CAREER: Quadruplet Codon Decoding - Mechanistic Studies and Application in Cellular Genetic Code Expansion

职业：四联体密码子解码 - 机制研究及其在细胞遗传密码扩展中的应用

• 批准号: 1553041
• 负责人: Jiantao Guo
• 金额: $ 62.23万
• 依托单位: University of Nebraska-Lincoln
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-04-01 至 2022-03-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1553041&HistoricalAwards=false
• 关键词: CAREER; Quadruplet; Codon; Decoding; Mechanistic

All living organisms transcribe and translate the information encoded by DNA by well-understood processes known as transcription and translation respectively. The process converts the information stored in DNA into protein functions that are essential catalysts for life supporting reactions. In this process, every three nucleotides in a gene are read as a specific code for the serial incorporation of the natural building blocks protein synthesis. This study aims at refactoring the translational machinery to evolve novel transfer-RNA species that will recognize not a three-, but four-nucleotide code for the specific translational incorporation of unnatural amino acids. The objective is to create proteins that feature novel catalytic functions and chemical properties to aid biochemical and biomedical research. In addition, this research will shed light on fundamental questions about detailed mechanisms of translation. The project will attract, train, and transform today's young talents into tomorrow's leaders in sciences at the interface of chemistry and biology. The scientific insights and new technologies obtained under the proposed research program will be valuable to a broad biochemistry and synthetic biology research community.While the triplet codon is the predominant form of the current genetic code, non-canonical reading of a quadruplet codon is a natural process and can be achieved under experimental conditions. Transfer RNA (tRNA) plays important roles in the quadruplet codon decoding process. The work will use a directed evolution approach to identify tRNA mutants that are able to efficiently decode quadruplet codons in a genomically recoded bacterium. A systematic study of quadruplet codon decoding mechanism will be conducted, which is likely contribute to the understanding on how ribosomes recognize the reading frame. This research is further strengthen by expanding the cellular genetic code with multiple and mutually orthogonal quadruplet codons. This would allow for the incorporation of t unnatural amino acids with different physical and chemical properties. Applications of this technology could enable unprecedented in vivo study of proteins in living cells, and could enable novel synthetic biology applications.

所有生物体都通过众所周知的转录和翻译过程转录和翻译DNA编码的信息。这个过程将存储在DNA中的信息转化为蛋白质功能，这些功能是生命支持反应的必要催化剂。在这个过程中，基因中的每三个核苷酸被读取为一个特定的代码，用于连续掺入天然构建模块蛋白质合成。这项研究的目的是重构翻译机制，发展新的transfer-RNA物种，将识别非天然氨基酸的特定翻译掺入的不是三个，而是四个核苷酸的代码。其目标是创造具有新的催化功能和化学性质的蛋白质，以帮助生物化学和生物医学研究。此外，这项研究将揭示有关翻译的详细机制的基本问题。该项目将吸引，培养和转化今天的年轻人才成为未来的领导者在化学和生物学的接口科学。 该研究计划所获得的科学见解和新技术将对广泛的生物化学和合成生物学研究社区具有价值。虽然三联体密码子是当前遗传密码的主要形式，但四联体密码子的非规范阅读是一个自然过程，可以在实验条件下实现。转运RNA（transfer RNA，tRNA）在四联体密码子的解码过程中起着重要的作用。 该工作将使用定向进化方法来鉴定能够有效解码基因组重新编码细菌中四联体密码子的tRNA突变体。对四联体密码子的解码机制进行系统的研究，将有助于理解核糖体如何识别阅读框架。 这一研究进一步加强了扩大细胞遗传密码与多个和相互正交的四联体密码子。这将允许掺入具有不同物理和化学性质的非天然氨基酸。这项技术的应用可以实现对活细胞中蛋白质的前所未有的体内研究，并可以实现新的合成生物学应用。