CROSS-REFERENCE TO A SEQUENCE LISTING

This application includes a sequence listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. This ASCII copy, created on Jun. 6, 2017, is named CPHDP012WO_2017-06-06_SeqList.txt and is 20795 bytes in size.

TECHNICAL FIELD

This disclosure relates generally to 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

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.

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 thermostable polymerase, 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.”

Hot start may be accomplished by various physical, chemical, or biochemical methods. In a physical hot start, the DNA polymerase or one or more reaction components that are essential for DNA polymerase activity is not allowed to contact the sample DNA until all the components required for the reaction are at a high temperature (Horton, R. M. et al., Biotechniques, 16; 42-43, 1994). Chemical hot start refers to a method which involves use of a DNA polymerase which is inactivated chemically but reversibly, such that the polymerase is inhibited at ambient temperatures and most active at higher temperatures (U.S. Pat. Nos. 5,773,258 and 6,183,998). Another way of implementing a hot start is to combine the DNA polymerase enzyme with an anti-DNA polymerase antibody before adding it to the reagent. The antibody inhibits the polymerase activity at ambient temperature but at the high polymerase reaction temperature, the antibody is denatured and dissociates from the polymerase, allowing activation of the polymerase (Sharkey et al., Bio/Technology, 12:506-509 (1994); Kellogg et al., Biotechniques, 16: 1134-1137 (1994)).

Another method for inhibiting thermophilic DNA polymerase at ambient temperatures involves the use of a nucleic acid aptamer which binds to and inhibits the DNA polymerase (U.S. Pat. No. 6,183,967). Aptamers are nucleic acid molecules having specific binding affinity to molecules through interactions other than classic Watson-Crick base pairing. Various aptamers have been selected and designed to exhibit specificity and affinity for thermostable polymerases and have been shown to be capable of reversibly inhibiting the polymerases. As with other reversible inhibitors of thermostable polymerases, aptamers are designed to bind and inhibit a thermostable polymerase at ambient temperatures. A subsequent increase in temperature results in the dissociation of the aptamer from the thermostable polymerase to allow primer extension and/or nucleic acid amplification at high reaction temperatures.

When designing and selecting an inhibitory aptamer for use in a reaction requiring thermostable polymerase activity, it is important to generate aptamers that bind and inhibit the polymerase in the appropriate temperature range, such as the temperature range for optimal polymerase activity, but which also dissociate from the polymerase at temperatures which allow optimal thermostable polymerase activity at temperatures when selected primers are hybridized to a target nucleic acid substrate. Described herein are aptamers which can form a secondary structure and which can 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.

BRIEF SUMMARY

In one aspect, an aptamer is provided, wherein the aptamer comprises in a 5′ to 3′ direction: a first nucleotide sequence comprising TATAATTGCAAAATAA (SEQ ID NO:1) or a variant thereof or TTATTTTGCAATTATA (SEQ ID NO:2) or a variant thereof; a second nucleotide sequence comprising TTCTTAGCGTTT (SEQ ID NO:3) or a variant thereof, a third nucleotide sequence comprising SEQ ID NO:1 or a variant thereof or SEQ ID NO:2 or a variant thereof; and a fourth nucleotide sequence comprising SEQ ID NO:3 or a variant thereof, wherein the first sequence is complementary to the second sequence.

In some embodiments, the aptamer has a secondary structure wherein the first and third nucleotide sequences hybridize at temperatures below about 45° C., 42° C., 40° C., 39° C., or 38° C. to form a stem and each of the second and third nucleotide sequences remain single stranded to form a loop, wherein the stem is positioned between the loops formed by the second and third nucleotide sequences.

In some embodiments, a covalent bond is present between the 5′ end of the first sequence and the 3′ end of the fourth sequence. In other embodiments, the covalent bond is a phosphodiester bond.

In some embodiments, the variant of SEQ ID NO:1 comprises 1, 2, 3 or 4 nucleotide substitutions and the third nucleotide sequence is complementary to the variant of SEQ ID NO:2 wherein there are no mismatches between SEQ ID NO:1 and SEQ ID NO:2.

In some embodiments, the variant of SEQ ID NO:2 comprises 1, 2, 3 or 4 nucleotide substitutions and the first nucleotide sequence is complementary to the variant of SEQ ID NO:2 wherein there are no mismatches between SEQ ID NO:1 and SEQ ID NO:2.

In some embodiments, each of the first sequence and the third sequence is 14-18, 15-16 or 16-17 nucleotides in length. In still other embodiments, each of the first sequence and the third sequence is 14, 15, 16, 17 or 18 nucleotides in length. In a particular embodiment, each of the first sequence and the third sequence is 16 nucleotides in length.

In some embodiments, the first sequence comprises SEQ ID NO:1 and the second sequence comprises SEQ ID NO:2. In still other embodiments, the first sequence consists of SEQ ID NO:1 and the second sequence consists of SEQ ID NO:2.

In some embodiments, the first sequence is selected from the group consisting of SEQ ID NO:11-41 or a variant thereof, wherein the variant comprises 1, 2, 3 or 4 nucleotide substitutions. In other embodiments, the third sequence is the same length as the first sequence, and the third sequence is complementary to the first sequence, wherein there are no mismatches between the first sequence and the third sequence. In still other embodiments, the first sequence is selected from the group consisting of SEQ ID NO:11-41.

In some embodiments, the second sequence is identical to the fourth sequence along the entire length of the second and fourth sequences.

In some embodiments, each of the second and fourth sequences is 12 nucleotides in length.

In some embodiments, the second sequence comprises SEQ ID NO:3 and the fourth sequence comprises SEQ ID NO:3. In other embodiments, the second sequence consists of SEQ ID NO:3 and the fourth sequence consists of SEQ ID NO:3.

In some embodiments, the aptamer does not have a 5′ end or a 3′ end.

In some embodiments, the aptamer comprises a 5′ end and a 3′ end, wherein there is no covalent bond between 2 nucleotides within the first sequence or within the second sequence. In other embodiments, there is no covalent bond between nucleotides 1 and 2, 2 and 3, 3 and 4, 4 and 5, 5 and 6, 6 and 7, 7 and 8, 8 and 9, 9 and 10, 10 and 11, 11 and 12, 12 and 13, 13 and 14, 14 and 15 or 15 and 16 of SEQ ID NO:1 or of SEQ ID NO:2.

In a some embodiments, the first sequence comprises SEQ ID NO:1 and the third sequence comprises SEQ ID NO:2, and there is no phosphodiester bond between nucleotides 8 and 9 of SEQ ID NO:1. In other embodiments, the first sequence comprises SEQ ID NO:2 and the third sequence comprises SEQ ID NO:1, and there is no phosphodiester bond between nucleotides 8 and 9 of SEQ ID NO:2.

In some embodiments, the first and third sequences of the aptamer are hybridized to each other in a double stranded configuration. In other embodiments, the first and third sequences of the aptamer are hybridized to each other in a double stranded configuration when the aptamer is in a solution at 25° C. or at 37° C. In still other embodiments, the solution has a pH of about 6 to 8.

In some embodiments, both the second sequence and the fourth sequence are in a single stranded configuration. In other embodiments, both the second sequence and the fourth sequence are in a single stranded configuration when the aptamer is in a solution at 25° C. or at 37° C. In still other embodiments, the solution has a pH of about 6 to 8.

In some embodiments, the aptamer has a melting temperature ranging from about 40° C. to 50° C., 45° C. to 65° C., 45° C. to 60° C., 45° C. to 55° C., 45° C. to 50° C., 50° C. to 65° C. or 50° C. to 60° C.

In some embodiments the aptamer comprises a sequence selected from the group consisting of SEQ ID NO:42-53 or a variant thereof wherein the variant comprises 1, 2, 3 or 4 nucleotide substitutions. In other embodiments, embodiments the aptamer comprises a sequence selected from the group consisting of SEQ ID NO:42-53. In still other embodiments, the aptamer has a 5′ end and a 3′ end.

In some embodiments, the 5′ end of the aptamer comprises a phosphate group. In other embodiments, the 3′ end of the aptamer comprises a phosphate group. In still other embodiments, the 5′ and the 3′ end each comprise a phosphate group.

In still other embodiments, the 3′ end of the aptamer is linked to a 3′ capping moiety. In yet another embodiment, the 5′ end of the aptamer is linked to a 5′ capping moiety. In still other embodiments, the 3′ end of the aptamer is linked to the 5′ capping moiety and the 3′ end of the aptamer is linked to the 5′ capping moiety.

In some embodiments, the 5′ capping moiety is selected from the group consisting of an amine group and an inverted deoxythymidine cap.

In some embodiments, the 3′ capping moiety linked to the 3′ end is selected from the group consisting of a phosphate, an inverted deoxythymidine cap and a propanediol spacer (C3).

In some embodiments, the aptamer comprises CAAAATAATTCTTAGCGTTTTTATTTTGCAATTATATTCTTAGCGTTTTATAATT G-C3 (SEQ ID NO:106); CAAAATAATTCTTAGCGTTTTTATTTTGGAATTATATTCTTAGCGTTTTATAATT C-C3 (SEQ ID NO:107); GAAAATAATTCTTAGCGTTTTTATTTTCGAATTATATTCTTAGCGTTTTATAATT C-C3 (SEQ ID NO:108); CAATTATATTCTTAGCGTTTTATAATTGGAAAATAATTCTTAGCGTTTTTATTTT C-C3 (SEQ ID NO:109); CAATTATATTCTTAGCGTTTTATAATTGCAAAATAATTCTTAGCGTTTTTATTTT G-C3 (SEQ ID NO:110); or GAATTATATTCTTAGCGTTTTATAATTCGAAAATAATTCTTAGCGTTTTTATTTT C-C3 (SEQ ID NO:111). In other embodiments, there is no covalent bond between the first nucleotide and the last nucleotide of the aptamer comprising the sequence of one of SEQ ID NO:106-111.

In another aspect, an aptamer is provided wherein the aptamer comprises in a 5′ to 3′ direction a first polynucleotide comprising SEQ ID NO:4 or a variant thereof or SEQ ID NO:5 or a variant thereof, a second polynucleotide comprising SEQ ID NO:3 or a variant thereof, a third polynucleotide comprising SEQ ID NO:5 or a variant thereof or SEQ ID NO:4 or a variant thereof, and a fourth polynucleotide comprising SEQ ID NO:3 or a variant thereof or SEQ ID NO:5 or a variant thereof, wherein each of the second and fourth sequences are 14-18 nucleotides in length, are equal in length, and are complementary to each other along their entire length; and wherein the aptamer comprises a 5′ end and a 3′ end.

In some embodiments, the first polynucleotide and the third polynucleotide anneal to each other to form a stem.

In some embodiments, each of the second and fourth polynucleotides do not anneal to another region of the aptamer and each of the second and fourth polynucleotides form a first and a second loop.

In some embodiments, the stem is positioned between the first and second loops.

In some embodiments, the 3′ end of the fourth sequence is covalently bound to the 5′ end of the first sequence and there is no phosphodiester bond between 2 nucleotides within the fourth sequence.

In some embodiments, the 3′ end of the fourth sequence is covalently bound to the 5′ end of the first sequence and there is no phosphodiester bond between the last nucleotide of the third sequence and the first nucleotide of the fourth sequence.

In some embodiments, there is no phosphodiester bond between nucleotides 1 and 2, 2 and 3, 3 and 4, 4 and 5, 5 and 6, 6 and 7, 7 and 8, 8 and 9, 9 and 10, 10 and 11 or 11 and 12 of SEQ ID NO:3 in the fourth sequence.

In some embodiments, the first polynucleotide comprises the sequence selected from the group consisting of SEQ ID NOs:54-77.

In some embodiments, the first polynucleotide and the third polynucleotide are the same length. In still other embodiments, there is no mismatch between the first polynucleotide and the third polynucleotide.

In some embodiments, the first sequence is identical to the third sequence along the entire length of the first and third sequences.

In some embodiments, each of the first and third sequences is 12 nucleotides in length.

In some embodiments, the first sequence comprises SEQ ID NO:3 and the third sequence comprises SEQ ID NO:3. In other embodiments, the first sequence consists of SEQ ID NO:3 and the third sequence consists of SEQ ID NO:3.

In some embodiments, the aptamer comprises a sequence selected from the group consisting of SEQ ID NOs:82-105.

In some embodiments, the 5′ end of the aptamer comprises a phosphate group. In other embodiments, the 3′ end of the aptamer comprises a phosphate group. In still other embodiments, the 5′ and the 3′ end each comprise a phosphate group.

In still other embodiments, the 3′ end of the aptamer is linked to a 3′ capping moiety. In yet another embodiment, the 5′ end of the aptamer is linked to a 5′ capping moiety. In still other embodiments, the 3′ end of the aptamer is linked to the 5′ capping moiety and the 3′ end of the aptamer is linked to the 5′ capping moiety.

In some embodiments, the 5′ capping moiety is selected from the group consisting of an amine group and an inverted deoxythymidine cap.

In some embodiments, the 3′ capping moiety linked to the 3′ end is selected from the group consisting of a phosphate, an inverted deoxythymidine cap and a propanediol spacer (C3).

In one aspect, a composition is provided comprising a thermostable polymerase and an aptamer according to any of the foregoing aspects and embodiments, wherein the aptamer inhibits the activity of the polymerase.

In some embodiments, the molar ratio of aptamer:polymerase in the composition ranges from about 1:1 to 20:1, 2:1 to 15:1, 5:1 to 20:1, 10:1 to 20:1, 12:1 to 18:1, 14:1 to 16:1, 1:1 to 10:1, 5:1 to 10:1, or 12:1 to 18:1. In other embodiments, the molar ratio of aptamer:polymerase in the composition is about 1:1, 2:1, 4:1, 5:1, 8:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1 or 20:1.

In some embodiments, the polymerase is selected from the group consisting ofThermus aquaticus, Thermus thermophilus, orThermus maritima.

In some embodiments, the composition further comprises deoxyribonucleotide triphosphates and a divalent metal cation.

In some embodiments, the composition further comprises a forward primer. In other embodiments, the composition comprises both the forward primer and a reverse primer. In still other embodiments, the forward and/or reverse primer is specific for a target nucleic acid.

In some embodiments, the composition is a dried composition.

In some embodiments, the composition is a liquid composition.

In one aspect, a method for extending a primer is provided wherein the method comprises mixing the primer with a thermostable polymerase and an aptamer according to any of the foregoing aspects and embodiments to form a reaction mixture.

In some embodiments, the primer is specific for a target nucleic acid sequence.

In some embodiments, the method further comprises adding a reverse primer. In other embodiments, the reverse primer is specific for the target nucleic acid sequence.

In some embodiments, the method further comprises adding to the reaction mixture deoxyribonucleotide triphosphates and a divalent metal cation.

In some embodiments, the aptamer inhibits, reduces or eliminates the polymerase activity at a temperature below 45° C., 40° C., 37° C., 35° C., 32° C., or 30° C.

In one aspect, a kit is provided wherein the kit comprises a composition comprising an aptamer according to any of the foregoing aspects and embodiments and a thermostable polymerase.

In some embodiments, the kit further comprises a forward and a reverse primer, each of which is specific for a target nucleic acid sequence.

In some embodiments, the kit comprises primers and at least one probe for detecting and/or determining a sequence of the target nucleic acid sequence.

In some embodiments, the composition further comprises a forward oligonucleotide primer which is specific for the target nucleic acid sequence. In other embodiments, the composition further comprises a reverse oligonucleotide primer, which is specific for the target nucleic acid sequence.

In some embodiments, the kit further comprises a reverse transcriptase.

In some embodiments, the composition further comprises a reverse transcriptase.

In some embodiments, the composition is a dried composition.

In some embodiments, the composition is a liquid composition.

These and other objects and features of the invention will become more fully apparent when read in conjunction with the following detailed description.

