Stabilized 5' Cap Analogue for Optochemical Activation of mRNA Translation.

The 5' cap is a distinguishing feature of transcripts made by polymerase II and characterized by an N7-methylated guanosine (m7G) linked to the first transcribed nucleotide by a 5'-5' triphosphate bridge. It stabilizes eukaryotic mRNAs and plays a crucial role in translation initiation. Its importance in mRNA processing, translation, and turnover makes the 5' cap a privileged structure for engineering by non-natural modifications. A photocleavable group at the 5' cap of guanosine was recently used to mute translation of exogenous mRNAs. Its removal by light enabled direct control of protein production at the posttranscriptional level. Modifications in the triphosphate bridge impede degradation by specific decapping enzymes and maintain translation. Here, we combined 5' cap modifications at different positions and investigated how they impact 5' cap-dependent processes in distinct manners. We synthesized 5' cap analogues with a photocleavable group at the N2-position of m7G in addition to a medronate in the triphosphate bridge to obtain a photoactivatable 5' cap analogue featuring a methylene group between the ß and γ phosphates. The resulting Medronate-FlashCap transiently or permanently impeded distinct crucial interactions of the 5' cap required for translation and degradation. We show that the Medronate-FlashCap is compatible with in vitro transcription to generate muted mRNA and that light can be used to activate translation in cells. After light-induced removal of the photocleavable group, the Medronate-FlashCap remained stable against degradation by the decapping enzyme DcpS. The additional methylene group renders the 5' cap resistant to DcpS, while maintaining the interaction with cap-binding proteins.

Optochemical Control of mRNA Translation in Eukaryotes.

A major stage in the expression of genes is the translation of messenger RNA (mRNA), and the regulation of this process is essential for protein production in cells. How tightly controlled gene expression can be spatially and temporally, is particularly evident in polar cells and embryonic development. We need tools to dissect these complex processes, if we wish to understand the underlying links, especially the difficulties brought on by malfunction. External bioorthogonal triggers are very helpful in this area, if they let us precisely control where and when a process is started. Equipping nucleic acids with light-responsive groups has proven to be an effective approach to examine the dynamic regulatory route of mRNA translation in living cells. In this review, we present an overview of the most recent methods for optochemically controlling translation, focusing on cis-acting technologies.

Photocaged 5' cap analogues for optical control of mRNA translation in cells.

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.

Tag-Free Internal RNA Labeling and Photocaging Based on mRNA Methyltransferases.

The mRNA modification N6 -methyladenosine (m6 A) is associated with multiple roles in cell function and disease. The methyltransferases METTL3-METTL14 and METTL16 act as "writers" for different target transcripts and sequence motifs. The modification is perceived by dedicated "reader" and "eraser" proteins, but not by polymerases. We report that METTL3-14 shows remarkable cosubstrate promiscuity, enabling sequence-specific internal labeling of RNA without additional guide RNAs. The transfer of ortho-nitrobenzyl and 6-nitropiperonyl groups allowed enzymatic photocaging of RNA in the consensus motif, which impaired polymerase-catalyzed primer extension in a reversible manner. METTL16 was less promiscuous but suitable for chemo-enzymatic labeling using different types of click chemistry. Since both enzymes act on distinct sequence motifs, their combination allowed orthogonal chemo-enzymatic modification of different sites in a single RNA.

Covalent labeling of nucleic acids.

Labeling of nucleic acids is required for many studies aiming to elucidate their functions and dynamics in vitro and in cells. Out of the numerous labeling concepts that have been devised, covalent labeling provides the most stable linkage, an unrivaled choice of small and highly fluorescent labels and - thanks to recent advances in click chemistry - an incredible versatility. Depending on the approach, site-, sequence- and cell-specificity can be achieved. DNA and RNA labeling are rapidly developing fields that bring together multiple areas of research: on the one hand, synthetic and biophysical chemists develop new fluorescent labels and isomorphic nucleobases as well as faster and more selective bioorthogonal reactions. On the other hand, the number of enzymes that can be harnessed for post-synthetic and site-specific labeling of nucleic acids has increased significantly. Together with protein engineering and genetic manipulation of cells, intracellular and cell-specific labeling has become possible. In this review, we provide a structured overview of covalent labeling approaches for nucleic acids and highlight notable developments, in particular recent examples. The majority of this review will focus on fluorescent labeling; however, the principles can often be readily applied to other labels. We will start with entirely chemical approaches, followed by chemo-enzymatic strategies and ribozymes, and finish with metabolic labeling of nucleic acids. Each section is subdivided into direct (or one-step) and two-step labeling approaches and will start with DNA before treating RNA.