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Probes for biomolecules

Mise à jour 17 janvier 2018

Fig. 8. Color response of an environment-sensitive probe to a biomolecular interaction.

In our early works (2005-2014) in the group of Yves Mely (UMR7213, Strasbourg), using environment-sensitive probes, mainly those undergoing ESIPT, we developed general concepts for monitoring interactions of peptides with other biomolécules 1 : nucleic acids 2,4 proteins 5 and lipids (membranes) 6. Interaction of a peptide with its target commonly changes its local nano-environment, which can be detected by the environment-sensitive probe though change of its emission color. Since 2015 we focused on solvatochromic and fluorogenic probes for sensing interaction of peptide ligands with membrane receptors.

Turn-on probes for membrane receptors

The classical fluorescence-based approaches to monitor ligand-receptor interactions are generally hampered by the background signal of the unbound ligand that must be removed by tedious washing steps. In collaboration with D. Bonnet and M. Hibert (UMR 7200) we initially developed fluorescent turn-on probe (NR-PEG-CBT) based on Nile Red for a G protein-coupled receptor (oxytocin) at the surface of living cells 7. Although the new probe significantly decreased the background, its brightness and photostability were limited. To address these issues, we continued collaboration with D. Bonnet and M. Hibert, following two concepts.

Within the first concept, we proposed fluorogenic dye, based on a squaraine dimer , that unfolds on changing environment from aqueous to organic, and thus turns on its fluorescence 8. In aqueous media, the dimers displayed a short-wavelength band characteristic of an H-aggregate that was poorly fluorescent, whereas in organic media, they displayed a strong fluorescence, like squaraine monomer (Fig. 9). For the best dimer, which contained a PEGylated squaraine core, we obtained a very high turn-on response (organic vs. aqueous) up to 82 (Fig. 9). To apply these fluorogenic dimers for targeted imaging, we grafted them to carbetocin (Fig. 9), a ligand of the oxytocin G protein-coupled receptor. A strong receptor-specific signal was observed for all three conjugates at nanomolar concentrations. The probe derived from the core-PEGylated squaraine showed the best specificity to the target receptor together with minimal non-specific interactions (Fig. 9). The obtained dimers can be considered as the brightest polarity-sensitive fluorogenic molecules reported to date, having 660,000 M-1cm-1 extinction coefficient and up to 40% quantum yield, whereas far-red operation region enables both in vitro and in vivo applications 8. The proposed concept can be extended to other dye families and other membrane receptors, opening the route to new ultrabright fluorogenic dyes.

Fig. 9. Probes operating by aggregation-caused quenching mechanism. (a) Principle and examples of these probes. (b) Absorption of squaraine dimer dSQ1 in water with addition of methanol (from 0 till 100%). (c) Fluorescence intensities (F/Fwater) of dyes in H2O-MeOH mixtures with respect to their intensity in water. (d,e) Confocal images of HEK cells expressing the oxytocin receptor with 100 nM of dSQ2L (d) or rhodamine-carbetocin conjugate (e). (f) Specificity of the probes (10 nM) : intensity ratio before and after addition of nonlabeled carbetocin (2 µM). Adapted from 8.

Fig. 10. Structure and rotor properties of dioxaborine molecular rotors. (a) Structure of the molecular rotor DXB Red. (b) Fluorescence response of the molecular rotor DXB Red to the change of the environment from fluid (MeOH, black) to viscous (glycerol, red). (c) Structure of the carbetocin conjugate DXB-CBT. (d) Correlation of the fluorescence quantum yield with the viscosity of the medium according to the Förster-Hoffmann equation. (e) Photostability of DXB Red and of Nile Red in toluene. Excitation wavelength was 525 nm ; emission was detected at 600 nm ; illumination power density was 1 mW cm-2. Adapted from 9.

In our second approach, we introduced a push-pull boron-bridged (dioxaborine) dye DXB Red that presents unique spectroscopic behavior combining solvatochromism and molecular rotor properties (Fig. 10) 9. In comparison to solvatochromic and molecular rotor dyes, dioxaborine derivative shows exceptional extinction coefficient (120,000 M-1 cm-1), high fluorescence quantum yields and red/far-red operating spectral range. DXB Red also displays much higher photostability in apolar media as compared to Nile Red (Fig. 10), a fluorogenic dye of similar color. Its carboxy derivative has been successfully grafted to carbetocin (DXB-CBT, Fig. 11). This conjugate exhibits >1000-fold turn on between apolar 1,4-dioxane and water. It targets specifically the oxytocin receptor at the cell surface, which enables receptor imaging with excellent signal-to-background ratio (>130) (Fig. 11) 9. We expect that presented push-pull dioxaborine dye opens a new page in the development of fluorogenic probes for bioimaging applications.

Fig. 11. Confocal images of OTR cells with 10 nM of DXB-CBT (a), 10 nM of DXB-CBT and 2 µM of CBT competitor (b), or 100 nM of DXB-CBT (c). Fluorogenic properties of DXB-CBT : average membrane and background fluorescence for all the images (d). Adapted from 9.

Probes for nucleic acids

In collaboration with a group of Michaël RYCKELYNCK (UPR9002, Strasbourg), we currently develop fluorogenic probes for detection of nucleic acids. The concept is based on “spinatch”-type assay, where initially non-fluorescent dye turns on its emission under recognition of nucleic acid aptamer. This work is under development (supported by ANR BrightRiboProbes).

References :

1. Klymchenko, A. S. ; Mely, Y. : Fluorescent Environment-Sensitive Dyes as Reporters of Biomolecular Interactions. In Fluorescence-Based Biosensors : From Concepts to Applications ; Morris, M. C., Ed., 2013 ; Vol. 113 ; pp 35-58.

2. Klymchenko, A. S. ; Shvadchak, V. V. ; Yushchenko, D. A. ; Jain, N. ; Mely, Y. : Excited-state intramolecular proton transfer distinguishes microenvironments in single- and double-stranded DNA. Journal of Physical Chemistry B 2008, 112, 12050-12055.

3. Shvadchak, V. V. ; Klymchenko, A. S. ; de Rocquigny, H. ; Mely, Y. : Sensing peptide-oligonucleotide interactions by a two-color fluorescence label : application to the HIV-1 nucleocapsid protein. Nucleic Acids Research 2009, 37.

4. Strizhak, A. V. ; Postupalenko, V. Y. ; Shvadchak, V. V. ; Morellet, N. ; Guittet, E. ; Pivovarenko, V. G. ; Klymchenko, A. S. ; Mely, Y. : Two-Color Fluorescent L-Amino Acid Mimic of Tryptophan for Probing Peptide-Nucleic Acid Complexes. Bioconjugate Chemistry 2012, 23, 2434-2443.

5. Enander, K. ; Choulier, L. ; Olsson, A. L. ; Yushchenko, D. A. ; Kanmert, D. ; Klymchenko, A. S. ; Demchenko, A. P. ; Mely, Y. ; Altschuh, D. : A peptide-based, ratiometric biosensor construct for direct fluorescence detection of a protein analyte. Bioconjugate Chemistry 2008, 19, 1864-1870.

6. Postupalenko, V. Y. ; Zamotaiev, O. M. ; Shvadchak, V. V. ; Strizhak, A. V. ; Pivovarenko, V. G. ; Klymchenko, A. S. ; Mely, Y. : Dual-Fluorescence L-Amino Acid Reports Insertion and Orientation of Melittin Peptide in Cell Membranes. Bioconjugate Chemistry 2013, 24, 1998-2007.

7. Karpenko, I. A. ; Kreder, R. ; Valencia, C. ; Villa, P. ; Mendre, C. ; Mouillac, B. ; Mely, Y. ; Hibert, M. ; Bonnet, D. ; Klymchenko, A. S. : Red Fluorescent Turn-On Ligands for Imaging and Quantifying G Protein-Coupled Receptors in Living Cells. Chembiochem 2014, 15, 359-363.

8. Karpenko, I. A. ; Collot, M. ; Richert, L. ; Valencia, C. ; Villa, P. ; Mely, Y. ; Hibert, M. ; Bonnet, D. ; Klymchenko, A. S. : Fluorogenic Squaraine Dimers with Polarity-Sensitive Folding As Bright Far-Red Probes for Background-Free Bioimaging. Journal of the American Chemical Society 2015, 137, 405-412.

9. Karpenko, I. A. ; Niko, Y. ; Yakubovskyi, V. P. ; Gerasov, A. O. ; Bonnet, D. ; Kovtun, Y. P. ; Klymchenko, A. S. : Push-pull dioxaborine as fluorescent molecular rotor : far-red fluorogenic probe for ligand-receptor interactions. Journal of Materials Chemistry C 2016, 4, 3002-3009.