Comprehensive chemical modification interference and nucleotide substitution analysis of an RNA pseudoknot inhibitor to HIV-1 reverse transcriptase.

We had previously used in vitro RNA selection techniques to describe a consensus RNA pseudoknot that binds and inhibits HIV-1 reverse transcriptase (HIV-RT). In this work we constructed variants of this consensus pseudoknot in order to evaluate the contributions of individual nucleotide identities and secondary structure to affinity for HIV-RT. We have also used chemical modification of ligand RNAs to corroborate the predicted structure of the pseudoknot, to discover which modifiable groups are protected from chemical attack when bound to HIV-RT, and to find which modifications interfere with binding to HIV-RT. A novel interference study is presented which involves selection of ligands from a pool created by mixed reagent oligonucleotide synthesis in order to rapidly determine allowed substitutions of 2'-OCH3 groups for the usual 2'-OH group in such RNA ligands.