Analysis of Compacted Semiflexible Polyanions Visualized by Atomic Force Microscopy: Influence of Chain Stiffness on the Morphologies of Polyelectrolyte Complexes ² Gjertrud Maurstad, Signe Danielsen, and Bjørn T. Stokke* Biophysics and Medical Technology, Department of Physics, The Norwegian UniVersity of Science and Technology, NTNU, NO-7491 Trondheim, Norway ReceiVed: October 11, 2002; In Final Form: December 30, 2002 The morphologies of the compacted semiflexible biological polyanions alginate, acetan, circular plasmid DNA, and xanthan were investigated using tapping mode atomic force microscopy followed by quantitative image analysis. A shape factor was calculated for each of the observed polyelectrolyte complexes and used as a basis for dividing the structures into ensembles of morphologically linear, toroidal, and globular structures for subsequent quantitative analysis. Compaction of polyanions with chitosan yielded a small fraction of the torus morphology when the persistence length, L p , of 25 nm (acetan) was reached. For both DNA, L p ) 50 nm, and xanthan, L p ) 120 nm, it was found that the toroids make up a substantial fraction of the complexed structures formed by the given chitosan and at room temperature. Rodlike complexes were additionally observed within DNA-chitosan complexes, whereas they do not appear as a significant fraction of chitosan-complexed high-molecular-weight xanthan. The average height of the condensates was observed to be ∼2 nm for the compacted xanthan toroids, while it was determined to be ∼5 nm for compacted DNA toroids. Reducing the degree of polymerization of xanthan yielded a decrease in the fraction of toroids. Compacted xanthan at room temperature displays a number of racquets and other morphologies similar to the reported intermediate, metastable states by simulations. The reduced abundance of such structures following annealing supports the interpretation of their metastable nature. Introduction Linear polymers can undergo a transition from extended to a compact state driven by intersegment attraction and repulsion. In general, the detailed geometry of the compacted (bio)polymer structure varies from the sequentially organized polypeptide chains folding into structurally well-defined functional proteins, via clearly recognizable toroidal and linear compacted DNA, to compacted flexible polymers where the structure is not as clearly defined. The intersegment attraction in the self-assembly- driven compaction of polyelectrolytes, in particular studied for DNA, is mediated by multi- or polyvalent cations, 1-7 or cations used in combination with crowding or dehydrating com- pounds. 8,9 The formation of the polyanion-polycation (polyelectrolyte) complexes is mainly driven by an electrostatic mechanism, where the exchange reaction of counterions with different valences is important. 10 Charge neutralization and possible local overcompensation or bridging mediated by a multivalent coun- terion induces attraction between topologically separated seg- ments of the polyelectrolytes. 11 The valence of the counterion is a crucial factor for the efficiency of the complexation, concomitant compaction, and eventual stability of the compacted structure. The chain stiffness is identified as one important factor influencing the occurrence and abundance of the toroidal morphology of compacted semiflexible polymers 12,13 by op- posing the formation of sharp bends. The sequence-unspecific compaction of high-molecular- weight DNA observed using trivalent, e.g., cobalt hexamine, 2 or polyvalent, e.g., poly-L-lysine (PLL), 14 cations is reported to yield mainly toroidal and linear morphologies. The differences in stability of the toroidal state relative to the linear condensate and globular state is suggested to be due to the flexibility of the polymer at the same intersegment attraction energy. This has been investigated using theoretical methods as well as numerical simulations. 12,13,15-17 In contrast to the relatively large parameter space of the intersegment attraction energy and chain stiffness explored in the theoretical and numerical work, there is less data available from the experimental side. There is a recent report of actin filaments forming toroidal condensates, 18 but apart from this most experimental work has focused on DNA compaction. DNA condensation has additionally been a strong motivation for many of the theoretical studies of polymer compaction. Here, we report on the compacted structures of different semiflexible polyanions chosen to span a range of persistence lengths, from 10 to 120 nm. The polyanions (alginate, acetan, plasmid DNA, and xanthan) are compacted using a polycation (chitosan or PLL). The structures are observed by tapping mode atomic force microscopy (AFM) and analyzed using the asymmetric proper- ties of the individual species similar to that used in the numerical simulation of self-assembled polymers. 12,16 This approach is the basis for collecting complexes into different characteristic ensembles. Although selection of various polysaccharides allows control of the persistence lengths, the intersegment attraction energy associated with the complexation can vary concomitant with the chemical structure. The experimental conditions are selected in a range of polyanion-polycation concentrations, ionic strengths I, and pH values where complex formation is dominating the behavior. From simulations it is also suggested that compaction into toroidal condensates occurs through ² Part of the special issue “International Symposium on Polyelectrolytes”. * To whom correspondence should be addressed. Fax: +47 73 59 77 10. E-mail: bjorn.stokke@phys.ntnu.no. 8172 J. Phys. Chem. B 2003, 107, 8172-8180 10.1021/jp0271965 CCC: $25.00 © 2003 American Chemical Society Published on Web 06/25/2003