Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152, Planegg, Germany.
Faculty of Physics and Center for Nanoscience, Ludwig Maximilian University, Geschwister-Scholl-Platz 1, 80539, Munich, Germany.
Small. 2023 Mar;19(12):e2206347. doi: 10.1002/smll.202206347. Epub 2023 Jan 15.
Super-resolution microscopy has revolutionized how researchers characterize samples in the life sciences in the last decades. Amongst methods employing single-molecule localization microscopy, DNA points accumulation for imaging in nanoscale topography (DNA-PAINT) is a relatively easy-to-implement method that uses the programmable and repetitive binding of dye-labeled DNA imager strands to their respective docking strands. Recently developed Peptide-PAINT replaces the interaction of oligonucleotides by short coiled-coil peptide sequences leading to an improved labeling scheme by reducing linkage errors to target proteins. However, only one coiled-coil pair is currently available for Peptide-PAINT, preventing multiplexed imaging. In this study, the initial Peptide-PAINT E/K coil is improved by modifying its length for optimized binding kinetics leading to improved localization precisions. Additionally, an orthogonal P3/P4 coil pair is introduced, enabling 2-plex Peptide-PAINT imaging and benchmarking its performance and orthogonality using single-molecule and DNA origami assays. Finally, the P3/P4 peptide pair is used to image the human epidermal growth factor receptors 2 (ErbB2/Her2) in 2D and 3D at the single receptor level using genetically encoded peptide tags.
在过去的几十年中，超分辨率显微镜彻底改变了研究人员对生命科学样本进行特征描述的方式。在采用单分子定位显微镜的方法中，DNA 点积累用于纳米级形貌成像（DNA-PAINT）是一种相对容易实现的方法，它利用染料标记的 DNA 成像链与各自的对接链之间的可编程和重复结合。最近开发的 Peptide-PAINT 取代了寡核苷酸的相互作用，使用短的卷曲螺旋肽序列，通过减少与靶蛋白的连接错误来改善标记方案。然而，Peptide-PAINT 目前只有一对卷曲螺旋对可用，这阻碍了多重成像。在这项研究中，通过修改初始的 Peptide-PAINT E/K 卷曲螺旋的长度来改善其结合动力学，从而提高定位精度，对其进行了改进。此外，引入了一个正交的 P3/P4 卷曲螺旋对，实现了 2 重 Peptide-PAINT 成像，并使用单分子和 DNA 折纸测定对其性能和正交性进行了基准测试。最后，使用 P3/P4 肽对在 2D 和 3D 水平上使用遗传编码的肽标签以单受体水平对人类表皮生长因子受体 2（ErbB2/Her2）进行成像。
