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Access Type

WSU Access

Date of Award

January 2022

Degree Type


Degree Name




First Advisor

Mary T. Rodgers


This thesis focuses primarily on studying on how both naturally occurring, and chemically engineered modifications could be explored for fine tuning and modulating the conformational characteristics and intrinsic stability of functional domains that fold into nucleic acid i-motif structures. While the impact of DNA methylation, in the form of epigenetics, is widely studied, much less is known about the role of 5-methylation, 5-halogenation and 2'-modification on the stability of i-motif conformations. In this work, we first examined the influence of the 2′- and 3′-hydroxy substituents of the sugar moieties and 5-methylation of the cytosine nucleobases on the base-pairing interactions of protonated cytidine nucleoside analogue base pairs, (xCyd)H+(xCyd), and show that the 2'- and 3'-hydroxy substituents of the sugar moieties have very little influence on the strength of the base-pairing interactions, whereas 5-methylation of the cytosine nucleobases is found to enhance the strength of the base-pairing interactions. The increase in stability resulting from 5-methylation is only modest but is more than twice as large for the DNA than RNA protonated cytidine base pair, suggesting that canonical DNA i-motif conformations should be more stable than analogous RNA i-motif conformations, and that 5-methylation of cytosine residues, a significant epigenetic marker, provides additional stabilization to DNA than RNA i-motif conformations. Next, we sought to determine the impact of 5-halogenation on the structure and stability of protonated cytidine base pairs and their potential impact on the stability of genomic i-motif conformations. Our results indicate that 5-halogenation of the canonical RNA nucleoside base pairs decrease the base-pairing energies. In contrast, 5-halogenation of the canonical DNA nucleoside base pairs increases the base-pairing energies and would therefore tend to stabilize DNA i-motif conformations. Finally, the potential to deploy 2'-modified cytidine chemistries as model systems for tuning i-motif stability is meticulously examined. To address a knowledge gap, we examine the effects of 2'-modifications including: methylation, fluorination and stereochemical inversion on the base-pairing interactions of protonated cytidine nucleoside analogue base pairs, (xCyd)H+(xCyd). All 2'-modifications examine are found to enhance the base-pairing interactions relative to the canonical cytidine nucleoside base pairs with the greatest enhancement arising from 2'-O-methylation and 2',2'-difluorination, suggesting that these modifications should well be tolerated in the narrow grooves of i-motif conformations. Overall, the results presented provide fascinating evidence, that modified cytidine nucleoside analogues are excellent mimics for studying the conformational dynamics, structures, and intrinsic energetics of emerging nucleic acid higher order structures. The ability of the various modifications to strengthen the base-pairing interactions provides an opportunity to correlate structural and functional information of i-motif architecture into possible biological function and nanotechnological applications.

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