Gabriella Caminati

Phospholipid Membranes Decorated by Cholesterol-Based Oligonucleotides as Soft Hybrid Nanostructures

DNA monomers and oligomers are currently showing great promise as building blocks for supramolecular arrays that can self-assemble in a fashion preprogrammed by the base pairing code. The design and build-up of hybrid DNA/amphiphilic self-assemblies can expand the range of possible architectures and enhance the selectivity toward a well-specified geometry. We report on the self-assembly properties in aqueous solution of a cholesteryl-tetraethylenglycol single stranded 18-mer oligonucleotide (ON 1TEG-Chol) and on its spontaneous insertion in fluid phospholipid membranes. Up to 500 units of these lipophilic ss-oligonucleotides can be incorporated in the outer leaflet of 350 A radius POPC vesicle. The insertion and hybridization with the complementary oligonucleotide are monitored through light scattering as an increase of hydrodynamic thickness, which is interpreted in terms of average distance between anchoring sites. The conformation of the ss-oligonucleotidic portion is strongly dependent on surface coverage, passing from a quasi-random coil to a more rigid configuration, as concentration increases. Interestingly, conformational details affect in a straightforward fashion the hybridization kinetics. Liposomes with single- and double-strand decorations remain stable within the experimental time window (about one week). The structure represents an example of successful and stable amphiphile/DNA supramolecular hybrid, where a DNA guest is held in a membrane by hydrophobic interactions. The lipophilic oligonucleotide under investigation is therefore a suitable building block that can effectively serve as a hydrophobic anchor in the fluid bilayer to assemble supramolecular constructs based on the DNA digital code.

Langmuir-Blodgett films of alkylpyridines as metal ion sensors

Abstract Monolayers of nonadecylpyridine (NDP) were studied at the water-air interface by measuring surface pressure-and surface potential-area isotherms. The conditions of preparation of Langmuir-Blodgett (LB) film of NDP on quartz were studied and the physicochemical properties of the film then examined. UV-visible and FTIR/ATR spectroscopy were used to characterize the dry LB film. Ellipsometry was also used to measure the layer thickness as a function of the number of transferred layers, the monolayer composition and the compression of the film. Transfer of the film from pure water subphase on substrates of different natures and hydrophobicity, i.e. quartz, glass, chromium, germanium and polymers, was also studied comparatively. The interactions of the monolayer with the metal cation Ni 2+ solubilized in the aqueous subphase were investigated by recording the surface pressure and surface potential at the water-air interface both in the presence and in the absence of the metal ion in subphases of the same ionic strength. The transfer was then performed using subphases containing the metal ion and the resulting LB films were characterized by ESCA spectroscopy and reported techniques. Absorption spectra were recorded for LB films of NDP immersed in water or in contact with the metal ion solutions. Complexation of metal cations from the subphase was studied by monitoring the absorption spectra of solutions containing the metal ion when the LB film was immersed in the solution.

Modulation of density and orientation of amphiphilic DNA anchored to phospholipid membranes. I. Supported lipid bilayers.

In the present series of papers, we describe the results of a systematic study on the anchoring of cholesterol-tagged oligonucleotides to phospholipids bilayers followed by membrane-assisted hybridization of the complementary strand in solution. This paper describes the anchoring of novel cholesterol-modified DNA-18mers in supported lipid bilayers (SLB) of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine: we compared the behavior of two anchoring functionalities differing in the number of cholesterol units and in the length of a spacer group. Quartz Crystal Microbalance with impedance monitoring (QCM-Z) measurements showed that both oligonucleotides insert into the bilayer membrane through cholesterol anchoring; however, dramatic differences, in terms of surface organization and thickness, are found as the number of anchoring units increases. In the case of multiple cholesterol units, a peculiar three-regimes concentration dependence was revealed and correlated to the effective size of the adsorbing units. Interestingly, for high oligonucleotide concentration, the adsorption process was rationalized in terms of a compaction model of amphiphilic DNA molecules. QCM-Z temperature cycles of the SLB-anchored double strands provided clear evidence for reversible hybridization at the bilayer interface.

Closed nanoconstructs assembled by step-by-step ss-DNA coupling assisted by phospholipid membranes

We report experimental evidence of the construction of closed pseudo-hexagonal DNA nanostructures grafted onto lipid membranes (supported planar bilayers or liposomes). The anchoring agent is a cholesteryl tetraethylenglycol-18-mer oligonucleotide, which recruits the complementary sequences added in a sequential fashion. A closed rather than an open nanostructure was chosen as a test for possible future applications in nanoelectronics and for the fact that a more rigid and defined structure can offer many advantages in terms of addressability and spatial control. The so-built hybrid soft nanomaterial has never been reported previously and merges the unique features offered by DNA building blocks in nanotechnology, to the characteristics of amphiphilic self-assembly, in terms of responsiveness and further hierarchical aggregation in functional arrays of nano-units. The liposomes decorated with DNA pseudo-hexagons are structurally stable for weeks and can thus be further exploited or specifically addressed. The structural features of the final nanoobjects are independent on the sequence of preparation, i.e. step-wise on the membrane or addition of preformed hexagons, up to a threshold of density on the surface or vesicle number density.

Adsorption Isotherms of Aqueous C12E6 and Cetyltrimethylammonium Bromide Surfactants on Solid Surfaces in the Presence of Low Molecular Weight Coadsorbents

In this work, we evaluate the effects of the low molecular weight compounds toluene, phenol, and 1-hexanol on the adsorption of two surfactants on one solid surface. The surfactants are cetyltrimethylammonium bromide (CTAB, cationic) and hexaethylene glycol monododecyl ether (C12E6, nonionic). The surface is gold, although X-ray photoelectron spectroscopic analysis reveals the presence of a large number of oxygenated sites that render the surface hydrophilic (contact angle 10 degrees). Adsorption isotherms are measured using a quartz crystal microbalance with dissipation monitoring (QCM-D). Although our measurements do not allow the determination of the morphology of the aggregates directly, we rationalize our results by referring to AFM images from the literature. On the basis of primarily the dissipative signal and on AFM studies done by others, our results are consistent with CTAB forming a patchy cylindrical structure and C12E6 likely yielding a monolayer structure. The presence of cosolutes almost doubles the mass of surface aggregates and increases the rigidness of the aggregates for CTAB, consistent with a morphological change from cylinders to flat bilayers. Part of the increase in adsorbed mass is likely due to increased surface area covered by admicelles. For C12E6, cosolutes cause small changes in the mass adsorption and essentially no change in the flexibility of surface aggregates.

Polystyrene sulfonate adsorption at water—graphon and water—air interfaces

Abstract The adsorption behaviour of a series of sodium polystyrene sulfonates with a narrow distribution of molecular weight was studied at the solid—liquid interface. Graphitized carbon black (graphon) was chosen as a model surface for real coal used in the preparation of coal—water slurries. Graphon has a highly homogeneous surface with virtually no polar groups and with a large specific area. Preliminary studies on the kinetics of adsorption were performed in order to establish attainment of steady-state conditions. The kinetic results were analyzed considering several models and the results were interpreted with a two-step process involving diffusion towards the interface and reorientation of the macromolecules at the surface. Adsorption at the graphon—water interface was found to follow the Langmuir equation for every sample with similar adsorption energies. The dependence of the maximum of adsorption on molecular weight was used to obtain information on the macromolecular conformation at the interface and using a simplified fractal approach, on the graphon surface. Adsorption isotherms at the water—air interface were also recorded. The dependence of surface tension on polymer concentration and on polymer molecular weight was used to obtain information on the structure of the adsorbed layer and on the energetics of the process. The results obtained at the water—air interface were correlated to the polyelectrolyte behaviour at the liquid—solid interface.

The Precise Chemical−Physical Nature of the Pharmacore in FK506 Binding Protein Inhibition: ElteX, a New Class of Nanomolar FKBP12 Ligands

Due to its central role in immunosuppression and cell proliferation and due to its specific peptidyl-prolyl-isomerase (PPI) function, the FKBP protein family is at the crossroad of several important metabolic pathways. Members of this family, and notably FK506 binding protein (FKBP12), are thought to be involved in neurodegenerative diseases such as Alzheimer disease, Parkinson disease, multiple sclerosis, amyotrophic lateral sclerosis, as well as in proliferation disorders and cancer. Using an interdisciplinary approach based on computational, synthetic, and experimental techniques, we show that the best potential binders for FKBP proteins optimally expose the two contiguous carbonyl oxygen in the proline-mimetic chain for FKBP docking and are characterized by the abundance of rigid quasi-cyclic structures stabilized in aqueous solution by intraligand hydrophobic interactions mimicking the macrolide structure of the natural FKBP binders FK506 and Rapamycin. These peculiar structural and chemical-physical features define at the same time an ElteX compound and the minimal pharmacore in the FKBP family, shedding new light on the isomerization mechanism of the PPI domain. On the basis of the above hypothesis, we have successfully designed and synthesized several nanomolar ElteX FKBP12 ligands. Among these, ElteN378 is a new low atomic weight ligand with affinity comparable to that of the macrolide Rapamycin.

DNA closed nanostructures: a structural and Monte Carlo simulation study

DNA nanoconstructs are obtained in solution by using six unique 42-mer DNA oligonucleotides, whose sequences have been designed to form a pseudohexagonal structure. The required flexibility is provided by the insertion of two non-base-paired thymines in the middle of each sequence that work as flexible hinges and constitute the corners of the nanostructure when formed. We show that hexagonally shaped nanostructures of about 7 nm diameter and their corresponding linear open constructs are formed by self-assembly of the specifically designed linear oligonucleotides. The structural and dynamical characterization of the nanostructure is obtained in situ for the first time by using dynamic light scattering (DLS), a noninvasive method that provides a fast dynamic and structural analysis and allows the characterization of the different synthetic DNA nanoconstructs in solution. A validation of the LS results is obtained through Monte Carlo (MC) simulations and atomic force microscopy (AFM). In particular, a mesoscale molecular model for DNA, developed by Knotts et al., is exploited to perform MC simulations and to obtain information about the conformations as well as the conformational flexibilities of these nanostructures, while AFM provides a very detailed particle analysis that yields an estimation of the particle size and size distribution. The structural features obtained by MC and AFM are in good agreement with DLS, showing that DLS is a fast and reliable tool for characterization of DNA nanostructures in solution.

Spectroscopic investigation of tetracycline interaction with phospholipid Langmuir–Blodgett films

Abstract A widely used veterinary antibiotic, tetracycline (TC), has been incorporated in Langmuir–Blodgett (LB) films of dipalmitoylphosphatidic acid (DPPA) by means of two different procedures: co-transfer and incubation in solution. The resulting structures were characterized by means of contact angle and ellipsometric measurements. The presence of the antibiotic in the phospholipid film was evidenced by means of UV–Vis electronic absorption and infrared vibrational spectroscopy. The two sets of measurements unambiguously indicated the presence of the drug in the LB layer films obtained with both methods, although incubation led to a smaller content of immobilized tetracycline. In both cases, the drug was found to reside in the hydrophilic portion of the layers due to specific interactions of the dimethylamino group of the molecule with the polar head groups of the phospholipid.

A new lipophilic fluorescent probe for interaction studies of bioactive lipopeptides with membrane models

The new fluorescent lipophilic moiety 11-[(7-amino-4-methyl-2-oxo-2H-1-benzopyran-3-acetyl)amino]undecanoic acid (AMCA-omegaAud-OH) was introduced by SPPS at the N-terminus of the immunodominant epitope GpMBP(74-85). FRET experiments using the new fluorescent lipopeptide demonstrate that the peptide interacts with much more affinity with the membrane compared to the lipid free analogue.