single molecule in vivo analysis of toll-like receptor 9 and cpg dna interactiontoll样受体的单分子体内分析9和cpg dna的相互作用.pdf

Single Molecule In Vivo Analysis of Toll-Like Receptor 9 and CpG DNA Interaction 1 2 3 1 Jiji Chen , Suman Nag , Pierre-Alexandre Vidi , Joseph Irudayaraj * 1 Bindley Bioscience Center and Birck Nanotechnology Center, Department of Agricultural and Biological Engineering, Purdue University, West Lafayette, Indiana, United States of America, 2 Department of Chemical Sciences, Tata Institute of Fundamental Research, Mumbai, India, 3 Department of Basic Medical Sciences, Purdue University, West Lafayette, Indiana, United States of America 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 69 nM) compared to non CpG DNA (153 nM626 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