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

Abstract

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.

Base-Pairing Energies of Proton-Bound Dimers and Proton Affinities of 1-Methyl-5-Halocytosines: Implications for the Effects of Halogenation on the Stability of the DNAi-Motif

“…This behavior is consistent with fragmentation via IRMPD [27,28] and CID [29][30][31] of similar proton-bound dimers. Production of the protonated nucleobase having the higher PA is energetically favored over production of the protonated nucleobase with the lower PA.…”

“…As discussed above, the stable tautomeric conformations of neutral cytosine, C, and protonated cytosine, H + (C), and various 1-and 5-methylated and 5-halogenated derivatives have previously been examined at the B3LYP/6-31G*, B3LYP/def2-TZVPPD, MP2(full)/6-31G* and MP2(full)/def2-TZVPPD levels of theory in the IRMPD and TCID studies we previously reported [29][30][31]. These calculations are expanded here to include structures for 1Me5FC and 1Me5BrC as well as the proton-bound dimers, (1Me5FC)H + (1Me5FC), (1Me5BrC)H + (1Me5BrC), (C)H + (1Me5FC), and (C)H + (1Me5BrC), and were optimized at the same levels of theory as described in the theoretical calculations section.…”

“…Theoretical calculations at the B3LYP/def2-TZVPPD level of theory predict the base-pairing energy (BPE) of the proton-bound dimer of cytosine (C + •C) as 170.1 kJ/mol, 79% and 163% greater than those of the canonical G•C and neutral C•C base pairs, respectively, indicating that the stronger base-pairing interactions in the C + •C homodimer are likely the major factor that stabilizes noncanonical DNA i-motif conformations [29][30][31]. Given the important biological roles that DNA i-motif conformations may play in several human diseases and cancer, including lung carcinoma [32], breast carcinoma [33], and Burkitt's lymphomas [34], a comprehensive study is needed to determine the influence of halogenation on the strengths of base-pairing interactions in proton-bound cytosine dimers.…”

“…A previous X-ray crystallography study suggests that cytosine protonation, required for the formation of the proton-bound C + •C dimers, is affected by a decrease of pK a of cytosine upon halogenation [35]. Quantitative determination of the BPEs of proton-bound homo-and heterodimers of C, 1MeC, and 5xC, where x = F, Br, and I, was performed using threshold collision-induced dissociation techniques (TCID) [29][30][31], It was determined that 1-methylation exerts very little influence on the BPE, whereas theory suggests that 1-methylation should lead to a decrease in the BPE. Halogenation of one or both cytosine residues at the 5-position decreases the BPE and should therefore destabilize DNA i-motif conformations.…”

“…Relative N3 proton affinities (PAs) of the 1-methyl-5-halocytosines are also extracted from the experimental data from competitive analyses of the two primary dissociation pathways that occur in parallel for the proton-bound heterodimers of cytosine and the 1-methyl-5-halocytosines. Absolute N3 PAs of the 1-methyl-5-halocytosines are also obtained using the relative PAs determined here and the PA of C [29,[36][37][38] reported in the literature. The measured values are compared with theoretical results calculated at the B3LYP and MP2(full) levels of theory to evaluate the ability of each level of theory for predicting accurate energetics.…”