Abstract
Super-resolution microscopy is transforming research in the life sciences by enabling the visualization of structures and interactions on the nanoscale. DNA-PAINT is a relatively easy-to-implement single-molecule-based technique, which uses the programmable and transient interaction of dye-labeled oligonucleotides with their complements for super-resolution imaging. However, similar to many imaging approaches, it is still hampered by the subpar performance of labeling probes in terms of their large size and limited labeling efficiency. To overcome this, we here translate the programmability and transient binding nature of DNA-PAINT to coiled coil interactions of short peptides and introduce Peptide-PAINT. We benchmark and optimize its binding kinetics in a single-molecule assay and demonstrate its super-resolution capability using self-assembled DNA origami structures. Peptide-PAINT outperforms classical DNA-PAINT in terms of imaging speed and efficiency. Finally, we prove the suitability of Peptide-PAINT for cellular super-resolution imaging by visualizing the microtubule and vimentin network in fixed cells.
Highlights
Super-resolution microscopy is transforming research in the life sciences by enabling the visualization of structures and interactions on the nanoscale
We have implemented a single-molecule assay to quantitatively characterize the binding kinetics of these programmable coiled coil interactions and observed that Peptide-points accumulation in nanoscale topography (PAINT) exhibits faster association kinetics compared to classical DNA-PAINT, eventually allowing for faster imaging
We found that Peptide-PAINT yielded an approximately 20% improved imaging efficiency compared to DNA-PAINT
Summary
The PAINT process requires a dynamic equilibrium between bound and unbound states of the fluorescently labeled imager probe. A negatively charged E19-Cy3B imager peptide was used akin to the DNA-PAINT concept, where negatively charged imager strands bind to their targets This assay enabled us to precisely characterize the kinetic properties and imaging efficiency of transient coiled coil interactions in direct comparison to DNA-PAINT. We observed transient binding kinetics for Peptide-PAINT imaging, similar to DNA-PAINT, indicating that these coiled coil interactions are suitable for super-resolution imaging (Figure 1d, see Table S1 for a binding kinetics overview), yielding comparable localization precisions (3.6 nm for DNAPAINT and 4 nm for Peptide-PAINT). In a negative control, where the surface-immobilized DNA molecules lacked the E22 peptide, no detectable binding of K19 imagers to the DNA was observed (Figure S5), once again indicating that the coiled coil interactions are specific and do not interact nonspecifically with DNA. The reconstructed super-resolution images clearly demonstrate the faithful visualization of the microtubule (Figure 3a) and vimentin filament network (Figure 3b) with high specificity and spatial resolution, similar to previous DNA-PAINT imaging of those targets,[10,33,40,41] which were not visible in the negative control (Figure S15)
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