DNA-Programmed Chemistry in Rapid Homogeneous Assays for DNA and Protein Targets
| Autor: | Christopher G.M. Wilson, Benjamin K. Benton, James M. Coull, Yumei Huang, Lawrence A. Haff, Julian F Bond, Andrew M. Stern, Richard F. Begley |
|---|---|
| Rok vydání: | 2006 |
| Předmět: |
Platelet-Derived Growth Factor
Tris Azides Aqueous solution Fluorophore Oligonucleotide Aptamer Biochemistry (medical) Clinical Biochemistry Becaplermin DNA Proto-Oncogene Proteins c-sis Aptamers Nucleotide Fluorescence Combinatorial chemistry Melting curve analysis Reaction rate chemistry.chemical_compound Organophosphorus Compounds chemistry Coumarins Humans Transition Temperature Organic chemistry Fluorescent Dyes |
| Zdroj: | Clinical Chemistry. 52:2147-2148 |
| ISSN: | 1530-8561 0009-9147 |
| DOI: | 10.1373/clinchem.2006.072751 |
| Popis: | DNA-programmed chemistry (DPC) is a novel technology for synthesis of a wide variety of organic compounds at nanomolar concentrations under physiologic conditions (1). Annealing of small molecule-oligonucleotide precursors to DNA templates, each with an attached chemical moiety, can generate high amounts of effective molarities of these reactants. This phenomenon can enhance, by nearly 1-million-fold, the rate of a reaction, while increasing its specificity. We have developed a specific chemical process that generates a fluorescent product on annealing of 2 oligonucleotides to each other. Oligonucleotides containing a 3′-terminal azidocoumarin (AzC)1 and a 5′-terminal triphenylphosphine (TPP) will react in aqueous solution to form a fluorescent product, 7-aminocoumarin. The reaction rate is very slow at submicromolar concentrations of the single-stranded TPP and AzC groups in free solution. However, if these fluorophore precursor-containing oligonucleotides anneal to each other or to a common DNA target, as long as the 3′ and 5′ fluorophore precursors are annealed in close proximity, their localized high concentration supports their reaction to yield a fluorescent product. We have found that this principle can also be exploited for detection of proteins (and other high molecular weight analytes), with the advantages of a simple, homogeneous phase assay with potentially very low background. We modified this architecture for detecting proteins and other non–nucleic-acid targets. The model target selected was platelet-derived growth factor (PDGF-BB), a protein that contains 2 identical B subunits, for which tight-binding aptamers have been previously identified. We obtained human PDGF-BB and mouse monoclonal antihuman PDGF-BB from R&D systems. Except as indicated, all reaction and melting curve solutions contained, in a volume of 100 μL: 50 mmol/L Tris/HCl buffer at pH 8.0, 10 mmol/L magnesium chloride, 40 pmol of detection oligonucleotides, 40 pmol of PDGF-BB, and typically, 350 mL/L formamide by volume. We determined melting curves by measuring fluorescence … |
| Databáze: | OpenAIRE |
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