Synthesis, detection, and quantification of modified nucleotides in RNA

Baron, Francis (2020) Synthesis, detection, and quantification of modified nucleotides in RNA. PhD thesis, University of Nottingham.

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Background: N6-methyladenosine (m6A) is the most abundant internal modification of mRNA. m6A regulates almost every stage in the mRNA life cycle with essential roles in splicing, polyadenylation, nuclear export, stability, translation, and degradation. m6A is essential for normal development of eukaryotic organisms, and abnormal levels of m6A have been associated with the development of disease including various types of cancer. Many research groups are working on artificially manipulating m6A levels for the therapeutic treatment of disease.

In order to fully understand the m6A modification and develop associated therapeutics, methods to accurately detect and quantify m6A are required. MeRIP-seq and miCLIP are the most commonly used methods of m6A sequencing, however, they are severely limited by (a) their inability to reliably quantify m6A levels, and (b) their use of an anti m6A antibody which is known to have problems with off-target binding. There is a significant demand for new methods of m6A sequencing and quantification.

m6A modified oligonucleotides are useful tools for the study of m6A. They can be used for various applications including the identification and study of m6A writer, reader, and eraser proteins, and can be used as substrates to test methods of m6A sequencing. Synthesis of m6A modified oligonucleotides requires an m6A phosphoramidite which is currently produced via a complicated seven-step chemical synthesis. Alternate methods for the synthesis of the m6A phosphoramidite will reduce the cost and increase availability of m6A modified oligonucleotides.

Research aims: The aims of this thesis are to (a) synthesise m6A modified oligonucleotide probes to study the role of the modification in mRNA, and (b) develop new methods for the detection and quantification of m6A.

Results: We have developed a method for the single step synthesis of m6A phosphoramidite and synthesised a number of m6A modified oligonucleotides which will prove to be valuable tools for research into m6A.

We have developed various new methods for the detection and quantification of m6A. We have developed a method involving site specific radiolabelling and thin layer chromatography (TLC) which we have used to probe m6A levels in a number of RNA transcripts. We have identified a modified thymidine nucleoside that selectively crosslinks with adenosine but not m6A in both mononucleotide and interstrand contexts. This molecule was unsuitable for m6A sequencing due to the unreliable nature of the crosslinking reaction. However, we have demonstrated that a modified nucleoside triphosphate is able to selectively inhibit reverse transcription reactions at sites of m6A in the RNA template.

Conclusions: Due to the significant challenge of detecting and quantifying m6A, the methods we have developed have had varying levels of success. The reverse transcription-based method using a modified nucleoside triphosphate is by far the most promising. Efforts to develop this technology as a transcriptome wide method of m6A sequencing are currently in progress.

Item Type: Thesis (University of Nottingham only) (PhD)
Supervisors: Hayes, Christopher
Fray, Rupert
Keywords: Oligonucleotides, Synthesis; Nucleotides; Ribonucleic acid
Subjects: Q Science > QP Physiology
Faculties/Schools: UK Campuses > Faculty of Science > School of Biosciences
UK Campuses > Faculty of Science > School of Chemistry
Item ID: 60070
Depositing User: Baron, Francis
Date Deposited: 31 Jan 2023 08:52
Last Modified: 31 Jan 2023 08:53

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