Abstract
A novel approach for detecting nucleic acid in solution has been adopted for real-time imaging of native mRNAs in living cells. This method utilizes hybridization probes, called “molecular beacons,” that generate fluorescent signals only when they are hybridized to a complementary target sequence. Nuclease-resistant molecular beacons are designed to efficiently hybridize to accessible regions within RNAs and then be detected via fluorescence microscopy. The target regions chosen for probe binding are selected using two computer algorithms, mfold and OligoWalk, that predict the secondary structure of RNAs and help narrow down sequence stretches to which the probes should bind with high affinity in vivo. As an example, molecular beacons were designed against regions of oskar mRNA, microinjected into living Drosophila melanogaster oocytes and imaged via confocal microscopy.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Tyagi, S. and Kramer, F. R. (1996) Molecular beacons: probes that fluoresce upon hybridization. Nature Biotechnol. 14, 303–308.
Bratu, D. P., Cha, B. J., Mhlanga, M. M., Kramer, F. R., and Tyagi, S. (2003) Visualizing the distribution and transport of mRNAs in living cells. Proc. Natl. Acad. Sci. USA 100, 13,308–13,313.
Matsuo, T. (1998) In situ visualization of messenger RNA for basic fibroblast growth factor in living cells. Biochem. Biophys. Acta 1379, 178–184.
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. USA 95, 11,538–11,543.
Southern, E. M., Milner, N., and Mir, K. U. (1997) Discovering antisense reagents by hybridization of RNA to oligonucleotide arrays. Ciba Found. Symp. 209, 38–44; discussion 44–46.
Ho, S. P., Bao, Y., Lesher, T., et al. (1998) Mapping of RNA accessible sites for antisense experiments with oligonucleotide libraries. Nature Biotechnol. 16, 59–63.
Lima, W. F., Mohan, V., and Crooke, S. T. (1997) The influence of antisense oligonucleotide-induced RNA structure on Escherichia coli RNase H1 activity. J. Biol. Chem. 272, 18,191–18,199.
Milner, N., Mir, K. U., and Southern, E. M. (1997) Selecting effective antisense reagents on combinatorial oligonucleotide arrays. Nature Biotechnol. 15, 537–541.
Zuker, M. (2003) Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res. 31, 1–10.
Mathews, D. H., Burkard, M. E., Freier, S. M., Wyatt, J. R., and Turner, D. H. (1999) Predicting oligonucleotide affinity to nucleic acid targets. RNA 5, 1458–1469.
Peyret, N., Seneviratne, P. A., Allawi, H. T., and SantaLucia, J., Jr. (1999) Nearestneighbor thermodynamics and NMR of DNA sequences with internal A.A, C.C, G.G, and T.T mismatches. Biochemistry 38, 3468–3477.
SantaLucia, J., Jr. (1998) A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics. Proc. Natl. Acad. Sci. USA 95, 1460–1465.
SantaLucia, J., Jr., Allawi, H. T., and Seneviratne, P. A. (1996) Improved nearestneighbor parameters for predicting DNA duplex stability. Biochemistry 35, 3555–3562.
Sugimoto, N., Nakano, S., Katoh, M., et al. (1995) Thermodynamic parameters to predict stability of RNA/DNA hybrid duplexes. Biochemistry 34, 11,211–11,216.
Xia, T., SantaLucia, J., Jr., Burkard, M. E., et al. (1998) Thermodynamic parameters for an expanded nearest-neighbor model for formation of RNA duplexes with Watson-Crick base pairs. Biochemistry 37, 14,719–14,735.
Marras, S. A., Kramer, F. R., and Tyagi, S. (2002) Efficiencies of fluorescence resonance energy transfer and contact-mediated quenching in oligonucleotide probes. Nucleic Acids Res. 30, e122.
Bratu, D. P. (2003) Imaging Native mRNAs in Living Drosophila Oocytes Using Molecular Beacons. New York University, UMI Dissertation Service, New York, New York.
Tyagi, S., Bratu, D. P., and Kramer, F. R. (1998) Multicolor molecular beacons for allele discrimination. Nature Biotechnol. 16, 49–53.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2006 Humana Press Inc.
About this protocol
Cite this protocol
Bratu, D.P. (2006). Molecular Beacons. In: Taatjes, D.J., Mossman, B.T. (eds) Cell Imaging Techniques. Methods in Molecular Biology™, vol 319. Humana Press. https://doi.org/10.1007/978-1-59259-993-6_1
Download citation
DOI: https://doi.org/10.1007/978-1-59259-993-6_1
Publisher Name: Humana Press
Print ISBN: 978-1-58829-157-8
Online ISBN: 978-1-59259-993-6
eBook Packages: Springer Protocols