TY - JOUR
T1 - Single molecule In Vivo analysis of toll-like receptor 9 and CpG DNA interaction
AU - Chen, Jiji
AU - Nag, Suman
AU - Vidi, Pierre Alexandre
AU - Irudayaraj, Joseph
PY - 2011
Y1 - 2011
N2 - 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.
AB - 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.
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U2 - 10.1371/journal.pone.0017991
DO - 10.1371/journal.pone.0017991
M3 - Article
C2 - 21483736
AN - SCOPUS:79953679912
SN - 1932-6203
VL - 6
JO - PloS one
JF - PloS one
IS - 4
M1 - e17991
ER -