Yeast genetic selections to optimize RNA decoys for transcription factor NF-kappa B.
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-kappaB. This RNA aptamer competitively inhibits DNA binding by NF-kappaB 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-kappaB in the eukaryotic nucleus. Selection for improved binding to NF-kappaB 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-kappaB 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.
Cassiday
Laura A
LA
Department of Biochemistry and Molecular Biology, Mayo Foundation, Rochester, MN 55905, USA.
Maher
L James
LJ
D001483:Base Sequence; D003001:Cloning, Molecular; D007700:Kinetics; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D012331:RNA, Fungal / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics; D012641:Selection, Genetic
D016328:NF-kappa B; D012331:RNA, Fungal; D029701:Saccharomyces cerevisiae Proteins