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Towards the Parallel, Accurate, and High-throughput Mapping of RNA Modifications by Liquid Chromatography Tandem Mass Spectrometry

Lobue, Peter
http://rave.ohiolink.edu/etdc/view?acc_num=ucin1595005836099446

Abstract Details

2020, PhD, University of Cincinnati, Arts and Sciences: Chemistry.
Ribonucleic acids (RNA) play critical roles in regulating the flow of genetic information inside a cell. Ribonucleosides, the building blocks of RNA (adenosine - A, guanosine - G, cytidine - C and uridine – U), store another layer of information in the form of post-transcriptional modifications (PTMs) in almost all types of RNA, including messenger RNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), long non-coding RNA (lncRNA), and micro RNA (miRNA). These nucleoside modifications do not change the amino acid sequence of the encoded protein, but can affect the stability, localization, translational accuracy and the function of RNA. More than 160 different kinds of chemically diverse PTMs have been reported in RNA, including those produced both enzymatically and synthetically. In addition to the previously stated roles of RNA modifications, the absence or presence of a modification (or set of modifications) may also play an important role in the development in human diseases. Transfer RNA (tRNA) is the most densely modified of class of RNAs, containing chemical modifications that range from simple base and ribose methylations, to more complex so-called “hyper-modifications” that result from multiple-step enzymatic transformations. Correlation between tRNA modification profiles and human diseases continues to emerge, with special focus recently on inherited disease caused by mitochondrial DNA mutations and altered modification profiles of mitochondrial tRNAs. Though mitochondrial abnormalities have been linked to prevalent central nervous system (CNS) disorders, very little is known about the modification profiles in normal cells (only 3 of the 22 normal human mt-tRNA sequences have been reported). Therefore, mapping the complete set of modifications in human mt-tRNA may provide important biomarkers and drug targets with the ultimate therapeutic goal of restoring normal CNS function. The most commonly understood tRNA mutation in mt-tRNA Leu (UUR) is known to lead to MELAS (Mitochondrial Encephalomyopathy, Lactic Acidosis and Stroke-like episodes), affecting at least 1 in 6000 individuals – for which there is currently no approved drug treatment. The most commonly applied RNA modification mapping approach involves isolation of the target RNA (e.g. mt-tRNA) and its subsequent enzymatic hydrolysis to generate oligonucleotides that are amenable to LC-MS/MS analysis. The sequence of the oligonucleotide including the potential modification will be deciphered from this tandem mass spectrum. Thus, the intact modified RNA sequence is reconstructed from the sequences of oligonucleotide digestion products in a bottom-up approach. Though this approach has shown its utility for over two decades, acquiring complete modification maps of mt-tRNA requires state-of-the-art analytical techniques like LC-MS/MS due to the challenges of isolating sufficiently pure amounts of sample and the complex mixture of oligonucleotide digestion products that must be separated and sequenced. Therefore, the goal of the research presented in this dissertation is to develop liquid chromatography, tandem mass-spectrometry, and data processing methods that allow for the efficient, accurate, and comprehensive modification mapping of human tRNA samples.
Patrick Limbach, Ph.D. (Committee Chair)
Thomas Beck, Ph.D. (Committee Member)
Ryan White, Ph.D. (Committee Member)
135 p.
Chemistry
tRNA; RNA; chemistry;

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Citations

  • Lobue, P. (2020). Towards the Parallel, Accurate, and High-throughput Mapping of RNA Modifications by Liquid Chromatography Tandem Mass Spectrometry [Doctoral dissertation, University of Cincinnati]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1595005836099446

    APA Style (7th edition)

  • Lobue, Peter. Towards the Parallel, Accurate, and High-throughput Mapping of RNA Modifications by Liquid Chromatography Tandem Mass Spectrometry. 2020. University of Cincinnati, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=ucin1595005836099446.

    MLA Style (8th edition)

  • Lobue, Peter. "Towards the Parallel, Accurate, and High-throughput Mapping of RNA Modifications by Liquid Chromatography Tandem Mass Spectrometry." Doctoral dissertation, University of Cincinnati, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1595005836099446

    Chicago Manual of Style (17th edition)

ucin1595005836099446
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This open access ETD is published by University of Cincinnati and OhioLINK.