Sergii Rudiuk

UV-Induced Bursting of Cell-Sized Multicomponent Lipid Vesicles in a Photosensitive Surfactant Solution

We study the behavior of multicomponent giant unilamellar vesicles (GUVs) in the presence of AzoTAB, a photosensitive surfactant. GUVs are made of an equimolar ratio of dioleoylphosphatidylcholine (DOPC) and dipalmitoylphosphatidylcholine (DPPC) and various amounts of cholesterol (Chol), where the lipid membrane shows a phase separation into a DPPC-rich liquid-ordered (Lo) phase and a DOPC-rich liquid-disordered (Ld) phase. We find that UV illumination at 365 nm for 1 s induces the bursting of a significant fraction of the GUV population. The percentage of UV-induced disrupted vesicles, called bursting rate (Yburst), increases with an increase in [AzoTAB] and depends on [Chol] in a non-monotonous manner. Yburst decreases when [Chol] increases from 0 to 10 mol % and then increases with a further increase in [Chol], which can be correlated with the phase composition of the membrane. We show that Yburst increases with the appearance of solid domains ([Chol] = 0) or with an increase in area fraction of Lo phase (with increasing [Chol] ≥ 10 mol %). Under our conditions (UV illumination at 365 nm for 1 s), maximal bursting efficiency (Yburst = 53%) is obtained for [AzoTAB] = 1 mM and [Chol] = 40 mol %. Finally, by restricting the illumination area, we demonstrate the first selective UV-induced bursting of individual target GUVs. These results show a new method to probe biomembrane mechanical properties using light as well as pave the way for novel strategies of light-induced drug delivery.

Importance of the dynamics of adsorption and of a transient interfacial stress on the formation of aggregates of IgG antibodies

It is common knowledge that aggregation of proteins may occur in aqueous solutions under mechanical stress (shaking or high shear), even in solutions that are stable at rest. Addition of surfactants is a practical generic means to prevent this stress-induced aggregation (e.g. in formulations of therapeutic proteins), which suggests that interfaces contribute to destabilization. We studied here the role of interfacial stress by applying brief mechanical impacts on the air–water interface, in the presence or absence of surfactants, in solutions of immunoglobulin G (IgG), a class of proteins of high importance to the developments of new therapeutics. A variety of surfactants was tested including the neutral ones Tween80, C10–C14 fos-cholines, alkylaminoxide, surfactin, and two ionic ones, TTAB and lauroylsarcosine sodium salt. We determined the presence of aggregates in solution by light scattering. Irrespective of the type of antibody, either human polyclonal or a monoclonal one, we show that the amount of aggregated IgG increases in proportion to the number of impacts on the interface. In the absence of stress, we recorded images of oblate aggregates of IgG (ca. 12 nm height and 200–1200 nm diameter) present at the air–water interface (fluorescence microscopy using anti-Fab or anti-Fc markers, and AFM scans after transfer on freshly cleaved mica). Our results evidence that aggregates are formed at the air–water interface, and are brought in solution by transient stresses applied on the water surface. Rupture of interfacial films is an important source of aggregates in solution. Finally, the role of surface dynamics in the protection brought by surfactants is discussed based on the comparison of protective efficiencies with dynamic surface tension properties (measured by the maximum bubble pressure method). Our work indicates that better protection is conferred by surfactants showing the faster interfacial dynamics, which corresponds also to conditions of faster lowering of the interfacial energy at a short time scale.

Enhancement and Modulation of Enzymatic Activity through Higher‐Order Structural Changes of Giant DNA–Protein Multibranch Conjugates

Conjugating an oligonucleotide to a protein is a widely used strategy to combine the protein function with the recognition ability provided by the short DNA fragment. On the other hand, genomic DNA molecules, owing to their extremely large size, have the unique ability to undergo dramatic higherorder structural changes upon addition of compaction/unfolding agents, but the influence of these structural changes on a conjugated protein has never been explored. Herein, we describe the first preparation of giant DNA–protein multibranch conjugates and study how regulated higher-order structural changes of DNA can control the function of the protein. These conjugates are composed of a b-lactamase enzyme attached to one to four 48.5 kbp lambda phage DNA (l DNA) “branches”. We show that the conjugation of giant DNA increases the enzymatic activity, which can be decreased by compaction with spermine and recovered by unfolding with NaCl. Conjugating DNA to a protein is a common strategy in modern biotechnology. It is however usually done with DNA in the form of short oligonucleotides (up to several tens of bases), which are used to recognize complementary nucleic acid targets. Such protein–oligonucleotide conjugates have led to many biosensing applications as probes for the presence of nucleic acids or proteins 2] and to the preparation of protein microarrays 3] or protein complexes by DNAmediated assembly. Additionally, DNA scaffolds with welldefined 2D and 3D nanostructures, for instance made by using DNA origami, have been used to precisely organize conjugated proteins at the nanometer scale. 7] In this approach, the DNA scaffold is usually made of a highly ordered DNA structure with characteristic dimensions up to a few hundred nanometers. However, to our knowledge, the behavior of proteins, and more specifically of enzymes, conjugated to giant (greater than 10 kbp) duplex DNA molecules has never been investigated. We prepared for the first time giant DNA–protein hybrid conjugates where a blactamase enzyme was included in the center of a branched complex composed of up to four l DNA (48.5 kbp) molecules. We studied the activity of the enzyme inside these conjugates. Since giant DNA molecules can undergo a dramatic and reversible higher-order structural transition between an unfolded, elongated coil state and a very dense, compact state, we investigated how the enzymatic activity was regulated through such higher-order structural changes. First, we studied the possibility of attaching a relatively short duplex DNA to b-lactamase using the streptavidin– biotin interaction. 12] This was done by assembling a streptavidin–b-lactamase conjugate (S-bLac) with a monobiotinylated 541 bp DNA (B-DNA541, Figure 1 a). S-bLac had a hydrodynamic diameter of about 6 nm according to DLS measurements (Supporting Information, Figure S1) while BDNA541 was expected to have a diameter of 2 nm and a contour length of about 184 nm. Atomic force microscopy (AFM) observations after deposition on mica revealed the presence of mainly four kinds of structures composed of a higher central part surrounded by one to four branches with a length of 173 9 nm (Figure 1 a, right panels). The central part and branches were thus attributed to S-bLac and conjugated DNA molecules, respectively. We also observed a few other structures with a larger number (5–14) of branches (Figure S2), which could be attributed to the presence of aggregates or streptavidin/b-lactamase ratios different than 1:1 in the starting material. Our results show that the streptavidin–biotin interaction allows for the attachment of one to four duplex DNAs to S-bLac, in agreement with previously reported work using unconjugated streptavidin. Next, we applied a similar strategy with biotinylated l DNA instead of B-DNA541. In this case, we could not observe any branched structures, neither by AFM nor by fluorescence microscopy. The difficulty to directly conjugate several biotinylated l DNAs to S-bLac could be attributed to steric hindrance or electrostatic repulsion between the giant l DNA molecules. We thus devised a two-step conjugation method (Figure 1 b). First, a short, 12 nt biotinylated oligonucleotide (B-oligo) complementary to one of the sticky ends of l DNA was attached to S-bLac. The resulting conjugates (bLac-oligo) were then mixed with a fourfold excess of [*] Dr. S. Rudiuk, A. Venancio-Marques, Prof. D. Baigl Department of Chemistry, Ecole Normale Sup rieure 24 rue Lhomond, 75005 Paris (France) and Universit Pierre et Marie Curie Paris 6 75005 Paris (France) and UMR 8640, CNRS (France) E-mail: damien.baigl@ens.fr Homepage: http://www.baigllab.com/

Photosensitive polyamines for high-performance photocontrol of DNA higher-order structure.

Polyamines are small, ubiquitous, positively charged molecules that play an essential role in numerous biological processes such as DNA packaging, gene regulation, neuron activity, and cell proliferation. Here, we synthesize the first series of photosensitive polyamines (PPAs) and demonstrate their ability to photoreversibly control nanoscale DNA higher-order structure with high efficiency. We show with fluorescence microscopy imaging that the efficiency of the PPAs as DNA-compacting agents is directly correlated to their molecular charge. Micromolar concentration of the most efficient molecule described here, a PPA containing three charges at neutral pH, compacts DNA molecules from a few kilobase pairs to a few hundred kilobase pairs, while subsequent 3 min UV illuminations at 365 nm triggers complete unfolding of DNA molecules. Additional application of blue light (440 nm for 3 min) induces the refolding of DNA into the compact state. Atomic force microscopy reveals that the compaction involves a global folding of the whole DNA molecule, whereas UV-induced unfolding is a modification initiated from the periphery of the compacted DNA, resulting in the occurrence of intermediate flower-like structures prior to the fully unfolded state.

Light-Directed Particle Patterning by Evaporative Optical Marangoni Assembly

Controlled particle deposition on surfaces is crucial for both exploiting collective properties of particles and their integration into devices. Most available methods depend on intrinsic properties of either the substrate or the particles to be deposited making them difficult to apply to complex, naturally occurring or industrial formulations. Here we describe a new strategy to pattern particles from an evaporating drop, regardless of inherent particle characteristics and suspension composition. We use light to generate Marangoni surface stresses resulting in flow patterns that accumulate particles at predefined positions. Using projected images, we generate a broad variety of complex patterns, including multiple spots, lines and letters. Strikingly, this method, which we call evaporative optical Marangoni assembly (eOMA), allows us to pattern particles regardless of their size or surface properties, in model suspensions as well as in complex, real-world formulations such as commercial coffee.

Protein Adsorption and Reorganization on Nanoparticles Probed by the Coffee-Ring Effect: Application to Single Point Mutation Detection

The coffee-ring effect denotes the accumulation of particles at the edge of an evaporating sessile drop pinned on a substrate. Because it can be detected by simple visual inspection, this ubiquitous phenomenon can be envisioned as a robust and cost-effective diagnostic tool. Toward this direction, here we systematically analyze the deposit morphology of drying drops containing polystyrene particles of different surface properties with various proteins (bovine serum albumin (BSA) and different forms of hemoglobin). We show that deposit patterns reveal information on both the adsorption of proteins onto particles and their reorganization following adsorption. By combining pattern analysis with adsorption isotherm and zeta potential measurements, we show that the suppression of the coffee-ring effect and the formation of a disk-shaped pattern is primarily associated with particle neutralization by protein adsorption. However, our findings also suggest that protein reorganization following adsorption can dramatically invert this tendency. Exposure of hydrophobic (respectively charged) residues can lead to disk (respectively ring) deposit morphologies independently of the global particle charge. Surface tension measurements and microscopic observations of the evaporating drops show that the determinant factor of the deposit morphology is the accumulation of particles at the liquid/gas interface during evaporation. This general behavior opens the possibility to probe protein adsorption and reorganization on particles by the analysis of the deposit patterns, the formation of a disk being the robust signature of particles rendered hydrophobic by protein adsorption. We show that this method is sensitive enough to detect a single point mutation in a protein, as demonstrated here by the distinct patterns formed by human native hemoglobin h-HbA and its mutant form h-HbS, which is responsible for sickle cell anemia.

Enhancement of DNA compaction by negatively charged nanoparticles. Application to reversible photocontrol of DNA higher-order structure

We report for the first time that negatively charged silica nanoparticles (NPs) enhance the ability of cationic surfactants to induce genomic DNA compaction. Single-chain compaction of duplex DNA molecules was studied by fluorescence microscopy in the presence of dodecyltrimethylammonium bromide (DTAB) and NPs. We found that very small amounts of NPs (∼10−4 to 10−2 wt%) significantly decreased the concentration of the surfactant at which DNA is compacted. This effect was maximal at intermediate NP concentration (here, 1.5 × 10−3 wt%) where the concentration of DTAB necessary for DNA compaction was 5-fold smaller than that in the absence of NPs. As a possible mechanism, we suggest that negatively charged NPs, by inducing the aggregation of DTAB molecules through electrostatic interactions, promote cooperative binding to DNA and thus enhance the ability of DTAB to compact DNA. By applying this phenomenon to a photosensitive cationic surfactant (AzoTAB), we could achieve reversible control of DNA higher-order structure using light at a much lower AzoTAB concentration than what has been reported up to now.

Modulation of photo-oxidative DNA damage by cationic surfactant complexation.

The natural packaging of DNA in the cell by histones provides a particular environment affecting its sensitivity to oxidative damage. In this work, we used the complexation of DNA by cationic surfactants to modulate the conformation, the dynamics, and the environment of the double helix. Photo-oxidative damage initiated by benzophenone as the photosensitizer on a plasmid DNA complexed by dodecyltrimethylammonium chloride (DTAC), tetradecyltrimethylammonium chloride (TTAC), cetyltrimethyammonium chloride (CTAC) and bromide (CTAB) was detected by agarose gel electrophoresis. By fluorescent titration in the presence of ethidium bromide (EB) and agarose gel electrophoresis, we experimentally confirmed the complexation diagrams with a critical aggregation concentration on DNA matrix (CAC DNA) delimiting two regions of complexation, according to the DNA-phosphate concentration. The study of the photo-oxidative damage shows, for the first time, a direct correlation between the DNA complexation by these surfactants and the efficiency of DNA cleavage, with a maximum corresponding to the CAC DNA for DTAC and CTAC, and to DNA neutralization for CTAC and CTAB. The localization of a photosensitizer having low water solubility, such as benzophenone, inside the hydrophobic domains formed by the surfactant aggregated on DNA, locally increases the photoinduced cleavage by the free radical oxygen species generated. The inefficiency of a water-soluble quencher of hydroxyl radicals, such as mannitol, confirmed this phenomenon. The detection of photo-oxidative damage constitutes a new tool for investigating DNA complexation by cationic surfactants. Moreover, highlighting the drastically increased sensitivity of a complexed DNA to photo-oxidative damage is of crucial importance for the biological use of surfactants as nonviral gene delivery systems.

Photodependent Melting of Unmodified DNA Using a Photosensitive Intercalator: A New and Generic Tool for Photoreversible Assembly of DNA Nanostructures at Constant Temperature

External control of DNA melting and hybridization, a key step in bio- and DNA nanotechnology, is commonly achieved with temperature. The use of light to direct this process is a challenging alternative, which has been only possible with a DNA modification, such as covalent grafting or mismatch introduction, so far. Here we describe the first photocontrol of DNA melting that relies on the addition of a molecule that noncovalently interacts with unmodified DNA and affects its melting properties in a photoreversible and highly robust manner, without any prerequisite in the length or sequence of the target DNA molecule. We synthesize azobenzene-containing guanidinium derivatives and show that a bivalent molecule with a conformation-dependent binding mode, AzoDiGua, strongly increases the melting temperature (Tm) of DNA under dark conditions because its trans isomer intercalates in the DNA double helix. Upon UV irradiation at 365 nm, the trans-cis isomerization induced the ejection of AzoDiGua from the intercalation binding sites, resulting in a decrease in Tm up to 18 °C. This illumination-dependent Tm shift is observed on many types of DNA, from self-complementary single-stranded or double-stranded oligonucleotides to long genomic duplex DNA molecules. Finally, we show that simply adding AzoDiGua allows us to photoreversibly control the assembly/disassembly of a DNA nanostructure at constant temperature, as demonstrated here with a self-hybridized DNA hairpin. We anticipate that this strategy will be the key ingredient in a new and generic way of placing DNA-based bio- and nanotechnologies under dynamic control by light.

Enhancement of DNA compaction by negatively charged nanoparticles: effect of nanoparticle size and surfactant chain length.

We study the compaction of genomic DNA by a series of alkyltrimethylammonium bromide surfactants having different hydrocarbon chain lengths n: dodecyl-(DTAB, n=12), tetradecyl-(TTAB, n=14) and hexadecyl-(CTAB, n=16), in the absence and in the presence of negatively charged silica nanoparticles (NPs) with a diameter in the range 15-100 nm. We show that NPs greatly enhance the ability of all cationic surfactants to induce DNA compaction and that this enhancement increases with an increase in NP diameter. In the absence of NP, the ability of cationic surfactants to induce DNA compaction increases with an increase in n. Conversely, in the presence of NPs, the enhancement of DNA compaction increases with a decrease in n. Therefore, although CTAB is the most efficient surfactant to compact DNA, maximal enhancement by NPs is obtained for the largest NP diameter (here, 100 nm) and the smallest surfactant chain length (here, DTAB). We suggest a mechanism where the preaggregation of surfactants on NP surface mediated by electrostatic interactions promotes cooperative binding to DNA and thus enhances the ability of surfactants to compact DNA. We show that the amplitude of enhancement is correlated with the difference between the surfactant concentration corresponding to aggregation on DNA alone and that corresponding to the onset of adsorption on nanoparticles.