Composition of TLR3-dsRNA complexes. (A) The stoichiometry of complexes formed by TLR3ecd and 90-bp dsRNA was measured by titrating TLR3ecd against a fixed dsRNA concentration (1 μM) and analyzing the mixture by gel filtration (GF) at pH 6.0. For this ligand, the stoichiometry is 4:1 (TLR3ecd:dsRNA), because the only peak observed at this ratio corresponds to the complex, and, at higher and lower ratios, unbound TLR3ecd and dsRNA were apparent. (B) The stoichiometries of complexes formed by dsRNA of different lengths as determined by GF. Ligands smaller than 48 bp did not form stable complexes (SI Fig. 6). Sedimentation equilibrium (C) and sedimentation velocity analyses (D) of TLR3ecd and dsRNA (48 bp) mixtures at pH 5.5. At a 2:1 loading ratio, a single species is observed with the molecular mass expected for a complex that consists of two TLR3ecd and one dsRNA molecule (SI Appendix 1). Standard errors are indicated in C. Note that in A and D, peak heights reflect the fact that TLR3ecd's coefficient of absorption is less than that of dsRNA. Mixture compositions in A, C, and D are reported as the molar ratio of TLR3ecd:dsRNA. The predicted molecular mass of 48-bp dsRNA is 31.1 kDa.