Small-Molecule Binding to DNA under Tension and Twist Studied by Magnetic Tweezers

Abstract

DNA-binding small molecules are present in the cellular environment and are ubiquitously used in biochemistry and biotechnology. Here, we use single molecule magnetic tweezers experiments to study the effect of small-molecule ligands on DNA mechanical properties.Using the magnetic tweezers ability to control both the applied stretching force and torque, we have systematically characterized the mechanical properties of DNA in the presence of Ethidium Bromide (EtBr), a well-known intercalator, and Netropsin, an anti-microbial drug and known minor groove binder. In addition, we have characterized the interactions of Topotecan, a clinically used anti-tumor drug, with bare DNA.Our results show a lengthening of DNA upon EtBr intercalation to an extension of ∼1.5 times the initial contour length and a decrease in DNA twist by 29 +- 3 degrees per intercalation event, in agreement with previous estimates from bulk experiments. Further effects of intercalation are a stabilization of the double strand under high forces and negative torques and a decrease in both the bending and twisting persistence lengths. In contrast, minor groove binding by Netropsin does not change the contour or persistence length significantly, but increases the twist per base of the DNA.For Topotecan, both intercalative and minor-groove binding have been postulated. Our magnetic tweezers results indicate that drug binding at > 50 micromolar concentrations has consequences qualitatively similar to the changes observed for EtBr, in support of a intercalative binding mode for Topotecan.These results point to important consequences of intercalative ligand binding for DNA torsional behavior, which we characterized at the single-molecule level using magnetic tweezers. Ultimately, such insights can help elucidate the effects of small molecule drugs in the cellular environment and their interference with DNA transcription and replication.