Nanopore single-molecule analysis of DNA-doxorubicin interactions

Anticancer activity and toxicity of doxorubicin (Dox) are associated with its DNA intercalation. To understand the role in gene regulation and the drug mechanism, it is a challenge to detect the DNA-Dox interaction at the single-molecule level without the use of laborious, time-consuming labeling assays and an error-prone amplification method. Here, we utilized the simplest and cheapest, yet highly sensitive, single-molecule nanopore technology to investigate the DNA-Dox interaction and explore in situ the intercalative reaction kinetics. Distinctive electronic signal patterns between DNA and the DNA-Dox complex allow protein nanopore to readily detect the changes in structure and function of DNA. After Dox insertion, nanopore unzipping time of DNA was elevated 10-fold while the blocking current decreased, demonstrating the higher affinity of the DNA-Dox complex (formation constant K(f) = 3.09 × 10(5) M(-1)). Continuous rapid nanopore detection in real time displayed that Dox intercalation in DNA is a two-state dynamic process: fast binding and slow conformational adaption. The nanopore platform provides a powerful tool for studying small molecule-biomacromolecule interactions and paves the way for novel applications aimed at drug screening and functional analysis.