Analytical Methods to Evaluate RNA Circularization Efficiency

ABSTRACT Circular RNAs (circRNAs) have emerged as pivotal players in RNA therapeutics. Unlike linear counterparts, circRNAs possess a closed‐loop structure, conferring them with enhanced stability and resistance to degradation. Ribozyme‐based strategy stands out as the predominant method for synthetic circRNA production, by precisely cleaving and promoting the formation of a covalent circular structure. However, there is still a lack of analytical methods that can provide high‐throughput and quantitative analysis to facilitate the circRNA vector engineering process. In the report, we detail analytical methods to characterize and evaluate ribozyme‐based RNA circularization efficiency. Our approach will capture the attention of researchers interested in optimizing RNA circularization efficiency, as well as those focused on exploring key elements for ribozyme catalytic activity.