Engineering translation machinery for a semi-synthetic organism

项目摘要 将非规范氨基酸(NCAA)结合到蛋白质中可以通过编码氨基酸来赋予新的性质 具有额外官能团和元素的酸，进而促进产生新的蛋白质 在化学生物学和蛋白质治疗方面的应用。目前的方法可以将两种方法结合起来 将非规范的氨基酸转化为蛋白质，并试图通过重新利用Stop或Stop来覆盖当前的遗传密码 通过引入重新调整用途的古生菌tRNA和氨基酰tRNA合成酶(AARs)，发现了稀有密码子。 尝试覆盖现有代码会导致与适应本机代码的机制竞争，在 转而降低翻译保真度。例如，从古生菌中重新调整tRNA/Aars对的用途已经导致了两个 本地细菌不能很好地识别的常用正交翻译单元(OTU) 翻译机制(包括EF-Tu和核糖体)。我们不是覆盖现有的密码子，而是寻求 创造新的。我们已经证明了非自然碱基对(UBP)可以在半复制中稳定复制 为此目的的合成有机体(SSO)。UBP的转录和翻译工作已经成功 使用目前可从古生菌获得的OTUS完成。随着更多的密码子包含不自然的 在现有基地的情况下，我们现在受到可用的OTUS数量和有效性的限制。因此，中心目标是 这项提议的一部分是开发用于非自然密码子翻译的OTUS。在具体目标1中，稳定和 在RNA中评估了UBP在所有序列上下文中的正交性，提供了对有希望的密码子的洞察 以供翻译。特殊目的2利用tRNAs反密码子中的非自然核苷酸作为识别基序 对大肠杆菌AARs重新编程以翻译UBP。最后，特异靶3会切换氨基酸的专一性 目标2中的AARSS从典型的氨基酸发展为与化学生物学和蛋白质相关的NCAA 治疗学。这些目标的成功完成将提供对RNA生物学的洞察，产生将 无缝集成到SSO的翻译机制中，并允许生产具有多个 全美大学生体育协会。这些OTU的创建和使用有可能通过以下方式使蛋白质治疗领域发生革命性变化 与几个NCAA一起为蛋白质生产提供了一个强大的平台。

Project Summary Incorporating noncanonical amino acids (ncAAs) into proteins can grant novel properties by encoding amino acids with additional functional groups and elements, in turn, facilitating the creation of novel proteins for applications in chemical biology and protein therapeutics. Current methods for doing so can incorporate two noncanonical amino acids into proteins and seek to overwrite the current genetic code by repurposing stop or rare codons through the introduction of repurposed archaeal tRNAs and aminoacyl tRNA synthetases (aaRSs). Attempting to overwrite an existing code results in competition with the machinery adapted to the native code, in turn reducing translational fidelity. For example, repurposing tRNA/aaRS pairs from archaea has resulted in two commonly used orthogonal translation units (OTUs) that are not recognized well by the native bacterial translational machinery (including EF-Tu and ribosomes). Instead of overwriting existing codons, we seek to create new ones. We have demonstrated that an unnatural base pair (UBP) can be stably replicated in a semi- synthetic organism (SSO) for this purpose. Transcription and translation of the UBP has been successfully accomplished using the current OTUs available from archaea. With many more codons containing an unnatural base available, we are now limited by the number and efficacy of the available OTUs. Therefore, the central goal of this proposal is to develop OTUs for the translation of unnatural codons. In Specific Aim 1, the stability and orthogonality of the UBP in all sequence contexts is evaluated in RNA, providing insight into promising codons for translation. Specific Aim 2 utilizes an unnatural nucleotide in the anticodon of tRNAs as a recognition motifs to reprogram E. coli aaRSs for translation of the UBP. Finally, Specific Aim 3 will switch amino acid specificity of the aaRSs developed in Aim 2 from a canonical amino acid to an ncAA relevant for chemical biology and protein therapeutics. Successful completion of these aims will provide insight into RNA biology, produce OTUs that will seamlessly integrate into the SSO’s translational machinery, and allow the production of proteins with multiple ncAAs. The creation and use of these OTUs has the potential to revolutionize the field of protein therapeutics by providing a robust platform for the production of proteins with several ncAAs.