mRNA is an emerging medical modality, however, approaches to control its activity lack behind other biologics. Bioorthogonal click-to-release reactions enable breaking chemical bonds at high reaction rates even in living cells to release a functionally active biomolecule ("uncaging"). We developed a 5' cap modified with a trans-cyclooctene (TCO-cap) that reacts with hydroxyaryl-tetrazines to efficiently release the native cap 0. This strategy is compatible with in vitro transcription and facilitates HPLC-based purification of the resulting TCO-capped mRNA, circumventing the need to digest uncapped mRNA produced in the process. Using eGFP- and luciferase-mRNAs in mammalian cells, we show that TCO-capped mRNAs are translationally muted and can be activated for translation by addition of cell-permeable, non-toxic sulfonamide-modified hydroxyphenyl-tetrazines. This work presents a new approach for small-molecule-induced translation in eukaryotes with potential to be applicable to any mRNA.

Translation of mRNA into protein is a fundamental process and tightly controlled during development. Several mechanisms acting on the mRNA level regulate when and where an mRNA is expressed. To explore the effects of conditional and transient gene expression in a developing organism, it is vital to experimentally enable abrogation and restoration of translation. We recently developed the FlashCaps technology allowing preparation of translationally muted mRNAs and their controlled activation by light. Here, we validate its functionality in vivo. We demonstrate that translation of FlashCap-eGFP-mRNA can be triggered in zebrafish embryos with spatiotemporal control. The injected FlashCap-mRNA is stable for hours and remains muted. Light-mediated activation up to 24 h post fertilization produces visible amounts of eGFP and can be restricted to distinct parts of the embryo. This methodology extends the toolbox for vertebrate models by enabling researchers to locally activate mRNA translation at different timepoints during development.

Internal modifications of mRNA have emerged as widespread and versatile regulatory mechanism to control gene expression at the post-transcriptional level. Most of these modifications are methyl groups, making S-adenosyl-L-methionine (SAM) a central metabolic hub. Here we show that metabolic labeling with a clickable metabolic precursor of SAM, propargyl-selenohomocysteine (PSH), enables detection and identification of various methylation sites. Propargylated A, C, and G nucleosides form at detectable amounts via intracellular generation of the corresponding SAM analogue. Integration into next generation sequencing enables mapping of N-methyladenosine (mA) and 5-methylcytidine (mC) sites in mRNA with single nucleotide precision (MePMe-seq). Analysis of the termination profiles can be used to distinguish mA from 2'-O-methyladenosine (A) and N1-methyladenosine (mA) sites. MePMe-seq overcomes the problems of antibodies for enrichment and sequence-motifs for evaluation, which was limiting previous methodologies. Metabolic labeling via clickable SAM facilitates the joint evaluation of methylation sites in RNA and potentially DNA and proteins.

The 5' cap is a hallmark of eukaryotic mRNA involved in the initiation of translation. Its modification with a single photo-cleavable group can bring translation of mRNA under the control of light. However, UV irradiation causes cell stress and downregulation of translation. Furthermore, complex processes often involve timed expression of more than one gene. The approach would thus greatly benefit from the ability to photo-cleave by blue light and to control more than one mRNA at a time. We report the synthesis of a 5' cap modified with a 7-(diethylamino)coumarin (CouCap) and adapted conditions for in vitro transcription. Translation of the resulting CouCap-mRNA is muted in vitro and in mammalian cells, and can be initiated by irradiation with 450 nm. The native cap is restored and no non-natural residues nor sequence alterations remain in the mRNA. Multiplexing for two different mRNAs was achieved by combining cap analogs with coumarin- and ortho-nitrobenzyl-based photo-cleavable groups.

The translation of messenger RNA (mRNA) is a fundamental process in gene expression, and control of translation is important to regulate protein synthesis in cells. The primary hallmark of eukaryotic mRNAs is their 5' cap, whose molecular contacts to the eukaryotic translation initiation factor eIF4E govern the initiation of translation. Here we report 5' cap analogues with photo-cleavable groups (FlashCaps) that prohibit binding to eIF4E and resist cleavage by decapping enzymes. These compounds are compatible with the general and efficient production of mRNAs by in vitro transcription. In FlashCap-mRNAs, the single photocaging group abrogates translation in vitro and in mammalian cells without increasing immunogenicity. Irradiation restores the native cap, triggering efficient translation. FlashCaps overcome the problem of remaining sequence or structure changes in mRNA after irradiation that limited previous designs. Together, these results demonstrate that FlashCaps offer a route to regulate the expression of any given mRNA and to dose mRNA therapeutics with spatio-temporal control.