Computational protocol: The effect of the neutral cytidine protonated analogue pseudoisocytidine on the stability of i-motif structures

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Protocol publication

[…] Samples for NMR experiments were dissolved (in Na+ form) in either D2O or 9:1 H2O/D2O (25 mM sodium phosphate buffer) in presence of 100 mM NaCl. Experiments were carried out at different pH values, ranging from 3.5 to 7. The pH was adjusted by adding aliquots of concentrated solution of either DCl or NaOD. All NMR spectra were acquired in Bruker spectrometers operating at 600 and 800 MHz, equipped with cryoprobes and processed with the TOPSPIN software. In the experiments in D2O, presaturation was used to suppress the residual H2O signal. A jump-and-return pulse sequence was employed to observe the rapidly exchanging protons in 1D H2O experiments. NOESY spectra in D2O and 9:1 H2O/D2O were acquired with mixing times of 150, 250 and 300 ms. TOCSY spectra were recorded with the standard MLEV-17 spin-lock sequence and a mixing time of 80 ms. In most of the experiments in H2O, water suppression was achieved by including a WATERGATE module in the pulse sequence prior to acquisition. The spectral analysis program SPARKY was used for semiautomatic assignment of the NOESY cross-peaks and quantitative evaluation of the NOE intensities. [...] Structures were calculated with the program DYANA and further refined with the SANDER module of the molecular dynamics package AMBER 12.0. Resulting DYANA structures were taken as starting points for the AMBER refinement, consisting of an annealing protocol in vacuo, followed by trajectories of 500 ps each in which explicit solvent molecules were included and using the Particle Mesh Ewald method to evaluate long-range electrostatic interactions. The specific protocols for these calculations have been describe elsewhere. The BSC1 force field was used to describe the DNA, and the TIP3P model was used to simulate water molecules. Charges for pseudoisocytosine were calculated using ab initio calculations with RESP/6-31G(d) in single base models. Analysis of the representative structures was carried out with the program MOLMOL. The refined structures are deposited in the PDB (code: 5NIP). […]

Pipeline specifications

Software tools Sparky, MOLMOL
Applications NMR-based proteomics analysis, Protein structure analysis
Chemicals Cytidine