Site-Specifically Modified Circular Ribonucleic Acid Serves as Multitarget miRNA Sponge with Low Immunogenicity.

miRNA sponges, which could regulate downstream gene expression through competitively binding to overexpressed intracellular miRNAs, have gained increasing attention in miRNA-based therapies. However, linear miRNA sponges typically exhibit low stability and high immunogenicity under physiological conditions. Although artificial circular RNAs (circRNAs) acting as miRNA sponges enhance biostability, immunogenicity still remains a challenge. The 2023 Nobel Prize in Physiology or Medicine highlighted that the incorporation of modified bases could reduce the immunogenicity of exogenous RNAs, yet achieving site-specific modifications in circRNAs remains difficult. Here, we developed a DNA-templated RNA ligation method for the synthesis of site-specifically modified circRNAs with multiple miRNA binding sites, which enabled long-term coregulation of multiple miRNAs. Based on this, we found that 30% pseudouridine modification effectively reduced the immunogenicity of synthetic circular miRNA sponges independent of modification sites or patterns. Taken together, the high yield and site-specific modification of our method enable the precise synthesis of modified circRNAs, which could balance the immunogenicity, biostability, and miRNA binding affinity through engineered modifications in linker regions. These advancements are instrumental for the development of a new generation of circRNA-based therapeutics.