Defining and Controlling Protein-RNA interactions in editing and interference pathways

这一最大化调查人员研究奖(MIRA)应用程序旨在支持 加州大学戴维斯分校的比尔实验室专注于定义和控制RNA编辑中的蛋白质-RNA相互作用 和RNA干扰途径。RNA编辑ADAR酶将腺苷(A)转化为肌苷 (I)在双链RNA中。由于我的行为类似于RNA中的鸟苷(G)，所以这种修饰可以具有 对修饰的RNA的结构和功能的深刻影响，包括但不限于， 特定密码子含义的变化(重新编码)。人类ADAR1基因突变导致 遗传性对称性色素异常症(DSH)和自身免疫性疾病Aicardi- 古铁雷斯综合征(AGS)。此外，还观察到ADAR1上调和超级编辑 几种不同的癌症。尽管这种形式的RNA结构调控和 功能，但在我们对A到I RNA编辑的理解中仍然存在关键差距。此外，鉴于ADAR的 能够改变RNA序列，人们对利用这一特性并指导它的兴趣与日俱增 以纠正与疾病相关的G-to-A突变。这一领域的关键问题将在 这个项目是：1)编辑途径中关键的蛋白质-RNA复合体的结构是什么？ 与不同RNA底物结合的全长人ADAR2的结构及其结构 ADAR1与RNA结合是充分理解底物识别和选择性所必需的 在RNA编辑中。2)我们能否开发出高效、选择性和低分子量的ADAR抑制剂？这类抑制剂 可作为ADAR1靶向癌症疗法开发的先导化合物。3)我们可以吗 开发新的策略来进化突变的编辑酶和新的底物RNA？这个 这些努力的结果将为设计高效和选择性的定向RNA试剂提供参考 编辑应用程序。 我们实验室对化学修饰的发展也有着长期的兴趣。 能够控制与RNA结合蛋白相互作用的RNA。我们最近在这方面所做的许多工作 专注于控制RNA与siRNA触发的组件或miRNA- 触发了基因沉默途径。利用RNAi途径研究基因功能已成为 是分子生物学的强大工具，并已被用于开发新的治疗方法。 然而，存在着限制其应用的具体问题。这些问题包括偏离目标的效果 产生于siRNA导向链作为miRNA的功能。此外，反义 靶向miRNAs(抗miRs)的寡核苷酸具有巨大的治疗潜力，需要 用于稳定性和有效性的化学修饰。到目前为止，新化学品的发展 治疗性RNA的修饰在很大程度上是一个特别的过程。解决的关键问题 在这方面提出的项目是：我们能否制定一种有效的系统方法来新 在干扰途径中调节蛋白质-RNA相互作用的RNA修饰？这个 这些研究的直接影响将是对siRNAs和抗miRs提供新的修饰， 提高效力和选择性。然而，我们继续改进一种使用 结合多种RNA修饰化学的计算筛选将通常是 适用于其他涉及用于治疗的化学修饰的RNA的项目。

This Maximizing Investigators Research Award (MIRA) application is proposed to support research in the Beal lab at UC Davis focused on defining and controlling protein-RNA interactions in RNA editing and RNA interference pathways. The RNA editing ADAR enzymes convert adenosines (A) to inosines (I) in duplex RNA. Since I can behave similarly to guanosine (G) in RNA, this modification can have profound effects on the structure and function of the modified RNA including, but not limited to, changes in the meaning of specific codons (recoding). Mutations in the human ADAR1 gene cause the skin disorder Dyschromatosis Symmetrica Hereditaria (DSH) and the autoimmune disease Aicardi- Goutieres Syndrome (AGS). Also, ADAR1 upregulation and hyper editing has been observed in several different cancers. Despite the significance of this form of regulation of RNA structure and function, there remain key gaps in our understanding of A to I RNA editing. In addition, given ADARs’ ability to change RNA sequence, there is growing interest in harnessing this property and directing it to correct disease-associated G-to-A mutations. Key questions in this field that will be addressed in this project are: 1) What are the structures of key protein-RNA complexes in editing pathways? Structures of full length human ADAR2 bound to different RNA substrates along with structures of ADAR1 bound to RNA are necessary for a full understanding of substrate recognition and selectivity in RNA editing. 2) Can we develop potent, selective and low MW ADAR inhibitors? Such inhibitors could serve as lead compounds in the development of ADAR1-targeted cancer therapies. 3) Can we develop new strategies to evolve mutant editing enzymes and novel substrate RNAs? The results of these efforts will inform the design of highly efficient and selective reagents for directed RNA editing applications. Our laboratory also has a long standing interest in the development of chemical modifications of RNA that can control the interaction with RNA-binding proteins. Much of our recent work in this area has focused on controlling the interaction of RNA with components of siRNA-triggered or miRNA- triggered gene silencing pathways. The use of the RNAi pathway to study gene function has become a powerful tool in molecular biology and has been exploited in the development of new therapeutics. However, specific issues exist that limit its application. These issues include off-target effects that arise from the ability of an siRNA guide strand to function as a miRNA. In addition, antisense oligonucleotides targeting miRNAs (anti-miRs) have significant therapeutic potential and require chemical modification for stability and efficacy. Up to this point, the development of new chemical modifications of therapeutic RNAs has been largely an ad hoc process. The key question addressed in this aspect of the proposed project is: Can we develop an effective systematic approach to new RNA modifications that modulate protein-RNA interactions in interference pathways? The immediate impact of these studies will be to provide new modifications to siRNAs and anti-miRs that improve potency and selectivity. However, our continued refinement of an approach that uses computational screening coupled with versatile RNA modification chemistry will be generally applicable other projects that involve chemically modified RNA for therapeutics.