Oligonucleotide-directed triple helix formation has been recognized as a potential tool for targeting genes with high specificity. Cystosine methylation in the 5′ position is both ubiquitous and a stable regulatory modification, which could potentially stabilize triple helix formation. In this work, we have used a combination of calorimetric and spectroscopic techniques to study the intramolecular unfolding of four triplexes and two duplexes. We used the following triplex control sequence, named Control Tri, d(AGAGAC₅TCTCTC₅TCTCT), where C₅ are loops of five cytosines. From this sequence, we studied three other sequences with dC → d(m⁵C) substitutions on the Hoogsteen strand (2MeH), Crick strand (2MeC) and both strands (4MeHC). Calorimetric studies determined that methylation does increase the thermal and enthalpic stability, leading to an overall favorable free energy, and that this increased stability is cumulative, i.e. methylation on both the Hoogsteen and Crick strands yields the largest favorable free energy. The differential uptake of protons, counterions and water was determined. It was found that methylation increases cytosine protonation by shifting the apparent pK_a value to a higher pH; this increase in proton uptake coincides with a release of counterions during folding of the triplex, likely due to repulsion from the increased positive charge from the protonated cytosines. The immobilization of water was not affected for triplexes with methylated cytosines on their Hoogsteen or Crick strands, but was seen for the triplex where both strands are methylated. This may be due to the alignment in the major groove of the methyl groups on the cytosines with the methyl groups on the thymines which causes an increase in structural water along the spine of the triplex.

寡核苷酸引导的三螺旋形成已被认为是高特异性靶向基因的潜在工具。 5'位的胱氨酸甲基化是普遍存在的且稳定的调节修饰，可能稳定三螺旋的形成。在这项工作中，我们结合使用量热和光谱技术来研究四个三链体和两个双链体的分子内展开。我们使用以下三链体控制序列，名为Control Tri , d(AGAGA C ₅ TCTCT C ₅ TCTCT)，其中C ₅是五个胞嘧啶的环。根据该序列，我们研究了 Hoogsteen 链 ( 2MeH )、Crick 链 ( 2MeC ) 和两条链 ( 4MeHC ) 上具有 dC → d(m ⁵ C) 取代的其他三个序列。量热研究确定甲基化确实增加了热稳定性和焓稳定性，从而产生总体有利的自由能，并且这种增加的稳定性是累积的，即Hoogsteen和Crick链上的甲基化产生最大的有利自由能。测定质子、抗衡离子和水的差异吸收。研究发现，甲基化通过将表观 p K _a值移至更高的 pH 值来增加胞嘧啶质子化；质子摄取的增加与三链体折叠过程中抗衡离子的释放同时发生，这可能是由于质子化胞嘧啶正电荷增加的排斥所致。 对于 Hoogsteen 或 Crick 链上具有甲基化胞嘧啶的三链体，水的固定不受影响，但对于两条链均甲基化的三链体来说，水的固定受到影响。这可能是由于胞嘧啶上的甲基的主沟与胸腺嘧啶上的甲基的排列导致沿三链体脊柱的结构水增加。