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.

Keywords: circular RNA; linear RNA; microfluidic capillary electrophoresis; vector engineering.

FIGURE 1

Analytical analysis workflow of synthetic RNA circularization. (A) A typical permuted intron–exon construct design for synthetic circRNA precursor. E1, E2: exon 1, exon 2; GOI: gene‐of‐interest; IRES: internal ribosome entry site; Catalytic intron as a preserved element critical for ribozyme folding is drawn as scribble lines and hairpin structures. (B) Schematic diagram showing fragment analysis of RNA species in RNA circularization on LabChip. Construct variants by circRNA vector engineering are labeled Vector‐Design 1 and Vector‐Design 2. (C) Real‐time PCR primer design and amplification curve for measuring ribozyme circularization. Top diagram represents results from linear precursor or samples with no or low circulation yield; bottom diagram represents results from samples with successful circularization. Red arrows represent junction primer set; black arrows represent body primer set.

FIGURE 2

In vitro transcription (IVT) and circularization. (A) Workflow of ribozyme‐based RNA circularization procedures. (B) LabChip gel confirmation of precursor RNA circularization. Construct_1 is not subjected to circularization process, and it only shows precursor RNA band representing the large molecular size. Construct_2 and Construct_3 are subject to circularization process, and two intron ends are spliced out as two small size bands (<200 nt).

FIGURE 3

Peak characterization and analytical test of circRNAs. (A) Splice site mutations (Construct_2_mut and Construct_3_mut) in circRNA vectors reduce circularization efficiency. (B) CircRNAs are enriched by RNase R treatment. One technical repeat result is used here for data display. (C) A relative circularization efficiency calculation by real‐time PCR between reference construct and mutant construct. ΔΔCt_{mut, ref}  < 0 means mutant construct improves circularization efficiency; ΔΔCt_{mut, ref}  > 0 means mutant construct decreases circularization efficiency. ΔΔCt_{mut, ref}  = 0 means mutant construct does not impact circularization efficiency. *: arbitrary mutations.

FIGURE 4

CircRNA peak patterns using RNA Pico reagents on LabChip. Top panel: One typical non‐purified RNA product by ribozyme‐based circularization. Bottom panel: RNA ladder from RNA Pico reagent. If size resolution of electrophoresis platform is optimal, assuming no 100% circularization efficiency, four bands are usually expected: Small introns’ peaks excised from 5′ end and 3′ end, one circRNA peak, and one RNA precursor peak. The sizes labeled below RNA species are theoretical value based on sequence design.