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Biophysical & crystallographic studies of DNA i-motifs

Gurung, S. P. (2018) Biophysical & crystallographic studies of DNA i-motifs. PhD thesis, University of Reading

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Intramolecular i-motifs of the form C3L3-8C3L3-8C3L3-8C3, where C3 denotes the cytosine stretch and L3-8 are “loop” regions containing any DNA base (L) including cytosine, were studied to understand the effects of loop length on i-motif stability. It contrast to the previously held notion that long-looped i-motifs are more stable, it was found that i-motif structures with short loops exhibit higher thermal stabilities and transitional pH values. The stability of long-looped i-motifs are then shown to increase with the addition of [Ru(phen)2dppz]2+ (phen = 1,10-phenanthroline, dppz = dipyrido [3,2-a:2',3'-c] phenazine); a polypyridyl complex that has a potential for photodynamic therapy. Addition of the complex enhances the stability of d(C3T838)3C3 but not that of d(C3T383)3C3, implying that loop lengths are important in defining i-motif-ligand interactions. The effects of loop base composition on the stability of i-motifs have also been presented. It is shown that when d(C3XYZ)3C3 sequences are used (where X and Z are adenine, thymine and guanine, and Y is any of the four DNA bases), pyrimidine-rich sequences form more stable i-motif structures. However, when guanine is X, only two out of 12 d(C3XYG)3C3 sequences were able to form i-motifs. Change in sequence direction also resulted in different thermal and pH stabilities; emphasising the role of loop base composition on not only the i-motif’s stability but also in its formation. X-ray crystallography was used to further understand the effects of loop bases on i-motif structures. The study focuses on four tetramolecular i-motifs; two of which were solved in the mid1990’s; (d(C4)4 and d(C3T)4 but have now been re-examined using improved experimental approaches. Two novel i-motif structures of d(C3A)4 and [d(C3A) + d(C3T)] are presented. Following the X-ray diffraction of d(C3T)4 crystals to 0.68 Å resolution (previously reported at 1.4 Å) at beamline I02 (Diamond Light Source Ltd.), a novel neutron diffraction study on the particular i-motif was conducted. Single crystal neutron diffraction was carried out at MaNDi beamline (Spallation Neutron Source) to find the distribution of the proton between the hemiprotonated cytosine+·cytosine base pairs and to understand the role that H-bonded water can play in stabilising the i-motif structure.

Item Type:Thesis (PhD)
Thesis Supervisor:Cardin, C., Brazier, J., Sorensen, T. and Winter, G.
Thesis/Report Department:School of Chemistry, Food and Pharmacy
Identification Number/DOI:
Divisions:Life Sciences > School of Chemistry, Food and Pharmacy > Department of Chemistry
ID Code:77642
Date on Title Page:2017
Additional Information:Offprints of the authors publications were bound into the physical thesis. This content has been removed from the electronic version which can be downloaded here as the content was copyright material.


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