Inhibition of HIV-1 reverse transcriptase by defined template/primer DNA oligonucleotides: effect of template length and binding characteristics.
The interaction of partially double stranded DNA oligonucleotides with HIV-1 RT was studied by investigating their ability to inhibit the homopolymeric poly(rC) directed (dG) synthesis reaction. A 20/18mer oligonucleotide, with a sequence based on the Lys3-tRNA primer region, showed stronger inhibition of the homopolymeric RT reaction than a G/C rich oligonucleotide series lacking or possessing a hairpin moiety. Interaction of the enzyme with the G/C rich oligonucleotides, as determined by IC50 measurements, was insensitive to the extent of the unpaired template region at the 3' or 5' position. Addition of a hairpin moiety, composed of four thymidine bases, onto G/C rich oligonucleotides increase their inhibitory potency (at least six times) and shifted the mode of inhibition of RT to competitive with respect to poly (rC).(dG), which was otherwise mixed (competitive/noncompetitive) for the linear G/C rich and 20/18mer oligonucleotides. The results indicate that interaction of the enzyme with the primer/template stem, but not with the unpaired template region, is an important step in complex formation.
Idriss
H
H
Wellcome Research Laboratories, Department of Molecular Sciences, Beckenham, Kent, England.
Stammers
D K
DK
D001665:Binding Sites; D017931:DNA Primers / Q000378:metabolism / Q000494:pharmacology*; D004277:DNA, Single-Stranded / Q000378:metabolism / Q000494:pharmacology; D054303:HIV Reverse Transcriptase; D007700:Kinetics; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000378:metabolism*; D011068:Poly G / Q000096:biosynthesis / Q000378:metabolism; D012357:RNA, Transfer, Lys / Q000378:metabolism; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism; D018894:Reverse Transcriptase Inhibitors; D016914:Ribonuclease H / Q000378:metabolism; D013329:Structure-Activity Relationship
D017931:DNA Primers; D004277:DNA, Single-Stranded; D009841:Oligonucleotides; D012357:RNA, Transfer, Lys; D018894:Reverse Transcriptase Inhibitors; D011068:Poly G; C026628:poly(dG); D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase; D016914:Ribonuclease H