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.

项目摘要多种RNA研究人员使用酶T7 RNA聚合酶合成其RNA，因此酶是强大的，可以在任何长度尺度上产生大量RNA。但是，不希望产品通常以不可预测的方式污染所需的RNA。这可能是最大的目前在mRNA治疗领域有影响力触发潜在的致命的先天免疫响应，但污染物几乎可以肯定会影响其他从细胞和分子生物学和生物化学/生物物理学的基础研究到研究，合成生物学，RNA纳米技术。凝胶净化是乏味的，低的产量和不完美的（的确，凝胶上最黑的频带可能不是正确的产品，甚至是正确的长度RNA 游泳池可能是异质的！）。较长的RNA杂质来自正确的RNA，在合成，可以分布在各种长度上，使凝胶分析有问题。基于新的机械理解，该项目将开发限制条件的系统这引起了这些杂质，特别是通过抑制越野反应，并会发展简单的亲和力净化方法 - 全部旨在实现单分散的RNA 定义的长度和序列。将开发敏感的分析和功能分析方法并应用于指导设计。将开发工具，以关注各种各样的广泛采用研究人员。