Evelyn McGown and Michael Su
Molecular beacons are fluorogenic oligonucleotide probes developed by Kramerand associates to detect specific nucleic acids in homogeneous solutions.1 4 They are single-stranded oligonucleotides that exist in a hairpin shape because of thecomplementary arms at the ends which bind together to form a stem. Thestrained loop portion contains the nucleotide that is complementary to theintended target. A fluorophore is covalently bound to one end of theoligonucleotide and a quencher to the other end. In the absence of target, thefluorophore and quencher are held in close proximity by the arms and thefluorescence is internally quenched. When the probe hybridizes to the targetmolecule, it undergoes a conformational change resulting in separation of thefluorophore and quencher, and restoration of fluorescence. Molecular beacons areappealing because they can be used in homogeneous solutions and obviate theneed to isolate probe/target hybrids from an excess of unbound hybridizationprobes. They have been used to monitor polymerase chain reactions.3
Many combinations of fluorophores and quenchers are possible. Kramers grouphas found that 4-(4-dimethylaminophenylazo) benzoic acid (DABCYL) can serveas a universal quencher for a number of fluorophores. Even if the fluorophoreemission spectrum does not overlap with the DABCYL absorption spectrum,quenching (with >95% efficiency) occurs by direct energy transfer fromfluorophore to quencher because of their close proximity.2
This application note describes measurement of probe/target complexes with theSPECTRAmax GEMINI microplate spectrofluorometer.
MATERIALS AND METHODS
Samples of molecular beacons and complementary targets were obtained fromResearch Genetics, Huntsville, Alabama. The oligonucleotide sequences are notimportant to this discussion. The microplates used were Nunc brand, black 384well microplates, surface-treated to minimize binding (cell culture-treated). Allsamples were prepared in 10 mM Tris buffer, pH 8, containing 1 mM MgCl2, and the final concentrations of beacon and target were 0.3 M and 1.6 M respectively, in a total volume of 54 L. The reaction mixtures were incubated at least 30 minutes at ambient temperature before measurement in a SPECTRAmax GEMINI microplate spectrofluorometer.
RESULTS AND DISCUSSION
Figure 1 shows the fluorescence spectrum of a tetrachlorofluorescein (TET)labelled probe in the presence and absence of complementary target. At the peak maximum (approximately 535 nm), the probe/target complex had approximately 35 times higher signal than did the probe alone.
The optimal wavelength settings for TET and several other molecular beacons were determined by examination of signal/blank ratios as previously described.5,6 The combination of excitation/emission wavelengths and emission cutoff filters giving the highest ratios are summarized in the table below.
There is considerable interest in measuring multiple targets (multiplexing) in a single sample. This can be done by using a different fluorophore for each probe, assuming the probes do not interfere with each other. If their emission spectra do not overlap, detection of one in the presence of the other is straightforward. Such is the case for fluorescein (FAM)- and tetramethylrhodamine (TAMRA)-labelled probes. Using the instrument settings intended for FAM, the TAMRA probe/ target complex does not interfere with the FAM signal ( Figure 2 ).
Conversely, when TAMRA settings are used, the FAM probe/target complex does not interfere with the TAMRA signal (Figure 3). There are many other 2-probe combinations which would be compatible. If two probes have partially overlapping emission spectra, they may be resolved by adjusting their excitation and/or emission wavelengths to minimize interference. The SPECTRAmax GEMINI systems dual monochromators facilitate the optimization process by making it easy to select different wavelengths.
There are also a number of 3-probe combinations that should be suitable for use in a multiplex assay, for example, FAM and CY5 with HEX, CR 6G, CY3, or TAMRA (see Table ). It is also possible that a 4-probe combination such as FAM, HEX, TAMRA, and CY5 could be resolved.
Molecular beacons, like other oligonucleotides, will readily adhere to untreated plastic. Therefore, it is important to use microplates which have been surface treated to minimize binding. We have found that plates treated for cell culture exhibit less binding than do untreated plates.
Molecular beacons, like other analytical tools, have potential pitfalls. Depending on the temperature, they will react with targets containing one or more nucleotide mismatches.24 Temperature control is especially important for discrimination between perfectly complementary targets and targets containing a single-nucleotide mismatch.3,4
Tyagi et al. state that fluorescence increases as much as 900-fold when probes bind to their target2. In practice, the ratios are typically much lower. A high ratio is obviously desirable because it affords high sensitivity. A low ratio can be due to a number of factors, including contamination with free fluorophore and contamination with oligonucleotides that are missing the quencher. At least three commercial suppliers guarantee a minimum ratio of 25 (Research Genetics; Synthegen, Houston, Texas; and Pacific Oligos, Toowong, Australia).
The reaction of molecular beacons with their targets can be measured in the SPECTRAmax GEMINI microplate spectrofluorometer. With its dual scanning monochromators, it easy to optimize excitation and emission wavelengths for specific fluorophores and to customize the settings for combinations of probes. Mixtures of 2, 3, and (probably) 4 probes in a single mixture can be resolved.
1. Tyagi, S. and F.R. Kramer. (1996) Molecular Beacons: Probes that Fluoresce upon Hybridization. Nature Biotechnology 14: 303308.
2. Tyagi, S., D.P. Bratu and F.R. Kramer. (1998) Multicolor molecular beacons for allele discrimination. Nature Biotechnology 16: 4953.
3. Marras, S.A.E., F.R. Kramer and S. Tyagi. (1999) Multiplex detection of single-nucleotide variations using molecular beacons. Genetic Analysis: Biomolecular Engineering 14: 151156.
4. Bonnet, G, S. Tyagi, A. Libchaber and F.R. Kramer. (1999) Thermodynamic basis of the enhanced specificity of structured DNA probes. Proc. Nat. Acad. Sci. 96, 61716176.
5. McGown, E.L. (1999) Selecting excitation and emission wavelengths using the SPECTRAmax GEMINI microplate spectrofluorometer basic principles. MAXline Application Note No. 30.
6. McGown, E.L. (1999) Optimizing excitation and emission wavelengths for narrow Stokes shift fluorophores using the SPECTRAmax GEMINI microplate spectrofluorometer. MAXline Application Note No. 31.