Enzymatic Incorporation of an Antibody‐Activated Blue Fluorophore into DNA

Kaufmann, Gunnar F.; Meijler, Michaël M.; Sun, Changqi; Chen, Dawei; Kujawa, David P.; Mee, Jenny M.; Hoffman, Timothy Z.; Wirsching, Peter; Lerner, Richard A.; Janda, Kim D.

doi:10.1002/ange.200461143

Cited by 7 publications

(5 citation statements)

References 22 publications

(11 reference statements)

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“…Nucleoside 6 was prepared according to the literature. [57] NMR spectra were recorded by using Bruker Avance 400 ( 1 H: 400, 13 C: 101, 32 P: 162 MHz) and Bruker DRX 600 ( 1 H: 600, 13 C: 151, 32 P: 243 MHz) spectrometers. Chemical shifts are given in parts per million and tetramethylsilane was used as external standard.…”

Section: Methodsmentioning

confidence: 99%

“…Due to hybridisation specificity and the ability of oligonucleotides to self-assemble, they have been used as tools to arrange precise structures in the nanoscale range. [1][2][3] Many different methods have been developed that use DNA as a scaffold to orientate and assemble inorganic [4][5][6][7][8][9][10] as well as organic [11][12][13] compounds on nanometer scales. DNA block copolymers with both a DNA segment and an organic polymer unit were reported recently.…”

Section: Introductionmentioning

confidence: 99%

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Enzymatic Synthesis of Organic‐Polymer‐Grafted DNA

Baccaro

Marx

2009

Chemistry A European J

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To create bioorganic hybrid materials, interdisciplinary work in the fields of chemistry, biology and materials science is conducted. DNA block copolymers are promising hybrid materials due to the combination of properties intrinsic to both the polymer and the nucleic acid blocks. Until now, the coupling of DNA and organic polymers has been exercised post-synthetically in solution or on solid support. Herein, we report the first enzyme-catalysed synthesis of DNA-organic polymer chimeras. For this purpose, four novel 2'-deoxyuridine triphosphates carrying polymer-like moieties linked to the nucleobase were synthesised. Linear polyethylene glycol monomethyl ethers of different sizes (1) and branched polyamido dendrons with varying terminal groups (2) were chosen as building blocks. We investigated the ability of DNA polymerases to accept the copolymers in comparison to the natural substrate and showed, through primer extensions, polymerase chain reactions and rolling circle amplification, that these building blocks could serve as a surrogate for the natural thymidine. By this method, DNA hybrid materials with high molecular weight, modification density, and defined structure are accessible.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Enzymatic Synthesis of Organic‐Polymer‐Grafted DNA

Baccaro

Marx

2009

Chemistry A European J

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“…While this technique tolerates an array of reporter species (13), we reasoned that the chosen reporter system should provide access to both affinity manipulation and optical analysis. An elegant choice for such a ligand was demonstrated with stilbene in its functional use with stilbene-binding antibodies (18)(19)(20)(21). Here, a stilbene hapten becomes a visible fluorophore as it binds to a complementary monoclonal antibody (mAb).…”

mentioning

confidence: 99%

“…Stilbene-labeled molecules have been shown to fluoresce when bound to mAb EP2-19G2 (18)(19)(20)(21). To verify the use of this protein modification scheme for both affinity and visualization applications, we quantified the fluorescence generated by antibody binding with stilbenetagged protein 1-CP in solution.…”

mentioning

confidence: 99%

“…Purified apo-VibB was modified by Sfp and stilbene-CoA 1. The labeled protein, stilbene-crypto-VibB, was purified via size-exclusion chromatography and concentrated to 11 µM via spin concentration, mixed with mAb EP2-19G2 (1.67 µM), and fluorescence intensity was analyzed at the optimal wavelengths for mAb EP2-19G2 (λ ex ) 327 nm, λ em ) 410 nm) (18)(19)(20)(21). Titration of mAb EP2-19G2 against stilbene-labeled crypto-VibB verified that maximal fluorescence arose from a 1:1 dependence of antibody binding sites to labeled protein.…”

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confidence: 99%

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A Site-Specific Bifunctional Protein Labeling System for Affinity and Fluorescent Analysis

et al. 2005

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Most covalent protein labeling schemes require a choice between visual and affinity properties, requiring the use of multiple fusion systems where both attributes are needed. While not disruptive at the single experiment level, this detail becomes critical when addressing high-throughput experimentation. Here we develop a uniform site-specific protein tag for use in both fluorescent and affinity screening. Covalent protein tagging with a stilbene reporter via promiscuous phosphopantetheinyltransferase (PPTase) modification enables a switchable, antibody-elicited fluorescent response in solution or on affinity resin. For demonstration purposes, VibB, a natural fusion protein harboring a carrier protein domain, was labeled with a stilbene tag through PPTase modification with a stilbene-labeled coenzyme A analogue. Analysis of the resulting stilbene-tagged VibB was accomplished by fluorescent and Western blot analysis with anti-stilbene monoclonal antibody EP2-19G2. The illustration of this method for general application to fusion protein analysis offers a dual role in assisting both solution-based fluorescent analysis and surface-based affinity detection and purification.

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Barcode‐modifizierte Nukleotide

2011

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Etiketten für DNA: Erstaunlicherweise sind DNA‐Polymerasen in der Lage, Oligodesoxynukleotid(ODN)‐modifizierte Nukleotide, die bis zu 40fach größer als ihre natürlichen Substrate sind, zu prozessieren, was zu Barcode‐markierter DNA führt (siehe Schema). Der Einbau der ODN‐modifizierten Nukleotide wurde in Lösung und an der festen Phase durch Hybridisierung mit einem fluoreszenzmarkierten DNA‐Strang komplementär zum ODN‐Strang nachgewiesen.

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Enzymatic Incorporation of an Antibody‐Activated Blue Fluorophore into DNA

Cited by 7 publications

References 22 publications

Enzymatic Synthesis of Organic‐Polymer‐Grafted DNA

Enzymatic Synthesis of Organic‐Polymer‐Grafted DNA

A Site-Specific Bifunctional Protein Labeling System for Affinity and Fluorescent Analysis

Barcode‐modifizierte Nukleotide

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