Locating cobalt-binding sites on dna using restriction endonucleases

Abstract

The interactions of simple metal complexes with DNA have been widely studied. The interaction specificities of these molecules range from simple outside binding, such as observed with [Co(NH 3 ) 6 ] 3+ , to covalent binding as observed with Pt(NH 3 ) 2 Cl 2 , to reactions involving DNA oxidative cleavage through Fenton or related chemistries. One important feature of such interactions, especially those leading to covalent binding, is the sequence specificity of the reaction. This chapter presents a facile and fast method that can be employed to assess the sequence specificity of a ligand's reaction with DNA. Although the reagent used here covalently binds to DNA, this method should also work for reversible interactions as well, as long as the ligand has a high affinity for the DNA. To assess the sequence specificity, if any, of the reaction of [Co(NH 3 ) 5 (OH 2 )] 3+ with DNA, a series of four different model DNA oligomers was synthesized and treated with the cobalt complex according to established protocols. Each oligomer is self-complementary and possesses a unique restriction endonuclease site containing one or two G bases. A thermodynamic approach was used to assess the sequence specificity of the interaction. To summarize the results of these studies, each oligomer was incubated with [Co(NH 3 ) 5 (OH 2 )] 3+ and then exhaustively dialyzed to remove nonspecifically bound cobalt complex.