Genetically encoded FRET-pair on the basis of terbium-binding peptide and red fluorescent protein

Arslanbaeva, L. R.; Zherdeva, Victoria V.; Ивашина, Т. В.; Vinokurov, Leonid M.; Русанов, А. Л.; Savitsky, Alexander P.

doi:10.1134/s0003683810020055

Cited by 9 publications

(11 citation statements)

References 26 publications

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“…As a result, we showed that there is an energy transfer in the Tb 3+ -TBP-19-TagRFP sensor from the terbium ion to the TagRFP chromophore. For the first time we obtained the spectrum of the sensitized TagRFP fluorescence with time delay for the previously described sensor [ 26 , 27 ]. Using the titration curve of TBP-19-TagRFP at 606 nm ( Figure 7 b), which was obtained by subtraction of intensity of the photobleached sample from the intensity of the corrected for reabsorption non-bleached sample, the dissociation constant of Tb 3+ and TBP-19-TagRFP complex was calculated.…”

Section: Resultsmentioning

confidence: 99%

“…Our laboratory has developed a genetically encoded FRET sensor for caspase-3 Tb 3+ -TBP-19-TagRFP [ 26 , 27 ], which consists of the complex of Tb 3+ with terbium-binding peptide YIDTNNDGWYEGDELLA [ 28 ] as a donor, flexible linker VDGGSGGDEVDGWGGSGLD [ 29 ] with the caspase-3 cleavage site DEVD [ 30 ], which was used earlier in the FRET-pair with a non-fluorescent acceptor [ 31 ], and red fluorescent protein TagRFP [ 26 , 32 ] as an acceptor. There are two donor-acceptor pairs in the resulting structure, which transfer energy using the fluorescence resonance mechanism.…”

Section: Introductionmentioning

confidence: 99%

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Genetically Encoded FRET-Sensor Based on Terbium Chelate and Red Fluorescent Protein for Detection of Caspase-3 Activity

Goryashchenko

Khrenova

Bochkova

et al. 2015

IJMS

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This article describes the genetically encoded caspase-3 FRET-sensor based on the terbium-binding peptide, cleavable linker with caspase-3 recognition site, and red fluorescent protein TagRFP. The engineered construction performs two induction-resonance energy transfer processes: from tryptophan of the terbium-binding peptide to Tb3+ and from sensitized Tb3+ to acceptor—the chromophore of TagRFP. Long-lived terbium-sensitized emission (microseconds), pulse excitation source, and time-resolved detection were utilized to eliminate directly excited TagRFP fluorescence and background cellular autofluorescence, which lasts a fraction of nanosecond, and thus to improve sensitivity of analyses. Furthermore the technique facilitates selective detection of fluorescence, induced by uncleaved acceptor emission. For the first time it was shown that fluorescence resonance energy transfer between sensitized terbium and TagRFP in the engineered construction can be studied via detection of microsecond TagRFP fluorescence intensities. The lifetime and distance distribution between donor and acceptor were calculated using molecular dynamics simulation. Using this data, quantum yield of terbium ions with binding peptide was estimated.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Genetically Encoded FRET-Sensor Based on Terbium Chelate and Red Fluorescent Protein for Detection of Caspase-3 Activity

Goryashchenko

Khrenova

Bochkova

et al. 2015

IJMS

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“…This is by far a simpler strategy than using systems in which chromophore-modified (fused) proteins are to be incorporated into cells in order to detect specific molecule interactions (7)(8)(9)30,31). Even though this in vivo approach represents a proof of concept, labeling of macrophages via FRET is expected to work also in other disease models related to spontaneous inflammatory processes, such as rheumatoid arthritis.…”

Section: Discussionmentioning

confidence: 99%

An In Vivo Spectral Multiplexing Approach for the Cooperative Imaging of Different Disease-Related Biomarkers with Near-Infrared Fluorescent Förster Resonance Energy Transfer Probes

Busch¹,

Schröter²,

Grabolle³

et al. 2012

J Nucl Med

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In recent years, much progress has been made in analyzing the molecular origin of many diseases in vivo. For most applications, attention has been devoted to the detection of single molecules only. In this study, we present a proof of concept for the straightforward monitoring of interactions between different molecules via Fö rster resonance energy transfer (FRET) in an in vivo spectral multiplexing approach using conventional small organic dyes covalently attached to antibodies. Methods: We coupled the fluorophores DY-682 (donor; absorption [abs]/ emission [em], 674/712 nm), DY-505 (control donor; abs/em, 498/529 nm), and DY-782 (acceptor; abs/em, 752/795 nm) to the model antibody IgG. The occurrence of FRET between these fluorophores was assessed in vitro for conjugate mixtures adsorbed onto membranes, after accumulation into the phagocytic compartment of macrophages (J774 cells), and in vivo in a mouse edema model using a whole-body animal imaging system with multispectral analysis features. Results: When the free acceptor DY-782 was combined with the DY-682 donor, FRET occurred as a consequence of small dye-to-dye distances, unlike the case for mixtures of the dyes DY-782 and DY-505. Our proof of concept was also transferred to living cells after internalization of the DY-682-IgG-DY-782-IgG pair into macrophages and finally to animals, where intermolecular FRET was observed after systemic probe application in vivo in edema-bearing mice. Conclusion: Our simple cooperative-imaging approach enables the noninvasive detection of the presence of two or principally even more neighboring disease-related biomarkers. This finding is of high relevance for the in vivo identification of complex biologic processes requiring strong spatial interrelations of target molecules in key pathologic activation processes such as inflammation, cancer, and neurodegenerative diseases.

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“…These include a study of the intra-and intermolecular distances in macromolecules (Vazquez-Ibar et al, 2002), and a study on protein-peptide interactions (Sculimbrene and Imperiali, 2006). More recently, Arslanbaeva et al have used LBPs and fluorescent proteins as FRET pairs, and proposed their use in time-resolved fluorescence (TRF) applications (Arslanbaeva et al, 2010;Arslanbaeva et al, 2011). Using this approach, there is considerable potential for the LBPs in bioaffinity assays and medical diagnostics.…”

Section: Lanthanide-binding Peptidesmentioning

confidence: 99%

Luminescent lanthanide reporters: new concepts for use in bioanalytical applications

Vuojola

Soukka

2014

Methods Appl. Fluoresc.

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Lanthanides represent the chemical elements from lanthanum to lutetium. They intrinsically exhibit some very exciting photophysical properties, which can be further enhanced by incorporating the lanthanide ion into organic or inorganic sensitizing structures. A very popular approach is to conjugate the lanthanide ion to an organic chromophore structure forming lanthanide chelates. Another approach, which has quickly gained interest, is to incorporate the lanthanide ions into nanoparticle structures, thus attaining improved specific activity and a large surface area for biomolecule immobilization. Lanthanide-based reporters, when properly shielded from the quenching effects of water, usually express strong luminescence emission, multiple narrow emission lines covering a wide wavelength range, and exceptionally long excited state lifetimes enabling time-gated luminescence detection. Because of these properties, lanthanide-based reporters have found widespread applications in various fields of life. This review focuses on the field of bioanalytical applications. Luminescent lanthanide reporters and assay formats utilizing these reporters pave the way for increasingly sensitive, simple, and easily automated bioanalytical applications.

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Genetically encoded FRET-pair on the basis of terbium-binding peptide and red fluorescent protein

Cited by 9 publications

References 26 publications

Genetically Encoded FRET-Sensor Based on Terbium Chelate and Red Fluorescent Protein for Detection of Caspase-3 Activity

Genetically Encoded FRET-Sensor Based on Terbium Chelate and Red Fluorescent Protein for Detection of Caspase-3 Activity

An In Vivo Spectral Multiplexing Approach for the Cooperative Imaging of Different Disease-Related Biomarkers with Near-Infrared Fluorescent Förster Resonance Energy Transfer Probes

Luminescent lanthanide reporters: new concepts for use in bioanalytical applications

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