@article{c517e7c8c12640d79653bc911ebaa14a,
title = "High-affinity RNA Aptamers Against the HIV-1 Protease Inhibit Both in Vitro Protease Activity and Late Events of Viral Replication",
abstract = "HIV-1 aspartyl protease (PR) plays a key role in virion morphogenesis, underscoring the effectiveness of protease inhibitors (PI). Despite their utility, side effects and drug-resistance remains a problem. We report the development of RNA aptamers as inhibitors of HIV-1 PR for potential use in anti-HIV gene therapy. Employing Systematic Evolution of Ligands by Exponential Enrichment (SELEX), we isolated four unique families of anti-HIV-1 PR RNA aptamers displaying moderate binding affinities (Kd = 92-140 nmol/l) and anti-PR inhibitory activity (Kis = 138-647 nmol/l). Second-generation RNA aptamers selected from partially randomized pools based on two of the aptamer sequences displayed striking enhancements in binding (Kds = 2-22 nmol/l) and inhibition (Kis = 31-49 nmol/l). The aptamers were specific in that they did not bind either the related HIV-2 protease, or the cellular aspartyl protease, Cathepsin D. Site-directed mutagenesis of a second-generation aptamer to probe the predicted secondary structure indicated that the stem-loops SL2 and SL3 and the stem P1 were essential for binding and that only the 3′-most 17 nucleotides were dispensable. Anti-PR aptamers inhibited HIV replication in vitro and the degree of inhibition was higher for second-generation aptamers with greater affinity and the inhibition was abrogated for a nonbinding aptamer variant.",
keywords = "HIV-1 PR inhibitor, HIV-1 inhibitor, RNA aptamer, RNA aptamer structure, anti-HIV-1 gene therapy",
author = "Sonald Duclair and Archana Gautam and Andrew Ellington and Prasad, {Vinayaka R.}",
note = "Funding Information: The authors would like to thank Brad Hall (University of Texas at Austin) for synthesizing the doped RNA pools, Na Li (University of Texas at Austin) and Carlos Garcia (University of Texas at Austin) for their assistance with the initial rounds of selections; Matthew Levy (Albert Einstein College of Medicine) for invaluable advice; Dhivya Ramgalingam (Albert Einstein College of Medicine) for the design of the pSilencer construction and technical help; Kimdar Kemal (Albert Einstein College of Medicine) for technical support; Scott Garforth (Albert Einstein College of Medicine) and William Drosopoulos (Albert Einstein College of Medicine) for critically reading the manuscript; and the Programme EVA Centre for AIDS Reagents, NIBSC, UK for providing recombinant HIV-1 PR (EVA630). S.D. was supported by an institutional NIH AIDS Training grant T32 AI007501 and a NIH MARC fellowship F31-GM78730. The research described in this report was supported by NIH grants P01 AI061797 and R37 AI030861 to V.R.P. S.D. performed a majority of the experimental work described in this manuscript, prepared the draft of the manuscript and all the figures except Figure 4 . A.G. performed the transfections and virus production assays. V.R.P. conceived of the project, designed, and supervised the overall approach and helped generate the final manuscript. The author(s) declare that they have no competing interests. Publisher Copyright: {\textcopyright} 2015 The American Society of Gene & Cell Therapy All rights reserved.",
year = "2015",
month = feb,
day = "17",
doi = "10.1038/mtna.2015.1",
language = "English (US)",
volume = "4",
pages = "e228",
journal = "Molecular Therapy - Nucleic Acids",
issn = "2162-2531",
publisher = "Nature Publishing Group",
number = "2",
}
