ISOLATION OF HIGH-AFFINITY RNA LIGANDS TO HIV-1 INTEGRASE FROM A RANDOM POOL

We were able to isolate high-affinity RNAs from a random pool that bin ds to integrase protein from the human immunodeficiency virus-type 1 u sing the procedure now known as SELEX. Generally, the RNAs fell into t hree different classes in binding buffer containing 250 mM NaCl: group I class of molecules binds integrase with a dissociation constant (K- d) on the order of 10 nM, group II molecules had a K-d of about 80 nM, and group III about 800 nM. The RNA with the highest affinity from th e group I class of molecules, designated P5, was characterized using c omputer modeling, chemical and enzymatic probing, and deletion analysi s. Our secondary structure model for this RNA suggests interactions be tween looped-out fixed nucleotides and nucleotides from the randomized region; a GNRA tetraloop is also in the structure. We showed that our integrase was able to process a U5 mimic in vitro. P5 competes effect ively for binding with the double-stranded DNA mimic of U5 at 180 mM N aCl concentration. (C) 1995 Academic Press, Inc.