Synthetic Biology Approaches to Elucidate and Exploit Nature's Selection of Lipid Modified RNA

阐明和利用自然选择的脂质修饰 RNA 的合成生物学方法

• 批准号: 1715234
• 负责人: Jia Sheng
• 金额: $ 50万
• 依托单位: SUNY at Albany
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-01 至 2021-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1715234&HistoricalAwards=false
• 关键词: Synthetic; Biology; Approaches; Elucidate; Exploit

Natural RNA molecules are heavily decorated by various functional groups to diversify and fine-tune their structures and biological functions. Studying these RNA modifications has great significance in understanding fundamental RNA biology, studying evolution and developing new therapeutics. This project is focused on a recently discovered lipid modification on bacterial tRNAs. tRNA (also known as transfer RNA or soluble RNA) is the RNA molecule that carries amino acids to ribosome for protein synthesis in a cell, therefore is a very critical life molecule. The research will use synthetic biology approaches to address the fundamental question of why nature uses such a hydrophobic lipid group in hydrophilic RNA systems by elucidating the metabolic pathways of this lipid modification, as well as the structural and functional features of the working enzyme called SelU. In addition, based on this special pathway, the research will also develop novel molecular tools to detect, label and monitor specific tRNAs in cells. This project will provide educational and research training opportunities for graduate and undergraduate students, as well as local K12 teachers to use cutting-edge multidisciplinary approaches to solve fundamental biological problems and advance their scientific careers.The systematic study of post-transcriptional chemical modifications of tRNAs have great significance in understanding RNA biology and its roles in evolution. Furthermore, many enzymes that mediate these processes have been demonstrated as targets for the development of new molecular tools or therapeutics. The main goal of this research is to explore tRNA geranylation, which is a recently discovered hydrophobic tRNA modification in several bacteria such as E. coli, E. aerogenes, P. aeruginosa and S. typhimurium. The geranylated 2-thiouridines are produced in the first anticodon position of tRNAs specific for lysine, glutamine, and glutamic acid by the enzyme SelU using geranyl pyrophosphate as the cofactor. It has been demonstrated that this hydrophobic terpene functional group affects the codon recognition patterns and reduces frameshifting errors during translation. Specifically, the geranyl group promotes wobble 2-thiouridine pairing with guanosine over adenosine and the long carbon chain of geranyl group is necessary to maintain this base pairing specificity in the context of RNA duplexes. This research will develop new chemical synthesis of RNA strands containing a series of geranyl-uridine analogues and identify the general rules for the in vitro codon specificity and ribosomal binding of these lipid modified tRNAs. In addition, this project will solve crystal structures of geranylated RNAs to elucidate their detailed structural features; and determine the structures of SelU-tRNA complexes for the mechanistic studies of this geranylation step. Furthermore, this research will develop novel molecular tools to detect, label and monitor specific tRNAs in cells.

天然RNA分子被各种官能团大量修饰，以使其结构和生物学功能多样化和微调。研究这些RNA修饰对于理解基础RNA生物学、研究进化和开发新的治疗方法具有重要意义。该项目的重点是最近发现的细菌tRNA的脂质修饰。tRNA（也称为转运RNA或可溶性RNA）是细胞中将氨基酸运送到核糖体进行蛋白质合成的RNA分子，因此是非常重要的生命分子。该研究将使用合成生物学方法来解决为什么自然界在亲水性RNA系统中使用这种疏水性脂质基团的基本问题，通过阐明这种脂质修饰的代谢途径，以及称为SelU的工作酶的结构和功能特征。此外，基于这一特殊途径，该研究还将开发新型分子工具来检测、标记和监测细胞中的特定tRNA。该项目将为研究生和本科生以及当地K12教师提供教育和研究培训机会，利用前沿的多学科方法解决基本生物学问题，并推动他们的科学事业。系统研究tRNA的转录后化学修饰对理解RNA生物学及其在进化中的作用具有重要意义。此外，许多介导这些过程的酶已被证明是开发新分子工具或治疗剂的靶点。本研究的主要目的是探索tRNA香叶基化，这是最近在几种细菌如大肠杆菌中发现的一种疏水性tRNA修饰。coli、大肠杆菌E.产气假单胞菌、铜绿假单胞菌和S.鼠伤寒。香叶基化的2-硫代尿苷是由酶SelU使用香叶基焦磷酸作为辅因子在对赖氨酸、谷氨酰胺和谷氨酸特异的tRNA的第一个反密码子位置产生的。已经证明，这种疏水萜烯官能团影响密码子识别模式，并减少翻译过程中的移码错误。具体地，香叶基促进摆动2-硫尿苷与鸟苷的配对超过腺苷，并且香叶基的长碳链对于在RNA双链体的情况下维持这种碱基配对特异性是必要的。本研究将开发新的化学合成的RNA链含有一系列的香叶基-尿苷类似物，并确定在体外密码子特异性和核糖体结合这些脂质修饰的tRNA的一般规则。此外，本项目将解析香叶基化RNA的晶体结构，以阐明其详细的结构特征;并确定SelU-tRNA复合物的结构，以研究香叶基化步骤的机理。此外，这项研究将开发新的分子工具来检测，标记和监测细胞中的特定tRNA。

项目成果

DNA Functionality with Photoswitchable Hydrazone Cytidine**

具有光开关腙胞苷的 DNA 功能**

• DOI: 10.1002/chem.202100742
• 发表时间: 2021
• 期刊: Chemistry – A European Journal
• 作者: Mao, Song;Chang, Zhihua;Ying Zheng, Ya;Shekhtman, Alexander;Sheng, Jia
• 通讯作者: Sheng, Jia

Cyano Modification on Uridine Decreases Base-Pairing Stability and Specificity through Neighboring Disruption in RNA Duplex

尿苷的氰基修饰通过 RNA 双链体中的相邻破坏降低碱基配对的稳定性和特异性

• DOI: 10.1002/cbic.201800399
• 发表时间: 2018
• 期刊: ChemBioChem
• 影响因子: 3.2
• 作者: Mao, Song;Ranganathan, Srivathsan V.;Tsai, Hsu-Chun;Haruehanroengra, Phensinee;Shen, Fusheng;Valsangkar, Vibhav A.;Han, Bo;Hassan, Abdalla E.;Chen, Alan;Sheng, Jia
• 通讯作者: Sheng, Jia

Synthesis of 5‐Cyanomethyluridine (cnm 5 U) and 5‐Cyanouridine (cn 5 U) Phosphoramidites and Their Incorporation into RNA Oligonucleotides

5-氰基甲基尿苷 (cnm 5 U) 和 5-氰基尿苷 (cn 5 U) 亚磷酰胺的合成及其掺入 RNA 寡核苷酸

• DOI: 10.1002/cpnc.114
• 发表时间: 2020
• 期刊: Current Protocols in Nucleic Acid Chemistry
• 作者: Mao, Song;Tsai, Hsu‐Chun;Sheng, Jia
• 通讯作者: Sheng, Jia

RNA Phosphorothioate Modification in Prokaryotes and Eukaryotes

• DOI: 10.1021/acschembio.0c00163
• 发表时间: 2020-06-19
• 期刊: ACS CHEMICAL BIOLOGY
• 影响因子: 4
• 作者: Wu, Ying;Tang, Yaning;Sheng, Jia
• 通讯作者: Sheng, Jia