Cytosine methylation, a common epigenetic modification, can regulate gene expression and lead to human diseases. Hypermethylation of cytosine in extended (CCG)n�(CGG)n trinucleotide repeats has been shown to cause fragile X syndrome, the most widespread inherited cause of mental retardation in humans. The extended (CCG)n�(CGG)n repeats adopt i-motif conformations that are preferentially stabilized by base-pairing interactions of intercalated proton-bound dimers of cytosine (C+�C). Thermochemical information regarding the strength of base pairing and the effects of methylation on the base-pairing energies (BPEs) of proton-bound cytosine base pairs is limited.Experimentally, base-pairing energies (BPEs) of the proton-bound homo- and heterodimers of cytosine (C), 1-methylcytosine (1MeC), 5-methlycytosine (5MeC), and 1,5-dimethylcytosine (15dMeC) are determined using threshold collision-induced dissociation techniques in a guided ion beam tandem mass spectrometer. The only dissociation pathways observed correspond to cleavage of the three hydrogen bonds responsible for the binding in these base pairs resulting in loss of one of the neutral nucleobases. Relative N3 proton affinities (PAs) of the methylated cytosines are also obtained from competitive analyses of the data for the proton-bound heterodimers. Structures of the proton-bound dimers, theoretical estimates for the BPEs, and relative N3 PAs are determined from DFT calculations at the B3LYP/def2-TZVPPD level of theory.Only 5-hypermethylation is found to increase the BPE, whereas all other modifications weaken the BPE. The increase/decrease in the BPE is found to directly correlate with the relative N3 PAs. However, the measured and calculated BPEs in all of the proton-bound dimers examined here significantly exceed those of Watson-Crick G�C and neutral C�C base pairs, indicating that C+�C mismatches are still energetically favored, and suggesting that i-motif conformations should be preserved upon methylation. The N3 PAs of cytosine and the methylated cytosines are determined to follow the order: 15dMeC > 5MeC > 1MeC > C.

Base-pairing energies of proton-bound dimers of cytosine and methylated cytosines: Implications for the stability of DNA i-motif conformations

248th National Meeting (2014)

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