Chemo-enzymatic modification of eukaryotic mRNA

Abstract

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.

Covalent labeling of nucleic acids

“…Furthermore, we would like to point out recent reviews about post-synthetic labeling of DNA and RNA. 29,33,[59][60][61][62][63] 2.1 Direct chemical labeling via solid-phase synthesis Solid-phase synthesis is the method of choice to make labeled RNA and DNA of up to 150-200 nucleotides. 64 This approach uses nucleoside phosphoramidites as key building blocks and is compatible with almost any natural or non-natural modification as long as undesired side reactions during synthesis are avoided and reactive hydroxyl and amino groups can be protected.…”

The multifaceted eukaryotic cap structure

“…A variant of Giardia lamblia trimethylguanosine synthase 2 (GlaTgs2) methylates the N2 position of the cap but is somewhat promiscuous in its substrate specificity. GlaTgs2 has been used to transfer alkyne, azido, and 4‐vinylbenzene groups to the N2 guanine position (Holstein, Stummer, & Rentmeister, 2015; Muttach, Muthmann, & Rentmeister, 2017; D. Schulz, Holstein, & Rentmeister, 2013). The derivatized guanines can then be further modified by biorthogonal reactions to append specific functional groups.…”

Chemo‐enzymatic treatment of RNA to facilitate analyses

“…It will be interesting to see whether the metabolic labeling with N 6 ‐allyladenosine can be combined with the chemical reaction and how this can be used to learn about the MTase target sites. An alternative way would be to start metabolic labeling from the AdoMet precursors, that is, methionine analogs as outlined below (Hartstock & Rentmeister, ; Muttach, Muthmann, & Rentmeister, ; Muttach & Rentmeister, , ).…”