Graphene Oxide-Modified Resin for Selective dsRNA Removal from In Vitro-Transcribed mRNA.

Abstract

Messenger RNA (mRNA) has proven to be an effective vaccine agent against unexpected pandemics, offering the advantage of rapidly producing customized therapeutics targeting specific pathogens. However, undesired byproducts, such as double-stranded RNA (dsRNA), generated during in vitro transcription (IVT) reactions may impede translation efficiency and trigger inflammatory cytokines in cells after mRNA uptake. In this study, we developed a facile method using PEGylated polystyrene resins that were further surface-modified with graphene oxide (GO@PEG-PS) for the removal of dsRNA from IVT mRNA. The GO@PEG-PS resin adsorbed mRNA due to the property of graphene oxide (GO), which preferentially adsorbs single-stranded nucleic acids over double-stranded nucleic acids in the presence of Mg²⁺. The resin-bound single-stranded (ss) RNA was readily desorbed with a mixture of EDTA and urea, possibly by chelating Mg²⁺ and disrupting hydrogen bonding, respectively. Spin-column chromatography with GO@PEG-PS for IVT mRNA eliminated at least 80% of dsRNA, recovering approximately 85% of mRNA. Furthermore, this procedure precluded the salt precipitation step after the IVT reaction, which fractionates mRNAs from the IVT components, including nucleotides and enzymes. The purified mRNA exhibited enhanced protein translation with reduced secretion of interferon (IFN)-β upon mRNA transfection. We anticipate that the mRNA purification chromatography system employing GO@PEG-PS resin will facilitate the removal of dsRNA contamination during mRNA production.