Ogawa Atsushi, Hayami Masayoshi, Sando Shinsuke, Aoyama Yasuhiro
Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan.
J Nucleic Acids. 2012;2012:538129. doi: 10.1155/2012/538129. Epub 2012 Jul 18.
Here is presented a concept for in vitro selection of suppressor tRNAs. It uses a pool of dsDNA templates in compartmentalized water-in-oil micelles. The template contains a transcription/translation trigger, an amber stop codon, and another transcription trigger for the anticodon- or anticodon loop-randomized gene for tRNA(Ser). Upon transcription are generated two types of RNAs, a tRNA and a translatable mRNA (mRNA-tRNA). When the tRNA suppresses the stop codon (UAG) of the mRNA, the full-length protein obtained upon translation remains attached to the mRNA (read-through ribosome display) that contains the sequence of the tRNA. In this way, the active suppressor tRNAs can be selected (amplified) and their sequences read out. The enriched anticodon (CUA) was complementary to the UAG stop codon and the enriched anticodon-loop was the same as that in the natural tRNA(Ser).
本文提出了一种体外筛选抑制性tRNA的概念。它使用了位于分隔的油包水微团中的双链DNA模板库。该模板包含一个转录/翻译触发序列、一个琥珀色终止密码子，以及用于tRNA（Ser）的反密码子或反密码子环随机化基因的另一个转录触发序列。转录后会产生两种类型的RNA，一种是tRNA，另一种是可翻译的mRNA（mRNA-tRNA）。当tRNA抑制mRNA的终止密码子（UAG）时，翻译后获得的全长蛋白质仍与包含tRNA序列的mRNA相连（通读核糖体展示）。通过这种方式，可以选择（扩增）活性抑制性tRNA并读出其序列。富集的反密码子（CUA）与UAG终止密码子互补，富集的反密码子环与天然tRNA（Ser）中的相同。
