Factors Controlling Phase Separation in Water−Salt Solutions of DNA and Polycations

Abstract

Factors affecting phase separation in water-salt solutions of polyelectrolyte complexes (PECs), formed by DNA and integral or pendant polycations with a quaternary amino group in every monomer unit, have been studied. When no salt was added, quantitative DNA precipitation occurred at a stoichiometric charge ratio, φ ) [+]/[-] ≈ 1. In DNA mixtures with poly(N,N′-dimethyldiallylammonium chloride) (PDMDAAC, a pendant polycation), insoluble PECs formed in the range 0.7 < φ < 2. This suggests the formation of soluble, negatively charged PECs at 0 < φ < 0.7 and soluble, positively charged PECs at φ > 2. For different aliphatic ionene bromides (integral polycations), the range of φ corresponding to insoluble PECs was significantly broader, mainly due to the poor ability of the ionenes to form soluble, positively charged PECs. The φ range was also relatively broad for poly(N-ethyl-4-vinylpyridinium bromide) (a pendant polycation) and became broader with decreasing degree of polymerization of the polycation. The formation of insoluble PECs was favored by the addition of salt (NaCl), and the effect was more pronounced when decreasing the relative content of the solubilizing component, i.e., the nucleic acid at φ < 1 and the polycation at φ > 1. At moderate ionic strength, 0.12 M < [NaCl] < 0.6 M, quantitative precipitation of DNA was attained by addition of PDMAAC in the whole region studied: 1 < φ < 4.5. The data obtained strongly suggest that phase separation in solutions of DNA-containing PECs follows general rules revealed by studying PECs formed by flexible vinyl polyanions. However, the high rigidity of the DNA double helix appears to be responsible for the key feature revealed in the phase diagrams, i.e., significant broadening of the region for insoluble PECs at the expense of the region in which soluble DNA-containing PECs are formed. This feature may severely limit the application of DNA-containing PECs in medicine and biology but could be beneficial in the development of simple and effective procedures for DNA separation in biotechnology.

Phase behavior and rheological properties of DNA–cationic polysaccharide mixtures

“…Izmrudov et al investigated dilute solutions of ''highly polymerized'' DNA from calf thymus in mixtures with highly charged aliphatic ionene bromides, poly(N,N 0 -dimethyldiallylammonium chloride) or poly(N-ethyl-4-vinylpyridinium bromide). Without addition of salt, they found insoluble complexes in the range 0.7 < r < 2 for the mixtures with poly(N,N 0 dimethyldiallylammonium chloride) and a broader range of phase separation in the mixtures of DNA with the other two polymers referred to above [35]. In mixtures of DNA with cationic surfactants, the immediate phase separation with addition of surfactant was also observed but a redissolution was never seen on further addition of surfactant [12].…”

Polyelectrolyte complexes as a tool for purification of plasmid DNABackground and development

“…The excess polycation chains added are incorporated into the PECs and act as solubilizing tails. It should be noted that in the case of DNA, no soluble PECs are formed at ϕ critical < ϕ < 1, since the maximum precipitation of DNA coincides with the amount of polycation added [29]. The addition of salt broadens the range of ϕ corresponding to the formation of insoluble PECs (Fig.…”

“…The double helix of DNA appears to be responsible for the revealed difference in the phase diagrams of PECs formed by synthetic polyanions and those formed by DNA [29]. The type of polycation (integral or pendant) and the type of counter ion, as well as the DP of the polycation and the DP of the nucleic acid were found to affect the precipitation efficiency [17,29]. In the presence of salt the precipitation was complete even at [+]/[−] > 1.…”

“…If this precipitation concept is to be incorporated in a plasmid purification process, it is extremely important to show that the polycation can be removed after solubilization of the pellet. It was earlier shown for highly polymerised DNA, that no soluble polycation-DNA complexes were formed above salt concentrations 0.7-0.8 M NaCl [29]. It was thus reasonable to perform precipitation of plasmid DNA (pBluescript) and re-dissolve the pellet in 2 M NaCl, i.e.…”

Precipitation by polycation as capture step in purification of plasmid DNA from a clarified lysate

“…Figure 4a and 4b shows the two plasmids (pBluescript 5.9 kbp and pJV4 6.1 kbp) behaved similarly to highly polymerized DNA and were completely precipitated in a broad B-region at 1M KAc. For a more mechanistic and fundamental discussion of the complex formation between polycations and nucleic acids the reader is directed to our previous publications (i.e., Izumrudov et al, 2003 andWahlund et al, 2003).…”