Combinatorial Selection Inhibition and Antiviral Activity of DNA Thioaptamers Targeting the RNase H Domain of HIV-1 Reverse Transcriptase

摘要

Despite the key role played by the RNase H of Human Immunodeficiency Virus-1 Reverse Transcriptase (HIV-1 RT) in viral proliferation, only a few inhibitors of RNase H have been reported. Using in vitro combinatorial selection methods and the RNase H domain of the HIV RT, we have selected double-stranded DNA thioaptamers (aptamers with selected thiophosphate backbone substitutions) that inhibit RNase H activity and viral replication. The selected thioaptamer sequences had a very high proportion of G residues. The consensus sequence for the selected thioaptamers showed G clusters separated by single residues at the 5′ end of the sequence. Gel electrophoresis mobility shift assays and nuclear magnetic resonance spectroscopy showed that the selected thioaptamer binds to the isolated RNase H domain, but did not bind to a structurally similar RNase H from E.coli. The lead thioaptamer, R12-2, showed specific binding to HIV-1 RT with a binding constant (Kd) of 70 nM. The thioaptamer inhibited the RNase H activity of intact HIV-1 RT. In cell culture, transfection of thioaptamer R12-2 (0.5 μg/ml) markedly reduced viral production and exhibited a dose response of inhibition with R12-2 concentrations ranging from 0.03 μg/ml to 2.0 μg/ml (IC50 <100 nM). Inhibition was also seen across a wide range of virus inoculum, ranging from multiplicity of infection (m.o.i) of 0.0005 to 0.05, with reduction of virus production by more than 50% at high m.o.i. Suppression of virus was comparable to that seen with AZT at m.o.i. ≦ 0.005.