The mRNA cap is a highly methylated 5′-modification that enhances the efficiency of mRNA translation. In nature, the mRNA cap initiates translation into proteins, protects mRNA from degradation, recruits protein complexes for processing mechanisms, and marks cellular mRNA to avoid immune system responses.1 Multiple cap structures exist based on the amount of methylation present (Figure 1). Cap 0 contains an N7-G cap connected by a 5′-5′-triphosphate bridge and initiates translation and protects RNA from degradation. Additional methylation at the 2′-position of the first transcribed nucleotide yields Cap 1. Cap 2 is present when the second transcribed nucleotide is methylated at the 2′- hydroxyl group. We would like to thank Dr. Chunping Xu and the TriLink team for their work and overview on mRNA capping reagents and their applications in mRNA studies.2
Figure 1. mRNA 5′-cap structures. In this rendering, the first transcribed nucleoside (adenosine) is further methylated to show m6Am.
To further support this research, we are introducing a 2′-OMe-N6-Me-A-CE (m6Am) phosphoramidite. This modification is crucial to the development of novel cap structures bearing the m6AmN dimer (in the case of CleanCap M6, N = G). In addition to its importance in the mRNA cap, m6Am has also been implicated in gene regulation.3 Several sequencing methods have been developed to detect and quantify m6Am levels. Results of m6Am mapping experiments have led to somewhat controversial findings. For example, the presence of m6Am enhances or reduces mRNA stability in certain genes. It’s likely that the effect of m6Am on stability depends on outside factors, such as tissue. In addition, internal m6Am modification was found to negatively impact splicing regulation. In a recent study, it was shown that downregulation of an m6Am writer, PCIF1, promoted cancer cell proliferation. In contrast, overexpression of the methyltransferase responsible for m6Am induces apoptosis.4
In our hands, the m6Am phosphoramidite is easy to use in oligonucleotide synthesis and deprotection. For coupling, a 6-minute coupling time was required using standard tetrazole activator. This monomer is protected with phenoxyacetyl (Pac) to minimize potential branching. If combined with other UltraMild phosphoramidites, use UltraMild Cap Mix A (40-4210-xx/40-4212-xx) to avoid exchange of the iPr-Pac group on G with acetyl. The m6Am can be deprotected using UltraMild conditions (0.05M Potassium Carbonate in Methanol, 4 h, RT or 30% Ammonium Hydroxide, 2 h, RT). This modification is also compatible with standard deprotection in AMA.
2′-OMe-N6-Me-A-CE Phosphoramidite
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