Enabling mass spectrometry-based mid-down sequencing of large modified RNAs

/Abstract This proposal aims at removing a series of enduring obstacles that have been thus far hampering the broader adoption of mass spectrometry (MS) for the analysis of large covalently-modified nucleic acids. This analytical platform has the potential to fill the gap created by the limited ability of traditional genomics techniques to identify the covalent modifications present on emerging nucleic acid biotherapeutics and regulatory non-coding RNAs. The fact that the practical size of oligonucleotides amenable to direct gas-phase sequencing falls in the 20-30 nt range implies that analytes spanning thousands of nts, such as mRNAs and viral genomes, must be cleaved into smaller products by using chemical or enzymatic reagents. However, the reach of divide-and-conquer approaches is affected by the complexity of the mixtures generated by classic nucleotide-specific reagents, which increases geometrically as a function of analyte size to challenge the capabilities of front-end separation, mass analysis, and data interpretation approaches. With the goal of minimizing these undesirable outcomes, we will capitalize on the superior sequence specificity of RNA-cleaving deoxyribozymes to control the size distribution of hydrolysis mixtures by attacking fewer sites on the target strand. We will assemble a kit of deoxyribozymes with different susceptible motifs to provide the flexibility necessary to generate products with sizes approaching the upper bound of the practical range, which will enable the mid-down characterization of analytes exceeding several thousands of nts. Sample complexity will be also approached from a different angle that will involve re- examining the coupling of capillary electrophoresis (CE) with MS detection for oligonucleotide analysis. We will specifically explore alternative chemistries for the separation surfaces and background electrolyte to increase the resolving power of capillary zone electrophoresis (CZE) while maintaining full compatibility with MS conditions. In the case of capillary gel electrophoresis (CGE), we will explore different sieving materials and methods for creating the desired separation matrix inside the lumen of the separation channel. We will test possible solutions to prevent such material from leaching into the ion source, which has traditionally hampered the effective coupling of CGE with MS. We will finally evaluate the ability of 2-dimensional mass spectrometry (2DMS) to accomplish the parallel analysis of progressively larger oligonucleotide precursors. We will assess the extent by which the ability of multiplexing mixture analysis with limited or no front-end separation may translate into greater sequencing capacity. The test-samples employed in this proposal will consist of cellular/viral samples containing natural RNA modifications with relatively low copy-number, as well as synthetic nucleic acid therapeutics containing pharmaceutical-type modifications. The challenges posed by such samples will allow us to identify the limitations of the approaches developed here and devise possible remedies. These samples will also help demonstrate the direct applicability of these approaches to the widest possible range of nucleic acids samples, natural and man-made, which will substantiate the broader impact of the proposed activities. Project Number: 1R01GM162759-01 | Fiscal Year: 2026 | NIH Institute/Center: National Institute of General Medical Sciences (NIGMS) | Principal Investigator: Daniele Fabris | Institution: UNIVERSITY OF CONNECTICUT STORRS, STORRS-MANSFIELD, CT | Award Amount: $607,470 | Activity Code: R01 | Study Section: Enabling Bioanalytical and Imaging Technologies Study Section[EBIT] View on NIH RePORTER: https://reporter.nih.gov/project-details/11277525