HIV-1 Reverse Transcriptase-Pseudoknot RNA Aptamer Interaction Has a Binding Affinity in the Low Picomolar Range Coupled with High Specificity*

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 lowK 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.

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