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.
Eukaryotic RNAs are heavily processed, including co‐ and post‐transcriptional formation of various 5′ caps. In small nuclear RNAs (snRNAs) or small nucleolar RNAs (snoRNAs), the canonical 7mG cap is hypermethylated at the N2‐position, whereas in higher eukaryotes and viruses 2′‐O‐methylation of the first transcribed nucleotide yields the cap1 structure. The function and potential dynamics of several RNA cap modifications have not been fully elucidated, which necessitates preparative access to these caps. However, the introduction of these modifications during chemical solid‐phase synthesis is challenging and enzymatic production of defined short and uniform RNAs also faces difficulties. In this work, the chemical synthesis of RNA is combined with site‐specific enzymatic methylation by using the methyltransferases human trimethylguanosine synthase 1 (hTgs1), trimethylguanosine synthase from Giardia lamblia (GlaTgs2), and cap methyltransferase 1 (CMTR1). It is shown that RNAs with di‐and trimethylated caps, as well as RNAs with caps methylated at the 2′‐O‐position of the first transcribed nucleotide, can be conveniently prepared. These highly modified RNAs, with a defined and uniform sequence, are hard to access by in vitro transcription or chemical synthesis alone.
Messenger RNA may not be very abundant in the cell but its central role in gene expression is indisputable. In addition to being the template for translation it can be subject for a variety of regulatory mechanisms affecting gene expression, ranging from simple structural changes to modifications and active transport. To elucidate and potentially control the underlying changes in vitro and in cells, site-specific modification and labeling strategies are required. In this perspective, we introduce chemo-enzymatic concepts for posttranscriptional modification focusing on eukaryotic mRNAs. We describe how eukaryotic mRNA can be enzymatically modified via its 5' cap. Directions towards chemo-enzymatic mRNA labeling and visualization inside cells are given, taking into account current developments in fluorophore design. Recent achievements and future perspectives will be highlighted in the framework of an honest discussion of existing challenges.
Modified nucleotides impact all aspects of eukaryotic mRNAs and contribute to regulation of gene expression at the transcriptional and translational level. At the 5' cap, adenosine as first transcribed nucleotide is often N 6 -methyl-2'-O-methyl adenosine (m 6 A m ). This modification is tissue dependent and reversible, pointing to a regulatory function. CAPAM was recently identified as methyltransferase responsible for m 6 A m formation, however, the direct assignment of its target transcripts proves difficult. Antibodies do not discriminate between internal N 6 -methyl adenosine (m 6 A) and m 6 A m . Here we present CAPturAM, an antibody-free chemical biology approach for direct enrichment and probing of physiological CAPAM-targets. We harness CAPAM's cosubstrate promiscuity to install propargyl groups on its targets. Subsequent functionalization with an affinity handle allows for their enrichment. Using wildtype and CAPAM À /À cells, we successfully applied CAPturAM to confirm or disprove CAPAM-targets, facilitating the verification and identification of CAPAM targets.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.