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

Abstract

Methylated cytosine within CpG dinucleotides is a key factor for epigenetic gene regulation. It has been revealed that methylated cytosine decreases DNA backbone flexibility and increases the thermal stability of DNA. Although the molecular environment is an important factor for the structure, thermodynamics, and function of biomolecules, there are few reports on the effects of methylated cytosine under a cell-mimicking molecular environment. Here, we systematically investigated the effects of methylated cytosine on the thermodynamics of DNA duplexes under molecular crowding conditions, which is a critical difference between the molecular environment in cells and test tubes. Thermodynamic parameters quantitatively demonstrated that the methylation effect and molecular crowding effect on DNA duplexes are independent and additive, in which the degree of the stabilization is the sum of the methylation effect and molecular crowding effect. Furthermore, the effects of methylation and molecular crowding correlate with the hydration states of DNA duplexes. The stabilization effect of methylation was due to the favorable enthalpic contribution, suggesting that direct interactions of the methyl group with adjacent bases and adjacent methyl groups play a role in determining the flexibility and thermodynamics of DNA duplexes. These results are useful to predict the properties of DNA duplexes with methylation in cell-mimicking conditions.

Natural selection acts on epigenetic marks

“…Providing “proof” for this is likely to require experimental evidence with directed evolution (Morselli et al, 2015; Finnegan et al, 2020). However, even for DNA methylation, whose effect on the mutation rate is strong and unequivocally established, it will be difficult to retain that effect while simultaneously blocking its potential effect on gene expression, since the mere methylation of CpGs changes the local thermodynamic properties of DNA (S Wang, Kool, 1995; Renciuk et al, 2013; Tsuruta et al, 2021) and can modulate transcription factor binding (Yin et al, 2017; Héberlé, Bardet, 2019).…”

Effect of DNA modifications on the transition between canonical and non-canonical DNA structures in CpG islands during senescence

“…Higher number of 5mC stabilise duplexes. [36][37][38] Single or double 5mC stabilise i-motif, whereas three or more 5mC and single 5hmC destabilise i-motif. 39,40 It has also been suggested that the position and not the number of 5mC modulates the stability of i-motif.…”

“…53 A few studies have been conducted to determine the effect of molecular crowding on the stability of duplex and imotif with modications. [38][39][40]44 It has been observed that molecular crowding does not change the tendency of (de)stabilisation and the structure of a duplex and an i-motif with modications. [38][39][40] It has also been shown that molecular crowding of 40 vol% acetonitrile does not remarkably change the stability of i-motifs with 5hmC modications.…”

“…[38][39][40]44 It has been observed that molecular crowding does not change the tendency of (de)stabilisation and the structure of a duplex and an i-motif with modications. [38][39][40] It has also been shown that molecular crowding of 40 vol% acetonitrile does not remarkably change the stability of i-motifs with 5hmC modications. 44 However, little is known about the effect of molecular crowding on the stability and structure of G-quadruplexes with modications.…”

Biophysical Study of the Structure, Dynamics, and Function of Nucleic Acids

“…This Special Issue contains three research articles that studied nucleic acid methylation, a prevalent regulatory modification in prokaryotes and eukaryotes . Tsuruta et al investigated the methylation effect within the CpG site on DNA duplexes’ thermodynamics under cell-mimicking molecular crowding conditions [ 3 ]. Lam et al revealed that the methylated adenine base, such as m 1 A and m 6 A, formed the Hoogsteen base pairs with the opposite T bases in a mini-dumbbell structure of two TTTA tetranucleotide repeats [ 4 ].…”