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Abstract Starting with the clinical application of two vaccines in 2020, mRNA therapeutics are currently being investigated for a variety of applications. Removing immunogenic uncapped mRNA from transcribed mRNA is critical in mRNA research and clinical applications. Commonly used capping methods provide maximum capping efficiency of around 80–90% for widely used Cap-0- and Cap-1-type mRNAs. However, uncapped and capped mRNA possesses almost identical physicochemical properties, posing challenges to their physical separation. In this work, we develop hydrophobic photocaged tag-modified cap analogs, which separate capped mRNA from uncapped mRNA by reversed-phase high-performance liquid chromatography. Subsequent photo-irradiation recovers footprint-free native capped mRNA. This approach provides 100% capping efficiency even in Cap-2-type mRNA with versatility applicable to 650 nt and 4,247 nt mRNA. We find that the Cap-2-type mRNA shows up to 3- to 4-fold higher translation activity in cultured cells and animals than the Cap-1-type mRNA prepared by the standard capping method.

The COVID-19 pandemic generated interest in the medicinal applications of messenger RNA (mRNA). It is expected that mRNA will be applied, not only to vaccines, but also to regenerative medicine. The purity of mRNA is important for its medicinal applications. However, the current mRNA synthesis techniques exhibit problems, including the contamination of undesired 5′-uncapped mRNA and double-stranded RNA. Recently, our group developed a completely capped mRNA synthesis technology that contributes to the progress of mRNA research. The introduction of chemically modified nucleosides, such as N1-methylpseudouridine and 5-methylcytidine, has been reported by Karikó and Weissman, opening a path for the practical application of mRNA for vaccines and regenerative medicine. Yamanaka reported the production of induced pluripotent stem cells (iPSCs) by introducing four types of genes using a retrovirus vector. iPSCs are widely used for research on regenerative medicine and the preparation of disease models to screen new drug candidates. Among the Yamanaka factors, Klf4 and c-Myc are oncogenes, and there is a risk of tumor development if these are integrated into genomic DNA. Therefore, regenerative medicine using mRNA, which poses no risk of genome insertion, has attracted attention. In this review, the author summarizes techniques for synthesizing mRNA and its application in regenerative medicine.

Employing the module strategy based on our recent finding that the recognition behavior of peptide ribonucleic acid (PRNA) with complementary DNA/RNA is effectively controlled by the anti-to-syn orientation switching of pyrimidine nucleobase induced by borate ester formation, we designed and synthesized PRNA-DNA and PRNA-PNA-DNA chimeras. In these chimeras, both of the PRNA (or PRNA-PNA) and DNA domains recognize the complementary DNA/RNA to form a stable complex, and the PRNA domain is simultaneously expected to play the dual role of switching the recognition behavior and inhibiting hydrolysis by exonucleases. The complexation and recognition control behaviors of these chimeras with DNA and RNA have been elucidated. [ABSTRACT FROM AUTHOR]