INCORPORATION OF SEQUENCE LISTING

The contents of the text file named “0102384-002US1 Sequence Listing.txt,” which was created on Jun. 12, 2020, and is 8 KB in size, are hereby incorporated by reference in their entirety.

BACKGROUND

Aspects of the present invention relate generally to improved methods of blocking DNA polymerase activity with oligonucleotide aptamers at low reaction temperatures, and restoring the enzyme activity upon raising the reaction temperature (e.g., hot-start methods).

DNA polymerases are enzymes used for synthesis of DNA strands by primer extension, wherein the polymerase-catalyzed DNA synthesis may be initiated by oligonucleotide primers hybridized to a complementary template DNA. Initiating DNA synthesis from this template-hybridized primer, DNA polymerases create complementary DNA strands in the presence of corresponding nucleotide 5′-triphosphates. Sequence specificity of nucleotide polymerization, when the oligonucleotide primers bind exclusively to the desired sites and nowhere else, is an important requirement in many applications wherein DNA synthesis is used. However, the efficiency and fidelity of DNA synthesis can be reduced when primers hybridize to non-complementary DNAs, leading to synthesis of incorrect DNA sequences.

Many so-called ‘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 a number of 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, if not impossible to achieve, and the aptamer structure usually represents a compromise between these two key requirements. New methods, therefore, are needed to improve control of aptamer activity in reaction mixtures containing DNA polymerases.

Particular aspects provide methods of activating an aptamer-inactivated DNA polymerase, comprising: providing a reaction mixture suitable for DNA synthesis, the reaction mixture comprising (i) a DNA polymerase, (ii) an endonuclease V-cleavable circular oligonucleotide aptamer that binds to the DNA polymerase, wherein the oligonucleotide aptamer is present in an amount effective to inhibit DNA synthesis activity of the DNA polymerase in the reaction mixture, and (iii) an endonuclease V enzymatic activity; and cleaving the circular aptamer by the endonuclease V enzymatic activity to reduce or eliminate binding of the circular oligonucleotide aptamer to the DNA polymerase, thereby activating the DNA synthesis activity of the DNA polymerase, to increase DNA synthesis in the reaction mixture. In the methods, cleaving may be facilitated using a reaction temperature that facilitates both DNA polymerase activity and the endonuclease V enzymatic activity. In the methods, cleaving may be facilitated by increasing the temperature of the reaction mixture from a first temperature to a second temperature that more strongly facilitates the endonuclease V enzymatic activity. In the methods, providing a reaction mixture suitable for DNA synthesis may comprise dissolving a dried form of at least one of said (i) DNA polymerase, (ii) endonuclease V-cleavable circular oligonucleotide aptamer, and (iii) endonuclease V enzymatic activity into an aqueous solution. DNA synthesis in the methods may result in DNA amplification in the reaction mixture (e.g., wherein the DNA amplification comprises PCR) DNA synthesis in the methods may comprise an isothermal amplification reaction. The methods may comprise detecting the presence of a target DNA and/or measuring an amount of a target DNA in the reaction mixture. In the methods, the circular oligonucleotide aptamer may comprise one or more deoxyinosine nucleotides. In the methods, the circular oligonucleotide aptamer may have a stem-loop structure, wherein one or more deoxyinosine nucleotides may be incorporated into the stem segment of the stem-loop structure, and/or wherein one or more deoxyinosine nucleotides may be incorporated into the loop segment of the stem-loop structure. In the methods, the loop of the stem-loop structure, may, for example, comprise a nucleotide sequence selected from the group consisting of 5′-TTCITAGCGTTT-3′ (SEQ ID NO:19), 5′-TTCTIAGCGTTT-3′ (SEQ ID NO:20), 5′-TTCTTAICGTTT-3′ (SEQ ID NO:21), 5′-TTCIIAGCGTTT-3′ (SEQ ID NO:22), 5′-TTCITAICGTTT-3′ (SEQ ID NO:23), 5′-TTCTIAICGTTT-3′ (SEQ ID NO:24), and 5′-TTCIITAICGTTT-3′ (SEQ ID NO:25). In the methods, the loop of the stem-loop structure may, for example, comprise one of the nucleotide sequences 5′-TTCITAGCGTTT-3′ (SEQ ID NO:19), 5′-TTCTIAGCGTTT-3′ (SEQ ID NO:20) or 5′-TTCTTAICGTTT-3′ (SEQ ID NO:21). In the methods, the endonuclease V enzymatic activity may comprise, for example,Thermotoga maritimaendonuclease V enzymatic activity.

Additional aspects provide kits for activating an aptamer-inactivated DNA polymerase, comprising: an endonuclease V enzymatic activity; and a DNA polymerase-binding circular oligonucleotide aptamer cleavable by an endonuclease V enzymatic activity. In the kits, the circular oligonucleotide aptamer may comprise one or more deoxyinosine nucleotides. In the kits, the circular oligonucleotide aptamer may comprise a stem-loop structure. In the kits, one or more deoxyinosine nucleotides may be located in the stem segment of the stem-loop structure, and/or one or more deoxyinosine nucleotides may be located in the loop segment of the stem-loop structure. In the kits, the loop of the stem-loop structure, may, for example, comprise a nucleotide sequence selected from the group consisting of 5′-TTCITAGCGTTT-3′ (SEQ ID NO:19), 5′-TTCTIAGCGTTT-3′ (SEQ ID NO:20), 5′-TTCTTAICGTTT-3′ (SEQ ID NO:21), 5′-TTCIIAGCGTTT-3′ (SEQ ID NO:22), 5′-TTCITAICGTTT-3′ (SEQ ID NO:23), 5′-TTCTIAICGTTT-3′ (SEQ ID NO:24), and 5′-TTCIITAICGTTT-3′ (SEQ ID NO:25). In the kits, the loop of the stem-loop structure may, for example, comprise one of the nucleotide sequences 5′-TTCITAGCGTTT-3′ (SEQ ID NO:19), 5′-TTCTIAGCGTTT-3′ (SEQ ID NO:20) or 5′-TTCTTAICGTTT-3′ (SEQ ID NO:21). In the kits, the endonuclease V enzymatic activity may comprise, for example,Thermotoga maritimaendonuclease V enzymatic activity.

Further aspects provide reaction mixtures for use in a method of DNA synthesis, which reaction mixture comprises: a DNA polymerase; an endonuclease V-cleavable circular oligonucleotide aptamer that binds reversibly to the DNA polymerase, wherein the circular oligonucleotide aptamer is present in an amount effective to inhibit DNA synthesis activity of the DNA polymerase in the reaction mixture; and an endonuclease V enzymatic activity capable of cleaving (or effective to cleave) the circular oligonucleotide aptamer to reduce or eliminate binding of the circular oligonucleotide aptamer to the DNA polymerase, thereby activating the DNA synthesis activity of the DNA polymerase. In the reaction mixtures, the DNA polymerase activity and/or the endonuclease V enzymatic activity may be temperature-dependent. In the reaction mixtures, the endonuclease V enzymatic activity may increase when the reaction mixture is heated from a first temperature value to a second temperature value that promotes the endonuclease V enzymatic activity. In the reaction mixtures, the DNA polymerase, oligonucleotide aptamer, and endonuclease V enzymatic activity may be in a dried state. In the reaction mixtures, the circular oligonucleotide aptamer may comprise one or more deoxyinosine nucleotides. In the reaction mixtures, the circular oligonucleotide aptamer may comprise a stem-loop structure. In the reaction mixtures, one or more deoxyinosine nucleotides may be located in the stem segment of the stem-loop structure, and/or one or more deoxyinosine nucleotides may be located in the loop segment of the stem-loop structure. In the reaction mixtures, the loop of the stem-loop structure, may, for example, comprise a nucleotide sequence selected from the group consisting of 5′-TTCITAGCGTTT-3′ (SEQ ID NO:19), 5′-TTCTIAGCGTTT-3′ (SEQ ID NO:20), 5′-TTCTTAICGTTT-3′ (SEQ ID NO:21), 5′-TTCIIAGCGTTT-3′ (SEQ ID NO:22), 5′-TTCITAICGTTT-3′ (SEQ ID NO:23), 5′-TTCTIAICGTTT-3′ (SEQ ID NO:24), and 5′-TTCITAICGTTT-3′ (SEQ ID NO:25). In the reaction mixtures, the loop of the stem-loop structure may, for example, comprise one of the nucleotide sequences 5′-TTCITAGCGTTT-3′ (SEQ ID NO:19), 5′-TTCTIAGCGTTT-3′ (SEQ ID NO:20) or 5′-TTCTTAICGTTT-3′ (SEQ ID NO:21). In the reaction mixtures, the endonuclease V enzymatic activity may comprise, for example,Thermotoga maritimaendonuclease V enzymatic activity. The reaction mixtures may further comprise one or more of dATP, dCTP, dGTP, and/or dTTP, and/or Mg2+ion.

Yet further aspects provide circular oligonucleotide aptamers, comprising a nucleic acid sequence that forms a hairpin structure, having a stem and a loop portion, that binds to a DNA polymerase, wherein the stem and/or the loop portion comprises one or more deoxyinosine nucleotides, and wherein the loop portion comprises a nucleotide sequence 5′-TTCTTAGCGTTT-3′ (SEQ ID NO:18) that may be substituted with deoxyinosine at one or more of positions 4, 5, and 7. In the oligonucleotide aptamers, the loop portion may comprise the nucleotide sequence 5′-TTCTTAGCGTTT-3′ (SEQ ID NO:18). In the oligonucleotide aptamers, one or more deoxyinosine nucleotides may be located in the stem segment of the stem-loop structure, and/or one or more deoxyinosine nucleotides may be located in the loop segment of the stem-loop structure. In the oligonucleotide aptamers, the loop of the stem-loop structure, may, for example, comprise a nucleotide sequence selected from the group consisting of 5′-TTCITAGCGTTT-3′ (SEQ ID NO:19), 5′-TTCTIAGCGTTT-3′ (SEQ ID NO:20), 5′-TTCTTAICGTTT-3′ (SEQ ID NO:21), 5′-TTCIIAGCGTTT-3′ (SEQ ID NO:22), 5′-TTCITAICGTTT-3′ (SEQ ID NO:23), 5′-TTCTIAICGTTT-3′ (SEQ ID NO:24), and 5′-TTCIITAICGTTT-3′ (SEQ ID NO:25). In the circular oligonucleotide aptamers, the loop of the stem-loop structure may, for example, comprise one of the nucleotide sequences 5′-TTCITAGCGTTT-3′ (SEQ ID NO:19), 5′-TTCTIAGCGTTT-3′ (SEQ ID NO:20) or 5′-TTCTTAICGTTT-3′ (SEQ ID NO:21). In the oligonucleotide aptamers, the one or more deoxyinosine nucleotides may render the circular oligonucleotide aptamer cleavable by an endonuclease V enzymatic activity (e.g., wherein the endonuclease V enzymatic activity may compriseThermotoga maritimaendonuclease V enzymatic activity).

Still further aspects provide compositions comprising a DNA polymerase complexed with a circular oligonucleotide aptamer as disclosed herein.

