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Long-lived bright red emitting azaoxa-triangulenium fluorophores.

Maliwal BP, Fudala R, Raut S, Kokate R, Sørensen TJ, Laursen BW, Gryczynski Z, Gryczynski I - PLoS ONE (2013)

Bottom Line: Despite the presence of significant local motions due to a flexible trimethylene linker, we successfully measured both intermediate nanosecond intra-protein motions and slower rotational correlation times approaching 100 ns.Their long lifetimes are unaffected by the cell membrane (hexadecyl-ADOTA) and the intra-cellular (DAOTA-Arginine) localization.ADOTA and DAOTA retain a long fluorescence lifetime when free, as protein conjugate, in membranes and inside the cell.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Immunology, University of North Texas Health Science Center, Fort Worth, Texas, United States of America. bpmal001@gmail.com

ABSTRACT
The fluorescence lifetimes of most red emitting organic probes are under 4 nanoseconds, which is a limiting factor in studying interactions and conformational dynamics of macromolecules. In addition, the nanosecond background autofluorescence is a significant interference during fluorescence measurements in cellular environment. Therefore, red fluorophores with longer lifetimes will be immensely helpful. Azaoxa-triangulenium fluorophores ADOTA and DAOTA are red emitting small organic molecules with high quantum yield, long fluorescence lifetime and high limiting anisotropy. In aqueous environment, ADOTA and DAOTA absorption and emission maxima are respectively 540 nm and 556 nm, and 556 nm and 589 nm. Their emission extends beyond 700 nm. Both probes have the limiting anisotropy between 0.36-0.38 at their absorption peak. In both protic and aprotic solvents, their lifetimes are around 20 ns, making them among the longest-lived red emitting organic fluorophores. Upon labeling of avidin, streptavidin and immunoglobulin their absorption and fluorescence are red-shifted. Unlike in free form, the protein-conjugated probes have heterogeneous fluorescence decays, with the presence of both significantly quenched and unquenched populations. Despite the presence of significant local motions due to a flexible trimethylene linker, we successfully measured both intermediate nanosecond intra-protein motions and slower rotational correlation times approaching 100 ns. Their long lifetimes are unaffected by the cell membrane (hexadecyl-ADOTA) and the intra-cellular (DAOTA-Arginine) localization. Their long lifetimes also enabled successful time-gating of the cellular autofluorescence resulting in background-free fluorescence lifetime based images. ADOTA and DAOTA retain a long fluorescence lifetime when free, as protein conjugate, in membranes and inside the cell. Our successful measurements of intermediate nanosecond internal motions and long correlations times of large proteins suggest that these probes will be highly useful to study slower intra-molecular motions and interactions among macromolecules. The fluorescence lifetime facilitated gating of cellular nanosecond autofluorescence should be of considerable help in in vitro and in vivo applications.

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Fluorescence excitation and emission spectra of hexadecyl derivatives in DOPC.(A) ADOTA (B) DAOTA.
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pone-0063043-g010: Fluorescence excitation and emission spectra of hexadecyl derivatives in DOPC.(A) ADOTA (B) DAOTA.

Mentions: Figures 10A and 10B show excitation and emission spectra of ADOTA and DAOTA hexadecyl derivatives in DOPC small vesicles. The probe concentrations were one probe for every 200 lipid molecules. The excitation and emission spectra for both ADOTA and DAOTA are red-shifted in the DOPC lipid vesicles, when compared to free butyric acid derivatives in phosphate buffer. The excitation and emission peaks for ADOTA in DOPC are at 545 nm–546 nm and 566 nm–567 nm representing red shifts of 5 nm in excitation and 11 nm in the emission spectrum. In the case of DAOTA excitation and emission peaks of 566 nm and 602 nm–603 nm represent red-shifts of 9 nm and 12 nm, when moving from an aqueous environment to the phospholipid head group/interface region. We did not characterize the probe location in the lipid vesicle. Due to the presence of a positive charge in the planar azaoxa-triangulenium molecules, the probes are unlike to penetrate into the lipid hydrocarbon core and are most likely present in strongly dipolar head group/interface region. As these red shifts are significantly larger than seen in 2-propanol, there are other factors involved beside simple polarity of the microenvironment. The local oriented dipoles of DOPC and counter ion effects are most likely the origin of these spectral shifts [30]–[31].


Long-lived bright red emitting azaoxa-triangulenium fluorophores.

Maliwal BP, Fudala R, Raut S, Kokate R, Sørensen TJ, Laursen BW, Gryczynski Z, Gryczynski I - PLoS ONE (2013)

Fluorescence excitation and emission spectra of hexadecyl derivatives in DOPC.(A) ADOTA (B) DAOTA.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3646960&req=5

pone-0063043-g010: Fluorescence excitation and emission spectra of hexadecyl derivatives in DOPC.(A) ADOTA (B) DAOTA.
Mentions: Figures 10A and 10B show excitation and emission spectra of ADOTA and DAOTA hexadecyl derivatives in DOPC small vesicles. The probe concentrations were one probe for every 200 lipid molecules. The excitation and emission spectra for both ADOTA and DAOTA are red-shifted in the DOPC lipid vesicles, when compared to free butyric acid derivatives in phosphate buffer. The excitation and emission peaks for ADOTA in DOPC are at 545 nm–546 nm and 566 nm–567 nm representing red shifts of 5 nm in excitation and 11 nm in the emission spectrum. In the case of DAOTA excitation and emission peaks of 566 nm and 602 nm–603 nm represent red-shifts of 9 nm and 12 nm, when moving from an aqueous environment to the phospholipid head group/interface region. We did not characterize the probe location in the lipid vesicle. Due to the presence of a positive charge in the planar azaoxa-triangulenium molecules, the probes are unlike to penetrate into the lipid hydrocarbon core and are most likely present in strongly dipolar head group/interface region. As these red shifts are significantly larger than seen in 2-propanol, there are other factors involved beside simple polarity of the microenvironment. The local oriented dipoles of DOPC and counter ion effects are most likely the origin of these spectral shifts [30]–[31].

Bottom Line: Despite the presence of significant local motions due to a flexible trimethylene linker, we successfully measured both intermediate nanosecond intra-protein motions and slower rotational correlation times approaching 100 ns.Their long lifetimes are unaffected by the cell membrane (hexadecyl-ADOTA) and the intra-cellular (DAOTA-Arginine) localization.ADOTA and DAOTA retain a long fluorescence lifetime when free, as protein conjugate, in membranes and inside the cell.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Immunology, University of North Texas Health Science Center, Fort Worth, Texas, United States of America. bpmal001@gmail.com

ABSTRACT
The fluorescence lifetimes of most red emitting organic probes are under 4 nanoseconds, which is a limiting factor in studying interactions and conformational dynamics of macromolecules. In addition, the nanosecond background autofluorescence is a significant interference during fluorescence measurements in cellular environment. Therefore, red fluorophores with longer lifetimes will be immensely helpful. Azaoxa-triangulenium fluorophores ADOTA and DAOTA are red emitting small organic molecules with high quantum yield, long fluorescence lifetime and high limiting anisotropy. In aqueous environment, ADOTA and DAOTA absorption and emission maxima are respectively 540 nm and 556 nm, and 556 nm and 589 nm. Their emission extends beyond 700 nm. Both probes have the limiting anisotropy between 0.36-0.38 at their absorption peak. In both protic and aprotic solvents, their lifetimes are around 20 ns, making them among the longest-lived red emitting organic fluorophores. Upon labeling of avidin, streptavidin and immunoglobulin their absorption and fluorescence are red-shifted. Unlike in free form, the protein-conjugated probes have heterogeneous fluorescence decays, with the presence of both significantly quenched and unquenched populations. Despite the presence of significant local motions due to a flexible trimethylene linker, we successfully measured both intermediate nanosecond intra-protein motions and slower rotational correlation times approaching 100 ns. Their long lifetimes are unaffected by the cell membrane (hexadecyl-ADOTA) and the intra-cellular (DAOTA-Arginine) localization. Their long lifetimes also enabled successful time-gating of the cellular autofluorescence resulting in background-free fluorescence lifetime based images. ADOTA and DAOTA retain a long fluorescence lifetime when free, as protein conjugate, in membranes and inside the cell. Our successful measurements of intermediate nanosecond internal motions and long correlations times of large proteins suggest that these probes will be highly useful to study slower intra-molecular motions and interactions among macromolecules. The fluorescence lifetime facilitated gating of cellular nanosecond autofluorescence should be of considerable help in in vitro and in vivo applications.

Show MeSH
Related in: MedlinePlus