Nature:用于蛋白质分析的单分子DNA技术
2014/11/28
11月27日,《自然》杂志期刊发表了George Church 及同事开发出用于并行蛋白相互作用分析、借助用于蛋白功能分析的单分子DNA方法的一个“单分子相互作用测序”(SMI-seq)技术。


George Church 及同事开发出用于并行蛋白相互作用分析、借助用于蛋白功能分析的单分子DNA方法的一个“单分子相互作用测序”(SMI-seq)技术。

SMI-seq的工作原理是,通过核糖体显示或酶结合将蛋白耦合到DNA识别符或“条码”上。

这些条码化的蛋白然后在水溶液中被一起分析,并在一个聚丙烯酰胺薄膜中被固定。

接下来,条码RNA被放大,并通过DNA测序被分析。该方法可以实现精确的蛋白量化以及对分子结合亲和力和特异性的同时测定。

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  • Multiplex single-molecule interaction profiling of DNA-barcoded proteins

    In contrast with advances in massively parallel DNA sequencing1, high-throughput protein analyses2, 3, 4 are often limited by ensemble measurements, individual analyte purification and hence compromised quality and cost-effectiveness. Single-molecule protein detection using optical methods5 is limited by the number of spectrally non-overlapping chromophores. Here we introduce a single-molecular-interaction sequencing (SMI-seq) technology for parallel protein interaction profiling leveraging single-molecule advantages. DNA barcodes are attached to proteins collectively via ribosome display6 or individually via enzymatic conjugation. Barcoded proteins are assayed en masse in aqueous solution and subsequently immobilized in a polyacrylamide thin film to construct a random single-molecule array, where barcoding DNAs are amplified into in situ polymerase colonies (polonies)7 and analysed by DNA sequencing. This method allows precise quantification of various proteins with a theoretical maximum array density of over one million polonies per square millimetre. Furthermore, protein interactions can be measured on the basis of the statistics of colocalized polonies arising from barcoding DNAs of interacting proteins. Two demanding applications, G-protein coupled receptor and antibody-binding profiling, are demonstrated. SMI-seq enables ‘library versus library’ screening in a one-pot assay, simultaneously interrogating molecular binding affinity and specificity.

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