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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

Anisotropy decays of ADOTA and DAOTA at room temperature.(A) ADOTA-ba (B). DAOTA-ba.
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pone-0063043-g008: Anisotropy decays of ADOTA and DAOTA at room temperature.(A) ADOTA-ba (B). DAOTA-ba.

Mentions: The anisotropy decays of ADOTA-ba in tris buffer, PG and 95% glycerol are shown in Figure8A and those for DAOTA-ba in triacetin and 95% glycerol in Figure 8B. As expected the very fast motions in buffer results in extremely rapid decay of anisotropy, while significantly slower motions in more viscous media can be seen as very slow decline of anisotropy with time. Same trend is visible between triacetin and 95% glycerol in case of DAOTA-ba. The measured rotational correlation times and associated anisotropy at room temperature are given in Table 3 for both the probes. It should be noted that our anisotropy decay analysis using tail fitting results in time gating of initial 100 ps to150 ps decay in case of diode laser (ADOTA-ba) and 1 ns to 1.2 ns (DAOTA-ba) in case of LED light source. The magnitude of missing fraction of total anisotropy will depend up on rotational correlation time values. If the time gate and rotational correlation times are comparable, we will be unable to recover a significant part of total anisotropy. On the other end when the rotational correlation time is >> the time gate we will recover almost all of the limiting anisotropy. As expected the recovered correlation times follow the bulk solvent viscosity which is expected to increase in the order acetonitrile < buffer << triacetin < PG << 95% glycerol. For ADOTA-ba in acetonitrile even after missing a significant part of anisotropy decay, we were able to recover a rotational correlation time of around 100 ps. The value is reasonable for a solvated ellipsoid of molecular weight (MW) of 370 Da. However, as expected from missing initial 100 ps of anisotropy decay the recovered anisotropy of 0.171 is significantly lower than the expected limiting anisotropy of 0.305. The correlation time for ADOTA-ba in buffer is 150 ps and as expected somewhat higher recovered anisotropy value of 0.268. The rotational correlation times are respectively 2.5 ns, 4 ns and 38 ns in more viscous triacetin, PG and 95% glycerol and follow the solvent bulk viscosity. With values of Ф >>100 ps–150 ps time gate the recovered associated anisotropy of 0.304–0.305 is now comparable to the limiting anisotropy measured in frozen glycerol at −40°C.


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)

Anisotropy decays of ADOTA and DAOTA at room temperature.(A) ADOTA-ba (B). DAOTA-ba.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0063043-g008: Anisotropy decays of ADOTA and DAOTA at room temperature.(A) ADOTA-ba (B). DAOTA-ba.
Mentions: The anisotropy decays of ADOTA-ba in tris buffer, PG and 95% glycerol are shown in Figure8A and those for DAOTA-ba in triacetin and 95% glycerol in Figure 8B. As expected the very fast motions in buffer results in extremely rapid decay of anisotropy, while significantly slower motions in more viscous media can be seen as very slow decline of anisotropy with time. Same trend is visible between triacetin and 95% glycerol in case of DAOTA-ba. The measured rotational correlation times and associated anisotropy at room temperature are given in Table 3 for both the probes. It should be noted that our anisotropy decay analysis using tail fitting results in time gating of initial 100 ps to150 ps decay in case of diode laser (ADOTA-ba) and 1 ns to 1.2 ns (DAOTA-ba) in case of LED light source. The magnitude of missing fraction of total anisotropy will depend up on rotational correlation time values. If the time gate and rotational correlation times are comparable, we will be unable to recover a significant part of total anisotropy. On the other end when the rotational correlation time is >> the time gate we will recover almost all of the limiting anisotropy. As expected the recovered correlation times follow the bulk solvent viscosity which is expected to increase in the order acetonitrile < buffer << triacetin < PG << 95% glycerol. For ADOTA-ba in acetonitrile even after missing a significant part of anisotropy decay, we were able to recover a rotational correlation time of around 100 ps. The value is reasonable for a solvated ellipsoid of molecular weight (MW) of 370 Da. However, as expected from missing initial 100 ps of anisotropy decay the recovered anisotropy of 0.171 is significantly lower than the expected limiting anisotropy of 0.305. The correlation time for ADOTA-ba in buffer is 150 ps and as expected somewhat higher recovered anisotropy value of 0.268. The rotational correlation times are respectively 2.5 ns, 4 ns and 38 ns in more viscous triacetin, PG and 95% glycerol and follow the solvent bulk viscosity. With values of Ф >>100 ps–150 ps time gate the recovered associated anisotropy of 0.304–0.305 is now comparable to the limiting anisotropy measured in frozen glycerol at −40°C.

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