Thursday, 5 May 2022

Exploiting and understanding cellular molecules: nucleic acids and proteins

Strategies for Constructing and Operating DNA Origami Linear Actuators

The authors discuss the protocol optimisation for the fabrication of a DNA origami rotaxane. The objective is to find the protocol that produces the highest yield of working rail/sliders systems to use as linear actuators on the nanometric scale. The use of these sliders, when combined, will allow the fabrication of materials with subnanometer precision using the slider as a “printing head”.

A tractable genotype–phenotype map modelling the self-assembly of protein quaternary structure

The polyomino model is introduced as a high-level model of assembly of protein sub-domains into larger complexes. The paper introduces the fundamental features of the polyomino model, starting with the genotype to the formation of individual assembly kits and finally the formation of complete structures from assembly kits. The paper investigates polyominos within the wider context of genotype-phenotype maps, with regards to genotype redundancy, phenotype bias, component disconnectivity, shape space covering, as well as phenotypic robustness and its relationship to evolvability, and finds that these are quite similar to the RNA folding GP map. From a GP map perspective, this raises the question of whether these traits are inherent to self-assembling systems. Eventually, the polyomino model could yield insights on artificial systems like DNA tiles.

Paper-based microfluidics for DNA diagnostics of malaria in low resource underserved rural communities

The researchers create a paper based lateral flow devices based on Loop-mediated isothermal amplification (LAMP) of DNA. Although, PCR-based amplification assays remain the gold-standard NAAT, the requirement for trained staff and external power has limited their application in areas with reduced resources. LAMP has recently emerged as easy-to-use alternatives to PCR, owing to greatly simplified hardware requirements.

The paper discusses use of paper origami techniques to prepare blood sample preparation (including magnetic beads on DNA molecules of interest), followed by the LAMP process in a small microfluidic chamber. A hand pressed button initiates lateral flow of the amplified DNA that travels along a small membrane where anti-FITC antibodies and immobilized streptavidin are present as test and control lines. Upon successful attachment of species-specific ligands to anti-FITC antibodies, a positive signal is generated thereby enabling detection of diseases.

The beauty of nucleic acids and the scope of their application

Continuous Cell-Free Replication and Evolution of Artificial Genomic DNA in a Compartmentalized Gene Expression System

In this study, they coupled DNA replication with gene expression in cell-free system. They performed the experiments in water-in-oil droplets in serial dilution cycles. Circular DNA is replicated through rolling-circle replication followed by homologous recombination catalysed by the proteins, phi29 DNA polymerase, and Cre recombinase expressed from the DNA. Isolated circular DNAs accumulated several common mutations that exhibited higher replication abilities than the original DNA due to its improved ability as a replication template, increased polymerase activity, and a reduced inhibitory effect of polymerization by the recombinase.

Fuel-Driven Dynamic Combinatorial Libraries 

The authors analyse the fuel-driven oligomerisation of isophthalic acid. They determine that while oligomer formation is mainly driven by fuel activation, its relaxation back to equilibrium (isophthalic acid monomers) is not symmetrical. Instead of hydrolysing, the relaxation is produced by the "reshuffling" of the longest oligomers with shorter ones to produce average length oligomers. They also demonstrate that oligomers longer than 3 units can produce some sort of feedback interaction, creating insoluble complexes that resist better the relaxation to equilibrium. Of course, they also have some leak reactions that produce an undesired subproduct with a constant rate. The paper presents an interesting view on a well established far-from-equilibrium assembly reaction as the oligomerisation of isophthalic acid. However, the control over the oligomerisation process is not impressive; the concentration of oligomers decreases exponentially with length.

A Comparison of Genotype-Phenotype Maps for RNA and Proteins

This paper attempts to identify differences and similarities in the RNA and HP-lattice Protein GP maps. To ensure appropriate comparison, the RNA GP Map has only 2 alphabets. Similarities include the tendency for some simple phenotypes to be highly overrepresented in genotype space. One interesting difference is that whereas most sequences in the RNA GP-Map tend to fold to a unique structure, only a small subset of sequences in the HP GP-Map do so. The average size of genotype sets are much smaller in the HP GP Map, and it takes more mutations from a given sequence to cover the whole phenotype space than in the RNA GP Map.

Exploiting cellular machinery for novel applications

Four different mechanisms for switching cell polarity

Cell polarity (asymmetric concentration profiles within the cell) plays a role in migration, division, differentiation, development and signalling. The mechanisms by which polarity is created and maintained is understood, but the dynamics of polarity are less well studied. Here they study a model in which the concentration profile of three interacting molecular species, a polarization marker, an antagonist, and a recruiter, change in response to signals of varying strength and duration. The signalling species either promote or suppress the rate constant for one reaction within the simple reaction network. This leads to altered phase space stability of the system in the presence or absence of a signal. Through phase space stability analysis and simulation, the authors exhaustively identify four distinct ways polarity can switch in response to a signal which could be tested in future experimental studies.

Recovery of Information Stored in Modified DNA with an Evolved Polymerase

DNA is used for digital information storage, but the potential information loss from degradation and associated issues with error during reading challenge its wide-scale implementation. To address this, the authors propose using degradation-resistant analogues of natural nucleic acids (xNAs) and they used direction evolution to create a polymerase capable of transforming 2’-O-methyl templates into double-stranded DNA with a fully functional proofreading domain to correct mismatches on DNA, RNA and 2’-O-methyl templates. In addition, they implemented a downstream analysis strategy that accommodates deletions to enable the large-scale use of nucleic acids for information storage.

Stretching of a fractal polymer around a disc reveals KPZ-like statistics

This paper aims to study the directed polymer model around a curved surface. This then has implications in biology for example wrapping DNA up into chromosomes as well as other situations where polymers are wrapped up around rods or similar. They use various scaling techniques to analyse the model around a surface with local radius of curvature R, where the two ends of the polymer are fixed a distance S apart. The key observations of this paper are that the typical distance the polymer goes away from the surface, Δ, scales as R^(1/3) for small radius of curvature and scales as S^α for large radius, with a cross over radius which scales as R^z. This is the same behaviour as surface roughness models mapping Δ to the roughness, R to time and S to the interface size. Further, they note that in a certain limit the exponents tend exactly to the 1+1D KPZ exponents.

Cooperative Branch Migration: A Mechanism for Flexible Control of DNA Strand Displacement

They basically demonstrate that if you have a strand that can sequester a displaced domain once it detaches, the reaction will proceed even if it was initially not favoured AG>0. They apply this to increase the rate of strand displacement reactions producing a bulge or a mismatch.

Wednesday, 4 May 2022

What are the odds?

Exact face-landing probabilities for bouncing objects: Edge probability in the coin toss and the three-sided die problem

The paper revisits the classical physics problem of what is the probability a thick coin lands on its side. They study the mechanics of a cylinder of a given thickness and radius, being given an initial random angular velocity and linear velocity. The cylinder is then allowed to bounce inelastically until it comes to rest either on one of the faces or on its edge. They then use the areas of phase space which correspond to each of the resting configurations in order to compute the respective probabilities as a function of the thickness to diameter ratio. They find that for example a £1 coin has a probability of landing on its edge of ~1/1000. Comparing to experimental and simulated data they find decent agreement. Further, they calculate the thickness to diameter ratio which would provide a 1/3 probability of landing on the edge. They calculate this to be ~0.831 which is much closer to experimental and numeric studies than previous theoretical suggestions.

Hamiltonian memory: An erasable classical bit

The authors consider a model of an information-carrying system in which the information is carried in the phase of a particle moving around a ring. They show that a (magnetic) Hamiltonian can be used to compress a uniform phase distribution to a highly-peaked one, apparently at the cost of no work input. It is unclear to me why this doesn't violate the second law - is this density in phase angle not exploitable as a non-equilibrium store of work? If not, why not?

A coarse-grained biophysical model of sequence evolution and the population size dependence

They present a coarse grained model of sequence evolution to ask questions about the speciation rate and how it differs due to effective population size. They rely on a framework analogous to thermodynamics, where the probability of a phenotype is dependent on a balance between its true fitness and the entropy of the phenotype. Using a DNA-protein binding co-evolving system as a framework, they show that, for smaller populations, the most likely phenotype is closer to inviability than for larger populations due to the greater entropic contribution in the former, and hence speciation is faster for smaller populations. This is consistent with experimental evidence, although theirs was a first attempt to explain this occurrence theoretically.

Monday, 29 November 2021

DNA in self-assembly, chemical reaction networks and more

DNA as a universal substrate for chemical kinetics

This paper discusses development of control circuitry within a chemical system to direct molecular events using strand displacement reactions. The authors show basic methods to construct unimolecular and bimolecular reactions (along with a short kinetic analysis associated with each reaction system). these 2 reaction types can then be used to construct any complex CRN (chemical reaction network). They show this by developing DNA reactions that recreate a Lotka-Volterra chemical oscillator, a limit cycle oscillator, a chaotic system, and a 2-bit pulse counter.

Undesired usage and the robust self-assembly of heterogeneous structures

This work introduces a formal description of the “principle of undesired usage”. This principle states that the yield of assembling a structure is not determined by ensuring a perfect stoichiometry between its components but by tuning the reagents chemical potentials, e.g. concentrations, to avoid undesired structures. They demonstrate this principle across several types of assembly processes, with several different modelling techniques.

SAT-assembly: A new approach for designing self-assembling systems

This paper presents a method of identifying patchy particle assembly components for a given structure. The foundation of the method is based on SAT, a well-known problem in computer science. The SAT problem consists of finding boolean values that solve a given set of boolean equations with a fixed number of variables. The paper goes into great detail on the variables and clauses that characterize patchy particle assembly as SAT problems. The method is performed on a cubic diamond lattice, and the resulting assembly kit is tested using an OxDNA simulation, which found that the correct structure was indeed formed.

Local time of random walks on graphs

This paper looks at finding expressions for averages of functions of the local time to be in a given state in a discrete state discrete time Markov process. The local time for a state is the number of times that state is visited in a given time window. The approach taken by the authors here is inspired by path integration techniques from quantum physics. The paper provides a method for finding the z-transform for the average of a given function of the local time. The z-transform is similar to a generating function for the averages of the function of local time in a time window n. Specifically, the average of the function of local time up to time n will be the coefficient of z^-n of the z transform expanded in powers of 1/z. This then does have the limitation, that finding the desired average given the z-transform can be a lot of work. However, overall it was nice to see a more interesting way for finding these functions of local time.

Imaging RNA polymerase III transcription using a photostable RNA-fluorophore complex.

Quantitative measurement of transcription rates in live cells is important for revealing mechanisms of transcriptional regulation. RNA Pol III is particulary challenging as this RNAP transcribes RNA molecules so it is not possible to use protein reporters. To address this issue, this group developed Corn RNA fluorescent aptamer that resembles the fluorophore found in red fluorescent protein. With this new tool, the authors were able to study and imaging the corn-tagged Pol III transcript levels.

Dissipation bounds the amplification of transition rates far from equilibrium

Kuznets-Speck and Limmer seek to demonstrate an idea that has long been gnawing at people working on the physics of computation. A system with two metastable states is capable of acting a bit. The lifetime of those metastable states determines how long the bit can reliably store information. Another related timescale is the time it takes to switch the bit, when such a switch is required. There is a general feeling that if you want both a long reliability time, and a short switching time, this should be costly (in terms of the energy you have to put in). However, such a tradeoff has not been found, in general, using the tools of modern stochastic thermodynamics. The title of this manuscript suggests that they have been able to identify a hard tradeoff; however, this tradeoff only appears when certain conditions are met. The authors argue that these conditions are quite general, but it is still unclear whether there is a limit on designing a reliable bit that can be switched quickly at low thermodynamic cost.

Saturday, 10 July 2021

The role of strand displacement: from the origin of life to current perspectives

Rolling-circle and strand-displacement mechanisms for non-enzymatic RNA replication at the time of the origin of life

Tupper and Higgs use basic chemical reasoning illustrated with ODE-level modelling to argue that pre-enzymatic replication of RNA templates would have been most successful in a "rolling circle displacement" mode, wherein a circular template is copied by a product that displaces its own tail as it goes round the template. This tail must eventually be cleaved by some kind of self-cleaving ribozyme, and ligated to form another circular template for the reaction to proceed to another generation. The authors argue that a displacement mechanism is the only way to avoid suppression of the reaction via product inhibition, wherein copies bind strongly to their templates. Going further than previous arguments, they claim that even in systems with time varying external conditions that allow separation and synthesis in different environments, products will still cause inevitable inhibition through rebinding at a critical concentration. This critical concentration is relatively low because pre-enzymatic extension of RNA on a template would have been slow. Moreover, rolling circle displacement is deemed to be better than displacement on a linear template, because linear templates suffer from the fact that the strand that is being synthesised can be easily displaced from the template at each (slow) synthesis step.

DNA computing: NOT logic gates see the light

This paper describes a NOT gate for DNA computation enabled by optical control of nucleic acid function via light-removable nucleobase caging groups. This temporal precise control using light allowed the authors the introduction of Boolean logic gates into single- and multilayer DNA circuits. The design was successfully integrated within NOT, NOR and NAND circuits demonstrating the potential of DNA circuitry.

Reactor design for minimizing product inhibition during enzymatic lignocellulose hydrolysis
II. Quantification of inhibition and suitability of membrane reactors

This review discusses the effect of product inhibition in the particular case of lignocellulose hydrolysis. The authors present ways of quantifying and experimentally characterise the inhibition and propose reactor designs that can minimise the product inhibition effect of the enzymatic reaction.

Perspective: Sloppiness and emergent theories in physics, biology, and beyond

This paper aims to utilise information geometry to simplify models with a large number of parameters. They make the point that in such models with many parameters, there are often multiple combinations of parameters which fit the model and that certain subsets of parameters make little difference to the predictions. This is made more formal by looking at the Fisher Information matrix (FIM) made from the parameters and finding that its eigenvalues have a roughly exponential structure, where the second largest is an order of magnitude smaller than the largest etc. This means only a few eigenvalues are relevant. Information geometry focusses on using the FIM as a Riemannian metric on the manifold of parameters. Due to the exponential structure of the eigenvalues the manifold has a ribbon like structure with boundaries corresponding to simplifications of the model. Certain combinations of parameters having little effect on the predictions translates into certain directions in parameter space being irrelevant. Following these directions to a boundary helps to simplify models. They give the specific example of a metabolic pathway being reduced from 48 parameters to 12.

Kinetics of heterochiral strand displacement from PNA-DNA heteroduplexes

DNA comes in two distinct enantiomers (L-DNA and D-DNA). These enantiomers can not form base pairs with each other. This paper develops a reaction known as heterochiral strand displacement which allows displacement of one enantiomer by the other using an achiral substrate strand made from PNA. This study undertakes an extensive characterisation of the kinetics of heterochiral strand displacement across a range of toehold lengths and mismatch positions. Heterochiral strand displacement is particularly useful when considering introduction of nanodevices into cells, as L-DNA will not interfere with native cellular molecules or be recognised by nucleases.

Room with a Re-view: A series of reviews on nucleic acid nanotechnology

 The i-Motif as a molecular target: More than a complementary DNA secondary structure

The i-motif is a DNA structure formed by C tetrads intercalated with small loops regions. In vitro this structure is formed at low Ph, since it requires the protonation of the C’s. However, this structures appear inside the cell at physiological pH. The present review discuss the functions of i-motifs as transcription regulators inside the cell and its uses in synthetic biology and nanotechnology. Special focus is given to the application of i-motifs knowledge in cancer therapy, since this motif is abundant in tumor cells.

In Vitro selection of RNA aptamers binding to nanosized DNA for constructing artificial riboswitches

The authors present a method to rationally construct artificial riboswitches using nanosized DNA aptamers. This particular aptamer allowed them to regulate the internal ribosome entry site-mediated translation in respond to a ligand (nanosized DNA). They proved that the induction ratio is much higher than the same type of riboswitch but using a different aptamer. They propose to use nanosized nucleic acid to build bacterial riboswitches as an alternative for other regulators such as toehold switches or small transcription activating RNAs (STARs)

Behaviour of information flow near criticality

In this paper the mutual information between two spins in a two-dimensional Ising model are explored. An input spin is chosen, and its value is set by a random telegraph process with a given timescale. An output spin, a distance, d, away from the input is then monitored. Two measures of the mutual information between the input and output spins are then measured: the instantaneous mutual information of the steady state, and the rate of increase of mutual information. For a given timescale of the input spin, both the instantaneous information and information rate were found to express a maximum close to, but not at, the critical temperature. Furthermore, the information rate maximum was found to be non-monotonic as a function of the timescale of the input. These maxima were explained to be due to the balance between thermal noise, which increases with temperature, and the response time of the system, which decreases with temperature.

Allosteric regulation of DNA circuits enables minimal and rapid biosensors of small molecules

This paper aimed to detect small molecule pollutants within environmental water samples, specifically two families of antibiotics. They employed the corresponding allosteric transcription factor to initially capture the ligand of interest e.g. TetR (tetracycline repressor). They exploited the competition between the allosteric transcription factor and an endonuclease to trigger a TMSD reaction and achieve signal amplification. In the presence of tetracycline, this can bind to TetR preventing binding of TetR to tetO (tet operator sequence) allowing the endonuclease to cleave and create a toehold. This can be accessed by a fluorescence reporter sequence. This is followed by cleavage cycles in order to get amplification of the signal. This system gives a broad linear range of detection.