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Wavelength-shifting molecular beacons

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Abstract

We describe wavelength-shifting molecular beacons, which are nucleic acid hybridization probes that fluoresce in a variety of different colors, yet are excited by a common monochromatic light source. The twin functions of absorption of energy from the excitation light and emission of that energy in the form of fluorescent light are assigned to two separate fluorophores in the same probe. These probes contain a harvester fluorophore that absorbs strongly in the wavelength range of the monochromatic light source, an emitter fluorophore of the desired emission color, and a nonfluorescent quencher. In the absence of complementary nucleic acid targets, the probes are dark, whereas in the presence of targets, they fluoresce—not in the emission range of the harvester fluorophore that absorbs the light, but rather in the emission range of the emitter fluorophore. This shift in emission spectrum is due to the transfer of the absorbed energy from the harvester fluorophore to the emitter fluorophore by fluorescence resonance energy transfer, and it only takes place in probes that are bound to targets. Wavelength-shifting molecular beacons are substantially brighter than conventional molecular beacons that contain a fluorophore that cannot efficiently absorb energy from the available monochromatic light source. We describe the spectral characteristics of wavelength-shifting molecular beacons, and we demonstrate how their use improves and simplifies multiplex genetic analyses.

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Figure 1: Principle of operation of wavelength-shifting molecular beacons.
Figure 2
Figure 3: Enhancement in the brightness of molecular beacons.
Figure 4: Multiplex detection of the products of PCRs containing conventional molecular beacons and wavelength-shifting molecular beacons.

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Acknowledgements

This work was supported by National Institutes of Health grants HL-43521 and ES-10536.

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Authors and Affiliations

  1. Department of Molecular Genetics, Public Health Research Institute, 455 First Avenue, New York, 10016, NY

    Sanjay Tyagi, Salvatore A.E. Marras & Fred Russell Kramer

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  1. Sanjay Tyagi
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  2. Salvatore A.E. Marras
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  3. Fred Russell Kramer
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Correspondence to Sanjay Tyagi.

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Tyagi, S., Marras, S. & Kramer, F. Wavelength-shifting molecular beacons. Nat Biotechnol 18, 1191–1196 (2000). https://doi.org/10.1038/81192

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