Analytical and Computational Methods for the Assessment of Biological Molecules and their Binding Interactions: Case Studies in DNA Aptamer-Target Binding and P450-P450 Dimerization

A DNA aptamer against atrazine, a small molecule target, was computationally predicted using mfold and RNA fold to generate secondary structure from sequence, and NAB and RNA Composer to subsequently generate tertiary structure, subsequently relaxed, and the most-representative structures docked (AutoDock) with atrazine and simazine. According to analysis with X3DNA, the best predicted structure had the majority of its base-pairing sugars in the 2\u27-endo conformation, contained multiple helical regions in the B-form. Constrained docking revealed no preference for the target molecule (atrazine) over a negative target (simaxine), in contrast to in vitro binding results, thus more calculations are needed to generate the correct structure.;The DNA sequence was synthesized, purified using ion exchange chromatography, and molecular weight confirmed by electrospray ionization mass spectrometry. Fluorescent equilibrium binding assays, circular dichroism and nuclear magnetic resonance spectroscopic analysis revealed strong binding to atrazine, B-form global conformation, 2\u27-endo sugar puckering, non-cooperative helical regions, and a thymine residue in a significantly different chemical environment than the other thymine in the aptamer. These observations were consistent with the predicted model. Multiple structural conformations were present when the aptamer was not bound to atrazine.;P450-P450 binding interactions were probed with the GRAMM-X protein-protein docking web server. Binding poses for CYP2C9 with enzyme partners CYP2C9, CYP2D6, CYP3A4, and CPR preferentially involved the CYP2C9 surface where the solvent entrance tunnel is located, which were identified using STAMP structural alignment. Binding affinity data derived from surface plasmon resonance experiments as well as MMGBSA calculations performed on P450-P450 computationally relaxed systems showed that CYP2C9 protein-protein interactions from strongest to weakest were with CYP2D6 (1.1 +/- 0.5 nM), CYP2C9 (2.6 +/- 1.0 nM) and CYP3A4 (18.1 +/- 3.0 nM). Results were consistent with observed changes in CYP2C9 metabolism of flurbiprofen