Systems for Dramatically Improved Synthetic RNA

显着改进合成 RNA 的系统

基本信息

批准号：
10331827
负责人：
Craig T Martin
金额：
$ 30.65万
依托单位：
UNIVERSITY OF MASSACHUSETTS AMHERST
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-04-01 至 2024-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10331827
关键词：
Adoption Affinity Affinity Chromatography Area Basic Science Biochemistry Biological Assay Biology Biophysics Biotechnology Cellular biology Complementary DNA DNA Dependence Development Discipline Double-Stranded RNA Enzymes Eye Feedback Fostering GTP-Binding Protein alpha Subunits, Gs Gel Genetic Transcription Guide RNA Health Immobilization In Vitro Innate Immune Response Intervention Length Life Messenger RNA Methods Mission Modeling Molecular Biology Nanotechnology Nature Pathway interactions Performance Polymerase Primer Extension Public Health RNA RNA Sequences RNA chemical synthesis Randomized Reaction Research Research Personnel Resistance Running Sodium Chloride Surveys System T7 RNA polymerase Technology Testing Therapeutic Time United States National Institutes of Health Work base catalyst design high standard improved innovation novel strategies prevent promoter success synthetic biology therapy design tool transcriptome sequencing

项目摘要

Project Summary A wide variety of RNA researchers synthesize their RNA using the enzyme T7 RNA polymerase, as this enzyme is robust and can yield large quantities of RNA, at any length scale. However, undesired products typically contaminate the desired RNA, in often unpredictable ways. This is perhaps most impactful currently in the mRNA therapeutics field, where contaminating double stranded RNAs can trigger a potentially lethal innate immune response, but contaminants almost certainly impact other studies as well, from basic research in cell and molecular biology and biochemistry/biophysics, to synthetic biology, to RNA nanotechnology. Gel purifications are tedious, low yield, and imperfect (indeed, the darkest band on the gel may not be the correct product, and even the correct length RNA pool can be heterogeneous!). Longer RNA impurities derive from correct RNA, reducing yields at synthesis, and can be distributed across a wide range of lengths, making gel analysis problematic. Building on new mechanistic understandings, this project will develop systems that limit the conditions that give rise to these impurities, specifically by inhibiting the off-pathway reactions, and will develop simple affinity purification approaches – all with an aim towards achieving monodisperse RNAs of defined length and sequence. Sensitive analytical and functional assays for success will be developed and applied to guide design. Tools will be developed with an eye towards broad adoption by a variety of researchers.

项目概要许多 RNA 研究人员使用 T7 RNA 聚合酶合成他们的 RNA，因为这酶是强大的并且可以产生大量的任意长度的RNA，然而，这是不希望的。产品通常会以不可预测的方式污染所需的 RNA，这可能是最常见的。目前在 mRNA 治疗领域具有影响力，污染双链 RNA 可以触发潜在致命的先天免疫反应，但污染物几乎肯定会影响其他研究也包括从细胞和分子生物学以及生物化学/生物物理学的基础研究到从合成生物学到RNA纳米技术，凝胶纯化过程繁琐、产量低且不完善。（事实上，凝胶上最暗的条带可能不是正确的产品，甚至是正确长度的RNA 池中的 RNA 杂质可能是异质的！）。合成，并且可以分布在很宽的长度范围内，这使得凝胶分析成为问题。基于新的机械理解，该项目将开发限制条件的系统会产生这些杂质，特别是通过抑制旁路反应，并且会发展简单的亲和纯化方法——所有这些方法的目的都是为了获得单分散的RNA 将开发成功的敏感分析和功能测定。并应用于指导设计的工具将着眼于被各种领域广泛采用。研究人员。