Yeast genetic selections to optimize RNA decoys for transcription factor NF-κB

Laura A. Cassiday, L James Maher III

Research output: Contribution to journalArticle

49 Scopus citations

Abstract

In vitro-selected RNA aptamers are potential inhibitors of disease-related proteins. Our laboratory previously isolated an RNA aptamer that binds with high affinity to human transcription factor NF-κB. This RNA aptamer competitively inhibits DNA binding by NF-κB in vitro and is recognized by its target protein in vivo in a yeast three-hybrid system. In the present study, yeast genetic selections were used to optimize the RNA aptamer for binding to NF-κB in the eukaryotic nucleus. Selection for improved binding to NF-κB from RNA libraries encoding (i) degenerate aptamer variants and (ii) sequences present at round 8 of 14 total rounds of in vitro selection yielded RNA aptamers with dramatically improved in vivo activity. Furthermore, we show that an in vivo-optimized RNA aptamer exhibits specific "decoy" activity, inhibiting transcriptional activation by its NF-κB target protein in a yeast one-hybrid assay. This decoy activity is enhanced by the expression of a bivalent aptamer. The combination of in vitro and in vivo genetic selections was crucial for obtaining RNA aptamers with in vivo decoy activity.

Original languageEnglish (US)
Pages (from-to)3930-3935
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume100
Issue number7
DOIs
StatePublished - Apr 1 2003

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ASJC Scopus subject areas

  • General
  • Genetics

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