Bioorthogonal Ligation-Activated Fluorogenic FRET Dyads

Abstract

An energy transfer-based signal amplification relay concept enabling transmission of bioorthogonally activatable fluorogenicity of blue-excitable coumarins to yellow/red emitting cyanine frames is presented. Such relay mechanism resulted in improved cyanine fluorogenicities together with increased photostabilities and large apparent Stokes-shifts allowing lower background fluorescence even in no-wash bioorthogonal fluorogenic labeling schemes of intracellular structures in live cells. These energy transfer dyads sharing the same donor moiety together with their parent donor molecule allowed three-color imaging of intracellular targets using one single excitation source with separate emission windows. Sub-diffraction imaging of intracellular structures using the bioorthogonally activatable FRET dyads by STED microscopy is also presented.

Keywords: FRET dyad; bioorthogonal; fluorogenic; multicolor; single excitation.

Figure 1

A) Representation of the bioorthogonally controlled fluorogenicity relay concept, B) Structures of bioorthogonal fluorogenic dyads 1–4 and further probes (5–7) used in this study.

Figure 2

Absorption (5 μM), excitation and emission spectra (1 μM) of (A) 7, λ_em: 560 nm, λ_ex: 405 nm; B) 1, λ_em: 620 nm, λ_ex: 405 nm; C) 2, λ_em: 690 nm, λ_ex: 405 nm and their BCN conjugates in PBS‐SDS; fluorescence spectra were normalized for each BCN conjugates.

Figure 3

Confocal microscopy images of Lamin‐HaloTag expressing HEK293T cells treated with Halo‐BCN (3 μM) and probes 1 (A), 2 (B) and 5 (C), 6 (D) (1 μM) following washing. Spectral detection: A) (dye 1): λ_exc: 405 nm/ λ_em: 560–800 nm; B) (dye 2): λ_exc: 405 nm/ λ_em: 560–800 nm; C) (5): λ_exc: 543 nm/ λ_em: 560–800 nm; D) (6): λ_exc: 633 nm/ λ_em: 650–800 nm.

Figure 4

Confocal microscopy images of Lamin‐L^TAG‐miRFP (A–F) or Lamin‐GFP^TAG (G–L) expressing HEK293T cells treated with Lys(BCN) and probes 1 (A–C), 2 (G–I) or 5 (D–F), 6 (J–L). Scale bar: 20 μm. Spectral detection: A,D) (miRFP/reporter channel): λ_exc: 633 nm/ λ_em: 645–800 nm; G,J) (GFP/reporter channel): λ_exc: 488 nm/ λ_em: 495–600 nm; B) (1): λ_exc: 405 nm/ λ_em: 560–600 nm; E) (5): λ_exc: 543 nm/ λ_em: 560–600 nm; H) (2): λ_exc: 405 nm/ λ_em: 620–800 nm; K) (6): λ_exc: 633 nm/ λ_em: 645–800 nm. Overlay pictures demonstrate the merged fluorescent signals of the reporter and dye channels.

Figure 5

Multicolor imaging of HepG2 cells. A) Lamin‐HaloTag treated with 7 tagged Halo‐substrate (Halo‐7), λ_exc: 405 nm/ λ_em: 420–500 nm); B) TOMM20 stained with 1‐tagged antibody, λ_exc: 405 nm/ λ_em: 570–600 nm); C) CK‐19 stained with 2‐tagged antibody, λ_exc: 405 nm/ λ_em: 660–800 nm; D) Overlay of A, B and C; E) magnification of D, Scale bar: 20 μm; F) Emission spectra of dyes 7 (cyan), 1 (yellow) and 2 (magenta) together with excitation wavelength (405 nm) and detection windows.

Figure 6

Confocal (A,B,E,F) and STED (C,D,G,H) images of COS‐7 cells immunostained for TOMM20 with secondary antibodies labeled with probe 1 (A–D) and probe 2 (E–H) (λ_exc: 405 nm/ λ_em: 560–755 nm; λ_STED=775 nm (pulsed)). Scale bar: 5 μm (A,C,E,G) and in magnified pictures (B,D,F,H): 1 μm. FWHM: full width at half maxima.