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Molecular Beacon-Equilibrium Cyclization Detection of DNA-Protein Complexes

Jason Vitko , Iulian Rujan ^*, Lagu Androga ^*, Ishita Mukerji  and Philip H. Bolton ^*

^* Department of Chemistry and Department of Molecular Biology and Biochemistry, Wesleyan University, Middletown, Connecticut 06459

Correspondence: Address reprint requests to Dr. Ishita Mukerji, Dept. of Molecular Biology and Biochemistry, Wesleyan University, Middletown, CT 06459. E-mail: imukerji{at}wesleyan.edu; or Dr. Philip H. Bolton, Dept. of Chemistry, Wesleyan University, Middletown, CT 06459. E-mail: pbolton{at}wesleyan.edu.

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.