Eukaryotic mRNA with its 5′-cap is of central importance for the cell. Many studies involving mRNA require reliable preparation and modification of 5′-capped RNAs. Depending on the length of the desired capped RNA, chemical or enzymatic preparation – or a combination of both – can be advantageous. We review state-of-the art methods and give directions for choosing the appropriate approach. We also discuss the preparation and properties of mRNAs with non-natural caps providing novel features such as improved stability or enhanced translational efficiency.

Eukaryotic mRNAs are emerging modalities for protein replacement therapy and vaccination. Their 5' cap is important for mRNA translation and immune response and can be naturally methylated at different positions by Sadenosyl-l-methionine (AdoMet)-dependent methyltransferases (MTases). We report on the cosubstrate scope of the MTase CAPAM responsible for methylation at the N 6 -position of adenosine start nucleotides using synthetic AdoMet analogs. The chemo-enzymatic propargylation enabled production of site-specifically modified reporter-mRNAs. These cap-propargylated mRNAs were efficiently translated and showed % 3fold increased immune response in human cells. The same effects were observed when the receptor binding domain (RBD) of SARS-CoV-2-a currently tested epitope for mRNA vaccination-was used. Site-specific chemo-enzymatic modification of eukaryotic mRNA may thus be a suitable strategy to modulate translation and immune response of mRNAs for future therapeutic applications.Scheme 1. mRNAs and chemo-enzymatic modifications used in this study. A) 5'-Clean Cap (5'-CC) mRNA is produced via IVT. B,C) N 6 -Modification of adenosine at the transcription start nucleotide using CAPAM and AdoMet (S-adenosyl-l-methionine) or AdoMet analogs yields methylated Clean Cap (5'-CC-N 6 mA m ) or propargylated Clean Cap (5'-CC-N 6 pA m ), respectively. D) Modification of the poly(A) tail of 5'-CC-mRNA using poly(A) polymerase and 2'-azido ATP followed by labeling with Cy5 yields 5'-CC-3'-Cy5-mRNA. E) 5'-ARCA (5'-AC) mRNA is produced via IVT. F) N 2 -Modification of m 7 G using GlaTgs and AdoMet results in N 2 methylated ARCA (5'-N 2 m-AC)-mRNA. G) Modification of the poly(A) tail of 5'-AC-mRNA using poly(A) polymerase and 2'-azido ATP followed by labeling with Cy5 yields 5'-AC-3'-Cy5-mRNA.

Benzylic AdoMet analogs enable highly efficient enzymatic transfer of norbornenes to nucleic acids and subsequent tetrazine ligation.

Labeling RNA is a recurring problem to make RNA compatible with state-of-theart methodology and comes in many flavors. Considering only cellular applications, the spectrum still ranges from site-specific labeling of individual transcripts, for example, for live-cell imaging of mRNA trafficking, to metabolic labeling in combination with next generation sequencing to capture dynamic aspects of RNA metabolism on a transcriptome-wide scale. Combining the specificity of RNAmodifying enzymes with non-natural substrates has emerged as a valuable strategy to modify RNA site-or sequence-specifically with functional groups suitable for subsequent bioorthogonal reactions and thus label RNA with reporter moieties such as affinity or fluorescent tags. In this review article, we will cover chemoenzymatic approaches (a) for in vitro labeling of RNA for application in cells, (b) for treatment of total RNA, and (c) for metabolic labeling of RNA.

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