Abstract
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. However, the mechanism of how TLR9 interacts with nucleic acid and becomes activated in live cells is not well understood. Here, we report on the successful implementation of single molecule tools, constituting fluorescence correlation/cross-correlation spectroscopy (FCS and FCCS) and photon count histogram (PCH) with fluorescence lifetime imaging (FLIM) to study the interaction of TLR9-GFP with Cy5 labeled oligonucleotide containing CpG or lacking CpG in live HEK 293 cells. Our findings show that i) TLR9 predominantly forms homodimers (80%) before binding to a ligand and further addition of CpG or non CpG DNA does not necessarily increase the proportion of TLR9 dimers, ii) CpG DNA has a lower dissociation constant (62 nM±9 nM) compared to non CpG DNA (153 nM±26 nM) upon binding to TLR9, suggesting that a motif specific binding affinity of TLR9 could be an important factor in instituting a conformational change-dependant activation, and iii) both CpG and non CpG DNA binds to TLR9 with a 1∶2 stoichiometry in vivo. Collectively, through our findings we establish an in vivo model of TLR9 binding and activation by CpG DNA using single molecule fluorescence techniques for single cell studies.
Highlights
Toll-like receptors (TLRs), one of the pattern-recognition receptors (PRRs), are the key sensors of microbial infection in mammals [1,2]
Through quantitative photon counting histogram (PCH) analysis, we find that TLR9 predominantly forms homodimers before binding to the nucleic acids and the percentage of dimers does not change upon interaction with either CpG or non CpG DNA
By quantitatively estimating the fraction of bound complex, we found that the percentage of dimers formed did not change irrespective of the interacting partner, either the CpG or non CpG DNA (Figure 2b)
Summary
Toll-like receptors (TLRs), one of the pattern-recognition receptors (PRRs), are the key sensors of microbial infection in mammals [1,2]. The prevailing paradigm attributes the activation of TLR9 to the recognition of CpG containing DNA, reports present different explanation on the ability of TLR9 to discriminate nucleic acids based on in vitro studies. It was further demonstrated that binding to CpG DNA could induce conformational changes and subsequently reduce the diameter of the extracellular domain [14]. All of these investigations relating to the interaction of TLR9 with nucleic acids are based upon in vitro assay, no report has examined such interactions in live cells. Tools to elucidate TLR9 activation or inactivation in vivo have the potential to broadly impact TLR9 biology and assist in the development of more effective therapeutics
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