Linker Molecules Convert Commercial Fluorophores into Tailored Functional Probes during Biolabelling

Abstract

Many life-science techniques and assays rely on selective labeling of biological target structures with commercial fluorophores that have specific yet invariant properties. Consequently, a fluorophore (or dye) is only useful for a limited range of applications, e.g., as a label for cellular compartments, super-resolution imaging, DNA sequencing or for a specific biomedical assay. Modifications of fluorophores with the goal to alter their bioconjugation chemistry, photophysical or functional properties typically require complex synthesis schemes. We here introduce a general strategy that allows to customize these properties during biolabelling with the goal to introduce the fluorophore in the last step of biolabelling. For this, we present the design and synthesis of 'linker' compounds, that bridge biotarget, fluorophore and a functional moiety via well-established labeling protocols. Linker molecules were synthesized via the Ugi four-component reaction (Ugi-4CR) which facilitates a modular design of linkers with diverse functional properties and bioconjugation- and fluorophore attachment moieties. To demonstrate the possibilities of different linkers experimentally, we characterized the ability of commercial fluorophores from the classes of cyanines, rhodamines, carbopyronines and silicon-rhodamines to become functional labels on different biological targets in vitro and in vivo via thiol-maleimide chemistry. With our strategy, we showed that the same commercial dye can become a photostable self-healing dye or a sensor for bivalent ions subject to the linker used. Finally, we quantified the photophysical performance of different self-healing linker-fluorophore conjugates and demonstrated their applications in super-resolution imaging and single-molecule spectroscopy.

Keywords: Biolabelling; Fluorophores; Metal-Chelating Fluorophores; Self-Healing Dyes; Super-Resolution Microscopy.

Figure 1

A) Schematic representation of the linker biolabelling strategy consisting of two steps: bioconjugation of the linker to a biotarget (e.g., for protein labeling via cysteine‐maleimide chemistry) and subsequent fluorophore attachment to the complex, e.g., via click‐chemistry. B) Retrosynthetic analysis of a possible linker structure.

Scheme 1

Synthesis route to linker compounds with photostabilizers (19, 20) or a metal‐chelating unit (21) as functional moiety. Compound 18 lacks a functional moiety and was synthesized as a negative control. Boc protection of compounds 8–11 was removed in the form of 2‐methylpropene and carbon dioxide under acidic conditions, yielding 12–15. DCM, dichloromethane; TFA, trifluoroacetic acid; Boc, tert‐butyloxycarbonyl.

Figure 2

Labeling performance of linker molecules 19 and 20 on different biotargets in comparison to commercially‐available maleimide fluorophore derivatives. A) Schematic view of biotargets and attachment strategy shown in the Figure. B) SEC‐image of a successful labeling procedure of SBD2‐T369C labeled with 19‐sCy5. The black curve shows absorption at 280 nm (protein absorbance) and the red curve shows absorption at 647Nm (sCy5 absorbance maximum). Comparison of the area below the elution profiles allowed to determine labeling efficiencies in (C) considering the extinction coefficient of the protein and the published values of the respective dyes (see Supporting Information, Part III for details). C) Quantitative comparison of the labeling efficiency of SBD2‐T369C with either maleimide‐functionalized fluorophore (protein‐maleimide dye) or with azide‐functionalized fluorophore clicked by 19 (protein‐19‐dye) or 20 (protein‐20‐dye). D) UV/Vis absorbance spectra showing the characteristic bands for dsDNA (≈260 nm) and the respective fluorophores (≈650 nm, data shown for sCy5). Control indicates that the labeling procedure of azide‐functionalized fluorophore was conducted in the absence of a linker. E) Brightfield and fluorescence microscopy images of live bacteria (E. coli K12), which were labeled with maleimide‐functionalized Cy3B (left column), azide‐functionalized Cy3B in the absence of a linker (middle column) and azide‐functionalized Cy3B clicked by compound 20 (right column).

Figure 3

Proof‐of‐concept experiments to demonstrate the ability of the linker to equip commercial sCy5 with self‐healing or blinking properties on model protein SBD2. A) Schematic view of protein surface immobilization strategy and linker/dye attachment. B) TIRF microscopy images show individual sCy5 molecules at the start of the movie and after 30 s (related to self‐healing sCy5). C)–F) Single‐molecule fluorescence time traces of (C) commercial sCy5 directly linked to SBD2 via maleimide and sCy5‐azide dyes linked to SBD2 via (E) 18, (D) 20 and (F) 21. C, D) Comparison of photostabilization between commercially available sCy5 maleimide without photostabilizer (C, left), with 2 mM COT in solution (C, right) and 20 in the absence of photostabilizer in solution, respectively under deoxygenated conditions using POC in PBS buffer at pH 7.4; see Methods for Details. E, F) Photoblinking induced by combination of 21 and Cu²⁺. E) Negative control of compound 18 in the absence of photostabilizer (left) and in buffer solution containing 2 mM TX and 5 μM CuSO₄ (right). F) Fluorescent transients of sCy5 linked via 21 under identical buffer conditions as in (E, right). E, F) Were recorded in MOPS buffer at pH 7.0 using PCA/PCD for oxygen removal with 100 ms (E/F, left) and 20 ms (F, right) exposure times, respectively. G) Quantitative blinking properties of sCy5 in the presence of 5 μM CuSO₄ on linker 18 (left) and on linker 21 (right). Values of the three white fields in the left panel are 0.7122 and 0.1171, and 0.1125 in the right panel, from top to down, respectively. (F, left) recorded at 100 ms and (F, right) recorded at 20 ms exposure time for clearer characterization of blinking behavior. For a direct comparison of blinking behavior between 18 and 21 under identical exposure times in presence and absence of CuSO₄, see Figure S37, Figure S38 and Figure S39. All measurements were done with 640 nm laser excitation with a power density of ≈150 W cm⁻².

Figure 4

Photophysical characterization of biotargets containing 19/20–dye conjugates with single‐molecule TIRF microscopy. Data was recorded in aqueous buffer at pH 7.4 in the absence of oxygen (POC) under continuous 640 nm excitation with ≈75 W cm⁻². A) Representative image frame (left panel) showing single fluorescent molecules (10×10 μm, exemplarily for ATTO647N). Subsequent images recorded over a period of 450 s showed an exponential decrease in the number of fluorescing molecules with a photobleaching lifetime τ _B as shown for SBD2‐ATTO647N (black, fitted in grey). The curves shown were obtained by averaging over 4 TIRF movies. The photobleaching times of 20‐ATT0647N on SBD2, ATTO647N+TX, ATTO647N+COT, 20‐ATTO647N on DNA are inaccurate and underestimated due to large fraction of fluorophores that did not show bleaching over the course of the experiment. B), C) Left panel: chemical structures and right panel: respective photophysical parameters obtained from background‐corrected fluorescence traces for ATTO647N (B, carbopyronines) and sCy5 (C, cyanines) on SBD2 protein or double strand DNA (hybridization of P1 with P2, sequences shown in the Supporting Information). Values and error bars (s.d.) in bar graphs obtained from >400 molecules. For further details of the experimental techniques, data acquisition and analysis, see the Methods section.

Figure 5

Applications of linker‐conjugated self‐healing dyes in single‐molecule FRET (μsALEX) and STED super‐resolution microscopy. A) 2D histograms of joint pair values of S* (labeling stoichiometry) and E* (FRET efficiency, that correlates to interprobe distance) of Cy3B (donor)/ATTO647N (acceptor) on dsDNA in the absence and presence of 2 mM TX and Cy3B (donor)/20‐Atto647N as the acceptor in PBS pH 7.4 buffer. B) Dependence of the donor‐acceptor population of the corresponding conditions in (A) for increasing excitation intensity. C, left half) confocal (top) and STED (bottom) images of tubulin stained with GFP‐binder‐20‐ATTO647N in HeLa H2B‐mRFP‐mEGPF‐αTubulin cells. Scalebar: 1 μm. (C, right half) intensity profile along the line indicated in (C top and bottom) by an arrow showing increase of resolution through STED. Lines indicate a gaussian fit for confocal (gray) and a double‐Lorentzian fit for STED data (black), respectively. The fit to the STED data reveals a line distance of 273 nm and a Full Width Half Maximum (FWHM) of 65 nm and 62 nm for the resolved peaks, respectively. D)–H) Images and bleaching analysis in Hela‐H2B‐GFP cells stained with GFP‐binder prelabeled with either ATTO647N or 20‐ATTO647N. D) Confocal images of nuclei labeled with GFP‐binder‐20‐ATTO647N before (top) and after (bottom) bleaching. Scalebar: 5 μm. E)–H) Bleaching analysis from stained cells as described in the Supporting Information (Part II, Immunostaining). E), F) Under Confocal Scanning Microscopy and (G, H) under STED Microscopy conditions. E), G) Normalized fluorescent time traces with exponential fit. Error bars correspond to one standard deviation of the data. F), H) Bleaching times under 20 % Confocal intensity (F, left), 50 % Confocal intensity (F, right), 20 % Confocal and 40 % STED intensity (H, left), 50 % Confocal and 40 % STED intensity (H, right). Error bars in (F), (H) correspond to one standard deviation of fitted bleaching times.