Editing Aptamer for Taq DNA Polymerase

TQ30 5'-ttctcggttggtctctggcggagcAAGACCAGACAATGTACAGTATTGGCCTGAtcttgtgtatgattcgcttttccc-3'

TQ21 5'-ttctcggttggtctctggcggagcGATCATCTCAGAGCATTCTTAGCGTTTTGTtcttgtgtatgattcgcttttccc-3'

four polymerases derived from Thermus species (Taq, Tth, Tbr and Tlf) were inhibited by both of the selected oligonucleotides, suggesting that these enzymes share a high degree of similarity. As stated above, Tth polymerase and Taq polymerase are reported to be 93% similar and 88% identical at the amino acid sequence level (

Tma polymerase from Thermotoga maritima and Tli polymerase from Thermococcus litoralis, on the other hand, were not inhibited by either of the ligands

Of the four reverse transcriptases tested, RTs from HIV-I and AMV (avian myeloblastosis virus) were not inhibited. On the other hand, RT from M-MLV (moloney murine leukemia virus) and its mutant lacking RNase H activity (SuperScript II) were inhibited by the two oligonucleotide ligands.

Mesophilic DNA polymerases, such as, Klenow fragment of E.coli DNAP1 (KF), T4 DNAP and T7 DNAP were not inhibited by either ligand at 0.5 μM concentration, despite the similarity of the polymerase domains of Taq polymerase and KF

the addition of either TQ21 (FIG. 10B, lanes 4-6) or TQ30 (FIG. 10B, lanes 7-9) carried out under standard conditions (without "hot start") eliminated the nonspecific bands without affecting the yield of the target-specific band

At 250 nM inhibitor concentration, Trnc.1-30 (SEQ ID NO:67) and Trnc.2-30 (SEQ ID NO:68) inhibited approximately 95% of the activity of Taq polymerase

IC50 values of the three truncates are Trnc.1-30=2.7 nM, Trnc.2-30=5.9 nM and Trnc.3-30=10.3 nM (FIG. 12). Overall, the three truncated forms of TQ30 are more effective in inhibiting the Stoffel fragment than Taq polymerase

The TQ21 sequence, which inhibits both Taq and Tth polymerase does not inhibit the Stoffel fragment

Trnc.21 showed increased affinity; the Kd of Trnc.21 for binding to Taq polymerase is 9 pM (FIG. 13A), about 4-fold higher affinity than the full-length sequence. The IC50 value of Trnc.21 for inhibition of Taq polymerase is 21 nM (FIG. 13B), about 3-fold lower than the value for the full-length sequence. The calculated IT50 values for Taq polymerase and Tth polymerase are 34° C. and 35.6° C., respectively

The affinity of D.30-D.30 dimer for binding to Taq polymerase is 40 pM (FIG. 14A), about 75-fold higher than its monomeric form. The IC50 value of the homodimer is 14 nM, about 3.5-fold lower than the monomeric form (FIG. 14B). Thus, the dimerization of the truncated TQ30 produced a more effective inhibitor for Taq polymerase.
[[Category:QPCR RT-PCR]] [[Category:Protocols]]