Single molecule in vivo analysis of toll-like receptor 9 and CpG DNA interaction.
Single molecule in vivo analysis of toll-like receptor 9 and CpG DNA interaction.
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DOI：
10.1371/journal.pone.0017991
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发表时间：
2011-04-04
期刊：
影响因子：
3.7
通讯作者：
Irudayaraj J
中科院分区：
文献类型：
--
作者：
Chen J;Nag S;Vidi PA;Irudayaraj J
Toll样受体9（TLR 9）激活先天免疫系统以响应富含CpG的寡核苷酸，而缺乏CpG的DNA可抑制其激活。然而，TLR 9如何与核酸相互作用并在活细胞中被激活的机制还不清楚。在这里，我们报告的单分子工具，构成荧光相关/互相关光谱（FCS和FCCS）和光子计数直方图（PCH）与荧光寿命成像（FLIM）的成功实施，研究TLR 9-GFP与Cy 5标记的寡核苷酸的相互作用含有CpG或缺乏CpG在活HEK 293细胞。我们的研究结果表明：i）TLR 9在与配体结合之前主要形成同源二聚体（80%），进一步加入CpG或非CpG DNA并不一定会增加TLR 9二聚体的比例，ii）CpG DNA具有较低的解离常数（62 nM±9 nM）与非CpG DNA相比（153 nM±26 nM），表明TLR 9的基序特异性结合亲和力可能是建立构象变化依赖性激活的重要因素，和iii）CpG和非CpG DNA在体内均以1：2的化学计量比与TLR 9结合。总的来说，通过我们的研究结果，我们建立了一个体内模型的TLR 9结合和激活的CpG DNA使用单分子荧光技术的单细胞研究。
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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影响因子：
2.4
作者：
Gannot, I;Ron, I;Gandjbakhche, A
通讯作者：
Gandjbakhche, A
影响因子：
30.5
作者：
Latz, Eicke;Verma, Anjali;Golenbock, Douglas T.
通讯作者：
Golenbock, Douglas T.
影响因子：
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作者：
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作者：
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通讯作者：
Fradin, C
DOI：
10.1073/pnas.0710779105
发表时间：
2008-01-08
影响因子：
11.1
作者：
Leonard, Joshua N.;Ghirlando, Rodolfo;Segal, David M.
通讯作者：
Segal, David M.