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

基本信息

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
项目类别：
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

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

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项目摘要

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.

项目摘要：针对癌症，癌症，神经变性和遗传疾病等难以药物疾病的新策略迫切需要。 RNA操纵是一种新兴的治疗范围，允许目标干预与吸毒蛋白的正交，没有基因编辑的持续性。多种工具已经开发了RNA操作，但它们依赖于核糖核蛋白，这些蛋白很难递送体内或效果范围有限。该建议旨在开发基于RNA的双功能分子（RBMS）它将由喜欢募集效应蛋白的小分子的RNA寡核苷酸组成，并将启用具有多种操作的RNA的模块化，可编程的靶向。提出的机制 RBMS基于小型干扰RNA（siRNA）寡核苷酸，该寡核苷酸被广泛用作研究工具和导致多种批准的治疗学。在细胞中，siRNA被加载到Argonaute（AGO）蛋白中这是RNA沉默复合物（RISC）的一部分。以前，然后将RISC引导到mRNA目标互补到其加载的siRNA指南，在结合后裂解，从而导致平移沉默。这 RBMS的寡核苷酸部分的功能与siRNA一样起作用，但将以与目标的关键不匹配 RNA。此类不匹配已被证明可以弥补RISC的裂解活动，同时保持目标结合。目标结合将诱导靶RNA和效应子蛋白之间的接近性 RBM的小分子配体，允许效应子对靶标作用。我将综合一个小库具有可变链接器长度和位置的RBM，并验证它们可以与AGO交互和模型效应器体外蛋白质（AIM 1）。接下来，我将使用AGO PULLDONN来证明这些相互作用可以在细胞，然后使用两个模型系统表明RBM可以实现对mRNA目标的转录后控制（目标2）。最后，我将证明针对核，长的非编码RNA的RBM可以控制基因表达（目标3）。总体而言，这将创建一个平台，其中siRNA范式扩展以启用各种各样的操作将实现新颖的研究工具和疗法。该项目将使用我现有的合成化学技能作为基础，然后让我分支在化学生物学领域。我的赞助商史蒂文·班尼克（Steven Banik）教授是一项支持性研究环境将使我能够成功学习执行该建议所需的新技能。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，这是一个高度的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资源，各种专业发展的机会以及与之合作的机会来自许多不同的科学家。