INHIBITORY POTENCY OF R-REGION SPECIFIC ANTISENSE OLIGONUCLEOTIDES AGAINST IN-VITRO DNA POLYMERIZATION AND TEMPLATE-SWITCHING REACTIONS CATALYZED BY HIV-1 REVERSE-TRANSCRIPTASE

Antisense oligonucleotides (AONs) targeted to the R-region near the 5' -LTR of HIV-1 genomic RNA inhibited both the synthesis of (-) strong s top DNA and the first template-switch reaction catalysed by HIV-1 reve rse transcriptase (RT) in vitro. The 18 nucleotide (nt) AONs used were identical in sequence but differed in the sugar component of the 3'-t erminal nucleotide, with either 2'-deoxy-D-ribose (DNA), 2'-deoxy-L-ri bose (L), or arabinose (ARA) in this position, All three AONs hybridiz ed to complementary 18 nt RNA (T-m approximate to 70 degrees C) and sp ecifically interacted with the target RNA HIV-1 sequence at 37 degrees C, L was unable to serve as primer for RT-catalysed DNA polymerizatio n, 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 t emplate-switch reaction products formed by RT, implying that elongatio n of the AONs did not enhance the inhibitory activity in vitro. A conc omitant increase in a truncated DNA product corresponding to polymeriz ation termination at the 5'-end of the AON was noted, indicating that RT was unable to displace the AON, Interestingly, near maximal inhibit ion in vitro an AON:target RNA template ratio of 1:1 was noted. Our re sults confirm the validity of our in vitro system for the analysis-of potential antisense oligonucleotide inhibitors, and suggest that antis ense oligonucleotides, directed to the R-region of HIV-1 RNA may be ef fective inhibitors of the initial stages of HIV-1 proviral DNA synthes is. (C) 1997 Elsevier Science Ltd. All rights reserved.