Computational protocol: Cytosine methylation alters DNA mechanical properties

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[…] Non-methylated DNA (nDNA), center-methylated DNA (cDNA) and fully-methylated DNA (fDNA), as employed in the experiments, were studied in steered molecular dynamics (SMD) simulations. In each simulation, a DNA was placed in a water box of size 300 Å×60 Å×60 Å and neutralized with 100 mM KCl, amounting to 87 K+ and 47 Cl− ions. The resulting system is shown in a. Each simulated system contained about 110 000 atoms. Simulations were performed using NAMD 2.6 (). The DNA models employed and simulation details are provided in Supplementary Data. Figure 3.SMD simulations, in which external forces are applied to a group of atoms, enable researchers to conduct single-molecule experiments in silico and see biomolecular mechanics in action. For a review of SMD simulations see (,,). In our SMD simulations, one 5′-end of DNA was fixed, and the other 5′-end was pulled, as shown in b. Three pulling conditions were applied: (i) constant velocity pulling at 10 Å/ns; (ii) constant velocity pulling at 1 Å/ns; and (iii) constant force pulling at 200 pN. In constant velocity SMD simulations, the 5′-end of DNA is attached to one end of a virtual spring; the other end of the spring is moved at a constant velocity v along the stretching x-direction and the force f applied on the 5′-end of DNA is determined through the extension of the virtual spring: f = −k[x(t)−x(t0)−v(t−t0)]. In constant force SMD simulations, a force along the stretching direction is continuously applied on the 5′-end of DNA. Five independent SMD simulations with 10 Å/ns-pulling velocity were conducted for nDNA, cDNA and fDNA; one SMD simulation with 1 Å/ns-pulling velocity covered 118 ns for each DNA; the constant force simulation covered 90 ns for nDNA and fDNA.Due to limited computational resources, we could not employ slower pulling velocities. Because of the high pulling velocity in our SMD simulations, the rupture force of DNA seen (∼1000 pN) is much higher than the experimental value (∼100 pN). Hence, we conducted the constant force pulling SMD simulation with a force value of 200 pN for nDNA and fDNA. Supplementary Table S1 lists all the simulations carried out. The relationship of simulated and measured rupture forces has been discussed extensively by Sotomayor and Schulten ().The interaction free energy of DNA, e.g. base pairing and base stacking interaction, stabilizes double stranded DNA against spontaneous dissociation of two strands. Subjected to external pulling force, B-form DNA undergoes a series of conformational changes, as shown in b. Monitoring the applied force and the length of stretched DNA in the simulations, we obtained the force–extension curve of DNA. The length of DNA is defined as the distance between the Cα atoms of the fixed 5′-end cytosine and the pulled 5′-end cytosine. To further characterize the thermodynamics of the force-induced dsDNA dissociation process, we monitored the time evolution of the number of base pairs and the stacking energy of DNA. A base pair is considered broken when the distance between hydrogen bond acceptor atom and donor atom exceeds 3 Å. Since the electrostatic contribution to DNA base stacking interaction is small (), the stacking energy of each nucleotide was obtained by just calculating the van der Waals energy between its neighbors and itself. Most of the analysis of MD results and respective figures were prepared using the software VMD (). […]

Pipeline specifications

Software tools NAMD, VMD
Application Protein structure analysis
Chemicals Cytosine