FIGURE 3: TL-532 is a well-defined dsRNA molecule. (A-B) Characterization of the sense strand 5′-rI(10)-rA(50)-rI(10)-3' and the antisense strand 5′-rC(10)-rU(50)-rC(10)-3' identities was performed by LC/MS. (C) The purity of the duplex RNA 5′-rI(10)-rA(50)-rI(10)-3' annealed with 5′-rC(10)-rU(50)-rC(10)-3' was evaluated by native SEC. (D) TL-532 70bp dsRNA was evaluated for its native gel profile in an 6% PAGE gel. 1 µg of each dsRNAs was deposited before visualization using bromide ethidium. (E) The double stranded RNA nature of TL-532 was evaluated by RNAse III limited digestion. 1 μg of each dsRNA was digested with 1 unit of RNAse III at 37°C for 10 min and deposited in a 6% native PAGE gel, before visualization using bromide ethidium. (F) The melting curve of TL-532 was evaluated using a Q-PCR melting curve program as described in Material and Methods section. (G) The half-life of TL-532 was evaluated in human serum. 2 μg of each dsRNA was incubated for increasing time in 50% human serum at 37°C and deposited in 2.5% agarose gel, before visualization using bromide ethidium. % = percentage of band intensity over the unincubated condition (0h). (H) Binding affinity of TL-532 to TLR3 Extra Cellular Domain (ECD) was determined by SPRi at pH=5.5. Data are representative of at least two (G) or three independent assays (A-F, H). Legend: LC/MS = Liquid Chromatography Mass Spectrometry; SEC = Size Exclusion Chromatography; SPRi = Surface Plasmon Resonance imaging.