FRET-based small-molecule fluorescent probes: rational design and bioimaging applications

Area of Science:

• Chemical Biology
• Biomedical Imaging
• Molecular Probes

Background:

• Fluorescence imaging offers high spatial and temporal resolution for monitoring biomolecules.
• Intensity-based fluorescent probes have limitations due to variations in concentration, environment, and excitation intensity.
• Ratiometric fluorescent probes, particularly those using Förster resonance energy transfer (FRET), can overcome these limitations.

Purpose of the Study:

• To review the rational design and biological applications of synthetic FRET probes.
• To highlight advancements in developing FRET platforms with favorable photophysical properties.
• To present strategies for creating versatile, sensitive, and synthetically accessible FRET probes.

Main Methods:

• Development of FRET platforms with well-resolved and separated spectra, comparable donor-acceptor brightness, rigid linkers, and high energy transfer efficiency.
• Modulation of donor-acceptor distance or spectral overlap integral in an analyte-dependent manner.
• Focus on modifying the molar absorption coefficient of donor dyes (e.g., rhodamine) via ring-opening/closing processes for spectral overlap tuning.

Main Results:

• Successful design of first-generation rhodamine-based FRET probes by modulating the acceptor's molar absorption coefficient.
• Proposal and demonstration of a second-generation probe design strategy to address limitations of earlier probes.
• Development of FRET imaging probes for diverse analytes including Cu²⁺, NO, HOCl, cysteine, and H₂O₂.

Conclusions:

• The rational design of FRET platforms is crucial for developing effective FRET probes.
• Modulating spectral overlap, particularly through changes in rhodamine dye absorption, is a viable strategy for FRET probe development.
• Further improvements in versatility, sensitivity, and synthetic accessibility are needed for advanced FRET probes as a molecular toolbox.