified Phi29 pol (lane 2). b) PCR with Taq pol-PC_oligo (left gel) and Pfu pol- PC_oligo (right gel) samples. In both cases, PCR product was not detected in nonilluminated samples (lane 2). Only irradiated samples (lane 3) showed the PCR product present in the unmodified enzyme samples (lane 1 ).
• Figure 4 Light-start applications, a), b) and c) failure-by-design experiments for Phi29 pol-PC_oligo, Pfu pol-PC_oligo, and Taq pol-PC_oligo, respectively, a) Whole genome amplification of human DNA by Phi29 pol-PC_oligo, and b) PCR amplification of E. coli Bir A gene by Pfu pol-PC_oligo.
• Figure 5 Tight-blockage of DNA polymerases and further failure-by-design assays, a) Tight blockage of the activity of Phi pol-PC_oligo. No amplification product was observed when 120 nM of Phi29 polPC_oligo was used (lane 1 ). Activity was recovered after a 10 s light pulse with 315 nm UV (lane 2). b), c), and d) Independent failure-by-design experiments were performed to corroborate the results shown in Figure 4. b) Whole genome amplification by Phi pol-PC_oligo. The hexamers concentration in reactions in lane 1 and 2 was 6.25 pM, and 3.12 pM in lane 3 and 4.
• Phi29 pol-Oligo as a specific nucleic acid sensor
• a) Amplification of T7 blue plasmid by Multiply-Primed RCA shows selective activation of Phi29 pol-Oligo only by the target sequence (complementary to the sensing oligo, GTGATGTAGGTGGTAGAGGAA, SEQ ID NO: 17). Reaction was performed for 2h with 150 nM polymerase at 30 °C.
• Mocking oligo is an oligo with a sequence not complementary to the blocking oligo (AGGGTCCACCAAACGTAATGC, SEQ ID NO: 18).
• Similar recovery of the activity was observed by a complementary RNA (right gel, lain 1 vs 2).
• Phi29 pol-AP_oligo an oligo-modified version of the Phi29 pol enzyme carrying an abasic site after the second nucleotide (Phi29 pol-AP_oligo), maintain the reversible blockage of the activity (see recovery of activity after incubation with the complementary RNA oligo, right gel lain 1 vs 2).
• Figure 7 Fidelity of Pfu pol-PC_oligo a) and Taq pol-PC_oligo b) enzymes.
• the Bir A gene from E. coli (GenBank: M15820.1 , SEQ ID NO: 11 ) was PCR amplified, gel-purified and sent to sequencing (Eurofins Genomics, Germany).
• the gene was amplified using unmodified Pfu pol, and Pfu pol-PC_oligo in a), unmodified Taq pol and Taq pol- PC_oligo in b), and the sequences obtained by the unmodified and oligo-modified versions compared (a pulse of 120 s 365 nm UV light was used for the activated enzymes).
• the first 50 and last 100-200 nucleotides of the sequencing reaction were omitted due to limitations of the sequencing reaction. In none of both cases, differences between the sequences retrieved by the unmodified enzymes and the oligo-modified ones were detected (see sequence alignment).
• sequence in a) shows 100% identity with nucleotides 58 to 906 of the Bir A deposited sequence (GenBank: M15820.1 , SEQ ID NO: 11 ) and the sequence in b) shows 100% identity with nucleotides 157 to 906 of the Bir A deposited sequence (GenBank: M15820.1 , SEQ ID NO: 11 ).
• Figure 8 miRNA and DNA detection. Schematics of a point of care (POC) assay.
• POC point of care
• the present invention provides of a new activatable DNA polymerase comprising a DNA polymerase protein unit coupled to a nucleic acid unit, optionally further comprising at least one linker unit between the polymerase protein unit and the nucleic acid unit, wherein the nucleic acid unit is coupled (a) by a covalent chemical bond or by streptavidin-biotin; and/or (b) by its terminal nucleotide.
• Coupled refers to a covalent chemical bond or to a non-covalent bond, such as streptavidin-biotin bonds (e.g., streptavidin-biotin coupling or interaction) or coiled coil. In the context of the present invention, it refers to the bond of the nucleic acid unit to the DNA polymerase protein unit, wherein the coupling may be direct (to the DNA polymerase protein unit) or indirect (e.g., via a linker). Moreover, typically the nucleic acid unit is coupled at one terminus (e.g., by it terminal nucleotide), the 5’ or the 3’ terminus of the nucleic acid unit.
• the nucleic acid unit is coupled by a covalent chemical bond or by streptavidin-biotin, more preferably by a covalent chemical bond.
• the term coupling is used in the context of connecting or tightly binding and the person skilled in the art will understand that the nucleic acid is coupled at a site of the DNA polymerase that is distinct and distant of the blocking site at the active center of the DNA polymerase.
• the coupling (direct or indirect) of the nucleic acid unit is to an amino acid of the DNA polymerase protein unit, i.e.
• the direct or indirect coupling of the nucleic acid unit is to an amino acid of the DNA polymerase protein unit which does not interfere with the active center, such as an amino acid which is outside of and/or distant to the active center of the DNA polymerase protein unit, preferably outside of and/or distant to the active center mediating the polymerase activity and/or nuclease activity of the DNA polymerase protein unit.
• the coupling of the nucleic acid unit is to a terminal amino acid of the DNA polymerase protein unit, such as the N-terminal or the C-terminal amino acid, preferably to the C-terminal amino acid.
• the nucleic acid unit blocks the activity of the DNA polymerase in a nonsequence specific manner.
• the coupling allows a high local nucleic acid concentration resulting in competitive binding of the nucleic acid to the active center of the DNA polymerase (at a site distinct from the coupling site of the DNA polymerase).
• the blocking mechanism by the nucleic acid unit is sequence unspecific.
• the nucleic acid unit does not inhibit the DNA polymerase unit when free in solution, i.e., not coupled to the DNA polymerase.
• the coupling according to the invention does not encompass binding of an aptamer to a DNA polymerase.
• Aptamers bind to specific DNA polymerases in a sequence specific manner, binding is dependent on the 3D-structure of the aptamer via multiple non- covalent interactions and is neither by the terminal nucleotide nor by covalent bonds.
• sequence unspecific or “non-sequence specific” as used herein in the context of the present invention means that nucleic acid units with different sequences block or inhibit the activity of a specific DNA polymerase protein unit and the same nucleic acid unit blocks or inhibits the activity of different DNA polymerase protein units, including DNA polymerases of different classes.
• the sequence of the nucleic acid unit may be a random sequence or may be a specific sequence, e.g., specific for a single stranded nucleic acid of interest in a method of diagnosing a disease, a medical condition or determining the presence of a pathogen in a sample and/or in a method of determining the presence or absence of a single stranded nucleic acid of interest according to the invention.
• the sequence of the nucleic acid unit is independent of or unspecific for the DNA polymerase protein unit of the activatable DNA polymerase according to the invention.
• the DNA polymerase protein unit is blocked via unspecific competition-based blockage by the nucleic acid unit coupled (e.g., covalently bound) to the DNA polymerase protein unit at its opposite end. This is also confirmed by the observation that the inhibition is released at low temperature (about 30°C) when the nucleic acid is cut from its attachment point to the enzyme, since a specific binder would remain bound thus blocking the enzyme at this low temperature.
• aptamer refers to a molecule comprising short sequence (about 25 to 70 bp) of artificial DNA or RNA that binds by specific non-covalent contact to a specific target molecule, such as a DNA polymerase.
• An aptamer therefore binds in a sequence-specific manner and is specific for a certain target molecule. It has a specific 3D-structure (secondary and tertiary structure) and binds to is specific target molecule similar to an antibody, via multiple non-covalent interactions.
• the activatable DNA polymerase comprises or consists of a DNA polymerase protein unit coupled to a nucleic acid unit, optionally further comprising at least one linker unit between the polymerase protein unit and the nucleic acid unit.
• the activatable DNA polymerase consists essentially of a DNA polymerase protein unit coupled to a nucleic acid unit, optionally further comprising at least one linker unit between the polymerase protein unit and the nucleic acid unit.
• the inventors unexpectedly observed that binding of a nucleic acid to a DNA polymerase blocked the activity of the DNA polymerase in a non-sequence-specific manner thereby allowing for universal applicability of the blocking mechanism without specific adaptation to a particular DNA polymerase. Without being bound by theory, it is believed that the nucleic acid blocks the activity of the DNA polymerase protein by an unspecific competition-based mechanism.
• the term “reversible” as used herein means that the activity of the DNA polymerase can be fully recovered. Specifically, in an inactive state of the activatable DNA polymerase, the nucleic acid unit blocks the activity of the DNA polymerase protein unit while in an active state of the activatable DNA polymerase the nucleic acid unit does not block the activity of the DNA polymerase protein unit, resulting in unblocking the DNA polymerase protein unit.
• unblocking the activity of the DNA polymerase is performed by dislocation of the nucleic acid unit from the active center of the DNA polymerase and hence inhibiting interaction with the DNA polymerase protein unit.
• Dislocation as used herein can be a removal of the nucleic acid unit from the activatable DNA polymerase or a steric modification of the nucleic acid unit which inhibits accessibility of the nucleic acid unit to the active center of the DNA polymerase protein unit.
• unblocking is performed by cleavage of at least a part of the nucleic acid unit at a cleavable site of the activatable DNA polymerase.
• the cleavable site is a photocleavable site.
• introduction of a photocleavable site near the terminus of the nucleic acid unit which is coupled to the DNA polymerase protein unit, optionally via a linker unit allows for unblocking of DNA polymerase protein unit by a light pulse. Prior to the light pulse, the DNA polymerase activity is tightly blocked while after the UV pulse the DNA polymerase activity is fully recovered ( Figure 1 ).
• the present invention provides a technically simple reversible blocking system which is independent of activation by temperature and/or the design of polymerase- tailored blocking means or mechanisms, and which is particularly useful for temperature-sensitive DNA polymerases, i.e. , mesophilic DNA polymerases, thereby enabling the application of the invention in a broad field of DNA synthesis processes such as PCR, isothermal amplification, and sequencing.
• unblocking is performed by exposing the activatable DNA polymerase to a nucleic acid, preferably a single stranded nucleic acid, which is sufficiently complementary for hybridizing to at least a part of the nucleic acid unit of the activatable DNA polymerase, preferably wherein the nucleic acid unit is at least partially single stranded as described herein.
• the rigidification derived from the transition of an at least partially single stranded nucleic acid to a double stranded nucleic acid inhibits accessibility of the double stranded nucleic acid to the active center of the DNA polymerase.
• the activity of the DNA polymerase can also be used as a read-out for the presence of DNA repair enzymes in a sample, in case at least one artificial or damaged nucleotide is incorporated into the nucleic acid unit resulting in cleavage of the nucleic acid unit in the presence of DNA repair enzymes and hence activation of the DNA polymerase.
• a “nucleic acid unit” as used herein is a nucleic acid which is a part of the activatable DNA polymerase of the invention.
• a “nucleic acid unit” as used herein is a macromolecule (e.g., a single or one nucleic acid strand) which is composed of monomers called nucleotides.
• a “nucleotide” as used herein is built of a nucleobase, a deoxyribose or ribose sugar and a phosphate group.
• a nucleic acid can be a single stranded or a double stranded macromolecule.
• a “double stranded” nucleic acid is formed by pairing of the bases of nucleotides of a first single strand with the bases of nucleotides of a second single strand.
• “Base pairing” as used herein refers to natural Watson-Crick base pairing wherein the bases guanine and cytosine or the bases adenine and thymidine (in DNA)/ uracil (in RNA) of the nucleotides bind to each other via hydrogen bonds, i.e., complementary base pairing.
• Base pairing as used herein might also comprise unnatural base pairing, i.e., binding, e.g., via hydrogen bonding, hydrophobic interaction, or metal coordination.
• the nucleic acid unit of the invention might be partially single stranded, i.e., it comprises at least a single stranded part.
• the term “partially single stranded” or “single stranded part” as used herein refers to at least 12, preferably at least 15, more preferably at least 20 consecutive nucleotides which are single stranded.
• Consecutive nucleotides as used herein means that the nucleotides are not separated by one or more double stranded nucleotides.
• the single stranded part of the nucleic acid unit is localized at a terminus of the nucleic acid unit which is coupled to the DNA polymerase protein unit or a linker unit if present.
• the nucleic acid unit comprises a single stranded part of at least 12, preferably at least 15, more preferably at least 20 consecutive nucleotides. In a further preferred embodiment, the nucleic acid unit is a single stranded nucleic acid unit, i.e., it comprises single stranded nucleotides only.
• the partially single stranded nucleic acid unit may comprise a terminal or an internal double stranded part, preferably wherein at least 12, preferably at least 15, more preferably at least 20 consecutive single stranded nucleotides are present which are preferably localized at a terminus of the nucleic acid unit which is coupled to the DNA polymerase protein unit or a linker unit if present.
• the internal or terminal double stranded part might preferably be generated by a self- complementary part, e.g., a hairpin, of a single stranded nucleic acid unit.
• the sequence of the nucleic acid unit does not have any predicted secondary structure.
• the nucleic acid unit is not an aptamer.
• the nucleic acid unit comprises deoxyribonucleotides (DNA), preferably the nucleic acid unit consists of deoxyribonucleotides.
• the nucleotides might be natural nucleotides, e.g., comprising the nucleobases adenine, guanine, thymine, and cytosine.
• the nucleic acid may comprise at least one non-naturally occurring or damaged nucleotide, e.g., a nucleotide which is damaged by oxidation, alkylation of bases, base loss, bulky adduct formation, or crosslinking.
• damaged nucleotides are deoxyinosine, 8- oxoguanine, thymidine glycol, deoxyuridine, 3-methyladenine, 5-hydroxymethyluridine or abasic nucleotides.
• the damaged nucleotides are recognized by DNA repair enzymes.
• the nucleic acid unit comprises 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 non-naturally and/or damaged nucleotides, more preferably 1 , 2 or 3.
• nucleotide sequence of the nucleic acid unit might vary and is not crucial for blocking the DNA polymerase protein unit.
• the blocking mechanism is sequence unspecific, i.e., the sequence of the nucleic acid unit is independent of or unspecific for the DNA polymerase protein unit of the activatable DNA polymerase according to the invention.
• the nucleic acid unit comprises or consists of a random nucleotide sequence.
• the inhibition is maintained at high temperature (up to 95°C). In contrast blocking aptamers are inactivated at high temperature and would no longer inhibit a DNA polymerase.
• inhibition is released at low temperature (about 30°C) when the nucleic acid is separated, e.g., by cleavage, such as photocleavage from its attachment point to the enzyme.
• the blocking and the coupling are at different sites of the DNA polymerase protein unit.
• the activatable DNA polymerase of the invention is activated by cleavage of at least a part of the nucleic acid unit as described herein.
• essentially the entire nucleic acid unit is cleaved off, wherein essentially means all or all but one, two or three nucleotides.
• the nucleotide sequence of the nucleic acid unit may be an artificial sequence, i.e., it is non-natural and cannot be isolated from an organism and/or does not hybridize to a genomic sequence.
• the nucleic acid unit preferably has a length which is sufficient to block activity of the DNA polymerase protein unit.
• the skilled person is aware that the exact length of the nucleic acid unit might vary. The exact length depends on various factors, e.g., the attachment site of the nucleic acid unit at the DNA polymerase protein unit relative to the active center of the DNA polymerase activity or the length and/or nature of additional units such as a linker unit. Further factors might be the length of a cleavable site as described herein which might be optionally present between the DNA polymerase protein unit and the nucleic acid unit and/or the length of the double stranded part of the nucleic acid unit which might be optionally present.
• the nucleic acid unit has a length of at least about 5 nucleotides. In a further embodiment, the nucleic acid unit has a length of about 10 to about 60 nucleotides, preferably about 12 to about 45 nucleotides, more preferably about 16 to about 40 nucleotides, most preferably about 18 to about 35 nucleotides. In a specific embodiment, the nucleic acid unit is an oligonucleotide, e.g., an oligonucleotide having about 15 to about 30 nucleotides.
• the nucleic acid unit comprises a nucleotide sequence selected from the group consisting of: ttcctctaccacctacatcac (SEQ ID NO: 1 ), cttcatcacactccatctcca (SEQ ID NO: 2), gcattacgtttggtggaccct (SEQ ID NO: 3), ttcctctaccacctacatcactcttct (SEQ ID NO: 4) and ttcctctaccacctacatcactcttctcattac (SEQ ID NO: 5).
• a “DNA polymerase protein unit” as used herein is a DNA polymerase which is a part of the activatable DNA polymerase of the invention.
• the DNA polymerase protein unit can be any DNA polymerase, preferably a DNA polymerase which is commonly used in biotechnological methods.
• the DNA polymerase protein unit is thermostable.
• thermostable DNA polymerases are known to the skilled person, e.g., Deep Vent polymerase (see Cline et al. (1996), Nucleic Acids Res, 24(18): 3546-51 ), Huang and Keohavong (1996), DNA and Cell Biology, 15(7): 589-94), KOD1 polymerase (Takagi et al. (1997), Appl.
• Tgo polymerase (Bonch-Osmolovskay et al. (1996)), TH polymerase (Cline et al. (1996), Nucleic Acids Res, 24(18): 3546-51 , Mattila et al. (1991 )), Tma polymerase (Diaz and Sabino(1998), Flaman et al. (1994), Nucleic Acids Res, 22(15): 3259-60), TNA1_polymerase (Kim et al. (2007), J Microbiol Biotechnol 17(7): 1090-7), Tne polymerase (Chatterjee et al.
• the DNA polymerase protein unit is mesophilic, such as the Phi29 DNA polymerase (Phi29 pol).
• the term “mesophilic” as used herein refers to a DNA polymerase which has a temperature optimum of activity in a range between about 20 °C to about 45 °C.
• the DNA polymerase is a deoxyribonucleic acid (DNA)-dependent DNA polymerase, i.e., the polymerase activity requires a pre-existing DNA strand for polymerase activity.
• DNA deoxyribonucleic acid
• Preferred DNA polymerases are Taq isolated or derived from Thermus aquaticus, Pfu isolated or derived from Pyrococcus furiosus, Bst isolated or derived from Bacillus stearothermophilus, Klenow fragment isolated or derived from Escherichia coli, or Phi29 isolated or derived from Bacillus phage phi29.
• “Derived” as used in this context means any modification of the DNA polymerase compared to the wild-type DNA polymerase isolated from the organism, e.g., a modification relating to expression, such as codon optimization, to activity, e.g., reduced or increased polymerase or (exo)nuclease activity, to thermostability or resistance to inhibitors from clinical or environmental samples. Modifications might be, e.g., spontaneous or directed mutations in the sequences encoding the DNA polymerase. The skilled person is aware of such modifications and mutations.
• the DNA polymerase protein unit is Phi29 derived from Bacillus phage phi29 wherein at amino acid position 59 the asparagine (asn, N) of the wild-type Phi29 DNA polymerase isolated from bacteriophage Phi29 has been replaced by an aspartic acid (asp, D), preferably comprising the amino acid sequence of SEQ ID NO: 6.
• the DNA polymerase protein unit is Taq, preferably comprising the amino acid sequence SEQ ID NO:7.
• the DNA polymerase protein unit is Pfu, preferably comprising the amino acid sequence SEQ ID NO: 8.
• the DNA polymerase protein unit is Klenow fragment, preferably comprising the amino acid sequence SEQ ID NO: 9.
• the DNA polymerase protein unit is Bst, preferably comprising the amino acid sequence SEQ ID NO: 10.
• the DNA polymerase protein unit is wild-type Phi, preferably comprising the amino acid sequence SEQ ID NO: 34.
• the DNA polymerase may be a ribonucleic acid (RNA)-dependent DNA polymerase, i.e., the polymerase activity requires a pre-existing RNA strand for polymerase activity.
• RNA-dependent DNA polymerases is the reverse transcriptase from viruses, eukaryotes, and prokaryotes such as plants or animals.
• the term “activity of the DNA polymerase protein unit” as used herein refers to a polymerase activity and/or a nuclease activity, preferably a polymerase activity and/or an exonuclease activity.
• Polymerase activity or DNA synthesis activity of the DNA polymerase is the activity wherein the DNA polymerase generates a new nucleic strand complementary to a pre-existing strand by incorporating nucleotides at the 3’ end of a primer hybridized to the pre-existing strand thereby moving in a 5’->3’ direction.
• the DNA polymerase incorporates a nucleotide comprising the base adenine while at positions in the pre-existing strand with a nucleotide comprising the base cytosine, the DNA polymerase incorporates a nucleotide comprising the base guanine and vice versa.
• Nuclease activity might by an exonuclease or an endonuclease activity, preferably an exonuclease activity such as a 3’->5’ exonuclease (proofreading) activity and/or a 5’->3’ exonuclease activity.
• the exonuclease activity of a polymerase is known in the art.
• Taq polymerase isolated from Thermus aquaticus which is frequently used in polymerase chain reaction (PCR) amplification possesses 5’->3’ exonuclease activity but no 3’->5’ exonuclease (proofreading) activity while others, such as Pfu polymerase which is also frequently used in PCR amplification has 3’->5’ exonuclease (proofreading) activity but no 5’->3’ exonuclease activity.
• the activatable DNA polymerase of the present invention may further comprise at least one cleavable site. Cleavage at the cleavable site separates the polymerase protein unit and at least a part of the nucleic acid unit of the activatable DNA polymerase.
• the skilled person would know that the exact localization of the cleavable site in the activatable DNA polymerase may vary.
• the cleavable site is localized at a position in the activatable DNA polymerase that allows removal of the entire or at least a part of the nucleic acid. Where at least a part of the nucleic acid unit is removed, the part of the nucleic acid unit which remains coupled to the DNA polymerase protein unit has a length which is insufficient for blockage of the activity of the DNA polymerase protein unit.
• the cleavable site is within the nucleic acid unit, i.e., the cleavable site is between two nucleotides (nt) of the nucleic acid unit, preferably at position 1 , 2, 3, 4, 5, 6, 7 or 8 of the nucleic acid unit from the terminus coupled directly or indirectly to the DNA polymerase protein unit.
• the cleavable site is at position 1 of the nucleic acid unit from the terminus coupled to the DNA polymerase protein unit.
• the cleavable site may be between the DNA polymerase protein unit and the nucleic acid unit or within a linker between the DNA polymerase protein unit and the nucleic acid unit.
• the cleavable site may be any cleavable site which upon cleavage removes the entire or at least a part of the nucleic acid unit.
• Specific cleavable sites which can be used in the context of the present invention are known to the skilled person and are commercially available.
• cleavage sites suitable for incorporation into nucleic acid units are known to the skilled person and are commercially available, e.g., from biomers-the biopolymer factory.
• the cleavable site is a photocleavable site.
• exemplary photocleavable sites without being limited thereto are an o-nitrobenzyl-based photocleavable site, a o-carbonyl-based photocleavable site or a benzyl-based photocleavable site,
• the photocleavable site is an o-nitrobenzyl-based photocleavable site selected from the group consisting of 1 -(2-Nitrophenyl)-1 ,3- propanediol and 1 -(2-Nitrophenyl)-1 ,3-butanediol.
• photocleavable sites are particularly suitable for incorporation into the nucleic acid unit, preferable close to the terminus directly or indirectly coupled to the DNA polymerase protein unit.
• photocleavable site and “photocleavable linker” as used herein are interchangeable.
• the activatable DNA polymerase of the present invention optionally further comprising at least one linker unit between the polymerase protein unit and the nucleic acid unit.
• the “linker unit” as used herein refers to any linker which is a part of the activatable DNA polymerase of the invention between the DNA polymerase protein unit and the nucleotide unit.
• the linker unit may be a peptide linker, a nucleic acid linker or any chemical linker known in the art.
• the linker is a flexible linker.
• a linker unit may serve as a spacer and reduce steric hinderance and/or increase flexibility of the coupled nucleic acid unit.
• the nucleic acid unit might be sterically hindered, and the blocking of the DNA polymerase protein activity might be hampered.
• the need for a linker may also depend on the attachment site of the nucleic acid unit to the DNA polymerase protein unit.
• a linker particularly a peptide linker expressed together with the DNA polymerase protein unit, may be used for coupling the nucleic acid unit to the DNA polymerase protein unit, e.g., for the introduction and use of a click chemistry unit.
• linker unit in the activatable DNA polymerase and the length of the linker unit might vary and depend on various factors, e.g., the attachment site of the nucleic acid unit at the DNA polymerase protein unit relative to the active center of the DNA polymerase activity and/or the length of the nucleic acid unit.
• linker units which can be used in the context of the present invention.
• the linker unit might be selected from the group consisting of a peptide linker and a polyethylene glycol (PEG) linker.
• the at least one linker unit comprises a peptide linker, more preferably a flexible linker, most preferably a glycine/serine (GS)-linker, preferably a GS-linker comprising a modified or unnatural amino acid which may be used for coupling as described herein (e.g., having the sequence of SEQ ID NOs: 12, 13, 14, 15 or 16).
• the modified or unnatural amino acid is a 4-azido-L-phenylalanine, such as at position 3 of the GS-linker.
• the linker unit consists of a peptide linker as described herein.
• the linker unit is a peptide linker, such as a GS-linker.
• the peptide linker has a length of about 1 to about 30 amino acid residues, preferably about 2 to about 20 amino acid residues, more preferably about 3 to about 15 amino acid residues most preferably about 4 to about 10.
• the nucleic acid unit is (directly or indirectly) coupled to the DNA polymerase protein unit (a) by a covalent chemical bond or by streptavidin-biotin; and/or (b) by its terminal nucleotide.
• the nucleic acid unit of the activatable DNA polymerase according to the invention does not comprise or consist of an aptamer.
• the nucleic acid unit is directly coupled to the DNA polymerase protein unit.
• the nucleic acid unit may further comprise a cleavage site, preferably close to the terminus of the nucleic acid unit coupled to the DNA polymerase protein unit.
• the nucleic acid unit is indirectly coupled to the DNA polymerase protein unit, e.g., by the cleavable site and optionally further by the at least one linker unit as described herein flanking the cleavable site on one or both sides.
• the cleavage site may be at one end of the linker or within the linker.
• the cleavable site may directly couple the DNA polymerase unit and the nucleic acid unit.
• the cleavable site is flanked by the at least one linker unit flanking the cleavable site on one side.
• the cleavable site is coupled to the DNA polymerase protein unit at its first end and to the linker unit at its second end.
• the photocleavable site is coupled to the nucleic acid unit at its first end and to the linker unit at its second end.
• the cleavable site is flanked by the linker unit on both sides, i.e. , the cleavable site is coupled to a first linker at its first end and to a second linker at its second end, i.e., the cleavable site is flanked by two linker units.
• the first and the second linker may be the same or different.
• the nucleic acid unit is indirectly coupled to the DNA polymerase protein unit by the at least one linker unit as described herein.
• the linker unit links the DNA polymerase unit to the nucleic acid unit.
• the activatable DNA polymerase may further comprise a cleavage site.
• the cleavage site may be within the linker or at either end of the linker.
• the cleavage site may be within the nucleic acid unit.
• the cleavage site is close to the terminus of the nucleic acid unit coupled to the DNA polymerase protein unit, preferably within 10, 8, 6, 4, 3, 2, 1 nucleotides of the terminus.
• the skilled person is aware of methods for coupling the nucleic acid unit and the DNA polymerase protein unit, or optionally the cleavable site or the linker.
• the coupling may be targeted or random, preferably the coupling is targeted, more preferably the coupling is at the C-terminus or the N-terminus of the DNA polymerase protein unit (or alternatively the linker), even more preferably the coupling is at the C-terminus of the DNA polymerase protein unit (or alternatively the linker).
• the linker unit may be fused to the DNA polymerase, e.g., by preparing an expression vector including an expression cassette encoding for the DNA polymerase protein unit fused to the linker.
• the sequence encoding for the linker may allow for the incorporation of a modified or unnatural amino acid which may be used for coupling.
• the incorporation might be accomplished e.g., via the amber codon suppression (TGA) strategy as described in the Examples (Section: Material and Methods, section 1.1 ; see also [17] which is incorporated herein by reference).
• nucleic acid unit might be coupled to the DNA polymerase protein unit (optionally to the DNA polymerase protein unit expressed together with the linker unit) by any suitable chemical coupling reaction known in the art.
• Chemical reactions for coupling are generally known to the skilled person and may comprise, e.g., click chemistry using a first click chemistry unit and a second click chemistry unit (see, e.g., [9-10] which are incorporated herein by reference).
• click chemistry units are known to the skilled person and are commercially available, often directly coupled to the nucleotide, the amino acid or to the functional groups of the linkers which allows for a simple reaction between the different units of the activatable DNA polymerase.
• click chemistry units may be selected from the group consisting of a propargyl group, an azide group, a tetrazine group, an alkyne group, a norbornene group, and a cyclooctyne group.
• the first click chemistry unit is an azide group.
• the azide group is present in an amino acid residue of the DNA polymerase protein unit or the linker unit.
• the azide group might be directly incorporated into the protein or peptide, e.g., the DNA polymerase protein unit or the peptide linker during synthesis by incorporating an azide-modified amino acid as described herein.
• a preferred modified amino acid in this regard is 4-Azido-L-phenylalanine.
• the at least one linker unit has a sequence selected from a group consisting of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, preferably the at least one linker unit has the sequence of SEQ ID NO: 14.
• the second click chemistry unit is a cyclooctyne moiety such as dibenzocyclooctyne (DBCO).
• DBCO dibenzocyclooctyne
• the second click chemistry unit is present in a nucleotide of the nucleic acid unit as described herein, more preferably a terminal nucleotide of the nucleic acid unit described herein.
• first and the second click chemistry units are coupled via cycloaddition.
• the nucleic acid unit can be coupled to the DNA polymerase protein unit, or optionally the linker unit, if present.
• the nucleic acid unit is coupled by its terminal nucleotide.
• the nucleic acid unit is single stranded, comprises a DBCO click chemistry unit in the terminal nucleotide and a photocleavable site at position 1 of a single stranded nucleic acid unit from the terminus coupled to the DNA polymerase protein unit.
• the nucleic acid unit is one as depicted in the Examples (see Material and Methods, Table 1 ).
• the orientation of the nucleic acid unit for coupling might vary and depend on various factors, e.g., the nuclease activity of the DNA polymer protein unit.
• the DNA polymerase protein unit has a 5’->3’ exonuclease activity and the nucleic acid unit is coupled at its 5’ terminal nucleotide and optionally the 3’ terminus is protected as described herein.
• the DNA polymerase protein unit has a 3’->5’ exonuclease activity and the nucleic acid unit is coupled at its 3’ terminal nucleotide and optionally the 5’ terminus is protected as described herein.
• the DNA polymerase has a 5’->3’ exonuclease activity and a 5’->3’ exonuclease activity.
• the nucleic acid unit is coupled at its 5’ terminal nucleotide or at its 3’ terminal nucleotide, preferably at its 3’ terminal nucleotide, and optionally the free terminus of the nucleic acid unit is protected as described herein.
• the nucleic acid unit is coupled at its 5’ terminal nucleotide and the 3’ terminus is protected.
• the nucleic acid unit is coupled at its 3’ terminal nucleotide and the 5’ terminus is protected.
• “Protection” or “protected “as used herein refers to a modification of a nucleotide and/or to the linkage between nucleotides, e.g., the phosphodiester bond.
• a “modification of a nucleotide” or a “modified nucleotide” as used herein might be any modification which protects a nucleic acid from degradation. Such modifications are known to the skilled person, e.g., a modification of the sugar of a nucleotide.
• a “modification of the sugar of a nucleotide” as used herein refers, e.g., to a replacement of the hydroxy group at the 2’- carbon atom of the ribose by another functional group, such as an amine group or a fluorine group; or to a replacement of the hydrogen of the hydroxyl group of the 2’ carbon atom of the ribose.
• the replacement of the hydrogen of the hydroxyl group may be by an alkyl group to generate a 2’-O-Methyl, 2’-O-Ethyl, or 2’-O-Propyl, preferably 2’-O-Methyl; an ether group, such as 2’-O-Methoxymethyl, 2’-O-Methoxyethyl, 2’-O- Methoxypropyl, preferably 2’-O-Methoxyethyl; an amine group to generate 2’-0-Amine, 2’-O-Methylamine, 2’-O-Ethylamine, 2’-O-Propylamine, preferably 2’-O-Propylamine; or an 2’-O-Proparagyl group (see also [30] and [31 ], which are incorporated herein by reference).
• a “modification of the linkage between nucleotides” as used herein refers to any modification of the backbone which increases stability of a nucleic acid, e.g., substitution of the phosphodiester bond by another linkage, e.g., a phosphorothioate, peptide nucleic acid oligomers (PNA) or morpholino oligomers such as phosphorodiamidate morpholino oligomers (PMO), (see also [30] and [31 ], which are incorporated herein by reference).
• PNA phosphorothioate
• PMO morpholino oligomers
• the terminal nucleotide of the free terminus of the nucleic acid unit is protected, preferably by a modification of a nucleotide as described herein.
• a modification of a nucleotide as described herein Preferably, at least a few of the nucleotides at the free terminus (two or more of the nucleotides at the free terminus, such as 2, 3 or 4) are protected.
• the free terminus is protected by a modification of a nucleotide and/or a modification of the linkage between nucleotides as described herein.
• the terminal nucleotide at the free terminus of the nucleic acid unit i.e., at least the nucleotide at position 1 , preferably the nucleotide at positions 1 and 2 from the free terminus, more preferably the nucleotide at positions 1 , 2 and 3 from the free terminus are protected.
• all nucleotides of the nucleic acid unit are protected, preferably by a modification of a nucleotide and/or by a modification of the linkage between nucleotides as described herein, more preferably by a modification of the linkage between nucleotides.
• the nucleic acid unit orientation for coupling and/or the protection of the free terminus of the nucleic acid unit as described herein may have the advantage that the nucleic acid unit is protected from the nuclease activity of the DNA polymerase protein unit.
• the nucleic acid unit can be coupled (directly or indirectly) as described herein to any amino acid of the DNA polymerase protein unit, e.g., a terminal amino acid or an internal amino acid, i.e. , an amino acid which is not localized at the terminus.
• the nucleic acid unit can be coupled to any functional group of the amino acid which is suitable for coupling.
• the nucleic acid unit can be coupled to a carboxyl-group, an amine-group or a side chain.
• the nucleic acid unit can be coupled to a side chain.
• the direct or indirect coupling of the nucleic acid unit is to an amino acid of the DNA polymerase protein unit which does not interfere with the active center, preferably an amino acid which is outside of and/or distant to the active center of the DNA polymerase protein unit, preferably outside of and/or distant to the active center mediating the polymerase activity and/or nuclease activity of the DNA polymerase protein unit.
• the (direct or indirect) coupling of the nucleic acid unit is to a terminal amino acid of the DNA polymerase protein unit, such as the N-terminal or the C-terminal amino acid, preferably to the C-terminal amino acid.
• the activatable DNA polymerase of the invention can be used or applied in any method using DNA polymerases.
• the embodiments described herein for the activatable DNA polymerase of the invention likewise apply to the methods, processes, and uses as described herein.
• the present invention provides a new reversible blocking mechanism for DNA polymerases.
• the activatable DNA polymerase of the present invention as described herein provides a blocking mechanism which is universally applicable to basically any DNA polymerase.
• the present invention is particularly useful for DNA polymerases which are temperature sensitive. To the inventor’s best knowledge there is no reversible blocking mechanism available for temperature-sensitive DNA polymerases to date.
• the present invention therefore provides a new field of applications for temperature sensitive, i.e. , mesophilic, DNA polymerases, e.g., in diagnostics.
• the invention relates to a method of synthesizing a nucleic acid comprising: a) providing a sample comprising at least one nucleic acid template, components for nucleic acid synthesis and the activatable DNA polymerase of the invention, b) activating the activatable DNA polymerase in the sample of step a), thereby generating an activated DNA polymerase, and c) subjecting the sample comprising the activated DNA polymerase of step b) to reaction conditions that allow synthesis of a nucleic acid complementary to the at least one nucleic acid template.
• the nucleic acid template might be any nucleic acid, e.g., a linear and/or a circular nucleic acid.
• the nucleic acid template might further be a single or double stranded nucleic acid.
• the DNA amplification might comprise polymerase-chain reaction (PCR), e.g., RT- PCR, qPCR, rolling circle amplification, whole genome amplification, or isothermal amplification, e.g., loop-mediated isothermal amplification (LAMP).
• PCR polymerase-chain reaction
• RT- PCR RT- PCR
• qPCR rolling circle amplification
• whole genome amplification e.g., qPCR
• isothermal amplification e.g., loop-mediated isothermal amplification (LAMP).
• LAMP loop-mediated isothermal amplification
• the nature and the amount of the components for nucleic acid synthesis and the reaction conditions might vary and depend on the type of the method of synthesizing a nucleic acid which are known to the skilled person.
• the DNA polymerase may be Bst as described herein, preferably Bst comprising the amino acid sequence of SEQ ID NO: 10.
• the DNA polymerases may be Taq or Pfu as described herein, preferably Taq or Pfu comprising the amino acid sequences of SEQ ID NOs: 7 or 8.
• the DNA polymerase might by Phi29, preferably Phi29 comprising the sequence of SEQ ID NO: 6 or SEQ ID NO: 34.
• the invention provides a method of DNA amplification, such as polymerase-chain reaction (PCR), comprising: a) providing a sample comprising at least one nucleic acid template, components for nucleic acid amplification and the activatable DNA polymerase of the invention as described herein, preferably wherein the DNA polymerase protein unit is selected from the group consisting of Taq and Pfu, more preferably of Taq comprising the amino acid sequence of SEQ ID NO: 7, and Pfu comprising the amino acid sequence of SEQ ID NO: 8, b) activating the activatable DNA polymerase in the sample of step a), thereby generating an activated DNA polymerase, and c) subjecting the sample with the activated DNA polymerase of step b) to reaction conditions that allow amplification of the at least one nucleic acid template.
• PCR polymerase-chain reaction
• the nucleic acid template might be any nucleic acid, e.g., a linear nucleic acid.
• the nucleic acid template might be a double stranded or single stranded nucleic acid, preferably a double stranded nucleic acid.
• the nucleic acid template is a circular nucleic acid which might by single stranded or double stranded.
• components for nucleic acid amplification might comprise a mixture of deoxyribonucleotides (dNTPs), a buffer which is suitable for carrying out nucleic acid synthesis, cofactors for the polymerase such as Mg 2+ , and primers.
• dNTPs deoxyribonucleotides
• the criteria for the choice of primers are known to the skilled person and might comprise degree of complementary to the nucleic acid template particularly at the 3’ end of the primer, annealing temperature (which is based on the length of the primers and the guanosine and cytidine content) and length of amplicon and location and orientation of the primers on the opposite nucleic acid strands.
• the reaction conditions that allow amplification of the at least one nucleic acid template are known to the skilled person and comprise for example a denaturation step at high temperatures, such as about 90 °C to about 95 °C for about 1 to about 5 minutes, followed by about 30 to about 50 cycles comprising a denaturation step at high temperatures, such as about 95 °C for about 15 seconds to about 1 minute, an annealing step at the primer-specific annealing temperature for about 30 seconds to about 1 minute, and an extension step or elongation step at about 72 °C for about 30 seconds to about 2 minutes depending on the length of the amplicon.
• the cycles are followed by a terminal elongation step at a temperature of about 72 °C for about 10 minutes and optionally subsequent cooling at about 8 °C until removal of the sample.
• the activatable DNA polymerase is preferably an activatable DNA polymerase which comprises a cleavable site as described herein.
• the activatable DNA polymerase comprises a photocleavable site as described herein.
• the DNA polymerase protein unit is directly coupled or indirectly coupled, e.g., by a linker; to the nucleic acid unit and the cleavable site, preferably the photocleavable site, is localized in the nucleic acid unit as described herein.
• the DNA polymerase protein unit is indirectly coupled to the nucleic acid unit by the cleavable site, preferably the photocleavable site, optionally wherein the cleavable site is flanked by at least one linker unit on one or both sites of the cleavable site as described herein.
• the photocleavable site is activated by irradiation with light, such as light of a wavelength which cleaves conventional photocleavable sites.
• Such a wavelength is known to the skilled person, e.g., a wavelength which is between about 300 nm to about 400 nm, preferably between about 310 nm to about 370 nm, more preferably between about 315 nm to about 365 nm, most preferably at about 315 or at about 365 nm may be used for a o- nitrobenzyl-based photocleavable site, e.g., 1-(2-Nitrophenyl)-1 ,3-propanediol or 1 -(2- Nitrophenyl)-1 ,3-butanediol.
• This activation (i.e., cleavage) of the photocleavable site results in cleavage of at least a part of the nucleic acid unit of the activatable DNA polymerase.
• the wavelength does not essentially interfere with the integrity of the nucleic acid template, the activity of the activatable DNA polymerase protein unit and/or is compatible with the assay method.
• the invention provides a method of rolling circle amplification comprising: a) providing a sample comprising at least one circular nucleic acid template, components for nucleic acid synthesis and the activatable DNA polymerase of the invention, preferably wherein the DNA polymerase protein unit is Phi29, more preferably comprising the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 34, b) activating the activatable DNA polymerase in the sample of step a), thereby generating an activated DNA polymerase, and c) subjecting the sample with the activated DNA polymerase of step b) to reaction conditions that allow amplification of the circular nucleic acid template.
• the conditions in step c) comprise incubation at an about constant temperature, preferably at temperature in a range between 28°C to 40°C, preferably between 30°C to 37°C.
• a “constant temperature” as used herein means a temperature of fixed value which might deviate from the fixed value within a range of 1 °C to 3 °C, preferably 1 °C to 2 °C.
• Buffers which might be used in step b) are known to the skilled person and might, e.g., comprise buffers which are suitable for DNA polymerase, e.g. a buffer which is suitable for carrying out nucleic acid synthesis comprising a salt and cofactors for the polymerase such as Mg 2+ .
• the activatable DNA polymerase is preferably an activatable DNA polymerase which comprises a cleavable site as described herein.
• the activatable DNA polymerase comprises a photocleavable site as described herein.
• the DNA polymerase protein unit is directly coupled or indirectly coupled, e.g., by a linker; to the nucleic acid unit and the cleavable site, preferably the photocleavable site, is localized in the nucleic acid unit as described herein.
• the DNA polymerase protein unit is indirectly coupled to the nucleic acid unit by the cleavable site, preferably the photocleavable site, wherein optionally the cleavable site is flanked by at least one linker unit on one or both sites of the cleavable site as described herein.
• the photocleavable site is activated (resulting in cleavage of at least a part of the nucleic acid unit of the activatable DNA polymerase) by irradiation with light, such as light of a wavelength which cleaves conventional photocleavable sites.
• Such a wavelength is known to the skilled person, e.g., a wavelength which is between about 300 nm to about 400 nm, preferably between about 310 nm to about 370 nm, more preferably between about 315 nm to about 365 nm, most preferably at about 315 or at about 365 nm may be used for a o-nitrobenzyl-based photocleavable site, e.g., 1 -(2-Nitrophenyl)-1 ,3- propanediol or 1 -(2-Nitrophenyl)-1 ,3-butanediol.
• the wavelength does not essentially interfere with the integrity of the nucleic acid template, the activity of the activatable DNA polymerase protein unit and/or is compatible with the assay method.
• the activatable DNA polymerase of this embodiment does not comprise a cleavable site as described herein.
• the nucleic acid of the activatable DNA polymerase is directly coupled to the DNA polymerase protein unit.
• the nucleic acid unit is indirectly coupled to the DNA polymerase protein unit by a linker unit as described herein.
• the DNA polymerase of this embodiment is activated by exposing the DNA polymerase to a single stranded nucleic acid which is sufficiently complementary for hybridizing to the nucleic acid unit, preferably wherein the nucleic acid unit is at least partially single stranded.
• the nucleic acid unit of the activatable DNA polymerase is at least partially single stranded or comprises at least a single stranded part as described herein.
• the nucleic acid unit comprises at least 12, preferably at least 15, more preferably at least 20 consecutive nucleotides which are single stranded.
• the single stranded part of the nucleic acid unit is localized at a terminus of the nucleic acid unit which is coupled to the DNA polymerase protein unit.
• the partially single stranded nucleic acid unit may comprise a terminal or an internal double stranded part, preferably wherein at least 12, preferably at least 15, more preferably at least 20 consecutive single stranded nucleotides are present which are preferably localized at a terminus of the nucleic acid unit which is coupled to the DNA polymerase protein unit.
• the internal or terminal double stranded part might preferably be generated by a self-complementary part, e.g., a hairpin, of a single stranded nucleic acid unit.
• the nucleic acid unit is a single stranded nucleic acid unit, i.e. , it comprises single stranded nucleotides only.
• the sequence of the nucleic acid unit does not have any predicted secondary structure.
• the nucleic acid unit is not an aptamer.
• the complementary sequence of the single stranded part of the nucleic acid unit has a sequence identity of at least about 80%, at least about 90%, at least about 95%, at least about 98% or at least about 100% to the complete sequence of the single stranded nucleic acid.
• the invention provides a method of nucleic acid sequencing comprising: a) providing a sample comprising a nucleic acid to be sequenced, components for nucleic acid sequencing and the activatable DNA polymerase of the invention as described herein, preferably wherein the DNA polymerase protein unit is Phi29, more preferably comprising the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 34, or Klenow fragment, more preferably comprising the amino acid sequence of SEQ ID NO: 9, b) activating the activatable DNA polymerase in the sample of step a), thereby generating an activated DNA polymerase, and c) subjecting the sample with the activated DNA polymerase of step b) to reaction conditions that allow sequencing of the nucleic acid to be sequenced.
• the DNA polymerase protein unit is Phi29, more preferably comprising the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 34, or Klenow fragment, more preferably comprising the amino acid sequence of SEQ ID NO: 9, b) activ
• the nucleic acid to be sequenced might be any nucleic acid, e.g., a linear nucleic acid and/or a circular nucleic acid.
• the nucleic acid template might be a single stranded or double stranded nucleic acid.
• the nucleic acid to be sequenced might be a genomic nucleic acid of an organism or a pathogen (e.g. a bacterial or viral pathogen).
• the components and amounts of the components for nucleic acid sequencing are known to the skilled person and are similar to the components described above for the DNA amplification method.
• the components might comprise a mixture deoxyribonucleotides (dNTPs), buffers which are suitable for carrying out synthesizing processes of nucleic acids, cofactors for the polymerase such as Mg 2+ , and primers.
• dNTPs mixture deoxyribonucleotides
• buffers which are suitable for carrying out synthesizing processes of nucleic acids
• cofactors for the polymerase such as Mg 2+
• primers primers.
• the basic principle of nucleic acid sequencing is the chain-termination method which comprises elongation of the primer with a dNTP mixture comprises nucleotides of each base which lead to a termination of the DNA polymerase activity, such as a 2’, 3’- dideoxynucleotide (ddNTP) and is described, e.g., in Sanger et al., 1977 [22] and 1975 [23], which are incorporated herein by reference.
• ddNTP dideoxynucleotide
• further sequencing methods which might be applicable in the present invention such as 454 pyrosequencing, Illumina (Solexa) sequencing or singlemolecule real-time (SMRT) sequencing (Pacific Biosciences) [24-28] which are incorporated herein by reference.
• the DNA polymerase protein unit may comprise any DNA polymerase known in the art, preferably an activatable DNA polymerase with a 3’->5’ exonuclease activity such as Klenow fragment or Phi29 as described herein, further comprising a cleavable site.
• the activatable DNA polymerase comprises a photocleavable site as described herein.
• the DNA polymerase protein unit is directly coupled or indirectly coupled, e.g., by a linker; to the nucleic acid unit and the cleavable site, preferably the photocleavable site, is localized in the nucleic acid unit as described herein.
• the DNA polymerase protein unit is indirectly coupled to the nucleic acid unit by the cleavable site, preferably the photocleavable site, wherein optionally the cleavable site is flanked by at least one linker unit on one or both sites of the cleavable site as described herein.
• the photocleavable site is activated by cleavage of the entire or at least a part of the nucleic acid unit by irradiation with light.
• the photocleavable site is activated (resulting in cleavage of at least a part of the nucleic acid unit of the activatable DNA polymerase) by irradiation with light, such as light of a wavelength which cleaves conventional photocleavable sites.
• Such a wavelength is known to the skilled person, e.g., a wavelength which is between about 300 nm to about 400 nm, preferably between about 310 nm to about 370 nm, more preferably between about 315 nm to about 365 nm, most preferably at about 315 or at about 365 nm may be used for a o- nitrobenzyl-based photocleavable site, e.g., 1-(2-Nitrophenyl)-1 ,3-propanediol or 1 -(2- Nitrophenyl)-1 ,3-butanediol.
• the wavelength does not essentially interfere with the integrity of the nucleic acid template, the activity of the activatable DNA polymerase protein unit and/or is compatible with the assay method.
• the invention provides a method of determining the presence or absence of a single stranded nucleic acid of interest comprising: a) providing a sample to be tested for comprising a single stranded nucleic acid of interest, b) adding the activatable DNA polymerase according to the invention and as described herein to the sample of step a), wherein at least a part of the nucleic acid unit is single stranded and complementary to the single stranded nucleic acid of interest, c) subjecting the sample with the activatable DNA polymerase of step b) to reaction conditions which allow hybridization, preferably hybridization between the single stranded part of the nucleic acid unit of the activatable DNA polymerase unit and the single stranded nucleic acid of interest thereby activating the activatable DNA polymerase of the invention, and d) determining the activity of the activatable DNA polymerase of step c), wherein the activity of the activatable DNA polymerase indicates the presence of the nucle
• the activatable DNA polymerase of this embodiment does not necessarily comprise a cleavable site as described herein.
• the nucleic acid of the activatable DNA polymerase is directly coupled to the DNA polymerase protein unit.
• the nucleic acid unit is indirectly coupled to the DNA polymerase protein unit by a linker unit as described herein.
• the activatable DNA polymerase may or may not comprise a cleavable site as this is not required for the method of determining the presence or absence of a single stranded nucleic acid of interest.
• the activatable DNA polymerase of the invention does not comprise a cleavable site.
• the activatable DNA polymerase comprises a mesophilic DNA polymerase protein unit as described herein, most preferably Phi 29 as described herein, e.g. Phi29 comprising the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 34.
• the nucleic acid unit of the activatable DNA polymerase is at least partially single stranded or comprises at least a single stranded part as described herein.
• the nucleic acid unit comprises at least 12, preferably at least 15, more preferably at least 20 consecutive nucleotides which are single stranded.
• the single stranded part of the nucleic acid unit is localized at a terminus of the nucleic acid unit which is coupled to the DNA polymerase protein unit.
• the partially single stranded nucleic acid unit may comprise a terminal or an internal double stranded part, preferably wherein at least 12, preferably at least 15, more preferably at least 20 consecutive single stranded nucleotides are present which are preferably localized at a terminus of the nucleic acid unit which is coupled to the DNA polymerase protein unit.
• the internal or terminal double stranded part might preferably be generated by a self-complementary part, e.g., a hairpin, of a single stranded nucleic acid unit.
• the nucleic acid unit is a single stranded nucleic acid unit, i.e. , it comprises single stranded nucleotides only.
• the sequence of the nucleic acid unit does not have any predicted secondary structure. According to the invention the nucleic acid unit is not an aptamer.
• the single stranded part of the nucleic acid unit of the activatable DNA polymerase comprises a sequence which is sufficient complementary to hybridize to the nucleic acid of interest.
• the complementary sequence of the single stranded part of the nucleic acid unit has a sequence identity of at least about 80%, at least about 90%, at least about 95%, at least about 98% or at least about 100% to the complete sequence of the single stranded nucleic acid of interest.
• the single stranded nucleic acid of interest can be any nucleic acid, e.g., DNA or RNA.
• the single stranded nucleic acid may be a full length or a truncated gene, a small RNA, such as a micro RNA, a fusion or tagged gene, and can be a cDNA, a genomic DNA, or a DNA fragment, preferably, a cDNA. It can be the native sequence, i.e., a wildtype sequence or can be mutated.
• the single stranded nucleic acid may be at least a part of a nucleic acid encoding a polypeptide or protein, such as a secreted, cytoplasmic, nuclear, membrane bound or cell surface polypeptide or might be a non-coding nucleic acid.
• the single stranded nucleic acid of interest is selected from the group consisting of a microRNA (small regulatory RNAs, related to ageing, neurodegeneration, and cancer and potentially useful as non-invasive biomarkers[16] which is incorporated herein by reference), a viral or bacterial nucleic acid, preferably from viruses or bacteria causing a disease, e.g., Steptococcus, Staphylococcus, Pneumcococcus, Orthomyxoviridae, Coronaviridae, gastrointestinal diseases, e.g., Salmonella, Campylobacteraceae, Norovirus, Rotavirus and a nucleic acid which is indicative of a disease or a disease state.
• a microRNA small regulatory RNAs, related to ageing, neurodegeneration, and cancer and potentially useful as non-invasive biomarkers[16] which is incorporated herein by reference
• a viral or bacterial nucleic acid preferably from viruses or bacteria causing a disease, e.g., Steptoc
• the single stranded nucleic acid of interest is a DNA generated by reverse transcription.
• the single stranded nucleic acid is an RNA generated by T7 RNA polymerase based on a nucleic acid, such as a PCR amplification product or a product from an isothermal amplification process, preferably after a few cycles of a PCR amplification process.
• the single stranded nucleic acid derives from or is a miRNA, such a miRNA which is expressed and/or a dysregulated in cancer, e.g., Iet-7a, let-7b and Iet7c, miR-17-3b, miR-1268b, miR- 6075.
• the single stranded nucleic acid derives from or is a viral nucleic acid, e.g., SARS-CoV such as SARS-CoV2.
• the single stranded nucleic acid of interest has length of between about 10 nucleotides to about 70 nucleotides, preferably between about 15 to about 50 nucleotides, more preferably between about 20 nucleotides to about 30 nucleotides.
• the sample might be a sample derived from an organism.
• the sample is a tissue or cell sample or sample from a body fluid.
• the sample may be a biopsy, a smear, a blood sample, e.g., serum or plasma, a urine sample, a feces sample, a liquor sample, a mucus sample or a pus sample.
• the conditions in step b) comprise incubation at an about constant temperature, preferably at temperature in a range between 28°C to 40°C, preferably between 30°C to 37°C.
• a “constant temperature” as used herein means a temperature of fixed value which might deviate from the fixed value within a range of 1 °C to 3 °C, preferably 1 °C to 2 °C.
• Buffers which might be used in step b) are known to the skilled person and might, e.g., comprise buffers which are suitable for DNA polymerase, e.g. a buffer which is suitable for carrying out nucleic acid synthesis comprising a salt and cofactors for the polymerase such as Mg 2+ .
• the detection of activity of the determination of activity of the activatable DNA polymerase in step d) may comprise any method for determination of DNA polymerase activity. Such methods are generally known to the skilled person.
• determination of activity of the activatable DNA polymerase may comprise determination of polymerase activity of the activatable DNA polymerase.
• determination of the activity of the DNA polymerase comprises amplification of a nucleic acid for detection and detection of said amplified nucleic acid for detection using, e.g., a labeled probe, e.g., a fluorescence-labeled probe, preferably comprising rolling circle amplification of a circular primed template.
• the circular primed template is single stranded and/or comprises a sequence which is identical to the single stranded nucleic acid unit of the activatable DNA polymerase.
• the DNA polymerase generates an amplification product of the circular template thereby generating a multitude of copies of the single stranded nucleic acid of interest. These copies may in turn activate further activatable DNA polymerases thereby rapidly amplifying the initial signal.
• Subsequent hybridizing of a labeled probe, e.g., fluorescence-labeled, to the amplification product, and measuring a signal generated by the label indicates the presence of the nucleic acid of interest.
• determination of activity of the activatable DNA polymerase activity may comprise determination of nuclease activity of the activatable DNA polymerase, such as degradation of a single stranded nucleic acid added to the activatable DNA polymerase wherein degradation creates a measurable signal, such as a fluorescence signal.
• nuclease activity of the activatable DNA polymerase such as degradation of a single stranded nucleic acid added to the activatable DNA polymerase wherein degradation creates a measurable signal, such as a fluorescence signal.
• a sequence which is complementary to a restriction enzyme site might be included in the circular template. This has the advantage that a restriction enzyme site is present in the amplification product. Cleaving of the amplification product at the restriction site by an appropriate restriction enzyme will allow to produce multiple smaller fragments of the amplification product which increases diffusivity and speeding up the exponential activation of the polymerases.
• sequence which is complementary to a restriction enzyme site in the circular template can be modified to protect the circular template from digestion by nicking and restriction enzymes. Suitable modifications are e.g., methylation of the sequence.
• sequences of restriction sites of methylation-sensitive enzymes are preferably used such as Stul and Nt BsmAI.
• the invention relates to methods of determining the presence or absence of a DNA repair enzyme of interest, the method comprising: a) providing a sample to be tested for comprising a DNA repair enzyme, b) contacting the sample of step a) with an activatable DNA polymerase of the invention as described herein wherein the nucleic acid unit comprises a damaged nucleotide, c) incubating the sample of step b) comprising the activatable DNA polymerase under conditions which allow nucleotide excision repair, preferably of cleavage of a at least a part of the nucleic acid unit, d) determining activity of the activatable DNA polymerase DNA polymer wherein the DNA polymerase activity indicates the presence of a DNA repair enzyme of interest.
• the sample might be a sample derived from an organism.
• the sample is a tissue or cell sample or sample from a body fluid.
• the sample might be a biopsy, a smear, a blood sample, e.g., serum or plasma, a urine sample, a feces sample, a liquor sample, a mucus sample or a pus sample.
• Conditions which allow nucleotide excision repair comprise incubation at a temperature of about 30 °C to about 39 °C, preferably about 37 °C.
• the skilled person would know that the incubation buffer needs to be adopted to be optimal for the activity of the specific repair enzyme to be tested.
• the activatable DNA polymerase of this embodiment does not necessarily comprise a cleavable site as described herein.
• the nucleic acid of the activatable DNA polymerase is directly coupled to the DNA polymerase protein unit.
• the nucleic acid unit is indirectly coupled to the DNA polymerase protein unit by a linker unit as described herein.
• the activatable DNA polymerase of the invention does not comprise a cleavable site.
• the activatable DNA polymerase comprises a mesophilic DNA polymerase protein unit as described herein, most preferably Phi 29 as described herein, e.g., Phi29 comprising the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 34.
• the nucleic acid unit of the activatable DNA polymerase is at least partially single stranded or comprises at least a single stranded part as described herein.
• the nucleic acid unit comprises at least 12, preferably at least 15, more preferably at least 20 consecutive nucleotides which are single stranded.
• the single stranded part of the nucleic acid unit is localized at a terminus of the nucleic acid unit which is coupled to the DNA polymerase protein unit.
• the partially single stranded nucleic acid unit may comprise a terminal or an internal double stranded part, preferably wherein at least 12, preferably at least 15, more preferably at least 20 consecutive single stranded nucleotides are present which are preferably localized at a terminus of the nucleic acid unit which is coupled to the DNA polymerase protein unit.
• the internal or terminal double stranded part might preferably be generated by a self-complementary part, e.g., a hairpin, of a single stranded nucleic acid unit.
• the nucleic acid unit is a single stranded nucleic acid unit, i.e. , it comprises single stranded nucleotides only.
• the sequence of the nucleic acid unit does not have any predicted secondary structure. According to the invention the nucleic acid unit is not an aptamer.
• the part of the nucleic acid unit which remains coupled to the DNA polymerase unit after nucleotide excision has a length which is not sufficient to block activity of DNA polymerase protein unit of the activatable DNA polymerase.
• the invention provides the use of a nucleic acid for reversibly inhibiting a DNA polymerase wherein the nucleic acid is coupled to the DNA polymerase (a) by a covalent chemical bond or by streptavidin-biotin; and/or (b) by its terminal nucleotide, preferably wherein the nucleic acid has a length which is sufficient to block activity of the DNA polymerase more preferably wherein the nucleic acid is at least partially single stranded as described herein.
• the invention provides the use of the activatable DNA polymerase of the invention in a nucleic acid synthesis process, preferably wherein the activatable DNA polymerase comprises a cleavable site wherein upon cleavage at least a part of the nucleic acid unit is removed and wherein the cleavable site is a photocleavable site.
• the nucleic acid synthesis process is preferably an in vitro nucleic acid synthesis process, more preferably selected from the group consisting of sequencing, preferably whole genome sequencing; and a nucleic acid amplification process, preferably PCR, RT-PCR, qPCR, rolling circle amplification, whole genome amplification or isothermal amplification, such as loop-mediated isothermal amplification (LAMP).
• a nucleic acid amplification process preferably PCR, RT-PCR, qPCR, rolling circle amplification, whole genome amplification or isothermal amplification, such as loop-mediated isothermal amplification (LAMP).
• LAMP loop-mediated isothermal amplification
• the invention provides the use of the activatable DNA polymerase of the invention for diagnosis as described herein, preferably for in vitro diagnosis.
• in vitro diagnosis refers to an in vitro use of said activatable DNA polymerase of the invention for diagnosis, such as diagnosis of a disease, a medical condition and/or detection of pathogens in a sample such as from a human or an animal (e.g., mammal or bird), food, drink, soil or water (e.g., a drinking water, a waste water or a hydrologic sample).
• the presence or absence of a single stranded nucleic acid of interest may be determined using the activatable DNA polymerase of the invention, such as using the method of determining the presence or absence of a single stranded nucleic acid of interest of the invention.
• the activatable DNA polymerase is activated by exposure of the nucleic acid unit of the activatable DNA polymerase to a nucleic acid of interest, preferably a single stranded nucleic acid of interest, wherein at least part of the nucleic acid unit is single stranded and sufficiently complementary to the nucleic acid of interest to hybridize to the nucleic acid of interest.
• the activation of the inventive DNA polymerases e.g., in DNA synthesis processes, is simple and also provides new apparatuses like PCR machines operating with a light pulse such as a UV light source and their uses in the methods involving DNA polymerases, such as methods and uses as described herein.
• the present invention provides an apparatus, in particular a thermal cycler, for performing a polymerase-chain reaction (PCR) comprising: a) means, in particular a thermal block, adapted for receiving at least one container suitable for the uptake of a sample comprising at least one nucleic acid template, components for nucleic acid amplification and the activatable DNA polymerase of the invention, preferably wherein the DNA polymerase protein unit is selected from the group consisting of Taq and Pfu, more preferably of Taq comprising the amino acid sequence of SEQ ID NO: 7 and Pfu comprising the amino acid sequence of SEQ ID NO: 8, b) at least one light source, in particular at least one LED, emitting light in a wavelength suitable for activating the activatable DNA polymerase in the at least one container of item a), thereby generating an activated DNA polymerase in the at least one container, c) means, in particular including at least one thermoelectric device, in particular a Peltier element, for subjecting the container with the activate
• PCR
• the apparatus of the invention is intended for use with the activatable DNA polymerase according to the invention comprising a photocleavable site.
• the photocleavable site is activated by irradiation with the at least one light source of item b) emitting light at a wavelength which cleaves conventional photocleavable sites, such as an o- nitrobenzyl-based photocleavable site, e.g., 1-(2-Nitrophenyl)-1 ,3-propanediol or 1 -(2- Nitrophenyl)-1 ,3-butanediol.
• Such a wavelength is known to the skilled person, e.g., a wavelength which is between about 300 nm to about 400 nm, preferably between about 310 nm to about 370 nm, more preferably between about 315 nm to about 365 nm, most preferably at about 315 or at about 365 nm.
• This activation results in cleavage of the at least a part of the nucleic acid unit of the activatable DNA polymerase.
• a thermal cycler including a thermal block (sample block), at least one light source, at least one thermoelectric device, and an electrical control device is generally known in the art, for example from the documents EP1386666A1 and EP3093649A2.
• An activatable DNA polymerase comprising a DNA polymerase protein unit coupled to a nucleic acid unit, optionally further comprising at least one linker unit between the polymerase protein unit and the nucleic acid unit, wherein the nucleic acid unit is coupled (a) by a covalent chemical bond or by streptavidin-biotin; and/or (b) by its terminal nucleotide.
• activatable DNA polymerase of any one of clauses 1 to 3 wherein the activatable DNA polymerase comprises a cleavable site wherein upon cleavage at least a part of the nucleic acid unit is removed.
• the activatable DNA polymerase of any one of clauses 1 to 11 wherein the nucleic acid unit has a length of at least about 5 nucleotides and/or wherein the nucleic acid unit is an oligonucleotide.
• the activatable DNA polymerase of clause 17 wherein the peptide linker has a length of about 1 to about 30 amino acid residues, preferably about 2 to about 20 amino acid residues, more preferably about 3 to about 17 amino acid residues.
• DBCO dibenzocyclooctyne
• Method of synthesizing a nucleic acid comprising: a) providing a sample comprising at least one nucleic acid template, components for nucleic acid synthesis and the activatable DNA polymerase of any one of clauses 1 to 30, b) activating the activatable DNA polymerase in the sample of step a), thereby generating an activated DNA polymerase, and c) subjecting the sample comprising the activated DNA polymerase of step b) to reaction conditions that allow synthesis of a nucleic acid complementary to the at least one nucleic acid template.
• Method of polymerase-chain reaction comprising: a) providing a sample comprising at least one nucleic acid template, components for nucleic acid amplification and the activatable DNA polymerase of any one of clauses 1 to 30, preferably wherein the DNA polymerase protein unit is selected from the group consisting of Taq and Pfu, more preferably of Taq comprising the amino acid sequence of SEQ ID NO: 7 and Pfu comprising the amino acid sequence of SEQ ID NO: 8, b) activating the activatable DNA polymerase in the sample of step a), thereby generating an activated DNA polymerase, and c) subjecting the sample with the activated DNA polymerase of step b) to reaction conditions that allow amplification of the at least one nucleic acid template.
• Method of nucleic acid sequencing comprising: a) providing a sample comprising a nucleic acid to be sequenced, components for nucleic acid sequencing and the activatable DNA polymerase of any one of clauses 1 to 30, preferably wherein the DNA polymerase protein unit is Phi29, more preferably comprising the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 34 or Klenow fragment, more preferably comprising the amino acid sequence of SEQ ID NO: 9, b) activating the activatable DNA polymerase in the sample of step a), thereby generating an activated DNA polymerase, and c) subjecting the sample with the activated DNA polymerase of step b) to reaction conditions that allow sequencing of the nucleic acid to be sequenced.
• the DNA polymerase protein unit is Phi29, more preferably comprising the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 34 or Klenow fragment, more preferably comprising the amino acid sequence of SEQ ID NO: 9, b) activating the activatable DNA
• Method of rolling circle amplification comprising: a) providing a sample comprising at least one circular nucleic acid template, components for nucleic acid synthesis and the activatable DNA polymerase of any one of clauses 1 to 30, preferably wherein the DNA polymerase protein unit is Phi29, more preferably comprising the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 34, b) activating the activatable DNA polymerase in the sample of step a), thereby generating an activated DNA polymerase, and c) subjecting the sample with the activated DNA polymerase of step b) to reaction conditions that allow amplification of the circular nucleic acid template.
• the activatable DNA polymerase comprises a cleavable site wherein upon cleavage at least a part of the single stranded nucleic acid unit is removed and wherein the cleavable site is a photocleavable site and activation in step b) comprises cleavage of at least a part of the nucleic acid unit of the activatable DNA polymerase by irradiation with light, preferably wherein the photocleavable site is an o-nitrobenzyl based photocleavable site and/or the light has a wavelength between about 300 nm to about 400 nm, preferably between about 310 nm to about 370 nm, more preferably between about 315 nm to about 365 nm, most preferably at about 315 or at about 365 nm.
• Method of determining the presence or absence of a single stranded nucleic acid of interest comprising: a) providing a sample to be tested for comprising a single stranded nucleic acid of interest, b) adding the activatable DNA polymerase according to any one of clauses 1 to 30 to the sample of step a), wherein at least a part of the nucleic acid unit is single stranded and complementary to the single stranded nucleic acid of interest,
• step c) subjecting the sample with the activatable DNA polymerase of step b) to reaction conditions which allow hybridization, preferably hybridization between the single stranded part of the nucleic acid unit of the activatable DNA polymerase unit and the single stranded nucleic acid of interest thereby activating the activatable DNA polymerase of the invention, and d) determining the activity of the activatable DNA polymerase of step c), wherein the activity of the activatable DNA polymerase indicates the presence of the single stranded nucleic acid of interest in the sample.
• the activatable DNA polymerase is Phi29, more preferably comprising the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 34.
• step b) comprises incubation at an about constant temperature, preferably at temperature in a range between about 28°C to about 40°C, preferably between about 30°C to about 37°C.
• determination of activity of the activatable DNA polymerase in step (d) comprises amplification of a nucleic acid for detection and detection of said amplified nucleic acid for detection using a labeled probe, e.g., a fluorescence-labeled probe, preferably comprising rolling circle amplification of a circular primed template thereby generating an amplification product, hybridizing a labeled probe, e.g., fluorescence-labeled, to the amplification product, and measuring a signal generated by the label.
• a labeled probe e.g., a fluorescence-labeled probe
• the single stranded nucleic acid of interest is selected from the group consisting of a microRNA, a viral or bacterial nucleic acid, and a nucleic acid which is indicative of a disease or a disease state.
• a nucleic acid for reversibly inhibiting a DNA polymerase wherein the nucleic acid is coupled (a) by a covalent chemical bond or by streptavidin-biotin; and/or (b) by its terminal nucleotide to the DNA polymerase.
• Claus 41 wherein the nucleic acid has a length which is sufficient to block activity of the DNA polymerase.
• activatable DNA polymerase of any one of clauses 1 to 30 in an in vitro nucleic acid synthesis process, preferably wherein the activatable DNA polymerase comprises a cleavable site wherein upon cleavage at least a part of the nucleic acid unit is removed and wherein the cleavable site is a photocleavable site.
• synthesizing a nucleic acid or the nucleic acid synthesis process is selected from the group consisting of sequencing, preferably whole genome sequencing; and a nucleic acid amplification process, preferably PCR, RT-PCR, qPCR, rolling circle amplification, whole genome amplification, or isothermal amplification such as loop-mediated isothermal amplification (LAMP).
• a nucleic acid amplification process preferably PCR, RT-PCR, qPCR, rolling circle amplification, whole genome amplification, or isothermal amplification such as loop-mediated isothermal amplification (LAMP).
• the activatable DNA polymerase comprises a photocleavable site wherein upon cleavage at least a part of the nucleic acid unit is removed, (preferably wherein the photocleavable site is an o-nitrobenzyl- based photocleavable site) and wherein the activatable DNA polymerase is activated by cleavage of at least a part of the nucleic acid unit of the activatable DNA polymerase, preferably wherein the cleavage is by irradiation with light, more preferably wherein the photocleavable site is an o-nitrobenzyl based photocleavable site and/or the light has a wavelength between about 300 nm to about 400 nm, preferably between about 310 nm to about 370 nm, more preferably between about 315 nm to about 365 nm, most preferably at about 315 or at about 365 nm.
• any one of clauses 1 to 30 for in vitro diagnosis, such as diagnosis of a disease, a medical condition and/or detection of pathogens in a sample such as from a human or an animal (e.g., mammal or bird), food, drink, soil or water (e.g., a drinking water, a waste water or a hydrologic sample).
• a human or an animal e.g., mammal or bird
• food, drink e.g., a drinking water, a waste water or a hydrologic sample.
• the activatable DNA polymerase is activated by exposure of an at least partially single stranded nucleic acid unit of the activatable DNA polymerase to a single stranded nucleic acid of interest, wherein the partially single stranded nucleic acid unit is sufficiently complementary to the single stranded nucleic acid of interest to hybridize to the single stranded nucleic acid of interest.
• the single stranded nucleic acid of interest is selected from a microRNA, a viral or bacterial nucleic acid, and a nucleic acid which is indicative of a disease or a disease state.
• An apparatus for performing a polymerase-chain reaction (PCR) comprising: a) means, in particular a thermal block, adapted for receiving at least one container suitable for the uptake of a sample comprising at least one nucleic acid template, components for nucleic acid amplification and the activatable DNA polymerase of any one of clauses 1 -30, preferably wherein the DNA polymerase protein unit is selected from the group consisting of Taq and Pfu, more preferably of Taq comprising the amino acid sequence of SEQ ID NO: 7 and Pfu comprising the amino acid sequence of SEQ ID NO: 8, b) at least one light source, in particular at least one LED, emitting light in a wavelength suitable for activating the activatable DNA polymerase in the at least one container of item a), thereby generating an activated DNA polymerase in the at least one container,
• Binding of the ssDNA to the DNA polymerases was achieved by site-specific incorporation of the unnatural amino acid 4-Azido-L-phenylalanine (azPhenylalanine).
• azPhenylalanine 4-Azido-L-phenylalanine
• TGA amber codon suppression
• the Taq and Pfu constructs were subcloned in the pET24-d plasmid (Novagen, Merck Millipore, Germany) for expression.
• the flanking Ncol and Xhol restriction sites were introduced in the Phi29 pol by PCR and finally subcloned in the expression vector pET21 -a (Novagen, Merck Millipore). Standard procedures for molecular cloning were used [3] and the E. coli strain XLI blue was used for all cloning steps.
• Enzymes for cloning were purchased from NE Biolabs (MA, USA).
• the resulting DNA polymerase protein units coupled to the linker unit have the sequences of SEQ ID NOs: 31 , 32 and 33.
• the inventors used the E. coli BI21 star (DE3) strain transformed with the plasmid pEVOL-pAzF (a gift from Prof. Peter Schultz, Addgene plasmid #31186) for incorporation of the unnatural amino acid 4-Azido-L-phenylalanine in the recombinant proteins.
• pEVOL-pAzF E. coli cells co-transformed with the expression plasmids were grown with vigorous shaking at 37 °C in LB medium with the adequate antibiotics until they reached an QD600 of « 1 .
• the cells were pelleted, washed with M9 minimal medium, and resuspended in M9 minimal medium supplemented with 0.2 mg/mL 4-azido-L-phenylalanine (Hycultec GmbH, Germany), 0.02% arabinose and antibiotics.
• the culture was incubated at 37 °C degree for one hour and finally induced with 1 mM IPTG overnight at 16°C.
• Cell pellets were first frozen at -80 °C and resuspended in 50 mM phosphate buffer pH 7.4, 500 mM NaCI, 50 mM Imidazole and 10% glycerol. Cells were lysed in this buffer supplemented with 1 mg/ml lysozyme, 5 pg/ml DNase I, 5 pg/ml RNase A and 1 % Triton® X-100 for 1 hour on ice. In the case of Taq and Pfu pol samples the lysate was heated to 75 °C for 20 min after the lysis to yield samples free of DNA contamination from E. coli.
• Proteins were purified by Ni 2+ -affinity chromatography using Histrap HP columns (GE Healthcare, USA) on an FPLC apparatus (AKTA Purifier, GE Healthcare) and eluted from the column in 50 mM phosphate buffer pH 7.4, 500 mM NaCI, 500 mM Imidazole buffer. All three enzymes were further purified by ion-exchange chromatography. Phi29 and Pfu pols were purified by cationic exchange in a HiScreen HP SP column (GE Healthcare), and Taq pol by anionic exchange in a HiScreen Q SP column (GE Healthcare). The purity of the proteins was higher than 95% as assessed by Coomassie staining in SDS-PAGE gels. The molar concentration was estimated spectrophotometrically using the theoretical extinction coefficient at 280 nm.
• DBCO was included in the 3' of the ssDNA in the case of Phi29 and Pfu pol (3' to 5' exonuclease activity) and the PC linker located before the last 3' terminal nucleotide (see table 1 ). In the case of Taq pol (5' to 3' exonuclease activity), DBCO was located at the 5' and the PC group between the first 5' nucleotide and the second one.
• Table 1 summarizes the details of the DBCO-modified oligos used and the DNA polymerase to which they were attached.
• Table 1 Sequence for the DBCO modified oligonucleotides based on SEQ ID NOs: 1 (ttcctctaccacctacatcac), 2 (cttcatcacactccatctcca) and 3 (gcattacgtttggtggaccct).
• the asterisk (*) indicates the position of the photocleavable linker.
• the DNA polymerases in the elution buffer from the ionic exchange were incubated for at least 3 hours with the DBCO-modified oligos at a molar ratio of 1 :1 ,3 or 1 :1 ,5 in light-tight tubes at room temperature. Afterwards, the samples were purified by ionic exchange, using a cationic exchange column followed by an anionic exchange column. The different charges of free polymerases, polymerase-ssDNA, and free oligos allowed for efficient separation with this purification scheme.
• the Phi29 and Taq pols samples were stored in 10 mM Tris-HCI, 100 mM KCI, 0.1 mM EDTA, 1 mM DTT, 0.5% Tween-20, 0.5 % IGEPAL CA-630 and 50 % glycerol.
• Pfu pol samples were stored in 25 mM Tris, 0.1 mM EDTA, 1 mM DTT, 0.5% Tween-20, 0.5 % IGEPAL CA- 630 and 50 % glycerol. All enzymes were stored at - 20 °C.
• the inventors estimated the concentration of the DNA polymerase-oligo samples building calibration curves using equimolar mixtures of purified polymerase and pure DBCO-oligo. For each polymerase-oligo construct the unmodified polymerase was mixed with the respective DBCO oligo at a molar ratio of 1 :1 , and the absorbance at 260 and 280 nm measured for several dilutions of this sample. As the concentration of the polymerase in the sample was known, the inventors performed a linear fit of absorbance vs enzyme concentration. Then, the absorbance at 260 and 280 nm of the polymerase-oligo samples were used to interpolate their concentration using the linear fits. Finally, the mean value of the estimated concentration at 260 and 280 nm was used as the sample concentration.
• UV pulses were applied using either a 45-watt 315 nm UV-Pad (Vilber, France) or a handheld 6-watt 365 nm lamp (Analytikjena GmbH, Germany). The samples were illuminated with UV light just before the polymerization or digestion reaction were started. 1.7 Multiply-primed amplification experiments
• the inventors used multiply-primed amplification [20] to test the activity of the Phi29 polymerase samples.
• the inventors amplified the T7 blue plasmid (Novagen, Merck Millipore) for the experiments in Figure 2 and 5a, and human genomic DNA (Roche Diagnostics, Germany) for the experiments in Figure 4a and 5b. Random hexamers (Thermofisher Scientific, USA) were first mixed with the DNA template, annealed at 95 °C for 3 min, and then cooled down for 5 min on ice.
• PCRs were carried out in 50 mM Tris-HCI, 10 mM KCI, 10 mM (NH4)2SO4, 2mM MgSO4, 0.1 % Triton® X-100, 0.1 mg/ml BSA pH 10.2 in a total volume of 25 pl.
• a plasmid carrying a codon-optimized version of the human Cyclophilin A gene (PCRs in figure 3b) and E.coli genomic DNA were used (PCRs in figures 4b, 4c, 5c and 5d).
• E.coli genomic material was prepared from 5 ml overnight saturated culture of XLI blue cells. The culture was centrifuged, and the pellet washed out twice with water.
• the cells were resuspended in 200 pl of molecular biology grade water and boiled at 99 °C for 5 min, afterwards the sample was centrifuged for 2 min at 15.000 g and the supernatant with the chromosomic DNA transferred to a fresh tube. This chromosomic sample was diluted 10 times and 0.5 pl was used for the PCRs.
• the inventors used 0.25 ng of the plasmid as template.
• Exonuclease tests for Pfu samples were performed likewise but in 20 mM Tris-HCI, 10mM KCI, 10mM (NH 4 )2SO4, 2mM MgSQ 4 , 0.1 % Triton® X-100 and 0.1 mg/ml BSA pH 8.8 at 45°C for 10 minutes.
• the 3' to 5' exonuclease proofreading activity of Pfu was probed using a dye-labeled dsDNA with a 5 bases mismatch at the 3' of the labeled oligonucleotide (FAM exo 3' reverse + Exo 3' mismatch forward, see table 4).
• the dye-labeled dsDNA was annealed forehand using a temperature gradient, and the reaction took place using 5 nM of enzyme and 50 nM of dsDNA at 45°C for 1 minute.
• the cleavage of single-stranded arms at the bifurcated end of base-paired duplexes by Taq pol (5'flap endonuclease activity) provides a convenient way to test the nuclease activity of this enzyme [21], A bifurcated junction was used as substrate, and a primer located 4 bases upstream of the bifurcation point was included as it is known to promote the nuclease activity of the enzyme [21 ].
• the oligonucleotide that forms the 5' protruding strand was labeled at its 3' with a fluorophore (see table 4), and the forklike structure assembled in a temperature gradient (Exo 5' taq fork FAM + Exo 5' taq template + Exo 5' taq 4pb gap, see table 4).
• the reaction took place for 20 min at 45 °C using 50 nM of enzyme and 50 nM of the substrate in 20mM Tris-HCI, 10 mM KCI, 10 mM (NH 4 ) 2 SO4, 2mM MgSO 4 , 0.1 % Triton® X-100 pH 8.8.
• the inventors covalently attached the oligo to the DNA polymerase using click chemistry [9], incorporating the unnatural amino acid 4-azido-L-phenylalanine [10] as in the enzyme and reacting with a DBCO-modified oligonucleotide.
• the unnatural amino acid was incorporated in an additional stretch of glycine-serine residues added at the c-terminus of the enzyme.
• the inventors reasoned that having the substrate of the DNA polymerase (DNA) attached to it, could produce the effective blockage of the enzyme by means of binding to the protein's cleft and competing for the accessibility to the active site (either obstructing the access to the active site or directly competing for it). In this case, the inactivation of the enzyme would be reversible, and controlled cleavage of the oligonucleotide would result in enzymatic reactivation (see Figure 1 ).
• an o-nitrobenzyl-based photocleavable (PC) linker (see Material and Methods section for details) between the first and the second nucleotide proximal to the anchoring point of the enzyme (see Figure 1 ).
• a short UV pulse will release the oligonucleotide from the enzyme and reactivate it.
• an SDS-PAGE gel shows the effect of UV pulses of different duration on the light-sensitive enzyme-oligo complex.
• a band corresponding to unmodified enzyme appeared in the irradiated samples (see lanes 3- 5 in Figure 2a), and as expected the proportion of this population correlated positively with duration of the light pulse.
• Phi29 pol bound to a regular oligo (Phi29 pol-oligo) and non-irradiated Phi29 pol attached to a PC oligo (Phi29 pol- PC_oligo) showed no detectable activity after two hours of reaction ( Figure 2b, lanes 2 and 4), as opposed to unmodified Phi29 pol enzyme, where an intense DNA band was observed in the agarose gel due to DNA polymerization (lane 1 ). This assures that enzymatic inhibition was also achieved for the PC variant.
• the Phi29 pol-PC_oligo samples that were irradiated recovered the enzymatic activity, and this reactivation was correlated with the intensity and duration of the light pulse ( Figure 2b, lanes 5-9). This reactivation was not observed in irradiated Phi29 pol_oligo (lane 3), confirming that the reactivation is specific to oligonucleotide cleavage.
• the inventors achieved a tight blockage of the enzymatic activity.
• the inventors did not detect residual activity in the non-illuminated samples, as there was no statistical difference (p ⁇ 0.01 ) with samples that are not able to polymerize (samples without dNTPs, see Figure 2e).
• Even experiments performed at high concentration of the enzyme (150 nM) did not show activity in the nonilluminated samples (see figure 5a), further confirming a severe inactivation of the enzyme.
• the inventors used a scrambled version (having the sequence of SEQ ID NO: 2) of the blocking oligonucleotide (having the sequence of SEQ ID NO: 1 ) used above, which has the same nucleobase composition but in a random order (Phi29 pol-PC_oligoScr, see Table 1 for details).
• This Phi29 pol-PC_oligoScr version also inhibited activity that was recovered with a light pulse ( Figure 2f, left).
• the inventors blocked the enzyme with a third oligonucleotide (having the sequence of SEQ ID NO: 3) with a completely different sequence (Phi29 pol_oligo2, Table 1 for details).
• This third type of modified Phi29 pol still displayed the same light-activated behavior ( Figure 2f, right). Similar results were also obtained with longer sequences, e.g., ttcctctaccacctacatcactcttct (SEQ ID NO: 4) and ttcctctaccacctacatcactcttctcattac (SEQ ID NO: 5) (data not shown).
• the proposed inhibition mechanism would also provide blockage of the 3’ to 5’ exonuclease (3'-5' exo) activity of the Phi29 pol, as the oligonucleotide bound to the enzyme might also successfully compete with other exonuclease substrates provided it still hampers the access to the active site.
• the inventors devised a test to characterize the 3'-5' exo activity of the Phi29 pol constructs using a 5’ fluorophore- labeled (6-Carboxyfluorescein, 6-FAM) single stranded DNA probe (FAM-labeled ssDNA).
• the position of the modification was not rationally designed, the inventors hypothesized that the same effect might be observed for other DNA polymerases as long as the oligonucleotide has enough flexibility to reach the active sites. Therefore, the inventors implemented the same strategy in two other DNA polymerases widely used in biotechnology, the Taq and the Pfu DNA polymerases (for a discussion on how exonuclease degradation of the blocking oligo was avoided see Material and Methods, section 1.4 Protein-oligo coupling).
• the Taq pol is the workhorse for PCR applications in all laboratories around the world and Pfu pol is a classical low error rate polymerase for application where high fidelity is desired [2b, 2c, 4b],
• the Pfu pol has 3’ to 5’ exo activity, which includes proofreading activity [2b, 2c] (3’ degradation in double stranded DNA, dsDNA, with 3’ terminal mismatches).
• Taq DNA pol possesses 5’ nuclease activity, including 5’ to 3’ exonuclease and 5’ flap nuclease activity [2c, 12],
• the nuclease activity of the Taq pol-PC_oligo was inhibited until the samples were photo-activated ( Figure 3d, see lanes 3 and 4 and comparison with the wild type enzyme in lane 1 ). Altogether, the results show that not only the polymerase activity was blocked in the enzymes, but also the nuclease activity.
• the data presented herein suggest that the inactivation of the enzymes is caused by obstructed access to the active site (or direct competition for it) by the oligonucleotide attached to the enzyme. It was hypothesized that a similar re-activation effect would be observed upon hybridization of a complementary oligonucleotide with the blocking ssDNA, as the rigidification derived from the transition from single to double stranded DNA could lead to hampered accessibility of the blocking DNA to the enzymatic active center. Indeed, the inventors observed such an effect when incubating the Phi29 pol-oligo with the complementary oligo, an effect that is dependent on the concentration of the latter (Fig 6a).
• the reactivation of the enzyme is sequence-specific, as the incubation with an oligo with different sequence (mocking oligo) does not produce a reactivation of the enzyme (Fig 6a).
• the enzymatic blocking mechanism is unspecific and can be attained by virtually any oligonucleotide sequence, the results show that the blocked enzyme can be used as a sensing device, where the blocking ssDNA is the sensing element and the polymerase the amplification unit.
• the inventive enzymes can be a used to develop a general approach for nucleic acid detection and the basis for a new molecular detection method for use in diagnostics.
• RNA sequences can also trigger the reactivation of the enzyme (see Figure 6b, left gel), showing therefore applications in the detection of, e.g., microRNA (small regulatory RNAs, related to ageing, neurodegeneration, and cancer and potentially useful as non- invasive biomarkers [16] ).
• microRNA small regulatory RNAs, related to ageing, neurodegeneration, and cancer and potentially useful as non- invasive biomarkers [16] ).
• the inventors also included base modifications in the blocking oligonucleotide bound to the enzyme and the activity of the enzyme remained reversibly blocked (see Figure 6b, right gel). Specifically, they substituted one of the bases for an abasic site.
• Example 3 the inventors showed that a DNA polymerase whose activity is blocked by a nucleic acid can be re-activated upon hybridization of a complementary oligonucleotide with the blocking nucleic acid. Based on this finding, the inventors propose a novel strategy for molecular detection (point of care, POC) of single stranded nucleic acids of interest such as miRNAs using, e.g., an activatable Phi29 DNA polymerase (Phi29 pol) in accordance with the invention, which is inactive and recovers activity only after the recognition of a single stranded nucleic acid of interest, e.g., a specific small nucleic acid (RNA or DNA, Fig 6a) and 6b), left gel).
• POC point of care
• Phi29 pol Due to its strand displacement activity, the Phi29 pol does not require thermal cycling to achieve amplification reactions, besides, it performs optimally at about 30 °C and when combined with rolling-circle amplification (RCA) can produce excellent signal amplifications in short times. Phi29 pol is therefore ideal to develop POC applications.
• the strategy proposed here is not limited to Phi29 polymerase. Unlike previous RCA-Phi29 pol assays, in the inventive approach the enzyme itself is the sensor. Phi29 pol is covalently modified with an at least partially single stranded DNA nucleic acid unit as described herein.
• the single stranded part of the DNA nucleic acid unit is complementary to the single stranded nucleic acid of interest, e.g., a target RNA sequence of interest, and after binding the enzyme recovers the activity due the rigidification associated with the transition from single to double strand (which hampers its accessibility to the active site).
• This initial activation (step 1 , Fig 8) is coupled to an RCA assay, producing thousands of DNA copies of the single stranded nucleic acid of interest, e.g., a target activator sequence (Step 2, Fig 8), and triggering a second wave of enzymatic activation (Step 3, Fig 8).
• the newly activated enzymes can repeat the process and an exponential activation of polymerases occurs (Step 4, Fig 8).
• the signal is further amplified by a nicking enzyme digesting of a fluorophore-quencher oligonucleotide complementary to the copied RCA template. Owing to the nicking enzyme, every copy of the template can participate in several rounds of fluorophore- quencher activation, producing a fluorescence signal.
• the assay can be implemented in a lateral flow strip format and read-out using commercially available fluorescence lateral flow readers [34], A restriction enzyme site is also included in the RCA template. Using an oligo complementary to the restriction enzyme site in the copied template, the enzymatic digestion would result in multiple smaller fragments of the latter, thus increasing diffusivity and speeding up the exponential activation of the polymerases.
• the restriction sites in the template can be methylated and methylation-sensitive enzymes used (such as Stul and Nt BsmAI, as restriction and nicking enzymes respectively).
• Possible competition for the target sequence by the RCA template (as it bears a complementary sequence to the activator RNA) can be avoided by keeping the molar ratio template/Phi29 pol low.
• Phi DNA polymerase with N59D (SEQ ID NO: 6)
• Taq DNA polymerase (SEQ ID NO: 7)
• Pfu DNA polymerase (SEQ ID NO: 8)
• Bst DNA polymerase (Geobacillus thermodenitrificans strain NG80-2) (SEQ ID NO: 10)
• Bir A gene (SEQ ID NO: 11 )
• Bir A gene forward primer (SEQ ID NO: 21 )
• Bir A gene reverse primer (SEQ ID NO: 22)
• Phi29 DNA polymerase N59D with C-terminal linker SEQ ID NO:31

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