50 research outputs found
Unzipping DNA with Optical Tweezers: High Sequence Sensitivity and Force Flips
AbstractForce measurements are performed on single DNA molecules with an optical trapping interferometer that combines subpiconewton force resolution and millisecond time resolution. A molecular construction is prepared for mechanically unzipping several thousand-basepair DNA sequences in an in vitro configuration. The force signals corresponding to opening and closing the double helix at low velocity are studied experimentally and are compared to calculations assuming thermal equilibrium. We address the effect of the stiffness on the basepair sensitivity and consider fluctuations in the force signal. With respect to earlier work performed with soft microneedles, we obtain a very significant increase in basepair sensitivity: presently, sequence features appearing at a scale of 10 basepairs are observed. When measured with the optical trap the unzipping force exhibits characteristic flips between different values at specific positions that are determined by the base sequence. This behavior is attributed to bistabilities in the position of the opening fork; the force flips directly reflect transitions between different states involved in the time-averaging of the molecular system
Pulling a polymer out of a potential well and the mechanical unzipping of DNA
Motivated by the experiments on DNA under torsion, we consider the problem of
pulling a polymer out of a potential well by a force applied to one of its
ends. If the force is less than a critical value, then the process is activated
and has an activation energy proportinal to the length of the chain. Above this
critical value, the process is barrierless and will occur spontaneously. We use
the Rouse model for the description of the dynamics of the peeling out and
study the average behaviour of the chain, by replacing the random noise by its
mean. The resultant mean-field equation is a nonlinear diffusion equation and
hence rather difficult to analyze. We use physical arguments to convert this in
to a moving boundary value problem, which can then be solved exactly. The
result is that the time required to pull out a polymer of segments
scales like . For models other than the Rouse, we argue that Comment: 11 pages, 6 figures. To appear in PhysicalReview
Unzipping of DNA with correlated base-sequence
We consider force-induced unzipping transition for a heterogeneous DNA model
with a correlated base-sequence. Both finite-range and long-range correlated
situations are considered. It is shown that finite-range correlations increase
stability of DNA with respect to the external unzipping force. Due to
long-range correlations the number of unzipped base-pairs displays two widely
different scenarios depending on the details of the base-sequence: either there
is no unzipping phase-transition at all, or the transition is realized via a
sequence of jumps with magnitude comparable to the size of the system. Both
scenarios are different from the behavior of the average number of unzipped
base-pairs (non-self-averaging). The results can be relevant for explaining the
biological purpose of correlated structures in DNA.Comment: 22 pages, revtex4, 14 eps figures; reprinted in the June 15, 2004
issue of Virtual Journal of Biological Physics Researc
Single-molecule derivation of salt dependent base-pair free energies in DNA
Accurate knowledge of the thermodynamic properties of nucleic acids is
crucial to predicting their structure and stability. To date most measurements
of base-pair free energies in DNA are obtained in thermal denaturation
experiments, which depend on several assumptions. Here we report measurements
of the DNA base-pair free energies based on a simplified system, the mechanical
unzipping of single DNA molecules. By combining experimental data with a
physical model and an optimization algorithm for analysis, we measure the 10
unique nearest-neighbor base-pair free energies with 0.1 kcal mol-1 precision
over two orders of magnitude of monovalent salt concentration. We find an
improved set of standard energy values compared with Unified Oligonucleotide
energies and a unique set of 10 base-pair-specific salt-correction values. The
latter are found to be strongest for AA/TT and weakest for CC/GG. Our new
energy values and salt corrections improve predictions of DNA unzipping forces
and are fully compatible with melting temperatures for oligos. The method
should make it possible to obtain free energies, enthalpies and entropies in
conditions not accessible by bulk methodologies.Comment: Main text: 27 pages, 4 figures, 2 tables. Supporting Information: 51
pages, 19 figures, 4 table
Single Molecule Statistics and the Polynucleotide Unzipping Transition
We present an extensive theoretical investigation of the mechanical unzipping
of double-stranded DNA under the influence of an applied force. In the limit of
long polymers, there is a thermodynamic unzipping transition at a critical
force value of order 10 pN, with different critical behavior for homopolymers
and for random heteropolymers. We extend results on the disorder-averaged
behavior of DNA's with random sequences to the more experimentally accessible
problem of unzipping a single DNA molecule. As the applied force approaches the
critical value, the double-stranded DNA unravels in a series of discrete,
sequence-dependent steps that allow it to reach successively deeper energy
minima. Plots of extension versus force thus take the striking form of a series
of plateaus separated by sharp jumps. Similar qualitative features should
reappear in micromanipulation experiments on proteins and on folded RNA
molecules. Despite their unusual form, the extension versus force curves for
single molecules still reveal remnants of the disorder-averaged critical
behavior. Above the transition, the dynamics of the unzipping fork is related
to that of a particle diffusing in a random force field; anomalous,
disorder-dominated behavior is expected until the applied force exceeds the
critical value for unzipping by roughly 5 pN.Comment: 40 pages, 18 figure
Modeling DNA Structure, Elasticity and Deformations at the Base-pair Level
We present a generic model for DNA at the base-pair level. We use a variant
of the Gay-Berne potential to represent the stacking energy between neighboring
base-pairs. The sugar-phosphate backbones are taken into account by semi-rigid
harmonic springs with a non-zero spring length. The competition of these two
interactions and the introduction of a simple geometrical constraint leads to a
stacked right-handed B-DNA-like conformation. The mapping of the presented
model to the Marko-Siggia and the Stack-of-Plates model enables us to optimize
the free model parameters so as to reproduce the experimentally known
observables such as persistence lengths, mean and mean squared base-pair step
parameters. For the optimized model parameters we measured the critical force
where the transition from B- to S-DNA occurs to be approximately . We
observe an overstretched S-DNA conformation with highly inclined bases that
partially preserves the stacking of successive base-pairs.Comment: 15 pages, 25 figures. submitted to PR
Single-molecule experiments in biological physics: methods and applications
I review single-molecule experiments (SME) in biological physics. Recent
technological developments have provided the tools to design and build
scientific instruments of high enough sensitivity and precision to manipulate
and visualize individual molecules and measure microscopic forces. Using SME it
is possible to: manipulate molecules one at a time and measure distributions
describing molecular properties; characterize the kinetics of biomolecular
reactions and; detect molecular intermediates. SME provide the additional
information about thermodynamics and kinetics of biomolecular processes. This
complements information obtained in traditional bulk assays. In SME it is also
possible to measure small energies and detect large Brownian deviations in
biomolecular reactions, thereby offering new methods and systems to scrutinize
the basic foundations of statistical mechanics. This review is written at a
very introductory level emphasizing the importance of SME to scientists
interested in knowing the common playground of ideas and the interdisciplinary
topics accessible by these techniques. The review discusses SME from an
experimental perspective, first exposing the most common experimental
methodologies and later presenting various molecular systems where such
techniques have been applied. I briefly discuss experimental techniques such as
atomic-force microscopy (AFM), laser optical tweezers (LOT), magnetic tweezers
(MT), biomembrane force probe (BFP) and single-molecule fluorescence (SMF). I
then present several applications of SME to the study of nucleic acids (DNA,
RNA and DNA condensation), proteins (protein-protein interactions, protein
folding and molecular motors). Finally, I discuss applications of SME to the
study of the nonequilibrium thermodynamics of small systems and the
experimental verification of fluctuation theorems. I conclude with a discussion
of open questions and future perspectives.Comment: Latex, 60 pages, 12 figures, Topical Review for J. Phys. C (Cond.
Matt
Hysteresis in Pressure-Driven DNA Denaturation
In the past, a great deal of attention has been drawn to thermal driven denaturation processes. In recent years, however, the discovery of stress-induced denaturation, observed at the one-molecule level, has revealed new insights into the complex phenomena involved in the thermo-mechanics of DNA function. Understanding the effect of local pressure variations in DNA stability is thus an appealing topic. Such processes as cellular stress, dehydration, and changes in the ionic strength of the medium could explain local pressure changes that will affect the molecular mechanics of DNA and hence its stability. In this work, a theory that accounts for hysteresis in pressure-driven DNA denaturation is proposed. We here combine an irreversible thermodynamic approach with an equation of state based on the Poisson-Boltzmann cell model. The latter one provides a good description of the osmotic pressure over a wide range of DNA concentrations. The resulting theoretical framework predicts, in general, the process of denaturation and, in particular, hysteresis curves for a DNA sequence in terms of system parameters such as salt concentration, density of DNA molecules and temperature in addition to structural and configurational states of DNA. Furthermore, this formalism can be naturally extended to more complex situations, for example, in cases where the host medium is made up of asymmetric salts or in the description of the (helical-like) charge distribution along the DNA molecule. Moreover, since this study incorporates the effect of pressure through a thermodynamic analysis, much of what is known from temperature-driven experiments will shed light on the pressure-induced melting issue