Applications of Phosphate Modification and Labeling to Study (m)RNA Caps

Abstract

The cap is a natural modification present at the 5 0 ends of eukaryotic messenger RNA (mRNA), which because of its unique structural features, mediates essential biological functions during the process of gene expression. The core structural feature of the mRNA cap is an N7-methylguanosine moiety linked by a 5 0 -5 0 triphosphate chain to the first transcribed nucleotide. Interestingly, other RNA 5 0 end modifications structurally and functionally resembling the m 7 G cap have been discovered in different RNA types and in different organisms. All these structures contain the 'inverted' 5 0 -5 0 oligophosphate bridge, which is necessary for interaction with specific proteins and also serves as a cleavage site for phosphohydrolases regulating RNA turnover. Therefore, cap analogs containing oligophosphate chain modifications or carrying spectroscopic labels attached to phosphate moieties serve as attractive molecular tools for studies on RNA metabolism and modification of natural RNA properties. Here, we review chemical, enzymatic, and chemoenzymatic approaches that enable preparation of modified cap structures and RNAs carrying such structures, with emphasis on phosphate-modified mRNA cap analogs and their potential applications.M. Warminski and P. J. Sikorski have contributed equally to this work.

A brief review of mRNA therapeutics and delivery for bone tissue engineering

“…These modifications include O-to-NH imidodiphosphate, O-to-BH 3 boranophosphate, O-to-S phosphorothioate and non-bridging oxygen. 25 …”

Nanomedicine-Based Approaches for mRNA Delivery

“…Chemical modification of the mRNA structure is an effective way of promoting mRNAs that are more resistant to enzymatic degradation than their native counterparts, as well as mRNAs with decreased immunogenicity. Major strategies have focused on the introduction of an m7G 5′-Cap structure, , modifications through poly­(A) tails, , incorporation of untranslated regions (UTRs), , and insertion of modified nucleotides to the structure of mRNA (Figure ). In the following sections, we summarize the advantages provided by these features.…”

“…In cotranscriptional capping, chemical cap analogues are incorporated during transcription. This allows optimization of chemical analogues, a work well covered by Jemielity’s group, as reviewed elsewhere . During transcription, the m7G cap analogue can be incorporated either in the correct orientation with the methylated guanosine in 5′ (m 7 GpppGpN) or the inverted orientation (Gpppm 7 GpN), with a sharp decrease in translation efficiency of mRNA with an inverted cap…”

Solid‐Phase Synthesis of RNA 5′‐Azides and Their Application for Labeling, Ligation, and Cyclization Via Click Chemistry

“…We envisage that RNA CuAAC cyclization reactions might be of particular interest to a biological chemist, since such a class of naturally occurring RNAs is still not well characterized, mainly due to limited access to model compounds by classical chemical synthesis methods (Maria, Di Fabio, & Anna, 2012; Micura, 1999; Smietana & Kool, 2002). Another interesting class of RNA conjugates accessible via CuAAC with oligoribonucleotides 5′‐azides are mRNA 5′‐end fragments, containing 7‐methylguanosine cap structure (Warminski, Kowalska, & Jemielity, 2017; Warminski, Sikorski, Kowalska, & Jemielity, 2017).…”