Antoine Diguet

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.

Photosensitive surfactants with various hydrophobic tail lengths for the photocontrol of genomic DNA conformation with improved efficiency.

We report the synthesis and characterisation of photosensitive cationic surfactants with various hydrophobic tail lengths. These molecules, called AzoCx, are used as photosensitive nucleic acid binders (pNABs) and are applied to the photocontrol of DNA conformation. All these molecules induce DNA compaction in a photodependent way, originating in the photodependent polarity of their hydrophobic tails. We show that increasing hydrophobicity strongly enhances the compaction efficiencies of these molecules, but reduces the possibility of reversible photocontrol of a DNA conformation. Optimal performance was achieved with AzoC5, which allowed reversible control of DNA conformation with light at a concentration seven times smaller than previously reported.

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.

Preparation of phospholipid multilayer patterns of controlled size and thickness by capillary assembly on a microstructured substrate.

By dragging a phospholipid solution on microstructured silicon surfaces, phospholipid molecules are selectively deposited inside the microstructures to get regular phospholipid multilayer patterns of controlled thickness over a large scale ( approximately cm(2)). By varying the dragging speed, the thickness of the patterns varies between 28 and 100 nm on average (7 to 25 bilayers). Electroswelling of phospholipid multilayer patterns leads to the formation of giant liposomes of controlled size and narrow size distributions.

An intrinsically fluorescent glycoligand for direct imaging of ligand trafficking in artificial and living cell systems

Glycoligands, sugar-based molecules able to complex metal cations, constitute a new class of molecules with great potential for biological and biochemical applications. To analyze their behaviour in a biological environment, we have synthesized an intrinsically fluorescent glycoligand and analyzed its trafficking in both living (U937 human cancer cells) and artificial (giant unilamellar vesicles) cell systems. We have found that this ligand has moderate cytotoxicity accompanied by specific accumulation in both living and reconstituted membranes, which it can cross to reach inner compartments.

Modification-Free Photocontrol of β-Lactam Conversion with Spatiotemporal Resolution

β-Lactams can be converted into β-amino acids by β-lactamase, a bacterial enzyme, leading to significant change in the biological function of the substrate molecules. Here we describe a method for photocontrol of β-lactam conversion without gene nor enzyme modification. This is achieved by the addition of a cationic photosensitive surfactant, AzoTAB, to a gene expression medium containing DNA coding for β-lactamase, the enzyme capable of the desired conversion. In the absence of UV (365 nm) or after illumination by blue light (480 nm) for 4 min, conversion of β-lactam is strongly reduced while the application of UV for 4 min results in a strong enhancement of substrate conversion. Several cycles of activation/inhibition are obtained upon successive UV/blue light illuminations. When both reconstituted photoresponsive gene expression medium and β-lactamase substrate are encapsulated in independent microfluidic chambers, selective UV illumination results in spatially resolved activation of substrate conversion.

Control of the Compaction/Unfolding Transition of Genomic DNA by the Addition/Disruption of Lipid Assemblies

We studied the interaction between individual long genomic DNA molecules and cationic lipid assemblies. The assembly of cationic lipid molecules into small unilamellar vesicles (SUVs) of about 50 nm diameter led to the compaction of DNA whereas the addition of a neutral surfactant resulted in the disruption of SUVs and the unfolding of DNA. This reversible process does not require any chemical reaction or change in the ionic strength of the solution. It was applied to switch DNA repeatedly between a compact and an unfolded conformation in a dynamic manner.