In Vitro RNA Folding Optimization

The in vitro transcripts were initially purified by undergoing gel purification on a 5% urea-PAGE gel (200 V at room temperature for 1 h). Following this, in vitro RNA folding was performed according to previously described methods [19 (link)]. Briefly, the in vitro transcripts were first dissolved in 0.5× TE buffer at pH 8.0. They were then subjected to heating at 95 °C for 3 min, followed by cooling on ice for 5 min. Subsequently, the RNA was incubated at 37 °C for 5 min in a folding buffer consisting of 500 mM Tris–HCl at pH 7.5 and 500 mM NaCl. After adding MgCl₂ to a final concentration of 10 mM, the mixture was further incubated for 30 min. Electrophoresis was then performed using 5% native PAGE gel (80 V for 4 h at 4 °C), with each sample containing 50 ng of RNA. For comparison, PSTVd-C was included as a control [21 (link)]. Trans2K^® Plus II DNA Marker (Transgen Biotech, Peking, China) was also loaded as a size marker. Images were captured after ethidium bromide staining under UV light.