The hAGO2 protein and siRNA (17 bp) complex utilized for MD studies was obtained from the model reported in literature (18). The topology and coordinate inputs of protein and RNA complex were prepared using the xleap module in AMBER 14 (19). The protonation state of the amino acids was assigned using PDBPQR program at pH 7.0 in AMBER force field. The system was neutralized using KCl ions and excess ions (100mM) were added to mimic the physiological conditions. Mg²⁺ ions at the cleavage site were parameterized using a reported procedure (20,21). The partial charges for the 2′-5′-linked modifications were calculated at the nucleotide level using Gaussian 09 (HF/6-31G*) package (22). The calculated charges were then fitted using the RESP algorithm (23). The force field parameters for 2′-5′ linkage were derived from the previously reported crystal structure and MD studies (8). The force fields used for the RNA and the protein are bsc0_XOL3 (24–27) and ff12SB (19), respectively. Using TIP3P water molecules, the system was solvated up to 8 Å from any of the solute atoms. The equilibration and MD simulations were performed as reported earlier (28,29). Unrestrained production MD simulations were performed for 250 ns using CUDA version of pmemd (30) in a GPU accelerated version (31,32) of AMBER 14 (19).

Particle mesh Ewald method was utilized for calculating the contributions from the non-bonded interactions with a cut-off of 10 Å. SHAKE was used to treat the bonds involving hydrogen atoms. The unrestrained MD simulations were performed in NPT ensemble of 2 fs time step. One atmospheric constant pressure was maintained using Bendersen weak-coupling barostat in a time constant of 1 ps (33). The MD simulations temperature (300 K) was maintained by Bendersen thermostat of 4 ps. MD trajectories were saved for every ps and then extracted at every 5 ps time interval for further analysis. Root mean square deviations (RMSDs) were calculated for the backbone heavy atoms of protein (CA, C and N) and RNA (P, O5′, C5′, C4′, C3′/C2′ and O3′) using CPPTRAJ module in AMBER 14 (34). The X3DNA package was utilized to compute the RNA helical parameters and backbone dihedral angles (35). Hydrogen bonds were considered based upon the heavy atom distance (donor-acceptor) cut-off of ≤3.3 Å and an angle cut-off of ≥135. The distance between the two heavy atoms were calculated using PTRAJ module. MD trajectories were visualized using UCSF Chimera (36). The free energy of siRNA–hAGO2 complex binding was computed using Molecular Mechanics/Poisson–Boltzmann Surface Area (MM/PBSA) method using an earlier report (37).