Poster abstracts

Poster number 147 submitted by Adam Wier

The AUGCs: Learning the chemistry of RNA through a mass-spectrometry screen of electrophiles

Adam B. Wier (University of Notre Dame Department of Chemistry & Biochemistry), Marla Gravino (University of Notre Dame Department of Chemistry & Biochemistry), Adam Albritton (University of Notre Dame Department of Chemistry & Biochemistry), Brittany S. Morgan, Ph.D. (University of Notre Dame Department of Chemistry & Biochemistry)

Abstract:
RNA possesses multiple nucleophiles that can react with small-molecule electrophiles, such as 2’-hydroxyls that are acylated by SHAPE reagents.1 These nucleophile-electrophile reactions anchor methods of learning about the higher-order structures and small-molecule binding sites of the transcriptome, providing a readout that persists through RNA’s dynamic nature.2,3 Despite their applicability, only a narrow range of RNA-reactive electrophiles have been demonstrated compared to those that are known to react with nucleophilic amino acids. Furthermore, there are few validated nucleophile-specific electrophiles for RNA, whereas amino acid residue-specific electrophiles such as acrylamides for cysteine or hydroxysuccinimides for lysine are well known.4 To address this gap in knowledge, I have developed a liquid chromatography-mass spectrometry (LC-MS) platform for screening reactions between electrophilic fragments and 5’-monophosphate ribonucleotides. Electrophiles are sorted into those that produce a mass-spectrometry identifiable product and those that are unreactive for each canonical nucleotide. In the future, reactive electrophiles will be quantified for differences in percentage of covalent bond formation using high-performance liquid-chromatography coupled with UV-absorbance (HPLC-UV). Products will be identified using 1H and 13C NMR. This work is the first systematic census of electrophile reactivity with ribonucleotides, revealing the myriad chemical transformations RNA can experience. I expect the results of this screen to provide greater context for existing electrophilic chemistries used with RNA and to broaden the available tools for researchers looking to develop covalent strategies for studying the transcriptome.

References:
(1): Merino, E.J.; Wilkinson, K.A.; Coughlan, J.L.; Weeks, K.M. RNA Structure Analysis at Single Nucleotide Resolution by Selective 2‘-Hydroxyl Acylation and Primer Extension (SHAPE). JACS 2005, 127, 12, 4223-4231
(2): Chan, D.; Feng, C.; Spitale, R.C. Measuring RNA structure transcriptome-wide with icSHAPE. Methods 2017, 120, 85-90.
(3): Fang, L.; Velema, W.A.; Lee, Y.; Xiao, L.; Mohsen, M.G.; Kietrys, A.M.; Kool, E.T. Pervasive transcriptome interactions of protein-targeted drugs. Nature Chemistry 2023, 15, 1374-1383.
(4): Gehringer, M. and Laufer, S.A. Emerging and Re-emerging Warheads for Targeted Covalent Inhibitors: Applications in Medicinal Chemistry and Chemical Biology. J. Med. Chem. 2019, 62, 12, 5673-5724.

Keywords: Electrophile, Ribonucleotide, LCMS