Department of Chemistry, Humboldt-Universität zu Berlin, Berlin, Germany.
Department of Biology II and Center for Molecular Biosystems (BioSysM), Human Biology and BioImaging, Ludwig-Maximilians-Universität München, Munich, Germany.
Nat Chem. 2021 Jan;13(1):15-23. doi: 10.1038/s41557-020-00584-z. Epub 2020 Dec 7.
DNA nanotechnology is an emerging field that promises fascinating opportunities for the manipulation and imaging of proteins on a cell surface. The key to progress is the ability to create a nucleic acid-protein junction in the context of living cells. Here we report a covalent labelling reaction that installs a biostable peptide nucleic acid (PNA) tag. The reaction proceeds within minutes and is specific for proteins carrying a 2 kDa coiled-coil peptide tag. Once installed, the PNA label serves as a generic landing platform that enables the recruitment of fluorescent dyes via nucleic acid hybridization. We demonstrate the versatility of this approach by recruiting different fluorophores, assembling multiple fluorophores for increased brightness and achieving reversible labelling by way of toehold-mediated strand displacement. Additionally, we show that labelling can be carried out using two different coiled-coil systems, with epidermal growth factor receptor and endothelin receptor type B, on both HEK293 and CHO cells. Finally, we apply the method to monitor internalization of epidermal growth factor receptor on CHO cells.
DNA 纳米技术是一个新兴领域,有望为细胞表面蛋白质的操作和成像带来令人着迷的机会。取得进展的关键是能够在活细胞的背景下创建核酸-蛋白质连接。在这里,我们报告了一种共价标记反应,该反应可以安装生物稳定的肽核酸 (PNA) 标记物。该反应在数分钟内进行,并且对携带 2 kDa 卷曲螺旋肽标签的蛋白质具有特异性。一旦安装,PNA 标记物就可以作为通用的着陆平台,通过核酸杂交来招募荧光染料。我们通过招募不同的荧光团、组装多个荧光团以提高亮度以及通过发夹介导的链置换实现可逆标记,展示了这种方法的多功能性。此外,我们表明,该标记可以使用两种不同的卷曲螺旋系统,针对表皮生长因子受体和内皮素受体 B,在 HEK293 和 CHO 细胞上进行。最后,我们将该方法应用于监测 CHO 细胞中表皮生长因子受体的内化。