Nucleic Acid Probes:  Hybridization (annealing) of a nucleic acid sequence to its complement is a highly specific molecular recognition event. Unlike other highly specific recognition systems (such as antibody/antigen complex, for example), nucleic acid based probe systems can be rationally designed using simple rules to recognize and detect any desired target with almost any desired degree of specificity and, further, can be chemically produced with relative ease. Synthetic oligonucleotides can be made specific for any desired sequence; by varying length, sequence, and hybridization conditions a probe oligonucleotide can identify and quantify the presence of its complementary sequence within a heterogeneous mixture and can even discriminate single nucleotide polymorphisms (SNPs).

It is necessary that some modification be made to the probe nucleic acid to enable detection of the hybridization event. Traditionally, this has involved tagging with radioactive, fluorescent, or other small molecular labels (such as biotin or digoxigenin). In this case, both bound and unbound probe molecules are detectable and any unhybridized probe must be removed to eliminate background. Unbound probe can be removed by dilution (such as the post-hybridization wash steps used in Southern blot or Northern blot techniques). Unbound probe can also be removed by digestion (for example, use of nucleases in RNase protection assays or S1 Nuclease assays). While effective, these methods disturb the equilibrium state of the nucleic acid hybridization event and preclude dynamic, real-time detection of hybridization. Further, such methods are not suited for use in vivo.

Molecular Beacons are a recent improvement in oligonucleotide probe design that enables dynamic, real-time detection of nucleic acid hybridization events both in vitro and in vivo (Tyagi and Kramer, 1996; Kostrikis et al., 1998; Tyagi et al., 1998). Hybridization of a Molecular Beacon to its complementary target results in a detectable conformational change that inherently has low background and therefore eliminates the need for washing or probe degradation steps.

A Molecular Beacon is a dual-labeled oligonucleotide having a fluorescent reporter group at one end and a fluorescence quencher group at the other end. The oligonucleotide is further designed such that in the absence of target the molecule forms an internal hairpin that brings the reporter and quencher groups in physical proximity resulting in efficient quenching of the reporter. In the presence of target, the probe molecule unfolds and hybridizes; reporter and quencher are now physically separated and the reporter dye will emit fluorescence signal upon stimulation. The basic design elements of a molecular beacon are shown below (Fig. 1). In this example, the molecular beacon forms a stem-loop structure with the fluorophore EDANS on the 5'-end and the quencher Dabcyl on the 3'-end. In this state (i.e., the unimolecular configuration), the fluorophore is in close proximity to the quencher; light energy captured by the fluorophore is transferred to the quencher which dissipates this energy without emission of any fluorescent signal (i.e., light).

Figure 1

In the presence of the complementary target sequence, the probe domain hybridizes to the target resulting in disruption of the unimolecular stem-loop structure. In this new two-molecule conformation, the fluorescent reporter group is no longer in physical proximity with the quencher group and the reporter can emit light at the wavelength characteristic to that fluorophore (Fig. 2).

Figure 2

Design Considerations

A properly designed Molecular Beacon allows for the transition between two conformational states, the unimolecular hairpin and the bimolecular probe:target hybrid. With attention to the distinction between the thermodynamics of hairpin vs hybrid formation, the beacon will be dark (i.e., quenched) in the absence of target or bright (i.e., unquenched) in the presence of target.

Temperature and salt conditions can be designed such that a perfect match with target is required for hybrid formation. As a general rule, the melting temperature of the stem-loop structure should be 7-10°C higher than the detection temperature. Similarly, the melting temperature of the probe-target hybrid (formed when the loop sequence hybridizes to the complementary target sequence) should also be 7-10°C higher than the detection temperature. If the Molecular Beacon is used in real-time PCR, this would equal 7-10°C above the primer anneal temperature of the reaction. A fundamental feature of a molecular beacon is that probe-target hybrids cannot co-exist with stem hybrids due to the rigidity of DNA helices. A perfect match probe-target hybrid will be energetically more stable than the stem-loop structure whereas a mismatched probe-target hybrid will be energetically less stable than the stem-loop structure. This characteristic is the basis of the extraordinary specificity offered by Molecular Beacons.

If it is desirable to tolerate mismatches in the assay, specificity can be relaxed by making the probe sequence in the loop and the probe-target hybrid more stable.

In practice, stems of 5-6 bases and probe-loop sequences of 16-22 bases are most commonly used. These averages assume that the Molecular Beacon targets a genome having an average G/C content. For more G/C-rich target sequences, the probe length can be reduced to as few as 16 nucleotides and still retain high specificity. Similarly, for A/T-rich target sequences, the probe length can be increased to as many as 25 nucleotides.

Another consideration in molecular beacon design is the choice of fluorophore and quencher. Dabcyl (4-(4'-dimethylaminophenylazo)benzoic acid) has been found to be the optimal choice for the quencher. Dabcyl is a neutral, hydrophobic molecule that makes it ideal for pairing with a variety of fluorophores. Further, dabcyl must be close to or directly in contact with the fluorophore for energy-transfer quenching to be efficient. Thus, dabcyl has an operational range for quenching that is small compared to the total length of a beacon oligonucleotide (see Fig. 2). Thus a stem-loop beacon is quenched while a probe-target hybrid is not quenched.

Many different fluorescent reporter dyes can be used with dabcyl quencher. Among the most commonly used are Fluorescein (Fam), Tetrachloro-6-carboxyfluorescein (Tet), Hetrachloro-6-carboxyfluorescein (Hex) and Tetramethylrhodamine (Tamra) and Rhodamine-X (Rox). This flexibility in reporter dye options afford the option to conveniently multiplex Molecular Beacon detection reactions.

For ordering information, please consult the Catalog page for Molecular Beacons.

For more information on fluorescent dyes, please consult the Tech Bulletin on Fluorescence Excitation and Emission.

Applications

The versatile features of Molecular Beacons permit their use in many different quantitative and qualitative target detection assays. As a tool to detect amplified targets, Molecular Beacons have been adapted to both real-time and end-point PCR and RT-PCR assays. They have also been used in the detection of RNA species in a homogenous, real-time NASBA assay (Leone et al., 1998).

Historically, the first use of a molecular beacon was in real-time monitoring of DNA amplification during PCR (see Tyagi and Kramer, 1996). Exploiting the option to employ different dyes, molecular beacon assays can be multiplexed and have been used for real-time fluorescent genotyping (Kostrikis et al., 1998; Tyagi et al., 1998) and in the simultaneous detection of four different pathogenic retroviruses in clinical samples (Vet et al., 1999).

The specificity of Molecular Beacons allows for use in single nucleotide polymorphism (SNP) detection (Marras et al., 1999). Their simplicity and sensitivity enables use in thermodynamic studies of the state transitions of the probes themselves (Bonnet et al., 1999). Finally, the non-toxic, homogenous nature of the probes allows for their use in vivo. Molecular Beacons have been used to detect transcripts in tissue culture cells following microinjection (Sokol et al., 1998). Applications to FISH, chromosome painting, and even real-time visualization of mRNA migration are envisioned. Many other applications are sure to appear in the scientific literature as the full potential of this exciting new technology emerges.

Acknowledgments

We thank Drs. Fred Kramer and Sanjay Tyagi of the Public Health Research Institute in New York for reviewing and expanding this technical discussion of Molecular Beacon Technology.

References

Bonnet, G., Tyagi, S., Libchaber, A., and Kramer, F.R. (1999) "Thermodynamic basis of the chemical specificity of structured DNA probes." Proc. Natl. Acad. Sci. U.S.A., 96:6171-6176.

Fang, X., Liu, X., Schuster, S., and Tan, W. (1999) "Designing a novel molecular beacon for surface-immobilized DNA hybridization studies." J. Am. Chem. Soc.,121:2921-2922.

Kostrikis, L.G., Tyagi, S., Mhlanga, M.M., Ho, D.D., and Kramer, F.R. (1998) "Molecular beacons: spectral genotyping of human alleles." Science, 279:1228-1229.

Leone, G., van Schijndel, H., van Gemen, B., Kramer, F.R., and Schoen, C.D. (1995) "Molecular beacon probes combined with amplification by NASBA enable homogenous real-time detection of RNA." Nucleic Acids Res., 26:2150-2155.

Marras, S.A.E., Kramer, F.R., and Tyagi, S. (1999) "Multiplex detection of single-nucleotide variation using molecular beacons." Genet. Anal. Biomol. Eng., 14:151-156.

Sokol, D.L., Zhang, X., Lu, P., and Gewirtz, A.M. (1998) "Real time detection of DNA:RNA hybridization in living cells." Proc. Natl. Acad. Sci. U.S.A., 95:11538-11543.

Tyagi, S., and Kramer, F.R. (1996) "Molecular beacons: probes that fluoresce upon hybridization." Nature Biotechnology, 1414:303-308.

Tyagi, S., Bratu, D.P., and Kramer, F.R. (1998) "Multicolor molecular beacons for allele discrimination." Nature Biotechnology, 16:49-53.

Vet, J.A.M., Majithia, A.R., Marras, S.A.E., Tyagi, S., Dube, S., Poiesz, B.J., and Kramer, F.R. (1999) "Multiplex detection of four pathogenic retroviruses using molecular beacons." Proc. Natl. Acad. Sci. U.S.A., 96:6394-6399.

Eric J. Devor, Ph.D.
Senior Research Scientist
Mark A. Behlke MD, Ph.D.
Vice President, Molecular Genetics and Bioinformatics
Integrated DNA Technologies

May 2000