18 research outputs found
Efficient preparation of internally modified single-molecule constructs using nicking enzymes
Investigations of enzymes involved in DNA metabolism have strongly benefited from the establishment of single molecule techniques. These experiments frequently require elaborate DNA substrates, which carry chemical labels or nucleic acid tertiary structures. Preparing such constructs often represents a technical challenge: long modified DNA molecules are usually produced via multi-step processes, involving low efficiency intermolecular ligations of several fragments. Here, we show how long stretches of DNA (>50 bp) can be modified using nicking enzymes to produce complex DNA constructs. Multiple different chemical and structural modifications can be placed internally along DNA, in a specific and precise manner. Furthermore, the nicks created can be resealed efficiently yielding intact molecules, whose mechanical properties are preserved. Additionally, the same strategy is applied to obtain long single-strand overhangs subsequently used for efficient ligation of ss- to dsDNA molecules. This technique offers promise for a wide range of applications, in particular single-molecule experiments, where frequently multiple internal DNA modifications are required
Inferring hidden Markov models from noisy time sequences: a method to alleviate degeneracy in molecular dynamics
We present a new method for inferring hidden Markov models from noisy time
sequences without the necessity of assuming a model architecture, thus allowing
for the detection of degenerate states. This is based on the statistical
prediction techniques developed by Crutchfield et al., and generates so called
causal state models, equivalent to hidden Markov models. This method is
applicable to any continuous data which clusters around discrete values and
exhibits multiple transitions between these values such as tethered particle
motion data or Fluorescence Resonance Energy Transfer (FRET) spectra. The
algorithms developed have been shown to perform well on simulated data,
demonstrating the ability to recover the model used to generate the data under
high noise, sparse data conditions and the ability to infer the existence of
degenerate states. They have also been applied to new experimental FRET data of
Holliday Junction dynamics, extracting the expected two state model and
providing values for the transition rates in good agreement with previous
results and with results obtained using existing maximum likelihood based
methods.Comment: 19 pages, 9 figure
BMOG: boosted Gaussian Mixture Model with controlled complexity for background subtraction
The Helicase-Like Domains of Type III Restriction Enzymes Trigger Long-Range Diffusion Along DNA
Magnetic tweezers measurements of the nanomechanical stability of DNA against denaturation at various conditions of pH and ionic strength
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