PT  - JOURNAL ARTICLE
AU  - Dominika Strzelecka
AU  - Miroslaw Smietanski
AU  - Pawel J. Sikorski
AU  - Marcin Warminski
AU  - Joanna Kowalska
AU  - Jacek Jemielity
TI  - Functional and LC-MS/MS analysis of <em>in vitro</em> transcribed mRNAs carrying phosphorothioate or boranophosphate moieties reveal polyA tail modifications that prevent deadenylation without compromising protein expression
AID  - 10.1101/2020.07.02.184598
DP  - 2020 Jan 01
TA  - bioRxiv
PG  - 2020.07.02.184598
4099  - http://biorxiv.org/content/early/2020/07/03/2020.07.02.184598.short
4100  - http://biorxiv.org/content/early/2020/07/03/2020.07.02.184598.full
AB  - Chemical 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.