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On-Chip Expression of Transcription Factors and Force-Based Interaction Analysis with DNA

转录因子的芯片表达以及与 DNA 的基于力的相互作用分析

基本信息

批准号：
211006235
负责人：
Professor Dr. Hermann E. Gaub
金额：
--
依托单位：
Lehrstuhl für Experimentelle Physik - Biophysik
依托单位国家：
德国
项目类别：
Research Grants
财政年份：
2012
资助国家：
德国
起止时间：
2011-12-31 至 2017-12-31
项目状态：
已结题

来源：
https://gepris.dfg.de/gepris/projekt/211006235?language=en
关键词：
Chip Expression Transcription Factors Force

项目摘要

Proteins regulate gene expression and their products may in turn regulate other genes. A highly en-tangled multi-component network arises upon which life is built. Needless to mention that deciphering this network requires the tracing of an exceedingly growing number of interaction partners and their quantification in order to characterize the quality of their interactions. A variety of highly parallel assays were developed in the past and most of them were hampered by a lack of sufficient sensitivity and/or selectivity. More independent measured values of a given interaction are needed to expand the parameter space into which these interactions are projected. The proposal here suggests to measure the interaction forces between proteins and DNA and by means of this to provide an additional parameter that characterizes the interaction. A highly parallel chip format is proposed which combines the expression of the proteins in soft rubber micro-fluidics with fluorescence-based binding force measurements via molecular force balances. As proof of principle a set of transcription factors from yeast will be tested against a matrix of ten different target sequences and for six different unbinding forces and compared to literature results. An expansion by at least one order of magnitude in integration density is envisioned which then allows the quantitative screening of a large number of interaction partners in particularly also weak binders in parallel. We will then expand our collaborations to characterize drosophila transcription factors based on interaction forces.

蛋白质调节基因的表达，其产物可以反过来调节其他基因。一个高度纠缠的多成分网络出现了，生命就建立在这个网络上。不用说，破译这个网络需要跟踪数量急剧增加的互动伙伴，并对其进行量化，以表征其互动的质量。过去开发了多种高度平行的测定，并且它们中的大多数由于缺乏足够的灵敏度和/或选择性而受到阻碍。需要给定相互作用的更独立的测量值来扩展这些相互作用被投影到其中的参数空间。这里的建议建议是测量蛋白质和DNA之间的相互作用力，并通过这种方式提供一个额外的参数，表征相互作用。提出了一种高度并行的芯片格式，它结合了在软橡胶微流体与基于荧光的结合力测量通过分子力平衡的蛋白质的表达。作为原理证明，将针对十种不同靶序列的基质和六种不同的解结合力测试一组来自酵母的转录因子，并与文献结果进行比较。设想在整合密度上扩展至少一个数量级，这然后允许平行地定量筛选大量相互作用配偶体，特别是弱结合剂。然后，我们将扩大我们的合作，以确定果蝇转录因子的基础上相互作用力。