# Purification of Circular RNA by Ultrafiltration

> **NIH NIH R21** · CLEMSON UNIVERSITY · 2022 · $217,771

## Abstract

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 wo...

## Key facts

- **NIH application ID:** 10426638
- **Project number:** 1R21GM145980-01
- **Recipient organization:** CLEMSON UNIVERSITY
- **Principal Investigator:** Scott M Husson
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $217,771
- **Award type:** 1
- **Project period:** 2022-06-01 → 2024-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10426638

## Citation

> US National Institutes of Health, RePORTER application 10426638, Purification of Circular RNA by Ultrafiltration (1R21GM145980-01). Retrieved via AI Analytics 2026-06-05 from https://api.ai-analytics.org/grant/nih/10426638. Licensed CC0.

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