Molecular beacon-equilibrium cyclization detection of DNA-protein complexes.
Molecular beacon detection of equilibrium cyclization (MBEC) is a novel, high sensitivity technique that can allow DNA-protein complex formation to be studied under diverse conditions in a cost effective and rapid manner that can be adapted to high throughput screening. To demonstrate the ease and utility of applying MBEC to the investigation of the K(D) values of protein-DNA complexes, the sequence-specific Escherichia coli integration host factor (IHF) protein has been used as a test system. Competition between a labeled MBEC DNA construct and unlabeled duplex DNA for IHF binding allows the determination of K(D) values as a function of the DNA duplex sequence. This allows sequence specificity to be monitored while using only a single molecular beacon-labeled DNA. The robustness of MBEC for monitoring protein-DNA complex formation has been further demonstrated by determining the K(D) values as a function of salt concentration to investigate the net number of salt bridges formed in sequence-specific and -nonspecific IHF-DNA complexes. These MBEC results have been compared with those from other approaches.
Vitko
Jason
J
Department of Molecular Biology and Biochemistry, Wesleyan University, Middletown, Connecticut 06459, USA.
Rujan
Iulian
I
Androga
Lagu
L
Mukerji
Ishita
I
Bolton
Philip H
PH
D010582:Bacteriophage lambda; D010583:Bacteriophage mu; D001483:Base Sequence; D003500:Cyclization; D004269:DNA, Bacterial / Q000737:chemistry* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism; D034982:Integration Host Factors / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D011485:Protein Binding / Q000502:physiology
D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D034982:Integration Host Factors