Bindley Bioscience Center, Department of Agricultural and Biological Engineering, Purdue University, West Lafayette, Indiana, United States of America.
PLoS One. 2011 Apr 4;6(4):e17991. doi: 10.1371/journal.pone.0017991.
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.
Toll 样受体 9（TLR9）在富含 CpG 的寡核苷酸的刺激下激活先天免疫系统，而不含 CpG 的 DNA 可以抑制其激活。然而，TLR9 如何与核酸相互作用并在活细胞中被激活的机制尚不清楚。在这里，我们成功地实施了单分子工具，包括荧光相关/交叉相关光谱（FCS 和 FCCS）和带有荧光寿命成像（FLIM）的光子计数直方图（PCH），以研究 TLR9-GFP 与 Cy5 标记的含有 CpG 或不含 CpG 的寡核苷酸在活 HEK 293 细胞中的相互作用。我们的研究结果表明：i）TLR9 在与配体结合之前主要形成同源二聚体（80%），而进一步添加 CpG 或非 CpG DNA 不一定会增加 TLR9 二聚体的比例；ii）CpG DNA 与 TLR9 结合的解离常数（62 nM±9 nM）低于非 CpG DNA（153 nM±26 nM），这表明 TLR9 的特定基序结合亲和力可能是启动构象变化依赖的激活的一个重要因素；iii）CpG 和非 CpG DNA 都以 1∶2 的化学计量比在体内与 TLR9 结合。总之，通过我们的研究结果，我们使用单分子荧光技术为单细胞研究建立了一个 TLR9 与 CpG DNA 结合和激活的体内模型。
