Yeast genetic selections to optimize RNA decoys for transcription factor NF-κB
Yeast genetic selections to optimize RNA decoys for transcription factor NF-κB
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DOI：
10.1073/pnas.0736013100
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发表时间：
2003-04-01
影响因子：
11.1
通讯作者：
Maher, LJ
中科院分区：
文献类型：
--
作者：
Cassiday, LA;Maher, LJ
体外选择的RNA适体是疾病相关蛋白的潜在抑制剂。我们的实验室先前分离出一种RNA适体，其以高亲和力结合人转录因子NF-κ B。这种RNA适体在体外竞争性抑制NF-κ B与DNA的结合，并在酵母三杂交系统中被其体内靶蛋白识别。在本研究中，使用酵母遗传选择来优化RNA适体以结合真核细胞核中的NF-κ B。从编码（f）简并适体变体和（6）在总共14轮体外选择的第8轮存在的序列的RNA文库中选择改善的与NF-κ B的结合产生具有显著改善的体内活性的RNA适体。此外，我们表明，在体内优化的RNA适体表现出特定的“诱饵”活性，抑制转录激活的NF-κ B靶蛋白在酵母单杂交试验。这种诱饵活性通过二价适体的表达而增强。体外和体内遗传选择的结合对于获得具有体内诱饵活性的RNA适体至关重要。
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.