Fluorescence-Quenching Phenomenon by Photoinduced Electron Transfer between a Fluorescent Dye and a Nucleotide Base

Fluorescently labeled oligonucleotide probes have been widely used in biotechnology, and fluorescence quenching by the interaction between the dyes and a nucleobase has been pointed out. This quenching causes big problem in analytical methods, but is useful in some other cases. Therefore, it is necessary to estimate the fluorescence quenching intensity under various conditions. We focused on the redox properties of some commercially available fluorescent dyes, and investigated dye-nucleotide interactions between a free dye and a nucleotide in aqueous solution by electrochemical and spectroscopic techniques. Our results suggested that the quenching was accompanied by photoinduced electron transfer between a thermodynamically quenchable excited dye and a specific base. Several kinds of fluorescent dyes labeled to the 5'-end of oligonucleotide C10T6 were prepared, and their quenching ratios compared upon hybridization with the complementary oligonucleotide A6G10. The quenching was completely reversible and their efficiencies depended on the attached fluorophore types. The fluorescence of 5-FAM, BODIPY FL or TAMRA-modified probe was strongly quenched by hybridization.

[1]  A. Harriman,et al.  Photochemistry of intercalated methylene blue: Photoinduced hydrogen atom abstraction from guanine and adenine , 1993 .

[2]  O. Brede,et al.  FOURIER TRANSFORM EPR STUDY OF N-CENTERED PYRIMIDINE RADICALS IN THE NANOSECOND TIME-SCALE , 1997 .

[3]  R. Larsen,et al.  Singlet Excited State Dynamics of Tetrakis(4-N-methylpyridyl)porphine Associated with DNA Nucleotides , 1997 .

[4]  A. Weller.  Photoinduced Electron Transfer in Solution: Exciplex and Radical Ion Pair Formation Free Enthalpies and their Solvent Dependence , 1982 .

[5]  S. Greenfield,et al.  Dynamics of Photoinduced Charge Separation and Charge Recombination in Synthetic DNA Hairpins with Stilbenedicarboxamide Linkers , 2000 .

[6]  Seog K. Kim,et al.  MECHANISMS OF QUENCHING OF THE FLUORESCENCE OF A BENZO[a]PYRENE TETRAOL METABOLITE MODEL COMPOUND BY 2′‐DEOXYNUCLEOSIDES , 1993, Photochemistry and photobiology.

[7]  Michael R. Wasielewski,et al.  Direct measurement of hole transport dynamics in DNA , 2000, Nature.

[8]  P. Hagerman,et al.  Analysis of fluorescence energy transfer in duplex and branched DNA molecules. , 1990, Biochemistry.

[9]  N. Lin,et al.  Riboflavin (VB2) photosensitized oxidation of 2′-deoxyguanosine-5′-monophosphate (dGMP) in aqueous solution: a transient intermediates study , 2000 .

[10]  J. G. Chirikjian,et al.  A fluorometric assay for DNA cleavage reactions characterized with BamHI restriction endonuclease. , 1994, Analytical biochemistry.

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

[12]  D. Rehm,et al.  Kinetics of Fluorescence Quenching by Electron and H‐Atom Transfer , 1970 .

[13]  James J. P. Stewart,et al.  Application of localized molecular orbitals to the solution of semiempirical self‐consistent field equations , 1996 .

[14]  W. Jones,et al.  Determination of Excited-State Redox Potentials by Phase-Modulated Voltammetry , 1994 .

[15]  D. Millar,et al.  Distance distribution in a dye-linked oligonucleotide determined by time-resolved fluorescence energy transfer. , 1992, Biophysical chemistry.

[16]  Hiroshi Sugiyama,et al.  Photoinduced DNA Cleavage via Electron Transfer: Demonstration That Guanine Residues Located 5' to Guanine Are the Most Electron-Donating Sites , 1995 .

[17]  U. Göbel,et al.  Fluorescence in situ hybridization (FISH) for direct visualization of microorganisms. , 2000, Journal of microbiological methods.

[18]  G. Schuster,et al.  Intramolecular Photoinduced Electron Transfer to Anthraquinones Linked to Duplex DNA: The Effect of Gaps and Traps on Long-Range Radical Cation Migration , 1997 .

[19]  C. Brenner,et al.  Fhit-nucleotide Specificity Probed with Novel Fluorescent and Fluorogenic Substrates* , 2000, The Journal of Biological Chemistry.

[20]  N. Walter,et al.  Real-time monitoring of hairpin ribozyme kinetics through base-specific quenching of fluorescein-labeled substrates. , 1997, RNA.

[21]  R. Rigler,et al.  Conformational transitions monitored for single molecules in solution. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[22]  A. Watanabe,et al.  Distance dependence of electron transfer in acridine-intercalated DNA , 1999 .

[23]  M. Urdea,et al.  Chemical synthesis and characterization of branched oligodeoxyribonucleotides (bDNA) for use as signal amplifiers in nucleic acid quantification assays. , 1997, Nucleic acids research.

[24]  G. G. Stokes.  "J." , 1890, The New Yale Book of Quotations.

[25]  Markus Sauer,et al.  NUCLEOBASE-SPECIFIC QUENCHING OF FLUORESCENT DYES. 1. NUCLEOBASE ONE-ELECTRON REDOX POTENTIALS AND THEIR CORRELATION WITH STATIC AND DYNAMIC QUENCHING EFFICIENCIES , 1996 .

[26]  N. Geacintov,et al.  Proton-Coupled Photoinduced Electron Transfer, Deuterium Isotope Effects, and Fluorescence Quenching in Noncovalent Benzo[a]pyrenetetraol—Nucleoside Complexes in Aqueous Solutions , 1995 .

[27]  S. Féry-Forgues,et al.  Ferrocene and ferrocenyl derivatives in luminescent systems , 2000 .

[28]  B. Armitage,et al.  Anthraquinone Photonucleases: A Surprising Role for Chloride in the Sequence‐Neutral Cleavage of DNA and the Footprinting of Minor Groove‐Bound Ligands , 1997, Photochemistry and photobiology.

[29]  R. Hochstrasser,et al.  Nonexponential kinetics of a single tRNAPhe molecule under physiological conditions. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[30]  J. Kelly,et al.  Photoreactions of ruthenium(II) and osmium(II) complexes with deoxyribonucleic acid (DNA). , 1997, Journal of photochemistry and photobiology. B, Biology.

[31]  A. Zewail,et al.  Femtosecond linear dichroism of DNA-intercalating chromophores: solvation and charge separation dynamics of [Ru(phen)2dppz]2+ systems. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[32]  S. Burgard.  ISR , 1999 .

[33]  D. Lilley,et al.  Location of cyanine-3 on double-stranded DNA: importance for fluorescence resonance energy transfer studies. , 2000, Biochemistry.

[34]  T. Netzel,et al.  Synthesis and Photophysics of a 1-Pyrenylmethyl-Substituted 2‘-Deoxyuridine-5-Carboxamide Nucleoside: Electron-Transfer Product Lifetimes and Energies , 2000 .

[35]  Markus Sauer,et al.  Dynamics of the electron transfer reaction between an oxazine dye and DNA oligonucleotides monitored on the single-molecule level , 1998 .

[36]  Jerker Widengren,et al.  Fast interactions between Rh6G and dGTP in water studied by fluorescence correlation spectroscopy , 1997 .

[37]  C. Seidel,et al.  Monitoring conformational dynamics of a single molecule by selective fluorescence spectroscopy. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[38]  M. Fox,et al.  Photophysical quenching mediated by guanine groups in pyrenyl-N-alkylbutanoamide end-labeled oligonucleotides , 1999 .

[39]  N. Geacintov,et al.  Influence of adduct stereochemistry and hydrogen-bonding solvents on photoinduced charge transfer in a covalent benzo[a]pyrene diol epoxide-nucleoside adduct on picosecond time scales , 1994 .

[40]  I. Kochevar,et al.  THE ROLE OF GROUND STATE COMPLEXATION IN THE ELECTRON TRANSFER QUENCHING OF METHYLENE BLUE FLUORESCENCE BY PURINE NUCLEOTIDES , 1991, Photochemistry and photobiology.