A Benzophenone‐Based Photocaging Strategy for the N7 Position of Guanosine

Abstract

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 m7G and m1A, 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 m7G and m1A with spatiotemporal control.

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

“…To expand the substrate scope of ChMAT towards benzylic methionine analogs, we substituted the amino acids I122, V126 and I330 (that constitute the hydrophobic binding pocket) as well as Q121 (that is part of the gating loop) by less sterically demanding residues, generating eight ChMAT variants: I122A, I122V, I122G, V126G, V126A, I330A, I330V and Q121A. These variants were tested in an enzymatic cascade reaction with Ecm1, a highly promiscuous guanine N7 methyltransferase from Encephalitozoon cuniculi that efficiently converts benzylic AdoMet analogs [6c, 7a, 14] (Figure 2).…”

Conditionally Activated (“Caged”) Oligonucleotides

“…Less commonly, Anhäuser et al introduced caging groups to purine imine positions. Aryl ketones such as benzophenone and α-hydroxyalkyl ketone were chemically or enzymatically installed at the N 7 position of guanosine or the N 1 position of adenosine, which successfully blocked the binding of translation initiation factor eIF4E [ 16 ]. Zhang et al applied tRNA guanine transglycosylase (TGT) to introduce a bulky pre-queuosine1 (preQ1) derivative, biotin-Bac-PreQ1, which suppressed translation of mRNA [ 17 ].…”

Maßgeschneiderte SAM‐Synthetasen zur enzymatischen Herstellung von AdoMet‐Analoga mit Photoschutzgruppen und zur reversiblen DNA‐Modifizierung in Kaskadenreaktionen

“…Um das Substratspektrum der ChMAT in Richtung benzylischer Methionin‐Analoga zu erweitern, haben wir die Aminosäuren I122, V126 und I330 (welche die hydrophobe Bindungstasche bilden) sowie Q121 (Teil des “Gating‐Loop”) durch sterisch weniger anspruchsvolle Reste ersetzt, was zu acht ChMAT‐Varianten führte: I122A, I122V, 122G, V126G, V126A, V126A, I330A, I330V und Q121A. Diese Varianten wurden in einer enzymatischen Kaskadenreaktion mit Ecm1 getestet, einer hoch promiskuitiven Guanin‐ N7 ‐Methyltransferase aus Encephalitozoon cuniculi , die benzylische AdoMet‐Analoga effizient umwandelt [6c, 7a, 14] . (Abbildung 2).…”