One-pot synthesis of fluorescent cap1 mRNAs using labelled trinucleotide m7G-cap analogs for real-time in vitro and in vivo mRNA tracking

Visualizing synthetic labelled mRNAs in cells and Animals enables the study of mRNA functions/dynamics and also helps to evaluate the delivery, distribution, intracellular kinetics, and efficacy of mRNA-based therapeutics/vaccines. Current methods for synthesizing labelled mRNAs, suitable for in vitro and in vivo tracking, rely on tedious and strenuous post-transcription modification processes. Herein, we report a direct one-pot co-transcriptional synthesis of labelled cap1 mRNAs by employing a series of fluorescently tagged trinucleotide Cap Analogs. These synthesized and translatable mRNAs with a single labelled fluorescent tag at the 3' position of the ribose ring of the m7G cap enabled real-time tracking of mRNA in biological systems without the need for additional chemical modifications. Robust transcriptional yield, capping efficiency and transcript integrity were achieved by the standard enzymatic co-transcription protocol with these fluorophore-labelled m⁷GpppNN trinucleotide analogues. These synthesized labelled mRNAs retained their translational competence and activity, as confirmed by protein expression assays in mammalian cells. Further, we demonstrated that mRNAs with these labelled caps allowed direct visualization of mRNA uptake, intracellular trafficking and localization, and biodistribution using fluorescence microscopy and whole-animal imaging in mouse models. This strategy streamlines the generation of functional and trackable mRNAs, providing a powerful tool for the study of mRNA function and dynamics, for further evaluating mRNA vaccines and therapeutic delivery systems.