Systematic Mutation in the Third Strand of a Purine Motif DNA Triple Helix: a Story of a Molecule Which Hides Its Tail

Abstract

A DNA triple helix formed according to the Purine-motif can accommodate both purines and pyrimidines in the third strand in a pH independent manner. This motif is thus a more versatile means of targeting double stranded DNA than the pH dependent Pyrimidine motif. In this paper we assess the impact of systematically replacing thymine with adenine, inosine or cytosine in the third strand. To this aim we have designed a double length, 22—mer “purine” strand to target a 9-mer pyrimidine strand such that the extending tail acts as the third strand (reversed-Hoogsteen strand) which is antiparallel to the purine strand of the underlying WC duplex. By systematically replacing thymines with adenines in the reversed-Hoogsteen strand there is an increase in the stability (T _m) of the triplex, particularly when the sequence closest to the loop consists of a stack of purines. Further substitution towards the 3′ end of the third strand reverses the stability. Systematic mutations in the third strand next to the loop reveal that the stability of the triads can be ranked according to their effect on T_m in the following order. A-AT > T-AT = I-AT. > C-AT where C is considered a mismatch.