DNA polymerase inhibitor

Abstract

Described herein are aptamers for temperature-dependent reversible inhibition of thermostable polymerase activity in order to improve sensitivity and specificity of various reactions and assays involving hot start polynucleotide synthesis. Methods for use of the aptamers and related compositions and kits are also provided.

Description

DNA POLYMERASE INHIBITORCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is being filed on December 19, 2025, as a PCT International Patent Application and claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 737,343, filed on December 20, 2024, the disclosure of which is hereby incorporated by reference in its entirety.FIELD

[0002] The methods and compositions described herein relate generally to the area of nucleic acid amplification. In particular, described herein are nucleic acid inhibitors of thermostable polymerase activity, compositions comprising the inhibitors, and methods of their use. The nucleic acid inhibitors can adapt a secondary structure to reversibly inhibit thermostable polymerases, wherein the inhibitory activity is temperature dependent. The inhibitors are useful for methods and assays which include nucleic acid synthesis by the thermostable polymerase wherein the methods and assays benefit from utilizing a hot start method.BACKGROUND

[0003] In vitro synthesis of nucleic acid target sequences, the foundation of numerous research assays and diagnostic products, often relies in part on the use of thermostable DNA polymerases and at least one oligonucleotide primer which is designed to specifically bind to a target nucleic acid substrate in a sample suspected of containing the target. While assays using the polymerase and primer(s) are designed to generate a specific sequence, it is well known that if the assay requires a period of time at a lower or ambient temperature, the target-specific primers may hybridize to non-target sequences or may form primer dimers, resulting in mispriming and the subsequent generation of non-specific products. Such products are undesirable as they can mask the product of interest as well as prematurely deplete the reaction mixture of necessary reagents.

[0004] A primary means for reducing the effects of mispriming is to reversibly inhibit the thermostable polymerase activity at lower temperatures where such mispriming is more likely to occur. Upon increasing the temperature of the polymerase reaction mix to temperatures approximating the optimal reaction temperature of the thermostablepolymerase, the inhibitory activity is removed, and the thermostable polymerase extends primer(s) bound to substrate nucleic acid molecules. This method of reversibly inhibiting a thermophilic DNA polymerase to prevent primer extension at lower temperatures is referred to as "hot-start."

[0005] Many hot-start methods have been developed to avoid incorrect primer extension products (e.g., see Paul, N., et al. (2010), for review). One of the most common techniques is based on use of oligonucleotide aptamers (Jayasena S. D., 1999). Aptamers offer several advantages over other reported methods. Using a method of molecular evolution (SELEX), they can be quickly engineered in a test tube and then readily and inexpensively manufactured by chemical synthesis. Ideally, an aptamer should: (i) completely block DNA polymerase at low temperatures, and (ii) provide no blockage effect at the desired elevated reaction temperature. Unfortunately, this is very difficult to achieve, and the aptamer structure usually represents a compromise between these two key requirements. New aptamer compositions and methods, therefore, are needed to improve control of aptamer activity in reaction mixtures containing DNA polymerases.

[0006] All references cited herein are incorporated herein by reference in their entireties.BRIEF SUMMARY

[0007] There are two common designs of DNA aptamers for hot-start enzymes:Dumbbell and Hairpin. DNA aptamer binds to an active site of Taq polymerase domain. There is a need to find new factors of DNA aptamer affinity, design DNA aptamer with higher affinity to TaqPol, and destroy aptamer structure (affinity) completely or partially during first PCR cycles. Described herein are aptamers which can form a secondary structure, reduce generation of non-specific polymerase products in various assays which rely on thermostable polymerase activity, but which can dissociate from the polymerase to facilitate target product generation and detection. The aptamer structures disclosed herein include shifted location of the kink to form a substantially asymmetric structure, unique location of one or more propynyl modified dUs (lT), and a GC bp in the stem. Hot-start properties have been shown to increase by at least 12°C compared to dumbbell aptamers described in US Pat. No. 11,299,739. Hot-start properties of dU modified aptamer can be almost completely eliminated by 1min pretreatment with Uracil-DNA glycosylase (UDG) at PCR temperatures. Hot-start properties of CAT-A aptamer can be improved with incorporations of propynyl dUs.

[0008] In some embodiments of the present disclosure, aptamer comprising in a 5' to 3' direction: a 5' terminus sequence at least three nucleotides in length, a first stem sequence comprising SEQ ID NO.:1 or a variant thereof or SEQ ID NO.: 2 or a variant thereof, a loop sequence comprising SEQ ID NO.: 3 or a variant thereof, a second stem sequence comprising SEQ ID NO.:1 or a variant thereof or SEQ ID NO.:2 or a variant thereof, and a 3' terminus sequence at least three nucleotides in length, wherein each of the first and second stem sequences are 100% complementary to each other along their entire length, and wherein the aptamer comprises at least one unnatural nucleotide substitution, wherein the unnatural nucleotide substitution comprises a hydrophobically modified base, are disclosed. The hydrophobically modified base can be a modified uridine base such as a propynyl modification. In some examples, the aptamer comprises 1, 2, 3, or 4 hydrophobically modified bases.

[0009] The 5' terminus sequence and 3' terminus sequence of the aptamer can comprise SEQ ID NO.:4, SEQ ID NO.:5, or a combination thereof. In some embodiments, the 5' terminus sequence and / or 3' terminus sequence can comprise 1, 2, 3, or 4 hydrophobically modified uridine bases. In some examples, the 5' terminus sequence is not complementary or equal in length to the 3' terminus sequence. The first and second stem sequences of the aptamer are each 14-18 nucleotides in length. In some embodiments, the first stem sequence comprises T and G bases near or at its 5' end. In some examples, the first stem sequence consists essentially of SEQ ID NO.: 1 and the second stem sequence consists essentially of SEQ ID NO.:2. The loop sequence of the aptamer is 10-14 nucleotides in length. In some embodiments, the loop sequence comprises 1, 2, 3, or 4 hydrophobically modified uridine bases.

[0010] The aptamer can further comprise a 5' and / or a 3' capping moiety. In some examples, the 3' terminus is linked to a 3' capping moiety. In some examples, the 5' terminus is linked to a 5' capping moiety. In some examples, the 3' terminus is linked to a 3' capping moiety and the 5' terminus is linked to a 5' capping moiety. The capping moiety can include a propanediol spacer (C3), a phosphate, or an amino group.

[0011] The aptamers provided herein can be modified for activating the aptamer-deactivated DNA polymerases. In this embodiment, the aptamer can be modified by uracil-DNA glycosylase enzymatic activity to reduce or eliminate binding of the aptamer to the DNA polymerase, thereby activating DNA synthesis activity of theDNA polymerase in a reaction mixture. In some aspects, the aptamers comprise one or more deoxyuridine nucleotides providing for aptamer-specific recognition and modification of the aptamer by the uracil-DNA glycosylase enzymatic activity. In some examples, the aptamer comprises 1, 2, 3, or 4 deoxy uridine nucleotides. For example, the 5' terminus sequence and / or 3' terminus sequence comprises 1, 2, 3, or 4 nucleotide substitutions having deoxyuridine nucleotides. In other examples, the loop sequence comprises 1, 2, 3, or 4 nucleotide substitutions having deoxyuridine nucleotides.

[0012] The aptamer disclosed herein can comprise any one or more of SEQ ID NO.:55-70. In some embodiments, the aptamer comprises a 5' end and a 3' end, wherein there is no covalent bond between the 5' terminus sequence and / or 3' terminus sequence and as such, the aptamer preferably has a hairpin structure. The aptamer preferably does not inhibit reverse transcriptase activity.

[0013] Compositions comprising the aptamer disclosed herein and a thermostable polymerase are disclosed. The polymerase can be selected from the group consisting of Thermits aquaticus, Thermus thermophilus, or Thermus maritima. The composition can further comprise deoxyribonucleotide triphosphates, a divalent metal cation, a forward primer specific for a target nucleic acid, and a reverse primer specific for the target nucleic acid. The ratio of the aptamer to the polymerase is >1: 1 (such as from about 2: 1 to 20:1, from about 2:1 to 15:1, from about 3:1 to 15:1, or from about 5:1 to 20:1). In some embodiments, the composition is a dried composition. In other embodiments, the composition is a liquid composition.

[0014] Methods for extending a primer, comprising contacting a sample that may or may not comprise a target nucleic acid w ith a reagent composition to generate a reaction mixture, wherein the reagent composition comprises an aptamer disclosed herein, a thermostable DNA polymerase, a forward primer specific for the target nucleic acid, deoxyribonucleotide triphosphates, and a divalent metal cation, are disclosed. Methods for amplifying a target DNA sequence, the method comprising (a) providing a reaction mixture comprising: (i) a thermostable DNA polymerase, (ii) an aptamer disclosed herein, wherein the aptamer binds to the thermostable DNA polymerase to form a blocked thermostable DNA polymerase, (iii) at least one oligonucleotide primer, and (iv) one or more sample nucleic acids that may or may not comprise a target sequence complementary to the at least one oligonucleotide primer; (b) hybridizing the at least one oligonucleotide primer to the sample nucleic acids that comprise a target DNA sequence complementary to the at least one oligonucleotideprimer; (c) elevating the temperature to release the aptamer from the blocked thermostable DNA polymerase; and (d) initiating DNA polymerase activity and extending the primer with the DNA polymerase, are disclosed.

[0015] The elevated temperature to release the aptamer from the blocked thermostable DNA polymerase is greater than 45°C such as from 55-65°C. Initiating DNA polymerase activity and extending the primer with the DNA polymerase can be performed at a temperature of greater than 65°C. The reaction mixture can further comprise a buffer mixture and dNTPs. The buffer can comprise 10 mM Tris-HCl (pH 8.4 at 25° C), 1.5 mM MgCl₂, 110 mM KCl, 0.08% (w / v) Brij-58, 0.2% (w / v) PEG-8000, and 0.1% (v / v) propylene glycol); and the dNTPs comprise 0.8 mM dNTPs. The reaction mixture can further comprise a reverse primer specific for the target nucleic acid, a reverse transcriptase and wherein the method further comprises performing a reverse transcriptase reaction prior to heating the reaction mixture.

[0016] Kits comprising a composition comprising the aptamer and a thermostable polymerase are disclosed. The kits can further comprise deoxyribonucleotide triphosphates and a divalent metal cation.BRIEF DESCRIPTION OF SEVERAL VIEWS OF THE DRAWINGS

[0017] The invention will be described in conjunction with the following drawings in which like reference numerals designate like elements and wherein:

[0018] FIG. 1 is a graph comparing PCR melt curve analysis of real time PCR reactions in the presence of dumbbell, symmetrical, or asymmetrical hairpin aptamer according to an embodiment of the present disclosure. All figures describe enzyme activity at slowly rising temperature. The activity was measured using a specially designed substrate with two hairpins. The first hairpin with an open 3' end can initiate extension by active enzyme and eventually cleave 5' FAM located on opposite hairpin structure. The cleavage results in rising FAM signal. At lower temperatures the aptamer holds the enzyme and makes it inactive, that is no enzyme activity so no nsing signal. As the aptamer loses affinity at higher temperature, the signal rises. The activity of the aptamers at higher temperature varies depending on the specific aptamer. Higher affinity aptamers exhibit rising FAM signal detected at higher temperature.

[0019] FIG. 2 is a graph comparing PCR melt curve analysis of real time PCR reactions in the presence of unmodified hairpin aptamer and propynyl dU incorporatedin loop sequence of hairpin aptamer according to an embodiment of the present disclosure.

[0020] FIG. 3 is a graph comparing PCR melt curve analysis of real time PCR reactions in the presence of unmodified hairpin aptamer and two propynyl dUs incorporated in loop sequence of hairpin aptamer according to an embodiment of the present disclosure.

[0021] FIG. 4 is a graph comparing PCR melt curve analysis of real time PCR reactions in the presence of unmodified hairpin aptamer and two propynyl dUs in loop sequence as well as GC base pair in stem of hairpin aptamer according to an embodiment of the present disclosure.

[0022] FIG. 5 is a graph comparing PCR melt curve analysis of real time PCR reactions in the presence of unmodified hairpin aptamer and dU(s) in loop or terminal arm sequences of hairpin aptamer according to an embodiment of the present disclosure. dU incorporation can improve hot-start, be neutral or reduce hot-start performance.

[0023] FIG. 6 is a graph comparing PCR melt curve analysis of real time PCR reactions in the presence of unmodified hairpin aptamer and dU(s) in loop or terminal arm sequences of hairpin aptamer according to an embodiment of the present disclosure. At some locations dU catalysis demonstrates very effective suppression of the hot-start. FIG. 6 discloses SEQ ID NO: 69.DETAILED DESCRIPTION

[0024] Various aspects now will be described more fully hereinafter. Such aspects may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey its scope to those skilled in the art.

[0025] Where a range of values is provided, it is intended that each intervening value between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the disclosure. For example, if a range of 1 % to 8% is stated, it is intended that 2%, 3%, 4%, 5%, 6%, and 7% are also explicitly disclosed, as well as the range of values greater than or equal to 1 % and the range of values less than or equal to 8%.I. Definitions

[0026] Terms used in the claims and specification are defined as set forth below unless otherwise specified.

[0027] The term “nucleic acid” refers to a nucleotide polymer, and unless otherwise limited, includes analogs of natural nucleotides that can function in a similar manner (e.g., hybridize) to naturally occurring nucleotides. The term “nucleic acid” encompasses multi -stranded, as well as single-stranded molecules. In double- or triplestranded nucleic acids, the nucleic acid strands need not be coextensive (i.e., a doublestranded nucleic acid need not be double-stranded along the entire length of both strands). Nucleic acid templates described herein may be any size depending on the sample (from small cell-free DNA fragments to entire genomes), including but not limited to 50-300 bases, 100-2000 bases, 100-750 bases, 170-500 bases, 100-5000 bases, 50-10,000 bases, or 50-2000 bases in length. In some instances, templates are at least 50, 100, 200, 500, 1000, 2000, 5000, 10,000, 20,000 50,000, 100,000, 200,000, 500,000, 1,000,000 or more than 1,000,000 bases in length. Methods described herein provide for the amplification of nucleic acids, such as nucleic acid templates. Methods described herein additionally provide for the generation of isolated and at least partially purified nucleic acids and libraries of nucleic acids. Nucleic acids include but are not limited to those comprising DNA, RNA, circular RNA, cfDNA (cell free DNA), cfRNA (cell free RNA), siRNA (small interfering RNA). cffDNA (cell free fetal DNA), mRNA, tRNA, rRNA, miRNA (microRNA), synthetic polynucleotides, polynucleotide analogues, any other nucleic acid consistent w ith the specification, or any combinations thereof. The length of polynucleotides, when provided, are described as the number of bases and abbreviated, such as nt (nucleotides), bp (bases), kb (kilobases), or Gb (gigabases). The term nucleic acid includes any form of DNA or RNA, including, for example, genomic DNA; complementary DNA (cDNA), which is a DNA representation of mRNA, usually obtained by reverse transcription of messenger RNA (mRNA) or by amplification; DNA molecules produced synthetically or by amplification; mRNA; and non-coding RNA. The term nucleic acid encompasses double- or triple-stranded nucleic acid complexes, as well as single-stranded molecules. In double- or triple-stranded nucleic acid complexes, the nucleic acid strands need not be coextensive (i.e, a double-stranded nucleic acid need not be double-stranded along the entire length of both strands).

[0028] The term nucleic acid also encompasses any modifications thereof, such as by methylation and / or by capping. Nucleic acid modifications can include addition of chemical groups that incorporate additional charge, polarizability, hydrogen bonding, electrostatic interaction, and functionality to the individual nucleic acid bases or to the nucleic acid as a whole. Such modifications may include base modifications such as 2'-position sugar modifications, 5-position pyrimidine modifications, 8-position purine modifications, modifications at cytosine exocyclic amines, substitutions of 5-bromo-uracil, sugar-phosphate backbone modifications, unusual base pairing combinations such as the isobases isocytidine and isoguanidine, and the like. More particularly, in some embodiments, nucleic acids, can include polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), and any other type of nucleic acid that is an N- or C-gly coside of a purine or pyrimidine base, as well as other polymers containing nonnucleotidic backbones, for example, polyamide (e.g., peptide nucleic acids (PNAs)) and polymorpholino polymers (see, e.g., Summerton and Weller (1997) “Morpholino Antisense Oligomers: Design, Preparation, and Properties,’" Antisense & Nucleic Acid Drug Dev. 7: 1817-195; Okamoto et al. (2002) “Development of electrochemically gene-analyzing method using DNA-modified electrodes,” Nucleic Acids Res. Supplement No. 2:171-172), and other synthetic sequence-specific nucleic acid polymers providing that the polymers contain nucleobases in a configuration which allows for base pairing and base stacking, such as is found in DNA and RNA. The term nucleic acid also encompasses locked nucleic acids (LNAs), which are described in U. S. Pat. Nos. 6,794,499, 6,670,461, 6,262,490, and 6,770,748, which are incorporated herein by reference in their entirety for their disclosure of LNAs.

[0029] The nucleic acid(s) can be derived from a completely chemical synthesis process, such as a solid phase-mediated chemical synthesis, from a biological source, such as through isolation from any species that produces nucleic acid, or from processes that involve the manipulation of nucleic acids by molecular biology tools, such as DNA replication, PCR amplification, reverse transcription, or from a combination of those processes.

[0030] The term “complementary” refers to the ability of a nucleic acid to form hydrogen bond(s) with another nucleic acid sequence by either traditional Watson-Crick or other non-traditional types. Complementarity of nucleic acid strands means that the strands form a stabile duplex due to hydrogen bonding between theirnucleobase groups. The complementary bases are in DNA, A with T and C with G, and in RNA, C with G. and U with A. Nucleotides in respective strands are complementarity when they form one of these (Watson-Crick pairings) when the strands are maximally aligned. Nucleotides are mismatched when they do not form a complementarity pair when their respective strands are maximally aligned.Complementarity of strands can be perfect or substantial. Perfect complementarity between two strands means that the two strands can form a duplex in which every base in the duplex is bonded to a complementary base by Watson-Crick pairing.Substantial complementarity' means most but not necessarily all bases in strands form Watson-Crick pairs to form a stable hybrid complex in set of hybridization conditions (e.g.. salt concentration and temperature). A percent complementarity’ indicates the percentage of residues in a nucleic acid molecule which can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100% complementary). “Perfectly complementary" means that all the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence. Complementarity is “complete,” “fully,” or “100%” when there are no mismatches between the two single-stranded nucleotide sequences. “100% complementarity along the full length of the sequences” indicates that there are no mismatches between two nucleic acid strands which can hybridize and which are of identical length. “Substantially complementary” as used herein refers to a degree of complementarity' that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%. 97%, 98%, 99%, or 100% over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22. 23, 24, 25, 30, 35, 40, 45, 50, or more nucleotides, or refers to two nucleic acids that hybridize under stringent conditions.

[0031] The term “hybridize” as used herein refers to the binding, duplexing, or hybridizing of a nucleic acid molecule preferentially to a particular nucleotide sequence. Hybridization generally involves the formation of hydrogen bonds between two single strands of a polynucleotide.

[0032] “Specific hybridization” refers to the binding of a nucleic acid to a target nucleotide sequence in the absence of substantial binding to other nucleotide sequences present in the hybridization mixture under defined stringency conditions. Those of skill in the art recognize that relaxing the stringency of the hybridization conditions allows sequence mismatches to be tolerated.

[0033] In some embodiments, hybridizations are carried out under stringent hybridization conditions. The phrase ‘"stringent hybridization conditions” generally refers to a temperature in a range from about 5°C to about 20°C or 25°C below than the melting temperature (Tm) for a specific sequence at a defined ionic strength and pH. As used herein, the Tmis the temperature at which a population of double-stranded nucleic acid molecules becomes half-dissociated into single strands. Methods for calculating the Tmof nucleic acids are well known in the art (see, e.g., Berger and Kimmel (1987) METHODS IN ENZYMOLOGY, VOL. 152: GUIDE TO MOLECULAR CLONING TECHNIQUES, San Diego: Academic Press, Inc. and Sambrook et al. (1989) MOLECULAR CLONING: A LABORATORY MANUAL, 2ND ED., VOLS. 1-3, Cold Spring Harbor Laboratory), both incorporated herein by reference for their descriptions of stringent hybridization conditions). As indicated by standard references, a simple estimate of the Tmvalue may be calculated by the equation: Tm=81.5+0.41(% G+C), when a nucleic acid is in aqueous solution at 1 M NaCl (see, e.g., Anderson and Young, Quantitative Filter Hybridization in NUCLEIC ACID HYBRIDIZATION (1985)). The melting temperature of a hybrid (and thus the conditions for stringent hybridization) is affected by various factors such as the length and nature (DNA, RNA, base composition) of the primer or probe and nature of the target nucleic acid (DNA, RNA, base composition, present in solution or immobilized, and the like), as well as the concentration of salts and other components (e.g.. the presence or absence of formamide, dextran sulfate, polyethylene glycol). The effects of these factors are well known and are discussed in standard references in the art. Illustrative stringent conditions suitable for achieving specific hybridization of most sequences are: a temperature of at least about 60° C. and a salt concentration of about 0.2 molar at pH7. Tmcalculation for oligonucleotide sequences based on nearest-neighbors thermodynamics can be carried out as described in “A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics” John SantaLucia, Jr., PNAS Feb. 17, 1998 vol. 95 no. 4 1460-1465 (which is incorporated by reference herein for this description).

[0034] The term “oligonucleotide” as used herein refers to a sequence of nucleotide monomers, each bound to an adjacent nucleotide monomer by a covalent bond. The oligonucleotide is relatively short, generally shorter than 200 nucleotides, more particularly, shorter than 100 nucleotides, most particularly, shorter than 50 nucleotides. Typically, oligonucleotides are single-stranded DNA molecules. An“oligonucleotide” may also include a non-nucleotide subunit or a nucleotide analog within the sequence of nucleotide monomers wherein the non-nucleotide subunit or nucleotide analog is bound to an adjacent subunit, analog or nucleotide by a covalent bond, bound by means of a covalent bond. The covalent bond between two adjacent nucleotide monomers in an oligonucleotide is a phosphodiester bond.

[0035] As used herein, the term “thermostable polymerase” or “thermophilic polymerase” refers to an enzyme that is relatively stable to heat when compared, for example, to nucleotide polymerases from E. coll, and which catalyzes the templatedependent polymerization of nucleoside triphosphates. A “thermostable polymerase,” will, e.g., retain enzymatic activity for polymerization and exonuclease activities when subjected to the repeated heating and cooling cycles used in PCR. Preferably, a “thermostable nucleic acid polymerase” has optimal activity at a temperature above 45°C, or at a temperature ranging from 40°C to 80°C and more preferably from 55°C to 75°C. A representative thermostable polymerase enzyme isolated from Thermus aquaticus (Taq) is described in U.S. Pat. No. 4,889,818 and a method for using it in conventional PCR is described in Saiki et al., 1988, Science 239:487. Other thermostable DNA polymerases include, but are not limited to, DNA polymerases from thermophilic Eubacteria or Archaebacteria, for example, T. thermophilus, T. bockianus, T. flavus, T. rubber, Thermococcus litoralis, Pyroccocus furiousus, P. wosei, Pyrococcus spec. KGD. Thermatoga maritime. Thermoplasma acidophilus, and Sulfolobus spec. Reverse transcriptases functional between 55-60°C include, but are not limited to, MmLV reverse transcriptase, AMV reverse transcriptase, RSV reverse transcriptase, HIV-1 reverse transcriptase, and HIV -2 reverse transcriptase.

[0036] The term “aptamer” as used herein refers to a nucleic acid that has a specific binding affinity for a target molecule, such as a protein. Like all nucleic acids, a particular nucleic acid ligand may be described by a linear sequence of nucleotides (A, U, T, C and G), typically 30-75 nucleotides long. Aptamers may also be described in terms of regions of predicted secondary structure wherein a single strand portion of the aptamer is complementary to another single strand portion of the same aptamer and thereby can hybridize (anneal) to each other to form a duplex which is referred to herein as a “stem,” and portions of the aptamer which are predicted not to hybridize with other portions of the same aptamer are referred to herein as “loops.” Generally, each end of a loop is linked to an end of a stem thereby providing a loop configuration.

[0037] The term “capping moiety” refers to a moiety attached to the 3' or 5' end of an aptamer or other nucleic acid that changes the stability of the nucleic acid, prevents polymerase elongation of the nucleic acid, and / or increases the efficiency of nucleic acid dimer formation. By “cap structure” is meant chemical modifications, which have been incorporated into the ends of oligonucleotide (see, for example, Matulic- Adamic et al., U. S. Pat. No. 5,998,203). In non-limiting examples: a suitable 5'-cap can be one selected from the group comprising inverted abasic residue; 4',5'-methylene nucleotide; l-(beta-D-erythrofuranosyl) nucleotide, 4'-thio nucleotide; carbocyclic nucleotide; 1,5-anhydrohexitol nucleotide; L-nucleotides; alpha-nucleotides; modified base nucleotide; phosphorodithioate linkage; threo-pentofuranosyl nucleotide; acyclic 3', 4'-seco nucleotide; acyclic 3,4-dihydroxybutyl nucleotide; acyclic 3,5-dihydroxypentyl nucleotide, 3'-3'-inverted nucleotide moiety; 3' -3 '-inverted abasic moiety; 3'-2'-inverted nucleotide moiety; 3'-2'-inverted abasic moiety; 1,4-butanediol phosphate; 3'-phosphoramidate; hexylphosphate; aminohexyl phosphate; 3'- phosphate; 3'-phosphorothioate; phosphorodithioate; or bridging or non-bridging methylphosphonate moiety. In another non-limiting example, a suitable 3'-cap can be selected from a group comprising, 4',5'-methylene nucleotide; 1 -(beta-D-erythrofuranosyl) nucleotide; 4'-thio nucleotide, carbocyclic nucleotide; 5 '-amino-alkyl phosphate; l,3-diamino-2-propyl phosphate; 3-aminopropyl phosphate; 6-aminohexyl phosphate; 1,2-aminododecyl phosphate; hydroxypropyl phosphate; 1.5-anhydrohexitol nucleotide; L-nucleotide; alpha-nucleotide; modified base nucleotide; phosphorodithioate; threo-pentofuranosyl nucleotide; acyclic 3', 4'- seco nucleotide; 3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentyl nucleotide, 5'-5'-inverted nucleotide moiety; 5'-5'-inverted abasic moiety; 5'-phosphoramidate; 5'-phosphorothioate; 1,4-butanediol phosphate; 5'-amino; bridging and / or non- bridging 5'-phosphoramidate, phosphorothioate and / or phosphorodithioate, bridging or non bridging methylphosphonate and 5'-mercapto moieties. For more details, see Beaucage and Iyer, 1993, Tetrahedron 49: 1925, which is incorporated by reference herein.

[0038] A “variant” of a first nucleic acid sequence refers to a second nucleic acid sequence that has one or more nucleotide substitutions relative to the first nucleic acid sequence.

[0039] “Primers” refer to single-stranded oligonucleotides which are complementary to sequence portions on a template nucleic acid molecule separated by a variable number of nucleotides. Primers annealed to the template nucleic acid can be extended bycovalent bonding of nucleotide monomers during amplification or polymerization of a nucleic acid molecule catalyzed by the thermostable polymerases. Typically, primers are from 12 to 35 nucleotides in length and are preferably from 15 to 20 nucleotides in length. Primers are designed from known parts of the template, one complementary to each strand of the double strand of the template nucleic acid molecule, lying on opposite sides of the region to be synthesized. Primers can be designed and synthetically prepared as is well known in the art.

[0040] The term used herein “forward primer” means a primer complementary to a strand of a nucleic acid sequence aligned in a 3' to 5' direction. The “reverse primer” has a complementary sequence to the other strand of the nucleic acid sequence.

[0041] “Template” as used herein refers to a double-stranded or single-stranded nucleic acid molecule, which serves a substrate for nucleic acid synthesis. In the case of a double-stranded DNA molecule, denaturation of its strands to form a first and a second strand is performed before these molecules may be used as substrates for nucleic acid synthesis. A primer, complementary to a portion of a single-stranded nucleic acid molecule serving as the temple template is hybridized under appropriate conditions and an appropriate polymerase may then synthesize a molecule complementary to the template or a portion thereof. The newly synthesized molecule may be equal or shorter in length than the original template.

[0042] The “polymerase chain reaction” (“PCR”) is a reaction in which replicate copies are made of a target polynucleotide using one or more primers, and a catalyst of polymerization, such as a DNA polymerase, and particularly a thermally stable polymerase enzyme. Generally, PCR involves repeatedly performing a “cycle” of three steps: “melting,” in which the temperature is adjusted such that the DNA dissociates to single strands, “annealing,” in which the temperature is adjusted such that oligonucleotide primers are permitted to match their complementary base sequence using base pair recognition to form a duplex at one end of the span of polynucleotide to be amplified; and “extension” or “synthesis,” which may occur at the same temperature as annealing, or in which the temperature is adjusted to a slightly higher and more optimum temperature, such that oligonucleotides that have formed a duplex are elongated with a DNA polymerase. This cycle is then repeated until the desired amount of amplified polynucleotide is obtained. Methods for PCR amplification are taught, for example, in U. S. Pat. Nos. 4,683,195 and 4.683,202.

[0043] “Speci fici ty” in primer extension or PCR amplification refers to the generation of a single, “specific” PCR product with the size and sequence predicted from the sequences of the primers and the genomic or transcribed region of nucleic acid to which the primers were designed to anneal in a base- complementary ■ manner.“Nonspecific” PCR product has a size or sequence different from such prediction.

[0044] A “target” or “target nucleic acid” is that genomic or transcribed region of nucleic acid, the ends of which are base-complementary (with proper orientation) to primers included in a complete set of PCR reagents. A primer refers to a nucleic acid sequence, which is complementary to a known portion of a target nucleic acid sequence and which is necessary to initiate synthesis by DNA polymerase. “Proper orientation” is for the two primers to anneal to opposite strands of double- stranded target nucleic acid with their 3' ends pointing toward one another. Such primers are said to target the genomic or transcribed sequence to the ends of which they are base-complementary. An “appropriate temperature”, as referred to in the claims in regard to the PCR amplifications, indicates the temperature at which specific annealing between primers and a target nucleic acid sequence occurs.

[0045] A “probe” is a nucleic acid capable of binding to a target nucleic acid of complementary sequence through one or more types of chemical bonds, generally through complementary base pairing, usually through hydrogen bond formation, thus forming a duplex structure. The probe can be labeled with a detectable label to permit facile detection of the probe, particularly once the probe has hybridized to its complementary target. Alternatively, however, the probe may be unlabeled but may be detectable by specific binding with a ligand that is labeled, either directly or indirectly. Probes can vary significantly in size. Generally, probes are at least 7 to 15 nucleotides in length. Other probes are at least 20, 30, or 40 nucleotides long. Still other probes are somewhat longer, being at least 50, 60, 70, 80, or 90 nucleotides long. Yet other probes are longer still, and are at least 100, 150, 200 or more nucleotides long. Probes can also be of any length that is within any range bounded by any of the above values (e.g., 7-200 nucleotides in length).

[0046] The primer or probe can be perfectly complementary to the target nucleotide sequence or can be less than perfectly complementary’. In some embodiments, the primer has at least 65% identity to the complement of the target nucleotide sequence over a sequence of at least 7 nucleotides, more typically over a sequence in the range of 10-30 nucleotides, and in some embodiments, over a sequence of at least 14-25nucleotides, and in some embodiments, has at least 75% identity, at least 85% identity, at least 90% identity, or at least 95%, 96%, 97%, 98%, or 99% identity. It will be understood that certain bases (e.g., the 3' base of a primer) are generally desirably perfectly complementary to corresponding bases of the target nucleotide sequence. Primer and probes typically anneal to the target sequence under stringent hybridization conditions.

[0047] As used herein with reference to a portion of a primer or a nucleotide sequence within the primer, the term “specific for” a nucleic acid, refers to a primer or nucleotide sequence that can specifically anneal to the target nucleic acid under suitable annealing conditions.

[0048] Amplification according to the present teachings encompasses any means by which at least a part of at least one target nucleic acid is reproduced, typically in a template-dependent manner, including without limitation, a broad range of techniques for amplifying nucleic acid sequences, either linearly or exponentially. Illustrative means for performing an amplifying step include PCR, nucleic acid strand-based amplification (NASBA). two-step multiplexed amplifications, rolling circle amplification (RCA), and the like, including multiplex versions and combinations thereof, for example but not limited to, OLA / PCR, PCR / OLA, LDR / PCR, PCR / PCR / LDR, PCR / LDR, LCR / PCR, PCR / LCR (also known as combined chain reaction — CCR), helicase-dependent amplification (HD A), and the like. Descriptions of such techniques can be found in, among other sources, Ausubel et al.; PCR Primer: A Laboratory Manual, Diffenbach, Ed., Cold Spring Harbor Press (1995); The Electronic Protocol Book, Chang Bioscience (2002); Msuih et al., J. Clin. Micro. 34:501-07 (1996); The Nucleic Acid Protocols Handbook, R. Rapley, ed., Humana Press, Totowa, N. J. (2002); Abramson et al., Curr Opin Biotechnol. 1993 February; 4(1):41-7, U. S. Pat. Nos. 6,027,998; 6,605,451, Barany et al., PCT Publication No. WO 97 / 31256; Wenz et al., PCT Publication No. WO 01 / 112579; Day et al., Genomics, 29(1): 152-162 (1995), Ehrlich et al., Science 252:1643-50 (1991); Innis et al.. PCR Protocols: A Guide to Methods and Applications. Academic Press (1990); Favis et al.. Nature Biotechnology 18:561-64 (2000); and Rabenau et al., Infection 28:97-102 (2000); Belgrader, Barany, and Lubin, Development of a Multiplex Ligation Detection Reaction DNA Typing Assay, Sixth International Symposium on Human Identification, 1995 (available on the world wide web at: promega.com / geneticidproc / ussymp6proc / blegrad.html); LCR Kit Instruction Manual,Cat. #200520, Rev. #050002, Stratagene, 2002; Barany, Proc. Natl. Acad. Sci. USA 88:188-93 (1991); Bi and Sambrook, Nucl. Acids Res. 25:2924-2951 (1997); Zirvi et al., Nucl. Acid Res. 27: e40i-viii (1999); Dean et al., Proc Natl Acad Sci USA 99:5261-66 (2002); Barany and Gelfand, Gene 109:1-11 (1991); Walker et al., Nucl. Acid Res.20:1691-96 (1992); Polstra et al., BMC Inf. Dis. 2:18-(2002); Lage et al., Genome Res.2003 February; 13(2):294-307, and Landegren et al., Science 241:1077-80 (1988), Demidov, V., Expert Rev Mol Diagn. 2002 November; 2(6):542-8., Cook et al., J Microbiol Methods. 2003 May; 53(2): 165-74, Schweitzer et al., Curr Opin Biotechnol.2001 February; 12(1):21-7, U. S. Pat. Nos. 5,830,711, 6,027,889, 5,686,243, PCT Publication No. WO0056927A3, and PCT Publication No. WO9803673A1.

[0049] In some embodiments, amplification comprises at least one cycle of the sequential procedures of: annealing at least one primer with complementary or substantially complementary sequences in at least one target nucleic acid; synthesizing at least one strand of nucleotides in a template-dependent manner using a polymerase; and denaturing the newly-formed nucleic acid duplex to separate the strands. The cycle may or may not be repeated. Amplification can comprise thermocycling or can be performed isothermally.

[0050] A “multiplex amplification reaction"’ is one in which two or more nucleic acids distinguishable by sequence are amplified simultaneously.

[0051] The term “qPCR” is used herein to refer to quantitative real-time polymerase chain reaction (PCR), which is also known as “real-time PCR” or “kinetic polymerase chain reaction;” all terms refer to PCR with real-time signal detection.

[0052] A “reagent” refers broadly to any agent used in a reaction, other than the analyte (e.g.. nucleic acid being analyzed). Illustrative reagents for a nucleic acid amplification reaction include, but are not limited to, buffer, metal ions, polymerase, reverse transcriptase, primers, template nucleic acid, nucleotides, labels, dyes, nucleases, dNTPs, and the like. Reagents for enzyme reactions include, for example, substrates, cofactors, buffer, metal ions, inhibitors, and activators.

[0053] The term “marker” or “label,” as used herein, refers to any atom or molecule that can be used to provide a detectable and / or quantifiable signal. In particular, the label can be attached, directly or indirectly, to a nucleic acid or protein. Suitable labels that can be attached to probes include, but are not limited to, radioisotopes, fluorophores, chromophores, mass labels, electron dense particles, magnetic particles, spin labels, molecules that emit chemiluminescence, electrochemically activemolecules, enzymes, cofactors, and enzyme substrates. As used herein, the term label refers to an agent capable of detection, for example by ELISA, spectrophotometry, flow cytometry, immunohistochemistry, immunofluorescence, microscopy. Northern analysis or Southern analysis. For example, a marker can be attached to a nucleic acid molecule or protein, thereby permitting detection of the nucleic acid molecule or protein. Examples of labels include, but are not limited to, radioactive isotopes, nitroimidazoles, enzyme substrates, co-factors, ligands, chemiluminescent agents, fluorophores, haptens, enzymes, and combinations thereof. Methods for labeling and guidance in the choice of markers appropriate for various purposes are discussed for example in Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N. Y., 1989) and Ausubel et al. (In Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1998).

[0054] The naturally occurring bases adenine, thymine, uracil, guanine, and cytosine, which make up DNA and RNA, are described herein as “unmodified bases” or “unmodified forms.”

[0055] The term “modified base” is used herein to refer to a base that is not a canonical, naturally occurring base (e.g., adenine, cytosine, guanine, thymine, or uracil). Examples of modified bases are 2-thiothymine, 2-aminoadenine, or 5-propynyl uridine.

[0056] Nucleotides comprising modified bases are referred to herein as “modified nucleotides.”

[0057] A DNA polymerase is said to be “stable” at a particular temperature if it provides a satisfactory extension rate in a nucleic acid amplification reaction.

[0058] Oligonucleotides including one or more “modified nucleotides” (e.g., pseudo-complementary nucleotides) are referred to herein as pseudo-complementary oligonucleotides (e.g., pseudo-complementary blocker oligonucleotide).

[0059] As used herein, the terms “subject” and “patient” are used interchangeably. As used herein, the term “patient” refers to an animal, preferably a mammal such as a nonprimate (e.g., cows, pigs, horses, cats, dogs, rats etc.) and a primate (e.g., monkey and human), and most preferably a human. In some embodiments, the subject is a nonhuman animal such as a farm animal (e.g., a horse, pig, or cow) or a pet (e.g., a dog or cat). In a specific embodiment, the subject is an elderly human. In another embodiment, the subject is a human adult. In another embodiment, the subject is a human child. In yet another embodiment, the subject is a human infant. The patient or subject to bemedicated according to the compositions and methods as disclosed herein may be any animal or human. In certain embodiments, animals may include vertebrates. The terms vertebrate or animals in this context is understood to comprise, for example fish, amphibians, reptiles, birds, and mammals including humans. One preferred group of vertebrates or animals according to the invention comprises warm-blooded animals including farm animals, such as cattle, horses, pigs, sheep and goats, poultry such as chickens, turkeys, guinea fowls and geese, fur-bearing animals such as mink, foxes, chinchillas, rabbits and the like, as well as companion animals such as ferrets, guinea pigs, rats, hamster, cats and dogs. A further group of preferred vertebrates or animals according to the disclosure comprises fish including salmonids, for example salmon, trout or whitefish. The subject is preferably mammalian. In some embodiments the subject is a human. In other embodiments the subject is an animal, more preferably a non-human mammal. The non-human mammal may be a domestic pet, or animal kept for commercial purposes, e.g., a racehorse, or farming livestock or animals such as pigs, sheep or cattle. As such the disclosure may have veterinary applications. Non-human mammals include rabbits, guinea pigs, rats, mice or other rodents (including any animal in the order Rodentia), cats, dogs, pigs, sheep, goats, cattle (including cows or any animal in the order Bos), horse (including any animal in the order Equidae), donkey, and non-human primates. The subject may be male or female. The subject may be an adult or a child. The subject may be a patient.

[0060] As used herein, the term “about’’ when used in conjunction with a stated numerical value or range has the meaning reasonably ascribed to it by a person skilled in the art, i.e., denoting somewhat more or somewhat less than the stated value or range. When a group of substituents is disclosed herein, it is understood that all individual members of those groups and all subgroups and classes that can be formed using the substituents are disclosed separately. When a Markush group or other grouping is used herein, all individual members of the group and all combinations and subcombinations possible of the group are intended to be individually included in the disclosure. As used herein, “and / or” means that one, all, or any combination of items in a list separated by “and / or” are included in the list; for example, “1, 2 and / or 3” is equivalent to “1, 2, 3, 1 and 2, 1 and 3, 2 and 3, or 1, 2, and 3”.

[0061] As used herein, “comprising” is synonymous with “including,” “containing,” or “characterized by,” and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. As used herein, “consisting of’ excludes anyelement, step, or ingredient not specified in the claim element. As used herein, “consisting essentially of’ does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim. Any recitation herein of the term “comprising”, particularly in a description of components of a composition, in a description of a method, or in a description of elements of a device, is understood to encompass those compositions, methods, or devices consisting essentially of and consisting of the recited components or elements, optionally in addition to other components or elements. The disclosure as illustratively described herein suitably may be practiced in the absence of any element, elements, limitation, or limitations which is not specifically disclosed herein.

[0062] As used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a method” includes a plurality of such methods and reference to “the nanoparticle” includes reference to one or more nanoparticles and equivalents thereof known to those skilled in the art, and so forth. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope as disclosed herein claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.

[0063] As used herein, the term sample may be, for example, selected from tissue(s) samples, cells, biological fluid samples (e.g., blood, urine, saliva, lymphatic fluid, cerebrospinal fluid (CSF), amniotic fluid, pleural fluid, pericardial fluid, ascites, aqueous humor), bone marrow samples, semen samples, biopsy samples, cancer samples, tumor samples, cell lysate samples, forensic samples, archaeological samples, paleontological samples, infection samples, production samples, whole plants, plant parts, microbiota samples, viral preparations, soil samples, marine samples, freshwater samples, household or industrial samples, and combinations and isolates thereof. In one embodiment of the systems and methods as disclosed herein, the sample is a cell (e.g.. an animal cell [e.g., a human cell], a plant cell, a fungal cell, a bacterial cell, and aprotozoal cell). In one specific embodiment, the cell is lysed prior to the replication. In one specific embodiment, cell lysis is accompanied by proteolysis. In one specific embodiment, the cell is selected from a cell from a preimplantation embryo, a stem cell, a fetal cell, a tumor cell, a suspected cancer cell, a cancer cell, a cell subjected to a gene editing procedure, a cell from a pathogenic organism, a cell obtained from a forensic sample, a cell obtained from an archeological sample, and a cell obtained from a paleontological sample. In one embodiment of any of the systems and methods as disclosed herein, the sample is a cell from a preimplantation embryo (e.g., a blastomere. In one specific embodiment, the method further comprises determining the presence of disease predisposing germline or somatic variants in the embryo cell. In one embodiment of any of the systems and methods as disclosed herein, the sample is a cell from a pathogenic organism (e.g., a bacterium, a fungus, a protozoan). In one specific embodiment, the pathogenic organism cell is obtained from fluid taken from a patient, microbiota sample (e.g., GI microbiota sample, vaginal microbiota sample, skin microbiota sample, etc.) or an indwelling medical device (e.g., an intravenous catheter, a urethral catheter, a cerebrospinal shunt, a prosthetic valve, an artificial joint, an endotracheal tube, etc.). In one specific embodiment, the method further comprises the step of determining the identity of the pathogenic organism. In one specific embodiment, the method further comprises determining the presence of genetic variants responsible for resistance of the pathogenic organism to a treatment. In one embodiment of any of the systems and methods as disclosed herein, the sample is a tumor cell, a suspected cancer cell, or a cancer cell. In one specific embodiment, the method further comprises determining the presence of one or more diagnostic or prognostic mutations. In one specific embodiment, the method further comprises determining the presence of germline or somatic variants responsible for resistance to a treatment. In one embodiment of any of the systems and methods as disclosed herein, the sample is a cell subjected to a gene editing procedure. In one specific embodiment, the method further comprises determining the presence of unplanned mutations caused by the gene editing process. In one embodiment of any of the systems and methods as disclosed herein, the method further comprises determining the history of a cell lineage. In a related aspect, the invention provides a use of any of the systems and methods as disclosed herein for identifying low frequency sequence variants (e.g., variants which constitute ≥0.01% of the total sequences). The phrase “fragment library" refers to a collection of nucleic acid fragments, wherein one or more fragments are used as asequencing template. A fragment library can be generated in numerous ways that are known in the art. As an example, a fragment library can be generated by cutting, shearing, restricting, or otherwise subdividing a larger nucleic acid into smaller fragments. Fragment libraries can be generated from naturally occurring nucleic acids, such as, for example, from bacteria, cancer cells, normal cells, or solid tissue. Libraries comprising synthetic nucleic acid sequences can also be generated to create a synthetic fragment library.

[0064] All references throughout this application, for example patent documents, including issued or granted patents or equivalents and patent application publications, and non-patent literature documents or other source material are hereby incorporated by reference herein in their entireties, as though individually incorporated by reference. None is admitted to being prior art.II. Aptamer Inhibitors of Polymerase Activity

[0065] The present disclosure provides reversible inhibitors of thermostable polymerases for use in reactions which require primer extension by the polymerase. These reversible inhibitors are aptamers, nucleic acids which are designed to adopt a secondary structure comprising a stem and a loop. The aptamers’ secondary structure can further comprise a kink-tum which defines the symmetry of the aptamer and impact the binding affinities of each aptamer to the polymerase. The aptamers are useful in reactions and assays that benefit from incorporating hot start and can improve the sensitivity and specificity of nucleic acid synthesis.

[0066] In some embodiments described herein, the aptamer is designed to form a hairpin structure wherein the aptamer is predicted to comprise a single stem having two single strands which are complementary and terminating in a single loop. The single stem in the predicted hairpin structure as described herein comprises a first polynucleotide sequence which is annealed to a third polynucleotide sequence wherein the first and third polynucleotide sequences are complementary and are equal in length. The 3' end of the first polynucleotide sequence is covalently bound to the 5' end of a second polynucleotide sequence which is predicted to form a single stranded loop in the predicted hairpin aptamer, and the 3' end of the second polynucleotide sequence is covalently bound to the 5' end of the third polynucleotide sequence.

[0067] The stem region of the aptamer is a double-stranded structure (formed by hybridization or annealing of the first and third polynucleotide sequences as described above) which is about 12 to 20 base pairs in length. However, the length of the stemcan vary with the understanding that the variation may affect the temperature at which an aptamer dissociates from and no longer inhibits the polymerase activity. It is advantageous to have aptamers which vary with respect to the temperature at which they dissociate from the polymerase. Accordingly, the aptamers of the present disclosure include those that comprise a stem which is 12 to 18, 12 to 16, 14 to 20, 14 to 18, 14 to 17, 14 to 16. 15 to 16, 15 to 17, 15 to 18, or 15 to 20 base pairs in length or is about 12, 13, 14, 15, 16, 17, 18, 19, or 20 base pairs in length. The stem is comprised of two single-stranded oligonucleotides of equal length such that the presently described aptamers are polynucleotides which comprise two single-strand oligonucleotide sequences which are 12 to 18, 12 to 16, 14 to 20, 14 to 18, 14 to 17, 14 to 16, 15 to 16, 15 to 17. 15 to 18. or 15 to 20 nucleotides in length or about 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length and which are complementary to one another. In some embodiments, the two oligonucleotide sequences are hybridized via hydrogen bonds and no mismatches are present in the resultant stem of the aptamer.

[0068] The stem sequence, such as that comprised of SEQ ID NO.:1 paired with SEQ ID NO.:2, is AT-rich. For example, the stem sequence is 12-18 base pairs in length wherein at least 13, 14, 15, 16, 17, or 18 of those base pairs are A-T base pairs. In some embodiments, the stem is 16 base pairs in length and comprises 15 A-T base pairs and one G-C base pair. In some embodiments, the stem is 16 base pairs in length and comprises 14 A-T base pairs and two G-C base pair. In some embodiments, the stem is 16 base pairs in length and comprises 13 A-T base pairs and three G-C base pair. The one or more G-C base pairs can be present at any location within the stem sequence. In some embodiments, the one or more G-C base pairs can be present near or at the 5' end of the first polynucleotide sequence (i.e., near or adjacent to the 5' overhang). In some embodiments, the one or more G-C base pairs can be present near or at the 3' end of the first polynucleotide sequence (i.e., near or adjacent to the second polynucleotide / loop sequence). In some embodiments, the one or more G-C base pairs can be present near the 5' end, such as at the second and / or third nucleotide of the first polynucleotide sequence.

[0069] The stem sequence, such as that comprised of SEQ ID NO.:1 paired with SEQ ID NO.:2, may comprise a kink-tum or bend in its helical structure. The location of the kink / bend in the stem region forms a substantially symmetrical or asymmetrical aptamer. The sequence of the aptamers disclosed herein shifts the location of the kink to form an asymmetrical structure, compared to previously disclosed symmetricalhairpin structures described in US Pat. No. 11,299,739. Figure 1 compares hot-start temperature of dumbbell vs. symmetrical and asymmetrical hairpins.

[0070] Table 1 below provides some exemplary oligonucleotide sequences for the first and / or third polynucleotide sequences in the hairpin aptamer. It is understood that any of the sequences disclosed herein may have a nucleotide substitution at 1, 2, 3, or 4 positions in the sequence. The nucleotide substitution at 1, 2, 3. or 4 positions in the sequence can comprise a naturally occurring base (e.g., adenine, thymine, uracil, guanine, and cytosine) and / or a modified base (e.g., 2-thiothymine, 2-aminoadenine, or 5-propynyl uridine). In some examples, the sequences disclosed herein may have a nucleotide substitution at 1, 2, 3, or 4 positions in the sequence, wherein the nucleotide substitution is selected from a nucleotide comprising propynyl modified uridine, deoxyuridine, and / or a GC base pair.Table 1: Hairpin Aptamer Stem SequencesSEQ ID NO Stem Sequence 5’“^ 3’1 TTATTTTTTAAAAATA2 TATTTTTAAAAAATAA4 GTATTTTTTAAAAATA5 TATTTTTAAAAAATAC6 CTA TTTTTTAAAAA TA7 TA TTTTTAAAAAA TA G8 GCA TTTTTTAAAAA TA9 TA TTTTTAAAAAA TGC10 CGA TTTTTTAAAAA TA11 TATTTTTAAAAAATCG12 TCA TTTTTTAAAAA TA13 TA TTTTTAAAAAA TGA14 TGA TTTTTTAAAAA TA15 TA TTTTTAAAAAA TCA16 TTATTTTTAAAAAATA17 TATTTTTTAAAAATAA

[0071] The second polynucleotide sequence which is predicted to form a loop can comprise TTCTTAGCGTTT (SEQ ID NO.:3). The loop sequence takes part in binding to the polymerase and effecting inhibition of polymerase activity when bound. In some embodiments, the second polynucleotide loop sequence of SEQ ID NO.:3 has 1, 2, 3, or4 nucleotide substitutions as described herein. For example, the 1, 2, 3, or 4 nucleotide substitutions in the second polynucleotide loop sequence can comprise one or more modified bases. Modified bases useful in the aptamers, for example, in the second polynucleotide loop sequence include those wherein the modified base forms stable hydrogen-bonded base pairs with the natural complementary base but further includes a hydrophobically modified group. Hydrophobically modified aptamer can allow an increase in binding affinity with polymerase by improving their interaction. The hydrophobic modification preferably fits in a hydrophobic pocket of the DNA polymerase. In general, a sufficient number of modified nucleotides are incorporated into the aptamer to preferentially increase the binding of the aptamer to the polymerase. It is not necessary to replace each natural nucleotide of the aptamer with a modified nucleotide in order to accomplish this. In some examples, the modified nucleotide can include a hydrophobically modified dA, dT, dC, dG, or dU. In specific examples, the modified nucleotide can include a hydrophobically modified dU. The hydrophobic modification can include a C1-C5alkyl, a C2-C5alkenyl, a C2-C5alkynyl group. In some instances, the hydrophobic modification can include a C2-C5alkynyl group such as a propynyl group. In some examples, the modified nucleotide can include a 5-propynyl-2'-deoxyuridine. In general, the loop sequence consists essentially of SEQ ID NO.: 3 with the exception of 1, 2, 3, or 4 nucleotide substitutions dT by dU or U*.

[0072] As described herein, the aptamers provided herein can be modified for activating the aptamer-deactivated DNA polymerases. In these embodiments, the aptamer can be modified by uracil-DNA glycosylase enzymatic activity to reduce or eliminate binding of the aptamer to the DNA polymerase, thereby activating DNA synthesis activity of the DNA polymerase in a reaction mixture. In some aspects, the aptamers comprise one or more deoxyuridine nucleotides providing for aptamer-specific recognition and modification of the aptamer by the uracil-DNA glycosylase enzymatic activity. In some examples, the loop sequence comprises 1, 2, 3, or 4 nucleotide substitutions having deoxy uridine nucleotides that aids in degradation of the aptamer by an enzyme, such as UDG.

[0073] Uracil-DNA glycosylase (UDG) is a highly conserved repair enzyme that catalyzes the excision of uracil from uracil-containing single- and double-stranded DNA. Combining processive and distributive mechanisms, UDG slides along the DNA strands, recognizes uracil molecules and proceeds to base excision by flipping the base into its active site pocket. More precisely, UDG binds, kinks and compresses the DNAbackbone (also called "pinch-push-pull ’-mechanism) to actually scan the minor groove in sections for damage. Once it recognizes a uracil base, it actively flips the dU nucleotide into an extrahelical conformation and eliminates uracil from DNA by cleaving the N-glycosidic bond that results in the formation of an abasic site (AP-site). In some examples, the aptamers described herein include one or more deoxyuridine. In further examples, the aptamers described herein include, in addition to one or more modified nucleotides, one or more deoxyuridine. Exemplary methods employing uracil-DNA glycosylase enzymatic activity are provided in US Patent No. 10,724,017B1, which is hereby incorporated herein by reference in its entirety. The methods can be practiced using kits comprising a DNA polymerase-binding aptamer and at least one uracil-DNA glycosylase enzymatic activity having oligonucleotide aptamer-specific recognition to provide for specific modification of the aptamer by the uracil-DNA glycosylase enzymatic activity.

[0074] Table 2 below provides some exemplary oligonucleotide sequences for the second polynucleotide sequences in the hairpin aptamer. It is understood that any of these sequences may have a nucleotide substitution at 1, 2, 3, or 4 positions in the sequence. In preferred embodiments, the second polynucleotide sequence which is predicted to form a loop comprises TTCTTAGCGTTT (SEQ ID NO.: 3).Table 2: Hairpin Aptamer Loop Sequences. Nucleotides with 5-propynyl-2'-deoxyuridine are marked as U* and nucleotides with deoxyuridine are marked as U.SEQ ID NO Sequence 5’"^ 3’3 TTCTTAGCGTTT18 TU’CTTAGCGTTT19 TTU’TTAGCGTTT20 TTCU’TAGCGTTT21 TTCTU’AGCGTTT22 TTCTTU’GCGTTT23 TTCTTAU’CGTTT24 TTCTTAGU’GTTT25 TTCTTAGCU’TTT26 TTCTTAGCGU*TT27 TTCTTAGCGTU*T28 UTCTTAGCGTTT29 UU'CTTAGCGTTT30 TU’UTTAGCGTTT31 TU’CGTAGCGTTT32 TU’CTUAGCGTTT33 TU'CTTEGCGTTT34 TU’CTTAGCGTTT35 TU’CTTAGGGTTT36 TU*CTTAGCGTTT37 TU’CTTAGCGGTT38 TU'CTTAGCGTGT39 TU'CTTAGCGTTG40 UU’CTTAGCGTTG41 GTU’TTAGCGTTT42 CZTCU’TAGCGTTT43 UTCTU*AGCGTTT44 GTCTTU’GCGTTT45 GTCTTAU’CGTTT46 GTCTTAGU*GTTT47 GTCTTAGCU*TTT48 GTCTTAGCGU’TT49 GTCTTAGCGTU’T

[0075] In some embodiments, the hairpin aptamers have 5' and / or 3' overhangs which can comprise a full or partial sequence of SEQ ID NO.:3 or a variant thereof. Examples of hairpin aptamer overhang sequences are provided in Table 3. For example, according to SEQ ID No. 56, the hairpin aptamer has an overhang comprising a 3' portion of SEQ ID NO.:3 in which a 3' portion of SEQ ID NO.:3 (GTTT) is ligated to the 5' nucleotide of a stem strand comprising the sequence TTATTTTTTAAAAATA (SEQ ID NO.:1) and the remaining 5' portion of SEQ ID NO.:3 is ligated to the 3' nucleotide of a second stem strand comprising the sequence TATTTTTAAAAAATAA (SEQ ID NO.:2). Because there is no covalent (phosphodiester) bond between nucleotides of the overhang sequences, the oligonucleotides described herein are predicted to fold into a hairpin structure. In some embodiments, the 5' and / or 3' overhangs each has 1, 2, 3, or 4 nucleotide substitutions. The 1, 2, 3, or 4 nucleotide substitutions in the 5' and / or 3' overhangs can comprise one or more modified nucleotides as described herein. In some examples, the modified nucleotides can include a hydrophobically modified dU such as a propynyl modified dU. In someaspects, the aptamers comprise one or more deoxy uridine nucleotides in the 5' and / or 3' overhangs, providing for aptamer-specific recognition and modification of the aptamer by the uracil-DNA glycosylase enzymatic activity. In some examples, the 5' and / or 3' overhangs each comprises 0, 1, 2, 3, or 4 nucleotide substitutions having deoxyuridine nucleotides that aids in degradation of the aptamer by an enzyme, such as UDG.Table 3: Hairpin Aptamer Terminus / 5' and / or 3' Overhang Sequences.Nucleotides with 5-propynyl-2'-deoxyuridine are marked as U* and nucleotides

[0076] The aptamers as presently described are in an open form. In other words, the terminal overhang in the aptamer lacks a covalent bond between two of the nucleotides in only one of the strands. In these embodiments, the aptamer has a 5' end and a 3' end. As all nucleotides in the stem can remain hybridized to the opposite, fully complementary strand, the 5' and 3' ends remain adjacent to one-another when the aptamer is folded into its predicted secondary structure. In some embodiments, the 5' end of the aptamer comprises a phosphate group while the 3' end comprises a hydroxyl group. In alternative embodiments, the 5' end, the 3' end or both ends of the aptamer are linked to a capping moiety. The capping moiety may function to stabilize the aptamer and / or to prevent polymerization initiated at the 3' end of the aptamer. It is also possible that the lack of a covalent bond and / or the presence of a capping moiety may disrupt hydrogen bond(s) between nucleotides closest to the 5' and / or 3' end but this will not necessarily affect the inhibitory activity of the aptamer. Examples of a hairpin aptamer sequences are provided in Table 4. It is understood that any of these sequences may have a nucleotide substitution at 1, 2, 3, or 4 positions in the sequence. Acceptable substitutions in the sequences are those which do not affect the reversible inhibitory activity of the aptamer by more than about 5% or 10% relative the aptamer in which the stem sequences each have SEQ ID NO.: 1 and / or 2 and the loop sequence have SEQ ID NO.:3.jo aouanbas doo| qnj aqi SJUOSSJCIOJ Sireqiaxo c aqi TBqi qans aq two ajnioruis uidireq aqi ‘uiaiaq paquasap sy BAoqp p ajqni ui uMoqs saauanbas aqi uo paspq pazisaqjuAs Xqpanuaqa aq irea sapqoapnuoSqo daureicte uidireq aqi azisaqiuXs op [ AOO]d-39V11311VV poujaunnXs 1VVVVV111111V1111939V11311V1VVVVVV11111V111119 IZ.d-D9VI13,niVDIVVVVVVII. LIIVin [BoupuniiXsy iioDoviiD.nnvivvvvviiiiiivoiino OL d-DOVna.lUVOl [BoupiuutXsy VVVVVVIlIIlV172IIO3OVII3,niVlVVVVVlIIIIlVOinilO 69 d-39VI13,mV31 [BoupiuuiXsy VVVVVVIlIIIViailGOOVIlD.aiVlVVVVVllIlllVOIIllO 89 d-39VI13,mV31 [BoujaiuuiAsy VVVVVVIlIIIVlIinO39VII3.niVlVVVVVllIIllV9II119 £9 d-3OVI13,nnV3I {eoiipuiuiAsy VVVVVV. T.. T.. T.. T.. T. V. T.. T.. T. J.93OV. T. J.3,113 V. T. VVVVV. T.. T.. T.. T.. T.. T. VO. T.. T.. T.. T. O 99 d-3OV113.nnV3I [PoupuiuiXsy VVVVVVlllIlVllI1030VI13.niVlVVVVVllIlllV01I110 S9 d-39VI13.niV3I [PoupuiuiXsy vvvvvviiiiiviiiio3ovii3.nnvivvvvviiiiiivoiino 19 d-39VII3.niV31 poijpuiuiXsy S 09 VVVVVVlIIllVIIllO3OV113,niVIVVVVVIIllIIVOIIIIO £9 d-39V113.niV9I poupuiuiXsy VVVVVVIIIlIVIIlIO3OVlI3,niVIVVVVVIIlIIIV3IIIIO Z9 d-39VII3.rn.931 [BoupuruiXsy 19 d-39V113, H1391 [BoujoiuutXsy 09 d-39V113.m9Vl [BoupiuuiXsy 6S d-39V113.niavi [BoupiuutXsy VVVVVV11111V1111939V113.111V1VVVVV111111V191119 8S d-39V113,mVVl [BoujaiuuiAsy SYS VVVVVV11111V1111939V113. I11V1VVVVV111111V111119 £9 d-39V11311VVl {eoijpuiuiAsy VVVVVV. T.l. T.. T.. T. V. T.. T.. T.. T.939V. T.. T.3,n. T. V. T. VVVVVl. T.. T.. T.. T.. T. V. T.. T.. T.. T.. T.9 99 d-39V11311VV [BoupuiuiXsy 1VVVVVV11111V1111939V11311V1VVVVV111111V111119 SS (juasaad JI (3») suoi)B9ij!poiu jeuiuuaj Smpnpui) aauanbas apijoapnuoSjio Jeauiq wi ON <u 6asa si; poq.mm an: auipi.mXxoap qji w sappoapnu pin: q su paq.mm a. in auipi.inXxoap -. Z- Cu do.id-g qji v sapijoapn^E 'saauanbas jaurejdy uid.iiiqi:p <qqi’ISEQ ID N0.:3 or the 5' overhang represents the full loop sequence of SEQ ID NO.:3. SEQ ID NO.: 56 is an example of a hairpin aptamer with a 3' overhang in which a 5' portion of a sequence consisting of SEQ ID NO.:3 is covalently linked to the 3' end of one stem sequence, the remaining 3' portion of the sequence consisting of SEQ ID NO.:3 is covalently linked to the 5' end of the complementary stem sequence, and there is no covalent (e g., phosphodiester) bond between nucleotides of the overhang sequences consisting of SEQ ID NO.: 3. The synthetic oligonucleotide will fold under appropriate conditions to have a predicted secondary structure with a loop and a stem region. The 5 '-phosphate end can be attached to a capping moiety such as an amino group or other relevant capping moiety as described herein. The 3'-hydroxyl of the oligonucleotide can be attached to a capping moiety such as a propanediol spacer C3 or other relevant capping moiety as described herein. The aptamer can comprise a 5' cap, a 3' cap or both a 5' cap and a 3' cap. The presence of a 5' cap and / or 3' cap can inhibit ligation of the two ends of the oligonucleotide and can also inhibit poly merization of the oligonucleotide.

[0078] Aptamers which can bind to a polymerase and inhibit the polymerase activity' as described above were designed to have a loop comprising the sequence TTCTTAGCGTTT (SEQ ID NO.:3) as was determined by Yakimovich et al. (2003, Biochem (Moscow), 68:228-235) as being responsible for binding to the polymerase and effecting inhibition of polymerase activity when bound. In some embodiments, the loop sequence of SEQ ID NO.:3 has 1 or 2 nucleotide substitutions. The loop is covalently attached to a stem comprising two complementary sequences of equal length. While an exemplary single strand sequence of a stem is SEQ ID NO.: 1 and its complement is SEQ ID NO.:2, it is contemplated that there may be 1, 2 or 3 base pair substitutions within the stem. Importantly, a substitution in one strand is always coupled with a complementary substitution in the complementary strand of the stem.

[0079] As described above, the aptamer can lack a covalent bond between two nucleotides present in the overhang sequences. Accordingly, the aptamer which lacks a covalent bond between the 2 nucleotides comprises a single 5' terminus and a single 3' terminus. As noted above, an aptamer comprising a single 5' terminus and a single 3' terminus can be chemically synthesized as a single-stranded oligonucleotide comprising the entire sequence of the aptamer. In other words, the synthesized single strand oligonucleotide comprises a 5' portion of a first stem sequence comprising SEQ ID NO.: 1 or a variant thereof, a loop sequence comprising SEQ ID NO.:3 or a variantthereof, a second stem sequence comprising SEQ ID NO.:2 or a variant thereof. Once synthesized as a single strand nucleotide it can fold under appropriate conditions to have a loop and a single stem, wherein one strand of the stem does not have a covalent bond between 2 adjacent nucleotides.III. Aptamer Inhibition Activity

[0080] Also contemplated is a method for reversibly inhibiting polymerase activity’ comprising incubating an aptamer as described herein with a thermostable polymerase. The aptamers of the present disclosure can bind to a thermostable polymerase and inhibit the polymerase activity of that polymerase at lower temperatures, then dissociate from the polymerase as the reaction temperature is raised. In some embodiments, the thermostable polymerase is selected from but not limited to DNA polymerases from thermophilic Eubacteria or Archaebacteria, including but not limited to, Thermits aquaticus, T. thermophilus, T. bockianus, T. flavus, T. rubber, Thermococcus litoralis, Pyroccocus furiousus, P. wosei, Pyrococcus spec. KGD, Thermatoga maritime, Thermoplasma acidophilus, and Sulfolobus spec. In some embodiments the polymerase may be a reverse transcriptase functional between 55-60°C, including but not limited to, MmLV reverse transcriptase, AMV reverse transcriptase, RSV reverse transcriptase, HIV-1 reverse transcriptase, and HIV-2 reverse transcriptase.

[0081] The ability of the aptamer to bind to and inhibit, then dissociate from the polymerase depends in part upon its secondary structure in the reaction mixture.Aptamers of the present disclosure have melting temperatures ranging from about 45°C to 70°C. In some embodiments, the open hairpin aptamers have higher melting temperatures, ranging from about 60°C to 70°C, 60°C to 65°C or 65°C to 70°C.Melting temperatures are determined in some embodiments at an aptamer concentration of about 8 μM in a buffered solution having a pH of about 8. The buffered solution in some embodiments comprises 3 mM MgCl2, 15 KCl, 25 mM HEPES, pH 8.0. Melting temperatures for each aptamer can be measured using a UV-Vis-NIR spectrophotometer.

[0082] While the loss of secondary structure of an inhibitory aptamer can result in a decrease in the aptamer's ability’ to remain bound to and inhibit a polymerase, dissociation from the polymerase with increasing temperatures can depend on other factors such as the primary’ sequence of the aptamer and the interface between the aptamer and the polymerase. Accordingly, contemplated and described are aptamersthat inhibit, reduce or eliminate thermostable polymerase activity at temperatures below about 50°C, 45°C, 44°C, 43°C, 42°C, 41 °C, 40°C, 39°C or 38°C. In some embodiments, the aptamer and polymerase are in a solution having a pH of about 6-8, 6-9, 7-8, 7-9, or 8-9.

[0083] The ability of an aptamer to inhibit, reduce or eliminate the polymerase activity' of a thermostable polymerase can be measured and quantified using an assay which measures the polymerase activity in the presence or absence of the aptamer at varying temperatures. It is understood that several alternative methods may be used to measure the ability' of an aptamer to inhibit polymerase activity' in a temperature-dependent way, only some of which are briefly described here.

[0084] One method for measuring the inhibitory effects of an aptamer on the activity of a thermostable polymerase is described in Nikiforov et al., 2011, Analytical Biochemistry', 412: 229-236. Specifically, a hairpin template is provided in which a fluorescent label (e.g., fluorescein (FAM; 5'-dimethoxytrityloxy-5-[N-((3',6'-dipivaloylfluoresceinyl)-aminohexyl)-3-acrylimido]-2'-deoxyuridine-3'-[(2-cyanoethyl)-(N, N-diisopropyl)]-phosphoramidite) dT residue) is incorporated near the 3' end (e.g., 2, 3, 4 or 5 bases away from the 3' end of the oligonucleotide substrate). To a PCR reaction mixture (e.g., 50 mM Tris-HCl, pH 8.3, 50 mM NaCl, 5 mM MgCh, and 2 pL of 2 mM dATP), a polymerase preparation is added wherein the polymerase is in the presence or absence of the inhibitor aptamer. In some embodiments comprising both polymerase and inhibitor aptamer, about 0.8 pM polymerase is mixed with 8 pM aptamer (1:10 ratio). In other embodiments, the polymerase / aptamer ratio is about 1:5 to 1: 15 or about 1:5 or 1: 15. After addition of polymerase to the PCR reaction mix containing the labeled hairpin substrate, the reaction is heated from about 25°C to 75°C and the FAM signal is measure using a standard dissociation curve program (e.g., Mx3005P, Agilent Technologies) to determine the temperature at which the polymerase is active, and the temperature below which the polymerase is inhibited by the aptamer.

[0085] Another method for measuring thermostable polymerase activity in the presence and absence of an aptamer involves use of an oligonucleotide substrate for the polymerase which forms a hairpin structure having a stem and a loop, wherein the stem has a long single-stranded portion which terminates at the 3' end. A quencher (e.g., N, N'-tetramethyl-rhodamine, TAMRA) is attached to the stem near the 5' end of the loop and a fluorescent dye (e.g., carboxyfluorescein, FAM) is attached to the stem near the 3' end of the loop. The fluorescence of the dye is quenched through resonanceenergy transfer by the quencher when the aptamer substrate is in its predicted folded structure until a oligonucleotide primer is added and allowed to anneal to the 3' end of the stem single-stranded portion and is extended to synthesize the complementary strand by the polymerase, resulting in opening of the loop and spatial separation of the quencher and dye. The polymerase and aptamer are mixed in an appropriate solution of reagents at least including nucleotide triphosphates, a divalent metal cation, an appropnate buffer and primer and fluorescence is measured over time, with fluorescence increasing at a rate proportional to the polymerase activity. The kinetics of enzymatic DNA synthesis exhibit Michaelis-Menten dependence on substrate concentration. Accordingly, inhibition profiles for various aptamers can be generated by running a series of polymerase reactions in the presence of increasing amounts of aptamer. The resulting data can be used to determine an IC50 value for each aptamer (Summerer and Marx, Angew Chem Int, 2002, 41:3620-3622). Moreover, IC50 values for each aptamer can be determined over a range of temperatures.

[0086] A third alternative method for measuring the ability of an aptamer as described herein to inhibit a thermostable polymerase in a temperature-dependent manner is by using a template oligonucleotide DNA molecule which is labeled at its 5' end with32P-y-ATP and which forms a hairpin wherein a 5' portion (e.g., 12-24 nucleotides) of the oligonucleotide is single-stranded followed by a double-stranded stem and small loop. As above, the thermostable polymerase is incubated with varying concentrations of an aptamer as described herein, then added to the substrate and standard PCR reaction mixture and the PCR reaction is allow ed to proceed for a set period of time (e.g., 45 min. to 1 hour) at different temperatures (e.g., 25°C to 75°C), stopped by the addition of EDTA, and then resolved using polyacrylamide gel electrophoresis. Standard phosphorimager detection and quantitation methods can be used to determine polymerase activity level as only when the polymerase is active (not inhibited by the aptamer at a given temperature) will the 3' end of the hairpin substrate be extended to form a longer product which is radio-labeled and detected. As with the assays above, an IC50 for each aptamer at a given temperature can be determined by quantitation and analysis.

[0087] The use of the aptamers described herein for hot start polymerase applications w hich can decrease nonspecific product generation relies on the ability of the aptamer to dissociate from the polymerase as the temperature. Accordingly, an aptamer according to the present disclosure degrades or dissociates from a thermostablepolymerase when a reaction mixture containing the aptamer and the polymerase is raised to a temperature of at least about 45°C. 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, or 80°C. As a loss of inhibition of the polymerase is a likely indication of aptamer degradation or dissociation from the polymerase, in other embodiments, a reversibly inhibitory' aptamer of the present disclosure does not inhibit thermostable polymerase activity when the reaction mixture containing the aptamer and the polymerase is raised to a temperature of at least about 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, or 80°C. It is understood that the temperature above which an aptamer no longer inhibits polymerase activity7depends on the sequence, composition and inherent structure of the aptamer in solution with the polymerase. Accordingly, any aptamer sequence as described herein may be further characterized or defined by its lack of polymerase activity inhibition at or above a specified temperature.

[0088] Moreover, any aptamer sequence and predicted structure as described herein may be further characterized or defined by its ability' to inhibit polymerase activity at or below a specified temperature. In some embodiments, the aptamer inhibits the polymerase by 95%-100%, 98% to 100% or by about 100% relative to polymerase activity' in the absence of the aptamer at or below a temperature of about 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, or 65°C. As the temperature of the mixture containing the aptamer and the polymerase is raised, there will be a corresponding loss of inhibition. Experimental studies suggest that a transition from about 95% to 100% of polymerase activity to loss of substantially all inhibition can occur over a temperature range of about 4°C to 10°C, or 5°C to 7°C. Inhibition of polymerase activity by an aptamer as described herein can also be affected by the concentration of the aptamer and polymerase in a mixture or by the ratio of aptamer: polymerase. For example, the aptamer is effective in inhibiting polymerase activity when the aptamer is present in the mixture at a concentration of about 100 nM to 1000 nM, 100 nM to 500 nM, 25 nM to 750 nM, for 500 nM to 1000 nM. The aptamers described are useful for reversibly inhibiting thermostable polymerases including DNA polymerases, RNA polymerases and reverse transcriptases.

[0089] The ability of the aptamers provided herein to inhibit polymerase activity7at ambient temperatures or at temperatures lower than temperatures which are optimal for polymerase activity' is advantageous for at least the reason that there will be a decrease in or elimination of non-specific products generated at these lower temperatures. Theuse of the aptamers can reduce generation of non-specific products and increase production of the target amplicon(s).IV. Applications

[0090] As described above, aptamers of the present disclosure have the ability to reversibly inhibit thermostable polymerases at ambient temperatures or temperatures below the optimal temperatures for various thermostable polymerases. These aptamers thus can readily be applied to any applications wherein a thermostable nucleotide polymerase is used and wherein it is desirable to have the polymerase activity turned off at a lower temperature but then be able to regain its activity at a higher temperature (hot start). Such applications include but are not limited to standard PCR, reverse transcriptase PCR. in-situ PCR, quantitative PCR, and minisequencing or other reactions involving primer extension in which a thermostable polymerase present and a hot-start method is advantageous. Such methods are well known in the art. In some embodiments, the increased sensitivity and specificity provided by the disclosed compositions is useful during the amplification and analysis of DNA and RNA in medical genetics research and diagnosis, pathogen detection, forensic analysis, and animal and plant genetics applications. The methods and compositions of the present disclosure are useful in any polynucleotide synthesis reaction that requires thermostable polymerase to cycle anywhere between 40°C and 80°C.

[0091] In PCR experiments using Taq polymerase in the presence or absence of an aptamer according to the present disclosure, the aptamers can both decrease generation of non-specific amplification products as well as increase quantities of target amplicons in the reaction mix. It is contemplated that any of the aptamers described herein which have melting temperatures below the optimal temperature of the thermostable polymerase in the reaction mix can enhance target amplicon generation and detection. Accordingly, contemplated herein are methods which involve using a thermostable polymerase to extend a primer which is annealed to a nucleotide template or substrate molecule. As well understood in the art, wherein the primer is extended by the polymerase in the presence of nucleotides (e.g., the deoxy ribonucleotides, dATP, dGTP, dCTP, dTTP), a divalent metal cation such as Mg2+or Mn2+, a buffer, and at least one primer.

[0092] In some embodiments, the inhibitors described herein may be used in combination with other hot-start technologies such as antibody-based hot start. In otherembodiments, for example, two or more aptamers which dissociate from a polymerase at different temperatures may be combined in a single reaction.V. Polymerase

[0093] As described herein, the disclosed methods make the use of a polymerase for amplification. In some embodiments, the polymerase is a DNA polymerase that lacks a 5' to 3' exonuclease activity. Exemplary DNA polymerases include variants of Taq DNA polymerase that lack 5' to 3' exonuclease activity, e.g., the Stoffel fragment of Taq DNA polymerase (ABI), SD polymerase (Bioron), mutant Taq lacking 5' to 3' exonuclease activity described in U. S. Pat. No. 5,474,920, Bea polymerase (Takara), Pfx50 polymerase (Invitrogen), Tfu DNA polymerase (Qbiogene), modified, truncated Taq DNA polymerase such as Klentaq. Other exemplary DNA polymerases include inhibition resistant DNA polymerases such as OmniTaq, and reverse transcription and PCR in one enzy mes such as OmniTaq 2. If thermocycling is to be carried out (as in PCR), the DNA polymerase is preferably a thermostable DNA polymerase. In some embodiments, it can be advantageous to use a blend of two or more polymerases. For example, an illustrative polymerase blend includes a polymerase that is particularly proficient at initiating extension from a partially double-stranded DNA primer and a polymerase that is particularly proficient at strand displacement synthesis, since combining these properties may provide a net advantage in some embodiments.Alternatively or in addition, where it is desirable to use a Taqman-style probe to carry our real-time PCR, a polymerase blend can include a polymerase that has 5' to 3' exonuclease activity, provided the primer structure is designed so that it is not susceptible to “flap’" endonuclease activity; indeed, the structures described herein may be inherently less susceptible to this activity because of the double-stranded nature of the ‘‘flap.” Taq DNA polymerase can, for example, be employed in such polymerase blends because, although it is described as including a 5' to 3' exonuclease activity, Taq DNA polymerase operates more like a flap endonuclease. US 11,352,622B2, incorporated herein by reference, provides several examples thermostable Stand-Displacing Polymerases Lacking 5' to 3' Exonuclease Activity.

[0094] In some embodiments, the DNA polymerase comprises a fusion between Taq polymerase and a portion of a topoisomerase, e.g., TOPOTAQ™ (Fidelity Systems, Inc.). Illustrative polymerase concentrations range from about 20 to 200 units per reaction, e.g., for SD polymerase. In various embodiments, the polymerase concentration can be at least: 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140,150, 160, 170, 180, 190, or 200 or more units per reaction. In some embodiments, the polymerase concentration falls within a range bounded by any of these values, e.g., 10-200, 10-150, 10-100, 10-50, 20-150, 20-100, 20-50, 50-200, 50-150, 50-100, 100-200, 100-150, etc. units per reaction. When polymerase blends are used, the total, combined polymerase concentration can be any of these values or fall within any of these ranges. Strand displacement can also be facilitated through the use of a strand displacement factor, such as a helicase. Any DNA polymerase that can perform strand displacement in the presence of a strand displacement factor is suitable for use in the disclosed method, even if the DNA polymerase does not perform strand displacement in the absence of such a factor. Strand displacement factors useful in the methods described herein include BMRF1 polymerase accessory subunit (Tsurumi et al., J. Virology 67(12):7648-7653 (1993)), adenovirus DNA-binding protein (Zijderveld and van der Vliet, J. Virology 68(2): 1158-1164 (1994)), herpes simplex viral protein ICP8 (Boehmer and Lehman, J. Virology 67(2):711-715 (1993); Skaliter and Lehman, Proc. Natl. Acad. Sci. USA 91(22): 10665-10669 (1994)), single-stranded DNA binding proteins (SSB; Rigler and Romano, J. Biol. Chem. 270:8910-8919 (1995)), and calf thymus helicase (Siegel et al., J. Biol. Chem. 267:13629-13635 (1992)). Helicase and SSB are available in thermostable forms and therefore suitable for use in PCR.VI. Amplification

[0095] For amplification in any of the methods described herein, primers and any other appropriate oligonucleotides are contacted with sample nucleic acids under conditions wherein the primers anneal to their template strands, if present. In some embodiments, the amplification step is performed using PCR. Illustrative PCR reaction mixtures generally contain an aptamer, an appropriate buffer, a source of magnesium ions (Mg2+) in the range of about 1 to about 10 mM, e.g., in the range of about 2 to about 8 mM, nucleotides, and optionally, detergents, and stabilizers. An example of one suitable buffer is TRIS buffer at a concentration of about 5 mM to about 85 mM, with a concentration of 10 mM to 30 mM preferred. In one embodiment, the TRIS buffer concentration is 20 mM in the reaction mix double-strength (2X) form. The reaction mix can have a pH range of from about 7.5 to about 9.0, with a pH range of about 8.0 to about 8.5 as typical. Concentration of nucleotides can be in the range of about 25 mM to about 1000 mM, typically in the range of about 100 mM to about 800 mM. Examples of dNTP concentrations are 100, 200, 300, 400, 500. 600, 700. and 800 mM. Detergents such as Tween 20, Triton X 100, and Nonidet P40 may also be included inthe reaction mixture. Stabilizing agents such as dithiothreitol (DTT, Cleland's reagent) or 2-mercaptoethanol may also be included. In addition, master mixes may optionally contain dUTP as well as uracil DNA glycosylase (uracil-N-glycosylase, UNG). A master mix is commercially available from Applied Biosystems, Foster City, Calif, (TaqMan® Universal Master Mix, cat. nos. 4304437, 4318157, and 4326708).VII. Sample

[0096] In one embodiment of the systems and methods as disclosed herein, the sample is selected from tissue(s) samples, cells, biological fluid samples (e.g., blood, urine, saliva, lymphatic fluid, cerebrospinal fluid (CSF), amniotic fluid, pleural fluid, pericardial fluid, ascites, aqueous humor), bone marrow samples, semen samples, biopsy samples, cancer samples, tumor samples, cell lysate samples, forensic samples, archaeological samples, paleontological samples, infection samples, production samples, whole plants, plant parts, microbiota samples, viral preparations, soil samples, marine samples, freshwater samples, household or industrial samples, and combinations and isolates thereof. In one embodiment of the systems and methods as disclosed herein, the sample is a cell (e.g.. an animal cell [e.g., a human cell], a plant cell, a fungal cell, a bacterial cell, and a protozoal cell). In one specific embodiment, the cell is lysed prior to the replication. In one specific embodiment, cell lysis is accompanied by proteolysis. In one specific embodiment, the cell is selected from a cell from a preimplantation embryo, a stem cell, a fetal cell, a tumor cell, a suspected cancer cell, a cancer cell, a cell subjected to a gene editing procedure, a cell from a pathogenic organism, a cell obtained from a forensic sample, a cell obtained from an archeological sample, and a cell obtained from a paleontological sample. In one embodiment of any of the systems and methods as disclosed herein, the sample is a cell from a preimplantation embryo (e.g., a blastomere). In one specific embodiment, the method further comprises determining the presence of disease predisposing germline or somatic variants in the embryo cell. In one embodiment of any of the systems and methods as disclosed herein, the sample is a cell from a pathogenic organism (e.g., a bacterium, a fungus, a protozoan). In one specific embodiment, the pathogenic organism cell is obtained from fluid taken from a patient, microbiota sample (e.g., GI microbiota sample, vaginal microbiota sample, skin microbiota sample, etc.) or an indwelling medical device (e.g., an intravenous catheter, a urethral catheter, a cerebrospinal shunt, a prosthetic valve, an artificial joint, an endotracheal tube, etc.). In one specific embodiment, the method further comprises the step of determining the identity of thepathogenic organism. In one specific embodiment, the method further comprises determining the presence of genetic variants responsible for resistance of the pathogenic organism to a treatment. In one embodiment of any of the systems and methods as disclosed herein, the sample is a tumor cell, a suspected cancer cell, or a cancer cell. In one specific embodiment, the method further comprises determining the presence of one or more diagnostic or prognostic mutations. In one specific embodiment, the method further comprises determining the presence of germline or somatic variants responsible for resistance to a treatment. In one embodiment of any of the systems and methods as disclosed herein, the sample is a cell subjected to a gene editing procedure. In one specific embodiment, the method further comprises determining the presence of unplanned mutations caused by the gene editing process. In one embodiment of any of the systems and methods as disclosed herein, the method further comprises determining the history of a cell lineage. In a related aspect, the invention provides a use of any of the systems and methods as disclosed herein for identifying low frequency sequence variants (e.g., variants which constitute ≥0.01% of the total sequences).VIII. Kits

[0097] Kits for detecting a nucleic acid target sequence present in solid, semi- solid, or liquid biological samples are also provided. The kit includes a reagent mix comprising a thermostable polymerase and an aptamer as described herein that can reversibly inhibit activity of the polymerase. In some embodiments, the kit can be used for detection, quantify ing or sequencing any target nucleic acid. Alternatively, the kit includes one or more oligonucleotide primers (e.g., a forward and / or reverse primer) that specifically hybridize to a specified target nucleic acid and may also include labeled primers and / or probes as is routine in the art.

[0098] The kits may include instructions for obtaining biological samples and contacting them with sample buffer, for mixing the samples w ith sample buffer, placing labels on the apparatus and recording relevant test data; for shipping the apparatus, and the like. The kits may include instructions for reading and interpreting the results of an assay. The kits may further comprise reference samples that may be used to compare test results with the specimen samples.IX. Exemplary Automation and Systems

[0099] In some embodiments, a target nucleic acid sequence is copied or amplified, then detected using an automated sample handling and / or analysis platform. In someembodiments, commercially available automated analysis platforms are utilized. For example, in some embodiments, the GeneXpert® system (Cepheid, Sunnyvale, Calif.) is utilized. The present disclosure is illustrated for use with the GeneXpert system. Exemplar}7sample preparation and analysis methods are described below. However, the present disclosure is not limited to a particular detection method or analysis platform. One of skill in the art recognizes that any number of platforms and methods may be utilized.

[0100] The GeneXpert® utilizes a self-contained, single use cartridge. Sample extraction, amplification, and detection may all be carried out within this self-contained “laboratory in a cartridge.” (See e.g., U. S. Pat. Nos. 5,958,349; 6,403,037; 6,440,725; 6,783,736; and 6,818, 185; each of which is herein incorporated by reference in its entirety.) Components of the cartridge include, but are not limited to, processing chambers containing reagents, filters, and capture technologies useful to extract, purify, and amplify' target nucleic acids. A valve enables fluid transfer from chamber to chamber and contains nucleic acids lysis and filtration components. An optical window enables real-time optical detection. A reaction tube enables very rapid thermal cycling. In some embodiments, the GenXpert® system includes a plurality of modules for scalability. Each module includes a plurality7of cartridges, along w ith sample handling and analysis components.

[0101] After the sample is added to the cartridge, the sample is contacted with lysis buffer and released DNA is bound to a DNA-binding substrate such as a silica or glass substrate. The sample supernatant is then removed and the DNA eluted in an elution buffer such as a Tris / EDTA buffer. The eluate may then be processed in the cartridge to detect target genes as described herein. In some embodiments, the eluate is used to reconstitute at least some of the PCR reagents, which are present in the cartridge as lyophilized particles.

[0102] In some embodiments, PCR is used to amplify and detect the presence of the target nucleic acid sequence and / or a nucleic acid sequence that indicates genomic copy number. In some embodiments, the PCR uses Taq polymerase with hot start function imparted by use of an aptamer as provided herein.

[0103] The invention will be illustrated in more detail with reference to the following Examples, but it should be understood that the present invention is not deemed to be limited thereto.EXAMPLESExample 1. Oligonucleotide Synthesis

[0104] Aptamers as described herein were synthesized using standard oligonucleotide synthesis methods. Oligonucleotide synthesis was performed on aMerMade 12 DNA Synthesizer (BioAutomation). Standard phosphoramidite synthesis cycles were used, and coupling time was increased to 360 seconds for modified phosphorami dites.Cleavage from the solid support and deprotection was carried in concentrated aqueous ammonia at RT for 24 hrs. HPLC analyses were done on an Agilent 1100 instrument equipped with a quaternary pump, autosampler, and diode array detector.Oligonucleotides were analyzed using reversed-phase HPLC (RP HPLC) on a C 18 Gemini column (4.6 mm x 250 mm. 5 um, Phenomenex) eluting with a linear gradient of acetonitrile / 0.1 M triethylammonium bicarbonate, pH 7: 16-23% acetonitrile over 20 min for DMT-on oligonucleotides and 7-14% acetonitrile for oligonucleotides with DMT groups removed (DMT-off). DMT-on oligonucleotides were purified using reverse phase HPLC, DMT groups were cleaved, and the final oligonucleotide products were isolated by ethanol precipitation and quantified by UV.

[0105] Where indicated, some aptamers were modified at the 5' and / or 3' end after synthesis. 5' and 3' phosphorylation was achieved using (3-(4.4'-Dimcthoxytrityloxy)-2,2-(dicarboxymethylamido)propyl-l-O-succinoyl-long chain alkylamino-CPG) (Glen Research Cat. No. 10-1901; U. S. Pat. No. 5,959,090 and EP Pat. No. EP0816368) and (3-(4,4'-Dimethoxytrityloxy)-2,2-dicarboxyethyl]propyl-(2-cyanoethyl)-(N, N-diisopropyl)-phosphoramidite) (Glen Research Cat. No. 20-2903; U. S. Pat. No.5,959,090). Modification of the 5' end with an amino group was achieved using 5'-amino-dT-CE phosphoramidite (Glen Research Cat. No. 10-1932), (5'-monomethoxytritylamino-2'-deoxy Thymidine, 3'-[(2-cyanoethyl)-(N, N-diisopropyl)]-phosphoramidite). Modification of the 3' end with a propanediol spacer CPG (-C3) was achieved using ((l-Dimethoxytrityloxy-propanediol-3-succinoyl)-long chain alkylamino-CPG) (Cat. No. 20-2913).Polymerase Inhibition

[0106] Studies are done to measure the inhibitory activity of aptamers described herein as a function of temperature. Polymerase activity of Taq polymerase in the presence of aptamers is assayed by a method described in the art (Nikiforov T., Analytical Biochemistry, 412: 229-236 (2011)) with the following modification. Because theaptamers of the present invention inhibit Taq polymerase activity above ambient temperature, the structure of the hairpin substrate is modified to increase its Tm.

[0107] To a solution of 150 nM of the hairpin oligonucleotide (Table 5) in 50 mMTris-HCl, 50 mM NaCl, 5 mM MgCl₂, and Taq polymerase, 2 pL of 2 mM dATP is added, and the FAM signal is monitored while the reaction mixture is heated from 25°C to 74°C using the standard dissociation curve program (Mx3005P, Agilent). The assays measure the reversible inhibition activity of the aptamers whereby polymerase activity is regained upon heating of the reaction mixture. The temperature at which inhibition is lost depends on the sequence and structure of the aptamer. Results are presented below in Table 5 and Figures 1-4.Table 5: Hairpin Aptamer Sequences. Nucleotides with 5-propynyl-2'-deoxyuridine are marked as U* and nucleotides with deoxyuridine are marked as U.SEQ ID NO Tm Linear Oligonucleotide Sequence 5’“^ 3’ (including terminal modifications (°C) if present)71GTTTTTATTTTTAAAAAATATTCTTAGCGTTTTATTTTTTAAAAATAATTCTSymmetrical TAGC-p55GTTTTTATTTTTTAAAAATATTCTTAGCGTTTTATTTTTAAAAAATAATTCTAsymmetrical 57.5 TAGC-p56GTTTTTATTTTTTAAAAATATU*CTTAGCGTTTTATTTTTAAAAAATAATTCAsymmetrical 57.5 TTAGC-p57GTTTTTATTTTTTAAAAATATU*CTTAGCGTTTTATTTTTAAAAAATAATU*C57.5Asymmetrical TTAGC-p58 GTTTGTATTTTTTAAAAATATU’CTTAGCGTTTTATTTTTAAAAAATACTU* Asymmetrical CTTAGC-p59 GTTTCTATTTTTTAAAAATATVCTTAGCGTTTTATTTTTAAAAAATAGTU* Asymmetrical CTTAGC-p60 GTTTGCATTTTTTAAAAATATU*CTTAGCGTTTTATTTTTAAAAAATGCTU* Asymmetrical CTTAGC-p61 GTTTCGATTTTTTAAAAATATU’CTTAGCGTTTTATTTTTAAAAAATCGTU* Asymmetrical CTTAGC-p62 GTTTTCATTTTTTAAAAATATU*CTTAGCGTTTTATTTTTAAAAAATGATU* Asymmetrical CTTAGC-p63 QTTTTGATTTTTTAAAAATAWCTTACTCCTTTTTATTTTTAAAAAATCATU* Asymmetrical 60.5 CTTAGC-p64 GTTTTGATTTTTTAAAAATATUU*CTTAGCGTTTTATTTTTAAAAAATCATU* Asymmetrical 60.5 CTTAGC-p65 GTTTTGATTTTTTAAAAATATU*CTTAGCGTTTTATTTTTAAAAAATCAUU* Asymmetrical 60.5 CTTAGC-p66 GTTTTGATTTTTTAAAAATATUU*CTTAGCGTTTTATTTTTAAAAAATCAUU* Asymmetrical 60.5CTTAGC-p67 GTTTTGATTTTTTAAAAATATU*CTTAGCGUTTTTATTTTTAAAAAATCATU* Asymmetrical 60.5 CTTAGC-p68 GTI ITGA 1TI ITI AAAAATAI I * Cl T AGCG'l 1 t 1 A Tl 1 T 1 AAAAAATCA 1 I ‘ Asymmetrical 60.5 CTTAGC-p69 GTTU TGATTTTTTAAAAATATU*CTTAGCGTTU TATTTTTAAAAAATCATU* Asymmetrical 60.5 CTTAGC-p70 GTTTTGATTTTTTAAAAATATUU*CTTAGCGTTU TATTTTTAAAAAATCATU* Asymmetrical 60.5CTTAGC-pApplication of Hairpin Aptamers Containing Deoxyuridine Nucleotide within the Loop and / or Arm Sequence to Control Polymerase Activity of Taq Polymerase

[0108] This working example shows application of hairpin-like aptamers containing deoxyuridine nucleotide within the loop sequence to control activity of Taq polymerase during PCR.

[0109] UDG catalyzes dU and creates an abasic site. UDG is a thermostable enzyme.Table 5 provides aptamer showing location of dU. dU can be located in the loop or / and in the arm sequences. Not all these dT to dU replacements are equal by efficiency of UDG catalysis and / or suppression of hot-start properties of the aptamer as shown in Figure 5 and 6. Figure 5 shows dU incorporation can improve hot-start, be neutral or reduce hot-start performance. At some locations dU catalysis demonstrates veryeffective suppression of the hot-start as shown in Figure 6. dU degradation was performed in the presence of 0.025 U / uL UDG, at a temperature of 55°C for 1 minute.The reaction was prepared and cooled on ice.

[0110] While the invention has been described in detail and with reference to specific examples thereof, it will be apparent to one skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope thereof.CLAUSES

[0111] The following clauses further define embodiments of the disclosure.1. An aptamer comprising in a 5' to 3' direction:a 5' terminus sequence at least three nucleotides in length,a first stem sequence comprising SEQ ID NO.:1 or a variant thereof, or SEQ ID NO.:2 or a variant thereof,a loop sequence comprising SEQ ID NO.:3 or a variant thereof,a second stem sequence comprising SEQ ID NO.:1 or a variant thereof or SEQ ID NO.:2 or a variant thereof, anda 3' terminus sequence at least three nucleotides in length,wherein each of the first and second stem sequences are 100% complementary to each other along their entire length, andwherein the aptamer comprises at least one unnatural nucleotide substitution, wherein the unnatural nucleotide substitution comprises a hydrophobically modified base.2. The aptamer of clause 1, wherein the hydrophobically modified base is a modified uridine base.3. The aptamer of clause 1 or 2, wherein the hydrophobically modified base comprises a C1-C5 alkyl modification, C2-C5 alkenyl modification, or C2-C5 alkynyl modification, preferably a propynyl modification.4. The aptamer of any one of clauses 1-3, wherein the aptamer comprises 1, 2. 3, or 4 hydrophobically modified bases.5. The aptamer of any one of clauses 1-4, wherein the 5' terminus sequence and / or 3' terminus sequence comprises 1, 2, 3, or 4 hydrophobically modified bases.6. The aptamer of any one of clauses 1-5, wherein the 5' terminus sequence and 3' terminus sequence comprises GTTT, TTCTTAGC, or a combination thereof.7. The aptamer of any one of clauses 1-6, wherein the 5' terminus sequence is not complementary or equal in length to the 3' terminus sequence.8. The aptamer of any one of clauses 1-7, wherein each of the first and second stem sequences are each 14-18 nucleotides in length.9. The aptamer of any one of clauses 1-8. wherein the first stem sequence comprises a deoxy guanidine nucleotide near or at its 5' end, preferably at positions 1, 2, or 3 of SEQ ID NO.:1, more preferably at position 2 of SEQ ID NO.:1.10. The aptamer of any one of clauses 1-9, wherein the first stem sequence comprises of SEQ ID NO.: 1 and the second stem sequence comprises of SEQ ID NO.:2.11. The aptamer of any one of clauses 1-10, wherein the loop sequence is 10-14 nucleotides in length.12. The aptamer of any one of clauses 1-11, wherein the loop sequence comprises 1, 2, 3, or 4 hydrophobically modified bases.13. The aptamer of any one of clauses 1-12, wherein the aptamer further comprises a 5' and / or a 3' capping moiety.14. The aptamer of any one of clauses 1-13, wherein the 3' terminus is linked to a 3' capping moiety.15. The aptamer of any one of clauses 1-14. wherein the 5' terminus is linked to a 5' capping moiety.16. The aptamer any one of clauses 1-15, wherein the 3' terminus is linked to a 3' capping moiety and the 5' terminus is linked to a 5' capping moiety.17. The aptamer of any one of clauses 13-16, wherein the 3' capping moiety is a propanediol spacer (C3), a phosphate, or an amino group.18. The aptamer of any one of clauses 1-17, wherein the aptamer is modifiable by uracil-DNA glycosylase enzymatic activity.19. The aptamer of clause 18, wherein the aptamer comprises a deoxyuridine nucleotide.20. The aptamer of clause 18 or 19, wherein one or more of SEQ ID NOs.:l-5 comprise 1, 2, 3. or 4 nucleotide substitutions having deoxy uridine nucleotide.21. The aptamer of any one of clauses 19-20, wherein the 5' terminus sequence and / or 3' terminus sequence comprises 1, 2, 3, or 4 nucleotide substitutions having deoxyuridine nucleotide.22. The aptamer of any one of clauses 19-21, wherein the loop sequence. SEQ ID NO.:3, comprises 1, 2, 3, or 4 nucleotide substitutions having deoxyuridine nucleotide.23. The aptamer of any one of clauses 1-22, wherein the aptamer comprises one or more of SEQ ID NOs.:55-70.24. The aptamer of any one of clauses 1-23, wherein the aptamer comprises a hairpin structure.25. The aptamer of any one of clauses 1-24, wherein the aptamer does not inhibit reverse transcriptase activity.26. A composition comprising an aptamer according to any one of clauses 1 -25 and a thermostable polymerase.27. The composition of clause 26, wherein the polymerase is selected from the group consisting of Thermus aquaticus, Thermus thermophilus, or Thermus maritima.28. The composition of clause 26 or 27, further comprising deoxyribonucleotide triphosphates and a divalent metal cation.29. The composition of any one of clauses 26-28, further comprising a forward primer specific for a target nucleic acid.30. The composition of any one of clauses 26-29, further comprising a reverse primer specific for the target nucleic acid.31. The composition of any one of clauses 26-30, wherein the ratio of the aptamer to the polymerase is >1:1 such as ranging from about 5:1 to 20:1.32. The composition of any one of clauses 26-31, wherein the composition is a dried composition.33. The composition of any one of clauses 26-31, wherein the composition is a liquid composition.34. A method for extending a primer, comprising:contacting a sample that may or may not comprise a target nucleic acid with a reagent composition to generate a reaction mixture, wherein the reagent composition comprises an aptamer according to any one of clauses 1-25, a thermostable DNA polymerase, a forward primer specific for the target nucleic acid, deoxyribonucleotide triphosphates, and a divalent metal cation.35. A method for amplifying a target DNA sequence, the method comprising: (a) providing a reaction mixture comprising:(i) a thermostable DNA polymerase,(ii) an aptamer according to any one of clauses 1-25, wherein the aptamer binds to the thermostable DNA polymerase to form a blocked thermostable DNA polymerase, (iii) at least one oligonucleotide primer, and(iv) one or more sample nucleic acids that may or may not comprise a target sequence complementary to the at least one oligonucleotide primer;(b) hybridizing the at least one oligonucleotide primer to the sample nucleic acids that comprise a target DNA sequence complementary to the at least one oligonucleotide primer;(c) elevating the temperature to release the aptamer from the blocked thermostable DNA polymerase; and(d) initiating DNA polymerase activity and extending the primer with the DNA polymerase.36. The method of clause 35, wherein the elevated temperature to release the aptamer from the blocked thermostable DNA polymerase is greater than 45°C.37. The method of clause 35 or 36, wherein the elevated temperature to release the aptamer from the blocked thermostable DNA polymerase is from 55-65°C.38. The method of any one of clauses 35-37, wherein the initiating DNA polymerase activity and extending the primer with the DNA polymerase is performed at a temperature of greater than 65°C.39. The method of any one of clauses 34-38. wherein the reaction mixture further comprises, a buffer mixture and dNTPs.40. The method of clause 39, wherein:the buffer mixture comprises: 10 mM Tris-HCl (pH 8.4 at 25° C), 1.5 mM MgCl₂, 110 mM KCl, 0.08% (w / v) Brij-58, 0.2% (w / v) PEG-8000, and 0.1% (v / v) propylene glycol); andthe dNTPs comprise 0.8 mM dNTPs.41. The method of any one of clauses 34-40, wherein the reaction mixture further comprises a reverse primer specific for the target nucleic acid.42. The method of any one of clauses 34-41, wherein the reaction mixture further comprises a reverse transcriptase and wherein the method further comprises performing a reverse transcriptase reaction prior to heating the reaction mixture.43. The method of any one of clauses 34-42, wherein the DNA polymerase is selected from the group consisting of Thermus aquaticus, Thermus thermophilus, or Thermus maritima.44. The method of any one of clauses 34-43, wherein the ratio of the aptamer to the polymerase is >1: 1 such as ranging from about 5: 1 to 20: 1.45. A kit comprising a composition comprising an aptamer according to any one of clauses 1-25 and a thermostable polymerase.46. The kit of clause 45, further comprising deoxyribonucleotide triphosphates and a divalent metal cation.47. The kit of clause 45 or 46, wherein the composition is a dried composition. 48. The kit of any one of clauses 45-47, further comprising an aqueous buffer. 49. The kit of any one of clauses 45-48, wherein the composition further comprises a forward primer specific for a target nucleic acid.50. The kit of any one of clauses 45-49, wherein the composition further comprises a reverse primer specific for the target nucleic acid.51. The kit of any one of clauses 45-50, further comprising a reverse transcriptase.

Claims

WHAT IS CLAIMED IS:

1. An aptamer comprising in a 5' to 3' direction:a 5' terminus sequence at least three nucleotides in length.a first stem sequence comprising SEQ ID NO: 1 or a variant thereof, or SEQ ID NO.:2 or a variant thereof,a loop sequence comprising SEQ ID NO.:3 or a variant thereof,a second stem sequence comprising SEQ ID NO.:1 or a variant thereof or SEQ ID NO.:2 or a variant thereof, anda 3' terminus sequence at least three nucleotides in length.wherein each of the first and second stem sequences are 100% complementary to each other along their entire length, andwherein the aptamer comprises at least one unnatural nucleotide substitution, wherein the unnatural nucleotide substitution comprises a hydrophobically modified base.

2. The aptamer of claim 1, wherein the hydrophobically modified base is a modified uridine base.

3. The aptamer of claim 1 or 2, wherein the hydrophobically modified base comprises a C1-C5 alkyl modification, C2-C5 alkenyl modification, or C2-C5 alkynyl modification, preferably a propynyl modification.

4. The aptamer of any one of claims 1-3, wherein the aptamer comprises 1, 2, 3, or 4 hydrophobically modified bases.

5. The aptamer of any one of claims 1-4. wherein the 5' terminus sequence and / or 3' terminus sequence comprises 1, 2, 3, or 4 hydrophobically modified bases.

6. The aptamer of any one of claims 1-5, wherein the 5' terminus sequence and 3' terminus sequence comprises GTTT, TTCTTAGC, or a combination thereof.

7. The aptamer of any one of claims 1-6, wherein the 5' terminus sequence is not complementary or equal in length to the 3' terminus sequence.

8. The aptamer of any one of claims 1-7, wherein each of the first and second stem sequences are each 14-18 nucleotides in length.

9. The aptamer of any one of claims 1-8, wherein the first stem sequence comprises a deoxy guanidine nucleotide near or at its 5' end, preferably at positions 1, 2, or 3 of SEQ ID NO.: 1, more preferably at position 2 of SEQ ID NO.: 1.

10. The aptamer of any one of claims 1-9, wherein the first stem sequence comprises of SEQ ID NO.:1 and the second stem sequence comprises of SEQ ID NO.:2.

11. The aptamer of any one of claims 1-10, wherein the loop sequence is 10-14 nucleotides in length.

12. The aptamer of any one of claims 1-11, wherein the loop sequence comprises 1, 2, 3, or 4 hydrophobically modified bases.

13. The aptamer of any one of claims 1-12, wherein the aptamer further comprises a 5' and / or a 3' capping moiety.

14. The aptamer of any one of claims 1-13, wherein the 3' terminus is linked to a 3' capping moiety.

15. The aptamer of any one of claims 1-14, wherein the 5' terminus is linked to a 5' capping moiety.

16. The aptamer any one of claims 1-15, wherein the 3' terminus is linked to a 3' capping moiety and the 5' terminus is linked to a 5' capping moiety.

17. The aptamer of any one of claims 13-1, wherein the 3' capping moiety is a propanediol spacer (C3), a phosphate, or an amino group.

18. The aptamer of any one of claims 1-17. wherein the aptamer is modifiable by uracil-DNA glycosylase enzymatic activity.

19. The aptamer of claim 18, wherein the aptamer comprises a deoxy uridine nucleotide.

20. The aptamer of claim 18 or 19, wherein one or more of SEQ ID NOs.:l-5 comprise 1, 2, 3, or 4 nucleotide substitutions having deoxyuridine nucleotide.

21. The aptamer of any one of claims 19-20, wherein the 5' terminus sequence and / or 3' terminus sequence comprises 1, 2, 3, or 4 nucleotide substitutions having deoxyuridine nucleotide.

22. The aptamer of any one of claims 19-21, wherein the loop sequence, SEQ ID NO.:3, comprises 1, 2, 3, or 4 nucleotide substitutions having deoxyuridine nucleotide.

23. The aptamer of any one of claims 1-22, wherein the aptamer comprises one or more of SEQ ID NOs.:55-70.

24. The aptamer of any one of claims 1-23, wherein the aptamer comprises a hairpin structure.

25. The aptamer of any one of claims 1-24, wherein the aptamer does not inhibit reverse transcriptase activity.

26. A composition comprising an aptamer according to any one of claims 1-25 and a thermostable polymerase.

27. The composition of claim 26. wherein the polymerase is selected from the group consisting of Thermus aquaticus, Thermus thermophilus, or Thermus maritima.

28. The composition of claim 26 or 27, further comprising deoxyribonucleotide triphosphates and a divalent metal cation.

29. The composition of any one of claims 26-28, further comprising a forward primer specific for a target nucleic acid.

30. The composition of any one of claims 26-29, further comprising a reverse primer specific for the target nucleic acid.

31. The composition of any one of claims 26-30, wherein the ratio of the aptamer to the polymerase is >1: 1 such as ranging from about 5: 1 to 20: 1.

32. The composition of any one of claims 26-31, wherein the composition is a dried composition.

33. The composition of claim of any one of claims 26-31, wherein the composition is a liquid composition.

34. A method for extending a primer, comprising:contacting a sample that may or may not comprise a target nucleic acid with a reagent composition to generate a reaction mixture, wherein the reagent composition comprises an aptamer according to any one of claims 1-25, a thermostable DNA polymerase, a forward primer specific for the target nucleic acid, deoxyribonucleotide triphosphates, and a divalent metal cation.

35. A method for amplifying a target DNA sequence, the method comprising: (a) providing a reaction mixture comprising:(i) a thermostable DNA polymerase,(ii) an aptamer according to any one of claims 1-25, wherein the aptamer binds to the thermostable DNA polymerase to form a blocked thermostable DNA polymerase, (iii) at least one oligonucleotide primer, and(iv) one or more sample nucleic acids that may or may not comprise a target sequence complementary to the at least one oligonucleotide primer;(b) hybridizing the at least one oligonucleotide primer to the sample nucleic acids that comprise a target DNA sequence complementary to the at least one oligonucleotide primer;(c) elevating the temperature to release the aptamer from the blockedthermostable DNA polymerase; and(d) initiating DNA polymerase activity and extending the primer with the DNA polymerase.

36. The method of claim 35, wherein the elevated temperature to release the aptamer from the blocked thermostable DNA polymerase is greater than 45°C.

37. The method of claim 35 or 36, wherein the elevated temperature to release the aptamer from the blocked thermostable DNA polymerase is from 55-65°C.

38. The method of any one of claims 35-37, wherein the initiating DNA polymerase activity and extending the primer with the DNA polymerase is performed at a temperature of greater than 65 °C.

39. The method of any one of claims 34-38, wherein the reaction mixture further comprises, a buffer mixture and dNTPs.

40. The method of claim 39, wherein:the buffer mixture comprises: 10 mM Tris-HCl (pH 8.4 at 25° C), 1.5 mM MgCl₂, 110 mM KCl, 0.08% (w / v) Brij-58, 0.2% (w / v) PEG-8000, and 0.1% (v / v) propylene glycol); andthe dNTPs comprise 0.8 mM dNTPs.

41. The method of any one of claims 34-40. wherein the reaction mixture further comprises a reverse primer specific for the target nucleic acid.

42. The method of any one of claims 34-41, wherein the reaction mixture further comprises a reverse transcriptase and wherein the method further comprises performing a reverse transcriptase reaction prior to heating the reaction mixture.

43. The method of any one of claims 34-42, wherein the DNA polymerase is selected from the group consisting of Thermits aquaticus, Thermus thermophilus, or Thermus maritima.

44. The method of any one of claims 34-43, wherein the ratio of the aptamer to the polymerase is >1: 1 such as ranging from about 5: 1 to 20: 1.

45. A kit comprising a composition comprising an aptamer according to any one of claims 1-25 and a thermostable polymerase.

46. The kit of claim 45, further comprising deoxyribonucleotide triphosphates and a divalent metal cation.

47. The kit of claim 45 or 46, wherein the composition is a dried composition.

48. The kit of any one of claims 45-47, further comprising an aqueous buffer.

49. The kit of any one of claims 45-48, wherein the composition further comprises a forward primer specific for a target nucleic acid.

50. The kit of any one of claims 45-49, wherein the composition further comprises a reverse primer specific for the target nucleic acid.

51. The kit of any one of claims 45-50, further comprising a reverse transcriptase.

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