COOPERATIVE DISORDERING OF SINGLE-STRANDED POLYNUCLEOTIDES THROUGH COPPER CROSSLINKING

The thermal transitions of single-stranded polynucleotides are noncooperative. In contrast, Cu2+ cooperatively disorders the single-stranded helical structures of poly(A) and poly(C), as demonstrated by ORD [optical rotatory dispersion] and UV spectral changes as a function of the Cu2+ activity and by a dramatic chain-length dependence of the spectral changes. Equilibrium dialysis binding studies indicate that the cooperative disordering is paralleled by a somewhat less cooperative binding process. The difference between the thermal and Cu2+-induced transition is explained by the following mechanism. Cu2+ initially binds in a noncooperative fashion to phosphate. The Cu2+ so bound forms a 2nd bond to a nonadjacent base site on the same polymer strand or another strand. These intramolecular and intermolecular crosslinks to the bases are responsible for the disordering. The initial crosslinks formed provide nuclei for the cooperative formation of additional crosslinks, producing cooperative spectral changes paralleled by cooperative binding. A comparison of the spectral and binding transitions indicates that there is appreciable noncooperative binding of Cu2+ to phosphate, which produces no spectral changes in the presence of added electrolyte. This comparison also indicates that each Cu2+ crosslink disorders several bases. The formation of intermolecular crosslinks is demonstrated by a polymer concentration dependence of the disordering. The formation of intramolecular crosslinks can be deduced from the fact that the cooperative unit required to explain the differences between the hexamer, which does not readily form intramolecular crosslinks, and the polymer is considerably larger than the cooperative unit determined from the polymer results. The poly(A) disordering transition is less symmetrical than that of poly(C), particularly at low polymer concentrations. These results, together with other phenomena, are explained by a greater flexibility of poly(A), which favors the formation of small intramolecular loops.