Poster abstracts
Poster number 157 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 R. Gravino (University of Notre Dame Department of Chemistry & Biochemistry), Adam J. Albritton (University of Notre Dame Department of Chemistry & Biochemistry), Nya S. Huff (University of Notre Dame Department of Chemistry & Biochemistry), Brittany S. Morgan, Ph.D. (University of Notre Dame Department of Chemistry & Biochemistry)
Abstract:
Covalent chemistry offers two unique dimensions to RNA chemical biology: 1) certain electrophilic small-molecules selectively react with a nucleophile, such as SHAPE reagents with the 2’-hydroxyl and 2) electrophile-nucleophile reactivities can change in supramolecular contexts, informing us about RNA’s higher-order structures (SHAPE), sites of interactions with RNA-binding proteins (CLIP), and potential binding pockets for small-molecule ligands (RBRP). These methods’ success depends on knowledge of each electrophile’s preference for individual nucleophiles. Yet, there has been no comprehensive census of electrophile reactivity with RNA nucleophiles and only a narrow range of RNA-reactive electrophiles have been demonstrated compared to nucleophilic amino acids.
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 as well as methylated nucleobases at pHs 7.4-9.0 to mimic standard and perturbed physiological pKas. Reactive electrophiles are then quantified for differences in percentage of covalent bond formation in physiological conditions using high-performance liquid-chromatography coupled with UV-absorbance (HPLC-UV); products are identified using 1H and 13C NMR. We have found that novel chemistries can display specificity for a given nucleotide, broad reactivity, or inertness. To explore how these reactivities change in a structural context, I have also designed a LC-MS platform for the separation and identification of reaction products with a small RNA hairpin loop. This is the first census of electrophile reactivity with ribonucleotides, providing greater context for chemistries used with RNA and broadening the available tools for researchers looking to develop covalent strategies for studying the transcriptome.
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Keywords: Electrophile, Mass Spectrometry, Covalent