Chemo-enzymatic production of base-modified ATP analogues for polyadenylation of RNA.

Base-modified adenosine-5'-triphosphate (ATP) analogues are highly sought after as building blocks for mRNAs and non-coding RNAs, for genetic code expansion or as inhibitors. Current synthetic strategies lack efficient and robust 5'-triphosphorylation of adenosine derivatives or rely on costly phosphorylation reagents. Here, we combine the efficient organic synthesis of base-modified AMP analogues with enzymatic phosphorylation by a promiscuous polyphosphate kinase 2 class III from an unclassified Erysipelotrichaceae bacterium (EbPPK2) to generate a panel of C2-, N6-, or C8-modified ATP analogues. These can be incorporated into RNA using template independent poly(A) polymerase. C2-halogenated ATP analogues were incorporated best, with incorporations of 300 to >1000 nucleotides forming hypermodified poly(A) tails.

MePMe-seq: antibody-free simultaneous m6A and m5C mapping in mRNA by metabolic propargyl labeling and sequencing.

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 N6-methyladenosine (m6A) and 5-methylcytidine (m5C) sites in mRNA with single nucleotide precision (MePMe-seq). Analysis of the termination profiles can be used to distinguish m6A from 2'-O-methyladenosine (Am) and N1-methyladenosine (m1A) 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.

Comparative S-adenosyl-L-methionine analogue generation for selective biocatalytic Friedel-Crafts alkylation.

Methyltransferases provide excellent specificity in late-stage alkylation of biomolecules. Their dependence on S-adenosyl-L-methionine (SAM) mandates efficient access to SAM analogues for biocatalytic applications. We directly compared halide methyltransferase (HMT) and methionine adenosyltransferase (MAT) to access SAM analogues and explored their utility in cascade reactions with NovO for regioselective, late-stage Friedel-Crafts alkylation of a coumarin. The HMT cascade efficiently provided SAM for methylation, while the MAT cascade also supplied high levels of SAM analogues for alkylation reactions.

Biomimetic S-Adenosylmethionine Regeneration Starting from Multiple Byproducts Enables Biocatalytic Alkylation with Radical SAM Enzymes.

S-Adenosylmethionine (SAM) is an enzyme cofactor involved in methylation, aminopropyl transfer, and radical reactions. This versatility renders SAM-dependent enzymes of great interest in biocatalysis. The usage of SAM analogues adds to this diversity. However, high cost and instability of the cofactor impedes the investigation and usage of these enzymes. While SAM regeneration protocols from the methyltransferase (MT) byproduct S-adenosylhomocysteine are available, aminopropyl transferases and radical SAM enzymes are not covered. Here, we report a set of efficient one-pot systems to supply or regenerate SAM and SAM analogues for all three enzyme classes. The systems' flexibility is showcased by the transfer of an ethyl group with a cobalamin-dependent radical SAM MT using S-adenosylethionine as a cofactor. This shows the potential of SAM (analogue) supply and regeneration for the application of diverse chemistry, as well as for mechanistic studies using cofactor analogues.

CAPturAM, a Chemo-Enzymatic Strategy for Selective Enrichment and Detection of Physiological CAPAM-Targets.

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 N6 -methyl-2'-O-methyl adenosine (m6 Am ). This modification is tissue dependent and reversible, pointing to a regulatory function. CAPAM was recently identified as methyltransferase responsible for m6 Am formation, however, the direct assignment of its target transcripts proves difficult. Antibodies do not discriminate between internal N6 -methyl adenosine (m6 A) and m6 Am . 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-/- cells, we successfully applied CAPturAM to confirm or disprove CAPAM-targets, facilitating the verification and identification of CAPAM targets.

Enzymatic Generation of Double-Modified AdoMet Analogues and Their Application in Cascade Reactions with Different Methyltransferases.

Methyltransferases (MTases) have become an important tool for site-specific alkylation and biomolecular labelling. In biocatalytic cascades with methionine adenosyltransferases (MATs), transfer of functional moieties has been realized starting from methionine analogues and ATP. However, the widespread use of S-adenosyl-l-methionine (AdoMet) and the abundance of MTases accepting sulfonium centre modifications limit selective modification in mixtures. AdoMet analogues with additional modifications at the nucleoside moiety bear potential for acceptance by specific MTases. Here, we explored the generation of double-modified AdoMets by an engineered Methanocaldococcus jannaschii MAT (PC-MjMAT), using 19 ATP analogues in combination with two methionine analogues. This substrate screening was extended to cascade reactions and to MTase competition assays. Our results show that MTase targeting selectivity can be improved by using bulky substituents at the N6 of adenine. The facile access to >10 new AdoMet analogues provides the groundwork for developing MAT-MTase cascades for orthogonal biomolecular labelling.

Sequence-specific targeting of RNA.

Post-transcriptional modifications play an important role in several processes, including translation, splicing, and RNA degradation in eukaryotic cells. To investigate the function of specific modifications it is of high interest to develop tools for sequence-specific RNA-targeting. This work focuses on two abundant modifications of eukaryotic mRNA, namely methylation of the guanine-N7 position of the 5'-cap and internal N6-methyladenosine (m6A). We describe the sequence-specific targeting of model RNA transcripts via RNA-binding proteins, such as nuclease-deficient RNA-targeting Cas9 (RCas9) and the Pumilio homology domain (PumHD) fused to two different effector enzymes, the dioxygenase FTO and the guanine-N7 methyltransferase Ecm1. With this tool, we were able to install and remove the methylation at the respective positions with high specificity.

Chemo-Enzymatic Modification of the 5' Cap To Study mRNAs.

The central dogma of molecular biology hinges on messenger RNA (mRNA), which presents a blueprint of the genetic information encoded in the DNA and serves as a template for translation into proteins. In addition to its fundamental importance in basic research, this class of biomolecules has recently become the first approved Covid vaccine, underscoring its utility in medical applications.Eukaryotic mRNA is heavily processed, including the 5' cap as the primary hallmark. This 5' cap protects mRNA from degradation by exoribonucleases but also interacts specifically with several proteins and enzymes to ensure mRNA turnover and processing, like splicing, export from the nucleus to the cytoplasm, and initiation of translation. The absence of a 5' cap leads to a strong immune response, and the methylation status contributes to distinguishing self from non-self RNA.Non-natural modifications of the 5' cap provide an avenue to label mRNAs and make them accessible to analyses, which is important to study their cellular localization, trafficking, and binding partners. They bear potential to engineer mRNAs, e.g., more stable or immunogenic mRNAs that are still translated, by impacting select interactions in a distinct manner. The modification of the 5' cap itself is powerful as it can be applied to make long mRNAs (∼1000 nt, not directly accessible by solid-phase synthesis) by in vitro transcription.This Account describes our contribution to the field of chemo-enzymatic modification of mRNA at the 5' cap. Our approach relies on RNA methyltransferases (MTases) with promiscuous activity on analogues of their natural cosubstrate S-adenosyl-L-methionine (AdoMet). We will describe how RNA MTases in combination with non-natural cosubstrates provide access to site-specific modification of different positions of the 5' cap, namely, the N2 and N7 position of guanosine and the N6 position of adenosine as the transcription start nucleotide (TSN) and exemplify strategies to make long mRNAs with modified 5' caps.We will compare the chemical and enzymatic synthesis of the AdoMet analogues used for this purpose. We could overcome previous limitations in methionine adenosyltransferase (MAT) substrate scope by engineering variants (termed PC-MATs) with the ability to convert methionine analogues with benzylic and photocaging groups at the sulfonium ion.The final part of this Account will highlight applications of the modified mRNAs. Like in many chemo-enzymatic approaches, a versatile strategy is to install small functional groups enzymatically and use them as handles in subsequent bioorthogonal reactions. We showed fluorescent labeling of mRNAs via different types of click chemistry in vitro and in cells. In a second line of applications, we used the handles to make mRNAs amenable for analyses, most notably next-generation sequencing. In the case of extremely promiscuous enzymes, the direct installation of photo-cross-linking groups was successful also and provided a way to covalently bind protein-interaction partners. Finally, the non-natural modifications of mRNAs can also modulate the properties of mRNAs. Propargylation of Am as the transcription start nucleotide at its N6 position maintained the translation of mRNAs but increased their immunogenicity. The installation of photocaging groups provides a way to revert these effects and control interactions by light.

Visible-Light Removable Photocaging Groups Accepted by MjMAT Variant: Structural Basis and Compatibility with DNA and RNA Methyltransferases.

Methylation and demethylation of DNA, RNA and proteins constitutes a major regulatory mechanism in epigenetic processes. Investigations would benefit from the ability to install photo-cleavable groups at methyltransferase target sites that block interactions with reader proteins until removed by non-damaging light in the visible spectrum. Engineered methionine adenosyltransferases (MATs) have been exploited in cascade reactions with methyltransferases (MTases) to modify biomolecules with non-natural groups, including first evidence for accepting photo-cleavable groups. We show that an engineered MAT from Methanocaldococcus jannaschii (PC-MjMAT) is 308-fold more efficient at converting ortho-nitrobenzyl-(ONB)-homocysteine than the wildtype enzyme. PC-MjMAT is active over a broad range of temperatures and compatible with MTases from mesophilic organisms. We solved the crystal structures of wildtype and PC-MjMAT in complex with AdoONB and a red-shifted derivative thereof. These structures reveal that aromatic stacking interactions within the ligands are key to accommodating the photocaging groups in PC-MjMAT. The enlargement of the binding pocket eliminates steric clashes to enable AdoMet analogue binding. Importantly, PC-MjMAT exhibits remarkable activity on methionine analogues with red-shifted ONB-derivatives enabling photo-deprotection of modified DNA by visible light.

Quantification of mRNA cap-modifications by means of LC-QqQ-MS.

Enzymatic modification of the 5'-cap is a versatile approach to modulate the properties of mRNAs. Transfer of methyl groups from S-adenosyl-l-methionine (AdoMet) or functional moieties from non-natural analogs by methyltransferases (MTases) allows for site-specific modifications at the cap. These modifications have been used to tune translation or control it in a temporal manner and even influence immunogenicity of mRNA. For quantification of the MTase-mediated cap modification, liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) provides the required sensitivity and accuracy. Here, we describe the complete workflow starting from in vitro transcription to produce mRNAs, via their enzymatic modification at the cap with natural or non-natural moieties to the quantification of these cap-modifications by LC-QqQ-MS.

Computational design and experimental characterization of a photo-controlled mRNA-cap guanine-N7 methyltransferase.

The spatial and temporal control of gene expression at the post-transcriptional level is essential in eukaryotic cells and developing multicellular organisms. In recent years optochemical and optogenetic tools have enabled the manipulation and investigation of many steps in the involved processes. However, examples for light-mediated control of eukaryotic mRNA processing and the responsible enzymes are still rare. In particular, methylation of the 5' cap of mRNA is required for ribosome assembly, and the responsible guanine-N7 methyltransferase (MTase) from E. cuniculi (Ecm1) proved suitable for activating translation. Here, we report on a photoswitchable MTase obtained by bridging the substrate-binding cleft of Ecm1 with a tetra-ortho-methoxy-azobenzene. This azobenzene derivative is characterized by efficient trans-to-cis isomerization using red light at 615 nm. Starting from a cysteine-free Ecm1 variant (ΔCys), we used a computational approach to identify suitable conjugation sites for the azobenzene moiety. We created and characterized the four best-ranked variants, each featuring two appropriately positioned cysteines close to the substrate-binding cleft. Conjugating and crosslinking the azobenzene between C149/C155 in a designed Ecm1 variant (VAR3-Az) enabled light-dependent modulation of the MTase activity and showed a 50% higher activity for the cis form than the trans-form of the azobenzene conjugated to VAR3-Az.

Chemoenzymatic labeling of RNA to enrich, detect and identify methyltransferase-target sites.

The RNA methyltransferase (MTase) complex METTL3-METTL14 transfers methyl groups from S-adenosyl-l-methionine (AdoMet) to the N6-position of adenosines within its consensus sequence, the DRACH motif (D=A, G, U; R=A, G; H=A, C, U). Interestingly, this MTase complex shows remarkable promiscuity regarding the cosubstrate. This can be exploited to install nonnatural modifications, like clickable or photocaging groups. Clickable groups are widely used for subsequent functionalization and open a broad range of possibilities for downstream applications. Here, we elaborate on click chemistry for coupling of RNA to biotin to enrich MTase targets via streptavidin-coated magnetic beads. Importantly, after clicking and coupling to beads the modification becomes sterically demanding and stalls reverse transcriptases, leading to termination adjacent to the MTase target site. Using radioactively labeled primers in the reverse transcription, the modified position can be precisely identified on a sequencing gel via phosphor imaging.

A novel 18F-labeled clickable substrate for targeted imaging of SNAP-tag expressing cells by PET in vivo.

Bioorthogonal covalent labeling with self-labeling enzymes like SNAP-tag bears a high potential for specific targeting of cells for imaging in vitro and also in vivo. To this end, fluorescent SNAP substrates have been established and used in microscopy and fluorescence imaging while radioactive substrates for the highly sensitive and whole-body positron emission tomography (PET) have been lacking. Here, we show for the first time successful and high-contrast PET imaging of subcutaneous SNAP-tag expressing tumor xenografts by bioorthogonal covalent targeting with a novel 18F-based radioligand in vivo.

Self-Assembled Cationic Polypeptide Supramolecular Nanogels for Intracellular DNA Delivery.

Supramolecular nanogels are an emerging class of polymer nanocarriers for intracellular delivery, due to their straightforward preparation, biocompatibility, and capability to spontaneously encapsulate biologically active components such as DNA. A completely biodegradable three-component cationic supramolecular nanogel was designed exploiting the multivalent host-guest interaction of cyclodextrin and adamantane attached to a polypeptide backbone. While cyclodextrin was conjugated to linear poly-L-lysine, adamantane was grafted to linear as well as star shaped poly-L-lysine. Size control of nanogels was obtained with the increase in the length of the host and guest polymer. Moreover, smaller nanogels were obtained using the star shaped polymers because of the compact nature of star polymers compared to linear polymers. Nanogels were loaded with anionic model cargoes, pyranine and carboxyfluorescein, and their enzyme responsive release was studied using protease trypsin. Confocal microscopy revealed successful transfection of mammalian HeLa cells and intracellular release of pyranine and plasmid DNA, as quantified using a luciferase assay, showing that supramolecular polypeptide nanogels have significant potential in gene therapy applications.

Multiresponsive hydrogels and organogels based on photocaged cysteine.

Here we present the readily accessible amino acid 4,5-dimethoxy-2-nitrobenzyl-l-cysteine (DNC), as an ultra-low molecular weight gelator (MW = 316 g mol-1). Sonication of DNC in water or organic solvents as well as pH adjustment in water trigger gelation. A diverse set of stimuli (UV irradiation, oxidation, heat or pH change) induce a gel-sol transition. Moreover, the photo-triggered gel-sol transition was used to obtain a controlled cysteine release from the hydrogel.

Harnessing methylation and AdoMet-utilising enzymes for selective modification in cascade reactions.

Enzyme-mediated methylation is a very important reaction in nature, yielding a wide range of modified natural products, diversifying small molecules and fine-tuning the activity of biomacromolecules. The field has attracted much attention over the recent years and interesting applications of the dedicated enzymes in biocatalysis and biomolecular labelling have emerged. In this review article, we summarise the concepts and recent advances in developing (chemo)-enzymatic cascades for selective methylation, alkylation and photocaging as tools to study biological methylation and as biotransformations to generate site-specifically alkylated products.

Chemo-Enzymatic Modification of the 5' Cap Maintains Translation and Increases Immunogenic Properties of mRNA.

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 S-adenosyl-l-methionine (AdoMet)-dependent methyltransferases (MTases). We report on the cosubstrate scope of the MTase CAPAM responsible for methylation at the N6 -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 ≈3-fold 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.

Engineered SAM Synthetases for Enzymatic Generation of AdoMet Analogs with Photocaging Groups and Reversible DNA Modification in Cascade Reactions.

Methylation and demethylation of DNA, RNA and proteins has emerged as a major regulatory mechanism. Studying the function of these modifications would benefit from tools for their site-specific inhibition and timed removal. S-Adenosyl-L-methionine (AdoMet) analogs in combination with methyltransferases (MTases) have proven useful to map or block and release MTase target sites, however their enzymatic generation has been limited to aliphatic groups at the sulfur atom. We engineered a SAM synthetase from Cryptosporidium hominis (PC-ChMAT) for efficient generation of AdoMet analogs with photocaging groups that are not accepted by any WT MAT reported to date. The crystal structure of PC-ChMAT at 1.87 Šrevealed how the photocaged AdoMet analog is accommodated and guided engineering of a thermostable MAT from Methanocaldococcus jannaschii. PC-MATs were compatible with DNA- and RNA-MTases, enabling sequence-specific modification ("writing") of plasmid DNA and light-triggered removal ("erasing").

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.

mRNA Therapies: New Hope in the Fight against Melanoma.

Melanoma is a life-threatening disease caused by mutations in pigment-producing cells. Numerous treatments for melanoma have been approved in the past several decades; however, they often cause severe side effects and in most cases do not result in a complete cure. mRNA (messenger RNA) as a therapeutic agent provides a new avenue for melanoma treatment and several advantages over conventional treatments. The first mRNA drugs for melanoma treatment are currently in clinical trials, and approval of mRNA drugs by the Food and Drug Administration seems to be within reach. This new class of drugs can be readily adapted to other diseases, raising the hope of providing a new therapeutic option for various diseases.