Secondary binding sites for heavily modified triplex forming oligonucleotides

Abstract

In order to enhance DNA triple helix stability synthetic oligonucleotides have been developed that bear amino groups on the sugar or base. One of the most effective of these is bis-amino-U (B), which possesses 5-propargylamino and 2'-aminoethoxy modifications. Inclusion of this modified nucleotide not only greatly enhances triplex stability, but also increases the affinity for related sequences. We have used a restriction enzyme protection, selection and amplification assay (REPSA) to isolate sequences that are bound by the heavily modified 9-mer triplex-forming oligonucleotide B(6)CBT. The isolated sequences contain A(n) tracts (n = 6), suggesting that the 5'-end of this TFO was responsible for successful triplex formation. DNase I footprinting with these sequences confirmed triple helix formation at these secondary targets and demonstrated no interaction with similar oligonucleotides containing T or 5-propargylamino-dU.

Figure 1.

(A) Chemical structures of bis-amino-U and propargylamino-dU. (B) DNase I footprint showing the interaction of B-TFO with the tyrT(43-59) fragment. The experiment was performed in 50 mM sodium acetate pH 5.0; TFO concentrations (µM) are shown at the top of each gel lane. The DNA was labelled at the 3′-end; the gel therefore runs 5′–3′ from top to bottom. The lane labelled control shows digestion of the DNA in the absence of oligonucleotide; GA is a marker specific for purines. The exact target site is shown by the filled box and the accompanying enhancement is indicated by an asterisks. Secondary binding sites are shown by the unfilled boxes and the enhancements are indicated by bracketed asterisks. (C) Sequence of the oligopurine tract in tyrT(43–59) and its interaction with the modified TFOs. The exact target site is boxed. (D) Sequence of a part of the tyrT(43–59) fragment showing the position of the secondary binding sites, which are underlined and the enhancements, which are shown in bold. The exact target site is boxed.

Figure 2.

DNase I footprints showing the interaction of B-TFO with REPSA-selected sequences 1–7. The experiments were performed in 50 mM sodium acetate pH 5.0; TFO concentrations (µM) are shown at the top of each gel lane. The DNA was labelled at the 3′-end; the gel therefore runs 5′–3′ from top to bottom. The lanes labelled ‘control’ show digestion of the DNA in the absence of oligonucleotide; tracks labelled ‘GA’ are markers specific for purines. The locations of the footprints are indicated by the filled boxes and any accompanying enhancements are indicated by asterisks.

Figure 3.

DNase I footprints showing the interaction of B-TFO with REPSA-selected sequences 8–14. The experiments were performed in 50 mM sodium acetate pH 5.0; TFO concentrations (µM) are shown at the top of each gel lane. The DNA was labelled at the 3′-end; the gel therefore runs 5′–3′ from top to bottom. The lanes labelled ‘control’ show digestion of the DNA in the absence of oligonucleotide; tracks labelled ‘GA’ are markers specific for purines. The locations of the footprints are indicated by the filled boxes and any accompanying enhancements are indicated by asterisks.

Figure 4.

DNase I footprints showing the interaction of P-TFO with REPSA-selected sequences 1–14 in the presence of 10 µM naphthylquinoline triplex-binding ligand. The experiments were performed in 50 mM sodium acetate pH 5.0; P-TFO concentrations (µM) are shown at the top of each gel lane. The lanes labelled ‘control’ show digestion of the DNA in the absence of oligonucleotide; tracks labelled ‘GA’ are markers specific for purines. The locations of the footprints are indicated by the filled boxes and any accompanying enhancements are indicated by asterisks.