Functional and LC-MS/MS analysis of in vitro transcribed mRNAs carrying phosphorothioate or boranophosphate moieties reveal polyA tail modifications that prevent deadenylation without compromising protein expression

ABSTRACTChemical modifications enable preparation of mRNAs with augmented stability and translational activity. In this study, we explored how chemical modifications of 5’,3’-phosphodiester bonds in the mRNA body and polyA tail influence the biological properties of eukaryotic mRNA. To obtain modified and unmodified in vitro transcribed mRNAs, we used ATP and ATP analogues modified at the α-phosphate (containing either O-to-S or O-to-BH3 substitutions) and three different RNA polymerases—SP6, T7 and polyA polymerase. To verify the efficiency of incorporation of ATP analogues in the presence of ATP, we developed a liquid chromatography–tandem mass spectrometry (LC-MS/MS) method for quantitative assessment of modification frequency based on exhaustive degradation of the transcripts to 5’-mononucleotides. The method also estimated the average polyA tail lengths, thereby providing a versatile tool for establishing a structure-biological property relationship for mRNA. We found that mRNAs containing phosphorothioate groups within the polyA tail were substantially less susceptible to degradation by 3’-deadenylase than unmodified mRNA and were efficiently expressed in cultured cells, which makes them useful research tools and potential candidates for future development of mRNA-based therapeutics.View Full Text