Glen Report 37-11: New Product- N3-Methyl-C-CE Phosphoramidite

N3-Methyl-C (m³C) is a naturally occurring ribonucleoside that is mostly found in tRNA but has recently been described in mRNA as well.¹ In tRNA, m³C is often located in the anticodon loop (position 32) where its absence can affect translation. In humans, four methyltransferases have thus far been identified for m³C. Methyltransferase-like 2A (METTL2A), METTL2B and METTL6 methylate tRNA while METTL8 methylates mRNA. In addition, human alkylation B (AlkB) proteins are possible demethylases for m³C, suggesting that m³C may be a dynamic modification. Under physiological conditions, m³C is positively charged (Figure 1). The methylation is on the base pairing face, and as such, base pairing is significantly disrupted. For a single substitution of m³C, melting temperatures can be reduced by as much as 20 °C and mismatch specificity can be lowered by 10-16 °C.²

Figure 1. m³C. Left, nucleoside; right, phosphoramidite.

While m³C was identified over 60 years ago, many questions remain regarding its function, and research on this modification continues to grow.¹ To facilitate these efforts, Glen Research is introducing the phosphoramidite of m³C (Figure 1). This phosphoramidite employs acetyl protection on the nucleobase and TBDMS protection on the 2′-OH and can be coupled using standard RNA coupling times. Of note, the recommended diluent is anhydrous acetonitrile/dichloromethane 1:1 by volume. For deprotection, UltraMild and standard deprotection conditions can be used. As reported earlier, even with acetyl protection, AMA deprotection is not compatible.³ Under such conditions, we also observed predominantly N4-methylated product (m^3,4C). As such, all deprotection options containing methylamine should be avoided.

References

1. K.E. Bohnsack, N. Kleiber, N. Lemus-Diaz, and M.T. Bohnsack, Trends Biochem Sci, 2022, 47, 596-608.
2. S. Mao, et al., ACS Chem Biol, 2021, 16, 76-85.
3. S. Moreno, L. Flemmich, and R. Micura, Monatsh Chem, 2022, 153, 285-291.

Product Information

N3-Methyl-C-CE Phosphoramidite (10-3013-xx)