Click chemistry with DNA

El‐Sagheer, Afaf H.; Brown, Tom

doi:10.1039/b901971p

Cited by 675 publications

(378 citation statements)

References 77 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 concept of "click chemistry" proposed by Kolb, Finn and Sharpless in 2001 [1] has revolutionized molecular engineering including applications to organic and medicinal chemistry [2][3][4][5][6][7][8][9][10][11], polymer science and materials science [12][13][14][15][16][17][18][19][20][21]. Among the various "click" reactions responding to the requirements of this concept, the most generally used one is the copper(I)-catalyzed reaction between terminal alkynes and azides (CuAAC) selectively yielding 1,4-disubstituted 1,2,3-triazoles, that was reported independently by the Sharpless-Fokin [22] and the Meldal groups in 2002 [ 23,24].…”

Section: Introductionmentioning

confidence: 99%

Metal-catalyzed azide-alkyne “click” reactions: Mechanistic overview and recent trends

Changlong

Ikhlef

Kahlal

et al. 2016

Coordination Chemistry Reviews

293

162

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Highlights-The review is focused on the mechanistic aspects and recent trends (since 2012) of the metal-catalyzed azide-alkyne cycloaddition (MAAC) so-called "click" reactions with catalysts based on various metals (Cu, Ru, Ag, Au, Ir, Ni, Zn, Ln), although Cu (I) catalysts are still the most used ones.-These MAAC reactions are by far the most common click reactions relevant to the "green chemistry" concept.-Mechanistic investigations are essential to reaction improvements and subsequent applications, and indeed as shown in this review the proposed mechanisms have been multiple during the last decade based on theoretical computations and experimental search of intermediates.-New trends are also presented here often representing both exciting approaches for various applications and new challenges for further mechanistic investigations.

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“…3-Iron bis(dicarbollide) redox label was covalently attached to DNA probe using "click chemistry" methodology (El-Sagheer and Brown, 2010;Wojtczak et al, 2008) in the manner providing its location close to the surface of gold electrode. This is in contrast to redox active labels and formats reported till now.…”

Section: Resultsmentioning

confidence: 99%

DNA probe modified with 3-iron bis(dicarbollide) for electrochemical determination of DNA sequence of Avian Influenza Virus H5N1

Grabowska

Stachyra

Góra-Sochacka

et al. 2014

Biosensors and Bioelectronics

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Click chemistry with DNA

Cited by 675 publications

References 77 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

Metal-catalyzed azide-alkyne “click” reactions: Mechanistic overview and recent trends

DNA probe modified with 3-iron bis(dicarbollide) for electrochemical determination of DNA sequence of Avian Influenza Virus H5N1

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