The development of single molecule force spectroscopy: from polymer biophysics to molecular machines.

The advent of single-molecule force spectroscopy represents the introduction of forces, torques, and displacements as controlled variables in biochemistry. These methods afford the direct manipulation of individual molecules to interrogate the forces that hold together their structure, the forces and torques that these molecules generate in the course of their biochemical reactions, and the use of force, torque, and displacement as tools to investigate the mechanisms of these reactions. Because of their microscopic nature, the signals detected in these experiments are often dominated by fluctuations, which, in turn, play an important role in the mechanisms that underlie the operation of the molecular machines of the cell. Their direct observation and quantification in single-molecule experiments provide a unique window to investigate those mechanisms, as well as a convenient way to investigate fundamental new fluctuation theorems of statistical mechanics that bridge the equilibrium and non-equilibrium realms of this discipline. In this review we have concentrated on the developments that occurred in our laboratory on the characterization of biopolymers and of molecular machines of the central dogma. Accordingly, some important areas like the study of cytoskeletal motors have not been included. While we adopt at times an anecdotal perspective with the hope of conveying the personal circumstances in which these developments took place, we have made every effort, nonetheless, to include the most important developments that were taking place at the same time in other laboratories.

Magnetic tweezers principles and promises.

Magnetic tweezers have become popular with the outbreak of single molecule micromanipulation: catching a single molecule of DNA, RNA or a single protein and applying mechanical constrains using micron-size magnetic beads and magnets turn out to be easy. Various factors have made this possible: the fact that manufacturers have been preparing these beads to catch various biological entities-the ease of use provided by magnets which apply a force or a torque at a distance thus inside a flow cell-some chance: since the forces so generated are in the right range to stretch a single molecule. This is a little less true for torque. Finally, one feature which also appears very important is the simplicity of their calibration using Brownian motion. Here we start by describing magnetic tweezers used routinely in our laboratory where we have tried to develop a device as simple as possible so that the experimentalist can really focus on the biological aspect of the biomolecules that he/she is interested in. We discuss the implications of the various components and their important features. Next, we summarize what is easy to achieve and what is less easy. Then we refer to contributions by other groups who have brought valuable insights to improve magnetic tweezers.

Torque-induced deformations of charged elastic DNA rods: thin helices, loops, and precursors of DNA supercoiling.

We study the deformations of charged elastic rods under applied end forces and torques. For neutral filaments, we analyze the energetics of initial helical deformations and loop formation. We supplement this elastic approach with electrostatic energies of bent filaments and find critical conditions for buckling depending on the ionic strength of the solution. We also study force-induced loop opening, for parameters relevant for DNA. Finally, some applications of this nano-mechanical DNA model to salt-dependent onset of the DNA supercoiling are discussed.

Determining Trap Compliances, Microsphere Size Variations, and Response Linearities in Single DNA Molecule Elasticity Measurements with Optical Tweezers.

We previously introduced the use of DNA molecules for calibration of biophysical force and displacement measurements with optical tweezers. Force and length scale factors can be determined from measurements of DNA stretching. Trap compliance can be determined by fitting the data to a nonlinear DNA elasticity model, however, noise/drift/offsets in the measurement can affect the reliability of this determination. Here we demonstrate a more robust method that uses a linear approximation for DNA elasticity applied to high force range (25-45 pN) data. We show that this method can be used to assess how small variations in microsphere sizes affect DNA length measurements and demonstrate methods for correcting for these errors. We further show that these measurements can be used to check assumed linearities of system responses. Finally, we demonstrate methods combining microsphere imaging and DNA stretching to check the compliance and positioning of individual traps.

Elastic Properties of Nucleic Acids by Single-Molecule Force Spectroscopy.

We review the current knowledge on the use of single-molecule force spectroscopy techniques to extrapolate the elastic properties of nucleic acids. We emphasize the lesser-known elastic properties of single-stranded DNA. We discuss the importance of accurately determining the elastic response in pulling experiments, and we review the simplest models used to rationalize the experimental data as well as the experimental approaches used to pull single-stranded DNA. Applications used to investigate DNA conformational transitions and secondary structure formation are also highlighted. Finally, we provide an overview of the effects of salt and temperature and briefly discuss the effects of contour length and sequence dependence.

DNA structure and function.

The proposal of a double-helical structure for DNA over 60 years ago provided an eminently satisfying explanation for the heritability of genetic information. But why is DNA, and not RNA, now the dominant biological information store? We argue that, in addition to its coding function, the ability of DNA, unlike RNA, to adopt a B-DNA structure confers advantages both for information accessibility and for packaging. The information encoded by DNA is both digital - the precise base specifying, for example, amino acid sequences - and analogue. The latter determines the sequence-dependent physicochemical properties of DNA, for example, its stiffness and susceptibility to strand separation. Most importantly, DNA chirality enables the formation of supercoiling under torsional stress. We review recent evidence suggesting that DNA supercoiling, particularly that generated by DNA translocases, is a major driver of gene regulation and patterns of chromosomal gene organization, and in its guise as a promoter of DNA packaging enables DNA to act as an energy store to facilitate the passage of translocating enzymes such as RNA polymerase.