Computational protocol: The solution structure of double helical arabino nucleic acids (ANA and 2′F-ANA): effect of arabinoses in duplex-hairpin interconversion

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Protocol publication

[…] Sample gapmer design [gap(FA)] () was suspended in 500 µL of either D2O or H2O/D2O 9:1 in 25 mM phosphate buffer, 100 mM NaCl, pH 7. NMR spectra were acquired in Bruker spectrometers operating at 600, 700 or 800 MHz and were processed with Topspin software. 19F and 1H 1D melting experiments, double quantum filtered correlation scpectroscopy (DQF-COSY), total correlation spectroscopy and nuclear overhauser effect spectroscopy (NOESY), were recorded in D2O and H2O/D2O 9:1. The NOESY spectra were acquired with mixing times of 50, 100, 150, 250 and 300 ms, and the total correlation spectroscopy spectra were recorded with standard MLEV-17 spin-lock sequence at 80 ms mixing time. For highly concentrated samples, NOESY spectra in H2O were acquired with 50 and 100 ms mixing times. For 2D experiments in H2O, water suppression was achieved by including a WATERGATE () module in the pulse sequence before acquisition. 2D experiments in D2O were carried out at temperatures ranging from 5°C to 35°C, whereas spectra in H2O were recorded at 5°C to reduce the exchange with water. 19F resonances were assigned from 19F detected using heteronuclear overhauser effect spectroscopy (HOESY) spectra (τm = 200 ms) (). The spectral analysis program Sparky () was used for semiautomatic assignment of the NOESY cross-peaks and quantitative evaluation of the nuclear overhauser effect (NOE) intensities. [...] Structures were calculated with the SANDER module of the MD package AMBER 7.0 (). Starting models of the arabino duplexes were built in the A- and B- canonical structures using SYBYL. These structures were taken as starting points for the AMBER refinement, which started with a short run in vacuo (using hexahydrated Na+ counterions placed near the phosphates to neutralize the system). The resulting structures from in vacuo calculations were refined, including explicit solvent, periodic boundary conditions and the particle-mesh Ewald method, to evaluate long-range electrostatic interactions (). Thus, the structures obtained in the previous step were placed in the center of a water-box with around 4000 water molecules and 20 sodium counterions to obtain electroneutral systems. We used the parmbsc0 () revision of the parm99 force field (,), including suitable parameters for the arabino and 2′F-arabino derivatives extracted from Noy et al. (). The TIP3P model was used to describe water molecules (). The protocol for the MD refinement consisted of an equilibration period of 160 ps using a standard equilibration process (), followed by 10 independent 500 ps runs. Final structures were obtained by averaging the last ps of individual trajectories and further relaxation of the structure. Analysis of the representative structures and the MD trajectories was carried out with the programs CURVES V5.1 (), MOLMOL (), the analysis tools of AMBER and SYBYL and additional ‘in–house’ programs. […]

Pipeline specifications

Software tools Sparky, MOLMOL
Applications NMR-based proteomics analysis, Protein structure analysis
Diseases Neurofibromatosis 1
Chemicals Arabinonucleotides, Fluorine, Nucleotides