title: Characterization of anti-NF-kappaB RNA aptamer-binding specificity in vitro and in the yeast three-hybrid system.
abstract: RNA aptamers offer a potential therapeutic approach to the competitive inhibition of DNA-binding transcription factors. In previous reports we described in vitro selection and characterization of anti-NF-kappaB p50 and p65 RNA aptamers. We now describe the further characterization of these aptamers in vitro and in vivo. We show that sub-saturating concentrations of certain anti-p50 RNA aptamers promote complex formation with NF-kappaB p50 tetramers, whereas anti-p65 R1 RNA aptamers bind NF-kappaB dimers under all conditions tested. Yeast three-hybrid RNA aptamer specificity studies corroborate previous in vitro results, verifying that anti-p50 and anti-p65 R1 RNA aptamers are highly specific for NF-kappaB p50(2) and p65(2), respectively. These studies introduce a novel T-cassette RNA transcript that improves RNA display from a four-way RNA junction. Mutagenesis of the anti-p65 R1 aptamer reveals tolerated substitutions, suggesting a complex tertiary structure. We describe in vivo selections from a yeast three-hybrid RNA library containing sequences present early in the R1 SELEX process to identify novel anti-p65 RNA aptamers, termed Y1 and Y3. These aptamers appear to be compact bulged hairpins, reminiscent of anti-p50. Y1 competitively inhibits the DNA-binding domain of NF-kappaB p65(2) in vitro.
authors.affiliation: Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, MN 55905, USA.
authors.forename: Susan E
authors.initials: SE
authors.lastname: Wurster
authors.forename: John Paul
authors.initials: JP
authors.lastname: Bida
authors.forename: Yeng F
authors.initials: YF
authors.lastname: Her
authors.forename: L James
authors.initials: LJ
authors.lastname: Maher
mesh_terms: D052157:Aptamers, Nucleotide / Q000737:chemistry*; D016296:Mutagenesis; D016328:NF-kappa B / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D052002:NF-kappa B p50 Subunit / Q000037:antagonists & inhibitors; D009690:Nucleic Acid Conformation; D055503:Protein Multimerization; D052156:SELEX Aptamer Technique; D051996:Transcription Factor RelA / Q000037:antagonists & inhibitors; D020798:Two-Hybrid System Techniques; D015003:Yeasts / Q000235:genetics
chemical_list: D052157:Aptamers, Nucleotide; D016328:NF-kappa B; D052002:NF-kappa B p50 Subunit; D051996:Transcription Factor RelA
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