Combinatorial selection, inhibition, and antiviral activity of DNA thioaptamers targeting the RNase H domain of HIV-1 reverse transcriptase

Anoma Somasunderam, Monique Ferguson, Daniel R. Rojo, Varatharasa Thiviyanathan, Xin Li, William A. O'Brien, David G. Gorenstein

Research output: Contribution to journalArticle

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Abstract

Despite the key role played by the RNase H of human immunodeficiency virus-1 reverse transcriptase (HIV-1 RT) in viral proliferation, only a few inhibitors of RNase H have been reported. Using in vitro combinatorial selection methods and the RNase H domain of the HIV RT, we have selected double-stranded DNA thioaptamers (aptamers with selected thiophosphate backbone substitutions) that inhibit RNase H activity and viral replication. The selected thioaptamer sequences had a very high proportion of G residues. The consensus sequence for the selected thioaptamers showed G clusters separated by single residues at the 5′-end of the sequence. Gel electrophoresis mobility shift assays and nuclear magnetic resonance spectroscopy showed that the selected thioaptamer binds to the isolated RNase H domain, but did not bind to a structurally similar RNase H from Escherichia coli. The lead thioaptamer, R12-2, showed specific binding to HIV-1 RT with a binding constant (Kd) of 70 nM. The thioaptamer inhibited the RNase H activity of intact HIV-1 RT. In cell culture, transfection of thioaptamer R12-2 (0.5 μg/mL) markedly inhibited viral production and exhibited a dose response of inhibition with R12-2 concentrations ranging from 0.03 to 2.0 μg/mL (IC50 < 100 nM). Inhibition was also seen across a wide range of virus inoculum, ranging from a multiplicity of infection (moi) of 0.0005 to 0.05, with a reduction of the level of virus production by more than 50% at high moi. Suppression of virus was comparable to that seen with AZT when moi ≤ 0.005.

Original languageEnglish (US)
Pages (from-to)10388-10395
Number of pages8
JournalBiochemistry
Volume44
Issue number30
DOIs
StatePublished - Aug 2 2005

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Ribonuclease H
Antiviral Agents
DNA
Viruses
Infection
Nucleotide Aptamers
Consensus Sequence
Electrophoretic Mobility Shift Assay
Human immunodeficiency virus 1 reverse transcriptase
Electrophoresis
Cell culture
Escherichia coli
Nuclear magnetic resonance spectroscopy
Inhibitory Concentration 50
Transfection
Assays
Substitution reactions
Magnetic Resonance Spectroscopy
Cell Culture Techniques
Gels

ASJC Scopus subject areas

  • Biochemistry

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Combinatorial selection, inhibition, and antiviral activity of DNA thioaptamers targeting the RNase H domain of HIV-1 reverse transcriptase. / Somasunderam, Anoma; Ferguson, Monique; Rojo, Daniel R.; Thiviyanathan, Varatharasa; Li, Xin; O'Brien, William A.; Gorenstein, David G.

In: Biochemistry, Vol. 44, No. 30, 02.08.2005, p. 10388-10395.

Research output: Contribution to journalArticle

Somasunderam, Anoma ; Ferguson, Monique ; Rojo, Daniel R. ; Thiviyanathan, Varatharasa ; Li, Xin ; O'Brien, William A. ; Gorenstein, David G. / Combinatorial selection, inhibition, and antiviral activity of DNA thioaptamers targeting the RNase H domain of HIV-1 reverse transcriptase. In: Biochemistry. 2005 ; Vol. 44, No. 30. pp. 10388-10395.
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