Triple helix-tetraplex equilibrium for G-rich oligonucleotide N3′→P5′ phosphoramidates: role of molecular concentration and counterions

The formation of a triple helix by a third N3′→P5′ phosphoramidate GT-rich strand 5′-d(T9G5)NP-3′ has been studied by FTIR, CD and UV absorbance spectroscopies. We show that in presence of sodium counterions, in conditions of molecular crowding induced by high DNA concentrations, the self-association of the phosphodiester analog into a tetrameric structure is favored and prevents the formation of the triple helix. The use of a phosphoramidate third strand in the same conditions allows to minimize self-association and to form the triple helix with a 5′-d(T9G5)NP-3′ third strand. Characteristic signatures of T*A·T and G*G·C base triplets have been obtained by FTIR spectroscopy. Formation of a triple helix with the unmodified GT third strand can be observed in dilute solution, in presence of lithium and divalent magnesium ions (1 M LiCl, 50 mM MgCl2). In that case a biphasic UV melting profile (TM at 40 and 60 °C) and a characteristic CD spectrum of a parallel stranded GT triple helix have been obtained.