Single-Molecule Interactions of a Monoclonal Anti-DNA Antibody with DNA.
Single-Molecule Interactions of a Monoclonal Anti-DNA Antibody with DNA.
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DOI：
10.1007/s12668-016-0303-0
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发表时间：
2017-03
期刊：
影响因子：
3
通讯作者：
Litvinov RI
中科院分区：
文献类型：
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作者：
Nevzorova TA;Zhao Q;Lomakin YA;Ponomareva AA;Mukhitov AR;Purohit PK;Weisel JW;Litvinov RI
DNA与蛋白质的相互作用是关键的生物学过程所必需的，具有基础和实际意义。特别是，DNA与抗DNA抗体的结合是自身免疫病理中的一种致病机制，如系统性红斑狼疮。在这里，我们在单分子水平上测量了红斑狼疮小鼠表面附着的DNA和抗DNA单抗MRL4的结合和强制解结合。在基于光学陷阱的力光谱中，微观的抗体包被乳胶珠被聚焦的激光捕获，并反复接触到包被DNA的表面。在仔细辨别非特异性相互作用后，我们发现DNA-抗体断裂力谱有两个区域，反映了较弱(20-40 pN)和较强(>；40 pN)免疫复合体的形成，这意味着至少存在两个机械稳定性不同的结合态。其二维无力关闭速率约为2.2×10−3 S−1，过渡态距离约为0.94 nm，表观开启速率约为5.2 6 S−1，弹性常数为0.0021 pN/nm，表明该复合物是一种相对稳定但柔软易变形的大分子结构.DNA分子的拉伸弹性是单链DNA的特征，这表明MRL4抗体优先结合到一条DNA上。总之，这些结果提供了DNA-抗体复合体形成和强制解离的基本特征，有助于理解DNA-蛋白质相互作用的原理，并揭示了伴随抗DNA抗体形成的自身免疫性疾病的分子基础。
Interactions of DNA with proteins are essential for key biological processes and have both a fundamental and practical significance. In particular, DNA binding to anti-DNA antibodies is a pathogenic mechanism in autoimmune pathology, such as systemic lupus erythematosus. Here we measured at the single-molecule level binding and forced unbinding of surface-attached DNA and a monoclonal anti-DNA antibody MRL4 from a lupus erythematosus mouse. In optical trap-based force spectroscopy, a microscopic antibodycoated latex bead is trapped by a focused laser beam and repeatedly brought into contact with a DNA-coated surface. After careful discrimination of non-specific interactions, we showed that the DNA-antibody rupture force spectra had two regimes, reflecting formation of weaker (20–40 pN) and stronger (>40 pN) immune complexes that implies the existence of at least two bound states with different mechanical stability. The two-dimensional force-free off-rate for the DNA-antibody complexes was ~2.2 × 10−3 s−1, the transition state distance was ~0.94 nm, the apparent on-rate was ~5.26 s−1, and the stiffness of the DNA-antibody complex was characterized by a spring constant of 0.0021 pN/nm, suggesting that the DNA-antibody complex is a relatively stable, but soft and deformable macromolecular structure. The stretching elasticity of the DNA molecules was characteristic of single-stranded DNA, suggesting preferential binding of the MRL4 antibody to one strand of DNA. Collectively, the results provide fundamental characteristics of formation and forced dissociation of DNA-antibody complexes that help to understand principles of DNA-protein interactions and shed light on the molecular basis of autoimmune diseases accompanied by formation of anti-DNA antibodies.