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Fluorescent DNA nanotags featuring covalently attached intercalating dyes: synthesis, antibody conjugation, and intracellular imaging.

ABSTRACT: We have synthesized fluorescent DNA duplexes featuring multiple thiazole orange (TO) intercalating dyes covalently attached to the DNA via a triazole linkage. The intercalating dyes stabilize the duplex against thermal denaturation and show bright fluorescence in the green region of the spectrum. The emission color can be changed to orange or red by addition of energy-accepting Cy3 or Cy5 dyes attached covalently to the DNA duplex. The dye-modified DNA duplexes were then attached to a secondary antibody for intracellular fluorescence imaging of centrosomes in Drosophila embryos. Bright fluorescent foci were observed at the centrosomes in both the donor (TO) and acceptor (Cy5) channels, because the energy transfer efficiency is moderate. Monitoring the Cy5 emission channel significantly minimized the background signal because of the large shift in emission wavelength allowed by energy transfer.

SUBMITTER: Stadler AL

PROVIDER: S-EPMC3163149 | biostudies-literature | 2011 Aug

REPOSITORIES: biostudies-literature

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Fluorescent DNA nanotags featuring covalently attached intercalating dyes: synthesis, antibody conjugation, and intracellular imaging.

Stadler Andrea L AL Delos Santos Junriz O JO Stensrud Elizabeth S ES Dembska Anna A Silva Gloria L GL Liu Shengpeng S Shank Nathaniel I NI Kunttas-Tatli Ezgi E Sobers Courtney J CJ Gramlich Philipp M E PM Carell Thomas T Peteanu Linda A LA McCartney Brooke M BM Armitage Bruce A BA

Bioconjugate chemistry 20110722 8

We have synthesized fluorescent DNA duplexes featuring multiple thiazole orange (TO) intercalating dyes covalently attached to the DNA via a triazole linkage. The intercalating dyes stabilize the duplex against thermal denaturation and show bright fluorescence in the green region of the spectrum. The emission color can be changed to orange or red by addition of energy-accepting Cy3 or Cy5 dyes attached covalently to the DNA duplex. The dye-modified DNA duplexes were then attached to a secondary ...[more]

PMID: 21755981

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Project description:Because the degree of labeling (DOL) of cell-bound antibodies, often required in quantitative fluorescence measurements, is largely unknown, we investigated the effect of labeling with two different fluorophores (AlexaFluor546, AlexaFluor647) in a systematic way using antibody stock solutions with different DOLs. Here, we show that the mean DOL of the cell-bound antibody fraction is lower than that of the stock using single molecule fluorescence measurements. The effect is so pronounced that the mean DOL levels off at approximately two fluorophores/IgG for some antibodies. We developed a method for comparing the average DOL of antibody stocks to that of the isolated, cell-bound fraction based on fluorescence anisotropy measurements confirming the aforementioned conclusions. We created a model in which individual antibody species with different DOLs, present in an antibody stock solution, were assumed to have distinct affinities and quantum yields. The model calculations confirmed that a calibration curve constructed from the anisotropy of antibody stocks can be used for determining the DOL of the bound fraction. The fluorescence intensity of the cell-bound antibody fractions and of the antibody stocks exhibited distinctly different dependence on the DOL. The behavior of the two dyes was systematically different in this respect. Fitting of the model to these data revealed that labeling with each dye affects quantum yield and antibody affinity differentially. These measurements also implied that fluorophores in multiply labeled antibodies exhibit self-quenching and lead to decreased antibody affinity, conclusions directly confirmed by steady-state intensity measurements and competitive binding assays. Although the fluorescence lifetime of antibodies labeled with multiple fluorophores decreased, the magnitude of this change was not sufficient to account for self-quenching indicating that both dynamic and static quenching processes occur involving H-aggregate formation. Our results reveal multiple effects of fluorophore conjugation, which must not be overlooked in quantitative cell biological measurements.

Dataset Information

Fluorescent DNA nanotags featuring covalently attached intercalating dyes: synthesis, antibody conjugation, and intracellular imaging.

Publications

Fluorescent DNA nanotags featuring covalently attached intercalating dyes: synthesis, antibody conjugation, and intracellular imaging.

Similar Datasets

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