Multiplex single-molecule interaction profiling of DNA-barcoded proteins.
Multiplex single-molecule interaction profiling of DNA-barcoded proteins.
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DOI：
10.1038/nature13761
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发表时间：
2014-11-27
期刊：
影响因子：
64.8
通讯作者：
Church, George M.
中科院分区：
文献类型：
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作者：
Gu, Liangcai;Li, Chao;Aach, John;Hill, David E.;Vidal, Marc;Church, George M.
与大规模并行DNA测序的进展相比，高通量蛋白质分析通常受到整体测量、单个分析物纯化的限制，因此质量和成本效益受到影响。使用光学方法实现的单分子（SM）蛋白质检测受到光谱不重叠发色团数量的限制。在这里，我们介绍了一个单一的分子相互作用测序（SMI-Seq）技术的并行蛋白质相互作用分析利用SM的优势。DNA条形码经由核糖体展示共同地或经由酶促缀合单独地附接至蛋白质。将条形码化蛋白质在水溶液中体外测定，随后固定在聚丙烯酰胺（PAA）薄膜中以构建随机SM阵列，其中将条形码化DNA扩增到原位聚合酶菌落（polonies）中并通过DNA测序进行分析。该方法允许精确定量各种蛋白质，其理论最大阵列密度超过每平方毫米一百万个波隆尼。此外，蛋白质相互作用可以基于由相互作用蛋白质的条形码化DNA产生的共定位的polonies的统计来测量。两个要求苛刻的应用，G-蛋白偶联受体（GPCR）和抗体结合分析，被证明。SMI-Seq能够在一锅法测定中进行“文库与文库”筛选，同时询问分子结合亲和力和特异性。
In contrast with advances in massively parallel DNA sequencing, high-throughput protein analyses are often limited by ensemble measurements, individual analyte purification and hence compromised quality and cost-effectiveness. Single-molecule (SM) protein detection achieved using optical methods is limited by the number of spectrally nonoverlapping chromophores. Here, we introduce a single molecular interaction-sequencing (SMI-Seq) technology for parallel protein interaction profiling leveraging SM advantages. DNA barcodes are attached to proteins collectively via ribosome display or individually via enzymatic conjugation. Barcoded proteins are assayed en masse in aqueous solution and subsequently immobilized in a polyacrylamide (PAA) thin film to construct a random SM array, where barcoding DNAs are amplified into in situ polymerase colonies (polonies) and analyzed by DNA sequencing. This method allows precise quantification of various proteins with a theoretical maximum array density of over one million polonies per square millimeter. Furthermore, protein interactions can be measured based on the statistics of colocalized polonies arising from barcoding DNAs of interacting proteins. Two demanding applications, G-protein coupled receptor (GPCR) and antibody binding profiling, were demonstrated. SMI-Seq enables “library vs. library” screening in a one-pot assay, simultaneously interrogating molecular binding affinity and specificity.
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