Single-molecule detection and manipulation is a powerful tool for unraveling dynamic biological processes. Unfortunately, success in such experiments is often challenged by tethering the biomolecule(s) of interest to a biocompatible surface. Here, we describe a robust surface passivation method by dense polymer brush grafting, based on optimized polyethylene glycol (PEG) deposition conditions, exactly at the lower critical point of an aqueous biphasic PEG-salt system. The increased biocompatibility achieved, compared with PEG deposition in sub-optimal conditions away from the critical point, allowed us to successfully detect the assembly and function of a large macromolecular machine, a fluorescent-labeled multi-subunit, human RNA Polymerase II Transcription Pre-Initiation Complex, on single, promoter-containing, surface-immobilized DNA molecules. This platform will enable probing the complex biochemistry and dynamics of large, multi-subunit macromolecular assemblies, such as during the initiation of human RNA Pol II transcription, at the single-molecule level.

单分子检测和操作是揭示动态生物过程的强大工具。不幸的是，此类实验的成功常常受到将感兴趣的生物分子束缚在生物相容性表面上的挑战。在这里，我们描述了一种稳健的表面钝化方法，通过密集聚合物刷接枝，基于优化的聚乙二醇 (PEG) 沉积条件，恰好在水性双相 PEG-盐系统的下临界点。与在远离临界点的次优条件下的 PEG 沉积相比，获得的生物相容性增加，使我们能够成功检测大型大分子机器、荧光标记的多亚基、人 RNA 聚合酶 II 转录前的组装和功能。起始复合物，在单个、含有启动子、表面固定的 DNA 分子上。