Identification of two-step chemical mechanisms using small temperature oscillations and a single tagged species.

In order to identify two-step chemical mechanisms, we propose a method based on a small temperature modulation and on the analysis of the concentration oscillations of a single tagged species involved in the first step. The thermokinetic parameters of the first reaction step are first determined. Then, we build test functions that are constant only if the chemical system actually possesses some assumed two-step mechanism. Next, if the test functions plotted using experimental data are actually even, the mechanism is attributed and the obtained constant values provide the rate constants and enthalpy of reaction of the second step. The advantage of the protocol is to use the first step as a probe reaction to reveal the dynamics of the second step, which can hence be relieved of any tagging. The protocol is anticipated to apply to many mechanisms of biological relevance. As far as ligand binding is considered, our approach can address receptor conformational changes or dimerization as well as competition with or modulation by a second partner. The method can also be used to screen libraries of untagged compounds, relying on a tracer whose concentration can be spectroscopically monitored.

Identification of two-step chemical mechanisms and determination of thermokinetic parameters using frequency responses to small temperature oscillations.

Increased focus on kinetic signatures in biology, coupled with the lack of simple tools for chemical dynamics characterization, lead us to develop an efficient method for mechanism identification. A small thermal modulation is used to reveal chemical dynamics, which makes the technique compatible with in cellulo imaging. Then, the detection of concentration oscillations in an appropriate frequency range followed by a judicious analytical treatment of the data is sufficient to determine the number of chemical characteristic times, the reaction mechanism, and the full set of associated rate constants and enthalpies of reaction. To illustrate the scope of the method, dimeric protein folding is chosen as a biologically relevant example of nonlinear mechanism with one or two characteristic times.

Unfolding of cytochrome C upon interaction with azobenzene-modified copolymers.

Hydrophilic or amphiphilic macromolecules are common organic matrices used to encapsulate and protect fragile drugs such as proteins. Polymer cargoes are in addition designed for remote control of protein delivery, upon imparting the macromolecules with stimuli-responsive properties, such as light-triggered polarity switches. The effect of interaction between polymers and proteins on the stability of the proteins is, however, rarely investigated. Here we studied the unfolding/folding equilibrium of cytochrome c (cyt c) under its oxidized or reduced forms, in the presence of various amphiphilic copolymers (by circular dichroism and intrinsic fluorescence measurements). As models of stimuli-responsive amphiphilic chains, we considered poly(acrylic acid) derivatives, modified to contain hydrophobic, light-responsive azobenzene moieties. These copolymers are, thus, capable to develop both ionic (under their sodium forms at pH > 8) and hydrophobic associations with the basic protein cyt c (isoelectric point of 10.0). In aqueous buffer upon increasing urea concentrations, cyt c underwent unfolding, at [urea] of 9-10 M, which was analyzed under the framework of the equilibrium between two states (native-unfolded). In the presence of polymers, the native folding of cyt c was preserved at low concentrations of urea (typically <4M). However, the presence of polymers facilitated unfolding, which occurred at urea concentrations lowered by 2-4 M as compared to unfolding in the absence of polymers (polymer/cyt c ratio of 1:1 g/g). The predominant contribution of coulombic interactions was shown by both the lack of significant impact of the amount of (neutral) azobenzene moieties in the copolymers and the disappearance of destabilization at ionic strength higher than 150 mM. In addition, stability was similar to that of an isolated cyt c, in the presence of a neutral chain bearing acryloyl(oligoethyleneoxide) units instead of the ionized sodium acrylate moieties. DSC measurements showed that in the presence of polymers, cyt c is thermally unfolded in aqueous buffer at temperatures lowered by >20 °C as compared to thermal unfolding in the absence of polymers. Upon exposure to UV light, properties of the polymers chains were perturbed in situ, upon cis/trans isomerization of the azobenzene groups. In polymers displaying a photoresponsive polarity and hydrophobicity switch (conventional azobenzene), the stability of cyt c was not affected by the exposure to light. In contrast, when photoionization occurred (using an hydroxyl-azobenzene whose pK(a) can be photoshifted), unfolding was initiated upon exposure to light. Altogether, these results show that coulombic binding is a predominant driving force that facilitates unfolding in water/urea solutions. In regard to the design of light-responsive systems for protein handling and control of folding, we conclude that remote control of the coulombic interaction upon photoionization of chromophores can be more efficient than the more conventional photomodulation of polarity.

Chemical mechanism identification from frequency response to small temperature modulation.

The description of interactions between biochemical species and the elucidation of the corresponding chemical mechanisms encounter an increasing interest both for the comprehension of biological pathways at the molecular scale and for the rationalization of drug design. Relying on powerful experimental tools such as thermal microfluidics and fluorescence detection, we propose a methodology to determine the chemical mechanism of a reaction without fitting parameters. A mechanism consistent with the accessible knowledge is assumed, and the assumption is checked through an iterative protocol. The test is based on the frequency analysis of the response of a targeted reactive species to temperature modulation. We build specific functions of the frequency that are constant for the assumed mechanism and show that the graph of these functions can be drawn from appropriate data analysis. The method is general and can be applied to any complex mechanism. It is here illustrated in detail in the case of single relaxation time mechanisms.

Temperature modulation and quadrature detection for selective titration of two-state exchanging reactants.

Biological samples exhibit huge molecular diversity over large concentration ranges. Titrating a given compound in such mixtures is often difficult, and innovative strategies emphasizing selectivity are thus demanded. To overcome limitations inherent to thermodynamics, we here present a generic technique where discrimination relies on the dynamics of interaction between the target of interest and a probe introduced in excess. Considering an ensemble of two-state exchanging reactants submitted to temperature modulation, we first demonstrate that the amplitude of the out-of-phase concentration oscillations is maximum for every compound involved in a reaction whose equilibrium constant is equal to unity and whose relaxation time is equal to the inverse of the excitation angular frequency. Taking advantage of this feature, we next devise a highly specific detection protocol and validate it using a microfabricated resistive heater and an epifluorescence microscope, as well as labeled oligonucleotides to model species displaying various dynamic properties. As expected, quantification of a sought for strand is obtained even if interfering reagents are present in similar amounts. Moreover, our approach does not require any separation and is compatible with imaging. It could then benefit some of the numerous binding assays performed every day in life sciences.

Fluorescent thermometers for dual-emission-wavelength measurements: molecular engineering and application to thermal imaging in a microsystem.

To facilitate thermal imaging, particularly in microdevices, one has to favor molecular thermometers in which the response is independent of the probe concentration and of the observation setup imperfections. Hence, this paper introduces two temperature fluorescent probes for ratiometric dual-emission-wavelength measurements in aqueous solutions. They are based on a nonathermal chemical reaction, either a conformational transition or a protonation, that induces a modification of their emission spectra as the temperature changes. Relying on both a straightforward theoretical analysis and thorough photophysical, thermodynamic, and kinetic investigations, we demonstrate how the flexible design of these two thermometers can be optimized to face applications with various requirements in terms of operating temperature and wavelength ranges as well as temporal resolution. For instance, the present molecules, which can be used between 5 and 35 degrees C, provide a relative sensitivity up to approximately 9 x 10(-2) K(-1) and milli- to microsecond response times. Finally, we utilize a two-color molecular beacon, a probe belonging to the first series of thermometers, to image temperature profiles in a microfluidic cell heated by a resistive strip. The ratiometric analysis of the fluorescence emission at two different wavelengths is performed on a widely available dual-view microscope, illustrating both the simplicity and reliability of the thermal mapping protocol.

Resonant response to temperature modulation for enzymatic dynamics characterization.

We consider enzymes involved in a three-state Michaelis-Menten kinetics and submitted to well-chosen temperature modulations of small amplitude. From the first-order amplitudes of concentration oscillations, we design a response function that is maximum for targeted values of the chemical relaxation times. This resonant function can be used to screen a large set of enzymes and identify the one governed by the desired kinetics. The method gives access to all the dynamical parameters of the targeted enzyme without resorting to a fit. We show how to estimate the precision of this parameter determination and give some hints for experimental validation.

Fourier analysis to measure diffusion coefficients and resolve mixtures on a continuous electrophoresis chip.

We report a method to measure diffusion coefficients of fluorescent solutes in the 10(2)-10(6) Da molecular mass range in a glass-PDMS chip. Upon applying a permanent electric field, the solute is introduced through a narrow channel into a wide analysis chamber where it migrates along the injection axis and diffuses in two dimensions. The diffusion coefficient is extracted after 1D Fourier transform of the resulting stationary concentration pattern. Analysis is straightforward, requiring no numerical integration or velocity field simulation. The diffusion coefficients measured for fluorescein, rhodamine green-labeled oligonucleotides, and YOYO-1-stained dsDNA fragments agree with the literature values and with our own fluorescence correlation spectroscopy measurements. As shown for 151 and 1257 base pair dsDNA mixtures, the present method allows us to rely on diffusion to quantitatively characterize the nature and the composition of binary mixtures. In particular, we implement a DNA hybridization assay to illustrate the efficiency of the proposed protocol for library screening.

Temporal modulation of a spatially periodic potential for kinetically governed oriented motion.

This theoretical paper introduces an experimental protocol derived from the concept of Brownian motors in order to selectively confer an oriented motion to given charged reactants. Instead of maintaining permanently the system in nonequilibrium conditions, we propose a simple experimental trick to restore periodically a transient out-of-equilibrium regime: the reactive medium is alternately submitted to a sawtooth potential and to a potential ramp. The model provides approximate analytical expressions for the operating conditions allowing us to design the extraction from a mixture of any desired reactant characterized by its rate constants. The orders of magnitude suggest a possible implementation in microsystems where the present approach could be used for separation and analysis.

Response to a temperature modulation as a signature of chemical mechanisms.

We consider n reactive species involved in unimolecular reactions and submitted to a temperature modulation of small amplitude. We determine the conditions on the rate constants for which the deviations from the equilibrium concentrations of each species can be optimized and find the analytical expression of the frequency associated with an extremum of concentration shift in the case n=3. We prove that the frequency dependence of the displacement of equilibrium gives access to the number n of species involved in the mechanism. We apply the results to the case of the transformation of a reactant into a product through a possible reactive intermediate and find the order relation obeyed by the activation energies of the different barriers. The results typically apply to enzymatic catalysis with kinetics of Michaelis-Menten type.

Quadruplex-based molecular beacons as tunable DNA probes.

Molecular beacons (MBs) are fluorescent nucleic acid probes with a hairpin-shaped structure in which the 5' and 3' ends are self-complementary. Due to a change in their emissive properties upon recognition with complementary sequences, MBs allow the diagnosis of single-stranded DNA or RNA with high mismatch discrimination, in vitro and in vivo. Whereas the stems of MB hairpins usually rely on the formation of a Watson-Crick duplex, we demonstrate in this report that the preceding structure can be replaced by a G-quadruplex motif (G4). Intramolecular quadruplexes may still be formed with a central loop composed of 12 to 21 bases, therefore extending the sequence repertoire of quadruplex formation. G4-MB can efficiently be used for oligonucleotide discrimination: in the presence of a complementary sequence, the central loop hybridizes and forms a duplex that causes opening of the quadruplex stem. The corresponding G4-MB unfolding can be detected by a change in its fluorescence emission. We discuss the thermodynamic and kinetic opportunities that are provided by using G4-MB instead of traditional MB. In particular, the intrinsic feature of the quadruplex motif facilitates the design of functional molecular beacons by independently varying the concentration of monovalent or divalent cations in the medium.

Symmetry-broken reactant motion upon phase-related symmetrically modulated excitations: application to highly selective molecular sorting.

This paper introduces a separation protocol relying on affinity chromatography that exhibits unprecedented selectivities. We submit the mixture contained in the separative medium to the simultaneous action of two symmetrically modulated excitations. The first is a uniform periodic field (e.g., electric field) with zero mean value, whereas the second is the periodic modulation of a thermodynamic parameter such as the temperature. Under appropriate tuning of the modulations with the dynamics of the discriminating chemical reaction, we predict a symmetry breaking of molecular motion: the mixture components that are addressed by their rate constants exhibit an oriented motion for a particular phase relation between the modulations of the field and the thermodynamic parameter. The resulting velocity of the mixture components depends on the rate constants and on a conjugated thermodynamic value such as the standard enthalpy of the discrimination process in the case of a temperature modulation. In particular, it may be possible to separate mixture components with identical rate constants. We use the present approach to design a protocol to sort nucleic acids by their sequence.

Micelles of lipid-oligonucleotide conjugates: implications for membrane anchoring and base pairing.

This report examines the organization properties of new fluorescent DNA-lipids, either alone in water or in interaction with 1-octyl-beta-d-glucopyranoside micelles or egg phosphatidylcholine vesicles. We first describe the design and the syntheses of the conjugates. Then, we use UV-Vis absorption, steady-state fluorescence emission, electron microscopy, and fluorescence correlation spectroscopy after two-photon excitation to show that these DNA-lipids form spherical micelles in aqueous solution and incorporate much better in micelles than in vesicles. We also investigate the significance of the lipophilic chains of these DNA-lipids on the melting behavior of the double-stranded hybrids: in water melting curves are broadened whereas in amphiphilic assemblies duplexes melt as the unconjugated controls. This work is expected to be useful for improving the rational design of antisense medicines.

A hyperpolar, multichromophoric cyclodextrin derivative: synthesis, and linear and nonlinear optical properties.

A chiral, highly polar, multichromophoric supermolecule has been designed by gathering seven push-pull chromophores onto a beta-cyclodextrin assembling unit through covalent flexible linkers. The photophysical and nonlinear optical properties of this mutichromophoric conical bundle were investigated and compared with those of the monomeric chromophore. The strongly absorbing multichromophoric system combines interesting features: it has a high molecular first-order hyperpolarisability and a very large dipolar moment (u = 38 D) which reveal a self-arrangement of the dipolar chromophores within the supermolecule. The confinement of the push-pull chromophores within the nanoscopic bundle affects their optical properties and promotes interactions: the multichromophoric supermolecule is hypochromically and hypsochromically shifted with respect to its monomeric analogue. In addition, the close proximity promotes excitonic coupling, as well as excimer formation phenomena.

Tetrahedral onsager crosses for solubility improvement and crystallization bypass.

Pure organic molecules exhibiting a suitable concave rigid shape are expected to give porous glasses in the solid state. Such a feature opens new opportunities to avoid crystallization and to improve molecular solubility in relation to the high internal energy of these solid phases. To quantitatively explore the latter strategy, a series of rigid tetrahedral conjugated molecules nC and the corresponding models nR have been synthesized. Related to the present purpose, several properties have been investigated using UV absorption, steady-state fluorescence emission, differential scanning calorimetry, (1)H NMR translational self-diffusion, magic angle spinning (13)C NMR, and multiple-beam interferometry experiments. The present tetrahedral crosses are up to 8 orders of magnitude more soluble than the corresponding model compounds after normalization to the same molecular length. In addition, they give concentrated monomeric solutions that can be used to cover surfaces with homogeneous films whose thickness goes down to the nanometer range. Such attractive features make cross-like molecular architectures promising for many applications.

pH-dependent specific binding and combing of DNA.

Recent developments in the rapid sequencing, mapping, and analysis of DNA rely on the specific binding of DNA to specially treated surfaces. We show here that specific binding of DNA via its unmodified extremities can be achieved on a great variety of surfaces by a judicious choice of the pH. On hydrophobic surfaces the best binding efficiency is reached at a pH of approximately 5.5. At that pH a approximately 40-kbp DNA is 10 times more likely to bind by an extremity than by a midsegment. A model is proposed to account for the differential adsorption of the molecule extremities and midsection as a function of pH. The pH-dependent specific binding can be used to align anchored DNA molecules by a receding meniscus, a process called molecular combing. The resulting properties of the combed molecules will be discussed.

NMR analysis of carbohydrates with model-free spectral densities: the dispersion range revisited.

Over the past decade molecular mechanics and molecular dynamics studies have demonstrated considerable flexibility for carbohydrates. In order to interpret the corresponding NMR parameters, which correspond to a time-averaged or 'virtual' conformer, it is necessary to simulate the experimental data using the averaged geometrical representation obtained with molecular modelling methods. This structural information can be transformed into theoretical NMR data using empirical Karplus-type equations for the scalar coupling constants and the appropriate formalism for the relaxation parameters. In the case of relaxation data, the 'model-free' spectral densities have been widely used in order to account for the internal motions in sugars. Several studies have been conducted with truncated model-free spectral densities based on the assumption that internal motion is very fast with respect to overall tumbling. In this report we present experimental and theoretical evidence that suggests that this approach is not justified. Indeed, recent results show that even in the case of moderate-sized carbohydrates internal motions are occurring on the same timescale as molecular reorientation. Simulations of relaxation parameters (NOESY volumes, proton cross-relaxation rates, carbon T1 and nOe values) in the dispersion range (0.1 < tau(c) < 5 ns) show that rates of internal motion can be fairly precisely defined with respect to overall tumbling. Experimental data for a variety of oligosaccharides clearly indicate similar timescales for internal and overall motion.

Drug delivery: piercing vesicles by their adsorption onto a porous medium.

Experimental evidence is presented that supports the possibility of building a "molecular drill." By the adsorption of a vesicle onto a porous substrate (specifically, a lycopode grain), it was possible to increase the permeability of the vesicle by locally stretching its membrane. Molecules contained within the vesicle, which could not cross the membrane, were delivered to the porous substrate upon adsorption. This general process could provide another method for drug delivery and targeting.