Enhanced stabilization of the triplexes d(C(+)-T)(6):d(A-G)(6);d(C-T)(6), d(T)(21):d(A)(21);d(T)(21) and poly r(U:AU) by water structure-making solutes.

A variety of organic cations, cationic lipids, low molecular weight alcohols, sodium dodecylsulfate, trehalose, glycerol, low molecular weight polyethylene glycols, and DMSO were tested for their ability to modulate the stability of the triplexes d(C(+)-T)(6):d(A-G)(6);d(C-T)(6), d(T)(21):d(A)(21);d(T)(21), poly r(U:A U) and their respective core duplexes, d(A-G)(6);d(C-T)(6), d(A)(21);d(T)(21), poly r(A-U). Very substantial enhancement of triplex stability over that in a physiological salt buffer at pH 7 is obtained with different combinations of triplex and high concentrations of these additives, e.g. trimethylammonium chloride and d(C(+)-T)(6):d(A-G)(6);d(C-T)(6); 2-propanol and d(T)(21):d(A)(21);d(T)(21); ethanol and poly r(U:A;U). Triplex formation is even observed with a 1:1 strand mixture of d(A-G)(6) and d(C-T)(6) in the presence of dimethylammonium, tetramethylammonium, and tetraethylammonium-chloride, as well as methanol, ethanol, and 2-propanol. Triplex stability follows the water structure-making ability (and in some cases the duplex unwinding ability) of the organic cations, the low molecular weight alcohols and other neutral organic compounds, whereas water structure-breaking additives decrease triplex stability. These findings are consistent with those reported in the accompanying paper that triplex formation occurs with a net uptake of water. Since the findings suggest that third strand-binding is facilitated by unwinding of the target duplex, it is inferred that triplex formation may be enhanced by nucleic acid binding proteins operating similarly.