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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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Related in: MedlinePlus

Excitation and emission spectra of labeled proteins in Tris buffer pH 8.(A) ADOTA-IgG (B) DAOTA-Streptavidin.
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pone-0063043-g004: Excitation and emission spectra of labeled proteins in Tris buffer pH 8.(A) ADOTA-IgG (B) DAOTA-Streptavidin.

Mentions: As expected the excitation spectra for both probes are very similar though not an exact match to their absorption spectra. The excitation spectral peaks in buffer are at 541 nm and 556 nm in case of ADOTA-ba and DAOTA-ba, respectively. The emission peaks of free fluorophores in Tris buffer are at 555 nm–556 nm and 589 nm–590 nm for ADOTA-ba and DAOTA-ba, respectively. For both fluorophores the Stokes' shifts are small resulting in significant spectral overlap between absorption and emission. Of the two probes, DAOTA has the larger Stokes' shift and lesser spectral overlap. Upon labeling to avidin, streptavidin and IgG we observed a red-shift in both excitation and emission of the probes. The excitation peaks were between 544 nm to 548 nm and the emission peaks between 560 nm to 564 nm in case of ADOTA-avidin and IgG. In case of DAOTA labeled streptavidin and IgG the respective excitation and emission wavelengths are between 560 nm to 565 nm and 593 nm to 598 nm. In general, we found that higher the degree of the labeling, the redder the spectra. As examples fluorescence excitation and emission spectra of ADOTA labeled IgG are given in Figure 4A and that of DAOTA labeled IgG in Figure 4B. The excitation fluorescence spectra are not corrected for the instrument response. Furthermore, unlike in their absorption spectrum the fluorescence populations are weighted by respective quantum yields.


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)

Excitation and emission spectra of labeled proteins in Tris buffer pH 8.(A) ADOTA-IgG (B) DAOTA-Streptavidin.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0063043-g004: Excitation and emission spectra of labeled proteins in Tris buffer pH 8.(A) ADOTA-IgG (B) DAOTA-Streptavidin.
Mentions: As expected the excitation spectra for both probes are very similar though not an exact match to their absorption spectra. The excitation spectral peaks in buffer are at 541 nm and 556 nm in case of ADOTA-ba and DAOTA-ba, respectively. The emission peaks of free fluorophores in Tris buffer are at 555 nm–556 nm and 589 nm–590 nm for ADOTA-ba and DAOTA-ba, respectively. For both fluorophores the Stokes' shifts are small resulting in significant spectral overlap between absorption and emission. Of the two probes, DAOTA has the larger Stokes' shift and lesser spectral overlap. Upon labeling to avidin, streptavidin and IgG we observed a red-shift in both excitation and emission of the probes. The excitation peaks were between 544 nm to 548 nm and the emission peaks between 560 nm to 564 nm in case of ADOTA-avidin and IgG. In case of DAOTA labeled streptavidin and IgG the respective excitation and emission wavelengths are between 560 nm to 565 nm and 593 nm to 598 nm. In general, we found that higher the degree of the labeling, the redder the spectra. As examples fluorescence excitation and emission spectra of ADOTA labeled IgG are given in Figure 4A and that of DAOTA labeled IgG in Figure 4B. The excitation fluorescence spectra are not corrected for the instrument response. Furthermore, unlike in their absorption spectrum the fluorescence populations are weighted by respective quantum yields.

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