Primer extension assay

DNA-dependent DNA polymerase activities of RTs were performed as previously described (19). In brief, 20 nM of RT (in-house purified HXB2 and its mutant variants) was incubated with 100 nM of aptamer in extension buffer (50 mM Tris pH 7.5, 50 mM NaCl, 5 mM MgCl₂) on ice for 10 min, then a mixture of primer/template/dNTP was added (final concentration: 10 nM primer, 20 nM template and 100 μM of each dNTP) (19). DNA-dependent DNA polymerase activities of RTs were assayed using a 31-nt DNA template (5′-CCATAGATAGCATTGGTGCTCGAACAGTGAC-3′) and a complementary, 5′-Cy3-labeled 18-nt DNA primer (5′-Cy3-GTCACTGTTCGAGCACCA-3′). The extension reaction was incubated at 37°C for 10 min. Reaction was stopped by adding 2× polyacrylamide gel electrophoresis (PAGE) loading dye (95% formamide, 50 mM ethylenediaminetetraacetic acid (EDTA), xylene and bromophenol blue). Samples were separated on 10% denaturing (8M urea) PAGE. Gels were scanned on a Typhoon FLA9000 imager (GE Healthcare) and quantified using Multi Gauge software (Fujifilm). P-values were calculated using unpaired t-test computed by GraphPad Prism.