Optimization of SEC-HPLC Analytical and Purification Workflow for Circular RNA via Column Temperature Regulation

OBJECTIVE To optimize the size-exclusion high-performance liquid chromatography(SEC-HPLC) purification process for circular RNA(circRNA) by regulating column temperature, and to investigate the effects of column temperature on chromatographic behavior, separation resolution, product purity, recovery yield and RNA integrity of two coding circRNAs. The optimal temperature-controlled purification parameters were screened, and the in vitro translational activity of circRNA before and after purification was compared.

METHODS Two crude circularized samples, circRNA-enhanced green fluorescent protein(EGFP) and circRNA-firefly luciferase(Fluc), were used as research materials. Sepax SRT SEC-2000 column was adopted to perform SEC-HPLC separation at five gradient column temperatures of 5, 25, 37, 55 and 70 ℃. The chromatographic peak profiles and component retention time at each temperature were recorded, the resolution between linear precursors and target circRNA was calculated, purification recovery was statistically analyzed. Eluted circRNA fractions collected at each temperature were subjected to agarose gel electrophoresis to analyze the purity of circRNA. SEC-HPLC-purified products and unpurified crude samples were separately transfected into HEK 293T cells, and EGFP fluorescence intensity as well as Fluc expression level were determined to evaluate biological activity.

RESULTS The retention time of all components shortened synchronously as column temperature increased. At low temperature of 5 °C, RNA secondary structure remained stable, leading to extensive peak overlap between linear impurities and circRNA with the lowest resolution. After temperature elevation to 25 °C and 37 °C, linear precursors and circRNA could be preliminarily separated, accompanied by simultaneous improvement of resolution and product purity. Column temperature at 55 °C further improved the comprehensive purification performance, realizing effective separation of linear precursors, nicked RNA and target circRNA, and the electrophoretic purity of circRNA reached the maximum among all groups. High temperature of 70 °C failed to enhance separation resolution, instead triggered massive generation of degraded fragments and drastically reduced the yield of intact circRNA. The two coding circRNAs presented nearly consistent variation trends in chromatographic profile and product purity along with temperature changes. Cellular assays verified that intracellular EGFP fluorescence intensity and Fluc expression level of SEC-HPLC-purified circRNA were significantly higher than those of unpurified crude samples.

CONCLUSION Column temperature alters the discrepancy of hydrodynamic volume by regulating the denaturation degree of RNA secondary structure, thereby determining the purification performance of circRNA in SEC-HPLC system. The optimal column temperature for purification in this experimental system is 55 °C, which can simultaneously obtain circRNA with high separation resolution, high purity and acceptable recovery yield. This temperature-controlled SEC-HPLC protocol features simple operation and favorable compatibility with circRNAs carrying different coding sequences, which can provide references for the preparation and quality control of coding circRNA.