Netropsin binds to the minor groove of AT-rich sequences of double stranded DNA.[3] In contrast, netropsin does not bind single stranded DNA or double stranded RNA. Crystallographic structures of DNA-bound Netropsin have been obtained and elucidate details of how the drug binds in the minor groove.[4][5] In the bound structure, the drug makes hydrogen bonding interactions with four subsequent base pairs of the DNA duplex, locally displacing the water molecules of the spine of hydration.
Using gel mobility and analytical ultracentrifugation, it was shown that Netropsin binding to DNA increases the twist per base by similar to 9˚ per molecule bound.[6][7] Thus, it removes supercoils when interacting with positively supercoiled DNA and introduces (additional) negative supercoils when binding to relaxed or negatively supercoiled DNA. Netropsin's effect on supercoiled DNA was observed in detail on single molecules using a magnetic tweezers.[8]