DNA Aggregation Induced by Polyamines and Cobalthexamine

Abstract

We have studied the precipitation of short DNA molecules by the polycations spermidine, spermine, and cobalthexamine. The addition of these cations to a DNA solution leads first to the precipitation of the DNA; further addition resolubilizes the DNA pellet. The multivalent salt concentration required for resolubilization is essentially independent of the DNA concentration (between 1 g/ml and 1 mg/ml) and of the monovalent cation concentration present in the DNA solution (up to 100 mM). The DNA aggregates are anisotropic; those obtained in the presence of the polyamines spermidine and spermine generally contain a cholesteric liquid crystalline phase that flows spontaneously. In contrast this phase is never seen in the presence of cobalthexamine. We propose that the ability of polyamines to condense DNA in fluid structures is an essential feature of their biological functions.Multivalent cations with a charge of 3ϩ or greater induce the condensation of DNA in aqueous solution (reviewed in Ref. 1). In extremely dilute DNA solutions, one can observe the monomolecular collapse of long chains; with more concentrated DNA solutions (of short or long chains), aggregation sets in. Electrostatic forces appear to be predominant in DNA condensation. For highly charged polyelectrolytes there is a strong electrostatic repulsion between the chains. One expects the addition of multivalent cations to decrease this repulsion. DNA condensation by multivalent cations has been analyzed within the framework of the counterion condensation theory developed by Manning (2). This theory predicts the fraction of the DNA charges neutralized by a given cation: a saturating trivalent cation for instance should neutralize 92% of the DNA charges. It has been shown experimentally that approximately 90% of the DNA charges must be neutralized before DNA condensation can occur (3, 4). In addition, mono-and divalent cations compete with multivalent cations in the condensation process, in agreement with the proposal that the interactions are predominantly electrostatic. It is known, however, that a purely electrostatic model is insufficient to account for the experimental data; cobalthexamine is for instance five times more efficient as a condensing agent than spermidine, although these compounds have the same charge (3ϩ) (4).DNA condensation has been studied with the naturally occurring polyamines spermidine (3ϩ) and spermine (4ϩ), as well as with the inorganic cation cobalthexamine (3ϩ). Experimental data indicate that condensation is usually coupled with an isotropic to an anisotropic transition. In particular, high molecular weight DNA aggregates formed by spermidine, spermine, or cobalthexamine give a strong equatorial reflection when analyzed by x-ray diffraction (5, 6). Based on these data, several types of crystalline and liquid crystalline structures have been suggested for these DNA aggregates (5, 7). We have recently undertaken a study of these DNA aggregates using short (about 150 base pairs long) DNA molecules (8). In the case of the triv...

Spermine Condenses DNA, but Not RNA Duplexes

“…This agrees with previous work showing that a significant amount of spermine is required to precipitate DNA (13). Prior experiments using CoHex suggest that an even higher concentration is required to completely draw the DNA out of solution (19). It is interesting that the poly(rA):poly(rU) RNA condenses out of solution in the presence of small amounts of spermine, consistent with previous literature reports (15) and in contrast to our findings on the mixed-sequence duplex.…”

“…Previous studies of spermine-induced RNA condensation had a limited scope but showed that spermine can condense RNAs with nonphysiological sequences or under nonphysiological experimental conditions, e.g., long duplex poly(rA):poly(rU) (15), tRNA at nonphysiological pHs (16), and viral RNA in the complete absence of other salts (17). More extensive studies of DNA condensation by spermine showed that even small numbers of spermines condense both long and short, natural and synthetic DNA duplexes (18)(19)(20). Since spermine is such an effective condensing agent, it seems plausible that it could also condense RNA in cells.…”

Factors affecting flow karyotype resolution

“…This often results in the under-representation of the larger chromosomes. It has been reported that with DNA in solution, the concentration of NaCl affects the solubilization state of DNA in the presence of polyamines (31). We found an increase in chromosome yield and a decrease in debris when NaCl is excluded from the PAB buffer ( Fig.…”

Phase Behavior of Columnar DNA Assemblies

“…According to the Manning condensation theory, a fraction of the cations condenses into the Bjerrum layer near the molecular surface [1]. If some of the ions specifically adsorb onto DNA, its surface could be almost fully neutralized [2] or even overcharged [3]. Far from its axis, DNA can be apprehended as a charged cylinder.…”