title: ADLOC: an aptamer-displacement assay based on luminescent oxygen channeling.
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.
authors.affiliation: Life and Medical Sciences (LIMES) Institute, Chemical Biology and Medicinal Chemistry Unit, University of Bonn, Gerhard-Domagk-Strasse 1, 53121 Bonn, Germany.
authors.forename: Björn
authors.initials: B
authors.lastname: Niebel
authors.forename: Christian
authors.initials: C
authors.lastname: Lentz
authors.forename: Monika
authors.initials: M
authors.lastname: Pofahl
authors.forename: Günter
authors.initials: G
authors.lastname: Mayer
authors.forename: Achim
authors.initials: A
authors.lastname: Hoerauf
authors.forename: Kenneth M
authors.initials: KM
authors.lastname: Pfarr
authors.forename: Michael
authors.initials: M
authors.lastname: Famulok
mesh_terms: D052157:Aptamers, Nucleotide / Q000032:analysis / Q000737:chemistry*; D015374:Biosensing Techniques; D005454:Fluorescence Polarization / Q000379:methods; D008024:Ligands; D008163:Luminescent Measurements / Q000379:methods; D015335:Molecular Probes / Q000737:chemistry*; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009696:Nucleic Acids / Q000032:analysis / Q000737:chemistry*; D009841:Oligonucleotides / Q000737:chemistry*; D010100:Oxygen / Q000737:chemistry*
chemical_list: D052157:Aptamers, Nucleotide; D008024:Ligands; D015335:Molecular Probes; D009696:Nucleic Acids; D009841:Oligonucleotides; D010100:Oxygen