Detection of Short DNA Sequences with DNA Nanopores.

Several studies suggest strong correlation between different types of cancer and the relative concentration of short circulating RNA sequences (miRNA). Because of short length and low concentration, miRNA detection is not easy. Standard methods such as RT-PCR require both the standard PCR amplification step and a preliminary additional step of reverse transcription. In this paper, we investigate the use of DNA nanopores as a tool to detect short oligonucleotide sequences at the single molecule level. These nanostructures show two different conformations depending on the presence of DNA analogues of miRNA sequences. By monitoring current across a lipid bilayer, we show that this change of conformation translates to different levels of conductance.

Thickness determination in anisotropic media with plasmon waveguide resonance imaging.

This paper describes a simple procedure to determine the local thickness of a thin anisotropic layer. It also discriminates between isotropic and anisotropic regions, provided a smoothness hypothesis on the refractive index distribution is satisfied. The procedure is based on the analysis of surface plasmon resonance (SPR) data acquired in an imaging mode. The general arrangement of the setup is the Kretschmann configuration. We show, on an azobenzene modified polymer layer, good agreement between atomic force microscopy and optical measurements of thickness variation.

Signal replication in a DNA nanostructure.

Logic circuits based on DNA strand displacement reaction are the basic building blocks of future nanorobotic systems. The circuits tethered to DNA origami platforms present several advantages over solution-phase versions where couplings are always diffusion-limited. Here we consider a possible implementation of one of the basic operations needed in the design of these circuits, namely, signal replication. We show that with an appropriate preparation of the initial state, signal replication performs in a reproducible way. We also show the existence of side effects concomitant to the high effective concentrations in tethered circuits, such as slow leaky reactions and cross-activation.

G-Quadruplexes Light up Localized DNA Circuits.

DNA circuits tethered to nanoplatforms can perform cascade reactions for signal amplification. One DNA single strand activates a strand-displacement cascade generating numerous outputs, and therefore amplifying the signal. These localized circuits present, however, an important limitation: the spontaneous activation of the cascade reaction. Current methods to stabilize these circuits employ combination of protective DNA strands, which need to be removed to activate the device. This protection-deprotection process generates an important amount of unwanted side reactions. This is indeed an important limitation for the large potential application of these amplification circuits. In the present work, G-quadruplex DNA structures were used to stabilize localized DNA circuits. This new protocol generates nanoplatforms that no longer requires protective-deprotective systems and is therefore completely neutral to the sample. In addition, cations such as Pb(2+) or Ca(2+) can be also employed to activate the device enlarging the potential applications from biosensors devices to metal detector sensors.

Time-lapse scanning surface plasmon microscopy of living adherent cells with a radially polarized beam.

We report on a fibered high-resolution scanning surface plasmon microscope for long term imaging of living adherent cells. The coupling of a high numerical aperture objective lens and a fibered heterodyne interferometer enhances both the sensitivity and the long term stability of this microscope, allowing for time-lapse recording over several days. The diffraction limit is reached with a radially polarized illumination beam. Adherence and motility of living C2C12 myoblast cells are followed for 50 h, revealing that the dynamics of these cells change after 10 h. This plasmon enhanced evanescent wave microscopy is particularly suited for investigating cell adhesion, since it can not only be performed without staining of the sample but it can also capture in real time the exchange of extracellular matrix elements between the substrate and the cells.

Plasmon-based tomographic microscopy.

The imaging principle of the scanning surface plasmon microscope (SSPM) springs from the high sensitivity of surface plasmons to modifications of material properties near the dielectric-metal interface. In this paper, we show that tomographic techniques can be applied to SSPM imaging of dielectric objects to reach resolutions beyond the diffraction-limited half-wavelength scale. Furthermore, this high resolution is not limited to the multiple scattering regime. Finally, we conclude that SSPM is less sensitive to noise because it provides higher contrast ratio than other far-field microscopies.

Evidence for cardiolipin binding sites on the membrane-exposed surface of the cytochrome bc1.

The respiratory chain is located in the inner membrane of mitochondria and produces the major part of the ATP used by a cell. Cardiolipin (CL), a double charged phospholipid composing ~10-20% of the mitochondrial membrane, plays an important role in the function and supramolecular organization of the respiratory chain complexes. We present an extensive set of coarse-grain molecular dynamics (CGMD) simulations aiming at the determination of the preferential interfaces of CLs on the respiratory chain complex III (cytochrome bc(1), CIII). Six CL binding sites are identified, including the CL binding sites known from earlier structural studies and buried into protein cavities. The simulations revealed the importance of two subunits of CIII (G and K in bovine heart) for the structural integrity of these internal CL binding sites. In addition, new binding sites are found on the membrane-exposed protein surface. The reproducibility of these binding sites over two species (bovine heart and yeast mitochondria) points to an important role for the function of the respiratory chain. Interestingly the membrane-exposed CL binding sites are located on the matrix side of CIII in the inner membrane and thus may provide localized sources of proton ready for uptake by CIII. Furthermore, we found that CLs bound to those membrane-exposed sites bridge the proteins during their assembly into supercomplexes by sharing the binding sites.

Guided wave microscopy: mastering the inverse problem.

Surface plasmon microscopy is widely recognized for its high sensitivity to nanoscale dielectric or metallic structures confined in a close neighborhood of a gold surface. Recently, its coupling to high-numerical-aperture objective lenses pushed its resolution down to the diffraction limit. Here, we show that the same microscope configuration can be used to excite standing guided waves in asymmetric slabs, which definitely extends the range of applications of this type of microscopy from nano- to microscale structure imaging. We demonstrate experimentally on PPMA films that the V(Z) response of a scanning surface plasmon microscope can be Fourier inverted in order to obtain the reflectivity curve R(ν). When the guided waves are excited, R(ν) shows a finite number of sharp peaks corresponding to quantified guiding modes from which one can extract both the refractive index (RI) and the thickness of the layer at the point focused by the microscope. This device can thus be used to reconstruct RI and thickness contours of dielectric samples with a high spatial resolution.

Cooperativity in the annealing of DNA origamis.

DNA based nanostructures built on a long single stranded DNA scaffold, known as DNA origamis, offer the possibility to organize various molecules at the nanometer scale in one pot experiments. The folding of the scaffold is guaranteed by the presence of short, single stranded DNA sequences (staples), that hold together separate regions of the scaffold. In this paper, we modelize the annealing-melting properties of these DNA constructions. The model captures important features such as the hysteresis between melting and annealing, as well as the dependence upon the topology of the scaffold. We show that cooperativity between staples is critical to quantitatively explain the folding process of DNA origamis.

Mechanical DNA Origami to Investigate Biological Systems.

The ability to self-assemble DNA nanodevices with programmed structural dynamics that can sense and respond to the local environment can enable transformative applications in fields including mechanobiology and nanomedicine. The responsive function of biomolecules is often driven by alterations in conformational distributions mediated by highly sensitive interactions with the local environment. In this review, the current state-of-the-art in constructing complex DNA geometries with dynamic and mechanical properties to enable a molecular scale force measurement is first summarized. Next, an overview of engineering modular DNA devices that interact with cell surfaces is highlighted detailing examples of mechanosensitive proteins and the force-induced dynamic molecular interaction on the downstream biochemical signaling. Finally, the challenges and an outlook on this promising class of DNA devices acting as nanomachines to operate at a low piconewton range suitable for a majority of biological effects or as hybrid materials to achieve higher tension exertion required for other biological investigations, are discussed.

Reverse engineering DNA origami nanostructure designs from raw scaffold and staple sequence lists.

Designs for scaffolded DNA origami nanostructures are commonly and minimally published as the list of DNA staple and scaffold sequences required. In nearly all cases, high-level editable design files (e.g. caDNAno) which generated the low-level sequences are not made available. This de facto 'raw sequence' exchange format allows published origami designs to be re-attempted in the laboratory by other groups, but effectively stops designs from being significantly modified or re-purposed for new future applications. To make the raw sequence exchange format more accessible to further design and engineering, in this work we propose the first algorithmic solution to the inverse problem of converting staple/scaffold sequences back to a 'guide schematic' resembling the original origami schematic. The guide schematic can be used to aid the manual re-input of an origami into a CAD tool like caDNAno, hence recovering a high-level editable design file. Creation of a guide schematic can also be used to double check that a list of staple strand sequences does not have errors and indeed does assemble into a desired origami nanostructure prior to costly laboratory experimentation. We tested our reverse algorithm on 36 diverse origami designs from the literature and found that 29 origamis (81 %) had a good quality guide schematic recovered from raw sequences. Our software is made available at https://revnano.readthedocs.io.

Direct visualization of transient thermal response of a DNA origami.

The DNA origami approach enables the construction of complex objects from DNA strands. A fundamental understanding of the kinetics and thermodynamics of DNA origami assembly is extremely important for building large DNA structures with multifunctionality. Here both experimental and theoretical studies of DNA origami melting were carried out in order to reveal the reversible association/disassociation process. Furthermore, by careful control of the temperature cycling via in situ thermally controlled atomic force microscopy, the self-assembly process of a rectangular DNA origami tile was directly visualized, unveiling key mechanisms underlying their structural and thermodynamic features.

Stability and structure of protein-lipoamino acid colloidal particles: toward nasal delivery of pharmaceutically active proteins.

To circumvent the painful intravenous injection of proteins in the treatment of children with growth deficiency, anemia, and calcium insufficiency, we investigated the stability and structure of protein-lipoamino acid complexes that could be nasally sprayed. Preparations that ensure a colloidal and structural stability of recombinant human growth hormone (rhGH), recombinant human erythropoietin (rhEPO), and salmon calcitonin (sCT) mixed with lauroyl proline (LP) were established. Protein structure was controlled by circular dichroism, and very small sizes of ca. 5 nm were determined by dynamic light scattering. The colloidal preparations could be sprayed with a droplet size of 20-30 µm. The molecular structure of aggregates was investigated by all-atom molecular dynamics. Whereas a lauroyl proline capping of globular proteins rhGH and rhEPO with preservation of their active structure was observed, a mixed micelle of sCT and lipoamino acids was formed. In the latter, aggregated LP constitutes the inner core and the surface is covered with calcitonins that acquire a marked α-helix character. Hydrophobic/philic interaction balance between proteins and LP drives the particles' stability. Passage through nasal cells grown at confluence was markedly increased by the colloidal preparations and could reach a 20 times increase in the case of EPO. Biological implications of such colloidal preparations are discussed in terms of furtiveness.

Folding of small origamis.

A model that preserves the known thermodynamic properties of double stranded DNA is introduced to study the formation of more complex DNA constructions, such as small origamis or Holliday junctions. We show that the thermodynamic behaviour of these complex DNA constructions is not only given by their sequence but also by their topology.

Modeling of the scanning surface plasmon microscope.

We propose a family of exact solutions of Maxwell's equations to model some aspects of the imaging process involved in the scanning surface plasmon microscope (SSPM). More precisely, we compute the SSPM response of a spherical nanoparticle immobilized close to a thin gold layer and illuminated by a tightly focused spot. We discuss the influence of parameters such as the defocus and the width of the gold layer on the image contrast. We show that this microscopy combines a subwavelength spatial resolution together with high sensitivity to small changes in dielectric properties on the nanoparticle.

Pore formation induced by an antimicrobial peptide: electrostatic effects.

We investigate the mode of action of Cateslytin, an antimicrobial peptide, on zwitterionic biomembranes by performing numerical simulations and electrophysiological measurements on membrane vesicles. Using this natural beta-sheet antimicrobial peptide secreted during stress as a model we show that a single peptide is able to form a stable membrane pore of 1 nm diameter of 0.25 nS conductance found both from calculation and electrical measurements. The resulting structure does not resemble the barrel-stave or carpet models earlier predicted, but is very close to that found in the simulation of alpha-helical peptides. Based on the simulation of a mutated peptide and the effects of small external electric fields, we conclude that electrostatic forces play a crucial role in the process of pore formation.

Spectral dependence of plasmon-enhanced fluorescence in a hollow nanotriangle assembled by DNA origami: towards plasmon assisted energy transfer.

The precise positioning of plasmonic nanoscale objects and organic molecules can significantly boost our ability to fabricate hybrid nanoarchitectures with specific target functionalities. In this work, we used a DNA origami structure to precisely localize three different fluorescent dyes close to the tips of hollow gold nanotriangles. A spectral dependence of plasmon-enhanced fluorescence is evidenced through co-localized AFM and fluorescence measurements. The experimental results match well with explanatory FDTD simulations. Our findings open the way to the bottom-up fabrication of plasmonic routers operating through plasmon energy transfer. They will allow one to actively control the direction of light propagation.

Spectroscopic data for the G-quadruplex DNA to duplex DNA reaction.

This article describes additional data related to a research article entitled "Kinetics of Quadruplex to Duplex Conversion" (Mendoza et al. 2015 [1]). We followed the opening reaction of a series of intramolecular G-quadruplex structures by the addition of their corresponding complementary strand. Fluorolabeled complementary strands allowed to monitor the reaction in real-time. An adapted kinetic model was then applied in order to obtain the kinetic parameters of this reaction. We present a series of kinetic traces providing raw data of the G4 opening reaction and the fitting model applied in every case. In addition CD spectra and UV melting data is also provided to confirm the stability of all the DNA structures considered (G-quadruplex and duplex DNA).

Kinetics of quadruplex to duplex conversion.

The equilibrium between G-quadruplex (G4) and duplex DNA structures is a fundamental question whenever G4 are considered within a genomic context. In this study, we performed a detailed thermodynamic and kinetic analysis of this equilibria using a new fast and high throughput technique for the screening of G4 structures. This assay examines the isothermal stability of G-quadruplexes in the presence of complementary strands monitoring the unwinding process by fluorescence techniques. Unlabelled G4 structures were used in order to avoid any thermodynamic effect that fluorophores could have on the G4 stability. The assay was applied to investigate the effect that flanking sequences can have on the thermodynamic stability of the quadruplex motifs. Interestingly, the presence of adjacent bases to the G4 structure facilitates the recognition of the complementary strand accelerating the G4 unfolding reaction. The simplicity of the systems employed and the use of fluorescence emission allowed the use of high throughput techniques and to monitor the opening reaction in real time in a "pseudo" label-free method. This G4 opening reaction may be easily implemented as a new isothermal assay for the screening of G4 structures, G4 ligands or G4-binding proteins.

Connecting localized DNA strand displacement reactions.

Logic circuits based on DNA strand displacement reactions have been shown to be versatile enough to compute the square root of four-bit numbers. The implementation of these circuits as a set of bulk reactions faces difficulties which include leaky reactions and intrinsically slow, diffusion-limited reaction rates. In this paper, we consider simple examples of these circuits when they are attached to platforms (DNA origamis). As expected, constraining distances between DNA strands leads to faster reaction rates. However, it also induces side-effects that are not detectable in the solution-phase version of this circuitry. Appropriate design of the system, including protection and asymmetry between input and fuel strands, leads to a reproducible behaviour, at least one order of magnitude faster than the one observed under bulk conditions.