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  ADLOC: an aptamer-displacement assay based on luminescent oxygen channeling

Niebel, B., Lentz, C., Pofahl, M., Mayer, G., Hoerauf, A., Pfarr, K. M., et al. (2010). ADLOC: an aptamer-displacement assay based on luminescent oxygen channeling. Chemistry, 16(36), 11100-7. doi:10.1002/chem.201001192.

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Item Permalink: http://hdl.handle.net/11858/00-001M-0000-0028-6432-F Version Permalink: http://hdl.handle.net/11858/00-001M-0000-0028-6433-D
Genre: Journal Article

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Niebel, B., Author
Lentz, C., Author
Pofahl, M., Author
Mayer, G., Author
Hoerauf, A., Author
Pfarr, K. M., Author
Famulok, M.1, Author
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Free keywords: Aptamers, Nucleotide/analysis/*chemistry Biosensing Techniques Fluorescence Polarization/methods Ligands Luminescent Measurements/methods Molecular Probes/*chemistry Molecular Sequence Data Molecular Structure Nucleic Acids/analysis/*chemistry Oligonucleotides/*chemistry Oxygen/*chemistry
 Abstract: Functional nucleic acids, such as aptamers and allosteric ribozymes, can sense their ligands specifically, thereby undergoing structural alterations that can be converted into a detectable signal. The direct coupling of molecular recognition to signal generation enables the production of versatile reporters that can be applied as molecular probes for various purposes, including high-throughput screening. Here we describe an unprecedented type of a nucleic acid-based sensor system and show that it is amenable to high-throughput screening (HTS) applications. The approach detects the displacement of an aptamer from its bound protein partner by means of luminescent oxygen channeling. In a proof-of-principle study we demonstrate that the format is feasible for efficient identification of small drug-like molecules that bind to a protein target, in this case to the Sec7 domain of cytohesin. We extended the approach to a new cytohesin-specific single chain DNA aptamer, C10.41, which exhibits a similar binding behavior to cytohesins but has the advantage of being more stable and easier to synthesize and to modify than the RNA-aptamer M69. The results obtained with both aptamers indicate the general suitability of the aptamer-displacement assay based on luminescent oxygen channelling (ADLOC) for HTS. We also analyzed the potential for false positive hits and identified from a library of 18,000 drug-like small molecules two compounds as strong singlet-oxygen quenchers. With full automation and the use of commercially available plate readers, we estimate that the ADLOC-based assay described here could be used to screen at least 100,000 compounds per day.

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 Dates: 2010
 Publication Status: Published in print
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 Identifiers: Other: 20690121
DOI: 10.1002/chem.201001192
ISSN: 1521-3765 (Electronic)
ISSN: 0947-6539 (Linking)
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Title: Chemistry
  Alternative Title : Chemistry
Source Genre: Journal
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Pages: - Volume / Issue: 16 (36) Sequence Number: - Start / End Page: 11100 - 7 Identifier: -