Teresa Kral

The application of fluorescence correlation spectroscopy in detecting DNA condensation

We report the application of fluorescence correlation spectroscopy (FCS) in characterizing conformational changes (condensation) of chemically well-defined DNA plasmids. The plasmids: pHbetaAPr-1-neo (10 kbp, contour length 3.4 microm) and pBluescript SKt (2.96 kbp, contour length 1.02 microm) were imaged by a confocal fluorescence microscope using two fluorescent probes: ethidium bromide (EtBr) and propidium iodide (PrIo). It became clear that the DNA molecule exhibits discrete conformational change between the coil and globule states with the addition of a small amount (the order of magnitude being 10(-5) M) of cationic surfactant, spermine and hexadecyltrimethyl ammonium bromide (HTAB). When the concentrations of both condensing agents are smaller than 6.0x10(-6) M and 2.0 x 10(-6) M for the 10 and 2.96 kbp, both plasmids are in the extended coil state with diffusion constants D(10 kbp)=9.6 x 0(-13) m(2) s(-1) and D(2.96 kbp)=2.5x10(-12) m(2) s(-1), respectively. When the condensing agent in a concentration higher than 1.10 x 10(-5) M is added to pHbetaAPr-1-neo (10 kbp), plasmids are in the condensed globular state and their diffusion constants are D(10 kbp)=8.0 x 10(-12) m(2) s(-1) (spermine) and D(10 kbp)=5.5x10(-12) m(2) s(-1) (HTAB). The globular state of the pBluescript SKt (2.96 kbp) plasmids is characterized by diffusion constants equal to D(2.96 kbp)=9.2x10(-12) m(2) s(-1) (spermine) and D(2.96 kbp)=8.2x10(-12) m(2) s(-1) (HTAB).

A comprehensive study in triblock copolymer membrane interaction

Poloxamers are triblock copolymers made of poly(ethylene glycol)-(poly(propylene glycol))-poly(ethylene glycol). They have been shown to enhance gene transfer in the muscle, and co-administration of polymers and DNA appeared to be crucial to obtain this effect. It is questionable then if some interaction occurs between polymers and DNA. Polymer interaction with membranes represents a second crucial point due to the central hydrophobic part of the triblock copolymers. Besides, the question of the polymer spanning or adsorbing to the surface has not been solved by now. We addressed these issues by means of sensitive techniques that allowed working in diluted conditions and gaining in comprehension of gene transfection. By means of simultaneous time-correlated single-photon counting and fluorescence correlation spectroscopy, we have shown that the diffusion time of a single DNA molecule and PicoGreen lifetime was not altered in the presence of the triblock copolymer L64. Polypropylene (glycol) interactions with dodecylphosphocholine micelles were shown to occur at a deep level by (1)H NMR using doxyl probes located at the head or the lipid extremity of the micelles. The polypropylene (glycol) also interacted with lipid bilayers in a manner dependent on the cholesterol content, as shown by differential scanning calorimetry using liposomes. This interaction destabilised the membrane and allowed the release of small molecules. Finally, molecular dynamic simulation of the copolymer L64 in the presence of dodecylphosphocholine showed that the hydrophobic core of the polymer formed an extremely tight cluster, whose dimensions excluded the possibility of polymer spanning across the lipidic micelles. The simulation positively correlated with the destabilising effect observed on the liposomal membrane models.

Propidium Iodide and PicoGreen as Dyes for the DNA Fluorescence Correlation Spectroscopy Measurements

Many experimental designs, in which nucleic acid conformational changes are of interest, require reliable fluorescence labeling. The appropriate fluorescence probe should have suitable optical properties and, more importantly, should not interfere with the investigated processes. In order to avoid chemical modifications the fluorescence label needs to be associated with nucleic acid via weak non-covalent interactions. There are a number of fluorescent probes that change their fluorescent properties (i.e. their quantum yield and/or spectral characteristics) upon association with nucleic acid. Such probes are frequently used to detect, visualize and follow processes involving nucleic acid and its conformational changes. In order to obtain reliable data regarding macromolecule or aggregate topology a detailed knowledge of probe–nucleic acid interactions on the molecular level is needed. In this paper we show that the association of propidium iodide with DNA alters its conformation and that it selectively labels plasmid fragments and/or its subpopulations in a concentration-dependent meaner. Another dye, PicoGreen, exhibits better properties. It labels nucleic acid uniformly and without any concentration-dependent artifacts.

The effect of spermine on plasmid condensation and dye release observed by fluorescence correlation spectroscopy.

Abstract We demonstrate that fluorescence correlation spectroscopy (FCS) can be employed to follow the conformational changes of DNA molecules induced by the addition of a cationic condensing compound (spermine). In our experiments the plasmid pHβAPr-1-neo (10 kbp; contour length 3.4 m) was labeled with propidium iodide (PrIo) and then titrated with spermine to induce its condensation. When spermine was applied at concentrations above 5 M (spermine/DNAphosphate =0.375), the diffusion time of the labeled plasmid dropped from 15 ms down to 3 ms (its diffusion coefficient, D, increased from 1.010 exp 12 square m/s to 6.010 exp 12 square m/s). The application of spermine was also accompanied by decreasing count rate and particle number, reflecting the dyes dissociation. The data presented show that FCS may become a valuable tool in studying supramolecular aggregate formation, especially when association is followed by a change in the hydrodynamic size of the resulting complex.

Lipopolythiourea/DNA interaction: A biophysical study

Lipopolythioureas (LPT) are original non cationic systems representing an alternative to cationic lipids. Their high transfection efficiency prompted us to investigate further their biophysical properties, and in particular how thiourea lipids interact with DNA. The interaction of lipopolythiourea with DNA was investigated by fluorescence correlation microscopy (FCS). Influence of the lipid length and nature of the thiourea head on the thiourea/DNA interaction were studied. FCS revealed a strong interaction between lipopolythiourea and DNA, occurring at 1 equivalent of a thiourea lipid by a DNA phosphate group, and leading to a condensed plasmid state. From previous in vitro experiments, we could conclude that the lipid leading to the more condensed state of DNA was also the more efficient to transfect cells.

New gluconamide-type cationic surfactants: Interactions with DNA and lipid membranes.

New linear cationic surfactants - 2-(alkyldimethylammonio)ethylgluconamide bromides, denoted as CnGAB, n=10, 12, 14 and 16 - were synthesized from natural resources and characterized with respect to their potential as gene-delivery agents in gene therapy applications. Interactions with plasmid DNA and with model membranes were studied both experimentally and theoretically. The compounds with n=12, 14 and 16 show exponentially increasing ability to fully condense DNA. C16GAB condenses DNA at 1:1 surfactant to nucleotide molar ratio. Furthermore, CnGABs interact with model membrane, slightly lowering the temperature of the main phase transition Tm of the DPPC bilayer. C10GAB is found to interact only at the membrane surface. C16GAB reduces Tm less than C12GAB and C14GAB, and forms domains in the bilayer at the surfactant/DPPC molar ratio of 0.1 and higher. The results suggest that C16GAB can be a promising candidate for building gene-delivery carrier systems.

Protonation of lipids impacts the supramolecular and biological properties of their self-assembly.

We assessed in this work how a chemical structure difference could influence a supramolecular organization and then its biological properties. In our case study, we considered two amphiphilic lipidic gene vectors. The chemical difference was situated on their hydrophilic part which was either a pure neutral thiourea head or a mixture of three thiourea function derivatives, thiourea, iminothiol, and charged iminothiol. This small difference was obtained thanks to the last chemical deprotection conditions of the polar head hydroxyl groups. Light, neutron, and X-ray scattering techniques have been used to investigate the spatial structure of the liposomes and lipoplexes formed by the lipids. The chemical structure difference impacts the supramolecular assemblies of the lipids and with DNA as shown by fluorescence correlation spectroscopy (FCS), X-ray, and neutron scattering. Hence the structures formed were found to be highly different in terms of liposomes to DNA ratio and size and polydispersity of the aggregates. Finally, the transfection and internalization results proved that the differences in the structure of the lipid aggregates fully affect the biological properties of the lipopolythiourea compounds. The lipid containing three functions is a better gene transfection agent than the lipid which only contains one thiourea moiety. As a conclusion, we showed that the conditions of the last chemical step can influence the lipidic supramolecular structure which in turn strongly impacts their biological properties.

Fluorescence Correlation Spectroscopic Studies of a Single Lipopolyamine–DNA Nanoparticle

We have studied lipopolyamine–DNA complex formation by fluorescence correlation spectroscopy(FCS). Two lipopolyamines, N 4,N 9-dioleoylspermineand N 1-cholesteryl spermine carbamate, wereused to condense linear calf thymus DNA and two plasmid DNAs: pGL3 (5.3 kilobase pairs) and pEGFP(4.7 kilobase pairs). PicoGreen

Different Effects of Di- and Triphenyltin Compounds on Lipid Bilayer Dithionite Permeabilization

Abstract Phenyltins are chemicals widely used in industry, hence their occurrence in the human environment is frequent and widespread. Such compounds include hydrophobic phenyl rings bonded to positively charged tin. This molecular structure makes them capable of adsorbing onto and penetrating through biological membranes, hence they are potentially hazardous. Two such compounds, diphenyltin and triphenyltin, show different steric constraints when interacting with the lipid bilayer. It has been demonstrated that these compounds are positioned at different locations within model lipid bilayers, causing dissimilarity in their ability to affect membrane properties. In this paper we present a study regarding the ability of these two phenyltins to facilitate the transport of S2O4-2 ions across the lipid bilayer, evaluated by a fluorescence quenching assay. In concentration range of up-to 60 μm those compounds do not affect lipid bilayer topology, when evaluated by vesicle size distribution. Both phenyltins facilitate the transfer of S2O4-2 across the model lipid bilayer, but the dependence of dithionite transport on phenyltin concentration is different for both. In principle, above 20 μm triphenyltin is more efficient in transfering ions across the lipid bilayer than diphenyltin.

Interaction of new butyltin citrate complex with lipid model membrane and DNA

Organotin(IV) complexes show a wide variety of biological activity: bactericidal, fungicidal, caricidal, and pesticidal. Many of them are more effective than traditional heavy metal anticancer drugs. The aim of the present study was to investigate the interaction of a new dibutyltin complex with citric acid (DBTC) with lipid membrane and plasmid DNA. The effect of this compound on the multilamellar liposomes formed with dipalmitoylphosphatidylcholine, dimyristoylphosphatidylcholine, and dimyristoylphosphatidylethanolamine was studied mainly by the means of differential scanning calorimetry and additionally by the steady-state fluorimetry. Calorimetrical results together with fluorescence spectroscopy suggest that investigated complex interacts with lipids and probably locates itself in the hydrophilic part of the membrane. The effect of DBCT on plasmid DNA was investigated using single-molecule florescence technique and time-correlated single photon counting fluorescence correlation spectroscopy. We could conclude then that DBTC–DNA interaction occurs due to the interaction with the DNA phosphate group and charge neutralization which then leads to DNA compaction.