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Updated: Jul 19, 2026
07:10
Split Hybridization Probe Utilizing a DNA Fluorescent Light-up Aptamer as a Signal Reporter for Sequence-Specific Nucleic Acid Analysis
Published on: July 8, 2025
Combinatorial fluorescence energy transfer molecular beacons for probing nucleic acid sequences
Xiaoxu Li1, Zengmin Li, Angel A Martí
1Columbia Genome Center, Columbia University College of Physicians and Surgeons, New York, NY 10032, USA.
Summary
We developed novel molecular beacons (MB) using three fluorophores for unique DNA detection. MBs change fluorescence signatures upon binding to target DNA, enabling distinct "bar code" identification.
Area of Science:
- Biochemistry
- Molecular Biology
- Nanotechnology
Background:
- Molecular beacons (MBs) are DNA/RNA sensors utilizing fluorescence.
- Existing MBs often rely on single or dual-color systems.
- Developing multi-fluorophore systems can enhance detection specificity.
Purpose of the Study:
- To design and synthesize a novel three-fluorophore molecular beacon.
- To investigate the energy transfer dynamics in closed and open MB states.
- To establish a unique fluorescence signature for target DNA detection.
Main Methods:
- Synthesis of molecular beacons with 6-carboxyfluorescein (Fam), N,N,N ',N '-tetramethyl-6-carboxyrhodamine (Tam), and Cyanine-5 (Cy5).
- Steady-state and time-resolved fluorescence spectroscopy.
- Fluorescence depolarization studies.
Main Results:
- A fluorescence energy transfer cascade (Fam -> Tam -> Cy5) was observed in the stem-closed MB.
- Target DNA binding induced MB opening, disrupting the Fam-Tam energy transfer.
- Distinct fluorescence signatures (ratios of emissions) were generated for closed and open MB states, forming a 'bar code'.
Conclusions:
- The three-fluorophore MB system generates unique, target-dependent fluorescence signatures.
- This combinatorial fluorescence energy transfer approach offers a novel method for DNA detection.
- The system's fluorescence signature can be tuned by adjusting fluorophore spacing.
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