Poly(L–lysine)–DNA interactions in NaCl solutions: B → C and B → ψ Transitions

Abstract

SynopsisThermal denaturation and circular dichroism (CD) properties of poly(L-1ysine)-DNA complexes vary greatly when these complexes are prepared differently, that is, whether by NaC1-gradient dialysis starting from 2.0 M NaCl or by direct mixing a t low salt. These differing properties were investigated in more detail by examining complexes, made by direct mixing in the presence of various concentrations of NaCl, both before and after the NaCl was dialyzed out of the complex solution. The precipitation curves of DNA due to polylysine binding indicate that such binding is noncooperative a t zero salt; from 0.1 up to 1.0 M NaCl they exhibit varying degrees of cooperativity. Starting from zero salt, as the NaCl concentration used for complex formation is increased, both the CD and the melting properties of the complexes are shifted from those of directly mixed a t zero salt to those of reconstitution: in the CD spectra there is a gradual shift from a B -C transition to a B -$transition; thermal denaturation results show a gradual increase in the melting temperatures of both free DNA (t,) and polylysine-bound DNA (Pm). The progressive shift from B -C to B -$suggests a close relationship between these two transitions. Large aggregates of the complexes do not warrant the appearance of $-type CD spectra: $-spectra have been obtained in the supernatants of polylysine-DNA complexes made and measured a t 1.0 M NaCl while slightly perturbed CD spectra in B -C transition have been observed in turbid solutions of fully covered complexes made at very low salt. If the complexes are made a t intermediate salts and dialyzed to a very low salt, although up to 60% of the DNA is still bound by polylysine, the CD spectra of the complexes are shifted back to the B-type CD characteristic of pure DNA.

Biological Properties of Poly-l-lysine-DNA Complexes Generated by Cooperative Binding of the Polycation

“…1A). These curves agree with the results of poly-L-lysine-induced DNA precipitation reported previously (28,37). Therefore, the concentration of NaCl at which selective binding occurred, i.e.…”

“…Cooperative binding of poly-L-lysine to DNA is a specific feature of poly-L-lysine⅐DNA complexes prepared at high concentrations of NaCl. Random, noncooperative binding occurs when poly-L-lysine⅐DNA complexes were prepared at a relatively low NaCl concentration (18,19,23,26,28,30). Cooperativity reflects the influence of one bound poly-L-lysine on the binding affinity of a second poly-L-lysine (38).…”

“…Thus, the simple electrostatic mixing of DNA and poly-L-lysine produces DNA particles with various structures. The two modes of binding of poly-L-lysine to DNA have been described: (a) poly-Llysine processively binds to specific DNA molecules in a selective manner (cooperative binding), or (b) poly-L-lysine binds to a population of DNA molecules randomly in a nonselective way (noncooperative binding) (18,19,23,26,28,30).In this paper, we evaluated the effect of NaCl on the binding mode of poly-L-lysine to DNA and the resulting structural motifs of the condensed DNA particles using DNA precipitation, DNase I resistance assays, and electron microscopy (EM). …”

“…Condensation of the DNA into small particles is a crucial step for successful gene transfer. The process of DNA condensation has been examined widely (17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28). For example, if electrostatic interactions between poly-L-lysine and the DNA phosphate backbone occur in a rapid fashion, mixing basic polypeptides with DNA at a stoichiometric charge ratio will result in uncontrolled precipitation of the DNA (18,20).…”

Differential scanning calorimetric studies of the thermal stability of plasmid DNA complexed with cationic lipids and polymers

“…Thermal perturbation combined with measurements of circular dichroism (CD), 1 UV absorption, 2,3 and differential scanning calorimetry (DSC) 4 have been commonly used to probe the structural and energetic changes of linear polynucleotides (which melt below 1008C) and to evaluate the effects of polycations and ionic strength upon nucleic acid stability. Recent improvements in the sensitivity and temperature range (up to 1258C) of modern pressurized differential scanning calorimeters, 5 however, allow the evaluation of the thermal stability of supercoiled (SC) DNA (which can unfold above 1008C) without the use of chaotropic agents to lower its denaturation temperature.…”