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Nucleic Acids Research Advance Access originally published online on August 20, 2009
Nucleic Acids Research 2009 37(18):6214-6224; doi:10.1093/nar/gkp670
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Nucleic Acids Research, 2009, Vol. 37, No. 18 6214-6224
© The Author 2009. Published by Oxford University Press.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses?by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

RNA

Characterization of anti-NF-{kappa}B RNA aptamer-binding specificity in vitro and in the yeast three-hybrid system

Susan E. Wurster, John Paul Bida, Yeng F. Her and L. James Maher, III*

Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, MN 55905, USA

*To whom correspondence should be addressed. Tel: +1 507 284 9041; Fax: +1 507 284 2053; Email: maher{at}mayo.edu

Received May 12, 2009. Revised July 29, 2009. Accepted July 30, 2009.

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-{kappa}B 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-{kappa}B p50 tetramers, whereas anti-p65 R1 RNA aptamers bind NF-{kappa}B 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-{kappa}B p502 and p652, 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-{kappa}B p652 in vitro.


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