2,623 research outputs found
Model-guided design of ligand-regulated RNAi for programmable control of gene expression
Progress in constructing biological networks will rely on the development of more advanced components that can be predictably modified to yield optimal system performance. We have engineered an RNA-based platform, which we call an shRNA switch, that provides for integrated ligand control of RNA interference (RNAi) by modular coupling of an aptamer, competing strand, and small hairpin (sh) RNA stem into a single component that links ligand concentration and target gene expression levels. A combined experimental and mathematical modelling approach identified multiple tuning strategies and moves towards a predictable framework for the forward design of shRNA switches. The utility of our platform is highlighted by the demonstration of fine-tuning, multi-input control, and model-guided design of shRNA switches with an optimized dynamic range. Thus, shRNA switches can serve as an advanced component for the construction of complex biological systems and offer a controlled means of activating RNAi in disease therapeutics
Designer cell signal processing circuits for biotechnology
Microorganisms are able to respond effectively to diverse signals from their environment and internal metabolism owing to their inherent sophisticated information processing capacity. A central aim of synthetic biology is to control and reprogramme the signal processing pathways within living cells so as to realise repurposed, beneficial applications ranging from disease diagnosis and environmental sensing to chemical bioproduction. To date most examples of synthetic biological signal processing have been built based on digital information flow, though analogue computing is being developed to cope with more complex operations and larger sets of variables. Great progress has been made in expanding the categories of characterised biological components that can be used for cellular signal manipulation, thereby allowing synthetic biologists to more rationally programme increasingly complex behaviours into living cells. Here we present a current overview of the components and strategies that exist for designer cell signal processing and decision making, discuss how these have been implemented in prototype systems for therapeutic, environmental, and industrial biotechnological applications, and examine emerging challenges in this promising field
Lipid vesicles chaperone an encapsulated RNA aptamer.
The organization of molecules into cells is believed to have been critical for the emergence of living systems. Early protocells likely consisted of RNA functioning inside vesicles made of simple lipids. However, little is known about how encapsulation would affect the activity and folding of RNA. Here we find that confinement of the malachite green RNA aptamer inside fatty acid vesicles increases binding affinity and locally stabilizes the bound conformation of the RNA. The vesicle effectively 'chaperones' the aptamer, consistent with an excluded volume mechanism due to confinement. Protocellular organization thereby leads to a direct benefit for the RNA. Coupled with previously described mechanisms by which encapsulated RNA aids membrane growth, this effect illustrates how the membrane and RNA might cooperate for mutual benefit. Encapsulation could thus increase RNA fitness and the likelihood that functional sequences would emerge during the origin of life
Mean-field cooperativity in chemical kinetics
We consider cooperative reactions and we study the effects of the interaction
strength among the system components on the reaction rate, hence realizing a
connection between microscopic and macroscopic observables. Our approach is
based on statistical mechanics models and it is developed analytically via
mean-field techniques. First of all, we show that, when the coupling strength
is set positive, the model is able to consistently recover all the various
cooperative measures previously introduced, hence obtaining a single unifying
framework. Furthermore, we introduce a criterion to discriminate between weak
and strong cooperativity, based on a measure of "susceptibility". We also
properly extend the model in order to account for multiple attachments
phenomena: this is realized by incorporating within the model -body
interactions, whose non-trivial cooperative capability is investigated too.Comment: 25 pages, 4 figure
Measuring the energy landscape roughness and the transition state location of biomolecules using single molecule mechanical unfolding experiments
Single molecule mechanical unfolding experiments are beginning to provide
profiles of the complex energy landscape of biomolecules. In order to obtain
reliable estimates of the energy landscape characteristics it is necessary to
combine the experimental measurements with sound theoretical models and
simulations. Here, we show how by using temperature as a variable in mechanical
unfolding of biomolecules in laser optical tweezer or AFM experiments the
roughness of the energy landscape can be measured without making any
assumptions about the underlying reaction oordinate. The efficacy of the
formalism is illustrated by reviewing experimental results that have directly
measured roughness in a protein-protein complex. The roughness model can also
be used to interpret experiments on forced-unfolding of proteins in which
temperature is varied. Estimates of other aspects of the energy landscape such
as free energy barriers or the transition state (TS) locations could depend on
the precise model used to analyze the experimental data. We illustrate the
inherent difficulties in obtaining the transition state location from loading
rate or force-dependent unfolding rates. Because the transition state moves as
the force or the loading rate is varied it is in general difficult to invert
the experimental data unless the curvature at the top of the one dimensional
free energy profile is large, i.e the barrier is sharp. The independence of the
TS location on force holds good only for brittle or hard biomolecules whereas
the TS location changes considerably if the molecule is soft or plastic. We
also comment on the usefulness of extension of the molecule as a surrogate
reaction coordinate especially in the context of force-quench refolding of
proteins and RNA.Comment: 44 pages, 7 figure
Stochastic model for nucleosome sliding in the presence of DNA ligands
Heat-induced mobility of nucleosomes along DNA is an experimentally
well-studied phenomenon. A recent experiment shows that the repositioning is
modified in the presence of minor-groove binding DNA ligands. We present here a
stochastic three-state model for the diffusion of a nucleosome along DNA in the
presence of such ligands. It allows us to describe the dynamics and the steady
state of such a motion analytically. The analytical results are in excellent
agreement with numerical simulations of this stochastic process.With this
model, we study the response of a nucleosome to an external force and how it is
affected by the presence of ligands.Comment: 10 pages, 8 figures, submitted to Eur. Phys. J.
Modelling the regulation of thermal adaptation in Candida albicans, a major fungal pathogen of humans
Peer reviewedPublisher PD
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