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、神经变性和遗传性疾病等难治性疾病的新策略 是迫切需要的。RNA操作是一种新兴的、具有治疗吸引力的范例，其允许靶向RNA。 干预正交于药物蛋白质，而没有基因编辑的永久性。各种工具， RNA操纵已经开发出来，但它们依赖于核糖核蛋白，这是很难交付的。 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） 其将由与募集效应蛋白的小分子连接的RNA寡核苷酸组成，并且将 使模块化，可编程的RNA靶向与各种操作。的拟议机制 RBM基于小干扰RNA（siRNA）寡核苷酸，其被广泛用作研究工具， 已经产生了多种被批准的治疗方法。在细胞中，siRNA被装载到Argonaute（AGO）蛋白中， RNA沉默复合物（RISC）AGO然后引导RISC到mRNA靶点互补 与其加载的siRNA引导物结合，所述siRNA引导物在结合时被切割，导致翻译沉默。的 RBM的寡核苷酸部分的功能与siRNA非常相似，但其特征在于与靶分子的关键错配 核糖核酸已经显示这种错配使RISC的切割活性变小，同时维持靶向的细胞分裂。 约束力靶结合将诱导靶RNA与由靶RNA募集的效应蛋白之间的接近。 RBM的小分子配体，允许效应物作用于靶标。我会合成一个小型的 RBM与可变接头长度和位置，并验证它们可以与AGO和模型效应物相互作用 体外蛋白质（目的1）。接下来，我将使用AGO下拉来说明这些交互可以在 细胞，然后使用两个模型系统，以表明RBM可以使转录后控制的mRNA目标 (Aim 2）。最后，我将展示RBM靶向细胞核，长的非编码RNA可以使基因控制， 表达式（目标3）。总的来说，这将创建一个平台，其中siRNA范例被扩展以使得能够实现对siRNA的扩增。 更广泛的操作，这将使新的研究工具和疗法。 这个项目将利用我现有的合成化学技能作为基础，然后让我分支出来 在化学生物学领域。我的赞助人Steven Banik教授的实验室是一项支持性研究， 环境，这将使我能够成功地学习新的技能所需的执行这一建议。教授 Banik是斯坦福大学的化学工程和人类健康医学（ChEM-H）的成员，这是一个非常重要的研究领域。 合作和跨学科研究所。斯坦福大学和ChEM-H会提供我所有必要的研究经费 资源，各种专业发展的机会，以及与之合作和学习的机会 来自许多不同的科学家