Purification of Circular RNA by Ultrafiltration

项目摘要 环状RNA（circRNA）在生理学和疾病中具有重要的，未充分研究的作用。此外，本发明还 蛋白质编码RNA的环化是一种有前途的策略，用于增加蛋白质编码RNA的持续时间和数量。 相对于线性信使RNA（mRNA），如 就像开发第一批COVID-19疫苗时使用的一样。不幸的是，circRNA的合成 含有导致细胞免疫的污染性线性RNA前体和切口RNA的溶液 反应;并且没有有效的纯化方法已被证明。本项目的目标是测试 这是一种新概念的可行性，以高纯度和高产量纯化环状RNA。拟议的研究已 推进生物医学研究的强大潜力，旨在回答有关circRNA在 疾病和生物功能，其中高样品纯度是必不可少的。它也有可能成为 通过展示高产量，对circRNA疗法的开发和获得产生重大影响 这是一种目前尚不存在的净化方法。我们的研究假设是， 超滤可用于基于形状和大小分离RNA。我们的理论是RNA传输 将开始在临界通量（流速和膜面积的商）下发生 由于流动引起的伸长。RNA构象异构体之间延伸特性的差异将导致 临界通量的差异，这为从线性RNA杂质中纯化circRNA提供了基础。 具体目的是（1）定量circRNA、线性RNA前体和切口RNA的超滤临界通量。 （2）开发用于纯化自剪接反应产生的circRNA的方案，以及（3） 证明超滤纯化的circRNA在细胞中的蛋白质产生。在目标1中，我们将生产纯化的 不同RNA构象的组分，并通过超滤准确测量其透射率 膜，以量化其基于膜性能的临界通量。新技术知识 使用纯化的RNA构象异构体产生的通量将鉴定可用于分离RNA构象异构体的通量条件。 自剪接反应溶液中线性污染物的circRNA。在目标2中，我们将建立一个简单的 实施利用这些临界通量差异从自剪接反应中纯化circRNA的方案 解决方案这一目标将促进对影响circRNA纯度和产量的关键因素的了解。在目标3中， 我们将对超滤纯化的细胞中蛋白质产量进行测量并评估其稳定性 circRNA相对于使用高效液相色谱法纯化的circRNA，其代表 最先进的方法该项目将为基于超滤的净化提供明确的概念证明 高纯度（95%）和高产率（>70%）的circRNA，这将是对 最先进的技术

Project Summary Circular RNA (circRNA) has important, understudied roles all across physiology and disease. Additionally, circularization of protein-coding RNA is a promising strategy for increasing the duration and quantity of therapeutic protein production and decreasing immunogenicity relative to linear messenger RNA (mRNA), such as those used to develop the first COVID-19 vaccines. Unfortunately, the synthesis of circRNA produces solutions that contain contaminating linear RNA precursors and nicked RNA that cause cellular immune responses; and no effective purification method has been demonstrated. The goal of this project is to test the feasibility of a novel concept to purify circular RNA with high purity and high yield. The proposed study has strong potential to advance biomedical research aimed at answering questions about the roles of circRNA in disease and biological function, where high sample purity is essential. It also has the potential to have a significant impact on the development of and access to circRNA therapies by demonstrating a high yield approach for their purification that does not exist today. Our research hypothesis is that membrane ultrafiltration can be used to separate RNA based on shape and size. We theorize that RNA transmission through ultrafiltration membranes will begin to occur at a critical flux (quotient of flowrate and membrane area) due to flow-induced elongation. Differences in elongational properties among RNA conformers will lead to differences in critical fluxes, which provides a basis for the purification of circRNA from linear RNA impurities. The Specific Aims are to (1) quantify ultrafiltration critical fluxes for circRNA, linear RNA precursor, and nicked RNA; (2) develop a protocol for purification of circRNA generated from self-splicing reactions, and (3) demonstrate protein production in cells for ultrafiltration-purified circRNA. In Aim 1, we will produce purified fractions of the different RNA conformers and accurately measure their transmission through ultrafiltration membranes to quantify their critical fluxes based on membrane properties. New technical knowledge generated using purified RNA conformers will identify flux conditions that can be used for the separation of circRNA from linear contaminants in self-splicing reaction solutions. In Aim 2, we will establish an easy to implement protocol that exploits differences in these critical fluxes to purify circRNA from self-splicing reaction solutions. This aim will advance knowledge on the key factors influencing circRNA purity and yield. In Aim 3, we will measure the quantity and evaluate the stability of protein production in cells for ultrafiltration-purified circRNA relative to circRNA purified using high-performance liquid chromatography, which represents the state-of-the-art approach. This project will provide a clear proof of concept for ultrafiltration-based purification of circRNA with high purity (95%) and high yield (>70%), which would be a significant advancement over the state of the art.