Effect of a Triplex-Binding Ligand on Parallel and Antiparallel DNA Triple Helixes Using Short Unmodified and Acridine-Linked Oligonucleotides

Abstract

We have used DNase I footprinting to investigate the effect of a triplex-binding ligand on the formation of intermolecular DNA triple helices at target sites that have been cloned into longer DNA fragments. In the presence of a triplex-binding ligand (N-[2-(dimethylamino)ethyl]-2-(2-naphthyl)quinolin-4-ylamine ), the concentrations of T5C5 and C5T5 required to generate DNase I footprints at the target sites A6G6.C6T6 and G6A6.T6C6, respectively, are reduced by at least 100-fold. Complexes with the acridine-linked oligonucleotides Acr-T5C5 and Acr-C5T5 are stabilized to a much lesser extent and produce footprints at concentrations similar to those of the unmodified oligonucleotides in the presence of the ligand. The stabilizing effects of acridine modification or the addition of a triplex-binding ligand are not additive. The position and length of the footprints produced by Acr-T5C5 and T5C5 at the target sequence A6G6.C6T6 are unaffected by the ligand. In contrast, footprints at the target site G6A6.T6C6 appear 3-4 bases shorter in the presence of the ligand, when viewed from the pyrimidine strand, and 1-2 bases longer on the purine strand. These results are explained by suggesting that the compound binds at T.AT triplets and prevents the transmission of any DNA structural changes into the flanking duplex. The compound has a smaller stabilizing effect on short antiparallel triplexes consisting of G.GC and T.AT triplets. Binding of Acr-G5T5 to A6G6.C6T6 is enhanced slightly by the compound, which increases the apparent footprinting site, probably by preventing fraying at the 3'-end of the third strand. The compound does not promote the binding of G5T5 to A6G6.C6T6 or that of Acr-T5G5 and T5G5 to G6A6.T6C6.