Rapid and high recovery isolation of mRNA from in vitro transcription system by ammonium sulphate precipitation at room temperature

Abstract

Messenger RNA (mRNA) technologies have shown great potential in prophylactic vaccines and therapeutic medicines. Rapidly and effectively separating mRNA from in vitro transcription (IVT) products is important to reduce the accumulation of byproducts and avoid degradation of the mRNA. In this study, the salting out of mRNA encoding enhanced green fluorescent protein (EGFP) by ammonium sulfate (AS) that was widely employed in protein precipitation was examined. Effects of precipitation conditions, including AS concentration, pH, temperature, and time, on the precipitation rate and re-dissolution yield of mRNA were first investigated by the design of experiments (DOE) method, using LiCl-purified mRNA. The optimal condition was determined to be 2 M AS, pH 7.0, 20 °C, and 2 h of precipitation time, under which almost 100 % precipitation rate and nearly 100 % dissolution rate could be reached. This optimal condition was then applied to separate mRNA from IVT and co-transcriptional capping system, overall recovery of about 80 % of mRNA was obtained; meanwhile, more than 80 % DNA template and 99.0 % residual NTP were removed. In co-transcriptional capping system, >90 % dsRNA was removed. The mRNA purified by AS precipitation was transfected into HEK293T cells to successfully express EGFP with efficiency of 45.65 % which was similar to that by LiCl-purified mRNA (43.85 %). Though the removal rate for T7 polymerase was low (about 30 %) due to protein-mRNA interaction induced co-precipitation in 2 M AS, our results demonstrated significant advantages of AS precipitation over conventional LiCl precipitation in terms of biosafety and energy cost. Thus, the AS precipitation provides a cost-effective method to rapidly separate, concentrate, and store the mRNA from the IVT system, as well as during subsequent downstream processes.