Molecular Beacon-Equilibrium Cyclization Detection of DNA-Protein Complexes
Jason Vitko 1, Iulian Rujan 1, Lagu A. Androga 1, Ishita Mukerji 1* and Philip H. Bolton 1
1 Wesleyan University
* To whom correspondence should be addressed. E-mail: imukerji{at}wesleyan.edu.
Submitted on September 19, 2006
Revised on December 5, 2006
Accepted on 19 June 2007
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Abstract |
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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 KD values of protein-DNA complexes the sequence specific E. coli integration host factor protein, IHF, 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 KD 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 KD values as a function of salt concentration to investigate the net number of salt bridges formed in sequence specific and non-specific IHF-DNA complexes. These MBEC results have been compared with those from other approaches.
Key Words:
equilibrium cyclization, fluorescence, molecular beacons, protein-DNA interactions
