High-throughput screening and structure-guided optimization of oligonucleotides for site-directed RNA editing by ADARs.

通过 ADAR 进行定点 RNA 编辑的寡核苷酸的高通量筛选和结构引导优化。

基本信息

批准号：
10636547
负责人：
ANDREW J FISHER
金额：
$ 33.2万
依托单位：
UNIVERSITY OF CALIFORNIA AT DAVIS
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-04-01 至 2027-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10636547
关键词：
ADAR1 Adenosine Amino Acids Base Pairing Binding Cells Clustered Regularly Interspaced Short Palindromic Repeats Code Codon Nucleotides Complex Cryoelectron Microscopy Cystic Fibrosis DNA DRADA2b protein Data Deaminase Deamination Disease Double-Stranded RNA Enzymes Future Genetic Diseases Genome Goals Guanosine Guide RNA Half-Life Human Inosine Knowledge Lead Length Location Messenger RNA Metabolic Methods Minor Molds Muscular Dystrophies Mutate Mutation Nonsense Mutation Nucleotides Oligonucleotides Parkinson Disease Pathogenicity Point Mutation Positioning Attribute Proteins RNA RNA Editing RNA Sequences Reaction Regulation Reporting Resolution Sequence Analysis Single Nucleotide Polymorphism Site Structure Syndrome Techniques Terminator Codon Transcript Translations Variant X-Ray Crystallography analog base base editing design dsRNA adenosine deaminase experience experimental study genome editing high throughput screening improved innovation insight lead optimization novel nucleobase nucleotide analog rational design repaired side effect tool

项目摘要

Project Summary The largest class of genetic alterations that cause disease are single point mutations. Most of these disease-causing errors can be remedied if a specific adenosine is changed to guanosine on the RNA transcript. This proposal aims to repurpose an RNA editing enzyme and direct it to selectively edit targeted adenosines in mRNA to treat genetic disorders. The RNA editing enzyme Adenosine Deaminase acting on RNA (ADAR) can convert adenosine to inosine (A-to-I) by catalyzing a deamination reaction on the nucleobase. Inosine is read as guanosine by the cellular translation machinery providing the ability to alter codons in mRNA. This proposal will focus on selectively editing disease-causing nonsense mutations, by directing ADAR to edit the adenosine in the stop codon thus allowing the transcript to continue translation, producing functional full- length protein. Because ADARs selectively edit adenosines in regions of dsRNA, disease-causing nonsense mutations can be selectively targeted by furnishing an appropriate guide oligonucleotide to create a dsRNA substrate. Canonical Watson-Crick complementarity of duplex RNA does not produce efficient substrates for ADARs, making it challenging to design effective guide oligonucleotides to target specific nonsense mutations. A high-throughput assay is proposed to search all sequence space of guide RNA oligonucleotides to identify lead sequences displaying high editing efficiency of the targeted nonsense transcript using endogenous ADARs. These lead sequences can be further optimized by structure-guided rational design methods. The lab has significant experience in determining ADAR-RNA structures to atomic resolution and leveraging this knowledge to improve editing efficiency. Both X-ray crystallography and Cryo-EM techniques are proposed for ADAR1 or ADAR2 complexed with dsRNA of the lead sequence bound to its targeted mRNA segment. These structures will provide the basis for rational design adjustments to develop nucleotide analogs to incorporate into guide oligonucleotides that can fashion structural features for improved editing and increased metabolic stability. This method of site-directed RNA editing (SDRE) to treat genetic disorders offers many advantages over current editing tools, which often require addition of sizable proteins (e.g., CRISPR/Cas). When fully developed, this method would permit the simple administration shorter oligonucleotides allowing the cell’s endogenous ADARs to recode the nonsense mutation to treat many genetic disorders.

项目概要导致疾病的最大一类基因改变是单点突变。如果将特定的腺苷更改为鸟苷，则可以纠正这些引起疾病的错误该提案旨在重新利用 RNA 编辑酶并将其引导至 RNA 转录本。选择性编辑 mRNA 中的目标腺苷以治疗遗传性疾病。作用于 RNA 的腺苷脱氨酶 (ADAR) 可以通过以下方式将腺苷转化为肌苷（A-to I）：催化核碱基上的脱氨基反应，被细胞读取为鸟苷。翻译机制提供了改变 mRNA 密码子的能力。通过指导 ADAR 编辑腺苷来选择性编辑致病的无义突变在终止密码子中，从而允许转录本继续翻译，产生功能完整的因为 ADAR 选择性地编辑 dsRNA 区域中的腺苷，从而导致疾病。通过提供适当的指导，可以选择性地靶向无义突变寡核苷酸以创建双链体的 Canonical Watson-Crick 互补性。 RNA 不能产生 ADAR 的有效底物，因此设计有效的 ADAR 具有挑战性引导寡核苷酸靶向特定的无义突变是一种高通量测定。建议搜索向导RNA寡核苷酸的所有序列空间以识别前导序列使用内源 ADAR 显示目标无义转录本的高编辑效率。这些先导序列可以通过结构引导的合理设计方法进一步优化。实验室在确定 ADAR-RNA 结构至原子分辨率方面拥有丰富的经验，利用这些知识提高 X 射线晶体学和冷冻电镜的编辑效率。提出了 ADAR1 或 ADAR2 与前导序列 dsRNA 复合的技术这些结构将为合理设计提供基础。调整以开发核苷酸类似物以掺入引导寡核苷酸中，这种方法可以改善编辑并提高代谢稳定性。用于治疗遗传性疾病的定点 RNA 编辑 (SDRE) 与目前的技术相比具有许多优势编辑工具，通常需要添加大量蛋白质（例如 CRISPR/Cas）。开发后，该方法将允许简单地施用较短的寡核苷酸，从而允许细胞的内源性 ADAR 可以重新编码无义突变，从而治疗许多遗传性疾病。