Base-Pairing Energies of Protonated Nucleobase Pairs and Proton Affinities of 1‑Methylated Cytosines: Model Systems for the Effects of the Sugar Moiety on the Stability of DNA i‑Motif Conformations

Expansion of (CCG) n ·(CGG) n trinucleotide repeats leads to hypermethylation of cytosine residues and results in Fragile X syndrome, the most common cause of inherited intellectual disability in humans. The (CCG) n ·(CGG) n repeats adopt i-motif conformations that are preferentially stabilized by base-pairing interactions of noncanonical protonated nucleobase pairs of cytosine (C+·C). Previously, we investigated the effects of 5-methylation of cytosine on the base-pairing energies (BPEs) using threshold collision-induced dissociation (TCID) techniques. In the present work, we extend our investigations to include protonated homo- and heteronucleobase pairs of cytosine, 1-methylcytosine, 5-methylcytosine, and 1,5-dimethylcytosine. The 1-methyl substituent prevents most tautomerization processes of cytosine and serves as a mimic for the sugar moiety of DNA nucleotides. In contrast to permethylation of cytosine at the 5-position, 1-methylation is found to exert very little influence on the BPE. All modifications to both nucleobases lead to a small increase in the BPEs, with 5-methylation producing a larger enhancement than either 1-methyl or 1,5-dimethylation. In contrast, modifications to a single nucleobase are found to produce a small decrease in the BPEs, again with 5-methylation producing a larger effect than 1-methylation. However, the BPEs of all of the protonated nucleobase pairs examined here significantly exceed those of canonical G·C and neutral C·C base pairs, and thus should still provide the driving force stabilizing DNA i-motif conformations even in the presence of such modifications. The proton affinities of the methylated cytosines are also obtained from the TCID experiments by competitive analyses of the primary dissociation pathways that occur in parallel for the protonated heteronucleobase pairs.