Studies of multistranded DNA structures and their interactions with metal ions using NMR spectroscopy

DNA molecules can adopt several higher order structures besides the well-known right-handed B-type duplex including triplex as well as quadruplex structures. The latter have been observed in the studies of guanine rich DNA oligonucleotide sequences and were named G-quadruplexes. The interest in G-quadruplex structures has been recently renewed due to the emerging evidence that such structures might participate in several important biological processes. This has at the same time kindled interest in G-quadruplex structures as possible therapeutics and therapeutic targets. In the current project we propose to incorporate modified nucleotides in oligonucleotide sequences in order to gain deeper insights into the folding and stability of G-quadruplexes. The use of chemically modified nucleotides will enable us to evaluate the importance of particular hydrogen bond on the stability of G-quadruplex structure. The residue specific 15N labeled oligonucleotides will be used in the modern NMR methods for measuring coupling constants across hydrogen bonds in base pairs as well as for measuring residual dipolar constants in partially oriented media. We expect to get additional insights into structural features of G-quadruplex structures and their sensitivity to the nature of present monovalent cationic species. It is known that metal ions greatly influence the structure and consequently the nucleic acid function. G-quadruplexes with their strong metal ion binding represent a good model system for studying metal ion interactions with DNA and method development for studies of metal ion interactions with nucleic acids in solution. The isotopically 15N labeled ammonium ions, whose exchange is slow on the NMR time-scale, will be used to localize binding sites for monovalent cations inside G-quadruplex structures. The influence of incorporation of chemically modified nucleotides on the binding and dynamics of ion exchange will also be assessed.