SELECTION OF STRUCTURE-SPECIFIC INHIBITORS OF THE HIV REV-REV RESPONSE ELEMENT COMPLEX

We devised a single-step selection method to identify short sequences within the folded Rev response element (RRE) of the human immunodefici ency virus (HIV) RNA genome that are proximal and able to bind short o ligonucleotides. The method employed a library of partially randomized tethered oligonucleotide probes (TOPs) complementary to all regions o f the RRE and an RNase H cleavage assay which identified those RRE reg ions preferred by the TOPs. Six short sequences were identified. Two T OPs synthesized on the basis of this selection, S1-5-S2 and S1-1-S2, w ere potent, concentration-dependent inhibitors of a RRE function in vi tro and abolished interaction of the RRE with the HIV regulatory prote in Rev at nanomolar concentration (25-degrees-C, 0.8 nM RRE, 45 nM Rev ). TOPs S1-5-S2 and S1-1-S2 exhibited greater potency and faster assoc iation kinetics than traditional oligonucleotides targeted to the same regions. The random TOP selection procedure described here allows rat ional design of oligonucleotide-based ligands for RNA that exploit (ra ther than avoid) the complex structure of the RNA target.