期刊
MOLECULAR THERAPY-NUCLEIC ACIDS
卷 4, 期 -, 页码 -出版社
NATURE PUBLISHING GROUP
DOI: 10.1038/mtna.2015.1
关键词
anti-HIV-1 gene therapy; HIV-1 inhibitor; HIV-1 PR inhibitor; RNA aptamer structure; RNA aptamer
资金
- NIH AIDS Training grant [T32 AI007501]
- NIH MARC fellowship [F31-GM78730]
- NIH [P01 AI061797, R37 AI030861]
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 (K-d = 92-140 nmol/l) and anti-PR inhibitory activity (K(i)s = 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 (K(d)s = 2-22 nmol/l) and inhibition (K(i)s = 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.
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