Color-Changing Fluorescent Barcode Based on Strand Displacement Reaction Enables Simple Multiplexed Labeling

Area of Science:

• Biomolecular imaging
• Molecular diagnostics
• Nucleic acid nanotechnology

Background:

• Fluorescence imaging is crucial for biological research but limited by spectral overlap, hindering multiplexing.
• Existing DNA-based multiplexing strategies are often complex, limiting their widespread adoption.
• Overcoming spectral overlap is key to advancing high-throughput biological detection.

Purpose of the Study:

• To develop a simplified, DNA-based multiplexed labeling system for enhanced biological imaging.
• To create a color-changing fluorescent barcode (CCFB) approach using strand displacement reactions.
• To enable simultaneous detection of multiple targets with minimal design complexity.

Main Methods:

• Sequential toehold-mediated strand displacement reactions to create color-changing fluorescent barcodes (CCFBs).
• Design of simple and small nucleic acid structures for CCFB complex formation.
• Attachment of CCFB labels to polystyrene beads and antibodies for target labeling.

Main Results:

• Developed 27 distinct CCFB labels using only 14 oligonucleotides.
• Demonstrated successful multiplexed labeling of targets on polystyrene beads.
• Showcased CCFB application for simultaneous intracellular protein detection when conjugated to antibodies.

Conclusions:

• The CCFB approach offers a practical, user-friendly, and scalable solution for multiplexed biomolecular imaging.
• This method overcomes limitations of spectral overlap in fluorescence detection.
• CCFB technology holds promise for widespread adoption in comprehensive cellular imaging.