Modular Reagents for Programmable RNA Manipulation by Endogenous Proteins

用于内源蛋白可编程 RNA 操作的模块化试剂

• 批准号: 10605050
• 负责人: Robert Follett Lusi
• 金额: $ 6.87万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-02-27 至 2026-02-26
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10605050
• 关键词: Ablation; Address; Adenosine; Affect; Base Pair Mismatch; Binding; Biological Assay; Biological Models; Biology; CCND1 gene; Cells; Chemicals; Chemistry; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Complementary RNA; Complex; Development; Disease; Down-Regulation; Engineering; Environment; Event; FOXM1 gene; Foundations; Gel; Gene Expression; Genes; Genetic Diseases; Genetic Transcription; Goals; Guide RNA; Health; Human; Immunoprecipitation; In Vitro; Incidence; Intervention; Laboratories; Learning; Length; Libraries; Ligands; Luciferases; Malignant Neoplasms; Mammals; Mediating; Medicine; Messenger RNA; Methylation; Methyltransferase; Modeling; Modification; N-terminal; Nerve Degeneration; Nuclear; Oligonucleotides; Pharmaceutical Preparations; Positioning Attribute; Post-Transcriptional Regulation; Primary carcinoma of the liver cells; Proliferating; Proteins; RNA; RNA Interference; RNA library; Reagent; Reporter; Research; Resources; Scientist; Signal Transduction; Small Interfering RNA; Structure-Activity Relationship; Synthesis Chemistry; System; Therapeutic; Transcript; Translations; Untranslated RNA; Validation; Work; chemical synthesis; design; experimental study; falls; in vivo; member; novel; posttranscriptional; prevent; recruit; side effect; skills; small molecule; tool; transcriptome sequencing

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.

项目概要： 针对癌症、神经退行性疾病和遗传性疾病等难以用药的疾病的新策略 是迫切需要的。 RNA操作是一种新兴的、具有治疗吸引力的范式，它允许靶标 与药物蛋白质正交的干预，并且没有基因编辑的永久性。各种工具用于 RNA操作已经开发出来，但它们依赖于核糖核蛋白，而核糖核蛋白很难在体内传递 体内或作用范围有限。该提案旨在开发基于RNA的双功能分子（RBM） 它由一个与小分子相似的RNA寡核苷酸组成，该小分子招募效应蛋白，并且将 通过各种操作实现模块化、可编程的 RNA 靶向。拟议的机制 RBM 基于小干扰 RNA (siRNA) 寡核苷酸，广泛用作研究工具和 已产生多种经批准的治疗方法。在细胞中，siRNA 被加载到 Argonaute (AGO) 蛋白中 它们是 RNA 沉默复合物 (RISC) 的一部分。然后，AGO 引导 RISC 实现互补的 mRNA 目标 到其加载的 siRNA 引导，结合后被切割，导致翻译沉默。这 RBM 的寡核苷酸部分的功能与 siRNA 非常相似，但具有与靶标关键不匹配的特征 核糖核酸。这种错配已被证明会削弱 RISC 的裂解活性，同时保持目标 绑定。靶标结合将诱导靶标RNA与由RNA招募的效应蛋白之间的接近。 RBM 的小分子配体，使效应器能够作用于靶标。我将合成一个小型库 具有可变接头长度和位置的 RBM，并验证它们是否可以与 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 Pulldown 来展示这些交互可以概括为 细胞，然后使用两个模型系统来证明 RBM 可以实现 mRNA 靶标的转录后控制 （目标 2）。最后，我将证明针对核长链非编码 RNA 的 RBM 可以实现基因控制 表达（目标 3）。总体而言，这将创建一个平台，在其中扩展 siRNA 范式以实现 更广泛的操作，这将使新的研究工具和疗法成为可能。 这个项目将以我在合成化学方面现有的技能为基础，然后让我能够扩展 在化学生物学领域。我的资助者 Steven Banik 教授的实验室是一项支持性研究 环境将使我能够成功学习执行该提案所需的新技能。教授。 巴尼克是斯坦福大学人类健康化学工程与医学 (ChEM-H) 的成员，该学院是一个高度 协作和跨学科研究所。斯坦福大学和 ChEM-H 将为我提供所有必要的研究费用 资源、各种职业发展机会以及合作和学习的机会 来自许多不同的科学家。