Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
Nat Biotechnol. 2011 Jun 26;29(7):659-64. doi: 10.1038/nbt.1882.
Several methods for characterizing DNA-protein interactions are available, but none have demonstrated both high throughput and quantitative measurement of affinity. Here we describe 'high-throughput sequencing'-'fluorescent ligand interaction profiling' (HiTS-FLIP), a technique for measuring quantitative protein-DNA binding affinity at unprecedented depth. In this approach, the optics built into a high-throughput sequencer are used to visualize in vitro binding of a protein to sequenced DNA in a flow cell. Application of HiTS-FLIP to the protein Gcn4 (Gcn4p), the master regulator of the yeast amino acid starvation response, yielded ~440 million binding measurements, enabling determination of dissociation constants for all 12-mer sequences having submicromolar affinity. These data revealed a complex interdependency between motif positions, allowed improved discrimination of in vivo Gcn4p binding sites and regulatory targets relative to previous methods and showed that sets of genes with different promoter affinities to Gcn4p have distinct functions and expression kinetics. Broad application of this approach should increase understanding of the interactions that drive transcription.
有几种方法可用于描述 DNA-蛋白质相互作用，但没有一种方法能够同时实现高通量和亲和力的定量测量。在这里，我们描述了“高通量测序-荧光配体相互作用分析”（HiTS-FLIP），这是一种用于以空前的深度测量定量蛋白质-DNA 结合亲和力的技术。在这种方法中，高通量测序仪内置的光学器件用于可视化在流池中的体外蛋白质与测序 DNA 的结合。将 HiTS-FLIP 应用于酵母氨基酸饥饿反应的主要调控因子 Gcn4 蛋白（Gcn4p），得到了约 4.4 亿次结合测量值，从而确定了所有具有亚毫摩尔亲和力的 12 -mer 序列的离解常数。这些数据揭示了基序位置之间的复杂相互依存关系，相对于以前的方法，提高了对体内 Gcn4p 结合位点和调控靶标的区分能力，并表明具有不同启动子与 Gcn4p 亲和力的基因集具有不同的功能和表达动力学。这种方法的广泛应用应该会增加对驱动转录的相互作用的理解。
