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Scaling Up Sequential Stepwise DNA Displacement-Based Signal Exchange for Rapid Ultraplex Fluorescent Imaging in

Yanju Chen1, Ryan N Delgado2,3, Ethan Xu1

  • 1Department of Chemistry, University of Florida, Gainesville, Florida 32603, United States.

Journal of the American Chemical Society
|January 20, 2026
PubMed
Summary

This study introduces a novel DNA displacement reaction method for highly multiplexed fluorescent imaging, overcoming spectral overlap limitations. The technique enables massive target visualization in cells and tissues with unprecedented multiplexity and speed.

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Area of Science:

  • Biotechnology
  • Molecular Biology
  • Genomics

Background:

  • Multiplexed fluorescent imaging is crucial for cellular and tissue analysis, but current methods are limited to 3-5 targets due to spectral overlap.
  • Existing techniques struggle to visualize numerous biomolecules simultaneously, hindering comprehensive biological studies.

Purpose of the Study:

  • To develop a highly multiplexed fluorescent imaging method overcoming spectral overlap limitations.
  • To enable visualization of massive numbers of targets in situ using DNA nanotechnology.

Main Methods:

  • Utilized scaled-up sequential DNA displacement reactions to encode fluorescent signals of targets in DNA probes.
  • Developed 25 DNA probes with 50 selected displacement sequences after screening 144 in situ reactions.
  • Employed sequential DNA displacement reactions to activate and remove fluorescent signals, preventing overlap.

Main Results:

  • Achieved 25-plex RNA imaging in fixed cells within 20 minutes using a single fluorophore channel.
  • Demonstrated 24-plex RNA imaging in retinal tissues, successfully resolving different cell types.
  • Showcased the potential for theoretically unlimited multiplexity due to DNA probe sequence design space.

Conclusions:

  • The novel DNA displacement method significantly simplifies high-plex fluorescent imaging.
  • This technique offers broad biotechnical applications for future medicine and diagnostics.
  • The method enables rapid, highly multiplexed imaging of massive targets in situ.