The mRNACalc webserver accounts for the hypochromicity of modified nucleosides and enables the accurate quantification of nucleoside-modified mRNA.

Nucleoside-modified 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 (A). 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 A of the mRNA sample to enable accurate quantification of nucleoside-modified mRNAs. The webserver is freely available at .