Molecular Beacon-Equilibrium Cyclization Detection of DNA-Protein Complexes

Abstract

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.

Applying 6-Methylisoxanthopterin-Enhanced Fluorescence To Examine Protein–DNA Interactions in the Picomolar Range

“…The binding affinity measured was comparable to previously reported values (Table 4). 45 Similarly, minimal perturbations were observed when the TFA15ds sequence was used to investigate the HU-DNA interaction, as shown by the K d value (Table 4). The HU_TFA15ds construct consisted of a single-stranded and duplex portion where the pentamer sequence is located at the junction of the duplex and single strand - the point at which HU putatively binds with nanomolar affinity.…”

“… 1 Measured with constant [DNA] = 2000 pM using fluorescence anisotropy. 2 Measured with constant [DNA]=1000 pM using fluorescence anisotropy. 3 Measured with constant [DNA]=500 pM using fluorescence intensity. 4 Measured with constant [DNA]=50 pM using fluorescence intensity. 5 From Vitko et al 45 6 From Zhai and Hingorani. 43 …”

Molecular Dissection of Mycobacterium tuberculosis Integration Host Factor Reveals Novel Insights into the Mode of DNA Binding and Nucleoid Compaction

“…51). (58,59). At low salt concentrations below 100 mM, the specificity of IHF is low, and the specificity increases as the salt concentration increases, up to 250 mM.…”

Characteristics of IHF Binding to Holliday Junction DNA

“…The dissociation constant K d calculated for the binding of IHF to H1 duplex is 38 ±27 nM. This value is consistent with that discovered in previous work: 10 ± 4 nM (Buzovetsky, O., 2010), and 31 ± 14 nM(Vitko et al, 2007).…”