Poster abstracts
Poster number 45 submitted by Olivia Miller
Direct Sequencing and Modification Mapping of Small RNA Mixtures by Liquid Chromatography Mass Spectrometry
Olivia F. Miller (Ohio State Biochemistry Graduate Program ), Cameron W. Divoky (OSU, Biological Chemistry and Pharmacology ), Michael A. Freitas (OSU, Biological Chemistry and Pharmacology )
Abstract:
Small RNAs (smRNAs) are key regulators of gene expression and translation, having profound roles in disease biology and cancer development making them powerful biomarkers for diagnosis and prognosis. Because of their stability and specificity, smRNAs are emerging as promising candidates for therapeutic drug development in oncology and other diseases caused by aberrant proteins. Current sequencing methods can detect unmodified RNAs but are limited in the amount of modifications that can be resolved in any one experiment. To address this limitation, liquid chromatography-tandem mass spectrometry (LC-MS/MS) techniques are being developed for the identification and mapping of modifications. Here, we have optimized LC-MS/MS-based sequencing and modification mapping of smRNAs, focusing on improving liquid chromatography separation, and mass spectrometry ionization efficiency and detection sensitivity. Using synthetic 21-nt piRNA sequences with and without internal m⁶A modification, as well as a deaminated version, we confirmed a low detection limit (0.1 pmol) and separation of the RNAs using a Q-Exactive Plus LC-MS/MS system. Using HFIP and TEA as mobile phases, we established an elution gradient that provides a clean and strong separation for oligonucleotides of similar molecular weights and sizes. To increase resolution and ion capacity we switched to the Orbitrap Fusion LC-MS/MS system. Multiple fragmentation activation methods were evaluated to obtain a tandem MS spectra with minimal secondary fragmentation while achieving full sequencing coverage. Using this optimized approach, we accurately separated and sequenced complex mixtures of synthetic oligonucleotides. Paired with our improved bioinformatic software, we determined the precise location of each modification. Overall, this optimized methodology provides a more sensitive and accurate method for identifying and characterizing smRNAs and their modifications.
References:
Xiao, J. MicroRNA : from bench to bedside; Academic Press,, 2022.
Helm, M.; Motorin, Y. Detecting RNA modifications in the epitranscriptome: predict and validate. Nat Rev Genet 2017, 18 (5), 275-291. DOI: 10.1038/nrg.2016.169 From NLM Medline.
Wein, S.; Andrews, B.; Sachsenberg, T.; Santos-Rosa, H.; Kohlbacher, O.; Kouzarides, T.; Garcia, B. A.; Weisser, H. A computational platform for high-throughput analysis of RNA sequences and modifications by mass spectrometry. Nat Commun 2020, 11 (1), 926. DOI: 10.1038/s41467-020-14665-7 From NLM Medline.
Keywords: LC-MSMS, Oligonucleotide, Direct Sequencing