Alternate-strand DNA triple-helix formation using short acridine-linked oligonucleotides.

Abstract

We have used DNAse I footprinting to examine the formation of intermolecular DNA triple helices at sequences containing adjacent blocks of purines and pyrimidines. The target sites G6T6.A6C6 and T6G6.C6A6 were cloned into longer DNA fragments and used as substrates for DNAse I footprinting, which examined the binding of the acridine (Acr)-linked oligonucleotides Acr-T5G5 and Acr-G5T5 respectively. These third strands were designed to incorporate both G.GC triplets, with antiparallel Gn strands held together by reverse Hoogsteen base pairs, and T.AT triplets, with the two T-containing strands arranged antiparallel to each other. We find that Acr-T5G5 binds to the target sequence G6T6.-A6C6, in the presence of magnesium at pH 7.0, generating clear DNAse I footprints. In this structure the central guanine is not recognized by the third strand and is accessible to modification by dimethyl sulphate. Under these conditions no footprint was observed with Acr-G5T5 and T6G6.C6A6, though this triplex was evident in the presence of manganese chloride. Manganese also facilitated the binding of Acr-T5G5 to a second site in the fragment containing the sequence T6G6.C6A6. This represents interaction with the sequence G4ATCT6, located at the boundary between the synthetic insert and the remainder of the fragment, and suggests that this bivalent metal ion may stabilize triplexes that contain one or two mismatches. Manganese did not affect the interaction of either oligonucleotide with G6T6.A6C6.