8,614 research outputs found
Paradigms for computational nucleic acid design
The design of DNA and RNA sequences is critical for many endeavors, from DNA nanotechnology, to PCR‐based applications, to DNA hybridization arrays. Results in the literature rely on a wide variety of design criteria adapted to the particular requirements of each application. Using an extensively studied thermodynamic model, we perform a detailed study of several criteria for designing sequences intended to adopt a target secondary structure. We conclude that superior design methods should explicitly implement both a positive design paradigm (optimize affinity for the target structure) and a negative design paradigm (optimize specificity for the target structure). The commonly used approaches of sequence symmetry minimization and minimum free‐energy satisfaction primarily implement negative design and can be strengthened by introducing a positive design component. Surprisingly, our findings hold for a wide range of secondary structures and are robust to modest perturbation of the thermodynamic parameters used for evaluating sequence quality, suggesting the feasibility and ongoing utility of a unified approach to nucleic acid design as parameter sets are refined further. Finally, we observe that designing for thermodynamic stability does not determine folding kinetics, emphasizing the opportunity for extending design criteria to target kinetic features of the energy landscape
Refolding dynamics of stretched biopolymers upon force quench
Single molecule force spectroscopy methods can be used to generate folding
trajectories of biopolymers from arbitrary regions of the folding landscape. We
illustrate the complexity of the folding kinetics and generic aspects of the
collapse of RNA and proteins upon force quench, using simulations of an RNA
hairpin and theory based on the de Gennes model for homopolymer collapse. The
folding time, , depends asymmetrically on and
where () is the stretch (quench) force, and
is the transition mid-force of the RNA hairpin. In accord with
experiments, the relaxation kinetics of the molecular extension, , occurs
in three stages: a rapid initial decrease in the extension is followed by a
plateau, and finally an abrupt reduction in that occurs as the native
state is approached.
The duration of the plateau increases as decreases
(where is the time in which the force is reduced from to ).
Variations in the mechanisms of force quench relaxation as is altered
are reflected in the experimentally measurable time-dependent entropy, which is
computed directly from the folding trajectories. An analytical solution of the
de Gennes model under tension reproduces the multistage stage kinetics in
. The prediction that the initial stages of collapse should also be a
generic feature of polymers is validated by simulation of the kinetics of
toroid (globule) formation in semiflexible (flexible) homopolymers in poor
solvents upon quenching the force from a fully stretched state. Our findings
give a unified explanation for multiple disparate experimental observations of
protein folding.Comment: 31 pages 11 figure
Spectral rate theory for projected two-state kinetics
Classical rate theories often fail in cases where the observable(s) or order
parameter(s) used are poor reaction coordinates or the observed signal is
deteriorated by noise, such that no clear separation between reactants and
products is possible. Here, we present a general spectral two-state rate theory
for ergodic dynamical systems in thermal equilibrium that explicitly takes into
account how the system is observed. The theory allows the systematic estimation
errors made by standard rate theories to be understood and quantified. We also
elucidate the connection of spectral rate theory with the popular Markov state
modeling (MSM) approach for molecular simulation studies. An optimal rate
estimator is formulated that gives robust and unbiased results even for poor
reaction coordinates and can be applied to both computer simulations and
single-molecule experiments. No definition of a dividing surface is required.
Another result of the theory is a model-free definition of the reaction
coordinate quality (RCQ). The RCQ can be bounded from below by the directly
computable observation quality (OQ), thus providing a measure allowing the RCQ
to be optimized by tuning the experimental setup. Additionally, the respective
partial probability distributions can be obtained for the reactant and product
states along the observed order parameter, even when these strongly overlap.
The effects of both filtering (averaging) and uncorrelated noise are also
examined. The approach is demonstrated on numerical examples and experimental
single-molecule force probe data of the p5ab RNA hairpin and the apo-myoglobin
protein at low pH, here focusing on the case of two-state kinetics
Complete RNA inverse folding: computational design of functional hammerhead ribozymes
Nanotechnology and synthetic biology currently constitute one of the most
innovative, interdisciplinary fields of research, poised to radically transform
society in the 21st century. This paper concerns the synthetic design of
ribonucleic acid molecules, using our recent algorithm, RNAiFold, which can
determine all RNA sequences whose minimum free energy secondary structure is a
user-specified target structure. Using RNAiFold, we design ten cis-cleaving
hammerhead ribozymes, all of which are shown to be functional by a cleavage
assay. We additionally use RNAiFold to design a functional cis-cleaving
hammerhead as a modular unit of a synthetic larger RNA. Analysis of kinetics on
this small set of hammerheads suggests that cleavage rate of computationally
designed ribozymes may be correlated with positional entropy, ensemble defect,
structural flexibility/rigidity and related measures. Artificial ribozymes have
been designed in the past either manually or by SELEX (Systematic Evolution of
Ligands by Exponential Enrichment); however, this appears to be the first
purely computational design and experimental validation of novel functional
ribozymes. RNAiFold is available at
http://bioinformatics.bc.edu/clotelab/RNAiFold/.Comment: 17 pages, 2 tables, 7 figures, final version to appear in Nucleic
Acids Researc
Improving signal-to-noise resolution in single molecule experiments using molecular constructs with short handles
We investigate unfolding/folding force kinetics in DNA hairpins exhibiting
two and three states with newly designed short dsDNA handles (29 bp) using
optical tweezers. We show how the higher stiffness of the molecular setup
moderately enhances the signal-to-noise ratio (SNR) in hopping experiments as
compared to conventional long handles constructs (approximately 700 bp). The
shorter construct results in a signal of higher SNR and slower
folding/unfolding kinetics, thereby facilitating the detection of otherwise
fast structural transitions. A novel analysis of the elastic properties of the
molecular setup, based on high-bandwidth measurements of force fluctuations
along the folded branch, reveals that the highest SNR that can be achieved with
short handles is potentially limited by the marked reduction of the effective
persistence length and stretch modulus of the short linker complex.Comment: Main paper: 20 pages and 6 figures. Supplementary Material: 25 page
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