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 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 mM NaCl: group I class of molecules binds integrase with a dissociation constant (Kd) on the order of 10 nM, group II molecules had a Kd of 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 mM NaCl concentration.
Allen
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Department of Molecular, Cellular and Developmental Biology, University of Colorado at Boulder 80309-0347, USA.
Worland
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Gold
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D001483:Base Sequence; D003001:Cloning, Molecular; D003198:Computer Simulation; D004254:DNA Nucleotidyltransferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D017931:DNA Primers; D005814:Genes, Viral; D015497:HIV-1 / Q000201:enzymology* / Q000235:genetics; D019426:Integrases; D007700:Kinetics; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D012367:RNA, Viral / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D015678:Viral Structural Proteins / Q000235:genetics; D016662:Virus Integration
D017931:DNA Primers; D008024:Ligands; D012367:RNA, Viral; D011994:Recombinant Proteins; D015678:Viral Structural Proteins; D004254:DNA Nucleotidyltransferases; D019426:Integrases