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

Chembiochem. 2022 Sep 16;23(18):e202200395. doi: 10.1002/cbic.202200395. Epub 2022 Aug 16.

Abstract

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.

Keywords: FRET; STED microscopy; dyes / pigments; fluorescence; nanobodies.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Disulfides
  • Dithiothreitol
  • Fluorescence Resonance Energy Transfer* / methods
  • Fluorescent Dyes / chemistry
  • Maleimides
  • Microscopy, Fluorescence / methods
  • Single-Domain Antibodies*

Substances

  • Disulfides
  • Fluorescent Dyes
  • Maleimides
  • Single-Domain Antibodies
  • Dithiothreitol