Publication date: Available online 24 December 2017
Source:Biochimie
Author(s): Carolyn E. Carr, Rajkumar Ganugula, Ronald Shikiya, Ana Maria Soto, Luis A. Marky
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 pKa 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.
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