Studies on formation and stability of the d[G(AG)5]* d[G(AG)5]. d[C(TC)5] and d[G(TG)5]* d[G(AG)5]. d[C(TC)5] triple helices.

Abstract

We have targeted the d[G(AG)5]. d[C(TC)5] duplex for triplex formation at neutral pH with either d[G(AG)5] or d[G(TG)5]. Using a combination of gel electrophoresis, uv and CD spectra, mixing and melting curves, along with DNase I digestion studies, we have investigated the stability of the 2:1 pur*pur.pyr triplex, d[G(AG)5]*d[G(AG)5].d[C(TC)5], in the presence of MgCl2. This triplex melts in a monophasic fashion at the same temperature as the underlying duplex. Although the uv spectrum changes little upon binding of the second purine strand, the CD spectrum shows significant changes in the wavelength range 200-230 nm and about a 7 nm shift in the positive band near 270 nm. In contrast, the 1:1:1 pur/pyr*pur.pyr triplex, d[G(TG)5]*d[G(AG)5].d[C(TC)5], is considerably less stable thermally, melting at a much lower temperature than the underlying duplex, and possesses a CD spectrum that is entirely negative from 200 to 300 nm. Ethidium bromide undergoes a strong fluorescence enhancement upon binding to each of these triplexes, and significantly stabilizes the pur/pyr*pur.pyr triplex. The uv melting and differential scanning calorimetry analysis of the alternating sequence duplex and pur*pur.pyr triplex shows that they are lower in thermodynamic stability than the corresponding 10-mer d(G3A4G3). d(C3T4C3) duplex and its pur*pur.pyr triplex under identical solution conditions.