Specific Binding Constant and Stoichiometry Determination in Free Solution by Mass Spectrometry and Capillary Electrophoresis Frontal Analysis

Abstract

A free solution method was developed for evaluating the specific binding affinity and stoichiometry of small molecules with oligo DNA subsequent to cation-induced G-quadruplex formation. A nonlinear curve fitting equation capable of extracting specific binding constants in the presence of nonspecific binding without the need for reference compounds was proposed and tested. Electrospray ionization mass spectrometry was first used to rapidly screen the small molecule candidates; then, the stoichiometry and affinity constants of the native state binding pair in solution were obtained with capillary electrophoresis frontal analysis (CE-FA). The B cell lymphoma 2 (Bcl-2) oncogene is directly responsible for the expression of Bcl-2 protein, which plays a significant role in cell apoptosis. The binding of a G-quadruplex formed in the promoter region of the Bcl-2 oncogene with a small molecule could stabilize the quadruplex structure and potentially regulate the transcription of Bcl-2. Four natural product drug candidates were tested for their ability to bind the Bcl-2 promoter G-quadruplex. Using this reference-free method based on CE-FA data, jatrorrhizine and palmatine were found to bind specifically to the Bcl-2 promoter G-quadruplex with stoichiometries of 4:1 and 3:1, respectively.

Capillary Electrophoresis/Dynamic Frontal Analysis for the Enzyme Assay of 4-Nitrophenyl Phosphate with Alkaline Phosphatase

“…This format of CE is called as capillary electrophoresis/frontal analysis (CE/FA); the height of a plateau signal is used for the quantification of the unbound species, and CE/FA has been utilized for the equilibrium or affinity analysis. 1−4 Binding equilibria of drug-protein complexes, 1,5,6 peptide complexes, 2 liposome-analyte interactions, 3 and Alkaloid-DNA complexes 7 have been investigated by CE/FA. Inhibitors of protein-protein interaction were also investigated by CE/FA.…”

Pressure‐assisted capillary electrophoresis frontal analysis for faster binding constant determination

“…The practical applications of PACE‐FA in the analysis of interactions between oppositely charged species has been demonstrated in another work . The B cell lymphoma 2 (Bcl‐2) promoter DNA quadruplex and palmatine hydrochloride interaction was characterized.…”

“…With PACE‐FA, a more complete set of information are provided with the electropherogram. The ligand height can be used for the binding constant calculation, and other part of the electropherogram also provides the information to conduct further kinetics and thermodynamics studies …”

Quantitative characterization of human oncogene promoter G‐quadruplex DNA‐ligand interactions using a combination of mass spectrometry and capillary electrophoresis

“…100 nL) of the pre‐equilibrated host‐guest mixture, could result in a series of plateaus corresponding to the free guest and host–guest complex [33]. Using a reference‐free method based on CE‐FA, high‐order binding data from the systems involving various kinds of biomolecules (proteins, nucleic acids) could be obtained, including their specific binding with ligands [34].…”