Purification of Circular RNA by Ultrafiltration

超滤纯化环状 RNA

基本信息

  • 批准号:
    10629427
  • 负责人:
  • 金额:
    $ 17.91万
  • 依托单位:
  • 依托单位国家:
    美国
  • 项目类别:
  • 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

项目摘要

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.
项目总结

项目成果

期刊论文数量(0)
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会议论文数量(0)
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Scott M Husson其他文献

Scott M Husson的其他文献

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{{ truncateString('Scott M Husson', 18)}}的其他基金

Purification of Circular RNA by Ultrafiltration
超滤纯化环状 RNA
  • 批准号:
    10426638
  • 财政年份:
    2022
  • 资助金额:
    $ 17.91万
  • 项目类别:
Universal affinity membrane chromatography for rapid, one-step purification of proteins
用于快速、一步纯化蛋白质的通用亲和膜层析
  • 批准号:
    10250634
  • 财政年份:
    2021
  • 资助金额:
    $ 17.91万
  • 项目类别:
High-performance membrane chromatography for protein purification
用于蛋白质纯化的高性能膜层析
  • 批准号:
    7978738
  • 财政年份:
    2010
  • 资助金额:
    $ 17.91万
  • 项目类别:

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