Ribozymes for new genetic coding systems

描述（由申请人提供）： 本研究的长期目标是设计一种新颖实用的基于核酶的tRNA氨酰化系统，从而促进非天然氨基酸突变技术，从而使其成为一种更易于使用的技术。本实验室筛选的一种人工核酶对苯丙氨酸（Phe）类似物和含有不同无义密码子的抑制性tRNA具有广谱活性。通过将这种核酶（称为PheFlexizyme）固定在树脂上，极大地促进了装载有Phe类似物的抑制剂tRNA的合成;其中用户指定的tRNA和Phe类似物在这种树脂上反应，并且所得滤液（或上清液）含有所需的氨酰-tRNA。然后将这种带电荷的tRNA用于无细胞翻译系统中，以在单个位置掺入Phe类似物或在两个特定位置掺入两个Phe类似物。整个过程，包括tRNA充电，体外翻译和蛋白质纯化，可以在一天内完成。翻译效率通常为50-70 mu/g/mL，因此可以生产足够量的蛋白质用于进一步的生物学研究。应该注意的是，PheFlexizyme树脂可以重复使用超过10次，因此它也是经济的。在本申请中，我们将尝试开发更复杂的核酶氨酰化技术。具体目标是（1）扩大PheFlexizyme的Phe类似物的库，（2）基于PheFlexizyme支架的新Flexizyme的体外进化，（3）在无细胞转录偶联翻译装置中原位产生PheFlexizyme，以及（4）PheFlexizyme的应用。

DESCRIPTION (provided by applicant): The long-term goal of this research is to devise a novel and practical tRNA aminoacylation system based on ribozymes, which facilitates the technique of nonnatural amino acid mutagenesis and thus raises it to a more user-accessible technology. An artificial ribozyme selected in our laboratory exhibits a broad spectrum of activities toward phenylalanine (Phe) analogs and suppressor tRNAs bearing different nonsense codons. By immobilizing this ribozyme (called PheFlexizyme) on a resin, the synthesis of suppressor tRNAs charged with Phe analogs is largely facilitated; where a user-specified tRNA and a Phe analog are reacted on this resin and the resulting filtrate (or supernatant) contains the desired aminoacyl-tRNA. This charged tRNA is then used in a cell-free translation system to incorporate a Phe analog at a single position or two Phe analogs at two specific positions. The entire processes, including tRNA charging, in vitro translation, and purification of protein, can be done in one day. The translation efficiency is generally 50-70 mu/g/mL, thus it is feasible to produce a sufficient amount of protein for further biological studies. It should be noted that the PheFlexizyme-resin can be reused more than 10 times, and therefore it is also economical. In this application, we will attempt to develop more sophisticated ribozyme aminoacylation technologies. Specific aims are (1) expanding repertoire of Phe analogs for PheFlexizyme, (2) in vitro evolution of new Flexizymes based on the PheFIlexizyme scaffold, (3) in situ generation of PheFlexizyme in the cell-free transcription-coupled translation apparatus, and (4) applications of PheFlexizyme.