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Small Peptide-Protein Interaction Pair for Genetically Encoded, Fixation Compatible Peptide-PAINT.

ABSTRACT: Super-resolution microscopy via PAINT has been widely adopted in life sciences to interrogate the nanoscale architecture of many cellular structures. However, obtaining quantitative information in fixed cellular samples remains challenging because control of labeling stoichiometry is hampered in current approaches due to click-chemistry and additional targeting probes. To overcome these challenges, we have identified a small, PDZ-based, peptide-protein interaction pair that is genetically encodable and compatible with super-resolution imaging upon cellular fixation without additional labeling. Stoichiometric labeling control by genetic incorporation of this probe into the cellular vimentin network and mitochondria resulted in super-resolved 3D reconstructions with high specificity and spatial resolution. Further characterization reveals that this peptide-protein interaction is compatible with quantitative PAINT and that its binding kinetics remains unaltered upon fixation. Finally, by fusion of our probe to nanobodies against conventional expression markers, we show that this approach provides a versatile addition to the super-resolution toolbox.

SUBMITTER: Tas RP

PROVIDER: S-EPMC8631740 | biostudies-literature |

REPOSITORIES: biostudies-literature

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