BPS2026 - Force clamp experiments with optical tweezers confirm nanomolar affinity of doxorubicin to DNA

Doxorubicin (Doxo) is a prominent anti-cancer drug that has been used to treat various types of cancers, although it is commonly used as a last-resort option due to its severe side effects. Doxo intercalates between the base pairs of the DNA, which increases duplex DNA stability and limits its ability to replicate. Using dual beam optical tweezers, single DNA molecules were isolated and trapped, allowing us to study the interactions between DNA and Doxo. Previously reported slow stretching experiments, where the DNA was stretched in steps with a pause between each, allowing for the drug to reach binding equilibrium, provided us with nanomolar binding affinity measurements, which contrasted with the micromolar binding affinity reported in prior literature. To validate these results, constant force experiments were conducted where the DNA was stretched and held at a constant force while introducing Doxo until binding equilibrium had been reached. This allowed for the extension of the DNA as a function of time to subsequently be measured. The results of the constant force experiments suggested a zero-force binding affinity of 75 nM, which matched with the slow-stretching results within uncertainty, hence confirming Doxo’s nanomolar binding affinity to DNA. These experiments also provided the binding kinetics between Doxo and DNA. A better understanding of Doxo and its interactions with DNA will give valuable information regarding more impactful usage of the drug and could lead to a safer alternative in the future.