Active site binding and sequence requirements for inhibition of HIV-1 reverse transcriptase by the RT1 family of single-stranded DNA aptamers.
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.
Kissel
Jay D
JD
Department of Biology, Indiana University, Bloomington, IN 47405, USA.
Held
Daniel M
DM
Hardy
Richard W
RW
Burke
Donald H
DH
D019380:Anti-HIV Agents / Q000737:chemistry* / Q000494:pharmacology; D052157:Aptamers, Nucleotide / Q000737:chemistry* / Q000494:pharmacology; D001483:Base Sequence; D001665:Binding Sites; D004252:DNA Mutational Analysis; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000494:pharmacology; D054303:HIV Reverse Transcriptase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D017665:Hydroxyl Radical / Q000737:chemistry; D009690:Nucleic Acid Conformation; D009711:Nucleotides / Q000378:metabolism; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism; D018894:Reverse Transcriptase Inhibitors / Q000737:chemistry* / Q000494:pharmacology; D015302:Simian Immunodeficiency Virus / Q000201:enzymology
D019380:Anti-HIV Agents; D052157:Aptamers, Nucleotide; D004277:DNA, Single-Stranded; D009711:Nucleotides; C523301:RT1t49 aptamer; D018894:Reverse Transcriptase Inhibitors; D017665:Hydroxyl Radical; C072416:reverse transcriptase, Human immunodeficiency virus 2; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase