Systems for Dramatically Improved Synthetic RNA

显着改进合成 RNA 的系统

• 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
• 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; 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

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。