Molecular Therapy - Nucleic Acids In Silico Aptamer Docking Studies: From a Retrospective Validation to a Prospective Case Study'TIM3 Aptamers Binding  Obdulia Rabal, Fernando Pastor, Helena Villanueva, Mario M Soldevilla, Sandra Hervas-Stubbs, Julen Oyarzabal  Molecular Therapy - Nucleic Acids  Volume 5, (January 2016) DOI: 10.1038/mtna.2016.84 Copyright © 2016 Official journal of the American Society of Gene & Cell Therapy Terms and Conditions

Figure 1 Predicted binding mode for the 3D modelled structures of two study cases. (a) Docking of the 29-nt modeled RNA aptamer to NF-κB. (b) Docking of the 38-nt modeled RNA aptamer to 30S ribosomal protein S8. In both cases, green structure corresponds to the crystal structure, pink to the lowest RMSD in the most populated cluster (RMSD of 8.5 Å for 29-nt/NF-κB and 3.9 Å for 38-nt/30 S ribosomal), blue to the lowest scoring structure within the most populated cluster (RMSD of 20.7 Å for 29-nt/NF-κB and 13.0 Å for 38-nt/30 S ribosomal) and salmon to the most stable structure from all the 3,000 poses (RMSD of 28.6 Å and 42.9 Å for a and b, respectively). nt, nucleotide; RMSD, root mean square deviation. Molecular Therapy - Nucleic Acids 2016 5, DOI: (10.1038/mtna.2016.84) Copyright © 2016 Official journal of the American Society of Gene & Cell Therapy Terms and Conditions

Figure 2 Predicted binding mode of TIM3 aptamers. (a) Predicted binding of TIM3-Apt1 and TIM3-Apt2 to TIM3 showing the two possible binding modes: binding site 1 (TIM3-Apt1 in turquoise, TIM3-Apt2 in orange) with residues important for TIM3 ligand binding depicted (Arg112, Asp121 and Arg69), and binding site 2 (TIM3-Apt1 in blue and TIM3-Apt2 in green) with Lys86 and Lys99 in stick. The constant priming sequence is displayed in thin ribbon view. (b,c) Surface view of the selected predicted complex TIM3-Apt1 – TIM3 (b) and TIM3-Apt2 – TIM3 (c). Molecular Therapy - Nucleic Acids 2016 5, DOI: (10.1038/mtna.2016.84) Copyright © 2016 Official journal of the American Society of Gene & Cell Therapy Terms and Conditions

Figure 3 Predicted binding mode of the TIM3 aptamers to TIM3 (pink ribbon) for the lowest DECK-RP scoring pose within the most populated cluster. Priming sequences of the aptamers are depicted in thin ribbon view and the variable 25-nt sequence in thick ribbon. For each aptamer, the TIM3 residues involved in hydrogen bond or ionic interactions, listed in Table 5, are shown. Molecular Therapy - Nucleic Acids 2016 5, DOI: (10.1038/mtna.2016.84) Copyright © 2016 Official journal of the American Society of Gene & Cell Therapy Terms and Conditions