Vol. 186, No. 3, 1992 August 14, 1992 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages ]249-1256 TEMPLATE. PHOSPHOROTHIOATE OLIGONUCLEOTIDES DUPLEXES AS INHIBITORS OF HIV-1 REVERSE TRANSCRIPTASE Georges Mauryl, Abdelaziz El alaoui 1 , Francis Morvan 1 , Barbara MOiler 2, Jean-LouisImbach 1 and Roger S. Goody 2 1Universit~ de Montpellier II, URA 488 du CNRS, D~partement de Chimie Organique Fine, Place E. Bataillon, 34095 Montpellier C~dex 5, France 2Abteilung Biophysik, Max Planck Institut for medizinische Forschung Jahnstrasse 29, 6900 Heidelberg, Germany Received June 2, 1992 SUMMARY :We have investigated the interaction between a number of 14 mers phosphorothioate oligonucleotides and HIV-1 reverse transcriptase. Two methods were used to measure the affinity of the analogs for the enzyme. In the first, the oligonucleotide or its duplex with Poly(rl) were used as inhibitors of the enzyme using Poly(rA).(dT)14 as template primer. In the second, the oligonucleotides or their duplexes were used to displace a fluorescent template.primer complex of known affinity from its binding site on reverse transcriptase. The two methods gave the same relative order of affinitiy. Phosphorothioate oligodeoxyribonucleotides had a much higher affinity than oligo(dC)l 4 and it was increased on hybridization. Quantitatively similar results were obtained for S(dC)14 or its analog with bases in the m-configuration. Of the analogs tested, only S(dC)14 showed priming activity. ~ 1992 Academic Press, Inc. The reverse transcriptase (RNA dependent DNA poiymerase, EC 2.7.7.49) (RT) of the human immunodeficiency virus type 1 (HIV-1) is an essential enzyme for the replication of the virus. The enzyme molecule exists as a stable and active heterodimer composed of two subunits of 66 kilodaltons and 51 kilodaltons (p66.p51) (1,2). This dimer is now readily available through bacterial expression and purification (3-5). Consequently, it has become the target of an increasing number of inhibition studies involving analogs of substrates (nucleoside triphosphate or primer) (6-8) or non nucleoside inhibitors (9). From steady state kinetic studies, an ordered mechanism for RT catalyzed DNA synthesis has been proposed with initial interaction of the free enzyme with the template.primer substrate followed by binding with the nucleoside 5'-triphosphate (10). Phosphorothioate oligonucleotides (14 mers or upper homologs) have been shown to effectively inhibit HIV replication (11). Majundar et aL demonstrated that the phosphorothioate deoxycytidinate S(dC)28 presumably influences the first stage of the reaction and is a potent competitive inhibitor of RT against Poly (rA).(dT)l 4 as template.primer (12). Annealing the phosphorothioate oligonucleotide with Poiy (rl) did not change noticeably the inhibition, and it was deduced that the free enzyme recognizes the primer rather than the template in the first step of the reaction (12). However, these conclusions are questionable since the experiments with annealed and not annealed 1249 0006-291X/92 $4.00 Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.