Modular Reagents for Programmable RNA Manipulation by Endogenous Proteins

项目总结： 针对难治性疾病的新策略，如癌症、神经变性和遗传性疾病 都是迫切需要的。RNA操作是一种新兴的、具有治疗吸引力的范例，它允许靶向 干预与给蛋白质下药是正交的，而且不需要永久的基因编辑。各种工具可用于 RNA操纵已经被开发出来，但它们依赖于核糖核蛋白，而核糖核蛋白在体内很难传递 活体或作用范围有限。这项提议旨在开发基于RNA的双功能分子(RBM) 它将由一个类似于小分子的RNA寡核苷酸组成，它招募效应蛋白，并将 通过各种操作实现模块化、可编程的RNA靶向。建议的机制 RBMS是基于小干扰RNA(SiRNA)寡核苷酸的，这种寡核苷酸被广泛用作研究工具和 已经产生了多个批准的治疗方法。在细胞中，siRNAs被装载到ArgAerte(AGO)蛋白中 它们是RNA沉默复合体(RISC)的一部分。AGO然后引导RISC以互补的mRNA为靶点 与其装载的siRNA导向物结合后被切割，导致翻译沉默。这个 RBM的寡核苷酸部分的功能与siRNAs很相似，但与靶标的关键不匹配 核糖核酸。这种错配被证明在保持靶标的同时削弱了RISC的切割活性 有约束力的。靶标结合将诱导靶标RNA与由 RBM的小分子配体，允许效应器作用于靶标。我将合成一个小型图书馆 具有可变链接器长度和位置的RBM，并验证它们可以与AGO和模型效应器相互作用 体外蛋白质(目标1)。接下来，我将使用AGO下拉菜单来说明这些交互可以概括为 细胞，然后使用两个模型系统来证明RBMS可以实现对mRNA靶标的转录后控制 (目标2)。最后，我将展示针对核的、长的非编码RNA的RBM可以实现对基因的控制 表达(目标3)。总体而言，这将创建一个平台，在该平台中，siRNA范例得到扩展，从而能够 更多种类的操作，这将使新的研究工具和疗法成为可能。 这个项目将使用我在合成化学方面的现有技能作为基础，然后允许我扩展 在化学生物学领域。我的赞助人史蒂文·巴尼克教授的实验室是一项支持性研究 这将使我能够成功地学习执行这项建议所需的新技能。教授，教授。 巴尼克是斯坦福大学化学工程和人类健康医学(Chem-H)的成员，该组织是一个高度 协作性和跨学科的研究所。斯坦福大学和化学-H大学将为我提供所有必要的研究 资源，各种职业发展机会，以及与之共事和学习的机会 来自许多不同的科学家。

PROJECT SUMMARY: New strategies to targeted difficult-to-drug diseases such as cancers, neurodegeneration, and genetic disorders are urgently needed. RNA manipulation is an emerging, therapeutically attractive paradigm which allows target intervention orthogonal to drugging proteins and without the permanence of gene editing. A variety of tools for RNA manipulation have been developed, but they rely on ribonuclear proteins, which are difficult to deliver in vivo or are limited in scope of effect. This proposal aims to develop RNA-based bifunctional molecules (RBMs) which will consist of an RNA oligonucleotide liked to a small molecule which recruits an effector protein, and will enable modular, programmable targeting of RNA with a variety of manipulations. The proposed mechanism for RBMs is based on small interfering RNA (siRNA) oligonucleotides which are widely used as research tools and have resulted in multiple approved therapeutics. In cells, siRNAs are loaded into Argonaute (AGO) proteins which are part of the RNA silencing complex (RISC). AGO then guides RISC to mRNA targets complementary to its loaded siRNA guide, which are cleaved upon binding, resulting in translational silencing. The oligonucleotide portion of RBMs will function much like siRNAs but will feature key mismatches with the target RNA. Such mismatches have been shown to oblate the cleavage activity of RISC while maintaining target binding. Target binding will induce proximity between the target RNA and an effector protein recruited by the small molecule ligand of the RBM, allowing the effector to act on the target. I will synthesize a small library of RBMs with variable linker lengths and positions, and verify that they can interact with AGO and a model effector protein in vitro (Aim 1). Next, I will use AGO pulldown to show that these interactions can be recapitulated in cells, then use two model systems to show that RBMs can enable post-transcriptional control of mRNA targets (Aim 2). Finally, I will show that RBMs targeting nuclear, long non-coding RNAs can enable control of gene expression (Aim 3). Overall, this will create a platform in which the siRNA paradigm is expanded to enable a much wider variety of manipulations, which will enable novel research tools and therapies. This project will use my existing skills in synthetic chemistry as a foundation and then allow me to branch out in the field of chemical biology. The laboratory of my sponsor, Prof. Steven Banik, is a supportive research environment which will enable me to successfully learn the new skills required to execute this proposal. Prof. Banik is a member of Stanford's Chemistry Engineering and Medicine for Human Health (ChEM-H), a highly collaborative and interdisciplinary institute. Stanford and ChEM-H will afford me all necessary research resources, a variety of opportunities for professional development, and the opportunity to work with, and learn from many, different scientists.