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

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，这些氨基酸是典型的氨基酸。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的4个核苷酸碱基组成三联体。有64个三联体，其中61个三联体分别对应一个氨基酸，而三个三联体作为“终止密码子”，并发出蛋白质末端的信号。遗传密码是三联体密码子和氨基酸之间的联系。核苷酸序列的“解码”是通过转移RNA（tRNA）进行的。tRNA分子的一端含有三联密码子，另一端含有相应的氨基酸。氨基酸通过一种叫做氨酰-tRNA-合成酶（阿尔斯）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技术，通过这种技术，新的tRNA和阿尔斯被添加到细胞中，使其能够使用20多种典型氨基酸制造蛋白质。额外的氨基酸，称为非典型氨基酸（ncAA），是化学合成的“设计师”氨基酸，具有自然界中未发现的特性。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联体中。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之一来完成的。我们以前已经在动物中建立了这项技术，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。在这项提案中，我们的目标是迈出下一大步，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中实现。为此，我们将开发i）用于C. elegans，其可以与现有对一起使用而没有交叉反应，和ii）我们将建立新的“空白”核苷酸密码子以确定ncAA将在蛋白质中的何处并入。我们将探索除了广泛使用的UAG（琥珀色）终止密码子之外的三联终止密码子以及核苷酸四联体密码子的使用。我们最近发现，在动物中使用四联体密码子是可能的，并且可以接近传统三联体密码子观察到的掺入效率。然后，我们将使用该技术来升级我们先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，我们将能够彼此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C中单个神经细胞的活动。elegans和梳理除了C的功能。我们开发的技术也将对C以外的研究人员非常有用。研究从细菌到人类细胞的生命运作的基础知识。

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.