A Bioorthogonally Applicable, Fluorogenic, Large Stokes-Shift Probe for Intracellular Super-Resolution Imaging of Proteins

Abstract

Herein, we present the synthesis and application of a fluorogenic, large Stokes-shift (>100 nm), bioorthogonally conjugatable, membrane-permeable tetrazine probe, which can be excited at common laser line 488 nm and detected at around 600 nm. The applied design enabled improved fluorogenicity in the orange/red emission range, thus efficient suppression of background and autofluorescence upon imaging biological samples. Moreover, unlike our previous advanced probes, it does not require the presence of special target platforms or microenvironments to achieve similar fluorogenicity and can be generally applied, e.g., on translationally bioorthogonalized proteins. Live-cell labeling schemes revealed that the fluorogenic probe is suitable for specific labeling of intracellular proteins, site-specifically modified with a cyclooctynylated, non-canonical amino acid, even under no-wash conditions. Furthermore, the probe was found to be applicable in stimulated emission depletion (STED) super-resolution microscopy imaging using a 660 nm depletion laser. Probably the most salient feature of this new probe is that the large Stokes-shift allows dual-color labeling schemes of cellular structures using distinct excitation and the same detection wavelengths for the combined probes, which circumvents chromatic aberration related problems.

Fluorescence Quenching Effects of Tetrazines and Their Diels–Alder Products: Mechanistic Insight Toward Fluorogenic Efficiency

“…[5] Beyond the remarkable kinetics,anattractive feature of iedDAreactions involving tetrazines is the possibility of obtaining fluorescent products from non-emissive precursors,t hat is,afluorogenic reaction, given the right choice of reaction partners. [9][10][11][12][13][14][15][16][17][18][19][20][21][22] This feature can be particularly useful for in situ fluorescence labeling of cellular species to enable their visualization and tracking by various microscopy techniques.T he fluorogenic character stems from the strong fluorescence quenching effect that tetrazines can exert on tethered fluorophores,w hich leads to net fluorescence enhancement once the tetrazine core is broken.…”

Fluorescence Quenching Effects of Tetrazines and Their Diels–Alder Products: Mechanistic Insight Toward Fluorogenic Efficiency

“…With remarkably fast reaction rates, innocuous byproduct formation, and no catalyst requirement, these transformations offer an excellent alternative to azide‐alkyne cycloadditions and other “click” reactions, especially for bioconjugation reactions performed in cellulo [5] . Beyond the remarkable kinetics, an attractive feature of iedDA reactions involving tetrazines is the possibility of obtaining fluorescent products from non‐emissive precursors, that is, a fluorogenic reaction, given the right choice of reaction partners [9–22] . This feature can be particularly useful for in situ fluorescence labeling of cellular species to enable their visualization and tracking by various microscopy techniques.…”

“…[5] Beyond the remarkable kinetics,a na ttractive feature of iedDAr eactions involving tetrazines is the possibility of obtaining fluorescent products from non-emissive precursors,t hat is,afluorogenic reaction, given the right choice of reaction partners. [9][10][11][12][13][14][15][16][17][18][19][20][21][22] This feature can be particularly useful for in situ fluorescence labeling of cellular species to enable their visualization and tracking by various microscopy techniques.T he fluorogenic character stems from the strong fluorescence quenching effect that tetrazines can exert on tethered fluorophores,w hich leads to net fluorescence enhancement once the tetrazine core is broken.…”

IEDDA: An Attractive Bioorthogonal Reaction for Biomedical Applications

“…Fluorescence increase in the order of 10 3 to 10 4 was observed after TCO-activation of the probes. From a mechanistic point of view, the TBET-based quenching process can be applied to any type of fluorophore, and Kele’s group reported Tz-caged probes based on various fluorophores, including phenoxazine, coumarin, siliconrhodamine and rhodamine analogs [ 112 , 113 , 114 , 115 ]. The feasibility of applying the probes to cell-based imaging has also been demonstrated by confocal microscopy.…”