Triple helix stabilization by covalently linked DNA–bisbenzimidazole conjugate synthesized by maleimide–thiol coupling chemistry

Abstract

Tethering of BBZPNH 2, an analogue of the Hoechst 33258, with a 14 nucleotide long DNA sequence with the help of succinimidyl-4-( N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), a heterobifunctional crosslinking reagent, using DMF/ water as solvent yields a conjugate which effectively stabilizes the triple helix. The above conjugate was hybridized with 26 bp long double stranded (ds) DNA having 14 bp long polypurine–polypyrimidine stretch to form a pyrimidine motif triple helix. The above conjugate increases the thermal stability of both the transitions, that is, triple helix to double helix by 12 °C and double helix to single strand transition by 16 °C for the triple helix formed with conjugated TFO over the triple helix made from non-conjugated TFO. Fluorescence and circular dichroism spectra recorded at different temperatures confirm the presence of minor groove binding bisbenzimidazole in the AT-rich minor groove of dsDNA even after the major groove bound TFO separates out.