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

项目摘要 导致疾病的最大一类遗传改变是单点突变。大部分 如果将特定的腺苷变成鸟苷， 在RNA转录本上。这项提案旨在重新利用RNA编辑酶，并将其引导到 选择性编辑mRNA中的靶向腺苷以治疗遗传疾病。RNA编辑酶 作用于RNA的腺苷脱氨酶（阿达尔）可以通过以下方式将腺苷转化为肌苷（A至I）： 催化所述核碱基上的脱氨基反应。肌苷被细胞识别为鸟苷， 翻译机器提供改变mRNA中密码子的能力。该提案将重点关注 通过指导阿达尔编辑腺苷， 在终止密码子中，从而允许转录物继续翻译，产生功能完整的 长度蛋白质。由于ADAR选择性地编辑dsRNA区域中的腺苷， 无义突变可以通过提供适当的指导来选择性地靶向 在一个实施方案中，该方法包括使用寡核苷酸来产生dsRNA底物。双链体的典型Watson-Crick互补性 RNA不产生ADAR的有效底物，使得设计有效的ADAR具有挑战性。 引导寡核苷酸靶向特定的无义突变。一种高通量测定法， 建议搜索引导RNA寡核苷酸的所有序列空间以识别前导序列 使用内源性ADAR显示靶向无义转录物的高编辑效率。 这些前导序列可以通过结构导向的合理设计方法进一步优化。的 实验室在确定原子分辨率的ADAR-RNA结构方面具有丰富的经验， 利用这些知识来提高编辑效率。X射线晶体学和冷冻电镜 提出了用于与前导序列的dsRNA复合的ADAR 1或ADAR 2的技术 与其靶向mRNA片段结合。这些结构将为合理设计提供依据 调整以开发核苷酸类似物以掺入指导寡核苷酸中，所述指导寡核苷酸可以 时尚的结构特征，以改善编辑和增加代谢稳定性。的这种方法 定点RNA编辑（SDRE）治疗遗传性疾病提供了许多优于目前的优势。 编辑工具，其通常需要添加相当大的蛋白质（例如，CRISPR/Cas）。当完全 开发的，这种方法将允许简单的管理较短的寡核苷酸， 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.