MSACL 2019 EU Abstract

Analysis and Characterization Modified in vitro Transcribed RNA Using LC-MS/MS Method

Dominika Strzelecka (1), Miroslaw Smietanski (2), Marcin Warminskia (1), Paweł Sikorski (2), Jacek Jemielity (2), Joanna Kowalska (1)
(1) Division of Biophysics, Institute of Experimental Physics, Faculty of Physics, University of Warsaw, Pasteura 5, Warsaw, Poland (2) Centre of New Technologies, University of Warsaw, Banacha 2C, Warsaw, Poland

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Dominika Strzelecka (Presenter) Faculty of Physics, University of Warsaw >> POSTER (PDF)

Presenter Bio: I graduated in Application of Physic in Biology and Medicine, Molecular Biophysic at Faculty of Physic, University of Warsaw. Currently I am a PhD student at Division of Biophysics, Faculty of Physics, University of Warsaw.

Relevant Financial Disclosures (within past 24 months) No relevant financial relationship(s) to disclose. Conflict mitigation NOT REQUIRED.

Introduction: The stability of mRNA largely depends on the 3’- end and 5’- end structures which are targets for enzymatic degradation. Chemical modifications of those regions are introduced into in vitro transcribed mRNAs in order to confer on them resistance to degradation and increase their translation efficiency, thereby increasing their therapeutic potential. Chemically modified mRNA is a promising therapeutic agent with potential uses in cancer immunotherapy and gene replacement therapy.
Objectives: The primary objective of the study was to develop an LC-MS/MS method to analyze modified in vitro transcribed RNA using triple-quadrupole apparatus with electrospray ion source.
Methods: An LC-MS method for quantitative assessment of incorporation of ATP analogs modified at the α-phosphate and nucleobase into transcripts by RNA polymerases has been developed. Modified RNA was enzymatically degraded into single nucleotides followed by their quantification. To this end, synthetic internal standards labelled with [18O] within phosphate were synthesized. Ion pair chromatography coupled with ESI-QQQ was used for HPLC separation.
Results: We obtained chemically modified in vitro transcribed RNA by replacing standard NTPs with appropriate ATP analogs either containing various O-to-X substitutions, differing in stereochemistry (ATPαX; SP versus RP), or modified within nucleobase. A mixture of modified NTP and unmodified ATP at different ratios was used in the transcription reaction. To assess the efficiency and frequency of incorporation of the modified NTP versus natural NTP, conditions for complete RNA degradation using phosphodiesterase enzyme were determined to obtain exhaustive cleavage into single nucleotides. By using synthesized isotope-labelled nucleotides we were able to quantify modified and unmodified monophosphate adenine nucleotides. Thereby we were able to determine structure of RNA and RNA polymerases specificity.
Conclusions: Proposed LC-MS quantitative method can be use to analyse modifications within naturally occurring RNA and in vitro transcribed RNA thereby providing a tool for establishing RNA structure-biological properties relationship. Acknowledgement: This work is founded by National Science Centre, Preludium (UMO-2016/23/N/ST4/03186.