A NOVEL ASSAY FOR DRUG-DNA BINDING MODE, AFFINITY, AND EXCLUSION NUMBER - SCANNING FORCE MICROSCOPY

Determining the mode-of-binding of a DNA ligand is not always straight forward. Here, we establish a scanning force microscopic assay for mod e-of binding that is (i) direct: lengths of individual DNA-ligand comp lexes are directly measured; (ii) rapid: there are no requirements for staining or elaborate sample preparation; and (iii) unambiguous: an o bserved increase in DNA length upon addition of a ligand is definitive evidence for an intercalative mode-of-binding, Mode-of-binding, bindi ng affinity, and site-exclusion number are readily determined from sca nning force microscopy measurements of the changes in length of indivi dual drug-DNA complexes as a function of drug concentration. With this assay, we resolve the ambiguity surrounding the mode of binding of 2, 5-bis(4-amidinophenyl)furan (APF) to DNA and show that it binds to DNA by nonintercalative modes. APF is a member of an important class of a romatic dicationic drugs that show significant activity in the treatme nt of Pneumocystis carinii pneumonia, an opportunistic infection that is the leading cause of death in AIDS patients.