Ribozymes for new genetic coding systems

用于新遗传编码系统的核酶

• 批准号: 7012204
• 负责人: John P Richard
• 金额: $ 32.08万
• 依托单位: STATE UNIVERSITY OF NEW YORK AT BUFFALO
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-02-01 至 2008-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7012204
• 关键词: acylation; aminoacid tRNA ligase; aminoacyl tRNA; biotechnology; green fluorescent proteins; peptide chemical synthesis; phenylalanine analog; protein engineering; protein structure function; ribonuclease P; ribozymes; technology /technique development

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.

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氨基酰化系统，以促进非天然氨基酸突变技术的发展，从而使其成为一种更易于用户使用的技术。我们实验室选择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tRNAs显示了广泛的活性。通过将这种核酶(称为PheFlexizyme)固定在树脂上，很大程度上促进了带有Phe类似物的抑制子tRNA的合成；其中用户指定的tRNA和Phe类似物在树脂上反应，得到的滤液(或上清液)包含所需的氨基酰基tRNA。然后将这种带电的tRNA用于无细胞翻译系统，以在单个位置结合一个Phe类似物或在两个特定位置结合两个Phe类似物。包括tRNA充电、体外翻译和蛋白质纯化在内的整个过程可以在一天内完成。翻译效率一般为50-70mU/g/mL，因此生产足够数量的蛋白质用于进一步的生物学研究是可行的。值得注意的是，PheFlexizyme树脂可以重复使用10次以上，因此也是经济的。在这项应用中，我们将尝试开发更复杂的核酶氨基酰化技术。具体目标是(1)扩大PheFlexizyme类似物的库，(2)基于PheFIlexizyme支架的新Flexizyme的体外进化，(3)在无细胞转录耦合翻译装置中原位产生PheFlexizyme，以及(4)PheFlexizyme的应用。