Thio-modification effects on mRNA translation using a PureCap-based capping method

Controlling translational efficiency, stability, and innate immune recognition remains a critical challenge for the practical application of mRNA therapeutics, and chemical modification of nucleic acids is widely employed as an effective strategy to address these issues. Sugar modifications exemplified by 4'-thio substitution and phosphate-backbone modifications exemplified by phosphorothioate (PS) substitution can alter the behavior of nucleic acids. In this study, to introduce 4'-thio substitution and PS substitution at the +1 and +2 nucleotides of the 5' end of mRNA, we synthesized a series of cap analogs based on the PureCap method that we previously established, which enables preparation of highly pure capped mRNA for reliable evaluation of cap modification effects. Using the resulting cap analogs, mRNAs were prepared by in vitro transcription (IVT) with high-performance liquid chromatography (HPLC) purification, and even when modifications were introduced into the ribose moiety, IVT yields were not substantially reduced. In cultured HeLa cells, PS-modified mRNAs exhibited higher protein expression than the 4'-thio-modified counterparts for Cap 0, Cap 1, and Cap 2 structures. By contrast, for the Cap 2 structure, PureCap-derived mRNAs bearing either 4'-thio or PS modifications showed protein expression comparable to that of an N ¹-methylpseudouridine (m¹Ψ)-modified control mRNA prepared using the commercially available CleanCap AG, both at 72 h in the cell-based translation assay and at each measurement time point following subcutaneous administration in mice. Taken together, these results suggest that 4'-thio and PS modifications at the 5'-terminal +1 and +2 nucleotides can alter mRNA function. This work further demonstrates the utility of the PureCap platform and highlights its broad applicability for mRNA therapeutic development.