title: Combinatorial selection, inhibition, and antiviral activity of DNA thioaptamers targeting the RNase H domain of HIV-1 reverse transcriptase.
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 (K(d)) 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 microg/mL) markedly inhibited viral production and exhibited a dose response of inhibition with R12-2 concentrations ranging from 0.03 to 2.0 microg/mL (IC(50) < 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 <or= 0.005.
authors.affiliation: Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, Texas 77555, USA.
authors.forename: Anoma
authors.initials: A
authors.lastname: Somasunderam
authors.forename: Monique R
authors.initials: MR
authors.lastname: Ferguson
authors.forename: Daniel R
authors.initials: DR
authors.lastname: Rojo
authors.forename: Varatharasa
authors.initials: V
authors.lastname: Thiviyanathan
authors.forename: Xin
authors.initials: X
authors.lastname: Li
authors.forename: William A
authors.initials: WA
authors.lastname: O'Brien
authors.forename: David G
authors.initials: DG
authors.lastname: Gorenstein
mesh_terms: D019380:Anti-HIV Agents / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D020650:Combinatorial Chemistry Techniques / Q000379:methods; D011756:Diphosphates / Q000737:chemistry*; D024202:Electrophoretic Mobility Shift Assay; D015723:Gene Library; D054303:HIV Reverse Transcriptase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D015497:HIV-1 / Q000187:drug effects / Q000201:enzymology / Q000502:physiology; D009682:Magnetic Resonance Spectroscopy; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism; D016133:Polymerase Chain Reaction; D017434:Protein Structure, Tertiary; D018894:Reverse Transcriptase Inhibitors / Q000737:chemistry* / Q000378:metabolism; D016914:Ribonuclease H / Q000378:metabolism*; D013873:Thionucleotides / Q000737:chemistry* / Q000378:metabolism; D014779:Virus Replication / Q000187:drug effects
chemical_list: D019380:Anti-HIV Agents; D011756:Diphosphates; D009838:Oligodeoxyribonucleotides; D018894:Reverse Transcriptase Inhibitors; D013873:Thionucleotides; C046010:monothiopyrophosphoric acid; D054303:HIV Reverse Transcriptase; D016914:Ribonuclease H