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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 intensity and steady state anisotropy of ADOTA-C16 in DMPC between 10°C and 35°C.
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pone-0063043-g011: Fluorescence intensity and steady state anisotropy of ADOTA-C16 in DMPC between 10°C and 35°C.

Mentions: We also looked at the steady-state anisotropy and fluorescence emission of hexadecyl-ADOTA in DMPC small vesicle during the transition from gel to liquid crystalline state. The results are shown in Figure 11. The fluorescence intensity decreases by less than 5% between 15°C and 35°C. Even though it is possible to see the subtle signature of phase transition, the change in emission maxima is rather small (less than 4 nm). There is also a reproducible small decrease in the intensity between 15°C and 10°C. We suspect it is primarily due to inner filter effect as the vesicles become more opaque up on lowering of temperature. The steady-state anisotropy decreased from 0.219 at 10°C to 0.114 at 35°C, reflecting the phase transition of DMPC. Our results above phase transition are similar to those seen with anchored DPH derivatives [23, 24, and 32] and fluorescent fatty acids with long lifetime [33], [34]. It should be noted that the fluorophore in DPH derivatives and fluorescent fatty acids are present in the hydrocarbon region of lipid while the ADOTA and DAOTA moieties are more likely to be at interface/head-group region, with the hexadecyl moiety aligning with the hydrocarbon core of the lipid.


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 intensity and steady state anisotropy of ADOTA-C16 in DMPC between 10°C and 35°C.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0063043-g011: Fluorescence intensity and steady state anisotropy of ADOTA-C16 in DMPC between 10°C and 35°C.
Mentions: We also looked at the steady-state anisotropy and fluorescence emission of hexadecyl-ADOTA in DMPC small vesicle during the transition from gel to liquid crystalline state. The results are shown in Figure 11. The fluorescence intensity decreases by less than 5% between 15°C and 35°C. Even though it is possible to see the subtle signature of phase transition, the change in emission maxima is rather small (less than 4 nm). There is also a reproducible small decrease in the intensity between 15°C and 10°C. We suspect it is primarily due to inner filter effect as the vesicles become more opaque up on lowering of temperature. The steady-state anisotropy decreased from 0.219 at 10°C to 0.114 at 35°C, reflecting the phase transition of DMPC. Our results above phase transition are similar to those seen with anchored DPH derivatives [23, 24, and 32] and fluorescent fatty acids with long lifetime [33], [34]. It should be noted that the fluorophore in DPH derivatives and fluorescent fatty acids are present in the hydrocarbon region of lipid while the ADOTA and DAOTA moieties are more likely to be at interface/head-group region, with the hexadecyl moiety aligning with the hydrocarbon core of the lipid.

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