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Analyzing Protein Dynamics Using Hydrogen Exchange Mass Spectrometry
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Dynamic Binder Exchange Improves Protein Labeling Efficiency in DNA-PAINT up to 15-Fold.

Clemens Steinek1, Isabelle Pachmayr1, Sebastian Strauss1

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Dynamic Binder Exchange (DyBE) improves DNA-PAINT super-resolution microscopy by using reversible binders for enhanced target sampling. This method achieves higher labeling efficiency, enabling sensitive detection of protein interactions like HER2 homodimers and EGFR-HER2 heterodimers.

Keywords:
DNA‐PAINTLabelingSingle‐molecule ImagingSpatial proteomicsSuper‐resolution microscopy

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Area of Science:

  • Biophysics
  • Molecular Imaging
  • Cell Biology

Background:

  • DNA-PAINT (Point Accumulation for Imaging in Nanoscale Topography) is a super-resolution microscopy technique.
  • Transient, reversible binder-target interactions are crucial for efficient molecular imaging.
  • Limitations exist in detecting targets with high-off-rate binders in super-resolution microscopy.

Purpose of the Study:

  • To introduce and validate Dynamic Binder Exchange (DyBE) as an enhancement for DNA-PAINT microscopy.
  • To improve labeling efficiency and enable sensitive detection of previously inaccessible targets.
  • To investigate the spatial organization of receptor proteins, including HER2 and EGFR, at the single-molecule level.

Main Methods:

  • DyBE utilizes DNA-conjugated binders (e.g., nanobodies) with high off-rates for transient target binding.
  • The dual-kinetic scheme integrates binder off-rates with DNA-PAINT blinking dynamics.
  • Single-protein resolution imaging was performed to detect HER2 homodimers and EGFR-HER2 heterodimers.

Main Results:

  • DyBE increased labeling efficiency up to 15-fold compared to conventional methods.
  • Sensitive detection of pre-existing HER2 homodimers and ligand-induced EGFR-HER2 heterodimers was achieved.
  • High-fidelity imaging of receptor dimerization dynamics at the single-protein level was demonstrated.

Conclusions:

  • DyBE significantly enhances DNA-PAINT super-resolution microscopy by optimizing binder-target interactions.
  • The method expands the repertoire of usable binders for advanced spatial proteomics.
  • DyBE facilitates mechanistic drug studies by enabling detailed analysis of receptor organization and signaling pathways.