Weak-cooperative binding of a long single-stranded DNA chain on a surface.

Binding gene-wide single-stranded nucleic acids to surface-immobilized complementary probes is an important but challenging process for biophysical studies and diagnostic applications. The challenge comes from the conformational dynamics of the long chain that affects its accessibility and weakens its hybridization to the probes. We investigated the binding of bacteriophage genome M13mp18 on several different 20-mer probes immobilized on the surface of a multi-spot, label-free biosensor, and observed that only a few of them display strong binding capability with dissociation constant as low as 10 pM. Comparing experimental data and computational analysis of the M13mp18 chain structural features, we found that the capturing performance of a specific probe is directly related to the multiplicity of binding sites on the genomic strand, and poorly connected with the predicted secondary and tertiary structure. We show that a model of weak cooperativity of transient bonds is compatible with the measured binding kinetics and accounts for the enhancement of probe capturing observed when more than 20 partial pairings with binding free energy lower than -10 kcal mol-1 are present. This mechanism provides a specific pattern of response of a genomic strand on a panel of properly selected oligomer probe sequences.

Digital Detection of Single Virus Particles by Multi-Spot, Label-Free Imaging Biosensor on Anti-Reflective Glass.

Rapid detection of whole virus particles in biological or environmental samples represents an unmet need for the containment of infectious diseases. Here, an optical device enabling the enumeration of single virion particles binding on antibody or aptamers immobilized on a surface with anti-reflective coating is described. In this regime, nanoparticles adhering to the sensor surface provide localized contributions to the reflected field that become detectable because of their mixing with the interfering waves in the reflection direction. Thus, these settings are exploited to realize a scan-free, label-free, micro-array-type digital assay on a disposable cartridge, in which the virion counting takes place in wide field-of-view imaging. With this approach we could quantify, by enumeration, different variants of SARS-CoV-2 virions interacting with antibodies and aptamers immobilized on different spots. For all tested variants, the aptamers showed larger affinity but lower specificity relative to the antibodies. It is found that the combination of different probes on the same surface enables increasing specificity of detection and dynamic range.

Deformation profiles and microscopic dynamics of complex fluids during oscillatory shear experiments.

Oscillatory shear tests are widely used in rheology to characterize the linear and non-linear mechanical response of complex fluids, including the yielding transition. There is an increasing urge to acquire detailed knowledge of the deformation field that is effectively present across the sample during these tests; at the same time, there is mounting evidence that the macroscopic rheological response depends on the elusive microscopic behavior of the material constituents. Here we employ a strain-controlled shear-cell with transparent walls to visualize and quantify the dynamics of tracers embedded in various cyclically sheared complex fluids, ranging from almost-ideal elastic to yield stress fluids. For each sample, we use image correlation processing to measure the macroscopic deformation field, and echo-differential dynamic microscopy to probe the microscopic irreversible sample dynamics in reciprocal space; finally, we devise a simple scheme to spatially map the rearrangements in the sheared sample, once again without tracking the tracers. For the yield stress sample, we obtain a wave-vector dependent characterization of shear-induced diffusion across the yielding transition, which is accompanied by a three-order-of-magnitude speed-up of the dynamics and by a transition from localized, intermittent rearrangements to a more spatially homogeneous and temporally uniform activity. Our tracking free approach is intrinsically multi-scale, can successfully discriminate between different types of dynamics, and can be automated to minimize user intervention. Applications are many, as it can be translated to other imaging modes, including fluorescence, and can be used with sub-resolution tracers and even without tracers, for samples that provide intrinsic optical contrast.

Yield stress "in a flash": investigation of nonlinearity and yielding in soft materials with an optofluidic microrheometer.

Yield stress materials deform as elastic solids or flow as viscous liquids, depending on the applied stress, which also allows them to trap particles below a certain size or density threshold. To investigate the conditions for such a transition at the microscale, we use an optofluidic microrheometer, based on the scattering of an infrared beam onto a microbead, which reaches forces in the nN scale. We perform creep experiments on a model soft material composed of swollen microgels, determining the limits of linear response and yield stress values, and observe quantitative agreement with bulk measurements. However, the motion of the microbead, both below and above yielding, reflects distinctive microscale features of the surrounding material, whose plastic rearrangements were investigated by us using small, passive tracers.

Backbone-free duplex-stacked monomer nucleic acids exhibiting Watson-Crick selectivity.

We demonstrate that nucleic acid (NA) mononucleotide triphosphates (dNTPs and rNTPs), at sufficiently high concentration and low temperature in aqueous solution, can exhibit a phase transition in which chromonic columnar liquid crystal ordering spontaneously appears. Remarkably, this polymer-free state exhibits, in a self-assembly of NA monomers, the key structural elements of biological nucleic acids, including: long-ranged duplex stacking of base pairs, complementarity-dependent partitioning of molecules, and Watson-Crick selectivity, such that, among all solutions of adenosine, cytosine, guanine, and thymine NTPs and their binary mixtures, duplex columnar ordering is most stable in the A-T and C-G combinations.

A Bit Stickier, a Bit Slower, a Lot Stiffer: Specific vs. Nonspecific Binding of Gal4 to DNA.

Transcription factors regulate gene activity by binding specific regions of genomic DNA thanks to a subtle interplay of specific and nonspecific interactions that is challenging to quantify. Here, we exploit Reflective Phantom Interface (RPI), a label-free biosensor based on optical reflectivity, to investigate the binding of the N-terminal domain of Gal4, a well-known gene regulator, to double-stranded DNA fragments containing or not its consensus sequence. The analysis of RPI-binding curves provides interaction strength and kinetics and their dependence on temperature and ionic strength. We found that the binding of Gal4 to its cognate site is stronger, as expected, but also markedly slower. We performed a combined analysis of specific and nonspecific binding-equilibrium and kinetics-by means of a simple model based on nested potential wells and found that the free energy gap between specific and nonspecific binding is of the order of one kcal/mol only. We investigated the origin of such a small value by performing all-atom molecular dynamics simulations of Gal4-DNA interactions. We found a strong enthalpy-entropy compensation, by which the binding of Gal4 to its cognate sequence entails a DNA bending and a striking conformational freezing, which could be instrumental in the biological function of Gal4.

Non-Langmuir Kinetics of DNA Surface Hybridization.

Hybridization of complementary single strands of DNA represents a very effective natural molecular recognition process widely exploited for diagnostic, biotechnology, and nanotechnology applications. A common approach relies on the immobilization on a surface of single-stranded DNA probes that bind complementary targets in solution. However, despite the deep knowledge on DNA interactions in bulk solution, the modeling of the same interactions on a surface are still challenging and perceived as strongly system dependent. Here, we show that a two-dimensional analysis of the kinetics of hybridization, performed at different target concentrations and probe surface densities by a label-free optical biosensor, reveals peculiar features inconsistent with an ideal Langmuir-like behavior. We propose a simple non-Langmuir kinetic model accounting for an enhanced electrostatic repulsion originating from the surface immobilization of nucleic acids and for steric hindrance close to full hybridization of the surface probes. The analysis of the kinetic data by the model enables quantifying the repulsive potential at the surface, as well as retrieving the kinetic parameters of isolated probes. We show that the strength and the kinetics of hybridization at large probe density can be improved by a three-dimensional immobilization strategy of probe strands with a double-stranded linker.

Platinum-Based Drugs and DNA Interactions Studied by Single-Molecule and Bulk Measurements.

Platinum-containing molecules are widely used as anticancer drugs. These molecules exert cytotoxic effects by binding to DNA through various mechanisms. The binding between DNA and platinum-based drugs hinders the opening of DNA, and therefore, DNA duplication and transcription are severely hampered. Overall, impeding the above-mentioned important DNA mechanisms results in irreversible DNA damage and the induction of apoptosis. Several molecules, including multinuclear platinum compounds, belong to the family of platinum drugs, and there is a body of research devoted to developing more efficient and less toxic versions of these compounds. In this study, we combined different biophysical methods, including single-molecule assays (magnetic tweezers) and bulk experiments (ultraviolet absorption for thermal denaturation) to analyze the differential stability of double-stranded DNA in complex with either cisplatin or multinuclear platinum agents. Specifically, we analyzed how the binding of BBR3005 and BBR3464, two representative multinuclear platinum-based compounds, to DNA affects its stability as compared with cisplatin binding. Our results suggest that single-molecule approaches can provide insights into the drug-DNA interactions that underlie drug potency and provide information that is complementary to that generated from bulk analysis; thus, single-molecule approaches have the potential to facilitate the selection and design of optimized drug compounds. In particular, relevant differences in DNA stability at the single-molecule level are demonstrated by analyzing nanomechanically induced DNA denaturation. On the basis of the comparison between the single-molecule and bulk analyses, we suggest that transplatinated drugs are able to locally destabilize small portions of the DNA chain, whereas other regions are stabilized.

Evidence of liquid crystal-assisted abiotic ligation of nucleic acids.

The emergence of early life must have been marked by the appearance in the prebiotic era of complex molecular structures and systems, motivating the investigation of conditions that could not only facilitate appropriate chemical synthesis, but also provide the mechanisms of molecular selection and structural templating necessary to pilot the complexification toward specific molecular patterns. We recently proposed and demonstrated that these functions could be afforded by the spontaneous ordering of ultrashort nucleic acids oligomers into Liquid Crystal (LC) phases. In such supramolecular assemblies, duplex-forming oligomers are held in average end-to-end contact to form chemically discontinuous but physically continuous double helices. Using blunt ended duplexes, we found that LC formation could both provide molecular selection mechanisms and boost inter-oligomer ligation. This paper provides an essential extension to this notion by investigating the catalytic effects of LC ordering in duplexes with mutually interacting overhangs. Specifically, we studied the influence of LC ordering of 5'-hydroxy-3'-phosphate partially self-complementary DNA 14mers with 3'-CG sticky-ends, on the efficiency of non-enzymatic ligation reaction induced by water-soluble carbodiimide EDC as condensing agent. We investigated the ligation products in mixtures of DNA with poly-ethylene glycol (PEG) at three PEG concentrations at which the system phase separates creating DNA-rich droplets that organize into isotropic, nematic LC and columnar LC phases. We observe remarkable LC-enhanced chain lengthening, and we demonstrate that such lengthening effectively promotes and stabilizes LC domains, providing the kernel of a positive feedback cycle by which LC ordering promotes elongation, in turn stabilizing the LC ordering.

Liquid crystal self-assembly of random-sequence DNA oligomers.

In biological systems and nanoscale assemblies, the self-association of DNA is typically studied and applied in the context of the evolved or directed design of base sequences that give complementary pairing, duplex formation, and specific structural motifs. Here we consider the collective behavior of DNA solutions in the distinctly different regime where DNA base sequences are chosen at random or with varying degrees of randomness. We show that in solutions of completely random sequences, corresponding to a remarkably large number of different molecules, e.g., approximately 10(12) for random 20-mers, complementary still emerges and, for a narrow range of oligomer lengths, produces a subtle hierarchical sequence of structured self-assembly and organization into liquid crystal (LC) phases. This ordering follows from the kinetic arrest of oligomer association into long-lived partially paired double helices, followed by reversible association of these pairs into linear aggregates that in turn condense into LC domains.

Viscoelasticity of nematic liquid crystals at a glance.

Polarised microscopy is shown to be a powerful alternative to light scattering for the determination of the viscoelasticity of aligned nematic liquid crystals. We perform experiments in a wide range of temperatures by using an adapted version of the recently introduced differential dynamic microscopy technique, which enables us to extract scattering information directly from the microscope images. A dynamic analysis of the images acquired in different geometries provides the splay, twist and bend viscoelastic ratios. A static analysis allows a successful determination of the bend elastic constant. All our results are in excellent agreement with those obtained with the far more time-consuming depolarised light scattering techniques. Remarkably, a noteworthy extension of the investigated temperature-range is observed, owing to the lower sensitivity of microscopy to multiple scattered light. Moreover, we show that the unique space-resolving capacities of our method enable us to investigate nematics in the presence of spatial disorder, where traditional light scattering fails. Our findings demonstrate that the proposed scattering-with-images approach provides a space-resolved probe of the local sample properties, applicable also to other optically anisotropic soft materials.

Serum antibody fingerprinting of SARS-CoV-2 variants in infected and vaccinated subjects by label-free microarray biosensor.

Both viral infection and vaccination affect the antibody repertoire of a person. Here, we demonstrate that the analysis of serum antibodies generates information not only on the virus type that caused the infection but also on the specific virus variant. We developed a rapid multiplex assay providing a fingerprint of serum antibodies against five different SARS-CoV-2 variants based on a microarray of virus antigens immobilized on the surface of a label-free reflectometric biosensor. We analyzed serum from the plasma of convalescent subjects and vaccinated volunteers and extracted individual antibody profiles of both total immunoglobulin Ig and IgA fractions. We found that Ig level profiles were strongly correlated with the specific variant of infection or vaccination and that vaccinated subjects displayed a larger quantity of total Ig and a lower fraction of IgA relative to the population of convalescent unvaccinated subjects.

Propagation of chirality in mixtures of natural and enantiomeric DNA oligomers.

Concentrated solutions of ultrashort duplex-forming DNA oligomers may develop various forms of liquid crystal ordering among which is the chiral nematic phase, characterized by a macroscopic helical precession of molecular orientation. The specifics of how chirality propagates from the molecular to the mesoscale is still unclear, both in general and in the case of DNA-based liquid crystals. We have here investigated the onset of nematic ordering and its chiral character in mixtures of natural D-DNA oligomers forming right-handed duplex helices and of mirror symmetric (L-DNA) molecules, forming left-handed helices. Since the nematic ordering of DNA duplexes is mediated by their end-to-end aggregation into linear columns, by controlling the terminals of both enantiomers we could study the propagation of chirality in solutions where the D and L species form mixtures of homochiral columns, and in solutions of heterochiral columns. The two systems behave in markedly different fashion. By adopting a simple model based on nearest-neighbor interactions, we account for the different observed dependence of the chirality of these two systems on the enantiomeric ratio.

Right-handed double-helix ultrashort DNA yields chiral nematic phases with both right- and left-handed director twist.

Concentrated solutions of duplex-forming DNA oligomers organize into various mesophases among which is the nematic (N(∗)), which exhibits a macroscopic chiral helical precession of molecular orientation because of the chirality of the DNA molecule. Using a quantitative analysis of the transmission spectra in polarized optical microscopy, we have determined the handedness and pitch of this chiral nematic helix for a large number of sequences ranging from 8 to 20 bases. The B-DNA molecule exhibits a right-handed molecular double-helix structure that, for long molecules, always yields N(∗) phases with left-handed pitch in the µm range. We report here that ultrashort oligomeric duplexes show an extremely diverse behavior, with both left- and right-handed N(∗) helices and pitches ranging from macroscopic down to 0.3 µm. The behavior depends on the length and the sequence of the oligomers, and on the nature of the end-to-end interactions between helices. In particular, the N(∗) handedness strongly correlates with the oligomer length and concentration. Right-handed phases are found only for oligomers shorter than 14 base pairs, and for the sequences having the transition to the N(∗) phase at concentration larger than 620 mg/mL. Our findings indicate that in short DNA, the intermolecular double-helical interactions switch the preferred liquid crystal handedness when the columns of stacked duplexes are forced at high concentrations to separations comparable to the DNA double-helix pitch, a regime still to be theoretically described.

Twisted Structures in Natural and Bioinspired Molecules: Self-Assembly and Propagation of Chirality Across Multiple Length Scales.

Several biomolecules can form dynamic aggregates in water, whose nanometric structures often reflect the chirality of the monomers in unexpected ways. Their twisted organization can be further propagated to the mesoscale, in chiral liquid crystalline phases, and even to the macroscale, where chiral, layered architectures contribute to the chromatic and mechanical properties of various plant, insect, and animal tissues. At all scales, the resulting organization is determined by a subtle balance among chiral and nonchiral interactions, whose understanding and fine-tuning is fundamental also for applications. We present recent advances in the chiral self-assembly and mesoscale ordering of biological and bioinspired molecules in water, focusing on systems based on nucleic acids or related aromatic molecules, oligopeptides, and their hybrid stuctures. We highlight the common features and key mechanisms governing this wide range of phenomena, together with novel characterization approaches.

Sliding and rolling of yield stress fluid droplets on highly slippery lubricated surfaces.

HYPOTHESIS: Droplets of yield stress fluids (YSFs), i.e. fluids that can flow only if they are subjected to a stress above a critical value and otherwise deform like solids, hardly move on solid surfaces due to their high viscosity. The use of highly slippery lubricated surfaces can shed light on the mobility of YSF droplets, which include everyday soft materials, such as toothpaste or mayonnaise, and biological fluids, such as mucus. EXPERIMENTS: The spreading and mobility of droplets of aqueous solutions of swollen Carbopol microgels were studied on lubricant infused surfaces. These solutions represent a model system of YSFs. Dynamical phase diagrams were established by varying the concentration of the solutions and the inclination angle of the surfaces. FINDINGS: Carbopol droplets deposited on lubricated surfaces could move even at low inclination angles. The droplets were found to slide because of the slip of the flowing oil that covered the solid substrate. However, as the descending speed increased, the droplets rolled down. Rolling was favored at high inclinations and low concentrations. A simple criterion based on the ratio between the yield stress of the Carbopol suspensions and the gravitational stress acting on the Carbopol droplets was found to nicely identify the transition between the two regimes.

DNA-based soft phases.

This chapter reviews the state-of-the-art in the study of molecular or colloidal systems whose mutual interactions are mediated by DNA molecules. In the last decade, the robust current knowledge of DNA interactions has enabled an impressive growth of self-assembled DNA-based structures that depend crucially on the properties of DNA-DNA interactions. In many cases, structures are built on design by exploiting the programmable selectivity of DNA interactions and the modularity of their strength. The study of DNA-based materials is definitely an emerging field in condensed matter physics, nanotechnology, and material science. This chapter will consider both systems that are entirely constructed by DNA and hybrid systems in which latex or metal colloidal particles are coated by DNA strands. We will confine our discussion to systems in which DNA-mediated interactions promote the formation of "phases," that is structures extending on length scales much larger than the building blocks. Their self-assembly typically involves a large number of interacting particles and often features hierarchical stages of structuring. Because of the possibility of fine-tuning the geometry and strength of the DNA-mediated interactions, these systems are characterized by a wide variety of patterns of self-assembly, ranging from amorphous, to liquid crystalline, to crystalline in one, two, or three dimensions.

Phase separation and liquid crystallization of complementary sequences in mixtures of nanoDNA oligomers.

Using optical microscopy, we have studied the phase behavior of mixtures of 12- to 22-bp-long nanoDNA oligomers. The mixtures are chosen such that only a fraction of the sample is composed of mutually complementary sequences, and hence the solutions are effectively mixtures of single-stranded and double-stranded (duplex) oligomers. When the concentrations are large enough, such mixtures phase-separate via the nucleation of duplex-rich liquid crystalline domains from an isotropic background rich in single strands. We find that the phase separation is approximately complete, thus corresponding to a spontaneous purification of duplexes from the single-strand oligos. We interpret this behavior as the combined result of the energy gain from the end-to-end stacking of duplexes and of depletion-type attractive interactions favoring the segregation of the more rigid duplexes from the flexible single strands. This form of spontaneous partitioning of complementary nDNA offers a route to purification of short duplex oligomers and, if in the presence of ligation, could provide a mode of positive feedback for the preferential synthesis of longer complementary oligomers, a mechanism of possible relevance in prebiotic environments.

Copolymer Coatings for DNA Biosensors: Effect of Charges and Immobilization Chemistries on Yield, Strength and Kinetics of Hybridization.

The physical-chemical properties of the surface of DNA microarrays and biosensors play a fundamental role in their performance, affecting the signal's amplitude and the strength and kinetics of binding. We studied how the interaction parameters vary for hybridization of complementary 23-mer DNA, when the probe strands are immobilized on different copolymers, which coat the surface of an optical, label-free biosensor. Copolymers of N, N-dimethylacrylamide bringing either a different type or density of sites for covalent immobilization of DNA probes, or different backbone charges, were used to functionalize the surface of a Reflective Phantom Interface multispot biosensor made of a glass prism with a silicon dioxide antireflective layer. By analyzing the kinetic hybridization curves at different probe surface densities and target concentrations in solution, we found that all the tested coatings displayed a common association kinetics of about 9 × 104 M-1·s-1 at small probe density, decreasing by one order of magnitude close to the surface saturation of probes. In contrast, both the yield of hybridization and the dissociation kinetics, and hence the equilibrium constant, depend on the type of copolymer coating. Nearly doubled signal amplitudes, although equilibrium dissociation constant was as large as 4 nM, were obtained by immobilizing the probe via click chemistry, whereas amine-based immobilization combined with passivation with diamine carrying positive charges granted much slower dissociation kinetics, yielding an equilibrium dissociation constant as low as 0.5 nM. These results offer quantitative criteria for an optimal selection of surface copolymer coatings, depending on the application.

Integrated Optofluidic Chip for Oscillatory Microrheology.

We propose and demonstrate an on-chip optofluidic device allowing active oscillatory microrheological measurements with sub-µL sample volume, low cost and high flexibility. Thanks to the use of this optofluidic microrheometer it is possible to measure the viscoelastic properties of complex fluids in the frequency range 0.01-10 Hz at different temperatures. The system is based on the optical forces exerted on a microbead by two counterpropagating infrared laser beams. The core elements of the optical part, integrated waveguides and an optical modulator, are fabricated by fs-laser writing on a glass substrate. The system performance is validated by measuring viscoelastic solutions of aqueous worm-like micelles composed by Cetylpyridinium Chloride (CPyCl) and Sodium Salicylate (NaSal).