RNA denaturation underlies circular RNA separation.

In vitro-synthesized circular RNAs (circRNAs) have emerged as a promising drug modality for RNA therapeutics due to their improved stability and reduced immunogenicity. However, effective analysis and purification of circRNAs pose critical challenges arising from the insufficient separation of circRNAs and linear RNA byproducts. In this study, we systematically evaluate the effectiveness of gel electrophoresis and high-performance liquid chromatography-size exclusion chromatography (HPLC-SEC) for separating circRNAs synthesized through ligase- or ribozyme-based strategies. While the synthesis strategy dictates the purification complexity, we demonstrate that both techniques rely on RNA denaturation to successfully separate circRNAs. Additionally, when using HPLC-SEC, we show that even a trace amount of magnesium ions in RNA samples can significantly compromise circRNA separation. Under optimized denaturing conditions, HPLC-SEC enables circRNA purification directly from crude enzymatic reactions, thereby streamlining the purification process. Our study provides mechanistic insights into circRNA separation, advancing the purity and scalability of circRNA-based therapeutics.