Determination of Free Energy Landscapes via Computer Simulations and its Application to the DNA i-motif Vasileios A. Tatsis, Raghvendra P. Singh and Andreas Heuer Institute of Physical Chemsitry, WWU, Münster, Germany email: andheuer@uni-muenster.de Simulation system definitions MD Engine: GROMACS with PLUMED Unfolding simulations temperatures: 300 and 500 K Electro-neutral system: 21 Na+ (for backbone charges) Water model : TIP3P(≈ 6000 water molecules) Simulation Box: 6.7 nm3. Force-field: Amber03-bsc0 (for nucleic acids) 3D Periodic Boundary Conditions PME for electrostatic interactions Simulations times up to 1μs (variable) Acknowledgements Molecular Dynamic Simulations Figure 2: Upper panel (L to R): Free energy landscapes resulted from: (iM@300k) multiple unbiased MD simulations, at 300K; (A and B ) Two well-tempered metadynamics simulations. The distance between the center of mass for C1 and T22 to A11d[C1/T22)-A11] and the distance between C1 and T22 d[C1-T22] were used as reaction coordinates. Units: Energy in kJ/mol., time length in 1μs. Lower panel (L to R): Normalized histograms of RMSD between the modeled i-motif structure and the crystal structure (pdb code: 1ELN) and Heat map describing the average RMSD value using the two assisting collective variables. Data extracted from the first well-tempered metadynamics simulations of 700 ns. Reaction coordinates were chosen using COM of rC1/T22, rC1,T22/A11. Metadynamics shows deeper minimum while system seems stuck at starting coordinates in MD. The RMSD shows that metadynamics explored phase space efficiently. i-motif An atypical DNA formed at low pH and a high temperature, C-rich telomeric regions fold in a unique conformation1. The formation is facilitated by the N3 protonation of cytosine residues. i-motif DNA can used in many bio-nanotechnological applications2 and recently has been shown its significance in cancer treatment3. We present microsecond range of MD simulations of a single stranded deprotonated DNA i-motif in ambient temperature and in high temperature, focusing primarily on thermodynamic and energetically favorable conformations. Figure1 (L to R): Showing the ribbon and cartoon representation of folded structure of 22mer single stranded DNA and the observation of cytosine pairing in acidic solution. 11 22 1 References [1] Guéron, M., Leroy, J.L, Current Opinion in Structural Biology 10(3),326-331(2000) [2] Liu, D., Balasubramian, S., Angewandte Chemie International 42(46), 5734-5736 (2003) [3] Ren, J., Qu, X., Trent, J.O., Chaires, J.B. Nucleic Acids Research 30, 2307–2315 (2002) Published results [4] Smiatek, J.,Chen, C., Liu, D., Heuer, A. The Journal of Physical Chemistry B, 115, 13788-13795 (2011) [5] Smiatek, J., Liu, D., Heuer, Current Physical Chemistry, 2, 115-123 (2012) [6] Smiatek, J., Janssen-Mueller, D., Friederich, R., Heuer, A. Physica A, 394, 136-144 (2014) Work in progress Currently, we are applying Bias-exchange metadynamics with the flavor of replica-exchange to study the i-motif unfolding. Computational pulling experiments via Steered-MD in 1D and 2D free energy landscapes of unfolding mechanism. Free energy landscape for crowed assemblies of DNA i-motif. Studying the stability of DNA i-motif using nucleotide defects by mutating the sequence as well as the effects of hairpin length on structural stability and conformations. Effects of sequence defects on i-motif The probability matrix provides the vital information regarding the occurrence of i-motif, hairpin and/or extended hairpin conformation from the free energy landscapes of different defects of i-motif. The tendency is the same but the effect in the high pH-regime is much weaker (if we calculate probability based on the ΔG values under acidic conditions, the effect would be much stronger) because then the i-motif is further stabilized by the H-bonds. 3C-2G 3C-1A 3C-1T 3C-2T 3C-7A Figure 3 : Comparison of free energy landscapes to show the effect of induced alteration on i-motif structure. The defects were induced using the COM of rC1/T22, rC1,T22/A11 Probability sequence i-motif hairpin Ext hairpin ΔG (kcal/mol) 0.09 0.07 0.84 -4.9 3C-7G 0.10 0.11 0.78 -2.2 3C-2G 0.05 0.86 -0.6 3C-1pDA 0.06 0.83 -- 3C-1pDT 0.03 0.90