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

项目摘要 导致疾病的最大类别的基因改变是单点突变。大部分 如果将特定的腺苷改变为鸟苷，这些致病错误可以得到补救 在核糖核酸记录上。这项提议旨在重新调整RNA编辑酶的用途，并将其定向到 选择性地编辑信使核糖核酸中的靶向腺苷来治疗遗传性疾病。RNA编辑酶 作用于RNA的腺苷脱氨酶(ADAR)通过以下途径将腺苷转化为肌苷(A-to-I) 催化碱基上的脱氨基反应。肌苷被细胞解读为鸟苷。 翻译机器，提供改变信使核糖核酸密码子的能力。这项提案将重点放在 通过指示ADAR编辑腺苷，选择性地编辑导致疾病的无义突变 在终止密码子中，从而允许转录继续翻译，产生功能完整的 长度蛋白质。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，可以有选择地针对无意义突变 形成dsRNA底物的寡核苷酸。二重结构的正则Watson-Crick互补 RNA不能为ADAR产生有效的底物，这使得设计有效的变得具有挑战性 引导寡核苷酸针对特定的无义突变。一种高通量分析方法是 提出了搜索引导RNA寡核苷酸的所有序列空间来识别前导序列的方法 使用内源性ADAR显示目标无意义转录本的高编辑效率。 这些导联序列可以通过结构引导的合理设计方法进一步优化。这个 Lab在确定ADAR-RNA结构以原子分辨率和 利用这些知识来提高编辑效率。X射线结晶学和低温电子显微镜 提出了ADAR1或ADAR2与前导序列的dsRNA络合的技术 结合到它的靶基因片段上。这些结构将为合理设计提供依据。 调整开发核苷酸类似物，以将其并入指南中，从而能够 时尚的结构特征，以改进编辑和提高新陈代谢稳定性。这种方法 定点RNA编辑(SDRE)用于治疗遗传性疾病具有许多优点 编辑工具，通常需要添加相当大的蛋白质(例如CRISPR/CA)。当完全 开发出的这种方法将允许简单地给药较短的寡核苷酸 细胞的内源性ADAR重新编码无义突变，以治疗许多遗传性疾病。

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.