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.
Green
L
L
NeXagen, Inc., Boulder, CO 80301.
Waugh
S
S
Binkley
J P
JP
Hostomska
Z
Z
Hostomsky
Z
Z
Tuerk
C
C
D000998:Antiviral Agents / Q000737:chemistry*; D001483:Base Sequence; D001665:Binding Sites; D016384:Consensus Sequence; D054303:HIV Reverse Transcriptase; D015497:HIV-1 / Q000201:enzymology; D008024:Ligands; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012313:RNA / Q000737:chemistry / Q000494:pharmacology*; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism; D018894:Reverse Transcriptase Inhibitors; D013329:Structure-Activity Relationship
D000998:Antiviral Agents; D008024:Ligands; D018894:Reverse Transcriptase Inhibitors; D012313:RNA; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase