14 research outputs found
A dark intermediate in the fluorogenic reaction between tetrazine fluorophores and trans-cyclooctene
Fluorogenic labeling via bioorthogonal tetrazine chemistry has proven to be highly successful in fluorescence microscopy of living cells. To date, trans-cyclooctene (TCO) and bicyclonyne have been found to be the most useful substrates for live-cell labeling owing to their fast labeling kinetics, high biocompatibility, and bioorthogonality. Recent kinetic studies of fluorogenic click reactions with TCO derivatives showed a transient fluorogenic effect but could not explain the reaction sequence and the contributions of different intermediates. More recently, fluorescence quenching by potential intermediates has been investigated, suggesting their occurrence in the reaction sequence. However, in situ studies of the click reaction that directly relate these observations to the known reaction sequence are still missing. In this study, we developed a single-molecule fluorescence detection framework to investigate fluorogenic click reactions. In combination with data from ultra-performance liquid chromatography-tandem mass spectrometry, this explains the transient intensity increase by relating fluorescent intermediates to the known reaction sequence of TCO with fluorogenic tetrazine dyes. More specifically, we confirm that the reaction of TCO with tetrazine rapidly forms a fluorescent 4,5-dihydropyridazine species that slowly tautomerizes to a weakly fluorescent 1,4-dihydropyridazine, explaining the observed drop in fluorescence intensity. On a much slower timescale of hours/days, the fluorescence intensity may be recovered by oxidation of the intermediate to a pyridazine. Our findings are of importance for quantitative applications in fluorescence microscopy and spectroscopy as the achieved peak intensity with TCO depends on the specific experimental settings. They clearly indicate the requirement for more robust benchmarking of click reactions with tetrazine dyes and the need for alternative dienophiles with fast reaction kinetics and stable fluorescence emission to further applications in advanced fluorescence microscopy
A Bioorthogonal Click Chemistry Toolbox for Targeted Synthesis of Branched and Well-Defined Protein-Protein Conjugates
A highly efficient technology for protein
functionalization with commonly used bioorthogonal motifs for Diels-Alder
cycloaddition with inverse electron demand (DAinv). With the aim of
precisely generating branched protein chimeras, we systematically assessed the
reactivity, stability and side product formation of various bioorthogonal
chemistries directly at the protein level. We demonstrate the efficiency and
versatility of our conjugation platform using different functional proteins and
the therapeutic antibody trastuzumab. This technology enables fast and routine
access to tailored and hitherto inaccessible protein chimeras useful for a
variety of scientific disciplines
Petrus Mullerus, U. I. D- Et Pandectarum in Academia Salana Professor Publicus Lectori S. P.
PETRUS MULLERUS, U. I. D- ET PANDECTARUM IN ACADEMIA SALANA PROFESSOR PUBLICUS LECTORI S. P.
Petrus Mullerus, U. I. D- Et Pandectarum in Academia Salana Professor Publicus Lectori S. P. ([1]
Correlative 3D microscopy of single cells using super-resolution and scanning ion-conductance microscopy
High-resolution live-cell imaging is necessary to study complex biological phenomena. Modern fluorescence microscopy methods are increasingly combined with complementary, label-free techniques to put the fluorescence information into the cellular context. The most common high-resolution imaging approaches used in combination with fluorescence imaging are electron microscopy and atomic-force microscopy (AFM), originally developed for solid-state material characterization. AFM routinely resolves atomic steps, however on soft biological samples, the forces between the tip and the sample deform the fragile membrane, thereby distorting the otherwise high axial resolution of the technique. Here we present scanning ion-conductance microscopy (SICM) as an alternative approach for topographical imaging of soft biological samples, preserving high axial resolution on cells. SICM is complemented with live-cell compatible super-resolution optical fluctuation imaging (SOFI). To demonstrate the capabilities of our method we show correlative 3D cellular maps with SOFI implementation in both 2D and 3D with self-blinking dyes for two-color high-order SOFI imaging. Finally, we employ correlative SICM/SOFI microscopy for visualizing actin dynamics in live COS-7 cells with subdiffraction-resolution
Bio-orthogonal Red and Far-Red Fluorogenic Probes for Wash-Free Live-Cell and Super-resolution Microscopy
Small-molecule fluorophores enable the observation of biomolecules in their native context with fluorescence microscopy. Specific labeling via bio-orthogonal tetrazine chemistry combines minimal label size with rapid labeling kinetics. At the same time, fluorogenic tetrazine-dye conjugates exhibit efficient quenching of dyes prior to target binding. However, live-cell compatible long-wavelength fluorophores with strong fluorogenicity have been difficult to realize. Here, we report close proximity tetrazine-dye conjugates with minimal distance between tetrazine and the fluorophore. Two synthetic routes give access to a series of cell-permeable and -impermeable dyes including highly fluorogenic far-red emitting derivatives with electron exchange as the dominant excited-state quenching mechanism. We demonstrate their potential for live-cell imaging in combination with unnatural amino acids, wash-free multicolor and super-resolution STED, and SOFI imaging. These dyes pave the way for advanced fluorescence imaging of biomolecules with minimal label size. BN/Kristin Grussmayer LabBN/Afdelingsburea
Bio-orthogonal red and far-red fluorogenic probes for wash-free live-cell and super-resolution microscopy
[Image: see text] Small-molecule fluorophores enable the observation of biomolecules in their native context with fluorescence microscopy. Specific labeling via bio-orthogonal tetrazine chemistry combines minimal label size with rapid labeling kinetics. At the same time, fluorogenic tetrazineâdye conjugates exhibit efficient quenching of dyes prior to target binding. However, live-cell compatible long-wavelength fluorophores with strong fluorogenicity have been difficult to realize. Here, we report close proximity tetrazineâdye conjugates with minimal distance between tetrazine and the fluorophore. Two synthetic routes give access to a series of cell-permeable and -impermeable dyes including highly fluorogenic far-red emitting derivatives with electron exchange as the dominant excited-state quenching mechanism. We demonstrate their potential for live-cell imaging in combination with unnatural amino acids, wash-free multicolor and super-resolution STED, and SOFI imaging. These dyes pave the way for advanced fluorescence imaging of biomolecules with minimal label size