C‐Terminal Incorporation of Bio‐Orthogonal Azide Groups into a Protein and Preparation of Protein–Oligodeoxynucleotide Conjugates by Cu<sup>I</sup>‐Catalyzed Cycloaddition

Humeník, Martin; Huang, Yiwei; Wang, Yiran; Sprinzl, Mathias

doi:10.1002/cbic.200700070

Cited by 44 publications

(46 citation statements)

References 40 publications

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“…[65] The ligand effect could also differ depending on the counter ions involved in cluster formation as sometimes observed. [66,67] Similar effects are observed with the generally most popular ligand TBTA, 10 introduced by the group of Sharpless. [32] Proximity Effects and Reactivity of the Alkyne and Azide Substrates…”

Section: General Considerations In Cuaac Click Chemistry In Polymer Ssupporting

confidence: 69%

“…[4] With increasing size of both alkyne and azide counterparts in triazole-based polymerization of large macromonomers or conjugation of azide containing proteins with large fragments of DNA containing alkyne the reaction conditions become inherently more dilute and conversions have been observed to decrease. [66] This indicates that there could be limiting cases where the utility of this ligation reaction may not be optimal although new improved conjugation conditions are continuously being developed. [68] Organic azides are considerably more reactive than the azide anion itself.…”

Section: General Considerations In Cuaac Click Chemistry In Polymer Smentioning

confidence: 98%

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Polymer “Clicking” by CuAAC Reactions

Meldal

2008

Macromol. Rapid Commun.

318

151

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The CuAAC “click” reaction has developed as one of the most useful and widely employed reactions in ligation within polymer chemistry. This is due to the unique properties of the Cu(I) catalysis which renders the reaction quantitative even at low concentrations, orthogonal with other chemistries and extremely robust. The formed triazole on the other hand is of intermediate polarity and chemically and biochemically “invisible”, and the CuAAC provides the ideal “click” reaction for stitching together polymer architectures of unprecedended complexity as was it molecular LEGO. The CuAAC “clicking” in polymer chemistry is increasing exponentially and lead to highly defined polymer materials with novel properties.magnified image

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Section: General Considerations In Cuaac Click Chemistry In Polymer Ssupporting

confidence: 69%

Section: General Considerations In Cuaac Click Chemistry In Polymer Smentioning

confidence: 98%

Polymer “Clicking” by CuAAC Reactions

Meldal

2008

Macromol. Rapid Commun.

318

151

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“…Oligonucleotides can be conjugated to proteins through various chemistries, including disulfide bond formation (24), biotin streptavidin interaction (25), maleimide-thiol coupling (26), etc. Recently, Huisgen-Sharpless cycloaddition chemistry as a simple, mild, and generally applicable strategy has also been explored (27). In this approach, the azide moiety was incorporated via in vitro translation into an esterase and the alkyne group was chemically conjugated to DNA.…”

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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“…[25] The N and C termini of EST2 are exposed on the esterase surface, [26] and provide the potential for the protein to be fused with other polypeptides or conjugated with molecules, such as different labels and oligonucleotides, without altering the esterase native fold. [27][28][29] EST2 linked to polypeptide chains can serve as a versatile and sensitive reporter for monitoring of protein expression and at the same time as an affinity tag for isolation of A C H T U N G T R E N N U N G expressed proteins from cellular extracts. [30] AFM provides topological information about biomacromolecules attached at solid-liquid interfaces, [31] and in the last decade has become one of the most frequently practiced techniques in biological sciences for the study of biomacromolecule surface properties.…”

Section: Introductionmentioning

confidence: 99%

Ligand‐Directed Immobilization of Proteins through an Esterase 2 Fusion Tag Studied by Atomic Force Microscopy

Minařík¹,

Humeník²,

Li³

et al. 2007

ChemBioChem

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Atomically flat mica surfaces were chemically modified with an alkyl trifluoromethyl ketone, a covalent inhibitor of esterase 2 from Alicyclobacillus acidocaldarius, which served as a tag for ligand-directed immobilization of esterase-linked proteins. Purified NADH oxidase from Thermus thermophilus and human exportin-t from cell lysates were anchored on the modified surfaces. The immobilization effectiveness of the proteins was studied by atomic force microscopy (AFM). It was shown that ligand-esterase interaction allowed specific attachment of exportin-t and resulted in high-resolution images and coverage patterns that were comparable with immobilized purified protein. Moreover, the biological functionality of immobilized human exportin-t in forming a quaternary complex with tRNA and the GTPase Ran-GTP, and the dimension changes before and after complex formation were also determined by AFM.

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C‐Terminal Incorporation of Bio‐Orthogonal Azide Groups into a Protein and Preparation of Protein–Oligodeoxynucleotide Conjugates by Cu^I‐Catalyzed Cycloaddition

Cited by 44 publications

References 40 publications

Polymer “Clicking” by CuAAC Reactions

Polymer “Clicking” by CuAAC Reactions

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

Ligand‐Directed Immobilization of Proteins through an Esterase 2 Fusion Tag Studied by Atomic Force Microscopy

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C‐Terminal Incorporation of Bio‐Orthogonal Azide Groups into a Protein and Preparation of Protein–Oligodeoxynucleotide Conjugates by CuI‐Catalyzed Cycloaddition

Cited by 44 publications

References 40 publications

Polymer “Clicking” by CuAAC Reactions

Polymer “Clicking” by CuAAC Reactions

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

Ligand‐Directed Immobilization of Proteins through an Esterase 2 Fusion Tag Studied by Atomic Force Microscopy

Contact Info

Product

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C‐Terminal Incorporation of Bio‐Orthogonal Azide Groups into a Protein and Preparation of Protein–Oligodeoxynucleotide Conjugates by Cu^I‐Catalyzed Cycloaddition