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Nucleic Acids Res. 2007;35(15):5039-50. Epub 2007 Jul 21.

Active site binding and sequence requirements for inhibition of HIV-1 reverse transcriptase by the RT1 family of single-stranded DNA aptamers.

Author information

1
Department of Biology, Indiana University, Bloomington, IN 47405, USA.

Abstract

Nucleic acid aptamers can potentially be developed as broad-spectrum antiviral agents. Single-stranded DNA (ssDNA) aptamer RT1t49 inhibits reverse transcriptases (RT) from HIV-1 and diverse lentiviral subtypes with low nanomolar values of Kd and IC50. To dissect the structural requirements for inhibition, RT-catalyzed DNA polymerization was measured in the presence of RT1t49 variants. Three structural domains were found to be essential for RT inhibition by RT1t49: a 5' stem (stem I), a connector and a 3' stem (stem II) capable of forming multiple secondary structures. Stem I tolerates considerable sequence plasticity, suggesting that it is recognized by RT more by structure than by sequence-specific contacts. Truncating five nucleotides from the 3' end prevents formation of the most stable stem II structure, yet has little effect on IC50 across diverse HIV-1, HIV-2 and SIV(CPZ) RT. When bound to wild-type RT or an RNase H active site mutant, site-specifically generated hydroxyl radicals cleave after nucleotide A32. Cleavage is eliminated by either of two polymerase (pol)-active site mutants, strongly suggesting that A32 lies within the RT pol-active site. These data suggest a model of ssDNA aptamer-RT interactions and provide an improved molecular understanding of a potent, broad-spectrum ssDNA aptamer.

PMID:
17644816
PMCID:
PMC1976467
DOI:
10.1093/nar/gkm420
[Indexed for MEDLINE]
Free PMC Article

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