Biophysical & crystallographic studies of DNA i-motifs

• Main Author: Gurung, Sarah Prava
• Published: University of Reading 2018
• Online Access: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.749345

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.