Generation of precise and accurate engineered circRNAs using enzymatic ligation

messenger RNA-based therapeutics have revolutionized the treatment and prevention of infectious, neurological, and cancer diseases. However, their linear topology makes them susceptible to rapid degradation in vivo, which limits their therapeutic efficacy. Engineered circular RNAs (circRNAs) due to their closed ends and high stability are emerging as a promising alternative to linear RNA therapies. Engineered circRNAs are also increasingly used to mimic naturally occurring circRNAs in functional studies. Both applications, however, depend on production of precise circRNAs with homogenous sequences to enable accurate interpretation of biological outcomes. To address this, we developed and optimized methods for generating precise circRNAs. We employed enzymatic ligation of linear RNAs rather than autocatalytic splicing to produce circRNAs to minimize extraneous nucleotides remaining from the ribozymes. We carefully designed the DNA transcription template to maintain sequence and structural integrity. A permuted DNA template leveraging three internal guanosines (Gs) was synthesized and amplified using a reverse primer containing two 2'-O-methyl groups. This approach optimally produced the linear precursor RNA with correct 5' and 3' ends. After testing multiple workflows, we found that GMP-primed in vitro transcription, T4 RNA ligase 2-mediated circularization, and urea-polyacrylamide gel electrophoresis (PAGE) gel extraction produced the highest fidelity circRNAs.