Chemo‐enzymatic treatment of RNA to facilitate analyses

Abstract

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.

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.…”

Repurposing Antiviral Drugs for Orthogonal RNA‐Catalyzed Labeling of RNA

“…Covalent labeling with small organic fluorophores through bioorthogonal linkages has several advantages. While incorporation of modified nucleotides by solid‐phase synthesis is limited to short RNAs, several strategies are known to post‐synthetically address natural RNAs at terminal or internal positions . Selected examples include ribozymes for 3′‐terminal labeling, protein enzymes for labeling of the cap structure, and RNA–protein complexes to address specific nucleotides in a defined sequence context…”

Bioorthogonal mRNA labeling at the poly(A) tail for imaging localization and dynamics in live zebrafish embryos

“…Alternatively, by adding a suitable genetically encoded tag in the UTR, RNAs can be labeled in cells using RNA-modifying enzymes recognizing this structural element. [55][56][57][58][59][60][61] We showed previously that the hallmarks of eukaryotic mRNAnamely the 5 0 -cap and the poly(A) tailcan be site-specically labeled using chemo-enzymatic approaches. [62][63][64][65] Both labeling approaches rely on small uorescent dyes and do not alter the coding sequence or the UTR, and therefore do not interfere with coding or regulatory elements of the mRNA.…”