期刊
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
卷 100, 期 7, 页码 3930-3935出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.0736013100
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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 (f) degenerate aptamer variants and (6) 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.
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