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Beacons of light

Nature Biotechnology volume 24, pages 303–304 (2006)Cite this article

Sangi Tyagi and Fred Kramer of the Public Health Research Institute (Newark, NJ, USA) were working at the time on Qβ replicase amplification of RNA, which, like the polymerase chain reaction that later supplanted it, had unacceptably high backgrounds when working with low-abundance RNAs. They had the idea, based on Paul Lizardi's (now at Yale University) work on conformational changes of nucleic acids, to create probes that would allow amplification only when they changed their shape.

Thus, Tyagi, Kramer and Lizardi designed a probe with a stem-loop structure that unfolds when it reacts with its target sequence, the complement for which is encoded in the loop. In the first paper, Tyagi, Kramer and Lizardi showed how their fluorogenic probe, which they dubbed molecular beacon, could assay the accumulation of a target molecule in solution (a PCR fragment of the HIV invertase gene), one of the first real-time PCR assays in the literature1. The choice of the two dyes used in the original molecular beacons turned out to be providential, according to Tyagi. By scouring the literature, he found a fluorogenic peptide assay that used the fluorophore/quencher pair adenosine-5′-EDANS (5-[(2-aminoethyl)amino]naphthalene-1 sulfonic acid) and DABCYL (4-(4-dimethylaminophenylazo) benzoic acid), used in the design of a protease assay by scientists at Abbott Laboratories (Deerfield, IL, USA)2. Later, when he started experimenting with different dye combinations, he was able to create a palette of molecular beacons with different colored dyes, the fluorescence from which could all be quenched by a single 'universal' quencher. Thus, a few years after the first paper, Tyagi and Kramer, together with Diana Bratu, had a second Nature Biotechnology paper in which they described their palette, and showed how multicolored molecular beacons could be used simultaneously in a single tube3.

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References

  1. Tyagi, S. & Kramer, F.R. Molecular beacons: probes that fluoresce upon hybridization. Nat. Biotechnol. 14, 303–308 (1996).

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  2. Matayoshi, E.D., Wang, G.T., Krafft, G.A. & Erickson, J. Novel fluorogenic substrates for assaying retroviral proteases by resonance energy transfer. Science 247, 954–958 (1999).

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  3. Tyagi, S., Bratu, D.P. & Kramer, F.R. Multicolor molecular beacons for allele discrimination. Nat. Biotechnol. 16, 49–53 (1998).

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  4. Nitin, N., Santangelo, P.J., Kim, G., Nie, S. & Bao, G. Peptide-linked molecular beacons for efficient delivery and rapid mRNA detection in living cells. Nucleic Acids Res. 32, e58 (2004).

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Beacons of light. Nat Biotechnol 24, 303–304 (2006). https://doi.org/10.1038/nbt0306-303b

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