André Estevez-Torres

DNA compaction: fundamentals and applications

Compaction is the process in which a large DNA molecule undergoes a transition between an elongated conformation and a very compact form. In nature, DNA compaction occurs to package genomic material inside tiny spaces such as viral capsids and cell nuclei. In vitro, several strategies exist to compact DNA. In this review, we first provide a physico-chemical description of this phenomenon, focusing on the modes of compaction, the types of compaction agents and the chemical and physical parameters that control compaction and its reverse process, decompaction. We then describe three main kinds of applications. First, we show how regulated compaction/decompaction can be used to control gene activity in vitro, with a particular emphasis on the use of light to reversibly control gene expression. Second, we describe several approaches where compaction is used as a way to reversibly protect DNA against chemical, biochemical, or mechanical stresses. Third, we show that compact DNA can be used as a nanostructure template to generate nanomaterials with a well-defined size and shape. We conclude by proposing some perspectives for future biochemical and biotechnological applications and enumerate some remaining challenges that we think worth being undertaken.

Spatial Waves in Synthetic Biochemical Networks

We report the experimental observation of traveling concentration waves and spirals in a chemical reaction network built from the bottom up. The mechanism of the network is an oscillator of the predator-prey type, and this is the first time that predator-prey waves have been observed in the laboratory. The molecular encoding of the nonequilibrium behavior relies on small DNA oligonucleotides that enforce the network connectivity and three purified enzymes that control the reactivity. Wave velocities in the range 80-400 μm min(-1) were measured. A reaction-diffusion model in quantitative agreement with the experiments is proposed. Three fundamental parameters are easy to tune in nucleic acid reaction networks: the topology of the network, the rate constants of the individual reactions, and the diffusion coefficients of the individual species. For this reason, we expect such networks to bring unprecedented opportunities for assaying the principles of spatiotemporal order formation in chemistry.

Sequence-independent and reversible photocontrol of transcription/expression systems using a photosensitive nucleic acid binder.

To understand non-trivial biological functions, it is crucial to develop minimal synthetic models that capture their basic features. Here, we demonstrate a sequence-independent, reversible control of transcription and gene expression using a photosensitive nucleic acid binder (pNAB). By introducing a pNAB whose affinity for nucleic acids is tuned by light, in vitro RNA production, EGFP translation, and GFP expression (a set of reactions including both transcription and translation) were successfully inhibited in the dark and recovered after a short illumination at 365 nm. Our results indicate that the accessibility of the protein machinery to one or several nucleic acid binding sites can be efficiently regulated by changing the conformational/condensation state of the nucleic acid (DNA conformation or mRNA aggregation), thus regulating gene activity in an efficient, reversible, and sequence-independent manner. The possibility offered by our approach to use light to trigger various gene expression systems in a system-independent way opens interesting perspectives to study gene expression dynamics as well as to develop photocontrolled biotechnological procedures.

Photocontrol of single-chain DNA conformation in cell-mimicking microcompartments.

It has been well established that the regulation of gene activity is strongly dependent on the higher-order structure of genomic DNA molecules. Several strategies have thus been developed to control the higher-order structure of long DNA molecules. Most of them have been based on the use of chemical compounds that bind to DNA to neutralize its charge, such as polyamines, multivalent metal cations, cationic surfactants, cationic polymers, nanoparticles, or crowding agents such as hydrophilic polymers. Depending on the concentration of these additives, DNA exhibits a folded or unfolded conformation. Nevertheless, with all these strategies, it is impossible to act in a reversible way on the DNA higher-order structure under a constant chemical composition. Moreover, for transfection applications, compacting DNA is an essential step to allow the entry of DNA into the cell. In most cases, however, DNA remains in a compact conformation inside the cell, which can significantly alter the DNA gene expression. Using an external stimulus to control DNA higherorder structure within a cell-sized compartment has thus became an important challenge. On the other hand, motivated by the perspective of DNA vectorization, preparation of artificial cells or biochemical microreactors, many scientists have attempted to encapsulate DNA into cell-like microcompartments, for example, cellsized liposomes or phospholipid-coated microdroplets. Consequently, various successful strategies have been proposed to prepare DNA–liposome complexes or encapsulate DNA inside liposomes. In most cases encapsulated DNA molecules were typically smaller than a few thousands base pairs. However, in nature, genomic DNA molecules can be much larger, up to hundreds of kbp (kilo base pairs). To the best of our knowledge, no method has been proposed to encapsulate efficiently, in a controlled way, and without degradation, DNA molecules that are larger than 1 kbp into cell-sized liposomes. One paper reported the encapsulation of T4 DNA molecules, but the data were not sufficient to draw conclusions about the integrity of encapsulated DNA chains. Another strategy was to encapsulate DNA in a compact state, but DNA molecules remained in their compact state once they were encapsulated. Very recently, Le Ny and Lee made a breakthrough by proposing a system where DNA higher-structure can be controlled by light in a reversible manner. This was achieved by adding to a DNA solution a photosensitive cationic surfactant, azobenzene trimethylammonium bromide surfactant (AzoTAB). The apolar tail of the surfactant contains an azo group, which is mainly in the trans (more hydrophobic) conformation under visible conditions. Under UV illumination (365 nm), the azo group photoisomerizes into the cis (more hydrophilic) conformation. They demonstrated that there exists an AzoTAB concentration range for which DNA is in the compact state under dark/visible conditions but in the unfolded state under UV illumination, that is, DNA higher-order structure can be controlled by light. In this study, the authors mainly characterized the average property of many DNA chains in solution. Here, we characterized the single-chain conformational behavior of long genomic DNA as a function of AzoTAB concentration and time of UV illumination. We established that the transition has a first-order character at the single-chain level. We studied the single-chain unfolding upon UV illumination and evidenced two mechanisms of single-chain DNA unfolding. Then we applied this strategy to unfold genomic DNA molecules that are encapsulated in cell-mimicking microcompartments. To this end, DNA that was compacted by AzoTAB under visible conditions was encapsulated into cell-sized microdroplets that were coated with various phospholipids prior to UV light exposition. We studied the unfolding process of individual DNA molecules inside the microdroplets. We could successfully unfold most of the DNA molecules when the phospholipid was anionic (DOPS phospholipid). We thus demonstrated how an external stimulus, here light, can be used to control the higher-order structure of individual genomic DNA molecules within cell-sized phospholipid-coated microcompartments. By using fluorescence microscopy (FM), we characterized the conformation of individual T4 DNA molecules at a very low DNA concentration (0.1 mm) in Tris–HCl buffer (10 mm, pH 7.4). To the DNA solution, we added azobenzene trimethylammonium bromide (AzoTAB, Figure 1A) at various concentrations under dark conditions (most of AzoTAB molecules are in the trans conformation, that is, more hydrophobic). Depending on AzoTAB concentration, we distinguished three regimes with re[a] M. Geoffroy, Dr. D. Baigl Department of Chemistry, Ecole Normale Sup rieure 24 rue Lhomond, 75005 Paris (France) Fax: (+33)1-4432-2402 E-mail : damien.baigl@ens.fr [b] Prof. M. Sollogoub, S. Guieu Institut de Chimie Mol culaire (FR2769) Laboratoire de Chimie Organique (UMR CNRS 7611) UPMC Universit Paris 06 4, Place Jussieu, C. 181, 75005 Paris (France) [c] Dr. A. Yamada, Dr. A. Est vez-Torres, Prof. K. Yoshikawa Department of Physics, Kyoto University Kitashirakawa oiwake-cho, Sakyo-ku, Kyoto 606-8502 (Japan) [d] Dr. A. Est vez-Torres, Prof. K. Yoshikawa, Dr. D. Baigl Spatio-Temporal Order Project, ICORP JST (Japan Science and Technology Agency) Kyoto University, Kyoto 606-8502 (Japan) A video clip is available as Supporting information on the WWW under http://www.chembiochem.org or from the author.

A microfluidic device for continuous cancer cell culture and passage with hydrodynamic forces

We demonstrate a novel and robust microfluidic chip with combined functions of continuous culture and output of PC-3 prostate cancer cells. With digital controls, polydimethylsiloxane (PDMS) flexible diaphragms are able to apply hydrodynamic shear forces on cultures, detaching a fraction of attached cancer cells from the surface for output while leaving others for reuse in subsequent cultures. The fractions of detached cells and remaining cells can be precisely controlled. The system has not only the advantages of small size, high cell culture efficiency, and digital control, but also of simple fabrication at low cost, easy operation and robust performance. The chip performs 9 passages during 30 days of continuous culture and shows promise as a durable design suitable for long-term cell output.

High-throughput and long-term observation of compartmentalized biochemical oscillators

We report the splitting of an oscillating DNA circuit into ∼700 droplets with picoliter volumes. Upon incubation at constant temperature, the droplets display sustained oscillations that can be observed for more than a day. Superimposed to the bulk behaviour, we find two intriguing new phenomena - slow desynchronization between the compartments and kinematic spatial waves - and investigate their possible origin. This approach provides a route to study the influence of small volume effects in biology, and paves the way to technological applications of compartmentalized molecular programs controlling complex dynamics.

Microscopic agents programmed by DNA circuits

Information stored in synthetic nucleic acids sequences can be used in vitro to create complex reaction networks with precisely programmed chemical dynamics. Here, we scale up this approach to program networks of microscopic particles (agents) dispersed in an enzymatic solution. Agents may possess multiple stable states, thus maintaining a memory and communicate by emitting various orthogonal chemical signals, while also sensing the behaviour of neighbouring agents. Using this approach, we can produce collective behaviours involving thousands of agents, for example retrieving information over long distances or creating spatial patterns. Our systems recapitulate some fundamental mechanisms of distributed decision making and morphogenesis among living organisms and could find applications in cases where many individual clues need to be combined to reach a decision, for example in molecular diagnostics.

Pressure-assisted selective preconcentration in a straight nanochannel.

We investigate the preconcentration profiles of a fluorescein and bovine serum albumin derivatized with this fluorescent tag in a microfluidic chip bearing a nanoslit. A new preconcentration method in which a hydrodynamic pressure is added to both electroosmotic and electrophoretic contributions is proposed to monitor the location of the preconcentration frontline. A simple predictive model of this pressure-assisted electropreconcentration is proposed for the evolution of the flow profile along this micro/nano/microfluidic structure. We show with a small analyte such as fluorescein that the additional hydrostatic pressure mode enables to stabilize the concentration polarization (CP) effect, resulting in better control of the cathodic focusing (CF) peak. For BSA (bovine serum albumin), we exhibit that the variation of the hydrodynamic pressure can have an even more drastic effect on the preconcentration. We show that, depending on this hydrodynamic pressure, the preconcentration can be chosen, either in the cathodic side or in the anodic one. For the first time, we prove here that both anodic focusing (AF) and cathodic focusing (CF) regimes can be reached in the same structures. These results also open new routes for the detection and the quantification of low abundance biomarkers.

Observing and Controlling the Folding Pathway of DNA Origami at the Nanoscale

DNA origami is a powerful method to fold DNA into rationally designed nanostructures that holds great promise for bionanotechnology. However, the folding mechanism has yet to be fully resolved, principally due to a lack of data with single molecule resolution. To address this issue, we have investigated in detail, using atomic force microscopy, the morphological evolution of hundreds of individual rectangular origamis in solution as a function of temperature. Significant structural changes were observed between 65 and 55 °C both for folding and melting, and six structural intermediates were identified. Under standard conditions, folding was initiated at the edges of the rectangle and progressed toward the center. Melting occurred through the reverse pathway until the structures were significantly disrupted but ended through a different pathway involving out-of-equilibrium chainlike structures. Increasing the relative concentration of center to edge staples dramatically modified the folding pathway to a mechanism progressing from the center toward the edges. These results indicate that the folding pathway is determined by thermodynamics and suggest a way of controlling it.

Automated design of programmable enzyme-driven DNA circuits

Molecular programming allows for the bottom-up engineering of biochemical reaction networks in a controlled in vitro setting. These engineered biochemical reaction networks yield important insight in the design principles of biological systems and can potentially enrich molecular diagnostic systems. The DNA polymerase-nickase-exonuclease (PEN) toolbox has recently been used to program oscillatory and bistable biochemical networks using a minimal number of components. Previous work has reported the automatic construction of in silico descriptions of biochemical networks derived from the PEN toolbox, paving the way for generating networks of arbitrary size and complexity in vitro. Here, we report an automated approach that further bridges the gap between an in silico description and in vitro realization. A biochemical network of arbitrary complexity can be globally screened for parameter values that display the desired function and combining this approach with robustness analysis further increases the chance of successful in vitro implementation. Moreover, we present an automated design procedure for generating optimal DNA sequences, exhibiting key characteristics deduced from the in silico analysis. Our in silico method has been tested on a previously reported network, the Oligator, and has also been applied to the design of a reaction network capable of displaying adaptation in one of its components. Finally, we experimentally characterize unproductive sequestration of the exonuclease to phosphorothioate protected ssDNA strands. The strong nonlinearities in the degradation of active components caused by this unintended cross-coupling are shown computationally to have a positive effect on adaptation quality.