Stabilization of a bimolecular triplex by 3′-S-phosphorothiolate modifications: An NMR and UV thermal melting investigation

Triplexes formed from oligonucleic acids are key to a number of biological processes. They have attracted attention as molecular biology tools and as a result of their relevance in novel therapeutic strategies. The recognition properties of single stranded nucleic acids are also relevant in third strand binding. Thus there has been considerable activity in generating such moieties, referred to as triplex forming oligonucleotides (TFOs). Triplexes composed of Watson-Crick (W-C) base-paired DNA duplexes and a Hoogsteen base-paired RNA strand are reported to be more thermodynamically stable than those in which the third strand is DNA. Consequently synthetic efforts have been focused on developing TFOs with RNA-like structural properties. Here we describe structural and stability studies of such a TFO; composed of deoxynucleic acids, but with 3’-S-phosphorothiolate (3’-SP) linkages at two sites. The modification results in an increase in triplex melting temperature as determined by UV absorption measurements. 1H NMR analysis and structure generation for the (hairpin) duplex component and the native and modified triplexes revealed that the double helix is not significantly altered by the major groove binding of either TFO. However the triplex involving the 3’-SP modifications is more compact. The 3’-SP modification was previously shown to stabilise G-quadruplex and i-motif structures and therefore is now proposed as a generic solution to stabilising multi-stranded DNA structures.