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Reprinted with permission from ref. 56.

Reprinted with permission from ref. 56.

Pgl3 Plasmid

Fig. 17. Scanning force microscopy images showing the toroid structures of pGL3 plasmid DNA formed by incubation with 25 pMspermine (A), 5 pM3-3-3-3 (B), 2 pM3-4-3-4-3 (C), and the partly formed toroids, observed in the presence of 2 ^M3-4-3-4-3 (D). Scale bar is 200 nm. (Reprinted with permission from ref. 57.)

Fig. 17. Scanning force microscopy images showing the toroid structures of pGL3 plasmid DNA formed by incubation with 25 pMspermine (A), 5 pM3-3-3-3 (B), 2 pM3-4-3-4-3 (C), and the partly formed toroids, observed in the presence of 2 ^M3-4-3-4-3 (D). Scale bar is 200 nm. (Reprinted with permission from ref. 57.)

involves several molecules of DNA to produce bundles or amorphous precipitates. DNA condensation and aggregation are related phenomena and are governed by similar structural specificity effects. Schellman and Parthasarathy (59) demonstrated that the structural arrangement of DNA collapsed by spermidine homologs depended on the chemical structure of the polyamines. They used the same series of spermidine homologs, H2N(CH2)3NH(CH2)nNH2, that were used by Thomas and Bloomfield (44) for T measurements and Allison et al. (54) for condensation experiments. The Bragg spacing and the interhelical spacing for a hexagonal packing model for DNA varied systematically with the length of the methylene bridge of the homologs. Pelta et al. (60) showed that at high concentrations (~mM), aggregates of calf thymus DNA could assume a liquid crystalline state in the presence of spermidine and spermine. Hexagonal packaging of DNA was found in liquid crystalline textures formed by polyamine-DNA interactions. Structural specificity effects of polyamines were also evident in the stabilization of liquid crystalline textures of DNA (Fig. 18) (61).

Polyamine structural dependence on triplex DNA aggregation was studied by Musso et al. (47). They found that 6is(ethyl) substitution of polyamines decreased the ability of the parent compound to aggregate DNA. Triplex DNA was more labile to undergo aggregation than double helical DNA. For example, the ability of spermine to provoke DNA precipitation was in the following order: triplex DNA > duplex DNA > single-stranded DNA. The effective concentration of spermine to precipitate DNA increased with Na+ in the medium. An interesting observation related to the aggregation of DNA was the resolu-bilization of the aggregates at millimolar concentrations of polyamines (Fig. 19). T data indicated that polyamines stabilized DNA even in the resolubilization state. Chemical structural specificity of spermidine and spermine analogs were observed in the resolubi-lization of sonicated calf thymus DNA, with N4-methyl substitution of spermidine and a heptamethylene separation of the imino groups of spermine exerting the maximal difference in the precipitating DNA compared with spermidine and spermine, respectively.

Pelta et al. (60) studied the precipitation of short DNA molecules by spermidine, spermine, and cobalt hexamine, and found that the addition of these cations to a DNA solution led first to the precipitation of the DNA, and further addition led to resolubi-lization of the DNA pellet. The multivalent salt concentration required for resolubilization is essentially independent of the DNA concentration (between 1 |ig/mL and 1 mg/mL) and of the monovalent cation concentration (up to 100 mM) present in the DNA solution. Saminathan et al. (62) further showed that the aggregation and resolubilization of DNA by polyamines are essentially independent of the length of the DNA. CD spectroscopy revealed a series of sequential conformational alterations of duplex and triplex DNA, with the duplex form regaining the B-DNA conformation at high concentrations (approx 200 mM) of spermine (Fig. 20). The triplex DNA, however, remained in a ^-DNA conformation in the resolubilization state. The ^-DNA form is believed to be a tightly packaged, twisted liquid crystalline form of DNA. The CD spectrum of ^-DNA arises from the interaction of circularly polarized light with the highly ordered tertiary structure, which depends on adjacent superhelical turns. Although base sequence specificity of polyamine-induced DNA condensation and aggregation has not been well-characterized, CD studies and centrifugation studies of an oligonucleotide harboring

Fig. 18. Effects of higher homologs of spermine on the liquid crystalline phase transitions of calf thymus DNA. (A) DNA (25 mMin Na cacodylate buffer) was incubated with 1 mM 3-5-3 for 24 h at 37°C. A crystalline phase is observed (x100). (B) A myelin-like growth, which developed a striped appearance, is found after incubation of DNA with 1 mM 3-6-3 for 12 h at 37°C (x360). (C) A crystalline phase was observed on further incubating the DNA in (B) for 24 h at 37°C (x90). (D) Fingerprint textures are obtained on incubating DNA with 1 mM3-9-3 for 12 h at 37°C (x200). (Reprinted with permission from ref. 61.)

Fig. 18. Effects of higher homologs of spermine on the liquid crystalline phase transitions of calf thymus DNA. (A) DNA (25 mMin Na cacodylate buffer) was incubated with 1 mM 3-5-3 for 24 h at 37°C. A crystalline phase is observed (x100). (B) A myelin-like growth, which developed a striped appearance, is found after incubation of DNA with 1 mM 3-6-3 for 12 h at 37°C (x360). (C) A crystalline phase was observed on further incubating the DNA in (B) for 24 h at 37°C (x90). (D) Fingerprint textures are obtained on incubating DNA with 1 mM3-9-3 for 12 h at 37°C (x200). (Reprinted with permission from ref. 61.)

estrogen response element (ERE) and control sequences indicated that the sequence containing ERE is more prone to conformational transitions and aggregation (63,64). Thus, the first step in DNA condensation and aggregation appears to be a high-affinity, site-specific interaction between polyamines and DNA, followed by multisite interaction. Multiple sites of interaction can occur around a nucleation center in a single, long DNA molecule, resulting in toroid formation at low concentrations of DNA or a unique ordering of multiple molecules in the case of oligonucleotides.

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