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

Washbrook, E. and Fox, K. R. (1994) Alternate-strand DNA triple-helix formation using short acridine-linked oligonucleotides. Biochemical Journal, 301 (2), 569-575. (doi:10.1042/bj3010569).

Abstract

We have used DNAase 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 DNAase 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 G(n) 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 DNAase 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-T₅G₅to 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.

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Contributors

Author: K. R. Fox

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