Effect of dC → d(m5C) substitutions on the folding of intramolecular triplexes with mixed TAT and C+GC base triplets

Abstract

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(AGAGACTCTCTCTCTCT), where C are loops of five cytosines. From this sequence, we studied three other sequences with dC → d(mC) 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 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.

Combined Effects of Methylated Cytosine and Molecular Crowding on the Thermodynamic Stability of DNA Duplexes

“…Thus, the methylation effects on the thermodynamics of the DNA duplex are conserved and do not depend on the experimental conditions. The stabilization effect of methylation and its independence are consistent with previous studies for DNA i-motifs and triplex structures [ 32 , 39 ].…”

“…These results suggest that methylation does not have a large effect on the number of water molecules taken up upon folding or the hydration state of the DNA duplex. This is consistent with a previous study showing that methylation does not affect the immobilization of water molecules in a DNA duplex or a DNA triplex [ 32 ]. Therefore, the enthalpic contribution of methylation to stabilize the DNA duplex does not originate from hydrophobic interactions of methyl groups.…”

“…The plots of T m and Δ G ° vs. the number of methylations ( Figure S3 and Figure 4 A) show the slopes, which correspond to the average value of the T m increment (Δ T m ) and of the Δ G ° 37 decrement (ΔΔ G ° 37 ) per one methylation, which were 1.1 °C and −0.3 kcal/mol, respectively, under the dilute conditions. These values are consistent with previous reports showing that the Δ T m and of the ΔΔ G ° 37 per one methylation are 0.5 ~1.5 °C [ 54 , 67 ] and around −0.9 kcal/mol [ 32 ]. In addition, the linear relationship between T m and Δ G ° and the number of methylations suggest that thermal and thermodynamic stability from methylations are additive and mostly independent of sequence context [ 32 , 68 , 69 ].…”

“…They found that the melting temperature values were systematically upshifted by approximately 0.5 °C per methylated cytosine. This thermal stabilization is consistent with a study by Marky and colleagues using differential scanning calorimetry [ 32 ]. Methylation effects on the thermal stability of noncanonical DNA structures, triplexes, and i-motifs have also been reported [ 26 , 32 , 33 , 34 ].…”

“…This thermal stabilization is consistent with a study by Marky and colleagues using differential scanning calorimetry [ 32 ]. Methylation effects on the thermal stability of noncanonical DNA structures, triplexes, and i-motifs have also been reported [ 26 , 32 , 33 , 34 ]. However, most of these biophysical and biochemical studies on methylation effects have been carried out in test tubes with dilute buffer conditions that are totally different from those in living cells, where DNA folds into various structures to play critical roles in various biological processes.…”