Exciton-controlled fluorescence: application to hybridization-sensitive fluorescent DNA probe

Area of Science:

• Molecular Biology
• Biochemistry
• Cell Biology

Background:

• Nucleic acid detection is crucial for molecular diagnostics and biological research.
• Existing fluorescent probes often face limitations in sensitivity, reversibility, or real-time cellular application.
• Developing advanced probes is essential for accurate and dynamic monitoring of genetic material within living systems.

Purpose of the Study:

• To design and synthesize a novel hybridization-sensitive fluorescent probe for nucleic acid detection.
• To investigate the probe's fluorescence properties, including quenching mechanisms and target hybridization.
• To demonstrate the probe's utility for visualizing intracellular mRNA localization in living cells and enabling multicolor detection.

Main Methods:

• Synthesis of a doubly thiazole orange-labeled nucleotide as a fluorescent probe.
• Utilizing intramolecular excitonic interaction for fluorescence quenching in single-stranded states.
• Applying the probe to living HeLa cells via microinjection for intracellular mRNA visualization.
• Assessing probe response to target hybridization and competitor DNA addition.
• Developing multicolor capabilities for simultaneous detection of multiple nucleic acid sequences.

Main Results:

• The synthesized probe exhibits high fluorescence intensity upon hybridization with target nucleic acids.
• Effective fluorescence quenching was observed in the single-stranded state due to excitonic interaction.
• The probe successfully visualized intracellular mRNA localization in living HeLa cells immediately after microinjection.
• Rapid and reversible fluorescence changes were observed in response to target nucleic acid concentration and competitor DNA.
• Multicolor probe variants enabled simultaneous detection of multiple RNA targets.

Conclusions:

• The developed exciton-controlled fluorescent probe offers a sensitive and responsive platform for nucleic acid detection.
• The probe facilitates dynamic, spatiotemporal monitoring of intracellular RNA behavior in living cells.
• Its ability for multicolor detection expands its potential for complex biological analyses and diagnostics.