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

所有活着的生物体通过众所周知的转录和翻译分别转录和翻译DNA编码的信息。这个过程将储存在DNA中的信息转化为蛋白质功能，这些功能是维持生命反应的重要催化剂。在这个过程中，基因中每三个核苷酸被解读为一种特定的密码，用于序列结合天然构建块的蛋白质合成。这项研究旨在重构翻译机制，以进化出新的转移RNA物种，该物种将识别非天然氨基酸的特定翻译结合的三核苷酸编码，而不是四核苷酸编码。其目标是创造具有新的催化功能和化学性质的蛋白质，以帮助生化和生物医学研究。此外，这项研究还将有助于揭示有关具体翻译机制的基本问题。该项目将吸引、培养和转化当今的年轻人才，使其成为未来化学和生物学科的领军人物。在拟议的研究计划下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领域具有价值。虽然三重密码子是当前遗传密码的主要形式，但四重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。转移RNA(TRNA)在四联密码子解码过程中起着重要作用。这项工作将使用定向进化方法来识别tRNA突变体，这些突变体能够有效地解码基因组重新编码的细菌中的四重密码子。将对四重密码子的解码机制进行系统的研究，这可能有助于理解核糖体如何识别阅读框架。通过使用多个相互正交的四重密码子扩展细胞遗传密码，这项研究得到了进一步加强。这将允许加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。

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.