A poly(A) polymerase and oligo(dT)-dependent method for the purification of engineering circular RNAs that enhances protein expression in eukaryotic cells

ABSTRACT Circular RNA (circRNA) has garnered significant attention as a promising therapeutic tool due to its remarkable stability and resistance to exonuclease degradation. However, the development of scalable and efficient purification methods remains a critical challenge. In this study, we propose and evaluate a novel purification strategy for engineered circular RNA (PAPdt), which leverages poly(A) polymerase and oligo(dT) affinity chromatography. Using poly(A) polymerase, we add poly(A) tails of over 200 nucleotides to the 3’-OH ends of all linear RNA impurities in a circular RNA preparation. These linear RNAs are then separated from the circular RNA via oligo(dT) affinity chromatography. The linear impurities, bearing longer poly(A) tails, exhibit extended retention on the oligo(dT) column, whereas the circular RNA, with a shorter poly(A) sequence, elutes more quickly. By stepwise elution with ultrapure water, we effectively separate the circular RNA from the linear contaminants. This method offers a fundamental solution to the purification of circular RNA, combining high efficiency, purity, and cost-effectiveness.