Abstract
We have developed novel nucleic acid probes that recognize and report the presence of specific nucleic acids in homogeneous solutions. These probes undergo a spontaneous fIuorogenic conforma-tional change when they hybridize to their targets. Only perfectly complementary targets elicit this response, as hybridization does not occur when the target contains a mismatched nucleotide or a deletion. The probes are particularly suited for monitoring the synthesis of specific nucleic acids in real time. When used in nucleic acid amplification assays, gene detection is homogeneous and sensitive, and can be carried out in a sealed tube. When introduced into living cells, these probes should enable the origin, movement, and fate of specific mRNAs to be traced.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$209.00 per year
only $17.42 per issue
Rent or buy this article
Prices vary by article type
from$1.95
to$39.95
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Lomeli, H., Tyagi, S., Pritchard, C.G., Lizardi, P.M. and Kramer, F.R. 1989. Quantitative assays based on the use of replicatable hybridization probes. Clin. Chem. 36: 1826–1831.
Heller, M.J., Morrison, L.E., Prevatt, W.D. and Akin, C. 1982. Homogeneous nucleic acid hybridization diagnostics by nonradiative energy transfer. European Patent Application 82303699.1.
Morrison, L.E., Hadler, T.C. and Stols, L.M. 1989. Solution phase detection of polynucleotides using interacting fluorescent labels and competitive hybridization. Anal. Biochem. 183: 231–244.
Cardullo, R.A., Agrawal, S., Flores, C., Zamecnik, P.C. and Wolf, D.E. 1988. Detection of nucleic acids by nonradiative fluorescence resonance energy transfer. Proc. Natl. Acad. Sci. USA 85: 8790–8794.
Morrison, L.E. and Stols, L.M. 1993. Sensitive fluorescence-based thermody-namic and kinetic measurements of DNA hybridization in solution. Biochemistry 32: 3095–3104.
Sixou, S., Szoka Jr, F.C., Green, G.A., Giusti, B., Zon, G. and Chin, D.J. 1994. Intracellular oligonucleotide hybridization detected by fluorescence resonance energy transfer (FRET). Nucleic Acids Res. 22: 662–668.
Stryer, L. 1978. Fluorescence energy transfer as a spectroscopic ruler. Ann. Rev. Biochem. 47: 819–846.
Shore, D., Langowski, J. and Baldwin, R.L. 1981. DNA flexibility studied by covalent closure of short fragments into circles. Proc. Natl. Acad. Sci. USA 78: 4833–4837.
Matayoshi, E.D., Wang, G.T., Krafft, G.A. and Erickson, J. 1990. Novel fluoro-genic substrates for assaying retroviral proteases by resonance energy transfer. Science 247: 954–958.
Wang, G.T., Matayoshi, E.D., Huffaker, H.J. and Krafft, G.A. 1990. Design and synthesis of new fluorogenic HIV protease substrates based on resonance energy transfer. Tetrahedron Lett 31: 6493–6496.
Pörschke, D. and Eigen, M. 1971. Cooperative nonenzymic base recognition. III. Kinetics of the helix-coil transition of the oligoribouridylic-oligoriboadenylic acid system and of oligoriboadenylic acid alone at acid pH. J. Mol. Biol. 62: 361–381.
Craig, M.E., Crothers, D.M. and Doty, P. 1971. Relaxation kinetics of dimer formation by self complementary oligonucleotides. J. Mol. Biol. 62: 383–401.
Coutts, S.M. 1971. Thermodynamics and kinetics of G:C base pairing in the isolated extra arm of serine-specific tRNA from yeast. Biochim. Biophys. Acta 232: 91–106.
Higuchi, R., Fockler, C., Dollinger, G. and Watson, R. 1993. Kinetic PCR analysis: Real-time monitoring of DNA amplification reactions. Bio/Technology 11: 1026–1030.
Cooper, J.P. and Hagerman, J.P. 1990. Analysis of fluorescence energy transfer in duplex and branched DNA molecules. Biochemistry 29: 9261–9268.
Meldal, M. and Breddam, K. 1991. Anthranilamide and nitrotyrosine as a donor-acceptor pair in internally quenched fluorescent substrates for endopeptidases. Anal. Biochem. 195: 141–147.
Mergny, J.-L., Boutorine, A.S., Garestier, T., Belloc, F., Rougée, M., Bulychev, N.V., Koshkin, A.A., Bourson, J., Lebedev, A.V., Valeur, B., Thuong, N.T. and Hélène, C. 1994. Fluorescence energy transfer as a probe for nucleic acid structures and sequences. Nucleic Acids Res. 22: 920–928.
Selvin, P.R. and Hearst, J.E. 1994. Luminescence energy transfer using terbium chelate: improvements on fluorescence energy transfer. Proc. Natl. Acad. Sci. USA 91: 10024–10028.
Holland, P.M., Abramson, R.D., Watson, R. and Gelfand, D.H. 1991. Detection of specific polymerase chain reaction product by utilizing 5′→3′ exonuclease activity of Thermus aquaticus DNA polymerase. Proc. Natl. Acad. Sci. USA 88: 7276–7280.
Lee, L.G., Connell, C.R. and Bloch, W. 1993. Allelic discrimination by nick-translation PCR with fluorogenic probes. Nucleic Acids Res. 21: 3761–3766.
Livak, K.J., Flood, S.J.A., Marmaro, J., Giusti, W. and Deetz, K. 1995. Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system useful for detecting PCR product and nucleic acid hybridization. PCR Methods Applic. 4: 357–362.
Connolly, B.A. and Rider, P. 1985. Chemical synthesis of oligonucleotides containing a free sulfhydryl group and subsequent attachment of thiol specific probes. Nucleic Acids Res. 13: 4485–4502.
Tyagi, S., Landegren, U., Tazi, M., Lizardi, P.M. and Kramer, F.R., 1996. Sensitive, background-free gene detection using binary probes and QB replicase. Proc. Natl. Acad. Sci. USA 93: In press.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Tyagi, S., Kramer, F. Molecular Beacons: Probes that Fluoresce upon Hybridization. Nat Biotechnol 14, 303–308 (1996). https://doi.org/10.1038/nbt0396-303
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1038/nbt0396-303
This article is cited by
-
Real-time single-molecule imaging of transcriptional regulatory networks in living cells
Nature Reviews Genetics (2024)
-
Heteromultivalency enables enhanced detection of nucleic acid mutations
Nature Chemistry (2024)
-
Medical viruses: diagnostic techniques
Virology Journal (2023)
-
CRISPR-dCas13-tracing reveals transcriptional memory and limited mRNA export in developing zebrafish embryos
Genome Biology (2023)
-
RT-IVT method allows multiplex real-time quantification of in vitro transcriptional mRNA production
Communications Biology (2023)