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.

Loops in DNA: an overview of experimental and theoretical approaches.

DNA loop formation plays a central role in many cellular processes. The aim of this paper is to present the state of the art and open problems regarding the experimental and theoretical approaches to DNA looping. A particular attention is devoted to the effects of the protein bridge size and of protein induced sharp DNA bending on DNA loop formation enhancement.

Statistical determination of the step size of molecular motors.

Molecular motors are enzymatic proteins that couple the consumption of chemical energy to mechanical displacement. In order to elucidate the translocation mechanisms of these enzymes, it is of fundamental importance to measure the physical step size. The step size can, in certain instances, be directly measured with single-molecule techniques; however, in the majority of cases individual steps are masked by noise. The step size can nevertheless be obtained from noisy single-molecule records through statistical methods. This analysis is analogous to determining the charge of the electron from current shot noise. We review methods for obtaining the step size based on analysing, in both the time and frequency domains, the variance in position from noisy single-molecule records of motor displacement. Additionally, we demonstrate how similar methods may be applied to measure the step size in bulk kinetic experiments.

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.

Transverse fluctuations of single DNA molecules attached at both extremities to a surface.

We present a simple method to stretch DNA molecules close to a surface without any chemical modification of either the molecules or the surface. By adjusting the pH of the solution, only the extremities of DNA molecules are tethered to a glass coverslip made hydrophobic, while stretching is achieved using a hydrodynamic flow. These extended molecules provide a very favorable template for DNA-protein interaction studies by purely optical means. Pursuing these experiments requires first a full characterization of the thermally driven fluctuations of the tethered DNA molecules. For this purpose, these fluctuations were recorded by fluorescence microscopy and were analyzed in terms of normal modes. Our experimental results are well described by a model accounting for the nonlinear elastic behavior of the chain. Remarkably, the proximity of the molecules to a rigid surface does not alter the main features of their dynamics, and our results are in agreement with previous studies on extended DNA in viscous solutions.

Stress-induced structural transitions in DNA and proteins.

The ability to manipulate, stretch and twist biomolecules opens the way to an understanding of their structural transitions. We review some of the recently discovered stress-induced structural transitions in DNA as well as the application of single molecule manipulation techniques to DNA unzipping and to the study of protein folding/unfolding transitions.

Stretched and overwound DNA forms a Pauling-like structure with exposed bases.

We investigate structural transitions within a single stretched and supercoiled DNA molecule. With negative supercoiling, for a stretching force >0.3 pN, we observe the coexistence of B-DNA and denatured DNA from sigma approximately -0.015 down to sigma = -1. Surprisingly, for positively supercoiled DNA (sigma > +0.037) stretched by 3 pN, we observe a similar coexistence of B-DNA and a new, highly twisted structure. Experimental data and molecular modeling suggest that this structure has approximately 2.62 bases per turn and an extension 75% larger than B-DNA. This structure has tightly interwound phosphate backbones and exposed bases in common with Pauling's early DNA structure [Pauling, L. & Corey, R. B. (1953), Proc. Natl. Acad. Sci. USA 39, 84-97] and an unusual structure proposed for the Pf1 bacteriophage [Liu, D. J. & Day, L. A. (1994) Science 265, 671-674].

Behavior of supercoiled DNA.

We study DNA supercoiling in a quantitative fashion by micromanipulating single linear DNA molecules with a magnetic field gradient. By anchoring one end of the DNA to multiple sites on a magnetic bead and the other end to multiple sites on a glass surface, we were able to exert torsional control on the DNA. A rotating magnetic field was used to induce rotation of the magnetic bead, and reversibly over- and underwind the molecule. The magnetic field was also used to increase or decrease the stretching force exerted by the magnetic bead on the DNA. The molecule's degree of supercoiling could therefore be quantitatively controlled and monitored, and tethered-particle motion analysis allowed us to measure the stretching force acting on the DNA. Experimental results indicate that this is a very powerful technique for measuring forces at the picoscale. We studied the effect of stretching forces ranging from 0.01 pN to 100 pN on supercoiled DNA (-0.1 < sigma < 0.2) in a variety of ionic conditions. Other effects, such as stretching-relaxing hysteresis and the braiding of two DNA molecules, are discussed.

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.

The elasticity of a single supercoiled DNA molecule.

Single linear DNA molecules were bound at multiple sites at one extremity to a treated glass cover slip and at the other to a magnetic bead. The DNA was therefore torsionally constrained. A magnetic field was used to rotate the beads and thus to coil and pull the DNA. The stretching force was determined by analysis of the Brownian fluctuations of the bead. Here the elastic behavior of individual lambda DNA molecules over- and underwound by up to 500 turns was studied. A sharp transition was discovered from a low to a high extension state at a force of approximately 0.45 piconewtons for underwound molecules and at a force of approximately 3 piconewtons for overwound ones. These transitions, probably reflecting the formation of alternative structures in stretched coiled DNA molecules, might be relevant for DNA transcription and replication.