English: mRNA in vitro transcription and innate immunity activation. (A) mRNA in vitro transcription. Using DNA with the antigen-encoding sequence as template, mRNA in vitro transcription products contain single-stranded RNA (ssRNA), double-stranded RNA (dsRNA), etc. The ssRNA structure normally includes five-prime cap (5′ cap), five-prime untranslated region (5′ UTR), open reading frame (ORF) region, three-prime untranslated region (3′ UTR), and poly (A) tail structure. (B) RNA translation and antigen presentation. Through endocytosis, mRNAs enter the cytoplasm. Some mRNAs combine with ribosomes of the host cell and translate successfully. Antigen proteins can be degraded to antigenic peptides by proteasome in the cytoplasm and presented to cytotoxic T lymphocytes (CTLs) via major histocompatibility complex (MHC) I pathway. Or, they can be released out of the host cell and taken up by DCs. Then, they are degraded and presented to helper T cells and B cells via MHC-II pathway. B cells can also recognize released antigen proteins. (C) Self-adjuvant effect. Various of pattern recognition receptors (PRRs) can recognize mRNA in vitro transcription product. ssRNA can be recognized by endosomal innate immune receptors (e.g., Toll-like receptor 7 (TLR7), TLR8). dsRNA can be recognized by endosomal innate immune receptors (e.g., TLR3) and cytoplasmic innate immune receptors (e.g., protein kinase RNA-activated (PKR), retinoic acid-inducible gene I protein (RIG-I), melanoma differentiation-associated protein 5 (MDA5), and 2′-5′-oligoadenylate synthase (OAS). Based on those, mRNA products can stimulate the secretion of pro-inflammatory cytokines and type I interferon (IFN), which leads to antigen-presenting cells (APCs) activation and inflammatory reaction. However, they can also activate antiviral enzymes that cause stalled mRNA translation and mRNA degradation.