By the PAZ domain, docking output data obtained from 2 different sources (iGEMDOCK and docking server) were used. The validity of data were insured by several docking runs, repeated internal compounds as well as by using 2 different programs in obtaining the results. Interestingly, the data obtained from the two docking resources (iGEMDOCK and docking 
server) were in good agreement. For correlating the previously obtained data on the suppression of gene expression with the MedChemExpress ��-Sitosterol ��-D-glucoside results obtained from the docking server, the docking output data are plotted MedChemExpress 1934-21-0 against the Renilla luciferase normalized by firefly luciferase data (RL/FL) in Fig. 4. The lower RL/FL indicates lower gene expression and potent RNAi activity. In contrast, higher RL/FL indicates lower potency of siRNA. Correlation of free and total intermolecular energy: From Fig. 4A, we see that the lower values of free energy and total intermolecular energy (more negative) are accompanied by higher RL/FL ratio (lower RNAi efficiency). That is, lower free energy and total intermolecular energy promote better in vivo activity. We observe a considerable negative correlation between RNAi efficacy and the estimated free energy, albeit with a low Pearson’s correlation coefficient (R = 20.168). The total and free energy are indicators of binding strength. Strong binding is associated with the release of free energy, which means lower binding strength is associated with better RNAi efficiency. Gu et al., suggested that PAZ domain acts as a handle to peel 1662274 off the passenger strand in miRNA-like mismatch-containing duplexes [21], thus, suggesting a strong interaction of PAZ domain with siRNA. In this context, the PAZ domain was found to bind natural nucleotides with weakto-moderate binding affinity [37]. Furthermore, the binding data from Ago1 and Ago2 indicates that PAZ is not a high-affinity nucleic acids-binding module [37]. Inhibitory constant (Ki): The compounds with a lower inhibitory constant are proposed to bind more strongly than thoseTable 4. Correlation analysis for RNAi activity and the measured parameters produced by the docking server.(indicated by asterisk), while Y641, R652, Y681 and K704 (indicated by rectangles) were important residues for hydrogen bonding (Fig. 3B). Figure 3C is a 2 dimensional plotting of the interaction of compound tt with residues of PAZ domain generated by ligand interaction scripts DS visualizer. The main coordinates of interactions agree with iGEMDOCK output (as mentioned above).vDw + Hbond + Inhibition constant Ki desolvation energyTotal intermolecular Electrostatic energy energyEstimated free energyInteraction surface0.0.1.20.5278*20.20.20.21.0.9642*0.1.0.5828*1.siRNA Recognition by PAZ DomainFigure 2. Docked poses of compounds with PAZ domain. Fig. 2A is a representative diagram showing the docked poses in the binding site of PAZ domain. From the docked poses, the best pose with the lowest energy was used to align with other compounds in the binding 18204824 site of PAZ domain. The docking profiles of compounds ordered by total interaction energy are shown in Fig. 2B. The numerical values of data can be viewed in Table 1. The clustering of compounds is shown in Fig. 2C. The dendrogram is generated by iGEMDOCK post analysis tools and viewed by tree view software. doi:10.1371/journal.pone.0057140.gFigure 3. Interaction parameters of compounds with the PAZ domain. A Hierarchical clustering and the profile of interactions of various compounds with PAZ domain is repr.By the PAZ domain, docking output data obtained from 2 different sources (iGEMDOCK and docking server) were used. The validity of data were insured by several docking runs, repeated internal compounds as well as by using 2 different programs in obtaining the results. Interestingly, the data obtained from the two docking resources (iGEMDOCK and docking server) were in good agreement. For correlating the previously obtained data on the suppression of gene expression with the results obtained from the docking server, the docking output data are plotted against the Renilla luciferase normalized by firefly luciferase data (RL/FL) in Fig. 4. The lower RL/FL indicates lower gene expression and potent RNAi activity. In contrast, higher RL/FL indicates lower potency of siRNA. Correlation of free and total intermolecular energy: From Fig. 4A, we see that the lower values of free energy and total intermolecular energy (more negative) are accompanied by higher RL/FL ratio (lower RNAi efficiency). That is, lower free energy and total intermolecular energy promote better in vivo activity. We observe a considerable negative correlation between RNAi efficacy and the estimated free energy, albeit with a low Pearson’s correlation coefficient (R = 20.168). The total and free energy are indicators of binding strength. Strong binding is associated with the release of free energy, which means lower binding strength is associated with better RNAi efficiency. Gu et al., suggested that PAZ domain acts as a handle to peel 1662274 off the passenger strand in miRNA-like mismatch-containing duplexes [21], thus, suggesting a strong interaction of PAZ domain with siRNA. In this context, the PAZ domain was found to bind natural nucleotides with weakto-moderate binding affinity [37]. Furthermore, the binding data from Ago1 and Ago2 indicates that PAZ is not a high-affinity nucleic acids-binding module [37]. Inhibitory constant (Ki): The compounds with a lower inhibitory constant are proposed to bind more strongly than thoseTable 4. Correlation analysis for RNAi activity and the measured parameters produced by the docking server.(indicated by asterisk), while Y641, R652, Y681 and K704 (indicated by rectangles) were important residues for hydrogen bonding (Fig. 3B). Figure 3C is a 2 dimensional plotting of the interaction of compound tt with residues of PAZ domain generated by ligand interaction scripts DS visualizer. The main coordinates of interactions agree with iGEMDOCK output (as mentioned above).vDw + Hbond + Inhibition constant Ki desolvation energyTotal intermolecular Electrostatic energy energyEstimated free energyInteraction surface0.0.1.20.5278*20.20.20.21.0.9642*0.1.0.5828*1.siRNA Recognition by PAZ DomainFigure 2. Docked poses of compounds with PAZ domain. Fig. 2A is a representative diagram showing the docked poses in the binding site of PAZ domain. From the docked poses, the best pose with the lowest energy was used to align with other compounds in the binding 18204824 site of PAZ domain. The docking profiles of compounds ordered by total interaction 
energy are shown in Fig. 2B. The numerical values of data can be viewed in Table 1. The clustering of compounds is shown in Fig. 2C. The dendrogram is generated by iGEMDOCK post analysis tools and viewed by tree view software. doi:10.1371/journal.pone.0057140.gFigure 3. Interaction parameters of compounds with the PAZ domain. A Hierarchical clustering and the profile of interactions of various compounds with PAZ domain is repr.
