Department of Molecular Biology and NCCR program Frontiers in Genetics, University of Geneva, Geneva 4, Switzerland.
Proc Natl Acad Sci U S A. 2012 Oct 9;109(41):16540-5. doi: 10.1073/pnas.1206011109. Epub 2012 Sep 24.
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 × 10(-6) M to 2 × 10(-9) M, and dissociation rate constants of approximately 6 s(-1) to 8.5 × 10(-3) s(-1). Association rate constants were uniform across 3 TF families, ranging from 3.7 × 10(6) M(-1) s(-1) to 9.6 × 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.
定量生物学需要定量数据。目前还没有能够在单个实验中获得数百个独立动力学结合测量的高通量技术。我们提出了一种集成的微流控装置（k-MITOMI），用于同时对 768 种生物分子相互作用进行动力学特征分析。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 的详细动力学图谱。我们通过在单个设备上同时表达、纯化和表征 27 种额外的酵母转录因子，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 个结合和解离曲线，平衡解离常数范围从 2×10(-6) M 到 2×10(-9) M，解离速率常数约为 6 s(-1) 到 8.5×10(-3) s(-1)。结合速率常数在 3 个 TF 家族中是一致的，范围从 3.7×10(6) M(-1) s(-1) 到 9.6×10(7) M(-1) s(-1)，远低于扩散极限。我们预计 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。
