Molecular beacons are single stranded oligonucleotides that, unless bound to target, exist in a hairpin conformation. This importance of this conformation scheme is realized considering that the 5' end of the oligo contains a fluorescent dye, and a quencher dye is attached to the 3' end. Therefore, when the beacon is not bound to target, the hairpin structure positions the fluorophore and quencher in close proximity, such that no fluorescence can be observed (the quencher, dabcyl in figure 1, suppresses fluorescence by transferring the light energy from the fluorophore to heat energy). However, once the beacon hybridizes with target, the fluorophore and quencher are separated because the hairpin structure is disrupted, and fluorescence is detectable.(Devor, 2000) The hairpin structure also serves to give molecular beacons impressive specificity; they are able to discriminate single base pair mismatches.(Tyagi et al., 1998)

Figures 1, 2. Representations of molecular beacons in looped and hybridized conformations, respectively. Courtesy of <www.idtdna.com> and <www.stratagene.com>

Figure 3. Schematic of the change in conformation that takes place when a molecular beacon hybridizes with its specific target sequence. Courtesy of <www.idtdna.com>

Molecular beacons have a wide range of uses, including the following:

• Real-time PCR quantitation- Molecular beacons can measure the amplification of a polymerase chain reaction while the reaction takes place in a closed tube.(Research Genetics, 2000) This feature eliminates the need for electrophoresis, and the drawbacks that are associated with it.(Robinson and Meuller, 2000) Using molecular beacons takes less time and effort, while at the same time reducing the potential for contamination (since the reaction takes place in a single closed tube). Also, due to their specificity, molecular beacons report only complementary PCR product, as opposed to other real-time fluorescence methods that use DNA binding dyes.(Robinson and Meuller, 2000)
• Allelic discrimination- In light of their specificity, it is possible to use molecular beacons to differentiate between homozygotes and heterozygotes. To do this, one would simply attach two different dyes to the beacons complementary to the two alleles.(Robinson and Meuller, 2000)(Piatek et al., 1998)
  

Figure 4. Representation of how molecular beacons can be used for allelic discrimination. Courtesy of <www.isogen.nl>

• Multiplexing- This technique refers to using several molecular beacons with different colored fluorophores to detect numerous targets in a single sample. For instance, molecular beacons can be used to detect single nucleotide differences in a DNA sequence. The sequence to be tested is amplified with PCR in the presence of four molecular beacon probes, each differing only in the nucleotide in question (A, C, T, or G) and in the color of their fluorophores. The identity of the variant nucleotide is deduced by observing which of the molecular beacons fluoresces (binds to the PCR product).(Marras et al., 1999)
• Real time detection of DNA/RNA hybridization in living cells- After microinjecting molecular beacons that target a specific protooncogene (vav) into K562 human leukemia cells, Sokol et al. detected a 60-fold fluorescence difference in comparison to controls. These results suggest that real-time visualization and localization of probe/mRNA complexes are possible, thus allowing RNA processing, trafficking, and folding to be studied in living cells.(Sokol et al., 1998)(Tyagi and Kramer, 1996)
  
• Diagnostic clinical assays- Vet et al. demonstrated that molecular beacons, in conjunction with PCR can be used to detect the presence and abundance of four different retroviruses in a tissue or blood sample.(Vet et al., 1999) Similarly, Poddar utilized molecular beacons and PCR to detect Adenovirus.(Poddar, 1999) Using molecular beacons enables multiplexing, thereby simplifying and improving the reliability and speed of diagnostic clinical assays.(Vet et al., 1999)

Design of Molecular Beacons(Devor, 2000)(Research Genetics, 2000)

Molecular beacons consist of four components: loop, stem, 5' fluorophore, and 3' quencher. The loop consists of the complement of the target sequence, usually approximately 21 nucleotides in length. The stem is formed by adding 3-7 nucleotides to the 5' end of the loop, and its reverse complement to the 3' end. A standard stem sequence would be 6 nucleotides long, comprised of 5 C/G pairs and one A/T pair. The rationale behind this composition is to make the melting temperature of the stem 7°C-10°C higher than the annealing temperature of the PCR, ensuring that unhybridized probes remain in the loop conformation (and do not fluoresce). In addition, the melting temperature of the molecular beacon/target complex should also be 7°C-10°C higher than the annealing temperature of the PCR, making certain that complexes will form. When these conditions are met, the presence of perfectly complementary target sequences will induce binding of the molecular beacon, overcoming the loop structure and allowing fluorescence to occur. However, by increasing the length of the loop sequence, the stringency of the probe/target interaction can be reduced, permitting the toleration of mismatches in the assay. The melting temperature of molecular beacons can be determined using a computer program on the internet developed by Michael Zuker.

Once these thermodynamic issues have been addressed, the final considerations in designing a molecular beacon are the fluorophore and the quencher. Dabcyl is a common quencher because it is neutral and hydrophobic, two characteristics that make it well suited to pairing with many fluorophores. Common fluorophores that work particularly well when paired with dabcyl include fluorescein (FAM), rhodamine x (ROX), tetrachloro-6-carboxyfluorescein (TET), and tetramethylrhodamine (TAMRA).

Links to manufacturers of molecular beacons:

References

-Devor, EJ. Dual-Labeled Probes. <http://www.idtdna.com/html/catalog/catalog5.html> Accessed 2000 Feb 16.
-Marras SA, Kramer FR, Tyagi S. 1999. Multiplex detection of single-nucleotide variations using molecular beacons. Genet Anal 5-6:151-6.
-Piatek AS, Tyagi S, Pol AC, Telenti A, Miller LP, Kramer FR, Alland D. 1998. Molecular beacon sequence analysis for detecting drug resistance in Mycobacterium tuberculosis. Nat Biotechnol 4:359-63.
-Poddar SK. 1999. Detection of adenovirus using PCR and molecular beacon. J Virol Methods 1:19-26.
-Research Genetics. Molecular Beacon Custom Synthesis. <http://www.resgen.com/products/MOLBEA.php3> Accessed 2000 Feb 16.
-Robinson, JK, Meuller, R. The New Molecular Beacon Technology. <http://www.stratagene.com/q_pcr/_include/info.htm> Accessed 2000 Feb 19.
-Sokol DL, Zhang X, Lu P, Gewirtz AM. 1998. Real time detection of DNA.RNA hybridization in living cells. Proc Natl Acad Sci USA 20:11538-43.
-Tyagi S, Bratu DP, Kramer FR. 1998. Multicolor molecular beacons for allele discrimination. Nat Biotechnol 1:49-53.
-Tyagi S, Kramer FR. 1996. Molecular beacons: probes that fluoresce upon hybridization. Nat Biotechnol 3:303-8.
-Vet JA, Majithia AR, Marras SA, Tyagi S, Dube S, Poiesz BJ, Kramer FR. 1999. Multiplex detection of four pathogenic retroviruses using molecular beacons. Proc Natl Acad Sci USA 11:6394-9.

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