Nucleic Acids Research Advance Access originally published online on July 21, 2007
Nucleic Acids Research 2007 35(15):5039-5050; doi:10.1093/nar/gkm420
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Nucleic Acids Research, 2007, Vol. 35, No. 15 5039-5050
© 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Molecular Biology |
Active site binding and sequence requirements for inhibition of HIV-1 reverse transcriptase by the RT1 family of single-stranded DNA aptamers
1Department of Biology, Indiana University, Bloomington, IN 47405, 2Biosciences Division, SRI International, Menlo Park, CA 94025 and 3Department of Molecular Microbiology & Immunology and Department of Biochemistry, 471h Life Sciences Center, University of Missouri School of Medicine, Columbia, MO 65211, USA
*To whom correspondence should be addressed. Tel: (573) 884 1316; Fax: (573) 884 9676; Email: burkedh{at}missouri.edu
Received April 9, 2007. Revised May 4, 2007. Accepted May 8, 2007.
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 SIVCPZ 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.