Fluorescent hybridization probes for sensitive and selective DNA and RNA detection.

We outline the different approaches taken by our group in the design of fluorescent hybridization sensors. Molecular beacons (MBs) and binary probes (BPs) using two dyes (2d-MB and 2d-BP, respectively) have been synthesized; these sensors serve as switches in emission upon binding to target biomolecules, such as DNA. These sensors allow for ratiometric fluorescence detection of polynucleotides (PNs) by visualization of the probes when bound to a target PN. Additionally, three-dye MBs (3d-MB) and BPs (3d-BP) have been developed, where an energy-transfer cascade is employed to decrease the overlap between the fluorophore emission spectra, resulting in a low direct excitation of the acceptor fluorophore. Pyrene-based MB (Py-MB) and BP (Py-BP), which possess the advantage of long fluorescence lifetimes, have also been synthesized. Time-resolved fluorescence spectra (TRES) can be used to discriminate between short-lived background fluorescence and long-lived fluorescence of the pyrene probes. This technique was demonstrated by time-resolving the signal of a Py-BP from the background fluorescence in Aplysia californica cell extracts.

[1]  S. Jockusch,et al.  Design and characterization of two-dye and three-dye binary fluorescent probes for mRNA detection. , 2007, Tetrahedron.

[2]  S. Jockusch,et al.  Combinatorial fluorescence energy transfer molecular beacons for probing nucleic acid sequences , 2006, Photochemical & photobiological sciences : Official journal of the European Photochemistry Association and the European Society for Photobiology.

[3]  W. Tan,et al.  Hybrid molecular probe for nucleic acid analysis in biological samples. , 2006, Journal of the American Chemical Society.

[4]  Igor L. Medintz,et al.  Materials for fluorescence resonance energy transfer analysis: beyond traditional donor-acceptor combinations. , 2006, Angewandte Chemie.

[5]  M. Komiyama,et al.  Insertion of two pyrene moieties into oligodeoxyribonucleotides for the efficient detection of deletion polymorphisms. , 2006, Chemical communications.

[6]  Sathyanarayanan V. Puthanveettil,et al.  Pyrene binary probes for unambiguous detection of mRNA using time-resolved fluorescence spectroscopy , 2006, Nucleic acids research.

[7]  Matthew S. Tremblay,et al.  Phosphorylation state-responsive lanthanide peptide conjugates: a luminescence switch based on reversible complex reorganization. , 2006, Organic letters.

[8]  S. Jockusch,et al.  Spectroscopic investigation of a FRET molecular beacon containing two fluorophores for probing DNA/RNA sequences† , 2006, Photochemical & photobiological sciences : Official journal of the European Photochemistry Association and the European Society for Photobiology.

[9]  S. Jockusch,et al.  Molecular beacons with intrinsically fluorescent nucleotides , 2006, Nucleic acids research.

[10]  G. Bao,et al.  Nanostructured Probes for RNA Detection in Living Cells , 2006, Annals of Biomedical Engineering.

[11]  M. Levy,et al.  Quantum‐Dot Aptamer Beacons for the Detection of Proteins , 2005, Chembiochem : a European journal of chemical biology.

[12]  G. Kumarasinghe,et al.  Rapid detection of codon 460 mutations in the UL97 gene of ganciclovir-resistant cytomegalovirus clinical isolates by real-time PCR using molecular beacons. , 2005, Molecular and cellular probes.

[13]  Weihong Tan,et al.  Light-switching excimer probes for rapid protein monitoring in complex biological fluids. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[14]  Tom Brown,et al.  Fluorescence based strategies for genetic analysis. , 2005, Chemical communications.

[15]  Mitsunobu Nakamura,et al.  DNA mismatch detection using a pyrene-excimer-forming probe. , 2005, Chemical communications.

[16]  Adam P. Silverman,et al.  Quenched probes for highly specific detection of cellular RNAs. , 2005, Trends in biotechnology.

[17]  S. Jockusch,et al.  Two-photon Excitation Induced Fluorescence of a Trifluorophore-labeled DNA¶ , 2005, Photochemistry and photobiology.

[18]  H. Tanke,et al.  FISH and immunocytochemistry: towards visualising single target molecules in living cells. , 2005, Current opinion in biotechnology.

[19]  A. Okamoto,et al.  Nitroxide-labeled guanine as an ESR spin probe for structural study of DNA. , 2004, Bioorganic & medicinal chemistry letters.

[20]  Manzour Hernando Hazbón,et al.  Molecular beacons for multiplex detection of four bacterial bioterrorism agents. , 2004, Clinical chemistry.

[21]  Jared P. Stephens,et al.  Hybrid Inorganic-Organic Molecular Beacons , 2004 .

[22]  Kazuhisa Fujimoto,et al.  Unambiguous detection of target DNAs by excimer-monomer switching molecular beacons. , 2004, The Journal of organic chemistry.

[23]  Gang Bao,et al.  Dual FRET molecular beacons for mRNA detection in living cells. , 2004, Nucleic acids research.

[24]  A. Tong,et al.  Combinatorial fluorescence energy transfer tags: new molecular tools for genomics applications , 2002 .

[25]  S. Schacher,et al.  Target Interaction Regulates Distribution and Stability of Specific mRNAs , 2002, The Journal of Neuroscience.

[26]  Joydeep Lahiri,et al.  Method for detection of single-base mismatches using bimolecular beacons. , 2002, Journal of the American Chemical Society.

[27]  S. Jockusch,et al.  Triple fluorescence energy transfer in covalently trichromophore-labeled DNA. , 2001, Journal of the American Chemical Society.

[28]  T. Beck,et al.  Design of a Molecular Beacon DNA Probe with Two Fluorophores. , 2001, Angewandte Chemie.

[29]  A. Tsuji,et al.  Real-time monitoring of in vitro transcriptional RNA synthesis using fluorescence resonance energy transfer. , 2000, Nucleic acids research.

[30]  A. Tsuji,et al.  Direct observation of specific messenger RNA in a single living cell under a fluorescence microscope. , 2000, Biophysical journal.

[31]  M. Masuko,et al.  Optimization of excimer-forming two-probe nucleic acid hybridization method with pyrene as a fluorophore. , 1998, Nucleic acids research.

[32]  E. Kool,et al.  Probing DNA sequences in solution with a monomer-excimer fluorescence color change. , 1998, Nucleic acids research.

[33]  K. Takata,et al.  DNA Staining for Fluorescence and Laser Confocal Microscopy , 1997, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[34]  Kjell Carlsson,et al.  Confocal fluorescence microscopy using spectral and lifetime information to simultaneously record four fluorophores with high channel separation , 1997 .

[35]  Sanjay Tyagi,et al.  Molecular Beacons: Probes that Fluoresce upon Hybridization , 1996, Nature Biotechnology.

[36]  M. Masuko,et al.  NUCLEIC ACID HYBRIDIZATION ACCOMPANIED WITH EXCIMER FORMATION FROM TWO PYRENE‐LABELED PROBES , 1995, Photochemistry and photobiology.

[37]  B. Valeur,et al.  Fluorescence energy transfer as a probe for nucleic acid structures and sequences. , 1994, Nucleic acids research.

[38]  A. Lamond,et al.  Antisense oligonucleotides made of 2'‐O‐alkylRNA: their properties and applications in RNA biochemistry , 1993, FEBS letters.

[39]  N. Turro,et al.  Molecular Recognition and Chemistry in Restricted Reaction Spaces. Photophysics and Photoinduced Electron Transfer on the Surfaces of Micelles, Dendrimers, and DNA , 1991 .

[40]  N. Turro,et al.  Mixed-ligand complexes of ruthenium(II): factors governing binding to DNA , 1989 .

[41]  D. E. Wolf,et al.  Detection of nucleic acid hybridization by nonradiative fluorescence resonance energy transfer. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[42]  N. Turro.  Modern Molecular Photochemistry , 1978 .

[43]  Kendall N Houk,et al.  Accounts of Chemical Research. , 2008, Accounts of chemical research.

[44]  S. Schuster,et al.  Molecular beacons , 2007, Cell Biochemistry and Biophysics.

[45]  Sanjay Tyagi,et al.  Real-time assays with molecular beacons and other fluorescent nucleic acid hybridization probes. , 2006, Clinica chimica acta; international journal of clinical chemistry.

[46]  Gang Bao,et al.  Shedding light on health and disease using molecular beacons. , 2003, Briefings in functional genomics & proteomics.

[47]  M. E. Beall.  U.S. patent and trademark office , 1997 .

[48]  K. Yamana,et al.  Oligonucleotides with bis-pyrene adduct in the backbone: syntheses and properties of intramolecular excimer forming probe. , 1991, Nucleic Acids Symposium Series.

[49]  J. Lakowicz.  Principles of fluorescence spectroscopy , 1983 .