Systems for Dramatically Improved Synthetic RNA

项目摘要 各种各样的RNA研究人员使用T7RNA聚合酶来合成他们的RNA，就像这样 酶是强大的，可以产生大量的RNA，在任何长度的规模。然而，不受欢迎的 产品通常会以不可预测的方式污染所需的RNA。这可能是最多的 目前在信使核糖核酸治疗领域有影响，污染双链RNA可以 触发潜在的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，到 合成生物学，到RNA纳米技术。凝胶纯化过程繁琐，产率低，而且不完善 (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 池可以是异类的！)。更长的RNA杂质来自正确的RNA，降低了产量 合成，并且可以分布在广泛的长度范围内，这使得凝胶分析成为问题。 在新的机械理解的基础上，这个项目将开发限制条件的系统 这会引起这些杂质，特别是通过抑制旁路反应，并将发展 简单的亲和纯化方法-所有这些方法的目的都是实现单分散的RNA 定义的长度和顺序。将为成功开发灵敏的分析和功能分析方法 并将其应用于导轨设计。工具的开发将着眼于广泛采用各种 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.