Heterocyclic diamidines, effective agents against trypanosomes, have been previously shown to bind strongly to the minor groove of DNA with selectivity for AT regions. Using restriction enzyme activity assays employing nine restriction enzymes, that cleave the DNA once or twice, to probe the binding of three diamidines, DB75, DB293 and DB818 [provided by D.W Boykin and W.D. Wilson, Georgia State University] to a mixture of supercoiled and relaxed circular phiX174 RF DNA at drug/DNA base pair ratios from 0.18 – 3.5. The assays suggest differences in the selectivity of binding. For example, at drug/DNA bp less than 0.7 with Nru I [TCGCGA], DB75 produced inhibition of cleavage of supercoiled and relaxed DNA, DB293 produced enhancement of cleavage of both and DB818 had no effect. With Dra I [TTTAAA], at low drug/DNA ratios [less than 0.7], all three agents produced enhanced cleavage. These compounds were frequently observed to produce enhanced cleavage at low ratios, suggesting that their binding produces a conformational change in the DNA structure. When enhanced cleavage was observed at low drug/DNA bp, inhibition was observed at drug/DNA bp above 2.0. Binding of one agent affects the binding of another. For example, DB75 alone produces inhibition of cleavage by Stu I [AGGCCT] and DB293 enhances cleavage. When DB75 and DB 293 are combined, enhanced cleavage is observed – suggesting that DB293 dominates the interaction with DNA. With Bssh II [GCGCGC], separately DB75 and DB 293 inhibit cleavage. Combined, they produce enhanced cleavage – suggesting that their interactions “cancel out” and that the effects are sequence dependent. These results provide further indication that altering drug structures affects sequence specificity and the nature of the interactions with DNA.

Selectivity, cooperativity, and competition in the binding of heterocyclic diamidines to DNA

249th National Meeting (2015)

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