A Benzophenone-Based Photocaging Strategy for the N7 Position of Guanosine.

Selective modification of nucleobases with photolabile caging groups enables the study and control of processes and interactions of nucleic acids. Numerous positions on nucleobases have been targeted, but all involve formal substitution of a hydrogen atom with a photocaging group. Nature, however, also uses ring-nitrogen methylation, such as m7 G and m1 A, to change the electronic structure and properties of RNA and control biomolecular interactions essential for translation and turnover. We report that aryl ketones such as benzophenone and α-hydroxyalkyl ketone are photolabile caging groups if installed at the N7 position of guanosine or the N1 position of adenosine. Common photocaging groups derived from the ortho-nitrobenzyl moiety were not suitable. Both chemical and enzymatic methods for site-specific modification of N7G in nucleosides, dinucleotides, and RNA were developed, thereby opening the door to studying the molecular interactions of m7 G and m1 A with spatiotemporal control.

New AdoMet Analogues as Tools for Enzymatic Transfer of Photo-Cross-Linkers and Capturing RNA-Protein Interactions.

Elucidation of biomolecular interactions is of utmost importance in biochemistry. Photo-cross-linking offers the possibility to precisely determine RNA-protein interactions. However, despite the inherent specificity of enzymes, approaches for site-specific introduction of photo-cross-linking moieties into nucleic acids are scarce. Methyltransferases in combination with synthetic analogues of their natural cosubstrate S-adenosyl-l-methionine (AdoMet) allow for the post-synthetic site-specific modification of biomolecules. We report on three novel AdoMet analogues bearing the most widespread photo-cross-linking moieties (aryl azide, diazirine, and benzophenone). We show that these photo-cross-linkers can be enzymatically transferred to the methyltransferase target, that is, the mRNA cap, with high efficiency. Photo-cross-linking of the resulting modified mRNAs with the cap interacting protein eIF4E was successful with aryl azide and diazirine but not benzophenone, reflecting the affinity of the modified 5' caps.

Dual 5' Cap Labeling Based on Regioselective RNA Methyltransferases and Bioorthogonal Reactions.

The ability to detect and localize defined RNA strands inside living cells requires probes with high specificity, sensitivity, and signal-to-background ratio. To track low-abundant biomolecules, such as strands of regular mRNA, and distinguish fluorescence signal from the background after bioorthogonal reactions in cells, it is imperative to employ turn-on concepts. Here, we have presented a straightforward enzymatic approach to allow site-specific modification of two different positions on the 5' cap of eukaryotic mRNA with either identical or different small functional groups. The approach relies on two methyltransferases and analogues of their natural co-substrate, and it can be extended to a three-enzyme cascade reaction for their in situ production. Subsequent labeling by using bioorthogonal click reactions provided access to double labeling with identical fluorophores or dual labeling with two different reporter groups, as exemplified by a Cy5 dye, a FRET pair, and a fluorophore/biotin combination. Our dual-labeling strategy addresses the need for increased sensitivity and should improve the signal-to-background ratio after bioorthogonal reactions in cells.

Chemo-enzymatic modification of eukaryotic mRNA.

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.

One-pot modification of 5'-capped RNA based on methionine analogs.

This paper outlines chemically and enzymatically synthesized S-adenosylmethionine (AdoMet) analogs and their use in the site-specific modification of RNA by methyltransferases, enabling the facile attachment of clickable moieties to the nucleic acid. We then focus on methodological aspects of setting up a methyltransferase-based enzymatic cascade reaction starting from methionine analogs. This strategy is applied to the one-pot modification of the mRNA cap which is subsequently derivatized in copper-free and copper-catalyzed click reactions. We show that high transfer efficiencies to the cap are obtained using Se-propargyl-, hexenynyl- and azido-bearing methionine analogs. By switching to other methyltransferases our one-pot modification approach should be directly applicable to the regiospecific modification of other target molecules including nucleic acids, proteins and small molecules.

Synthetic mRNA capping.

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.

A Biocatalytic Cascade for Versatile One-Pot Modification of mRNA Starting from Methionine Analogues.

Methyltransferases have proven useful to install functional groups site-specifically in different classes of biomolecules when analogues of their cosubstrate S-adenosyl-l-methionine (AdoMet) are available. Methyltransferases have been used to address different classes of RNA molecules selectively and site-specifically, which is indispensable for biophysical and mechanistic studies as well as labeling in the complex cellular environment. However, the AdoMet analogues are not cell-permeable, thus preventing implementation of this strategy in cells. We present a two-step enzymatic cascade for site-specific mRNA modification starting from stable methionine analogues. Our approach combines the enzymatic synthesis of AdoMet with modification of the 5' cap by a specific RNA methyltransferase in one pot. We demonstrate that a substrate panel including alkene, alkyne, and azido functionalities can be used and further derivatized in different types of click reactions.

Phosphine-free Stille-Migita chemistry for the mild and orthogonal modification of DNA and RNA.

An optimized catalyst system of [Pd2 (dba)3 ] and AsPh3 efficiently catalyzes the Stille reaction between a diverse set of functionalized stannanes and halogenated mono-, di- and oligonucleotides. The methodology allows for the facile conjugation of short and long nucleic acid molecules with moieties that are not compatible with conventional chemical or enzymatic synthesis, among them acid-, base-, or fluoride-labile protecting groups, fluorogenic and synthetically challenging moieties with good to near-quantitative yields. Notably, even azides can be directly introduced into oligonucleotides and (deoxy)nucleoside triphosphates, thereby giving direct access to "clickable" nucleic acids.

Nucleoside-modified AdoMet analogues for differential methyltransferase targeting.

Methyltransferases (MTases) modify a wide range of biomolecules using S-adenosyl-l-methionine (AdoMet) as the cosubstrate. Synthetic AdoMet analogues are powerful tools to site-specifically introduce a variety of functional groups and exhibit potential to be converted only by distinct MTases. Extending the size of the substituent at the sulfur/selenium atom provides selectivity among MTases but is insufficient to discriminate between promiscuous MTases. We present a panel of AdoMet analogues differing in the nucleoside moiety (NM-AdoMets). These NM-AdoMets were efficiently produced by a previously uncharacterized methionine adenosyltransferase (MAT) from methionine and ATP analogues, such as ITP and N6-propargyl-ATP. The N6-modification changed the relative activity of three representative MTases up to 13-fold resulting in discrimination of substrates for the methyl transfer and could also be combined with transfer of allyl and propargyl groups.

Enzymatic Transfer of Photo-Cross-Linkers for RNA-Protein Photo-Cross-Linking at the mRNA 5'-Cap.

Photo-cross-linking moieties have proven invaluable for elucidating interactions of biomolecules. While methods for site-specific incorporation of those moieties into proteins have been developed, comparable methods for nucleic acids are lacking. Utilizing the inherent specificity of enzymes, methyltransferases (MTase) exhibiting relaxed cosubstrate specificity in combination with synthetic analogs of S-adenosyl-L-methionine (AdoMet) allow for the precise installation of reporter molecules or affinity tags in various biomolecules. In this chapter, we describe AdoMet analogs with photo-cross-linking moieties that-in combination with an MTase-are ideal for site-specific installation. The workflow for chemo-enzymatic installation of photo-cross-linking moieties at the mRNA cap based on AdoMet analogs is given in detail.

Reversible modification of DNA by methyltransferase-catalyzed transfer and light-triggered removal of photo-caging groups.

Methyltransferases are powerful tools for site-specific transfer of non-natural functional groups from synthetic analogs of their cosubstrate S-adenosyl-l-methionine (AdoMet). We present a new class of AdoMet analogs containing photo-caging (PC) groups in their side chain, enzymatic transfer of PC groups by a promiscuous DNA MTase as well as light-triggered removal from the target DNA. This strategy provides a new avenue to reversibly modulate the functionality of DNA at MTase target sites.