Optimized Coiled‐Coil Interactions for Multiplexed Peptide‐PAINT

• Published in: Small (Weinheim an der Bergstrasse, Germany) Vol. 19; no. 12; pp. e2206347 - n/a
• Main Authors: Eklund, Alexandra S., Jungmann, Ralf
• Format: Journal Article
• Language: English
• Published: Germany Wiley Subscription Services, Inc 01-03-2023
• Subjects: Binding; cell receptor imaging; coiled‐coil interactions; Coils; DNA - chemistry; DNA origami; DNA points accumulation for imaging in nanoscale topography; Genetic code; Growth factors; Humans; Imaging; Localization; Microscopy; Microscopy, Fluorescence - methods; Multiplexing; Nanotechnology; Nanotechnology - methods; Oligonucleotides; Oligonucleotides - chemistry; Orthogonality; peptide points accumulation for imaging in nanoscale topography; Peptides; Receptors; single molecules; Strands; super‐resolution imaging; DNA points accumulation for imaging in nanoscale topography; cell receptor imaging; DNA origami; peptide points accumulation for imaging in nanoscale topography; super-resolution imaging; coiled-coil interactions; single molecules
• ISSN: 1613-6810; 1613-6829
• DOI: 10.1002/smll.202206347

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.
Peptide points accumulation for imaging in nanoscale topography (PAINT) is a super‐resolution imaging method that relies on the repetitive binding of an imager coil to its docking coil on a target. Here, a newly developed orthogonal coil is benchmarked in single‐molecule and DNA origami experiments and used to image human epidermal growth factor receptors 2 (ErbB2/Her2) in 2D/3D in cells.