Purification of Circular RNA by Ultrafiltration

超滤纯化环状 RNA

• 批准号: 10629427
• 负责人: Scott M Husson
• 金额: $ 17.91万
• 依托单位: CLEMSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-06-01 至 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10629427
• 关键词: Address; Adopted; Area; Biological Process; Biology; Biomedical Research; COVID-19 prevention; COVID-19 vaccine; Cell Count; Cells; Code; DNA; Data; Development; Disease; Emerging Communicable Diseases; Flow Cytometry; Goals; Half-Life; High Pressure Liquid Chromatography; Hour; Immune response; Knowledge; Measures; Membrane; Messenger RNA; Methodology; Methods; Molecular Weight; Output; Pathology; Performance; Physiology; Play; Polymers; Predisposition; Production; Property; Proteins; Protocols documentation; RNA; RNA Conformation; RNA Precursors; RNA Splicing; RNA purification; Reaction; Recovery; Research; Resolution; Role; Sampling; Shapes; Structure; Technology; Therapeutic; Transfection; Ultrafiltration; Vaccine Antigen; Vaccine Production; circular RNA; conformer; cytotoxicity; experimental study; feasibility testing; immunogenicity; in vivo; innovation; live cell imaging; macromolecule; novel; novel strategies; plasmid DNA; prevent; protein expression; protein purification; public health relevance; theories; therapeutic RNA; therapeutic protein; therapeutic vaccine; transmission process

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.

项目摘要 圆形RNA（circrna）在生理和疾病中具有重要的，研究的作用。此外， 蛋白质编码RNA的循环是增加持续时间和数量的有前途的策略 相对于线性信使RNA（mRNA），治疗性蛋白质的产生和免疫原性降低，这样 正如那些用来开发第一种Covid-19疫苗的人。不幸的是，circrna的合成产生 包含污染的线性RNA前体和引起细胞免疫的昵称的RNA的溶液 回应；并且尚未证明有效的纯化方法。该项目的目的是测试 一种新颖概念的可行性，以纯化高纯度和高产量净化圆形RNA。拟议的研究有 促进生物医学研究的强大潜力，旨在回答有关Circrna在 疾病和生物学功能，其中高样本纯度至关重要。它也有可能拥有 通过证明高产量，对circrna疗法的发展和获取的重大影响 他们的净化方法今天不存在。我们的研究假设是膜 超滤可根据形状和尺寸分离RNA。我们将RNA传播理论化 通过超滤膜将开始在临界通量（流量和膜面积的商）处发生 由于流动诱导的伸长。 RNA构象体之间伸长特性的差异将导致 临界通量的差异，这为线性RNA杂质纯化CircRNA提供了基础。 具体目的是（1）量化circRNA，线性RNA前体和划痕的超滤临界通量 RNA； （2）制定一种用于纯化自裂反应产生的ciRCRNA的方案，（3） 证明了超滤细胞纯化的circrna的细胞中蛋白质产生。在AIM 1中，我们将产生纯化 不同RNA构象体的分数，并通过超滤准确测量其传播 膜基于膜特性来量化其临界通量。新的技术知识 使用纯化的RNA构象异构体生成的将识别可用于分离的通量条件 从线性污染物中的ciRCRNA中的自剪接反应溶液中。在AIM 2中，我们将建立一个易于 实施利用这些关键通量差异以净化circrna的协议 解决方案。这个目标将促进有关影响Circrna纯度和产量的关键因素的知识。在AIM 3中， 我们将测量数量并评估细胞中蛋白质产生的稳定性超滤光度 circrna相对于使用高性能液相色谱法纯化的Circrna，代表 最先进的方法。该项目将为基于超滤的纯化提供明确的概念证明 具有高纯度（95％）和高产量（> 70％）的Circrna的占地 最先进的。