A light in the storm: what is a Molecular Beacon?

Updated : Mon, March 20, 2023 @ 9:54 PM

Molecular Beacons are a special type of dual-labeled oligonucleotide probe. Beacons are hairpin loop structures with a 5'-fluorophore and a 3'-quencher dye. The stem region is a short sequence of 5-7 complementary bases. The loop sequence is complementary to the target sequence. In the absence of target or prior to amplification, the stem anneals to form a closed hairpin conformation which holds the reporter and quencher close together to enable efficient FRET quenching and to promote contact or static quenching. The beacon is engineered such that the probe-target hybrid is more stable than the closed hairpin conformation, which is respectively more stable than a probe-mismatch hybrid. Therefore it is only in the event of a perfect-match hybridisation that signal occurs, allowing beacons to discriminate mismatches as small as a single nucleotide polymorphism (SNP).

Structure

What parts are similar to dual-labeled Black Hole Quencher™ (BHQ) probes?
A 5'-fluorophore and a 3'-BHQ dye covalently bound to the oligonucleotide termini.

What parts are unique?
A target-binding region comprised of a stem-loop structure resembling a "closed hairpin".

What is its native conformation at melting and annealing temperatures?
Melted - random coil conformation
Annealed (perfect match) - stable double helix, probe-target hybrid (high signal to noise ratio)
Annealed (mismatch) - hairpin conformation (FRET quenched, no signal).

Mechanism

How does it yield signal?
The target-binding region is located within the loop portion of the hairpin conformation and sets atop an annealed stem region. When a perfectly matched complementary sequence is available, the probe region hybridises to the complementary sequence. Conformational changes associated with hybridisation force the hairpin stem region open, separating the fluorophore and quencher, decreasing FRET quenching and releasing fluorescence.

What happens with each consecutive PCR cycle?

1. At high temperatures target DNA duplexes and Molecular Beacons are melted and maintain random coil conformations.
2. As the reaction cools to temperatures appropriate for the binding of primers and targets, the beacon will reach an equilibrium between the hairpin and hybridised states. This equilibrium depends on the availability of a perfect-match target, whose binding by the probe is thermodynamically favoured, by design, over the hairpin conformation. This ensures that the molecular beacon can hybridise when appropriate target is available. In the hybridised conformation the reporter and quencher dyes are separated, releasing fluorescence.
3. During polymerisation, the beacon is dislodged from the target sequence, refolds into and maintains the hairpin conformation until the next cycle of amplification. The increase in fluorescence intensity with repeated PCR cycles indicates the accumulation of product and allows for accurate quantification of template.

What if the available target is a mismatch?
Mismatched hybrids are less stable than reformation of the hairpin stem. Therefore signal is only produced when the target binding region hybridises to a perfect-match target sequence. This enables specificity of signal generation to be accurate at the level of single nucleotide polymorphisms (SNP).

Advantages of Molecular Beacons

• High signal to noise ratio - the efficiency of FRET quenching, when in the unhybridised state, decreases errant fluorescence and enables a high signal to noise ratio with hybridisation.
• High specificity - single base mismatches can be detected.
• Post-PCR melt curve analyses - Molecular Beacons do not require the enzymatic activity of a polymerase for fluorescent signal generation in qPCR and, under non-hydrolytic conditions, allow for post-PCR melt curve analyses.

Applications
Single and multiplexed, quantitative and qualitative, real-time and endpoint qPCR analyses; allelic discrimination; SNP analysis; DNA microarray-immobilised probes and biosensors and; as nuclease-resistant antisense probes for detection RNA in vivo.

Written by: Christina Ferrell, Ph.D., Technical Applications Specialist

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Recommended reading

1. Tyagi, S. and Kramer, F.R. "Molecular beacons: probes that fluoresce upon hybridization". Nature Biotechnology 14 (1996): 303-308.
2. Bonnet, G., Tyagi, S., Libchaber, A., Kramer, F.R. "Thermodynamic basis of the enhanced specificity of structured DNA probes". Proc Natl Acad Sci. 96 (1999): 6171-6176.
3. Marras, S.A.E., Kramer, F.R., Tyagi, S. "Multiplex detection of single-nucleotide variations using molecular beacons". Genet Anal, 14 (1999): 151-156.
4. Marras, S.A.E., Kramer F.R., and Tyagi, S. "Genotyping single nucleotide polymorphisms with molecular beacons". Single nucleotide polymorphisms: methods and protocols (v. 212). Ed. P.Y. Kwok. Totowa, NJ: The Humana Press, 2003. 111-128.
5. Vet, J.A.M. and Marras, S.A.E. "Design and optimization of molecular beacon real-time polymerase chain reaction assays". Oligonucleotide synthesis: Methods and Applications (v. 288). Ed. P. Herdewijn. Totowa, NJ: Humana Press, 2004. 273-290.
6. Bustin, S.A. A-Z of Quantitative PCR. (IUL Biotechnology Series). La Jolla, California: International University Line, 2004.
7. Broude, Natalia E. "Molecular Beacons and Other Hairpin Probes". Encyclopedia of Diagnostic Genomics and Proteomics. (2005): DOI: 10.1081/E-EDGP 120020717. 846-850
8. Public Health Research Institute. "Designing Molecular Beacons". www.molecular-beacons.org