mRNA—a bridge linking genetic information to functional proteins—is a novel drug modality with both informational (carrying genetic code) and functional (coding/non-coding RNA regulation) characteristics. In mRNA drug development, in vitro modification is critical. mRNA needs a proper 5' cap—like a well-fitted hat—to express stably in vivo.

Mature mRNA has several key structural elements: 5' cap, 5' UTR, ORF, 3' UTR, and poly(A) tail. The 5' cap—a 7-methylguanosine (m7G) structure—was discovered in the 1970s. It is where ribosomes initiate protein translation. Natural mRNA has three cap types: Cap0, Cap1, and Cap2.

The 5' cap has two main functions: stabilizing mRNA (protecting against exonuclease degradation) and facilitating translation (helping ribosome recognition and binding). In vitro-synthesized mRNA is generated by transcription from linearized plasmid DNA, with capping and poly(A) tailing added at the end.

Capping is essential for translation initiation, splicing, intracellular transport, and nuclear export. It provides a signal for ribosome recognition, protects the 5' end from degradation, participates in mRNA splicing, and ensures stable cytoplasmic transport. Various capping methods exist—two-step capping and co-transcriptional capping. As research on mRNA cap structure deepens, chemical modifications are increasingly explored to enhance expression, translation efficiency, and transfection efficiency.