Self-organized Structures of Polynucleotides on the Stearic Acid Monolayers

Transcription

1 WDS'05 Proceedings of Contributed Papers, Part III, , ISBN MATFYZPRESS Self-organized Structures of Polynucleotides on the Stearic Acid Monolayers S. Staritsyn and E. Dubrovin Moscow State University, Faculty of Physics. Lenin Gory, Moscow, , Russia. Abstract. Formation of polynucleotide complexes with Langmuir monolayers of stearic acid (SA) on the surface of synthetic polyadenine (polya) aqueous solution with different ionic strength has been studied. AFM topographic images of polya complexes with Langmuir monolayers deposited on the mica substrates were obtained. The complex structures and individual DNA molecules on the amphiphile monolayer surface were observed. The surface consists of domains of stretched parallel polya molecules, and each domain mostly has its own direction of alignment. The data obtained give evidence for the effectiveness of monolayer techniques for investigation the mechanisms of DNA/RNA complexation with amphiphilic anions and demonstrate its perspectives for creation of supramolecular planar RNA/DNA-based self-organized nanostructures with nanoscale structural ordering. Introduction Four main classes of biomolecules, namely, proteins, nucleic acids, polysaccharides and lipids are known to form highly organized molecular and supramolecular structures within the cell. Understanding the nature of nanoscale structural order in these systems and elucidation of the physicochemical mechanisms of formation of distinct functional molecular nanostructures is one of the most important fundamental problems in modern biophysics directly connected with a wide number of practical tasks in medicine biotechnology. DNA molecules are promising candidates for nanobiotechnology for fabrication of functional nanostructures and nanodevices due to the unique DNA recognition capabilities, physicochemical stability, mechanical rigidity, and possibilities to repeated denaturation-hybridization cycles. Langmuir-Blodgett (LB) technique (the molecular films at the liquid-gas interface and mono & multilayers transferred onto a sold substrate) is one of methods of nanofabrication such as photolithography or self-assembled monolayers (chemisorption). Furthermore, Langmuir monolayers are an ideal two-dimensional system and can be regarded as model systems for biomembranes. Budker et al. [1978, 1980] have shown the importance role of bivalent ions in the interactions between polynucleotides and zwitterionic lipid (phosphatidylcholine) membranes. His experiments provide a strong evidence that there is no direct electrostatic interaction between zwitterionic lipids and DNA, but the interaction mediated by divalent cations by formation of salt (Mg 2+ ) bridges between the phosphate residues of the polynucleotides and the phosphate groups in the phospholipids. But the mechanisms leading to the formation of polynucleotides and zwitterionic lipids complexes are still under consideration [McManus et al, 2003]. The characterization of many types of DNA-cationic lipid complexes has received much attention in recent years, since their potential for use in gene delivery applications was identified [Felgner et al, 1987]. A number of works utilized various techniques for preparation and investigation of the DNA/RNA complexes with cationic lipids and polycations, including LB technique and atomic force microscopy (AFM) [Sukhorukov et al, 1996; Antipina et al, 2003]. But there is a lack of investigating of nucleic acids anionic lipid complexes, although Patil et al [2004] show that transfection efficiency of anionic liposomes was similar to that of cationic ones, whereas their toxicity was significantly lower. It is possible to prepare aggregates from like-charged surfactants and polymers by mediating the electrostatic attraction using multivalent counterions. For example, it was demonstrated [Bhaumik et al, 2004] the immobilization of long dsdna on fatty acids monolayer with the help of Zn coordination and biological activity of immobilized DNA. Here, we report a system we have developed where long polynucleotide molecules are immobilized on a monolayer of stearic acid in a stretched manner. PolyA molecules are parallel each other, and form domains in which molecules have its own direction of alignment. We have applied the Langmuir-Blodgett technique to transfer polya/sa monolayer complexes onto the atomically flat mica and used AFM for its investigation. 535

2 Experimental Section Polyadenylic and stearic acids, MgCl 2 and EDTA were purchased from Sigma. The sodium chloride was USP-grade (>99.99%, Akzo Nobel). All reagents were used without additional purification. The polynucleotides have molecular mass of Da ( Sigma data) which corresponds to nucleotides in single-strand chain. The polynucleotide concentration in the subphase was mg/ml (molar concentration ~ 3*10-6 M of monomer units). The monolayers studied were obtained in Langmuir trough with dimensions cm. The deionized water (Millipore Milli-Q system) with specific conductivity no less than 18 MΩm cm -1 was used in all experiments. The bulk solutions of the RNA (the concentration of the RNA is 0.01 mg/ml) in the Milli-Q water, of the sodium chloride (the concentration of the salt is 2M) and of the magnesium chloride (100 mm) were prepared separately and stored in the refrigerator not more then 3 days. For the preparation of the final solutions the bulk solutions and Milli-Q water were mixed in the appropriate proportions just before use. Stearic acid was dissolved in chloroform (spectroscopic grade) to obtain 1mM solutions. The absence of pressure-area isotherm for chloroform itself after its evaporation from the water-air interface indicated the purity of the solvent. After surfactant deposition onto the subphase, the monolayer was stored during 10 minutes before its compression (30 minutes if the subphase contained a polynucleotide). In the case of polynucleotide free subphase, the shape of pressure-area curves for the surfactants did not depend on the time period of the monolayer storage (10 or 30 minutes) before compression. All experiments were done at 20 o C. For the AFM studies the monolayers were transferred by vertical dipping method onto the mica surfaces. The dipping was started when monolayer was compressed, and the surface pressure (18 mn/m) was kept constant during the monolayer transfer procedure. The substrate dipping speed for the monolayer transfer was 3 mm/min. The transfer ratio for the downstroke deposition was not more than 0.1 and for upstroke deposition it was 0.9±0.1. Hence a monolayer film was obtained onto the substrate. All the AFM measurements were provided in the air using Nanoscope IIIa microscope (Digital Instruments, USA) with the tapping mode of scanning. The commercial silicon cantilevers had 42 N/m stiffness; their resonance frequency lays at the range of khz. The scan frequency was about 2 Hz. Image processing was performed using FemtoScan software [Filonov et al, 2001]. Figure 1. AFM image of polyadenine molecules bound with stearic acid monolayer. Thread-like structures with dimensions typical for nucleic acid chains (height nm, width nm) are seen on the surface. The monolayer was created on the water subphase contained 1 M of NaCl and polya mg/ml. 536

3 Figure 2. AFM image of polyadenine molecules bound with stearic acid monolayer. Thread-like structures with dimensions typical for nucleic acid chains (height nm, width nm) are seen on the surface. The monolayer was created on the water subphase contained 100 mm of NaCl, 1 mm MgCl 2 and polya mg/ml. Results and Discussion Case 1 there are no guest molecules in the subphase. AFM image of stearic acid monolayer, transferred onto the mica substrate from the subphase free from the nucleic acids, represents a smooth, homogeneous surface with rare defects (holes). Monolayer defects (holes in the monolayer or artificial defects made by repeated scanning of the same region) testify the existence of the monolayer and can be used for the qualitative determination of its thickness. An additional evidence of the successful monolayer transfer is the change in the hydrophilic properties of the substrate it becomes hydrophobic, because the hydrocarbon chains of fatty acid molecules are placed on the film surface. Case 2 there are polya molecules and NaCl in the subphase. Samples obtained from the subphase containing polya and 1 mm of NaCl (or without any salt at all) looks just as stated above in Case 1 : a smooth surface without any traces of nucleic acids. Coulomb repulsion prevent adsorption of polynucleotides (anionic polymer) onto the like-charged fatty acid monolayer. In the presence of 10 mm of NaCl in the subphase some solitary fibers were visualized on the sample. The height ( nm) and the thickness of each fiber (10-20 nm) are typical for AFM images of RNA/DNA [Hansma H.G. et al, 1996], especially since polynucleotides are included in the lipid monolayer. The length of the fibers is up to some hundreds nanometers. Increase in concentration of sodium chloride in the subphase results in increasing of surface density of polya molecules adsorbed onto the monolayer. At 1000 mm of NaCl (we underline that none at all of bivalent ions were specially added) the surface of transferred monolayer consists of domains of stretched parallel polya molecules, and each domain mostly has its own direction of alignment (Figure 1). Addition of bivalent ions (Mg ++, 1мМ) into the subphase induced the intense complexation of polya with the fatty acids monolayer. Amount of adsorpted gene molecules is practically constant in the presence of Mg ++ regardless of concentration of NaCl at range mm (Figure 2). 537

4 When the concentration of the sodium chloride in the subphase was 1000 mm (and 1mM of the magnesium chloride), the quantity of adsorbed polya molecules drastic increased, but they were still placed randomly and there is no any ordering in its arrangement was found. Case 3 there are chelating agent in the subphase. In the case when 1M NaCl and chelating agent were in the subphase (EDTA sodium salt, 10 мм), polynucleotides were not found on the substrate (Figure 3). It s a strong evidence that in our experiments adsorption of polynucleotides onto the anionic surfactants is mediated by divalent cations by formation of salt (cationic) bridges between like-charged anionic molecules. Figure 3. AFM image of stearic acid LB monolayer transferred from the subphase containing poly(a) (0.001 mg/ml), NaCl (1M) and EDTA (10mM), represents a smooth, homogeneous surface. Conclusions We have demonstrated here a new promising method of creation of self-organized nanostructures of long single-stranded RNA using simple Langmuir-Blodgett (LB) technique and the transfer of the monolayer along with the polynucleotides onto a solid substrate. We observed that the gene molecules could be aligned in a stretched manner on the surface. The stretched parallel polya molecules forms domains, and each domain mostly has its own direction of alignment. Salt solution allows negatively charged polya molecules to reach stearic acid and to bind with it via bivalent ions mediation. NaCl salt also diminishes electrostatic interaction between polya molecules allowing them to be close to each other and to pack compactly. For the adsorption of polynucleotides onto the likely charged surface bivalent ions are needed, but a minute amount of them is sufficient (the total amount of bi- or polyvalent cations in NaCl used in our experiments for the preparation of the solutions was less then 5 mg/kg). Although in phosphatidylcholine DNA/RNA systems excess of monovalent cations inhibits the association of polynucleotides with lipid membranes [Budker et al, 1980], we ve demonstrated that binding of RNA onto the fatty acid monolayers via formation of salt bridges isn t disturbed by excess of sodium chloride. The data obtained give evidence for the effectiveness of monolayer techniques for investigation the mechanisms of DNA/RNA complexation with amphiphilic anions and demonstrate its perspectives for creation of supramolecular planar DNA-based selforganized nanostructures with nanoscale structural ordering. 538

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