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Hiv-1 reverse transcriptase-pseudoknot RNA aptamer interaction has a binding affinity in the low picomolar range coupled with high specificity

Lookup NU author(s): Professor Bernard Connolly


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Systematic evolution of ligands by exponential enrichment (SELEX) is a powerful method for the identification of small oligonucleotides that bind with high affinity and specificity to target proteins. Such DNAs/RNAs are a new class of potential chemotherapeutics that could block the enzymatic activity of pathologically relevant proteins. We have conducted a detailed biochemical study of the interaction of human immunodeficiency virus 1 (HIV- 1) reverse transcriptase (RT) with a SELEX-derived pseudoknot RNA aptamer. Electron paramagnetic resonance spectroscopy of site-directed spin-labeled RT mutants revealed that this aptamer was selected for binding to the 'closed' conformation of the enzyme. Kinetic analysis showed that the RNA inhibitor bound to HIV RT in a two-step process, with association rates similar to those described for model DNA/DNA and DNA/RNA substrates. However, the dissociation of the pseudoknot RNA from RT was dramatically slower than observed for model substrates. Equilibrium binding studies revealed an extraordinarily low K(d), of about 25 pM, for the enzyme-aptamer interaction, presumably a consequence of the slow off-rates. Additionally, this pseudoknot aptamer is highly specific for HIV-1 RT, with the closely related HIV-2 enzyme showing a binding affinity close to 4 orders of magnitude lower.

Publication metadata

Author(s): Connolly BA; Kensch O; Steinhoff H-J; McGregor A; Goody RS; Restle T

Publication type: Article

Publication status: Published

Journal: Journal of Biological Chemistry

Year: 2000

Volume: 275

Issue: 24

Pages: 18271-18278

ISSN (print): 0021-9258

ISSN (electronic): 1083-351X

Publisher: American Society for Biochemistry and Molecular Biology


DOI: 10.1074/jbc.M001309200

PubMed id: 10751399


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