The application of an immobilized molecular beacon for the analysis of the DNA binding domains from the ecdysteroid receptor proteins Usp and EcR's interaction with the hsp27 response element.
The nonstandard molecular beacon described in this article consists of 2 fragments, each built of a short single-stranded oligonucleotide sequence and a double-stranded sequence. One of these hybridization probes, labeled with a fluorescence donor (fluorescein), is solid phase immobilized. The second nonimmobilized probe is labeled with a fluorescence quencher (dabcyl). Annealing of both probes via single-stranded sequences was possible only in the presence of a specific protein molecule that recognized the response element sequence initially separated between the immobilized and nonimmobilized fragments. The system was applied successfully to detect the sequence-specific interaction of a natural hsp27 response element from the promoter of the hsp27 gene with the DNA binding domains of 2 nuclear receptor proteins: ultraspiracle Usp (UspDBD) and the ecdysone receptor EcR (EcRDBD). Measured in the absence of EcRDBD, the dissociation constant, K(d) of the UspDBD-hsp27 complex, was determined to be 3.26 nM, whereas for UspDBD devoid of the A-box (UspDBDDeltaA-hsp27 ), the dissociation constant was 4.81 nM. The respective K(d) values in the presence of EcRDBD were 2.43 nM and 10.80 nM. The results obtained with the immobilized molecular beacon technology were in agreement with those obtained by conventional fluorescence titrations and by fluorescence resonance energy transfer measurements with nonimmobilized beacons.
Krusiński
Tomasz
T
Faculty of Chemistry, Division of Biochemistry, Wroclaw University of Technology, Wroclaw, Poland.
Laskowska
Anna
A
Ozyhar
Andrzej
A
Dobryszycki
Piotr
P
D001483:Base Sequence; D001710:Biotin / Q000737:chemistry; D002467:Cell Nucleus / Q000378:metabolism; D004247:DNA / Q000737:chemistry*; D031541:Fluorescence Resonance Energy Transfer; D055551:HSP27 Heat-Shock Proteins / Q000737:chemistry*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000737:chemistry; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011987:Receptors, Steroid / Q000737:chemistry*; D020218:Response Elements
D055551:HSP27 Heat-Shock Proteins; D009841:Oligonucleotides; D011987:Receptors, Steroid; C034808:ecdysteroid receptor; D001710:Biotin; D004247:DNA