Unraveling the molecular mechanism of perchlorate anion effects on in vitro transcription of mRNA via molecular dynamics simulations.

The yield and quality of mRNA synthesized by in vitro transcription (IVT) are highly dependent on reaction components. While Mg2+ is a well-established cofactor, the role of anions remains less explored. This study investigates the regulatory effects and mechanisms of the chaotropic salt sodium perchlorate (NaClO4) on IVT process, focusing on mRNA yield and double-stranded RNA (dsRNA) formation. Experimentally, introducing 50 mM NaClO4 in IVT achieves up to 85% reduction in dsRNA, while doubling the mRNA yield. This regulatory performance is superior to high-concentration urea, which requires 400 mM to achieve a similar dsRNA inhibitory effect and provides negligible yield enhancement. To elucidate the distinct regulatory mechanisms of NaClO₄ and urea, molecular dynamics simulations were performed. Results indicate that urea molecules abundantly bind to RNA, blocking base pairing and inhibiting RNA folding, whereas ClO4- ions bind minimally but raise the electrostatic energy barrier for RNA folding, both mechanisms reduce the dsRNA formation. Furthermore, urea disrupts the hydration layer around the DNA template and interferes with its recognition by T7 RNA polymerase, while ClO4- has negligible impact. Conformational analysis on T7 RNA polymerase-DNA complex shows that low NaClO4 concentrations promote enzyme's active pocket contraction, enhancing binding; higher concentrations, however, lead to displacement of a key residue (Trp 422), impairing promoter recognition. This explains the non-monotonic variation in mRNA yield with increasing NaClO4 concentrations: beyond a critical concentration, transcription efficiency declines and may fail completely. These findings provide crucial insights for optimizing IVT systems by tailoring anion selection and concentration.