SELECTION AND DESIGN OF HIGH-AFFINITY RNA LIGANDS FOR HIV-1 REV

We have used in vitro selection to isolate minimal, high-affinity RNA ligands for the Rev protein of HIV-1. Sequence analysis reveals that t he tightest binding aptamers exhibit some similarity to a Rev-binding element (RBE) localized within the Rev-responsive element (RRE), but a lso contain novel sequence and structural motifs. A short helical stem and bulged nucleotides (nt) CUC...UYGAG that have no counterpart in t he wild-type (wt) element contribute to high-affinity binding. We have designed and synthesized a short (37 nt) RNA molecule that incorporat es this motif; this RNA ligand has from three- to fivefold tighter bin ding than the full-length wt element, and up to 16-fold tighter than m inimal wt RBEs. A guanosine:guanosine pairing that is postulated to oc cur in the wt element has been altered to other base pairings in the c ontext of our optimized minimal element. RNAs that contain non-Watson- Crick base pairings, that can be modeled as isosteric to the wt G:G pa ir, bind Rev up to 160-fold tighter than elements that contain canonic al Watson-Crick pairings or non-isosteric mismatches. These results su pport the hypothesis that Rev recognizes structural features associate d with a non-Watson-Crick base pair.