Distinguishing Dual DNA Binding Modes of Actinomycin D using Optical Tweezers

Abstract

Actinomycin D (ActD), the first antibiotic which exhibited anti-tumor activity, was initially believed to bind double stranded DNA (dsDNA) through intercalation. Later it was shown to bind single stranded DNA (ssDNA) with an order of magnitude higher affinity. ssDNA binding can be extremely important in inhibiting replication of viruses that replicate through ssDNA templates such as HIV. While these two binding modes can be separately quantified by studying binding to specific substrates, it is very difficult to determine the mode of binding to polymeric DNA. DNA stretching studies can precisely quantify intercalation by measuring the increase in DNA length upon intercalation. However, ssDNA binding also increases DNA length. Therefore, we have developed a method that combines the measured increase in DNA length with the overall DNA melting free energy change, allowing us to simultaneously determine ssDNA binding and intercalation as DNA is stretched. Using this method, we were able to distinguish between dual binding modes of ActD. We determined that the ssDNA binding of ActD (Kss ∼ 108 M−1) is much higher than its binding to dsDNA (Kds ∼ 106 M−1) for long polymeric DNA. We also determined the ssDNA and dsDNA binding site size, which are 3 bases and 6 base pairs, respectively.