The mRNACalc webserver accounts for the N1-methylpseudouridine hypochromicity to enable precise nucleoside-modified mRNA quantification

Esteban Finol; Sarah E Krul; Sean J Hoehn; Xudong Lyu; Carlos E Crespo-Hernández

doi:10.1016/j.omtn.2024.102171

The mRNACalc webserver accounts for the N1-methylpseudouridine hypochromicity to enable precise nucleoside-modified mRNA quantification

Mol Ther Nucleic Acids. 2024 Mar 11;35(2):102171. doi: 10.1016/j.omtn.2024.102171. eCollection 2024 Jun 11.

Authors

Esteban Finol¹, Sarah E Krul², Sean J Hoehn², Xudong Lyu¹, Carlos E Crespo-Hernández²

Affiliations

¹ Programme in Emerging Infectious Diseases, Duke-NUS Medical School, National University of Singapore, Singapore 169857, Singapore.
² Department of Chemistry, Case Western Reserve University, Cleveland, OH 44106, USA.

Abstract

Nucleoside-modified messenger RNA (mRNA) technologies necessarily incorporate N1-methylpseudouridine into the mRNA molecules to prevent the over-stimulation of cytoplasmic RNA sensors. Despite this modification, mRNA concentrations remain mostly determined through the measurement of UV absorbance at 260 nm wavelength (A₂₆₀). Herein, we report that the N1-methylpseudouridine absorbs approximately 40% less UV light at 260 nm than uridine, and its incorporation into mRNAs leads to the under-estimation of nucleoside-modified mRNA concentrations, with 5%-15% error, in an mRNA-sequence-dependent manner. We therefore examined the RNA quantification methods and developed the mRNACalc webserver. It accounts for the molar absorption coefficient of modified nucleotides at 260 nm wavelength, the RNA composition of the mRNA, and the A₂₆₀ of the mRNA sample to enable accurate quantification of nucleoside-modified mRNAs.

Keywords: MT: Bioinformatics; N1-methylpseudouridine; UV absorption; mRNA; modified-nucleoside; pseudouridine.