Ionic and structural specificity effects of natural and synthetic polyamines on the aggregation and resolubilization of single-, double-, and triple-stranded DNA

DNA condensation, precipitation, and aggregation are related phenomena invo lving DNA-DNA interactions in the presence of multivalent cations, and stud ied for their potential implications in DNA packaging in the cell. Recent s tudies have shown that the condensation/aggregation is a prerequisite for t he cellular uptake of DNA for gene therapy applications. To elucidate the i onic and structural factors involved in DNA aggregation, we studied the pre cipitation and resolubilization of high molecular weight and sonicated calf thymus DNA, two therapeutic oligonucleotides, and poly(dA).2Poly(dT) tripl er DNA in the presence of the tetravalent polyamine spermine using a centri fugation assay, T-m measurements, and CD spectroscopy. The ability of sperm ine to provoke DNA precipitation was in the following order: tripler DNA, d uplex DNA > single-stranded DNA. In contrast, their resolubilization at hig h polyamine concentrations followed a reverse order. The effective concentr ation of spermine to precipitate DNA increased with Na+ in the medium. T-m data indicated the DNA stabilizing effect of spermine even in the resolubil ized state. CD spectroscopy revealed a series of sequential conformational alterations of duplex and tripler DNA, with the duplex form regaining the B -DNA conformation at high concentrations (similar to 200 mM) of spermine. T he tripler DNA, however, remained in a Psi-DNA conformation in the resolubi lized state. Chemical structural specificity effects were exerted by spermi dine and spermine analogues in precipitating and resolubilizing sonicated c alf thymus DNA, with N-4-methyl substitution of spermidine and a heptamethy lene separation of the imino groups of spermine having the maximal differen ce in the precipitating ability of the analogues compared to spermidine and spermine, respectively. Therapeutically important bis(ethyl) substitution reduced the precipitating ability of the analogues compared to spermine. Th e effect of the cationicity of polyamines was evident with the pentamines b eing much more efficacious than the tetramines and triamines, These results provide new insights into the mechanism of DNA precipitation by polyamines , and suggest the importance of polyamine structure in developing gene deli very vehicles for therapeutic applications.