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

Kensch O, Connolly BA, Steinhoff HJ, McGregor A, Goody RS, Restle T
Physikalische Biochemie, MPI fuer molekulare Physiologie, Dortmund 44227.

SELEX is a powerful method for the identification of small oligonucleotides which bind
with high affinity and specificity to target proteins. Such DNAs/RNAs are a new class of
potential chemotherapeutics which could block the enzymatic activity of pathologically
relevant proteins. We have conducted a detailed biochemical study of the interaction of
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 similar association rates 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 Kd, 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.