Two-dimensional densely packed DNA nanostructure derived from DNA complexation with a low-generation poly(amidoamine) dendrimer

One of the keys for using deoxyribonucleic acid (DNA) as a nanomaterial relies on how the individual DNA chain can be aligned and how a multitude of DNA chains can be packed into ordered nanostructures. Here we present a simple method for constructing a 2-D densely packed DNA nanostructure using the electrostatic complex of DNA with a poly(amidoamine) (PAMAM) dendrimer of generation two. Ordered DNA arrays are formed by drop-casting an aqueous solution containing positively overcharged complexes onto mica followed by a prolonged incubation. During the incubation, the complexes tend to adsorb onto the negatively charged mica surface through electrostatic attraction. The rodlike complexes organize to form ordered arrays to increase the surface density of the adsorbed complexes and hence the attractive free energy of adsorption. The densely packed nanostructure obtained here is distinguished from the previously reported spheroid or toroid structure derived from DNA complexations with the higher-generation dendrimers.     

DNA single strands tethered to fused quartz/water interfaces studied by second harmonic generation

Second harmonic generation (SHG) is used to study oligonucleotides at aqueous/solid interfaces for the first time. Detailed thermodynamic state information for interfacial DNA single strands, namely, the interfacial charge density, the interfacial potential, and the change in the interfacial energy density, is obtained. The phosphate groups on the DNA backbone serve as intrinsic labels that do not require DNA modification other than surface attachment. This approach is broadly applicable for the investigation of DNA during its interaction with biological targets, as well as charged biopolymers in general, and has important implications for predicting and controlling macromolecular interactions, improving biodiagnostics, and understanding life processes.     

Understanding the recognition mechanism of protein-RNA complexes using energy based approach

Protein-RNA interactions perform diverse functions within the cell. Understanding the recognition mechanism of protein-RNA complexes is a challenging task in molecular and computational biology. In this work, we have developed an energy based approach for identifying the binding sites and important residues for binding in protein-RNA complexes. The new approach considers the repulsive interactions as well as the effect of distance between the atoms in protein and RNA in terms of interaction energy, which are not considered in traditional distance based methods to identify the binding sites. We found that the positively charged, polar and aromatic residues are important for binding. These residues influence to form electrostatic, hydrogen bonding and stacking interactions. Our observation has been verified with the experimental binding specificity of protein-RNA complexes and found good agreement with experiments. Further, the propensities of residues/nucleotides in the binding sites of proteins/RNA and their atomic contributions have been derived. Based on these results we have proposed a novel mechanism for the recognition of protein-RNA complexes: the charged and polar residues in proteins initiate recognition with RNA by making electrostatic and hydrogen bonding interactions between them; the aromatic side chains tend to form aromatic-aromatic interactions and the hydrophobic residues aid to stabilize the complex.     

Molecular dynamics study of DNA binding by INT‐DBD under a polarized force field

The DNA binding domain of transposon Tn916 integrase (INT‐DBD) binds to DNA target site by positioning the face of a three‐stranded antiparallel β‐sheet within the major groove. As the negatively charged DNA directly interacts with the positively charged residues (such as Arg and Lys) of INT‐DBD, the electrostatic interaction is expected to play an important role in the dynamical stability of the protein–DNA binding complex. In the current work, the combined use of quantum‐based polarized protein‐specific charge (PPC) for protein and polarized nucleic acid‐specific charge (PNC) for DNA were employed in molecular dynamics simulation to study the interaction dynamics between INT‐DBD and DNA. Our study shows that the protein–DNA structure is stabilized by polarization and the calculated protein–DNA binding free energy is in good agreement with the experimental data. Furthermore, our study revealed a positive correlation between the measured binding energy difference in alanine mutation and the occupancy of the corresponding residue's hydrogen bond. This correlation relation directly relates the contribution of a specific residue to protein–DNA binding energy to the strength of the hydrogen bond formed between the specific residue and DNA. © 2013 Wiley Periodicals, Inc.     

Production and characterization of oil-in-water emulsions containing droplets stabilized by multilayer membranes consisting of beta-lactoglobulin, iota-carrageenan and gelatin

The influence of environmental conditions (pH, NaCl, CaCl2, and temperature) on the properties and stability of oil-in-water (O/W) emulsions containing oil droplets surrounded by one-, two-, or three-layer interfacial membranes has been investigated. Three oil-in-water emulsions were prepared with the same droplet concentration and buffer (5 wt % corn oil, 5 mM phosphate buffer, pH 6) but with different biopolymers: (i) primary emulsion: 0.5 wt % beta-Lg; (ii) secondary emulsion: 0.5 wt % beta-Lg, 0.1 wt % iota-carrageenan; (iii) tertiary emulsion: 0.5 wt % beta-Lg, 0.1 wt % iota-carrageenan, 0-2 wt % gelatin. The secondary and tertiary emulsions were prepared by electrostatic deposition of the charged biopolymers onto the surfaces of the oil droplets so as to form two- and three-layer interfacial membranes, respectively. The stability of the emulsions to pH (3-8), sodium chloride (0-500 mM), calcium chloride (0-12 mM), and thermal processing (30-90 degrees C) was determined. We found that multilayer emulsions had better stability to droplet aggregation than single-layer emulsions under certain environmental conditions and that one or more of the biopolymer layers could be made to desorb from the droplet surfaces in response to specific environmental changes (e.g., high salt or high temperature). These results suggest that the interfacial engineering technology used in this study could lead to the creation of food emulsions with improved stability to environmental stresses or to emulsions with triggered release characteristics.     

Role of the coulombic interaction in ligand-induced biopolymer aggregation

The interaction mechanisms responsible for the binding between metal complexes and biopolymers in aqueous solution, as well as the consequent aggregation process of biopolymers themselves, involve many factors, from geometrical aspects and hydrophobic contributions, as examples, to the electrostatic potential. In this paper aqueous solutions of a polynucleotide, polyadenylic acid (PolyA), which mimics the helix arrangement of RNA or single-stranded DNA but has a simpler structure, are used as a model system. The role of the electrostatic interactions in the binding process between some platinum(II) complexes and PolyA and in the aggregation among PolyA molecules is investigated, by means of elastic and quasielastic light scattering and electrophoretic mobility. The results show that the presence of large, planar aromatic moiety in the dicationic platinum(II) complexes is essential for the binding with PolyA and suggest that the consequent lowering of the local electrostatic barrier between PolyA molecules can be involved in triggering the aggregation process.     

Design, synthesis, conformational analysis and nucleic acid hybridisation properties of thymidyl pyrrolidine-amide oligonucleotide mimics (POM)

Pyrrolidine-amide oligonucleotide mimics (POM) 1 were designed to be stereochemically and conformationally similar to natural nucleic acids, but with an oppositely charged, cationic backbone. Molecular modelling reveals that the lowest energy conformation of a thymidyl-POM monomer is similar to the conformation adopted by ribonucleosides. An efficient solution phase synthesis of the thymidyl POM oligomers has been developed, using both N-alkylation and acylation coupling strategies. 1H NMR spectroscopy confirmed that the highly water soluble thymidyl-dimer, T2-POM, preferentially adopts both a configuration about the pyrrolidine N-atom and an overall conformation in D2O that are very similar to a typical C3'-endo nucleotide in RNA. In addition the nucleic acid hybridisation properties of a thymidyl-pentamer, T5-POM, with an N-terminal phthalimide group were evaluated using both UV spectroscopy and surface plasmon resonance (SPR). It was found that T5-POM exhibits very high affinity for complementary ssDNA and RNA, similar to that of a T5-PNA oligomer. SPR experiments also showed that T5-POM binds with high sequence fidelity to ssDNA under near physiological conditions. In addition, it was found possible to attenuate the binding affinity of T5-POM to ssDNA and RNA by varying both the ionic strength and pH. However, the most striking feature exhibited by T5-POM is an unprecedented kinetic binding selectivity for ssRNA over DNA.     

DNA and RNA noncovalent interaction of platinum(II) polypyridine complexes

A comparative investigation of the noncovalent interaction of the platinum(II) polypyridine complexes [Pt(dipy)(n-Rpy)2]2+ and [Pt(4,4'-Me2dipy)(2-Rpy)2]2+ (dipy = 2,2'-dipyridine; Me = CH3; n = 2-4; R = H or CH3) with double-helical DNA (calf thymus) and RNA [poly(A).poly(U)] has been conducted. With the exception of [Pt(dipy)(2-Mepy)2]2+, all of the complexes interact strongly, by intercalation, with both nucleic acids giving rise to large changes in the electronic spectra and induced circular dichroism signals; in addition, viscosity experiments on rodlike DNA and RNA show that both biopolymers elongate upon interaction with the complexes. The binding constant values, KB, determined at 25 degrees C, indicate that, at 0.101 M ionic strength, the affinity for poly(A).poly(U) is strongly dependent on the complexes nature, while for DNA it is leveled off. [Pt(dipy)(2-Mepy)2]2+ binds to DNA but does not interact appreciably with poly(A).poly(U).     

Relationship between interfacial forces measured by colloid-probe atomic force microscopy and protein resistance of poly(ethylene glycol)-grafted poly(L-lysine) adlayers on niobia surfaces

Adsorbed layers of "comb-type" copolymers consisting of PEG chains grafted onto a poly(l-lysine) (PLL) backbone on niobium oxide substrates were studied by colloid-probe AFM in order to characterize the interfacial forces associated with coatings of varying architectures (PEG/PLL ratios and PEG chain lengths) and their relevance to protein resistance. The steric and electrostatic forces measured varied substantially with the architecture of the PLL-g-PEG copolymers. Varying the ionic strength of the buffer solutions enabled discrimination between electrostatic and steric-entropic contributions to the net interfacial force. For high PEG grafting densities the steric component was most prominent, but at low ionic strengths and high grafting densities, a repulsive electrostatic surface force was also observed; its origin was assigned to the niobia charges beneath the copolymer, as insufficient protonated amine groups in the PLL backbone were available for compensation of the oxide surface charges. For lower grafting densities and lower ionic strengths there was a substantial attractive electrostatic contribution arising from interaction of the electrical double layer arising from the protonated amine groups, with that of the silica probe surface (as under low ionic strength conditions, the electrical double layer was thicker than the PEG layer). For these PLL-g-PEG coatings the net interfacial force can thus be a markedly varying superposition of electrostatic and steric-entropic contributions, depending on various factors. The force curves correlate with protein adsorption data, demonstrating the utility of AFM colloid-probe force measurements for quantitative analysis of surface forces and how they determine interfacial interactions with proteins. Such characterization of the net interfacial forces is essential to elucidate the multiple types of interfacial forces relevant to the interactions between PLL-g-PEG coatings and proteins and to advance interpretation of protein adsorption or repellence beyond the oversimplified steric barrier model; in particular, our data demonstrate the importance of an ionic-strength-dependent minimum PEG layer thickness to screen the electrostatic interactions of charged interfaces.     

Strand invasion of mixed-sequence B-DNA by acridine-linked, gamma-peptide nucleic acid (gamma-PNA)

Peptide nucleic acid (PNA) is a synthetic mimic of DNA and RNA that can recognize double-stranded B-DNA through direct Watson-Crick base-pairing. Although promising, PNA recognition is presently limited to mostly purine- and pyrimidine-rich targets, because mixed-sequence PNA, in general, does not have sufficient binding free energy to invade B-DNA. In this Article, we show that conformationally preorganized gamma-peptide nucleic acid (gamma-PNA) containing an acridine moiety covalently linked at the C-terminus can invade mixed-sequence B-DNA in a sequence-specific manner. Recognition occurs through direct Watson-Crick base-pairing. This finding is significant because it demonstrates that the same principles that guide the recognition of single-stranded DNA and RNA can also be applied to double-stranded B-DNA.     

Influence of confinement on the electrostatic interaction between charged colloids: a (N,V,T) Monte Carlo study within hyperspherical geometry

Monte Carlo simulations within closed hyperspherical geometry are used to analyze the ionic distribution around two confined charged colloids to determine the origin of the net attraction recently reported in the literature. A scaling procedure is used to compare our numerical results obtained with small ideal colloids with the conclusion of the measurements performed with large silica colloids. Although no electrostatic attraction is detected under confinement, our simulations exhibit a significant reduction of the electrostatic repulsion between charged colloids confined between two weakly charged walls. After rescaling to reproduce the electrostatic repulsion between large confined colloids, our numerical results are qualitatively consistent with the reported attraction because we reasonably expect a reduction of the electrostatic force between such confined colloids below the order of magnitude of their van der Waals attraction.     

Sequence-specific binding of DNA to liposomes containing di-alkyl peptide nucleic acid (PNA) amphiphiles

We present a method to covalently attach peptide nucleic acid (PNA) to liposomes by conjugation of PNA peptide to charged amino acids and synthetic di-alkyl lipids ("PNA amphiphile," PNAA) followed by co-extrusion with disteroylphosphatidylcholine (DSPC) and cholesterol. Attachment of four Glu residues and two ethylene oxide spacers to the PNAA was required to confer proper hydration for extrusion and presentation for DNA hybridization. The extent of DNA oligomer binding to 10-mer PNAA liposomes was assessed using capillary zone electrophoresis. Nearly all PNAs on the liposome surface are complexed with a stoichiometric amount of complementary DNA 10-mers after 3-h incubation in pH 8.0 Tris buffer. No binding to PNAA liposomes was observed using DNA 10-mers with a single mismatch. Longer DNA showed a greatly attenuated binding efficiency, likely because of electrostatic repulsion between the PNAA liposome double layer and the DNA backbone. Langmuir isotherms of PNAA:DSPC:chol monolayers indicate miscibility of these components at the compositions used for liposome preparation. PNAA liposomes preserve the high sequence-selectivity of PNAs and emerge as a useful sequence tag for highly sensitive bioanalytical devices.     

The role of electrostatic interactions on the rejection of organic solutes in aqueous solutions with nanofiltration

The effects of electrostatic interactions on the rejection of organic solutes with nanofiltration membranes were investigated. For two different membranes, the rejection of selected organic acids, positively and negatively charged pharmaceuticals and neutral pharmaceuticals was investigated at different feed water chemistries (different ionic strengths and pH conditions, with and without the presence of NOM and divalent cations). It was concluded that for negatively charged membranes, electrostatic repulsion leads to an increase of the rejection of negatively charged solutes and electrostatic attraction leads to a decrease of the rejection of positively charged solutes, compared to neutral solutes. Neutral and positively charged solutes engage in hydrophobic interactions with negatively charged membranes, whereas negatively charged solutes do not engage in hydrophobic interactions since they cannot approach the membrane surface. This provides proof for the theory of an increased concentration of positively charged organic solutes and a decreased concentration of negatively charged organic solutes at the membrane surface compared to the bulk fluid. This concept may be denoted as “charge concentration polarisation”. The concept was further used as a modelling tool to predict the effects of electrostatic interactions on the rejection of trace organic solutes.     

Hydrogen/deuterium exchange on protein solutions containing nucleic acids: utility of protamine sulfate

Obtaining global hydrogen/deuterium (H/D) exchange data on proteins is an important first step in amide proton exchange experiments. Important information such as the mode of exchange, the cooperativity of folding/unfolding reactions, and the effects of ligand binding can be readily obtained in global exchange experiments. Many interesting biological systems are complexes containing both proteins and nucleic acids. The low pH conditions required to quench H/D exchange reactions result in the formation of stable protein/nucleic acid precipitates which interfere with the liquid chromatography step of the experiment and preclude obtaining mass spectrometric data. In this work we show that the precipitation of proteins and nucleic acids is electrostatic in nature and can be prevented by high ionic strength and by removing nucleic acids by protamine sulfate. Using protamine sulfate in quenching solution, we were able to obtain global H/D data with protein samples containing large amounts of DNA or RNA.     

Immunoblotting techniques with picogram sensitivity in cerebrospinal fluid protein detection

Agarose isoelectric focusing followed by blotting with nitrocellulose, nylon or polyvinylidene difluoride membranes, and immunochemical detection of cerebrospinal fluid IgG with various combinations of antisera, was evaluated. Polyvinylidene difluoride proved to be an easy-to-handle and reliable membrane for protein blotting. Among immunochemical visualization reactions, the most sensitive employed biotinylated goat anti-human IgG followed by streptavidin colloidal gold conjugate and silver enhancement in 20% w/v urea, allowing a sensitivity of less then 1 picogram IgG/band.     

Single-molecule studies on DNA and RNA.

DNA and RNA are the most individual molecules known. Therefore, single-molecule experiments with these nucleic acids are particularly useful. This review reports on recent experiments with single DNA and RNA molecules. First, techniques for their preparation and handling are summarised including the use of AFM nanotips and optical or magnetic tweezers. As important detection techniques, conventional and near-field microscopy as well as fluorescence resonance energy transfer (FRET) and fluorescence correlation spectroscopy (FCS) are touched on briefly. The use of single-molecule techniques currently includes force measurements in stretched nucleic acids and in their complexes with binding partners, particularly proteins, and the analysis of DNA by restriction mapping, fragment sizing and single-molecule hybridisation. Also, the reactions of RNA polymerases and enzymes involved in DNA replication and repair are dealt with in some detail, followed by a discussion of the transport of individual nucleic acid molecules during the readout and use of genetic information and during the infection of cells by viruses. The final sections show how the enormous addressability in nucleic acid molecules can be exploited to construct a single-molecule field-effect transistor and a walking single-molecule robot, and how individual DNA molecules can be used to assemble a single-molecule DNA computer.     

Amino acids attached to 2'-amino-LNA: synthesis and excellent duplex stability

The synthesis of 2'-amino-LNA (the 2'-amino derivative of locked nucleic acid) has opened up a number of exciting possibilities with respect to modified nucleic acids. While maintaining the excellent duplex stability inferred by LNA-type oligonucleotides, the nitrogen in the 2'-position of 2'-amino-LNA monomers provides an excellent handle for functionalisation. Herein, the synthesis of amino acid functionalised 2'-amino-LNA derivatives is described. Following ON synthesis, a glycyl unit attached to the N2'-position of 2'-amino-LNA monomers was further acylated with a variety of amino acids. On binding to DNA/RNA complements, the modified ONs induce a marked increase in thermal stability, which is particularly apparent in a buffer system with a low salt concentration. The increase in thermal stability is thought to be caused, at least in part, by decreased electrostatic repulsion between the negatively charged phosphate backbones when positively charged amino acid residues are appended. Upon incorporation of more than one 2'-amino-LNA modification, the effects are found to be nearly additive. For comparison, 2'-amino-LNA derivatives modified with uncharged groups have been synthesised and their effect on duplex thermal stability likewise investigated.     

Effects of mutational (Lys to Ala) surface charge changes on the redox properties of electrode-immobilized cytochrome c

Untrimethylated yeast iso-1-cytochrome c (cytc) and its single and multiple Lys to Ala variants at the surface lysines 72, 73, and 79 were adsorbed on carboxyalkanethiol self-assembled monolayers (SAMs) on gold, and the thermodynamics and kinetics of the heterogeneous protein-electrode electron-transfer (ET) reaction were determined by voltammetry. The reaction thermodynamics were also measured for the same species freely diffusing in solution. The selected lysine residues surround the heme group and contribute to the positively charged domain of cytc involved in the binding to redox partners and to carboxyl-terminated SAM-coated surfaces. The E degrees' (standard reduction potential) values for the proteins immobilized on SAMs made of 11-mercapto-1-undecanoic acid and 11-mercapto-1-undecanol on gold were found to be lower than those for the corresponding diffusing species owing to the stabilization of the ferric state by the negatively charged SAM. For the immobilized proteins, Lys to Ala substitution(s) do not affect the surface coverage, but induce significant changes in the E degrees' values, which do not simply follow the Coulomb law. The results suggest that the species-dependent orientation of the protein (and thereby of the heme group) toward the negatively charged SAM influences the electrostatic interaction and the resulting E degree' change. Moreover, these charge suppressions moderately affect the kinetics of the heterogeneous ET acting on the reorganization energy and the donor-acceptor distance. The kinetic data suggest that none of the studied lysines belong to the interfacial ET pathway.     

Native bromoperoxidases do not bind to nitrocellulose: use of DEAE-cellulose as an alternative in blotting

Bromoperoxidases were investigated by protein blotting after polyacrylamide gel electrophoresis. While the denatured proteins bound to nitrocellulose, the native enzymes did not. Instead, they could be transferred successfully to DEAE-cellulose. Procedures for immunostaining and glycoprotein detection with concanavalin A on DEAE-cellulose are described. The results indicate that binding of native proteins to nitrocellulose can not necessarily be assumed. DEAE-cellulose is suitable both to investigate this phenomenon or as a substitute for nitrocellulose in blotting of native proteins.