TEMPERATURE CONTROLLED DNA POLYMERASE INHIBITORS
The present disclosure provides polynucleotide-based inhibitors for reversible activation of DNA polymerases. Use of lower Tm polynucleotide-based inhibitors allow PCR reaction assembly at room temperature while activating polymerase at higher PCR primer annealing temperatures, where the reversible nature of the inhibition additionally improves priming specificity during each PCR cycle. Additionally, temperature controlled inactivation of polymerase activity after PCR or other polymerase based enzymatic incubation eliminates a purification step when needed for compatibility with subsequent enzymatic incubations. For this application, the T m of the polynucleotide-based inhibitor is higher than the desired reaction conditions of the subsequent enzymatic incubation.
1 - 183 . (canceled)
184 . A polymerase inhibitor comprising a synthetic nucleic acid molecule comprising:
a first oligonucleotide comprising a first complementary region; and
a second oligonucleotide comprising a second complementary region and a first single-stranded region positioned 5′ to the second complementary region,
wherein the first complementary region is sufficiently complementary to the second complementary region to form a double-stranded region at a temperature below a melting temperature of the polymerase inhibitor,
wherein the first oligonucleotide further comprises a second single-stranded region positioned 5′ to the first complementary region,
wherein the first single-stranded region comprises a first replication blocking sequence or the first oligonucleotide further comprises a first extension blocking group at a 3′ end of the first oligonucleotide, and
wherein the second single-stranded region comprises a second replication blocking sequence or the second oligonucleotide further comprises a second extension blocking group at a 3′ end of the second oligonucleotide.
185 . The polymerase inhibitor of claim 184 , wherein first complementary region and second complementary region are from about 6 to about 100 nucleotides.
186 - 187 . (canceled)
188 . The polymerase inhibitor of claim 184 , wherein the first complementary region is sufficiently complementary to the second complementary region to form a double-stranded region below the melting temperature, and wherein the melting temperature is selected from the group consisting of below 90° C., 80° C., 70° C., 60° C., and 50° C.
189 - 191 . (canceled)
192 . The polymerase inhibitor of claim 184 , wherein the first complementary region comprises a homopolymer sequence or a heteropolymeric sequence comprising a dinucleotide sequence.
193 - 199 . (canceled)
200 . The polymerase inhibitor of claim 184 , wherein the first single-stranded region comprises the first replication blocking sequence and the second single-stranded region comprises the second replication blocking sequence.
201 . The polymerase inhibitor of claim 184 , wherein the first oligonucleotide further comprises the first extension blocking group and the second oligonucleotide further comprises the second extension blocking group.
202 . The polymerase inhibitor of claim 184 , wherein the first single-stranded region comprises the first replication blocking sequence and the second oligonucleotide further comprises the second extension blocking group.
203 . The polymerase inhibitor of claim 184 , wherein the second single-stranded region comprises the second replication blocking sequence and the first oligonucleotide further comprises the first extension blocking group.
204 . The polymerase inhibitor of claim 184 , wherein the first single-stranded region comprises the first replication blocking sequence, the second single-stranded region comprises the second replication blocking sequence, the first oligonucleotide further comprises the first extension blocking group, and the second oligonucleotide further comprises the second extension blocking group.
205 . The polymerase inhibitor of claim 200 , wherein the first replication blocking sequence and the second replication blocking sequence are each selected from the group consisting of a stable abasic site, a carbon spacer, at least one 2′-O methyl nucleotide, a deoxyuridine base, a deoxyinosine base, and from about 2 to about 50 consecutive ribonucleotide bases.
206 . The polymerase inhibitor of claim 201 , wherein the first extension blocking group and the second extension blocking group are each selected from the group consisting of a 3′ phosphate, a carbon spacer, and a dideoxynucleotide.
207 . The polymerase inhibitor of claim 202 , wherein the first replication blocking sequence is selected from the group consisting of a stable abasic site, a carbon spacer, at least one 2′-O methyl nucleotide, a deoxyuridine base, a deoxyinosine base, and from about 2 to about 50 consecutive ribonucleotide bases, and wherein the second extension blocking group is selected from the group consisting of a 3′ phosphate, a carbon spacer, and a dideoxynucleotide.
208 . The polymerase inhibitor of claim 203 , wherein the second replication blocking sequence is selected from the group consisting of a stable abasic site, a carbon spacer, at least one 2′-O methyl nucleotide, a deoxyuridine base, a deoxyinosine base, and from about 2 to about 50 consecutive ribonucleotide bases, and wherein the first extension blocking group is selected from the group consisting of a 3′ phosphate, a carbon spacer, and a dideoxynucleotide.
209 . The polymerase inhibitor of claim 204 , wherein the first replication blocking sequence and the second replication blocking sequence are each selected from the group consisting of a stable abasic site, a carbon spacer, at least one 2′-O methyl nucleotide, a deoxyuridine base, a deoxyinosine base, and from about 2 to about 50 consecutive ribonucleotide bases, and wherein wherein the first extension blocking group and the second extension blocking group are each selected from the group consisting of a 3′ phosphate, a carbon spacer, and a dideoxynucleotide.
210 . The polymerase inhibitor of claim 201 , wherein the first oligonucleotide further comprises a first nuclease resistant linkage at the 3′ end of the first oligonucleotide.
211 . The polymerase inhibitor of claim 201 , wherein the second oligonucleotide further comprises a second nuclease resistant linkage at the 3′ end of the second oligonucleotide.
212 . The polymerase inhibitor of claim 201 , wherein the first oligonucleotide further comprises a first nuclease resistant linkage at the 3′ end of the first oligonucleotide, and wherein the second oligonucleotide further comprises a second nuclease resistant linkage at the 3′ end of the second oligonucleotide.
213 . The polymerase inhibitor of claim 206 , wherein the first oligonucleotide further comprises a first nuclease resistant linkage at the 3′ end of the first oligonucleotide.
214 . The polymerase inhibitor of claim 206 , wherein the second oligonucleotide further comprises a second nuclease resistant linkage at the 3′ end of the second oligonucleotide.
215 . The polymerase inhibitor of claim 206 , wherein the first oligonucleotide further comprises a first nuclease resistant linkage at the 3′ end of the first oligonucleotide, and wherein the second oligonucleotide further comprises a second nuclease resistant linkage at the 3′ end of the second oligonucleotide.
216 - 449 . (canceled)