Date of Award

1-1-2019

Language

English

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

College/School/Department

Department of Chemistry

Content Description

1 online resource (xiii, 191 pages) : illustrations.

Dissertation/Thesis Chair

Daniele Fabris

Committee Members

Cara Pager, Li Niu, Jan Halamek, Alexander Shekhtman

Keywords

Epitranscriptomics, Mass Spectrometry, Nucleic acid sequencing, Post transcriptional modifications, Ribonucleic acid, Non-coding RNA, Mass spectrometry, Genetic transcription

Subject Categories

Analytical Chemistry | Chemistry

Abstract

According to the results of the Human Genome Project, less than 5% of our genome consists of sequences that code for actual proteins. At the same time, however, ~90% of these non-coding sequences are transcribed into a myriad of classes of ribonucleic acids collectively known as ncRNAs, which have been recognized as essential regulatory factors. It has been recently shown that the activity of ncRNAs can be in turn modulated by post-transcriptional modifications (PTMs), covalent marks that can be established/removed by tightly regulated enzymatic pathways. Now, the race is on to understand the new set of controls of cellular functions enacted by these epitranscriptomics marks. This Dissertation addresses the exploding demands for effective, sensitive, robust approaches for PTM analysis required to sustain these types of studies. In particular, we explored new strategies for detecting PTMs by mass spectrometry (MS) and for obtaining their sequence positions in functional ncRNAs. Capitalizing on unique mass and fragmentation features, MS approaches afford the ability to identify any of the >160 covalent variant discovered thus far. These capabilities were leveraged to enable the comprehensive analysis of PTM samples obtained by hydrolysis of total RNA extracts into mono-nucleotide mixtures. The optimization of high-resolution affinity capture techniques allowed me to perform PTM profiling of selected RNA targets that were enriched from complex biological samples. The performance of these global and targeted profiling approaches was evaluated by investigating viral systems, which revealed for the first time the remarkable diversity of viral epitranscriptomes. With the goal of increasing the size of RNA strands amenable to direct gas-phase sequencing, I have explored different strategies to achieve denaturation of higher-order structure, which has been limiting the effectiveness of traditional fragmentation techniques. These approaches have the potential to become indispensable complements to the current genomics techniques employed in RNA analysis. Their widespread diffusion in the broader biomedical community will be expected to enable the discovery of regulatory mechanisms, putative targets for therapeutics, and diagnostic markers for human diseases.

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