Date of Award

5-1-2024

Language

English

Document Type

Master's Thesis

Degree Name

Master of Science (MS)

College/School/Department

Department of Chemistry

Dissertation/Thesis Chair

Alan Chen

Committee Members

Sweta Vangaveti, Thomas Begley

Subject Categories

Chemistry

Abstract

Nucleic acid modifications are pivotal in biological processes due to their multifaceted roles. They regulate gene expression by modulating transcription, with modifications in the nucleic acid, sugar group, or in its phosphate backbone. In our investigation, we explore the use of molecular dynamics simulations to study nucleic acid modifications for potential applications in RNA-based therapeutics. These modifications introduce nonstandard atoms, such as selenium, and new parameterizations must be performed to properly simulate in GROMACS. These parameters govern bonded (strength of bonds, angles and dihedrals) and non-bonded (size and charges) interactions for each of the atoms in the modification. The first part focuses on parameterizing and modeling 12 distinct modifications of the wobble uridine at the 34th position in bacterial transfer RNAs (tRNAs). These modifications are introduced by the mnm enzyme pathway and can shift the base-pairing preferences from A-ending to G-ending codons, specifically with the geranyl modification. Sulfur, selenium, and geranyl modifications are introduced at the second position of uridine and aminomethyl (nm), methylaminomethyl (mnm), and carboxy-methylaminomethyl (cmnm) modifications occur at the fifth position. All-atom molecular dynamics simulations (MDS) were carried out to characterize the hydrogen bonding patterns and base-pairing specificities of the modified uridine with A/C/G/U base pairs. The simulations revealed how modifications like thiolation, selenation, and introduction of a geranyl group impact hydrogen bonding and could modulate translation by altering codon recognition. The second part describes parameterization of antisense oligonucleotides (ASOs) targeting the insulin receptor (INSR) gene and their influence on alternative splicing relevant to myotonic dystrophy. A minigene reporter system containing INSR exon 11 was constructed along with wild-type and mutant sequences. Simulations assessed how ASO binding to this region could rescue aberrant splicing patterns caused by sequestration of muscleblind-like (MBNL) proteins in the disease state. Overall, this work provides molecular insights into RNA modifications and demonstrates the utility of simulations for developing RNA-targeting therapeutics.

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