Quantification of mRNA Using ³¹P NMR Spectroscopy and CRAFT

Messenger RNA (mRNA) has emerged as a promising therapeutic modality in vaccine development due to its safety, efficacy, and rapid development potential. Reliable measurement and comparison of mRNA concentration in different chemical environments are crucial for research and manufacturing purposes. Conventionally, UV spectroscopy is used for quantification. However, variability in solution conditions, such as ionic strength, may influence the measurement of the UV absorbance at 260 nm, A₂₆₀. This therefore necessitates accurate calibration of the extinction coefficient, ε, at a given set of solution conditions to the concentration of mRNA, as measured by an orthogonal and quantitative method. To that end, we utilized quantitative ³¹P NMR spectroscopy. We outline a general quantitative treatment of mRNA samples that may contain multiple polyadenylated mRNA sequences and show that the bias introduced by the method's assumptions into the measured mRNA concentrations is expected to be ≤ 4%. We also identified the limitations and subjectivities of accurate integration-based measurement of broad mRNA resonances in the frequency domain through a mini round-robin study. Quantification of broad mRNA signals using CRAFT in the time domain is shown to be superior to integration, as any subjectivity potentially introduced by the operator during spectral processing is obviated. This enables the use of ³¹P qNMR to accurately quantify total mRNA content and thus accurately calibrate mRNA extinction coefficients.