Heterodimeric DNA-binding dyes designed for energy transfer: synthesis and spectroscopic properties

Benson, Scott C.; Singh, Paramjit; Glazer, Alexander N.

doi:10.1093/nar/21.24.5727

Cited by 69 publications

(38 citation statements)

References 19 publications

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“…Thus, fluorescence resonance ET has been used extensively as a spectroscopic ruler for biological structures (13), and ET-coupled tandem phycobiliprotein conjugates have found wide application as unique fluorescent labels (14). Earlier, we showed that polycationic heterodimeric fluorophores that exploit ET and that have high affinities for double-stranded DNA offer advantages over monomeric fluorophores in multiplex fluorescence-labeling applications (15)(16)(17). We show here that the ET primers are markedly superior to single dye-labeled primers in DNA sequencing, and we anticipate that they will find broad use in multiplex PCR-based mapping and sizing protocols.…”

mentioning

confidence: 99%

Fluorescence energy transfer dye-labeled primers for DNA sequencing and analysis.

Ruan²,

Fuller³

et al. 1995

Proc. Natl. Acad. Sci. U.S.A.

253

140

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Fluorescent dye-labeled DNA primers have been developed that exploit fluorescence energy transfer (ET) to optimize the absorption and emission properties of the label. These primers carry a fluorescein derivative at the 5' end as a common donor and other fluorescein and rhodamine derivatives attached to a modified thymidine residue within the primer sequence as acceptors. Adjustment of the donoracceptor spacing through the placement of the modified thymidine in the primer sequence allowed generation of four primers, all having strong absorption at a common excitation wavelength (488 nm)

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mentioning

confidence: 99%

Fluorescence energy transfer dye-labeled primers for DNA sequencing and analysis.

Ruan²,

Fuller³

et al. 1995

Proc. Natl. Acad. Sci. U.S.A.

253

140

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“…For experiments using fluorescent intercalating dye for detection, 500-bp PvuII fragments from plasmids pBL4 and pBL6 (8) were used; pBL4 and pBL6 contain four and six NGAAN repeats, respectively. An aliquot of dimeric intercalating fluorescent dye (either TOTO (22) or TOTAB (15,16)) was added to the DNA-protein mixture at a DNA base pair to dye molar ratio of 40:1, and the reaction mixture incubated in the dark at room temperature for 30 min. After electrophoresis, gels with radiolabeled complexes were dried and scanned using a Molecular Dynamics PhosphorImager.…”

Section: Methodsmentioning

confidence: 99%

“…To measure the absolute quantities of protein and DNA in each complex, we developed a two-color mobility shift fluorescence assay with a mutant HSF DT engineered for site-specific labeling with fluorescein and target DNA labeled with thiazole orange-thiazole blue heterodimer (TOTAB), an "energy transfer" dye (15,16). A technical difficulty was encountered in this work.…”

mentioning

confidence: 99%

Environment-sensitive Labels in Multiplex Fluorescence Analyses of Protein-DNA Complexes

Drees¹,

Rye²,

Glazer

et al. 1996

Journal of Biological Chemistry

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Fluorescein is widely used for protein labeling because of its high extinction coefficient and fluorescence emission quantum yield. However, its emission is readily quenched by various pathways. We exploit these properties of fluorescein to examine the self-association of a DNA binding protein and determine the amount of the protein in gel-shifted complexes with specific DNA. A construct ( The "gel mobility shift" or "band-shift" assay is widely used to detect and analyze protein-DNA interactions. The most direct way to determine the stoichiometry of protein-DNA complexes resolved in such experiments is to measure independently the amounts of both DNA and protein in a band on a gel. Quantitation of DNA in the shifted complex is relatively straightforward with 32 P end-labeling, but accurate measurement of protein can be quite challenging. Protein quantities in shifted complexes have been measured in double-labeling experiments using Coomassie staining, Western blot, and radioisotopic labeling of the protein with 3 H, 125 I, and 35 S (1-7). Drawbacks of these methods include limitations on sensitivity, difficulty in ensuring accurately the specific activity of radiolabeled proteins, and the need to handle the gel after electrophoresis for staining or blotting, or to cut out gel bands for scintillation counting. Consequently, few accurate determinations of the molar ratios of protein-to-DNA have been reported.We have shown that multiplex fluorescence analysis is a superior alternative method of characterizing protein-DNA complexes (8). In our earlier work, we described the first detailed analysis of the application of fluorescent polycationic intercalating dyes with high affinity for double-stranded DNA to the study of protein-DNA interactions. Such intercalating dyes, in conjunction with high sensitivity laser-excited, confocal, fluorescence scanning systems, allow detection of doublestranded DNA in agarose or acrylamide gels with a sensitivity comparable to that of autoradiography (9 -12). We used such dyes in gel mobility shift assays to detect the multiple protein-DNA complexes formed by the heat shock transcription factor (HSF) 1 (8,13,14). Using a truncation of Kluyveromyces lactis HSF that contains the DNA-binding and trimerization domains (HSF DT ), we were able to detect fluorescent dye-labeled HSF DT -DNA complexes with a spatial resolution superior to that of conventional autoradiography, and therefore we were able to analyze multimer protein-DNA complexes that are not resolved by traditional methods. An analysis of the mobilities of the multiple HSF DT -DNA complexes, indicated that HSF forms multimeric complexes on DNA by the addition of trimeric units (8).To measure the absolute quantities of protein and DNA in each complex, we developed a two-color mobility shift fluorescence assay with a mutant HSF DT engineered for site-specific labeling with fluorescein and target DNA labeled with thiazole orange-thiazole blue heterodimer (TOTAB), an "energy transfer" dye (15, 16). A technical difficulty was enco...

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“…Interest in nonradioactive DNA stains that are stable under gel electrophoretic conditions has led to the synthesis and characterization of a family of homo-and heterodimeric DNA-binding dyes (1)(2)(3). Organic fluorescent probes (FPs) are, as a rule, polycyclic aromatic cations with the planar structure capable of incorporating (intercalating) between the planes of the DNA bases, pulling apart these planes and changing the structure of nucleic acids.…”

Section: Introductionmentioning

confidence: 99%

“…Bis-intercalating TOTO-1 dyes have a weak fluorescence in the free state, but they sharply (more than 1000 times) increase fluorescence due to binding with DNA (4) and, therefore, they are widely used in biological, medical and drug development areas as fluorescent labels and probes (8)(9)(10)(11)(12)(13)(14)(15). They found also applications in genetic studies and modern diagnostic methods (1)(2)(3), for examples, in the case of the polymerase chain reaction for cancer diagnostics at early stages of the disease; for diagnostics of infection diseases (1)(2)(3)(4), including AIDS; for the identification of DNA samples in criminal law (16,17); flow cytometry (18), DNA sequencing (19,20) and quantification of nucleic acids in capillary and gel electrophoresis (21)(22)(23). Besides, they are commonly applied to lasers (24), electronics (25), nonlinear optics (26) and solar cells (27).…”

Section: Introductionmentioning

confidence: 99%

Microwave synthesis and fluorescence properties of homo- and heterodimeric monomethine cyanine dyes TOTO and their precursors

Alganzory

El‐Sayed

Arief

et al. 2016

Green Chemistry Letters and Reviews

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A series of monomeric and dimeric cyanine dyes belonging to the thiazole orange family have been prepared via an improved synthetic procedure, by the reaction of the monomethine dye containing an iodoalkyl group with tertiary diamine linkers under microwave irradiation. The effects of microwave power and irradiation time on yield were examined. The electronic absorption and steady-state fluorescence spectra of prepared dyes have been investigated. Fluorescence properties indicate significance in singlet oxygen sensitization and make the present compounds potential candidates in the area of photodynamic therapy. ARTICLE HISTORY

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Heterodimeric DNA-binding dyes designed for energy transfer: synthesis and spectroscopic properties

Cited by 69 publications

References 19 publications

Fluorescence energy transfer dye-labeled primers for DNA sequencing and analysis.

Fluorescence energy transfer dye-labeled primers for DNA sequencing and analysis.

Environment-sensitive Labels in Multiplex Fluorescence Analyses of Protein-DNA Complexes

Microwave synthesis and fluorescence properties of homo- and heterodimeric monomethine cyanine dyes TOTO and their precursors

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