Rigid double-stranded DNA Linkers for Single Molecule Enzyme-Drug Interaction Measurements Using Molecular Recognition Force Spectroscopy.

Single-molecule studies can reveal the distribution of states and interactions between ligand-enzyme complexes not accessible for most studies that measure a large ensemble average response of many molecules. Furthermore, in some biological applications, the information regarding the outliers, not the average of measured properties, can be more important. The high spatial and force resolution provided by atomic force microscopy (AFM) under physiological conditions has been utilized in this study to quantify the force-distance relations of enzyme-drug interactions. Different immobilization techniques of the protein to a surface and the drug to AFM tip were quantitatively compared to improve the accuracy and precision of the measurement. Protein that is directly bound to the surface, forming a monolayer, was compared to enzyme molecules bound to the surface with rigid double-stranded (ds) DNA spacers. These surfaces immobilization techniques were studied with the drug bound directly to the AFM tip and drug bound via flexible poly(ethylene glycol) and rigid dsDNA linkers. The activity of the enzyme was found to be not significantly altered by immobilization methods relative to its activity in solution. The findings indicate that the approach for studying drug-enzyme interaction based on rigid dsDNA linker on the surface and either flexible or rigid linker on the tip affords straightforward, highly specific, reproducible, and accurate force measurements with a potential for single-molecule level studies. The method could facilitate in-depth examination of a broad spectrum of biological targets and potential drugs.