Double Incorporation of Non-Canonical Amino Acids in an Animal and its Application for Precise and Independent Optical Control of Two Target Genes

动物体内非规范氨基酸的双重掺入及其在两个靶基因精确独立光学控制中的应用

• 批准号: BB/Y006380/1
• 负责人: Sebastian Greiss
• 金额: $ 58.56万
• 依托单位: University of Edinburgh
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2024
• 资助国家: 英国
• 起止时间: 2024 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FY006380%2F1
• 关键词: Double; Incorporation; Non; Canonical; Amino

Proteins and the nucleic acids DNA and RNA are the most fundamental building blocks of life. Proteins are chains of amino acids made from 20 different building blocks, the canonical amino acids. The genetic code is the cipher that connects proteins to nucleic acids. When proteins are produced, the sequence of nucleic acids is translated into a sequence of amino acids. There are 4 nucleotide bases of DNA and RNA organized in triplets. There are 64 triplets of which 61 are each assigned to one amino acid, while three triplets act as 'stop codons' and signal the end of the protein.The genetic code is the link between triplet codons and amino acids. The 'decoding' of the nucleotide sequence happens using transfer RNA (tRNA). A tRNA molecule contains a triplet codon on one end and the corresponding amino acid at the other end. The amino acid is attached to tRNA by an enzyme called aminoacyl-tRNA-synthetase (aaRS). The tRNA triplet binds to an mRNA triplet and an amino acid is added to the growing protein chain.Genetic code expansion is a technology whereby new tRNAs and aaRS are added to the cell, allowing it to make proteins using more than the 20 canonical amino acids. The additional amino acids, called non-canonical amino acids (ncAA), are chemically synthesized 'designer' amino acids with properties not found in nature. To specify where the ncAA should go within the amino acid sequence of a protein it is necessary to assign them to a nucleic acid triplet. Since 61 triplets are already assigned, this is normally done by making use of one of the three stop codons.We have previously established this technology in animals and we have used it to develop powerful in vivo tools that employ proteins modified by the introduction of ncAA. In this proposal we aim to take the next big step and develop a method allowing the use of multiple ncAA together, the first time this will be made possible in an animal. For this we will develop i) additional aaRS/tRNA pairs for use in C. elegans, which can be used together with existing pairs without cross-reacting, and ii) we will establish new 'blank' nucleotide codons to determine where in the protein the ncAA will be incorporated. We will explore the use of triplet stop codons other than the widely used UAG (amber) stop, as well as nucleotide quadruplet codons. We have recently shown that the use of quadruplet codons is possible in animals and can approach incorporation efficiencies close to that observed with traditional triplet codons.We will then use the technology to upgrade a method we have previously developed for controlling gene expression with light. Using the new ability to employ two ncAA we will be able to switch on or off two genes independently of each other. This is an important breakthrough and will allow us and other researchers to, for example, control the activity of single nerve cells in C. elegans and tease apart the functioning of the C. elegans nervous system with a level of precision that is impossible using existing methods.The technology we develop will also be immensely useful for researchers outside of C. elegans to study the basics of how life works in anything from bacteria to human cells.

蛋白质和核酸DNA和RNA是生命最基本的组成部分。蛋白质是由20种不同的氨基酸组成的氨基酸链，这些氨基酸是标准氨基酸。遗传密码是连接蛋白质和核酸的密码。当蛋白质产生时，核酸序列被翻译成氨基酸序列。DNA和RNA有4个核苷酸碱基组成三胞胎。共有64个三胞胎，其中61个分别对应于一个氨基酸，而三个三胞胎则充当“终止密码子”，标志着蛋白质的终结。遗传密码是连接三联体密码子和氨基酸的纽带。核苷酸序列的“解码”是通过转运RNA （tRNA）进行的。tRNA分子的一端包含一个三联体密码子，另一端包含相应的氨基酸。这种氨基酸通过一种叫做氨基酰基-tRNA合成酶（aaRS）的酶附着在tRNA上。tRNA三联体与mRNA三联体结合，一个氨基酸被添加到生长的蛋白质链中。遗传密码扩展是一种将新的trna和aar添加到细胞中，使其能够使用20多种典型氨基酸制造蛋白质的技术。额外的氨基酸，称为非规范氨基酸（ncAA），是化学合成的“设计”氨基酸，具有自然界中不存在的特性。为了确定ncAA在蛋白质氨基酸序列中的位置，有必要将它们分配到核酸三重体中。由于已经分配了61个三联体，这通常是通过使用三个终止密码子中的一个来完成的。我们之前已经在动物身上建立了这项技术，我们已经用它来开发强大的体内工具，这些工具使用通过引入ncAA修饰的蛋白质。在这项提议中，我们的目标是迈出下一步，开发一种允许同时使用多种ncAA的方法，这将是第一次在动物身上实现。为此，我们将开发i)用于秀丽隐杆线虫的额外aaRS/tRNA对，这些aaRS/tRNA对可以与现有的aaRS/tRNA对一起使用，而不会发生交叉反应；ii)我们将建立新的“空白”核苷酸密码子，以确定ncAA在蛋白质中的结合位置。我们将探索除广泛使用的UAG（琥珀色）终止子之外的三联体终止密码子以及核苷酸四联体密码子的使用。我们最近表明，在动物中使用四联体密码子是可能的，并且可以接近与传统三联体密码子观察到的结合效率。然后，我们将利用这项技术升级我们之前开发的用光控制基因表达的方法。利用使用两个ncAA的新能力，我们将能够独立地打开或关闭两个基因。这是一个重要的突破，将使我们和其他研究人员能够控制秀丽隐杆线虫单个神经细胞的活动，并以现有方法无法达到的精度梳理秀丽隐杆线虫神经系统的功能。我们开发的技术也将对秀丽隐杆线虫以外的研究人员非常有用，他们可以研究从细菌到人类细胞的生命是如何运作的基础。