CAREER: Chemical Biology Approaches to Study 5-Methylcytidine Derivatives in RNA and Develop CRISPR-Tet2-Based Epitranscriptomic Tool for Gene Regulation

核苷酸构成核糖核酸（rna），并包含细胞用来制造蛋白质的遗传信息的关键“蓝图”。凭借这一奖项，化学系生命过程化学项目资助纽约州立大学奥尔巴尼分校的贾生博士开发工具，以高精度地将修饰的核苷酸整合到RNA中。这些修饰改变了RNA的结构和功能。这些变化帮助我们理解RNA在许多生物过程中的作用。研究重点是确定含有修饰核苷酸的RNA的三维结构，了解这些变化如何影响RNA的反应性，进而研究核苷酸在基因表达中的作用。盛博士将新的天然核苷酸及其3D结构提供给广泛的研究人员。在这个项目下开发的策略可能适用于针对不同RNA修饰的其他酶，导致RNA编辑的一种新的通用方法，因此，这项研究被认为是在理解生命规则计划的范围内。本研究的研究方法和成果被纳入新开设的《核酸化学》课程。盛博士是“化学世界”项目的领导者，该项目帮助化学专业的新生顺利过渡到大学。盛博士还在他的实验室组织一年一度的“RNA日”，为当地K-12学生提供实验演示和演讲，以唤醒他们内心的科学家。最后，课题组通过“大学在高中”项目招募学生和教师参加暑期研究。该奖项的研究重点是RNA中化学修饰的胞嘧啶相对较新的表观遗传学意义。合成5-甲基胞嘧啶（5mC）及其氧化变体（5xrC）被系统地整合到rna中，以确定它们对rna的稳定性和局部和全局构象的影响。此外，对5xrc修饰的rna的研究揭示了它们对酶识别的影响以及10 - 11易位（TET）加氧酶家族对5mC的酶促氧化机制。最后，将靶向聚集规律间隔短回文重复（CRISPR）酶的序列与TET加氧酶融合，以创建一种工具，可以选择性地氧化天然rna中的5mC。该工具用于确定5xrC核苷酸如何影响将细胞mrna翻译成蛋白质的效率。盛博士所获得的知识和开发的新的通用方法可能会使其他研究真核生物中信使RNA的表观转录组修饰和基因表达的转录后调控的科学家受益。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Nucleotides make up ribonucleic acids (RNAs) and contain the crucial "blueprint" of genetic information that is used by cells to make proteins. With this award, the Chemistry of Life Processes Program in the Division of Chemistry is funding Dr. Jia Sheng, from the State University of New York at Albany, to develop tools to incorporate modified nucleotides into RNA with high precision. These modifications change the RNA structure and function. These changes help us understand the role of RNA in numerous biological processes. The research focuses on the determining the three-dimensional structure of RNAs containing the modified nucleotides, understanding how these changes affect the reactivity of the RNA and then studying the study of the nucleotides' role in gene expression. The Dr. Sheng makes the new natural nucleotides and their 3D structures available to a wide range of researchers. The strategies developed under this project may be applicable to other enzymes targeting different RNA modifications, leading to a new general approach for RNA editing and thus, this research is considered to be within the Understanding the Rules of Life initiative. The research methodologies and outcomes of the study are integrated into a new course "Nucleic Acid Chemistry". Dr. Sheng is a leader in the "World of Chemistry" program which helps freshmen chemistry majors transition to college successfully. Dr. Sheng also organizes the annual "RNA Day" in his lab for local K-12 students offering experimental demonstrations and presentations to awaken their inner scientist. Finally, the research group recruits students and teachers to participate in summer research through the "University in the High School" program.The research studies made possible by this award focus on the relatively new epigenetic significance of chemically-modified cytosines in RNA. Synthetic 5-methylcytosine (5mC) and its oxidized variants (5xrC) are systematically incorporated into RNAs to determine their impact on the stability and local and global conformations of the RNAs. In addition, studies with 5xrC-modified RNAs reveal their effects on enzyme recognition and the mechanism of enzymatic oxidation of 5mC by the ten-eleven translocation (TET) family of oxygenases. Finally, the sequence targeting the clustered regularly interspaced short palindromic repeats (CRISPR) enzyme is fused with a TET oxygenase to create a tool that site-selectively oxidizes 5mC in natural RNAs. This tool is used to determine how 5xrC nucleotides affect the efficiency of translating cellular mRNAs into proteins. The knowledge gained and new general approaches developed by Dr. Sheng may benefit other scientists who study epitranscriptomic modifications of messenger RNA and posttranscriptional regulation of gene expression in eukaryotes.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.