Cleavable Linker Incorporation into a Synthetic Dye‐Nanobody‐Fluorescent Protein Assembly: FRET, FLIM and STED Microscopy

A bright and photostable fluorescent dye with a disulfide (S−S) linker and maleimide group (Rho594‐S2‐mal), as cleavable and reactive sites, was synthesized and conjugated with anti‐GFP nanobodies (NB). The binding of EGFP (FRET donor) with anti‐GFP NB labeled with one or two Rho594‐S2‐mal residues...

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Autores principales: Aktalay, Ayse, Ponsot, Flavien, Bossi, Mariano L., Belov, Vladimir N., Hell, Stefan W.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: John Wiley and Sons Inc. 2022
Materias:
Research Articles
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9804610/
https://www.ncbi.nlm.nih.gov/pubmed/35838445
http://dx.doi.org/10.1002/cbic.202200395
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author Aktalay, Ayse
Ponsot, Flavien
Bossi, Mariano L.
Belov, Vladimir N.
Hell, Stefan W.
author_facet Aktalay, Ayse
Ponsot, Flavien
Bossi, Mariano L.
Belov, Vladimir N.
Hell, Stefan W.
author_sort Aktalay, Ayse
collection PubMed
description A bright and photostable fluorescent dye with a disulfide (S−S) linker and maleimide group (Rho594‐S2‐mal), as cleavable and reactive sites, was synthesized and conjugated with anti‐GFP nanobodies (NB). The binding of EGFP (FRET donor) with anti‐GFP NB labeled with one or two Rho594‐S2‐mal residues was studied in vitro and in cellulo. The linker was cleaved with dithiothreitol recovering the donor (FP) signal. The bioconjugates (FP‐NB‐dye) were applied in FRET‐FLIM assays, confocal imaging, and superresolution STED microscopy.
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spelling pubmed-98046102023-01-03 Cleavable Linker Incorporation into a Synthetic Dye‐Nanobody‐Fluorescent Protein Assembly: FRET, FLIM and STED Microscopy Aktalay, Ayse Ponsot, Flavien Bossi, Mariano L. Belov, Vladimir N. Hell, Stefan W. Chembiochem Research Articles A bright and photostable fluorescent dye with a disulfide (S−S) linker and maleimide group (Rho594‐S2‐mal), as cleavable and reactive sites, was synthesized and conjugated with anti‐GFP nanobodies (NB). The binding of EGFP (FRET donor) with anti‐GFP NB labeled with one or two Rho594‐S2‐mal residues was studied in vitro and in cellulo. The linker was cleaved with dithiothreitol recovering the donor (FP) signal. The bioconjugates (FP‐NB‐dye) were applied in FRET‐FLIM assays, confocal imaging, and superresolution STED microscopy. John Wiley and Sons Inc. 2022-08-16 2022-09-16 /pmc/articles/PMC9804610/ /pubmed/35838445 http://dx.doi.org/10.1002/cbic.202200395 Text en © 2022 The Authors. ChemBioChem published by Wiley-VCH GmbH https://creativecommons.org/licenses/by-nc-nd/4.0/This is an open access article under the terms of the http://creativecommons.org/licenses/by-nc-nd/4.0/ (https://creativecommons.org/licenses/by-nc-nd/4.0/) License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made.
spellingShingle Research Articles
Aktalay, Ayse
Ponsot, Flavien
Bossi, Mariano L.
Belov, Vladimir N.
Hell, Stefan W.
Cleavable Linker Incorporation into a Synthetic Dye‐Nanobody‐Fluorescent Protein Assembly: FRET, FLIM and STED Microscopy
title Cleavable Linker Incorporation into a Synthetic Dye‐Nanobody‐Fluorescent Protein Assembly: FRET, FLIM and STED Microscopy
title_full Cleavable Linker Incorporation into a Synthetic Dye‐Nanobody‐Fluorescent Protein Assembly: FRET, FLIM and STED Microscopy
title_fullStr Cleavable Linker Incorporation into a Synthetic Dye‐Nanobody‐Fluorescent Protein Assembly: FRET, FLIM and STED Microscopy
title_full_unstemmed Cleavable Linker Incorporation into a Synthetic Dye‐Nanobody‐Fluorescent Protein Assembly: FRET, FLIM and STED Microscopy
title_short Cleavable Linker Incorporation into a Synthetic Dye‐Nanobody‐Fluorescent Protein Assembly: FRET, FLIM and STED Microscopy
title_sort cleavable linker incorporation into a synthetic dye‐nanobody‐fluorescent protein assembly: fret, flim and sted microscopy
topic Research Articles
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9804610/
https://www.ncbi.nlm.nih.gov/pubmed/35838445
http://dx.doi.org/10.1002/cbic.202200395
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