Yang Bo, Moehlig Aaron R, Frieler C E, Rodgers M T
Department of Chemistry, Wayne State University , Detroit, Michigan 48202, United States.
J Phys Chem B. 2015 Feb 5;119(5):1857-68. doi: 10.1021/acs.jpcb.5b00035. Epub 2015 Jan 21.
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.
(CCG)n·(CGG)n三核苷酸重复序列的扩增会导致胞嘧啶残基的高甲基化，并引发脆性X综合征，这是人类遗传性智力残疾的最常见原因。(CCG)n·(CGG)n重复序列呈现i-基序构象，这种构象通过胞嘧啶非经典质子化核碱基对（C(+)·C）的碱基配对相互作用而优先稳定。此前，我们使用阈值碰撞诱导解离（TCID）技术研究了胞嘧啶的5-甲基化对碱基配对能量（BPE）的影响。在本研究中，我们将研究范围扩大到包括质子化的胞嘧啶、1-甲基胞嘧啶、5-甲基胞嘧啶和1,5-二甲基胞嘧啶的同型和异型核碱基对。1-甲基取代基可阻止胞嘧啶的大多数互变异构过程，并可模拟DNA核苷酸的糖部分。与胞嘧啶在5位的全甲基化不同，发现1-甲基化对BPE的影响很小。对两个核碱基的所有修饰都会导致BPE略有增加，其中5-甲基化产生的增强作用比1-甲基化或1,5-二甲基化更大。相比之下，对单个核碱基的修饰会导致BPE略有降低，同样，5-甲基化产生的影响比1-甲基化更大。然而，此处研究的所有质子化核碱基对的BPE都显著超过了经典G·C和中性C·C碱基对的BPE，因此即使存在此类修饰，它们仍应提供稳定DNA i-基序构象的驱动力。甲基化胞嘧啶的质子亲和力也通过TCID实验，通过对质子化异型核碱基对平行发生的主要解离途径进行竞争分析而获得。
