Massively parallel measurements of molecular interaction kinetics on a microfluidic platform
Massively parallel measurements of molecular interaction kinetics on a microfluidic platform
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DOI：
10.1073/pnas.1206011109
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发表时间：
2012-10-09
影响因子：
11.1
通讯作者：
Maerkl, Sebastian J.
中科院分区：
文献类型：
--
作者：
Geertz, Marcel;Shore, David;Maerkl, Sebastian J.
定量生物学需要定量数据。不存在能够在单个实验中获得数百个独立的动力学结合测量的高通量技术。我们提出了一种集成微流体装置 (k-MITOMI)，用于同时表征 768 种生物分子相互作用的动力学。我们将 k-MITOMI 应用于转录因子 (TF)-DNA 相互作用的动力学分析，测量了小鼠 TF Zif268 以及酵母 TF Tye7p、Yox1p 和 Tbf1p 的详细动力学景观。我们通过在单个设备上并行表达、纯化和表征 27 个其他酵母转录因子，证明了 k-MITOMI 的集成性质。总体而言，我们获得了 223 种独特分子相互作用的 2,388 条缔合和解离曲线，平衡解离常数范围为 2 x 10(-6) M 至 2x 10(-9) M，解离速率常数约为 6 s(-1) 至 8.5 x 10(-3) s(-1)。 3个TF家族的关联速率常数是一致的，范围从3.7 x 10(6) M-1 s(-1)到9.6 x 10(7) M-1 s(-1)，并且远低于扩散极限。我们期望 k-MITOMI 将有助于我们对生物系统的定量理解，并加速工程系统的开发和表征。
Quantitative biology requires quantitative data. No high-throughput technologies exist capable of obtaining several hundred independent kinetic binding measurements in a single experiment. We present an integrated microfluidic device (k-MITOMI) for the simultaneous kinetic characterization of 768 biomolecular interactions. We applied k-MITOMI to the kinetic analysis of transcription factor (TF)-DNA interactions, measuring the detailed kinetic landscapes of the mouse TF Zif268, and the yeast TFs Tye7p, Yox1p, and Tbf1p. We demonstrated the integrated nature of k-MITOMI by expressing, purifying, and characterizing 27 additional yeast transcription factors in parallel on a single device. Overall, we obtained 2,388 association and dissociation curves of 223 unique molecular interactions with equilibrium dissociation constants ranging from 2 x 10(-6) M to 2x 10(-9) M, and dissociation rate constants of approximately 6 s(-1) to 8.5 x 10(-3) s(-1). Association rate constants were uniform across 3 TF families, ranging from 3.7 x 10(6) M-1 s(-1) to 9.6 x 10(7) M-1 s(-1), and are well below the diffusion limit. We expect that k-MITOMI will contribute to our quantitative understanding of biological systems and accelerate the development and characterization of engineered systems.