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.

Tuesday, 2 February 2021

Under pressure: DNA origami, hairpins and the effect of extreme pressure

Robust direct digital-to-biological data storage in living cells

This paper describes an engineered redox-responsive CRISPR adaptation system for direct storage of digital data in living cells. They encoded binary data in 3-bit units into CRISPR arrays using SoxRS system and proved that it can be maintained over many generations. This DNA-based cellular memory device can be used not only in digital data storage but also in other biological recording applications.

How we make DNA origami

A practical guide on making a DNA origami object. From designing a 3D objects, ordering to folding, purification and quantification.

DNA hairpin hybridization under extreme pressures: A single-molecule FRET study

The authors test the stability of small hairpins of DNA as a function of temperature, pressure (1-3000 bar) and stem length. The overall results show that, due to the increase in free volume of the hairpin, an increase in the media pressure destabilises the hairpins. In addition, it is shown that the thermodynamic parameters of the hairpin can be easily modelled by dividing the contribution of the stem and loop.

First-passage probabilities and mean number of sites visited by a persistent random walker in one- and two-dimensional lattices

This paper looks to solve for a few relevant statistics for persistent random walker models in 1 and 2 dimensions. A persistent random walk is a discrete time stochastic process and a simple example of a random walker with memory. The walker moves in a certain direction, one step per time and at any time has a certain probability to change direction. This paper utilises various methods, primarily generating functions and transforms of them, to calculate the first passage probability for a site, that is the probability that the walker reaches a certain site for the first time at a certain time; and the mean number of sites visited by the walker as a function of time. However, most of the equations required to solve to find analytic solutions were not soluble, so, instead the limiting behaviours were found. Further, the continuum limit of these were also found to be in agreement with previous calculations. This somewhat technical paper showcases various methods and theorems useful for studying random walk models.

An enzyme-free surface plasmon resonance biosensor for real-time detecting microRNA based on allosteric effect of mismatched catalytic hairpin assembly

This paper presents an alternative approach for miRNA detection with a potential diagnostic outlook. This particular study aimed to achieve enzyme-free and label-free detection. They made use of catalytic hairpin assembly to facilitate enzyme-free amplification, and surface plasmon resonance for label-free detection. This system realised a picomolar limit of detection, even in the presence of total cellular RNA. This platform also shows good reusability.

DNA-based stategies for site-specific doping 

In the present paper the authors propose two different strategies with which DNA origami could be used as a tool for doping in Silicon lithography. In the first one the adsorption of DNA constructs over the surface at high temperature results on the deposition of phosphate groups over the surface resulting in n-type doping, whereas in the second one, the DNA origami acts as a passive masking element that gets modified with functional groups and acts as mask element prior to the etching process. The authors demonstrate the viability of the process to build FET devices with the technique, but, although the technique presents advantages such as low cost, the minimum width of the device built is equivalent to fabrication standards from 12 years ago.

 Second-generation DNA-templated macrocycle libraries for the discovery of bioactive small molecules

Here the authors improve upon an earlier method in which DNA templated chemical synthesis is used to generate diverse DNA-tagged libraries of bioactive molecules from a few DNA-tagged building blocks. First, a library of 20x20x20x32=256000 DNA templates with orthogonal codons is generated. Then reagents, which have DNA tags complementary to the codons on the template, combine to generate the macrocycle molecule encoded by the DNA template. Effective molecules can be identified by selection (increased binding affinity to a target molecule and filtering) and then reading the DNA templates by dna sequencing.

Saturday, 26 December 2020

From monomers to polymers

 Kinetic roughening of the urban skyline

A neat little paper showcasing an application of a statistical mechanical model to city skylines. Kinetic roughening is a nice example of a simple model that showcases some of the main themes of modern statistical mechanics including scaling and continuum limits. These models, in the discrete regime, consist of a lattice of height vectors which evolve in time due to some rules relating them to their neighbours. The roughness is defined as the root mean square of the heights. This roughness displays scaling behaviour, in particular for this paper, the roughness scales as the system size to some power after sufficient time passes. This paper looks at a huge database of 10^7 buildings in the Netherlands and calculates this saturation exponent for many cities. Where there is significant enough data, they find that the cities can be grouped into two sets with different exponents. These exponents correspond to the two main universality classes for roughening models. Finally, they remark that it would be interesting to consider the buildings regulations for each city to explain why they would fall into a given universality class.

Characterization and mitigation of gene expression burden in mammalian cells

In this work, the authors investigate the burden imposed by synthetic circuits in mammalian cells and study transcriptional and translational burden caused by cellular resource sharing. They were able to mitigate the effects of resource limitations using a microRNA-based incoherent feedforward loop (iFFL) motif. They concluded that using burden-aware designs, synthetic circuits that rely on perturbations will be able to show more accuracy and predictability.

A basic introduction to large deviations: Theory, applications, simulations

This approachable set of lecture notes was written following a 2009 paper reviewing the theory of large deviations (or LDT). The techniques of LDT provide a framework for a rigorous formulation of statistical mechanics. Most physicists and engineers have used the techniques of LDT at some point or another, but might not have been aware that this was the case! In essence, LDT is used to quantify the rate at which the probability that the sample mean S (of a set of samples of n independent identically distributed random variables each with mean u) is exponentially suppressed with increasing sample size n, where S is not equal to the mean u. This is a generalisation of the central limit theorem.

Programmed spatial organization of biomacromolecules into discrete, coacervate-based protocells

The coacervate created in this work was able to recruit his-tagged enzymes with via interaction with Nickel ion in the protocell. The increased concentration of enzymes led to acceleration in the enzymatic process. The proteins could be released by cleaving specific site as well.

DNA programmed chemical synthesis of polymers and inorganic materials 

The present review offers a general perspective on how DNA programmable interactions have been exploited in the field of chemical synthesis, first referring to the possibility of using directed interactions to perform directed polymer synthesis analogously to how copying polymers work in living cells, as well as how DNA can direct the conjugation and directed arrangement of different polymeric materials with aims as diverse as functionalisation for in vivo applications or directed assembly of conducting polymers as well as the assembly of metallic nanoparticles in prescribed arrangements that can have applications for plasmon resonance-based sensor applications. While showing that DNA nanotechnology is a powerful tool for material science-based nanotechnology applications, this review fails to specify which challenges face the field in order to be applied to other other fields (such as MOF, COF or polyoxometalate synthesis) in which the chance to implement programmable interactions could become a paradigm shift.

Emergence of low-symmetry foldamers from single monomers

Molecular self-assembly of simpler components often give rise to complex features in dynamic combinatorial libraries. In this article, the authors describe the emergence of large molecules with low symmetry unlike most previously described systems. When the monomers, capable of forming disulfide bonds among themselves, are equilibrated for several days, different libraries show the formation of predominantly one product, or a very small family of products. In some cases, large molecules were generated with very low symmetry (17 or 23 monomers). Further analysis by ion mobility mass spectrometry, NMR and CD spectroscopy showed that these large molecules are not simple 2D circular molecules, rather they all have unique complex folded structures. This was confirmed by the X-ray crystal structures of the synthesised molecules. The authors conclude that the semi-rigid backbone structures of the molecules, and the presence of several diverse sites for non-covalent interaction, which can overcome the inherent instability of large macrocycles, are crucial for spontaneous formation of newer complex foldamers.

Fuel-driven transient DNA strand displacement circuitry with self-resetting function

The authors present an enzyme-driven mechanism to allow continuous cycling of nucleic acid strand displacement circuits. The basic idea is to have strands that are uncompetitive on their own at displacing an output from a complex, but which can be ligated to a helper duplex which in turn can be ligated to the complex, allowing displacement to proceed. The ligation is performed by specific ligase enzymes, and consumes ATP. Subsequently, restriction enzymes can cut the strands, allowing the system to revert back to its initial condition. In principle, such a system could maintain a dynamic steady state of constantly cycled outputs, but the evidence for that in this manuscript is limited. Rather, transient spikes are observed in response to a signal, since there is apparently not enough ATP to sustain the output for a long period. An interesting question to ask is whether the scheme presented can be generalised to a large strand displacement network.

Feedback regulation of crystal growth by buffering monomer concentration

Many reactions, like crystallisation, need to operate at a very specific reagent concentration regime. However, even if the concentration requisites are met, as the reaction proceeds, the reagents get consumed and the reaction regime will change. The authors propose a method for maintaining constant reagent concentration by using buffering species. The mechanism consists of a pool of inactive DNA bricks that are in equilibrium with its active form thanks to a toehold exchange reaction. This mechanism is then used to grow a population of DNA nanotubes of regular sizes. When the active bricks are consumed, the equilibrium is displaced to the formation of new active bricks. However, the buffering power of this method is still limited, and the desired concentration can only be maintained for a few hours. Further increments of the buffering species concentration would block the reaction sites of the active monomers, hindering nanotube formation.

Thursday, 3 December 2020

A communication problem: cellular signalling networks

Do we understand the mechanisms used by biological systems to correct their errors?

This paper is a review of kinetic proofreading in biological systems, presented in a mostly historical fashion. The original concept of kinetic proofreading as introduced by Hopfield and Ninio is recounted. Following this, the concept of trade-offs between speed, energy dissipation and accuracy is introduced. The key results that biological systems tend to get accuracy to within acceptable levels, then prioritise maximising speed, followed by minimising energy dissipation is presented. In the process of explaining this, a number of methods utilised are briefly mentioned including: first passage time analysis, asymptotic analysis, and thermodynamic uncertainty relations. Finally, the concept of energetic vs kinetic discrimination is briefly introduced.

A biomimetic DNA‐based membrane gate for protein‐controlled transport of cytotoxic drugs

The ability to design membrane nanopores with controllable channel opening has the potential to have many applications in biomedicine, biosensing and artificial cells. In this paper, the researchers have shown the design and synthesis of a membrane nanopore constructed of only seven oligonucleotides where the lid of the nanopore is a thrombin/binding aptamer (TBA). Therefore, passage of small molecules through the nanopore is only allowed in presence of thrombin. It is shown that the nanopores can be incorporated in lipid vesicles. Nanopore-incorporated vesicles containing fluorescent dye are observed to release the dye only in presence of thrombin which proves that the channel opening is tightly controlled. This approach is applied to release biologically relevant molecules for controlled killing of cells. Cytotoxic drug topotecan is filled in vesicles containing the nanopores on the membrane; and these vesicles are added to HeLa cervical cancer cells. Upon addition of thrombin, the drug is released to the cells, and cell viability drops to 20% after three days compared to 95% when no thrombin is added. This paper thus shows a simple approach for biocompatible membrane nanopore design which can be opened by the presence of a biologically relevant substance.

DNA origami guided self-assembly of plasmonic polymers with robust long-range plasmonic resonance

The production of 1D chains by assembling DNA origami tiles have been widely used in nanofabrication. The defined geometry of the DNA tiles is harnessed to control the distance between DNA-bound metallic nanoparticles, producing nanowires used for plasmon propagation. However, regular origami tile assemblies are flawed by its flexibility, which leads to defects in plasmon signal propagation due to the variable distance between nanoparticles. The authors of this paper optimise the rigidity of the DNA origami chains by exchanging the classical tile for a hashtag-shaped structure. The proposed hashtag-structure can be functionalised with nanoparticles in several ways, while increasing the persistence length of the produced nanowires by one order of magnitude.

Combinatorial signal perception in the BMP pathway

Cells can sense ligands in their environment which bind to receptors on their surface, and trigger a response through intra-cellular signalling pathways. There are many types of type 1 and type 2 receptors in the bone morphogen pathway (BMP) that a cell could express, even more types of dimeric ligands that could be sensed. Through a combination of an in vivo search and the construction of a minimal mathematical model, Antebi et al. create a framework through which we can understand the multifarious signal processing operations that occur at the boundaries of cells. Crucially, the concentration of ligands (which is assumed to greatly exceed the number of available receptors) and the affinities between receptors and ligands determine the equilibrium distribution of ligand-receptor configurations, but the activity (the strength with which a ligand receptor combo can catalyse a signal) of the configuration does not need correlate with its probability of occurrence. Therefore, in a system with two types of surface receptors and two types of ligands (each with an affinity and activity with each receptor), the signal transduced can quantitatively differ when the ligands are presented alone or in combination.

3D engine in DNA code

Turing-completeness of Chemical Reaction Networks as well as the capability to be mapped into DNA Strand Displacement systems is always brought up as a proof of their versatility. However, most of the academic literature focuses on the implementation of CRNs in the context of building controllers, cellular automata or other sorts of computer science model problems rather than software applications that are found in our daily life. The present work presents how to encode a rudimentary 3D shader engine into a CRN and how to emulate and execute it, the most interesting part being how does it display a mapping between a high level standard abstraction language (Like JavaScript and CRNs). While still being impractical when compared to silicon-based devices, it's easy to see how a bridge between regular coding skills and molecular programming can be built. But it also must be said that in terms of software conservation, being able to engineer a sort of CRN/DNA-based assembler code that an be retroengineered into a high level language can be a promising technology to preserve software in a format with a longer life than optical or magnetic devices. 

Computing signal transduction in signaling networks modeled as Boolean Networks, Petri Nets and hypergraphs 

Transduction networks are known for being the regular architecture used bicellular systems to perform signal processing and decision making. Back from the foundational work by Dennis Bray they have been described as distributed network computing elements and mappings with computational models (mainly neural networks) have been drawn. But alternative mappings with other computing models can be drawn. In this paper, the authors explore other networks computing models that can be used to model transduction such as graphs, hypergraphs, Boolean Networks or Petri Nets, establishing the conditions in which a model is isomorphic to another and highlighting the strengths and weaknesses of each one. However, some of the strengths attributed to the Boolean Network model are rarely found in a biological context and none of the models take into account explicitly the catalytic nature of the network elements and the implications of the system, thus leaving room to potential new formalisms.

Self-limiting polymerization of DNA origami subunits with strain accumulation

The polymerization of DNA origami was controlled by accumulated strain during the growth process. The DNA origami consist of three domains with the middle domain being shorter than others. As the three domains are connected by linkers, they can deform while they grow, accumulating the strain coming from short middle domain. By controlling the length of the middle domain, the authors precisely controlled the growth of the DNA origami objects without any external control.

Single cell characterization of a synthetic bacterial clock with a hybrid feedback loop containing dCas9-sgRNA

Oscillatory dynamics facilitates the temporal orchestration of metabolic and growth processes inside cells and organisms. In this work, they present a synthetic oscillator gene circuit (repressilator) in which one of the repressors was replaced by CRISPRi system and they monitored the oscillations in microfluidic reacts using single cell experiments. They found that the period of the oscillator is much slower since it depends on the irreversible binding of the CRISPR system that prolongs its dilution phase. They propose to use RNA aptamers and use CRISPRa (activation) to improve the dynamics of the oscillator and explore the potential applications.

A ratiometric electrochemical biosensor for the exosomal microRNAs detection based on bipedal DNA walkers propelled by locked nucleic acid modified toehold mediate strand displacement reaction

In this paper, the researchers attempted to develop a novel biosensor for exosomal miRNAs, which achieved high sensitivity, high specificity and high reproducibility, such that the biosensor could be used multiple times. Their system involved locked nucleic acid capture probe associated with a bipedal DNA walker. The presence of the target miRNA led to release of the bipedal DNA walker by toehold-mediated strand displacement. The DNA walker walking over the electrode surface facilitated amplification of the electrochemical signal, allowing a low signal-to-background ratio.

Reciprocal coupling in chemically fueled assembly: A reaction cycle regulates self-assembly and vice versa

The authors seek to develop a synthetic system that replicates the behaviour of natural biopolymers such as actin, in which the constituent monomers can adopt two states, one of which is favourable for polymerisation ("active") and the other of which is less so. Moreover, the environment of the polymer catalyses the deactivation of the monomers, leading to non-equilibrium "living" polymerisation. The resultant dynamics are essential to cytoskeletal mechanics and motion. In this paper, the assembling monomers are short peptides with a switachble chemical moiety which can be charged (inactive) or neutral (active). Differential assembly in the two regimes is demonstrated, as is a catalytic effect on activation/deactivation rates of the polymer environment. However, the paper doesn't demonstrate preferential deactivation in the polymer state, as is seen in the natural counterpart.

Saturday, 21 November 2020

From membrane to cytoplasm: cellular applications of nucleic acid nanotechnology

Unzipping of a double stranded block copolymer DNA by a periodic force

In this paper, a simple model for mechanical unzipping of double stranded DNA (dsDNA) is explored using Monte Carlo simulations. Block copolymer DNA is considered, consisting of repeated units of different length each having two hydrogen bond monomers and three hydrogen bond monomers. Results for a static force applied to the tip of the dsDNA are reproduced displaying a first order transition from zipped to unzipped states and is independent of sequence. Simulations for a periodic force are also made and these are found to depend greatly on the sequence. Hysteresis curves are obtained for different sequences and the scaling of their area with frequency and amplitude of the periodic force is studied.

Genetic circuit characterization by inferring RNA polymerase movement and ribosome usage

This paper focuses on the analysis of complete genetic circuits using -omics tools (RNA sequencing and ribosome profiling) with pre-characterized genetic parts. They characterized the performance of each part of the circuit as well as the impact of the genomic context. These characterization results were used to understand the circuit dynamics and to analyse the circuit impact on the host cell (genetic burden).

Noise-induced symmetry breaking far from equilibrium and the emergence of biological homochirality

All biological molecules, like amino acids and sugars, are mostly found with a single chirality i.e. the spatial arrangement of its atoms. A specular arrangement of the molecule (enantiomer) would be equally stable; however, they are significantly scarcer. A mechanism to explain this asymmetry is still unknown, but some reactions networks have been proposed to explain the homochirality of biological molecules. Previous models assumed that homochirality is an equilibrium state. These models rely on an opposite enantiomers annihilation reaction, which doesn't correspond with the real dynamics of living systems.

The authors of this paper propose a new model that can explain homochirality with non-equilibrium dynamics. In the model, a single enantiomer can be randomly formed due to the system noise. If the autocatalytic formation of that enantiomer is faster than its degradation, it can keep replicating itself if the system is continuously fed with the enantiomer precursor. Next steps will validate these results in an experimental system, like Joyce's self-replicative ribozyme.

Signalling-based neural networks for cellular computation

Samaniego et al. point out that the kinase/phosphatase signal transduction networks have properties that lend themselves towards imitating neural networks built of molecular perceptrons. In particular, the antagonistic nature of the phosphatase/kinase push-pull motif naturally allows the output level of a substrate to incorporate a weighted sum over inputs with positive and negative contributions. Moreover, mechanisms such as zero-order ultrasensitivity allow the activity level of the substrate to show almost switch-like behaviour relative to a threshold in this weighted sum. The authors prove certain properties of cellular neural nets constructed in this way, demonstrating the possibility of encoding non-linear functions in multi-layer networks. The question of how practical this would be - particularly due to the peculiar properties of zero-order ultrasensitivity - remains an open question.

Functional and morphological adaptation in DNA protocells via signal processing prompted by artificial metalloenzymes

A protocell made of pure DNA. This protocell is constructed by annealing two types of ssDNAs produced by rolling circle polymerization from circular DNA templates. The protocell encapsulates artificial metallozymes that produces DNA-intercalating chemical. As the intercalating molecule gets produced, the protocell undergoes morphological changes such as expansion.

A DNA-nanoassembly-based approach to map membrane protein nanoenvironments

Mapping the nano-environment and the expression levels of extracellular proteins can reveal a lot of details about the cellular conditions associated with particular diseases. Different cancer states have shown different levels of EGFR family proteins and their dimerization states, especially the formation of heterodimers with Her2 protein. In this article the researchers have devised an approach to hybridize affibody (small, engineered proteins for high target specificity)-oligomer conjugates with a nanocomb (a long DNA strand partially hybridized with shorter DNA oligomers with overhangs). This nanocomb contains a Her2 binding affibody anti-Her2 attached to one end as a reference. The other overhangs encode information of their relative position to the reference, and a common binding domain for the oligos conjugated with the protein-binding affibodies. The affibody-oligo conjugates library is added to the concerned protein, and the nanocomb then binds to the conjugates which are close to the bound reference. These partially hybridised overhang-oligo complexes are then polymerised and nicked by enzymes, and the resulting double stranded DNAs are analysed by NGS. This analysis provides information about which affibodies are bound relatively close to the reference, and the distance between them. The group has shown that this approach can distinguish between different cells with different expression levels and dimerization state of Her2 as a proof of principle.

Nucleic acid strand displacement with synthetic mRNA inputs in living mammalian cells

In this paper, the authors investigated the efficiency of a basic strand displacement reaction within living mammalian cells. Probes were designed and modified in order to minimise the egration of nucleases and to ensure that the probes localise to the cytoplasm. The cells are engineered to express a target mRNA with the probe target sequence. In the 3’ UTR. Detection of the target mRNA results in detectable fluorescence and estimation of the number of mRNA molecules per cell. Moreover, this system can be used to follow target mRNA localisation in real-time.

Wednesday, 4 November 2020

Storage Wars: A battle of nucleic proportions

Reading and writing digital information in TNA

DNA molecules have become a popular information storage system but are susceptible to degradation in a cell-like environment. In this paper, the authors suggest the use of the unnatural genetic framework based on TNA. The backbone structure of this unnatural genetic polymer is recalcitrant to nuclease digestion. 23 kilobytes of digital information were stored in TNA and recovered with perfect accuracy after exposure to biological nucleases that destroyed equivalent DNA messages.

Metastable hybridization-based DNA information storage to allow rapid and permanent erasure

The authors argue that data stored in DNA in the traditional manner is hard to wipe clean - it requires enzymes, UV or extreme heat. They propose a stable but easily-erasable memory, in which single strands carry a memory location domain and the actual contents of the memory. Multiple distinct strands with the same memory location domain are included, but only one is prepared with a "truth" flag (a strand that is complementary to the memory location domain) whilst the rest are prepared with "false" flags (chemically modified strands). Data can be read out by performing PCR on the system, since the modified strands that constitute the false flags cannot be extended. Data can be easily wiped through a short heating protocol, which will randomise the flags very quickly. While the method achieves this functionality pretty well, it is limited - there is no scope to erase and then re-write data, for example.

Exponential volume dependence of entropy-current fluctuations at first-order phase transitions in chemical reaction networks

In this paper, the divergence of the fluctuations of a chemical switch as a function of system size is quantified and a critical exponent is calculated. The chemical reaction scheme of Schlögl model is studied. Here, the steady state number of a certain chemical species X displays a first order phase transition as the reaction rates are varied. This paper uses various methods to analyse the chemical master equation (CME) at the transition and calculate the variance to find an exponential dependence on system size with a given exponent. This provides a very readable example of a few methods to analyse CMEs.

Single-particle cryo-EM at atomic resolution

Cryogenic electron microscopy is used to determine the atomic-scale structure of biomolecules without needing to form a crystal of the sample, as is required for x-ray crystallography. In single-particle cryo-EM, a sample containing a high concentration of the molecule of interest is flash frozen and imaged repeatedly with a beam of single electrons. The scattered electrons, that are captured by a camera, form an image of the sample which is used to reconstruct the shape of the molecule of interest. Here the authors report three improvements that brought the resolution of cryo-EM below 2 Angstroms; an electron source with a narrow energy spread that reduced chromatic aberrations, a more effective energy filter that can separate out useful elastically and useless inelastically scattered electrons, and an advanced camera and reconstruction algorithm capable of capturing scattered electrons with high spatio-temporal resolution. In a reconstruction of a 5-fold symmetric human membrane channel protein, small molecule coordination, alternative amino acid conformations and side chain variations were resolved. Further, using an advanced reconstruction method of a 24-fold symmetric mouse protein, the scattering potential of hydrogen atoms could be resolved in the most ordered parts of the structure, enabling analysis of the hydrogen-bonding network in the protein. Atomic resolution cryo-EM will provide insights into the mechanical function of biomolecules and improve structure-based drug discovery.

The evolution of DNA-templated synthesis as a tool for materials discovery

Control of reactivity and product structure is one of the main challenges during the design of chemical reactions. A method to introduce said control is DNA-templated synthesis (DTS), where DNA-bound chemicals are assembled over a complementary DNA strand that acts as a template. The focus of this review is the potential application of DTS for the directed evolution of chemicals, by several cycles of template mutation and selection. The authors enumerate all the possible DTS mechanisms that allow the implementation of selection cycles, the chemical reactions templated with said mechanisms, and the challenges they face.

Nanopore-based DNA hard drives for rewritable and secure data storage

Solid-state nanopores have been demonstrated to be powerful molecular sensors for detecting nanoscale objects. In this article, a long DNA scaffold is use as a “hard drive” to write, erase, and rewrite data on, and read it out by passing the strand through a solid-state nanopore. The data sites are functionalised with small DNA-overhangs which can be hybridised with unique complementary biotinylated strands. The unhybridised and hybridised strands generate weaker and stronger current signals respectively when passed through the nanopore which can be read as 0 or 1. Thus, a combination of data sites can encode meaningful data in binary format. To erase the data, another fuel strand is added which utilises a short toehold at the end of the biotynilated strands to detach it from the overhangs which can be reused again to encode different data. To refine this approach, the group has also demonstrated the utilization of two separate encoding sites (address sites and data sites) simultaneously to encode both the data and its sequence. Moreover, they showed the use of a physical “key” to encrypt the data on the DNA hard drive without which the readout does not match the actual encoded data.​​​​​​​

Toehold-mediated strand displacement reaction for dual-signal electrochemical assay of Apolipoprotein E genotyping

Apolipoprotein E (ApoE) is a polymorphic gene which has been identified as an important genetic determinant for Alzheimer's disease. In the human population six genotypes of ApoE exist, with distinct risks associated with each. In this paper, the authors developed a toehold-mediated strand displacement-based approach to genotype ApoE. Using an electrochemical detection system, the authors could detect the presence of absense of mutations at 2 codons within the gene. This system allowed for systematic characterisation of ApoE genotype.

Proton-driven transformable nanovaccine for cancer immunotherapy

They developed a polymer-peptide conjugate that forms nanoscale sphere at normal pH. The polymer gets uptaken by cancer cells. When the polymer is at the endosome, it changes conformation to micrometre sized sheet and destroys the endosome. Then the peptide gets presented outside of the cell to trigger an inflammation response.

Light-activated signaling in DNA-encoded sender–receiver architectures

In this last work from De Greef's lab, the authors present the last developments on their compartmentalized DNA strand displacement platform BIO-PC. More precisely, they show how with the introduction of a photo-cleaving group in a DNA duplex, combined with different tunable variables, this system allows them to implement localized spatial activation of strand displacement circuits with controlled diffusion.  Based on this, they demonstrate how catalytic activation can transmit a given signal further than conventional activation and that this system allows them to build a spatially-coded AND gate.