A methodological and mechanistic exploration to precise separation of circular RNA and nicked RNA by capillary gel electrophoresis with laser-induced fluorescence

As the 2.0 version of mRNA technology, circular RNA (circRNA) is advantageous over the linear mRNA in stability, offered by its covalently closed loop structure. In the in vitro circularization (IVC) system for circRNA synthesis, there exist linear precursor RNA and nicked RNA by-product as impurities. Both impurities are highly similar to the target circRNA in size and structure, making high-resolution separation and quantification of circRNA more challenging. In this study, we developed a capillary gel electrophoresis with laser-induced fluorescence (CGE-LIF) method, which could precisely separate the circRNA from its impurities in the IVC systems with high resolution (Rs > 5.8) and excellent reproducibility (RSD < 0.04%). We then focused on the different migration behaviors of circRNA in gel environments by systematically exploring the key influencing factors, including sample pretreatment, separation voltage and gel matrix. Our results demonstrated that the circular integrity of circRNA is a critical determinant of its electrophoretic migration behavior. Pretreating the circRNA with denaturant or prolonging the migration time increased the possibility of its digestion into the nicked species. Further mechanistic analysis revealed that circRNA underwent random, rather than sequence-dependent backbone cleavage during electrophoresis. These findings suggested that circRNA stability during electrophoretic separation may be influenced by cumulative mechanical stress arising from the combined effects of electrophoretic force and molecular sieving. The mechanism elucidating the determinants of circRNA mobility in different gel systems lays the foundation for developing more reliable approaches for circRNA purification and characterization.