Inhibitory potency of R-region specific antisense oligonucleotides against in vitro DNA polymerization and template-switching reactions catalysed by HIV-1 reverse transcriptase.

ABSTRACT

Antisense oligonucleotides (AONs) targeted to the R-region near the 5'-LTR of HIV-1 genomic RNA inhibited both the synthesis of (-) strong stop DNA and the first template-switch reaction catalysed by HIV-1 reverse transcriptase (RT) in vitro. The 18 nucleotide (nt) AONs used were identical in sequence but differed in the sugar component of the 3'-terminal nucleotide, with either 2'-deoxy-D-ribose (DNA), 2'-deoxy-L-ribose (L), or arabinose (ARA) in this position. All three AONs hybridized to complementary 18 nt RNA (T(m) approximately 70 degrees C) and specifically interacted with the target RNA HIV-1 sequence at 37 degrees C. L was unable to serve as primer for RT-catalysed DNA polymerization, whereas priming from ARA was about 30% that noted with DNA. Each of the three AONs resulted in similar 85-95% decreases in the amount of full length (-) strong stop DNA and up to 75% decreases in the first template-switch reaction products formed by RT, implying that elongation of the AONs did not enhance the inhibitory activity in vitro. A concomitant increase in a truncated DNA product corresponding to polymerization termination at the 5'-end of the AON was noted, indicating that RT was unable to displace the AON. Interestingly, near maximal inhibition in vitro an AONtarget RNA template ratio of 11 was noted. Our results confirm the validity of our in vitro system for the analysis of potential antisense oligonucleotide inhibitors, and suggest that antisense oligonucleotides directed to the R-region of HIV-1 RNA may be effective inhibitors of the initial stages of HIV-1 proviral DNA synthesis.