The mRNACalc web server 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 over-stimulation of cytoplasmic RNA sensors. Despite this modification, mRNA concentrations remain mostly determined through 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 a mRNA sequence dependent manner. We therefore examined the RNA quantification methods and developed the mRNACalc web server. It accounts for the molar absorption coefficient of modified nucleotides at 260 nm wavelength, the RNA composition of the mRNA, and the A₂₆₀of the mRNA sample to enable accurate quantification of nucleoside-modified mRNAs. The webserver is freely available at<ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="https://www.mrnacalc.com">https://www.mrnacalc.com</ext-link>.