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 A260 of the mRNA sample to enable accurate quantification of nucleoside-modified mRNAs. The webserver is freely available at https://www.mrnacalc.com. Graphical Abstract


Introduction:
The therapeutic use of messenger RNA (mRNA) has sparked great optimism in the development of novel vaccines and therapeutics against a myriad of infectious or yet incurable diseases (1).The mRNA technology enables the production of antigenic, functional, and/or therapeutic proteins by introducing mRNA into the human body and cells (2).Since mRNAs act in the cytoplasm transiently, they do not bear any risk of integration into the host cell genome.Most importantly, the mRNA technology enables rapid, cost-efficient and scalable production, which is free of cellular (cell cultures) or animal materials (3).Thus, mRNA technologies facilitate manufacturing and allow for a rapid response to emerging infectious diseases, as emphatically underscored by the rapid rollout of COVID-19 mRNA vaccines in many parts of the world.Modified nucleosides, such as pseudouridine (Ψ), N 1 -methylpseudouridine (m 1 Ψ) and 5methylcytidine (m 5 C), are often incorporated into the mRNA molecules.Such modifications reduce stimulation of cytoplasmic RNA sensors, such as toll-like receptor 3 and 7, for improved safety profiles and enhanced mRNA translation (4,5).
However, how modified nucleosides affect mRNA concentration measurements and potentially confound pre-clinical dosing, efficacy, and toxicology studies, which could make or break further clinical development of any therapeutic, remains undefined.
The determination of RNA concentration often relies on measurements of its UV absorbance at 260 nm wavelength (A260) and the implementation of the Beer-Lambert law (6).The accuracy of these measurements is scattered by the variable hypochromicity of RNA due to its sequence-dependent folding.The molar absorption coefficient (MAC or extinction coefficient, ε) of a folded RNA at 260 nm (ε260) is reduced as compared to its unfolded state (7).This difference is buffer-and concentrationdependent and arises from changes in the chemical environment of the nucleobases -the main chromophore, due to base-pairing, stacking, intermolecular interactions, and other conformational changes.Considering these variabilities, a rough estimation for the ε260 of any single stranded RNA (40 μg/ml per absorbance unit) is extensively used and its associated ±10-20 % error in the estimation of RNA concentration is widely accepted (6).This error range may suffice to assess dose-response for mRNA therapeutics across several orders of magnitude in celula or in vivo experiments.Yet it would be valuable to know concentrations at higher accuracy for the development of mRNA technologies.Our particular concern is in measurements of self-amplifying RNAs (saRNA) and nucleoside-modified mRNAs.The logarithmic amplification of saRNA can convert a 20 % accepted error in RNA concentration into a several fold differences in dose-response between one experiment and the subsequent replicates.
The chemical modifications on the nucleobases of mRNA can also induce profound changes in the mRNA MAC, hindering the accurate quantification of nucleosidemodified mRNA concentrations.
To attain greater accuracy in RNA quantification, RNA molecules are hydrolysed prior to UV absorbance determination using a combination of thermal and alkaline hydrolysis (6,8).The RNA hydrolysis shifts the hypochromic folded state of the RNA to the hyperchromic state of the single monophosphate nucleotides (9).Since the precise MAC of the four standard nucleotides in aqueous buffered solution are known, the molar absorption of any hydrolysed mRNA can be calculated as the sum of the molar absorption of its nucleotide compositions.Thus, upon the determination of the 260 nm UV absorbance (A260), the RNA concentration can be quantified with an error of ~ 4 % using these methods (6).The incorporation of modified nucleosides can alter the RNA molar absorption and increase the error of the measurements in an RNA sequence-dependent manner.Other non-UV-spectroscopic methods relying on the unspecific RNA binding of fluorophores (such as RiboGreen, Thermo Fisher Scientific) for the determination of RNA concentration may help to overcome any change in the MAC of modified nucleoside mRNA.However, the impact of RNA modifications on the binding affinity of these fluorophores also remains unknown.
Herein, we report our effort to revisit and determine the molar absorption coefficients of modified nucleosides (Ψ, m 1 Ψ and m 5 C).We also examined three different methods for RNA hydrolysis and provided them along with the mRNACalc web server.This web tool incorporates the most recently revised ε260 for standard, modified and mRNA capping nucleosides, allowing the accurate determination of standard and nucleosidemodified mRNAs using UV spectroscopy.Once the RNA sequence, the A260 and the RNA stock volume values are provided as input, the mRNACalc web server calculates the RNA stock concentration in nM and ng/μl and the total RNA mass in μmole and μg.

Results and discussion:
To assess the impact of chemical modifications on the spectrophotometric parameters of nucleosides for mRNA quantification, we examined the modified nucleosides that have recently been employed in nucleoside-modified mRNA technologies: pseudouridine (Ψ), N 1 -methyl-pseudouridine (m 1 Ψ), and 5-methylcytidine (m 5 C).
Pseudouridine is an isomer of uridine -the standard nucleoside in RNA.
Pseudouridine, as opposed to other nucleosides, is a carbon-carbon ribofuranosyl nucleoside, i.e., the uracil nucleobase is linked to the ribose through its fifth carbon, instead of a N 1 -linkage (10).This unique arrangement places the N 1 -imino group toward the so-called "C-H" edge of the pyrimidine ring and confers additional properties to this edge in pseudouridine.This imino hydrogen proton is susceptible to hydrogen bonding, chemical exchange, and chemical modifications such as N 1methylation.Thus, the N 1 -methyl-pseudouridine, as well as the m 5 C, represents a modification of the C-H edge of the pyrimidine nucleobase.A similar 5-methyl modification also differentiates uridine from thymidine.The influence of a 5-methyl substituent on the UV molar absorption of pyrimidine rings is well known since the 1940's when Sister Miriam Michael Stimson showed that it provokes a subtle reduction in molar absorbance and a shift of the peak maximum (εmax and λmax, respectively) to a longer wavelength (lower energy) -the so-called bathochromic or red shift (11)(12)(13)(14).
This red shift also leads to a substantial ε260 reduction for the methylated pyrimidine nucleosides.For the uridine to thymidine and cytidine to 5-methylcytidine comparisons, the respective peak shifts (∆λmax) are + 5 and + 7 nm with a 11.4 % and 20.8 % reduction in their ε260 (Fig. 1a and 1b).For the Ψ and m 1 Ψ curves, we have observed a similar bathochromic shift (∆λmax = + 9 nm, Fig. 1c), leading to a reduced molar absorption at 260 nm for m 1 Ψ (∆ε260 = -22.8%).More importantly, m 1 Ψ is hypochromic as compared to uridine at λmax (∆εmax = -21 %) and, due to the bathochromic shift, m 1 Ψ absorbs 39.8 % less than uridine at 260 nm (Fig. 1d).Thus, the substitution of uridine by m 1 Ψ in mRNA technologies can lead to substantial changes in the spectrophotometric properties of the mRNA and may lead to the underestimation of nucleoside-modified mRNA concentrations.) mRNA, three mRNAs were transcribed using either U, Ψ, or m 1 Ψ.These mRNA also encoded a double-Broccoli aptamer in their 3' untranslated region.Once the DFHBI-1T fluorophore was bound to the G-quadruplex in the Broccoli aptamer, the mRNA emitted green light upon excitation (15).The brightness, melting point and affinity of the DFHBI-1T-Broccoli complex were not significantly perturbed by the U-to-Ψ or Uto-m 1 Ψ substitutions (Supplementary Table 1 and Supplementary Figure 1).By normalizing the UV absorbance (A260) of each mRNA by its corresponding fluorescence (F507), it was observed that in practice the relative UV absorbance of the nucleoside-modified mRNA was significantly reduced as compared to the standard mRNA (∆Α260 = -10.6 %, Fig. 1e and 1f).The hypochromicity was slightly more pronounced in a second m 1 Ψ-mRNA with higher m 1 Ψ composition (∆Α260 = -11.8%,Supplementary figure 2).In principle, the modified nucleosides may also promote mRNA folding and reduce its UV absorption.This is particularly relevant for the pseudouridine modification.Its N1-hydrogen can engage in additional hydrogen bonds, promoting and stabilizing RNA folding.For instance, the U-to-Ψ substitution in tRNA stabilize the folded structure that is essential for translation (reviewed in ref. 16).
However, the m 1 Ψ nucleobase lacks this additional hydrogen bonding capability, and it is expected to have little or no effect on the RNA folding of low CG-content (< 45 %) and less-structured RNA molecules, such as our mRNAs.Considering that both Ψand m 1 Ψ-mRNAs followed the anticipated hypochromicity associated to the nucleoside hypochromicity at 260 nm wavelength, instead of their expected distinct contribution to RNA folding, these data suggest that the observed reduction in nucleoside-modified mRNA UV absorption is mainly determined by the nucleobase composition and the intrinsic MAC of the nucleosides in these mRNA.Importantly, the UV absorption spectrum of the m 1 Ψ mRNA also depicted a broad absorption peak and a red shift, which brings additional implications for the assessment of the RNA sample purity (Supplementary note).These findings indicate that, for accurate determination of nucleoside-modified mRNA concentrations and proper interpretation of doseranging preclinical studies, the reported UV spectroscopic changes must be accounted for to prevent under-estimating mRNA concentrations by 5 to 15%, depending on the proportion of m 1 Ψ in the mRNA composition.
To enable accurate measurement of nucleoside-modified mRNA, we also assessed different RNA hydrolysis and built an experimental workflow for the determination of mRNA concentrations.The modern analytical use of alkaline hydrolysis of RNA is known since 1922, when Steudel and Peiser demonstrated that 1 M NaOH hydrolysed yeast RNA, whereas thymus DNA resisted the NaOH hydrolysis (17).The alkalipromoted transesterification of RNA occurs due to the nucleophilic attack of the 2'-OH in the ribose to the 3',5'-phosphodiester bond, explaining the alkali-resistance of the 2'-deoxyribonucleotides (Fig. 2a) (18).This reaction is further catalysed with the introduction of heat.However, the combination of thermal and alkaline hydrolysis, e.g., 1 M NaOH at 95 °C, also catalyses the deamination of cytosine to uridine in a small percentage of residues (19,20).Thus, a compromise between the two methods is often applied.In our hands, three of such protocols showed a similar change in A260 upon hydrolysis of yeast RNA -a historical standard sample for these methods.(Fig. 2b).These protocols are provided in the mRNACalc web server along with a workflow that implement a linear regression model from multiple measurements at serial dilutions to reduce the impact of sample handling variation (Fig. 2c).
In the mRNACalc web server, the MACs of distinct modified nucleosides that form the capping nucleotide in the 5' mRNA cap were also implemented for the sake of completeness.The capping nucleotide only represents one nucleotide out of thousands of nucleotides in a mRNA molecule and its contribution to the mRNA molar absorption is rather negligible.
Taken together, by incorporating modified nucleoside MAC parameters, accounting for the actual mRNA nucleotide composition, and providing an experimental workflow, the mRNACalc web server represents a freely available and all-inclusive tool for the determination of nucleoside-modified mRNA concentrations using UV spectroscopy.a, Alkali-promoted transesterification allows RNA hydrolysis and mRNA quantification.Under alkaline conditions, the reactive -OH triggers the nucleophilic attack of the 2'-OH on the 3',5'phosphodiester linkage, converting the ground-state configuration of RNA into a pentacoordinated intermediate and leading to a 2'3'-cyclic phosphodiester.This cyclic form is then known to form 3' and 2' monophosphate nucleotides (not shown).b, Thermal and/or alkaline hydrolysis of RNA over time.Yeast RNA was hydrolysed using three different previously described methods and the ∆A260 was determined using an UV spectrophotometer at different intervals.For expedite RNA hydrolysis (1-or 2-hours incubation), a combination of thermal and alkaline hydrolysis can be used (dark blue dots, 0.8 M NaOH at 37 °C; red dots, 0.5 M Na2CO3 pH 8 at 95 °C).For overnight incubation, alkaline hydrolysis suffices (light blue dots, 0.8 M NaOH at 20 °C, the last four measurements were performed after an overnight incubation).Dots indicate the mean value of three measurements.Error bars correspond to standard deviations.c, Experimental workflow for the determination of RNA concentration using the mRNACalc web server.The coloured dots refer to the different RNA hydrolysis methods in 2.b.

Fig. 1 :
Fig. 1: The nucleobase methylation and its bathochromic effect on the UV molar absorption spectra of pyrimidines and nucleoside-modified mRNAs.

Fig. 2 :
Fig. 2: The mRNACalc webserver and the photometric measurement of RNA concentrations rely on the intrinsic UV molar absorption properties of nucleotides.