USING IN-VITRO SELECTION TO DIRECT THE COVALENT ATTACHMENT OF HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 REV PROTEIN TO HIGH-AFFINITY RNA LIGANDS

We have used an in vitro selection procedure called crosslinking SELEX (SELEX = systematic evolution of ligands by exponential enrichment) t o identify RNA sequences that bind with high affinity and crosslink to the Rev protein from human immunodeficiency virus type 1 (HIV-1). A r andomized TC?IA library substituted with the photoreactive chromophore 5-iodouracil was irradiated with monochromatic UV light in the presen ce of Rev. Those sequences with the ability to photocrosslink to Rev w ere partitioned from the rest of the RNA pool, amplified, and used for the next round of selection, Rounds of photocrosslinking selection we re alternated with rounds of selection for RNA sequences with high aff inity to Rev. This iterative, dual-selection method yielded RNA molecu les with subnanomolar dissociation constants and high efficiency photo crosslinking to Rer. Some of the RNA molecules isolated by this proced ure form a stable complex with Rev that is resistant to denaturing gel electrophoresis in the absence of UV irradiation. In vitro selection of nucleic acids by using modified nucleotides allows the isolation of nucleic acid molecules with potentially limitless chemical capacities to covalently attack a target molecule.