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DNA-barcoded labeling probes for highly multiplexed Exchange-PAINT imaging.

Sarit S Agasti1,2,3, Yu Wang1,2,4, Florian Schueder1,2,5,6

  • 1Wyss Institute for Biologically Inspired Engineering , Harvard University , Boston , Massachusetts , USA . Email: py@hms.harvard.edu ;

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Summary

Researchers developed a universal method for creating DNA-barcoded probes, enabling highly multiplexed super-resolution imaging. This breakthrough allows for nine-color imaging in cells, advancing the study of nanoscale protein interactions.

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Area of Science:

  • Biophysics
  • Molecular Biology
  • Nanotechnology

Background:

  • Super-resolution fluorescence imaging overcomes the diffraction limit for biological studies.
  • Limited multiplexing in current methods hinders understanding of nanoscale multi-protein interactions.
  • Exchange-PAINT offers spectrally-unlimited multiplexing but requires DNA-conjugated ligands.

Purpose of the Study:

  • To develop a universal approach for creating DNA-barcoded labeling probes for highly multiplexed Exchange-PAINT imaging.
  • To expand the repertoire of orthogonal imager strands for Exchange-PAINT.
  • To demonstrate the utility of these probes for advanced super-resolution microscopy in biological samples.

Main Methods:

  • Developed a universal conjugation strategy for DNA-barcoded labeling probes using antibodies, nanobodies, and small molecule binders.
  • Expanded the library of orthogonal imager strands for Exchange-PAINT to over 50 options.
  • Utilized a novel DNA origami-based assay to evaluate imager strand orthogonality and minimize crosstalk.
  • Optimized conjugation and labeling protocols for efficient probe preparation.

Main Results:

  • Successfully created DNA-barcoded probes compatible with a variety of affinity reagents.
  • Demonstrated over 50 orthogonal imager strands for Exchange-PAINT, significantly enhancing multiplexing potential.
  • Validated probe performance through a DNA origami-based crosstalk assay.
  • Achieved nine-color super-resolution imaging in situ in fixed cells, showcasing the system's capability.

Conclusions:

  • The developed universal approach significantly broadens the applicability of DNA-barcoded probes for multiplexed super-resolution imaging.
  • The expanded set of orthogonal imager strands and optimized protocols facilitate complex nanoscale biological investigations.
  • This work enables detailed, multi-protein interaction studies with unprecedented resolution and multiplexing.