In Vivo Binding Kinetics and Stoichiometry of Toll-Like Receptor 9 and CpG DNA Resolved by Multiparametric Single Molecule Techniques

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Toll-like receptor 9 (TLR9) activates the innate immune system in response to oligonucleotides rich in CpG whereas DNA lacking CpG could inhibit its activation. Although in vitro experiments demonstrate TLR9 binding to nucleic acids, the mechanism of how this receptor interacts with nucleic acid and becomes activated in live cells is far from behind understood. Here, we report on the successful implementation of single molecule tools, constituting fluorescence correlation spectroscopy (FCS), fluorescence cross-correlation spectroscopy (FCCS), photon count histogram (PCH) and fluorescence lifetime imaging (FLIM) to study the interaction of TLR9-GFP with Cy5 labeled oligonucleotide containing CpG or lacking CpG in live cells. We found that TLR9 predominantly forms homodimers (80%) before binding to a ligand and addition of CpG or non CpG DNA does not necessarily increase the proportion of TLR9 dimers. Experiments in living cells reveal that CpG DNA has a lower dissociation constant (62±9 nM) compared to non CpG DNA (153±26 nM) upon binding to TLR9 in live cells, suggesting that a motif specific binding affinity of TLR9 could be an important factor in its conformational change-dependant activation. Furthermore, it was observed that the CpG-TLR9 has a slower mobility compared to non CpG-TLR9 complex and both CpG and non CpG DNA bound to TLR9 with a 1:2 stoichiometry in vivo. Collectively, our findings establish an in vivo model for TLR9 activation by CpG DNA and highlight the significance of the need for an integrated and sensitive approach to obtain quantitative investigation of biomolecular interactions in live cells.