Salt concentration effects on equilibrium melting curves from DNA microarrays.

DNA microarrays find applications in an increasing number of domains where more quantitative results are required. DNA being a charged polymer, the repulsive interactions between the surface of the microarray and the targets in solution are increasing upon hybridization. Such electrostatic penalty is generally reduced by increasing the salt concentration. In this article, we present equilibrium-melting curves obtained from dedicated physicochemical experiments on DNA microarrays in order to get a better understanding of the electrostatic penalty incurred during the hybridization reaction at the surface. Various salt concentrations have been considered and deviations from the commonly used Langmuir adsorption model are experimentally quantified for the first time in agreement with theoretical predictions.

SPR-imaging based assays on an oligonucleotide-array to analyze DNA lesions recognition and excision by repair proteins.

An original oligonucleotide-array, coupled with SPR-imaging detection, has been developed to study biological interactions between DNA base lesions and DNA repair enzymes. This bioanalytical tool constitutes an efficient screening platform to quantify DNA repair activities and to search for new DNA repair inhibitors.

Point mutation detection by surface plasmon resonance imaging coupled with a temperature scan method in a model system.

The detection of point mutations in genes presents clear biological and medical interest. Various methods have been considered. In this paper, we take advantage of surface plasmon resonance imaging, a technique allowing detection of unlabeled DNA hybridization. Coupled with a temperature scan, this approach allows us to determine the presence of single-point mutations in oligonucleotide samples from the analysis of DNA's melting curves in either the homozygous or heterozygous case. Moreover, these experimental data are confirmed in good agreement with numerical calculations.

Temperature effects on DNA chip experiments from surface plasmon resonance imaging: isotherms and melting curves.

We present an analysis of hybridization experiments on a DNA chip studied by surface plasmon resonance imaging. The reaction constants at various temperatures and for different probe lengths are obtained from Langmuir isotherms and hybridization kinetics. The melting curves from temperature scans are also obtained without any labeling of the targets. The effects of the probe length on the hybridization thermodynamics, deduced from the temperature dependence of the reaction constants as well as from the melting curves, suggest dispersion in the length of the hybridization segments of the probes accessible to the targets. Those are, however, sufficient to suggest efficient point mutation detection from temperature scans.

Hybridization at a surface: the role of spacers in DNA microarrays.

Flexible spacer chains are utilized to enhance the hybridization of terminally anchored oligonucleotide probes of DNA microarrays. A polymer physics approach identifies an underlying mechanism and yields guidelines for the optimal spacer length in terms of the effect on the equilibrium state. For low grafting densities, the dominant effect arises because of the decimation in the number of accessible chain configurations due to the impenetrable surface. Opposing trends are found for long targets and for short targets. At higher grafting densities, different brush regimes introduce an extra hybridization penalty. A novel brush regime is obtained for long neutral spacers and short targets at intermediate ionic strength where the chain stretching is due to the electrostatic interactions between the probes.

Exact curvilinear diffusion coefficients in the repton model.

The Rubinstein-Duke or repton model is one of the simplest lattice model of reptation for the diffusion of a polymer in a gel or a melt. Recently, a slightly modified model with hardcore interactions between the reptons has been introduced. The curvilinear diffusion coefficients of both models are exactly determined for all chain lengths. The case of periodic boundary conditions is also considered.

Brush effects on DNA chips: thermodynamics, kinetics, and design guidelines.

In biology experiments, oligonucleotide microarrays are contacted with a solution of long nucleic acid targets. The hybridized probes thus carry long tails. When the surface density of the oligonucleotide probes is high enough, the progress of hybridization gives rise to a polyelectrolyte brush due to mutual crowding of the nucleic acid tails. The free-energy penalty associated with the brush modifies both the hybridization isotherms and the rate equations: the attainable hybridization is lowered significantly as is the hybridization rate. When the equilibrium hybridization fraction, x(eq), is low, the hybridization follows a Langmuir type isotherm, x(eq)/(1 - x(eq)) = c(t)K where c(t) is the target concentration and K is the equilibrium constant. K is smaller than its bulk value by a factor (n/N)(2/5) due to wall effects where n and N denote the number of bases in the probe and the target. At higher x(eq), when the brush is formed, the leading correction is x(eq)/(1 - x(eq)) = c(t)K exp - const'x(eq)(2/3) - x(B)(2/3) where x(B) corresponds to the onset of the brush regime. The denaturation rate constant in the two regimes is identical. However, the hybridization rate constant in the brush regime is lower, the leading correction being exp -const' x(2/3) - x(B)(2/3).

Effects of stacking on the configurations and elasticity of single-stranded nucleic acids.

Stacking interactions in single-stranded nucleic acids give rise to configurations of an annealed rod-coil multiblock copolymer. Theoretical analysis identifies the following resulting signatures for long homopolynucleotides: a nonmonotonic dependence of size on temperature, the corresponding effects on cyclization and a plateau in the extension force law. Explicit numerical results for polydeoxyadenylate [poly(dA)] and polyriboadenylate [poly(rU)] are presented.

Sensitivity, specificity, and the hybridization isotherms of DNA chips.

Competitive hybridization, at the surface and in the bulk, lowers the sensitivity of DNA chips. Competitive surface hybridization occurs when different targets can hybridize with the same probe. Competitive bulk hybridization takes place when the targets can hybridize with free complementary chains in the solution. The effects of competitive hybridization on the thermodynamically attainable performance of DNA chips are quantified in terms of the hybridization isotherms of the spots. These relate the equilibrium degree of the hybridization to the bulk composition. The hybridization isotherm emerges as a Langmuir isotherm modified for electrostatic interactions within the probe layer. The sensitivity of the assay in equilibrium is directly related to the slope of the isotherm. A simpler description is possible, in terms of c(50) values specifying the bulk composition corresponding to 50% hybridization at the surface. The effects of competitive hybridization are important for the quantitative analysis of DNA chip results, especially when used to study point mutations.

Crossover from fragile to strong glassy behavior in kinetically constrained systems.

We show the existence of fragile-to-strong transitions in kinetically constrained systems by studying the equilibrium and out-of-equilibrium dynamics of a generic constrained Ising spin chain that interpolates between the symmetric and fully asymmetric cases. We find that for large but finite asymmetry the model displays a crossover from fragile to strong glassy behavior at finite temperature, which is controlled by the asymmetry parameter. The relaxation in the fragile region presents stretched exponential behavior, with a temperature dependent stretching exponent that is predicted. Our results are confirmed by numerical simulations.

Extension of rod-coil multiblock copolymers and the effect of the helix-coil transition

The extension elasticity of rod-coil mutliblock copolymers is analyzed for two experimentally accessible situations. In the quenched case, when the architecture is fixed by the synthesis, the force law is distinguished by a sharp change in the slope. In the annealed case, where interconversion between rod and coil states is possible, the resulting force law is sigmoid with a pronounced plateau. This last case is realized, for example, when homopolypeptides capable of undergoing a helix-coil transition are extended from a coil state.