TLR9 senses single-stranded (ss) DNA. Compared with the relatively uniform shape of the A form dsRNA molecule, the TLR3 ligand, the molecular signature of ssDNA remains obscure. Agonistic ssDNA contains an unmethylated CpG motif that is common to bacterial and viral DNA, as well as a characteristic of apoptotic host DNA. The mechanisms for TLR9-mediated recognition of agonistic DNA have not been fully clarified. Different models have been proposed, such as the "induced-fit" mechanism where conformational changes in the TLR9-ECD are required to initiate TLR9 signaling upon DNA binding, or the "baseless" assumption where the sugar backbone alone in the DNA determine its interaction with TLR9-ECD. Other factors provide versatility for TLR9-mediated DNA recognition, such as HMGB1, a nuclear DNA-binding protein released from necrotic cells, or DNA-antibodies, or malarial hemozoin. Despite the enormous data of TLR9-mediated DNA recognition, important questions regarding the structural basis of DNA induced-TLR9 signaling remain open. Currently we are focusing on the TLR9-ECD consisting of 25 LRRs together with the N and C-terminal capping domains. Our goals are 1) to determine the structure of TLR9-ECD, 2) to define the DNA-binding site, and 3) to explain the observed versatility and specificity of TLR9-mediated DNA recognition at the molecular level. Our ongoing research could help answer some of these questions including if there is conformational change upon DNA binding, whether the binding is baseless, and how TLR9 dimerization occurs. [unreadable] [unreadable] In addition to the intact TLR9 ectodomain, we have also examined 13 human TLR9 chimeric constructs. The whole C-terminal cap and some of the C-terminal LRRs from TLR9 were truncated and the C-capping domain from human TLR3 was fused. Constructs #1 has 24 LRR from TLR9 and the LRRs were truncated one by one when the other constructs were made. The last, construct #13 has 12 LRR from TLR9. The TLR9 expression clones were made by two destination vectors, pDEST-8 and pDEST-612. The former has His and flag tags at the C-terminal and the latter has one more fused GFP tag besides His and flag tags. The expression clones of Construct #1 were transformed into E.coli DH10Bac cells and bacmid DNA was generated. I am now working on other TLR9 constructs and hope they will give better results.[unreadable] [unreadable] TLR8 recognizes ssRNA. We are attempting to express the TLR8 ectodomain in sufficient quantity to enable crystallization experiments. Different expression vectors are being explored.