Single-stranded model oligodeoxyribonucleotides, each containing a single protonatable base-cytosine, adenine, guanine, or 5-methylcytosine-centrally located in a background of non-protonatable thymine residues, were acid-titrated in aqueous solution, with UV monitoring. The basicity of the central base was shown to depend on the type of the central base and its nearest neighbours and to rise with increasing oligonucleotide length and decreasing ionic strength of the solution. More complex model oligonucleotides, each containing a centrally located 5-methylcytosine base, were comparatively evaluated in single-stranded and double-stranded form, by UV spectroscopy and high-field NMR. The N protonation of the 5-methylcytosine moiety in the double-stranded case occurred at much lower pH, at which the duplex was already experiencing general dissociation, than in the single-stranded case. The central guanine:5-methylcytosine base pair remained intact up to this point, possibly due to an unusual alternative protonation on O of the 5-methylcytosine moiety, already taking place at neutral or weakly basic pH, as indicated by UV spectroscopy, thus suggesting that 5-methylcytosine sites in double-stranded DNA might be protonated to a significant extent under physiological conditions.

Repetitive guanine-rich nucleic acid sequences play a crucial role in maintaining genome stability and the cell life cycle and represent potential targets for regulatory drugs. Recently, it has been demonstrated that guanine-based ligands with a porphyrin core can be used as markers of G-quadruplex assemblies in cell tissues. Herein, model systems of guanine-based ligands are explored by DFT methods. The energies of formation of modified guanine tetrads and those of modified tetrads stacked on the top of natural guanine tetrads have been calculated. The interaction energy has been decomposed into contributions from hydrogen bonding, stacking, and ion coordination and a twist-rise potential energy scan has been performed to find the individual local minima. Energy decomposition analysis reveals the impact of various substituents (F, Cl, Br, I, Me, NMe2 ) on individual energy terms. In addition, cooperative reinforcement in forming the modified and stacked tetrads, as well as the frontier orbitals participating in the hydrogen-bonding framework involving the HOMO-LUMO gap between the occupied σHOMO on the proton-accepting C=O and =N- groups and unoccupied σLUMO on the N-H groups, has been studied. The investigated systems are demonstrated to have a potential in ligand development, mainly due to stacking enhancement compared with natural guanine, which is used as a reference.