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基于核酶的新颖实用的tRNA氨酰化系统，促进非天然氨基酸诱变技术，从而将其提升为更易于用户使用的技术。我们实验室选择的人工核酶对苯丙氨酸 (Phe) 类似物和带有不同无义密码子的抑制 tRNA 表现出广泛的活性。通过将这种核酶（称为 PheFlexizyme）固定在树脂上，可以极大地促进带有 Phe 类似物的抑制性 tRNA 的合成；其中用户指定的 tRNA 和 Phe 类似物在该树脂上发生反应，所得滤液（或上清液）包含所需的氨酰基-tRNA。然后将该带电 tRNA 用于无细胞翻译系统，在单个位置掺入一个 Phe 类似物或在两个特定位置掺入两个 Phe 类似物。包括tRNA充电、体外翻译和蛋白质纯化的整个过程可以在一天内完成。翻译效率一般为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。值得注意的是，PheFlexizyme-树脂可以重复使用10次以上，因此也很经济。在此应用中，我们将尝试开发更复杂的核酶氨酰化技术。具体目标是（1）扩展 PheFlexizyme 的 Phe 类似物库，（2）基于 PheFIlexizyme 支架的新型 Flexizyme 体外进化，（3）在无细胞转录偶联翻译装置中原位生成 PheFlexizyme，以及（ 4）PheFlexizyme的应用。