Isolation of High-Affinity RNA Ligands to HIV-1 Integrase from a Random Pool

Autor: Allen, Patrick, Worland, Steve, Gold, Larry
Zdroj: Virology; June 1995, Vol. 209 Issue: 2 p327-336, 10p
Abstrakt: We were able to isolate high-affinity RNAs from a random pool that binds to integrase protein from the human immunodeficiency virus-type 1 using the procedure now known as SELEX. Generally, the RNAs fell into three different classes in binding buffer containing 250 mMNaCl: group I class of molecules binds integrase with a dissociation constant (Kd) on the order of 10 nM, group II molecules had a Kdof about 80 nM, and group III about 800 nM. The RNA with the highest affinity from the group I class of molecules, designated P5, was characterized using computer modeling, chemical and enzymatic probing, and deletion analysis. Our secondary structure model for this RNA suggests interactions between 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 effectively for binding with the double-stranded DNA mimic of U5 at 180 mMNaCl concentration.
Databáze: Supplemental Index