In situ click chemistry: probing the binding landscapes of biological molecules

Mamidyala, Sreeman K.; Finn, M. G.

doi:10.1039/b901969n

Cited by 452 publications

(227 citation statements)

References 99 publications

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“…For reaction with this, azide groups were introduced into goat anti-rat IgG antibodies by modifying lysine residues with commercially available 3-(azidotetra(ethyleneoxy))propionic acid succinimidyl ester, which required only a simple 30-min incubation of the antibody with 600 μM of the azide labeling compound in aqueous buffer. These azide-modified antibodies were subsequently conjugated to the six alkyne-functionalized ODFs via standard Cu(I)-mediated cycloaddition reactions (28)(29)(30) at 37°C overnight (Scheme S1). For comparison of spectral properties and coupling efficiency, we followed the same procedure to label the antibody with Alexa 488, a conventional organic dye, employing a commercially available alkyne-modified derivative of this fluorophore.…”

Section: Resultsmentioning

confidence: 99%

Multispectral labeling of antibodies with polyfluorophores on a DNA backbone and application in cellular imaging

Guo

Wang

Dai

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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Most current approaches to multiantigen fluorescent imaging require overlaying of multiple images taken with separate filter sets as a result of differing dye excitation requirements. This requirement for false-color composite imaging prevents the user from visualizing multiple species in real time and disallows imaging of rapidly moving specimens. To address this limitation, here we investigate the use of oligodeoxyfluoroside (ODF) fluorophores as labels for antibodies. ODFs are short DNA-like oligomers with fluorophores replacing the DNA bases and can be assembled in many colors with excitation at a single wavelength. A DNA synthesizer was used to construct several short ODFs carrying a terminal alkyne group and having emission maxima of 410-670 nm. We developed a new approach to antibody conjugation, using HuisgenSharpless cycloaddition, which was used to react the alkynes on ODFs with azide groups added to secondary antibodies. Multiple ODF-tagged secondary antibodies were then used to mark primary antibodies. The set of antibodies was tested for spectral characteristics in labeling tubulin in HeLa cells and revealed a wide spectrum of colors, ranging from violet-blue to red with excitation through a single filter (340-380 nm). Selected sets of the differently labeled secondary antibodies were then used to simultaneously mark four antigens in fixed cells, using a single image and filter set. We also imaged different surface tumor markers on two live cell lines. Experiments showed that all colors could be visualized simultaneously by eye under the microscope, yielding multicolor images of multiple cellular antigens in real time.bioconjugation | immunofluorescence | multiplex T o understand the complexity and dynamics of the molecular interactions in biological systems, the parallel analysis of multiple species, such as different proteins in a cell or cells in a tissue specimen, is often needed (1, 2). The most common mode of imaging for tracking and labeling such species is fluorescence microscopy. For multispecies imaging, this typically requires the use of various fluorophores having distinct excitation and emission wavelengths. Commonly available organic fluorophores are typically used to tag biomolecules for these purposes (3, 4), which allows the visualization of three, or occasionally more, species via the use of separate excitation and emission filters. Using this strategy, one can label multiple cellular antigens, for example, by use of different commercially available dye-labeled secondary antibodies.Although this approach is widely employed, some nonideal factors still exist. One of the major limiting issues of common organic dyes is that they have widely separated absorption spectra. This fact requires the researcher to use specialized filter sets and take a separate image for each dye; the final multicolor image is then constructed by overlaying false-color single-dye images. This approach enforces some restrictions on the researcher and equipment and places limitations on data acquisition. For ex...

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Section: Resultsmentioning

confidence: 99%

Multispectral labeling of antibodies with polyfluorophores on a DNA backbone and application in cellular imaging

Guo

Wang

Dai

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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“…The last dienophile used was diethyl azodicarboxylate (33). Its prolonged heating (50 h) with 1b in a 1 : 1 mixture of chloroform and acetonitrile gave a complex mixture from which symdiethyl hydrazinedicarboxylate 22 (34, 24%) and traces of 3 were isolated.…”

Section: Scheme 10 Proposed Intermediates Leading To Products 28-30mentioning

confidence: 99%

Reactivity of 1,2,3-triazole-substituted 1-azabutadienes (vinamidines)

Bátori¹,

Bokotey²,

Messmer³

2012

Arkivoc

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“…1). 到目前为止, Cue-AAC 反应已经被广泛应用于药物制备 [8,9] 、核苷衍生物的制备 [10] 虽然 Cue-AAC 反应一直被视为点击化学中的经典反应, 但是其也有不完美的地方. 其中, 主要原因是在反应过程中需要使用 Cu(I)作为催化剂.…”

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Construction of Nitrogen-Oxygen-Heterocycles via Copper-Free Click Reactions of Nitrile Oxides

Han¹,

Yi²,

Xiong³

2014

Chin. J. Org. Chem.

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In situ click chemistry: probing the binding landscapes of biological molecules

Cited by 452 publications

References 99 publications

Multispectral labeling of antibodies with polyfluorophores on a DNA backbone and application in cellular imaging

Multispectral labeling of antibodies with polyfluorophores on a DNA backbone and application in cellular imaging

Reactivity of 1,2,3-triazole-substituted 1-azabutadienes (vinamidines)

Construction of Nitrogen-Oxygen-Heterocycles via Copper-Free Click Reactions of Nitrile Oxides

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