Insights in the Organization of DNA-Surfactant Monolayers Using Cryo-Electron Tomography Matthijn R. J. Vos, ²,‡ Paul H. H. Bomans, ‡,§ Felix de Haas, ‡,¶ Peter M. Frederik, ‡,§ John A. Jansen, # Roeland J. M. Nolte, ²,£ and Nico A. J. M. Sommerdijk* ,²,‡ Laboratory for Macromolecular and Organic Chemistry, and Soft Matter Cryo-TEM Research Unit, EindhoVen UniVersity of Technology, EindhoVen UniVersity of Technology, P.O. Box 513, 5600 MB EindhoVen, The Netherlands, EM Unit, Department of Pathology, UniVersity of Maastricht, UniVersiteitssingel 50, 6229 ER Maastricht, The Netherlands, FEI Company, Achtseweg Noord 5, 5651 GG, EindhoVen, The Netherlands, Department of Periodontology and Biomaterials, Radboud UniVersity Nijmegen Medical Center, 6500 HB Nijmegen, The Netherlands, and IMM Supramolecular Chemistry, Radboud UniVersiteit Nijmegen, ToernooiVeld 1, 6525 ED, Nijmegen Received June 7, 2007; E-mail: n.sommerdijk@tue.nl The organization and complexation of DNA has been a subject of intensive research, not only to gain insight into biological processes 1 but also to understand its behavior in biomedical applications. 2,3 In particular, the behavior of DNA in the presence of cationic surfactants is of interest to understand phenomena involved in gene transfection, 2 DNA sensing, 3 and more recently the layer-by-layer synthesis of DNA-based biomaterial coatings. 4 In addition to the DNA-surfactant complexes studied in the bulk phase, 5 Langmuir monolayers are frequently used to obtain information about DNA-surfactant interactions. 6 The model for such a system generally involves nucleic acid molecules that are orderly bound via electrostatic interactions to a closely packed monolayer of cationic surfactants. Several techniques, such as Brewster angle microscopy (BAM), 7 in situ grazing incidence X-ray diffraction, and infrared reflection absorption spectroscopy (IR- RAS), have been applied to obtain information about the organiza- tion of these monolayer systems; however, from these studies, no generalizing conclusions could be drawn. 8 Whereas Mo ¨wald et al. concluded that DNA becomes aligned due to the compression of the complex, Okahata showed that the presence of an intercalating dye was required for the ordering of DNA under a surfactant monolayer. 9 Significantly, Yamaoka et al. demonstrated that the complex present at the air-water interface changes its structure upon deposition to a solid substrate. Here we present the first direct 3D in situ imaging of DNA molecules bound to a monolayer of a bisurea-stabilized surfactant (1) using cryo-electron tomography. It is demonstrated that, for the present system, individual DNA strands do not organize in an orderly fashion at the monolayer surface, but bind only partially with a part of the chain extending down into the subphase. Surface pressure versus surface area (Π-A) isotherms were recorded for the bisureido-based surfactant 1 10 spread on a PBS (pH 7.4) subphase (Figure 1). The isotherm was dominated by a liquid condensed state as was evidenced by a steep increase in surface pressure upon compressing the layer below 25 Å 2 /molecule. By extrapolation of the slope of the curve to zero pressure, a limiting molecular area of 22 Å 2 /molecule was deduced (Figure 1). These results are indicative for the preorganization of the surfactant dictated by the formation of strong intermolecular hydrogen bonds already at low degrees of compression (see Supporting Information). The Π-A isotherm of a cationic surfactant monolayer is known to change significantly when it is compressed on a DNA-containing subphase. 11 When 1 was spread and compressed on a buffered DNA-containing subphase ([DNA] ) 3 mg/mL), an increase in the surface pressure was indeed observed, indicating a liquid expanded phase from ∼100 to 30 Å 2 /molecule (Figure 1). Although the DNA alone also displayed some surface activity, summation of the curves recorded with pure 1 and pure DNA, respectively, demonstrated that this fact cannot account for the observed changes in the isotherms (see Supporting Information). This suggests that the presence of DNA in the subphase leads to the formation of complexes that no longer allow for a close packing of the surfactant molecules despite the strong H-bonding capacity of the bisurea units. In contrast, when DNA was injected under a preformed monolayer of 1 kept at a constant pressure of 35 mN/ m, no expansion of the monolayer was observed. This suggests that, once the monolayer is formed, the strong hydrogen bonds of the bisurea units prevent penetration of the DNA in between the surfactant molecules. Cryo-TEM images of a vitrified sample of a 3 mg/mL DNA solution ([DNA] in the subphase) without an applied monolayer clearly showed individual DNA chains (Figure 2A). ² Laboratory for Macromolecular and Organic Chemistry, Eindhoven University. ‡ Soft Matter Cryo-TEM Research Unit, Eindhoven University. § University of Maastricht. ¶ FEI Company. # Radboud University Nijmegen Medical Center. £ Radboud Universiteit Nijmegen. Figure 1. Top: The molecular structure of surfactant 1. Bottom: surface pressure versus surface area isotherms (Π-A) of bisurea surfactant spread and compressed on a PBS buffer (-) and on 3 mg/mL DNA/PBS subphase (- -). The arrows indicate the extrapolated mean molecular area (MMA) at zero pressure. Published on Web 09/11/2007 11894 9 J. AM. CHEM. SOC. 2007, 129, 11894-11895 10.1021/ja0736515 CCC: $37.00 © 2007 American Chemical Society