Systems for Dramatically Improved Synthetic RNA

项目摘要 各种各样的RNA研究人员使用酶T7 RNA聚合酶合成他们的RNA，因为 酶是稳健的，并且可以在任何长度尺度上产生大量RNA。然而，不希望 产物通常以不可预测的方式污染所需的RNA。这也许是最 目前在mRNA治疗领域是有影响的，其中污染的双链RNA可以 引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 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， 从合成生物学到RNA纳米技术。凝胶纯化是繁琐的，低产量，和不完善的 （事实上，凝胶上最暗的条带可能不是正确的产物，甚至不是正确长度的RNA。 池可以是异构的！）。较长的RNA杂质来自正确的RNA， 合成，并且可以分布在很宽的长度范围内，使得凝胶分析有问题。 基于新的机械理解，该项目将开发限制条件的系统， 特别是通过抑制非途径反应，产生这些杂质， 简单的亲和纯化方法-所有目的都是为了获得单分散的RNA， 定义长度和顺序。将开发成功的灵敏分析和功能测定 并应用于指导设计。开发工具时，将着眼于被各种 研究人员

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.