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

In vitro-selected RNA aptamers are potential inhibitors of diseaserelated proteins. Our laboratory previously isolated an RNA aptamer that binds with high affinity to human transcription factor NFB. This RNA aptamer competitively inhibits DNA binding by NFB 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 NFB in the eukaryotic nucleus. Selection for improved binding to NFB 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 NFB 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.