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.

https://arxiv.org/abs/2010.13367

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).

https://www.nature.com/articles/s41467-020-18630-2

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.

https://journals.aps.org/pre/abstract/10.1103/PhysRevE.95.032407

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.

https://www.biorxiv.org/content/10.1101/2020.11.10.377077v1

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.

https://www.nature.com/articles/s41565-020-0761-y

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.

https://www.nature.com/articles/s41565-020-00785-0

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.

https://pubs.acs.org/doi/pdf/10.1021/acssynbio.8b0028

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.

https://pubs.acs.org/doi/pdf/10.1021/acssynbio.0c00361

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. 

https://www.nature.com/articles/s41467-020-18842-6

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.

https://journals.aps.org/pre/abstract/10.1103/PhysRevE.102.022101

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.

https://www.nature.com/articles/s41586-020-2829-0

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.

https://pubs.acs.org/doi/10.1021/acs.accounts.7b00280

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.

https://pubs.acs.org/doi/full/10.1021/acs.nanolett.0c00755​​​​​​​

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. 

https://pubs.acs.org/doi/pdf/10.1021/acssensors.0c01511


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.

Friday, 17 July 2020

All about that base (or at least, more than one article about pH-triggered nucleic acid systems)

pH-Controlled Detachable DNA Circuitry and Its Application in Resettable Self-Assembly of Spherical Nucleic Acids
https://pubs.acs.org/doi/abs/10.1021/acsnano.0c02329

Through Hogsteen interactions, DNA can form triple helices that are sensitive to PH variations. Triplexes form when pH is low, and break when pH is high. In this paper, the authors harness the formation of triplexes to build toeholds for strand displacement reactions. The kinetics of the displacement reactions are dependent of the formation of the triple helix, and they can even be stopped or resumed by changing the pH in the solution. Finally, they build a system with 2 triple helices that activate at different pH and can aggregate or disaggregate gold nanoparticles, just by changing the pH, without producing waste duplexes.


Preparation of a Millimeter-Sized Supergiant Liposome That Allows for Efficient, Eukaryotic Cell-Free Translation in the Interior by Spontaneous Emulsion Transfer
https://pubs.acs.org/doi/pdf/10.1021/acssynbio.0c00173

This paper describes an improved protocol to increase the efficiency of eukariotic cell-free translation. The conventional protocols are limited by high concentrations of sucrose that affect protein translation inside vesicles. They optimized the preparation conditions to permit supergiant unilamellar vesicle (SGUV) formation at a much lower concentration of sucrose that has almost no effect on translation. Under the optimized conditions, they observed a high rate of succesful SGUV formation (>90%) and a decent stability of the formed SGUVs (>60 min). These SGUVs are expected to serve as research tools in cell-free synthetic biology and as foundations for artificial cell-based biosensors.


Rational design of aptamer switches with programmable pH response
https://www.nature.com/articles/s41467-020-16808-2

Non-conventional base-pairs (Hoogsteen, Wobble) can introduce additional degree of tuneability to standard Watson- Crick base-pairs. A change in the environmental pH, which changes the propensity of such base-pairs, can be utilised to fine tune DNA-based systems. In this paper, the authors describe a designed strand where a linker domain forms a DNA triple helix at lower pH, and therefore destabilises a duplex of an ATP aptamer and a displacement domain, and thereby allows the aptamer to bind ATP with a 1000 fold higher binding efficiency compared to an elevated pH. In a separate approach, they demonstrated another design where the aptamer binds ATP better at higher pH. By combining these two feature in orthogonal fashion, the authors demonstrate that it is indeed possible to design an aptamer which binds its target in a very narrow pH range. Furthermore, since the actual structure of the aptamer sequence is not altered during the pH change, and the added domains show the pH responsiveness, the authors argue that this strategy can turn any aptamer into a pH switch with the need minimal alterations of the sequences.


DNA Logic Circuits Based on Accurate Step Function Gate
https://ieeexplore.ieee.org/abstract/document/9121273

A step function gate is achieved by combining step activation and a threshold module. This module was used to construct AND, OR, and XOR gates. The result was simulated using DSD.


Aminoacyl-tRNA Synthetases 
https://rnajournal.cshlp.org/content/early/2020/04/17/rna.071720.119.abstract

This is a comprehensive review of Aminoacyl-tRNA synthetases (aaRS), the family of enzymes responsible for charging transfer-RNAs (tRNA) with their cognate amino acid (AA). There is a unique aminoacyl-tRNA synthetase for each of the 20 amino acids. The accuracy of protein synthesis relies on both the matching of mRNA:tRNA duplexes in the ribosome, and the accuracy of amino acid:tRNA charging in aaRSs. AaRSs function as templates for the specific dimerisation of the two types of genetic molecule, amino acids and nucleic acids. Therefore these are the only enzymes that really do the job of converting the genetic code from nucleic acids to amino acids.

 Charged tRNAs are synthesised in two phases (AA binding and attack, followed by tRNA binding, hybridisation, and release) coupled to the turnover of one molecule of ATP to AMP. Some aaRSs exhibit post-charging editing, indicating that kinetic proofreading may be used to increase specificity beyond the equilibrium limit.


Kinetic asymmetry allows macromolecular catalysts to drive an information ratchet
https://www.nature.com/articles/s41467-019-11402-7

The present paper details how, in molecular systems, due to the local lack of detailed balance between the fluxes in macromolecular machines, it is possible to describe the working of out-of-equilibrium systems via two different mechanisms: (a) energy ratchets (in which the driving force of the system is a change in the external conditions -such as redox potential light or pH-  that drives the system to a new equilibrium) and (b) information ratchets (in which a catalyst biases the system by controlling and favouring the energy barriers we want). The paper, besides making the distinction, and showing the criteria to clasify molecular machines, shows different examples of each coming from different parts of chemistry explaining what properties of the system arise from underlying kinetic asymmetries.


Programming and simulating chemical reaction networks on a surface
https://doi.org/10.1098/rsif.2019.0790

Clamons, Qian and Winfree present an exploration of a previously-introduced surface CRN model of molecular computing. They present an authoritative review of the topic, and then demonstrate how to deal with some of the challenges of implementing complex surface-based CRNs, in particular issues with asynchronicity. The authors then show how these strategies can be used to build an extremely broad range of interesting molecular circuits. I have no concerns about the accuracy of the results.
The examples and supporting code are extremely effective pedagogically and the paper is fun to read - it should inspire theorists and experimentalists alike.


Wednesday, 24 June 2020

Reading List: Rats on the latest celebrity diet

Caloric Restriction Reprograms the Single-Cell Transcriptional Landscape of Rattus Norvegicus Aging
 
Calorie Restriction (CR) leads to slow down of ageing in mammals. The statement has been passed around and been a cause for scientific debates. What is missing to substantiate the former claim is a CR cell atlas across body tissues. The research observes (in mice) the systemic effects of aging and CR on different tissues evaluated in terms of cell type composition, tissue-specific molecular programs, regulatory transcription factors (TFs), and cell-cell communication networks. It was observed that fewer lipid droplets accumulated in livers of the experimental group (excess lipids cause atherosclerosis: high cholesterol levels). An accumulation of senescent cells was found in the control group (senescence is caused by gradual telomere attrition at the ends of DNA reducing reproducibility) as compared to the experimental group. The number of immune cells in nearly every tissue studied dramatically increased as control rats aged but was not affected by age in rats with restricted calories. Levels of the transcription factor Ybx1 were altered by the diet in 23 different cell types (out of 40 chosen). The scientists believe Ybx1 may be an age-related transcription factor and are planning more research into its effects.


Interfacing gene circuits with microelectronics through engineered population dynamics

In this paper, the authors propose an alternative to fluorescent reporters to analyse bacterial population behaviours. They interfaced synthetic biology with microelectronics through engineered population dynamics that regulate the accumulation of charged metabolites. During bacterial growth, charged ions are naturally released because of metabolic processes and the environment becomes more conductive, which decreases the impedance to electrical current. To control bacterial populations, they engineered a genetic circuit to express a bacterial killing gene that is activated upon the addition of an external stimulus. Therefore the bacterial population resembles a resistor, which is controlled by a genetic circuit. The plan is to implement this regulatory system in a microelectronic platform where several chambers may contain unique genetic circuits, connected via electrodes to an impedance output system.


Isothermal digital detection of microRNAs using background-free molecular circuit

Micro-RNAs have emerged as a class of potential biomarkers due to an increasing amount of research identify their disregulation in several diseases. But detection of very low concentration of mi-RNA with high accuracy poses a challenge for the existing techniques. One of them is background noise and nonspecific amplification of products which renders low-concentration detection of microRNA practically impossible. This article describes the utilization of already existing PEN-DNA toolbox coupled with a leak absorption mechanism to eliminate background noise. This method generates a fluorescence signal specific for the amplification of the signal strand which is produced upstream by the microRNA. The overall system can detect microRNA concentration as low as 1fM.


Information-theoretical bound of the irreversibility in thermal relaxation processes

Relaxation processes must produce entropy as they cannot be quasistatic or reversible. Here Shiraishi dervied a stronger than 2nd law bound on the entropy production in for a relaxation process. The bound on the entropy production is given by the Kullback divergence between the initial and current time distribution of the system. Hence if the initial and final distributions are very different then there MUST be a large entropy production.
Brings up an interesting way of describing permissable trajectories using information geometry (interpret KL div as an euclidean distance).


Cancer Diagnosis with DNA molecular computation

A simplified winner takes all DNA computing scheme using 4 miRNA input was used to diagnose lung cancer cells. The network was trained in silico and realised with DNA.


The Synthesis Success Calculator: Predicting the Rapid Synthesis of DNA Fragments with Machine Learning

The efficiency of DNA synthesis is sequence-dependent; however, the effects of each sequence property is not well understood. To address this problem, the authors design a random forest classifier to quantify the effects of 38 sequence properties in the synthesis of more than one thousand DNA sequences. The conclusion is that only 9 properties are relevant, the most important being the length of the longest repetitive sequence within the DNA fragment (26 nt is the limit), followed by the strand GC content (between 29-63%). A predictive tool is available online to estimate the success of your DNA synthesis orders and suggest changes for synonym codons: https://salislab.net/software/


A Dynamical Biomolecular Neural Network 

Artificial neural networks (ANN) are amongst the most used computation models in machine learning and they have been proved to be extremely powerful for classification tasks in silico. Some DNA strand displacement implementations of these systems have been developed in the past, but this circuits were limited by its size, implementability and inability to be rebooted. In the present work, Ron Weiss and collaborators described a theoretical implementation of a perceptron (the functional unit of an ANN) in a biochemically feasable CRN. In the CRN two mutually sequestering chemical species with their production and degradation rates, encode the positive and negative weights of the perceptron.  Based in this design, the authors demonstrate that perceptrons can be extended into deeper networks and implement complex behaviours.


Small RNA driven feed-forward loop: Fine-tuning of protein synthesis through sRNA mediated cross-talk

Tej and Mukherji present an analysis of an sRNA-mediated feed-forward loop, in which a particular sRNA not only promotes translation of a protein, but also translation of a second protein (a sigma factor) that in turn promotes transcription of the mRNA of the first protein. They show that competition between the mRNA for the sRNA leads to a non-monotonic effect of sigma factor transcription rate on output protein levels, and that relative fluctuations are smallest at the point of maximal output protein levels.



Friday, 22 May 2020

New paper in Nature Communications introducing a new strategy to build synthetic DNA-based networks that function more like similar systems in living cells.

Natalie E. C. Haley, Thomas E. Ouldridge, Ismael Mullor Ruiz, Alessandro Geraldini, Ard A. Louis, Jonathan Bath & Andrew J. Turberfield 
In recent years, scientists have sought to construct molecular systems that reproduce the complexity of life in a synthetic (human-designed-and-built) setting. On the one hand, building a synthetic version of a natural system would help us to understand the natural systems more deeply, in the same way that actually building a walking robot demonstrates just how impressive locomotion is in the animal kingdom. On the other hand, synthetic molecular systems have great potential as an engineering platform of the future, adding control and designability to the power and versatility of nature.
The use of synthetic DNA as an engineering material has been particularly successful, leading to the growth of the field of DNA nanotechnology. Bespoke single strands of DNA can be ordered from chemical suppliers, as easily as personalised greetings cards. Sequences of the bases - the familiar A, C, G and T of the genetic code - can be specified at will. These bases interact in a highly specific and predictable way, with A-T and C-G base pairs allowing the formation of the famous DNA double helix. If a set of strands is well designed, they can spontaneously self-assemble into a complex structure, or implement a computational calculation, when mixed [1,2].
Although these results are impressive, we are a long way from the power and flexibility of the natural systems that inspire us. One important aspect is the following: a defining feature of life at the molecular scale is constant activity; a cell isn't a static structure that assembles once with all its components in place. Instead, the molecular circuits inside are constantly on the go, allowing for growth, replication and maintenance of the cell in a healthy state, ready to respond to changes in the outside world. Key components (such as enzymes) participate in reactions but are then recovered, rather than being consumed, allowing them to continue to operate.
Physicists would say that these living systems operate out of equilibrium, and must continuously consume chemical fuel such as ATP to do so [3]. These fuel molecules must be stable on their own, but provide a large energy boost when they are broken down - just like the fuel in a car. In this work we present a new strategy for designing similar behaviour in DNA-based systems: we place mismatched base pairs (not A-T or C-G) in the interior of double-stranded DNA reactants. These mismatches are eventually eliminated when the reactants are converted into products. However, the reactants are essentially stable, despite the overwhelming favourability of mismatch-free products, because the destabilizing mismatches are well hidden. The effect of the mismatches is only felt when additional DNA strands - the key (enzyme-like) species mentioned above - trigger the system. These key species are recovered, as in natural systems, and the elimination of hidden mismatches fuels the process in a controlled way, analogous to the role of ATP in natural systems.



Fig. 1. Analogy between hidden thermodynamic driving in our DNA-based system and ATP in a natural context. The breakdown of ATP releases energy, but is slow unless an enzyme is present to lower activation barriers. Similarly, the conversion of reactants to products in our DNA system eliminates a mismatch “X” and therefore releases energy; however, the hiding of the mismatch makes the reaction slow unless a triggering strand is present.

[1] Rothemund, P. W. K. Folding DNA to create nanoscale shapes and patterns. Nature 440, 297 –302 (2006).
[2] Cherry, K. M. & Qian, L. Scaling up molecular pattern recognition with DNAbased winner-take-all neural networks. Nature 559, 370–376 (2018).
[3] Ouldridge, T. E. The importance of thermodynamics for molecular systems, and the importance of molecular systems for thermodynamics. Nat. Comput. 17, 3-29 (2018). 

Thursday, 14 May 2020

2 weeks of the reading group - plenty of DNA nanotechnology, from assembly through detection to signalling

A fluorescence assay for microRNA let-7a by a double-stranded DNA modified gold nanoparticle nanoprobe combined with graphene oxide
https://pubs.rsc.org/en/content/articlelanding/2020/an/c9an02274k/unauth#!divAbstract  

The authors used a cascaded toehold-mediated strand displacement reaction as a biosensor for miRNA. This required both a fuel strand and a target strand and the target strand was recycled as part of the reaction, to amplify the signal for detection.


Orthogonal regulation of DNA nanostructure self-assembly and disassembly using antibodies
https://www.nature.com/articles/s41467-019-13104-6

Despite tremendous developments in DNA nanotechnology and antibody research, there have been very few examples of designing a DNA-based network specifically responsive to a particular biomarker. Here the researchers demonstrate the design of an antigen-conjugated split-input invader strand which increases the rate of a TMSD reaction when it binds a specific antibody. Different antibody-controlled reactions can be triggered orthogonally in a solution with several reaction components without any crosstalk. The output strands of these reactions can be specifically tuned to trigger a dynamic self-assembly of DNA tiles into a nanostructure or the disassembly of it into individual building blocks.


Availability-Driven Design of Hairpin Fuels and Small Interfering Strands for Leakage Reduction in Autocatalytic Networks
https://pubs.acs.org/doi/pdf/10.1021/acs.jpcb.0c01229

Enzymes are hard to use in detection and amplification circuits for eg. diagnostics. Nucleic acids provide an alternative, but are subject to unintended leaks in the absence of input. In this article, the authors seek to avoid leak reactions by sequestering nucleotides that are predicted - based on simple thermodynamic models - to trigger these leaks. However, success is limited because sequestering these nucleotides, if effective, also interferes with the intended reactions in the presence of a trigger.


Nicking-Assisted Reactant Recycle To Implement Entropy-Driven DNA Circuit
https://pubs.acs.org/doi/10.1021/jacs.9b07521

Molecular circuits implemented using nucleic acid nanotechnology typically produce double-stranded waste complexes when they run. In this work, the authors propose that these waste complexes can be reconverted into active reaction-ready multi-stranded "gates" through the action of a nicking enzyme that cleaves the backbone of one of the fuel strands. This approach means that, in the simplest of settings, only a supply of single-stranded molecules (rather than harder-to-produce gate complexes) is required to sustain circuit function.

Although impressive, these circuits show a fairly high level of unwanted leak reactions. Moreover, the recycling of waste does not occur indefinitely, and complex cascaded circuits cannot be produced due to sequence constraints. The article really emphasizes the need for in situ production of nucleic acid complexes.


ATP-Triggered, Allosteric Self-Assembly of DNA Nanostructures
https://pubs.acs.org/doi/10.1021/jacs.9b10272

Trigger-responsive DNA self-assembly is commonly observed in several biological processes and have potential application in sensing and smart biomaterials. In this article, the authors show the design of a double stranded DNA which, upon binding with ATP, can form T-junctions among themselves to make larger self-assembled structures. In the absence of ATP, such structures are not formed. It also demonstrated that the ATP-binding and subsequent change in the overall conformation of the DNA is the crucial part of stimulus-responsiveness.


Fluorogenic probe for fast 3D whole-cell DNA-PAINT
https://www.biorxiv.org/content/10.1101/2020.04.29.066886v1

DNA-PAINT is a super-resolution microscopy method that uses fluorophore-modified DNA labels to image some target DNA strands. However, DNA-PAINT requires a high concentration of labels, resulting in high levels of background fluorescence during the imaging. In addition, the binding speed of the labels can hinder DNA-PAINT by reducing its imaging speed. This research introduces a new type of label for DNA-PAINT. The new labels reduce its fluorescence emission in solution by attaching a dedicated quencher. Since no hairpin in the label is needed for quenching the fluorescence, the binding rate to the target is increased. The unbinding rate is increased as well by adding mismatches between target and label. The new label design results in a higher imaging speed while still producing a low background fluorescence.


Information Coding in Reconfigurable DNA Origami Domino Array
https://onlinelibrary.wiley.com/doi/abs/10.1002/anie.202003823

DNA origami domino arrays, whose building blocks adopts two different conformations, were used to encrypt information in their 2D pattern. Additionally, strand-displacement was used to reveal overhangs with specific sequence that encodes information.


Non-enzymatic primer extension with strand displacement
https://doi.org/10.7554/eLife.51888

Non-enzymatic template copying reactions are the precursor to biological self-replication. Separating the duplex that forms between parent and daughter strands after templated copying is key to ensuring independent function of the daughter and cyclical copying of the template. Cycling environmental conditions (hydration/pH/temperature) have been posed as solutions to the duplex separation problem. However, here an RNA template is copied by primer extension and simultaneously the previous daughter (blocker) is displaced from the template by a strand invasion reaction, enabling further extension.

Here a template RNA is occupied by a partially complete primer strand and a blocker strand (equivalent to an earlier daughter) with a large free toehold. The blocker strand prevents extension of the primer by binding to the next extension site thereby occluding the template. An invader strand is introduced that binds the blocker toehold and then invades the blocker-template bond at the extension site. The strand invasion interaction opens the template which triggers the extension of the primer. Increase in primer length is confirmed by PAGE.


Artificial molecular motors
https://pubs.rsc.org/en/content/articlelanding/2017/cs/c7cs00245a#!divAbstract

Living cells use a plethora of molecular motors to carry out key biological processes. Muscle contraction, production of ATP from ADP, DNA transcription are all examples of molecular motors at different scales. Development of synthetic motors is a contemporary field of research in nanoscience with one application being drug delivery to cancer cells. Molecular switches and motors are 2 different types of molecular machines. In both these machines, a change in relative position of components with respect to each other occurs; the cycle of a motor, however, can perform work. Present research explains the physics of these molecular machines utilizing the chemistry (steric interactions, effect of pH, acidity) of chemical compounds.

This review focuses on molecular devices constructed using organic chemistry, rather than biomolecules. Research groups have been developing nanocars and are actively working on making them unidirectional. Unidirectionality in the presence of light has been shown at microscale (rotation of an alkene doped glass rod) and macroscale (droplet along a photo-responsive surface). Molecular motors have evolved from elegant proof-of-principles to advanced designs, the main question remains is how to convert this motion into useful functionality?


Encoding multiple digital DNA signals in a single analog channel https://academic.oup.com/nar/advance-article/doi/10.1093/nar/gkaa303/5825621

DNA strand displacement systems' output readouts are normally limited by the different amount of fluorophores that can be implemented and read in the fluorescent reporter systems. This limitation usually results in systems with a very limited number of outputs: one possible output per fluorescent channel. In the present work the authors propose  method to overcome this limitation based on representing multiple discrete bits of information in a single analog fluorescent signal. With this method, optimizing the toehold and sequence design they are able to encode reliably a 4-bit signal in a single fluorescent signal - they could detect the presence of 4 different genes with a single fluorophore - as well as applying the methodology to two channels simultaneously, thus increasing remarkably the number of possible readable outputs of a circuit.


A Coculture Based Tyrosine-Tyrosinase Electrochemical Gene Circuit for Connecting Cellular Communication with Electronic Networks
https://pubs.acs.org/doi/abs/10.1021/acssynbio.9b00469

In this paper, authors reported a cell-based synthetic biology−electrochemical device. The system builds on the tyrosinase-mediated conversion of tyrosine to L-DOPA and L-DOPA quinone which are both redox active and can be detected by a gold electrode. The use of cell consortia allows for divisions of labor to lower any particular metabolic burden in the production of tyrosine and tyrosinase. To induce the expression of these molecules, they use quorum sensing signalling molecules and pyocyanin that are secreted by Pseudomonas aeruginosa.

Sunday, 12 April 2020

Reading group - lots of novel DNA systems, including cryptography!

Self-Assembly of DNA Origami Heterodimers in High Yields and Analysis of the Involved Mechanisms
https://onlinelibrary.wiley.com/doi/abs/10.1002/smll.201902979

DNA nanostructures can be formed of several different DNA origami units that bind between themselves with complementary extended strands. However, the binding reaction between two origamis doesn't have a perfect yield (80~90%), which decreases exponentially with the number of origami units added.

The paper demonstrates that the source of imperfect yield when binding origamis is not due to the stability of the binding, but all the possible competing reactions. Proper purification of each origami piece, especially with an agarose gel in low salt conditions, removes the excess of DNA strands used to build these origamis. The removal of excess strands helps to reduce the homodimers and other large structures, increasing binding yield up to 99%.


Coupling of DNA Circuit and Templated Reactions for Quadratic Amplification and Release of Functional Molecules
https://pubs.acs.org/doi/abs/10.1021/jacs.9b05688

By putting a four base-pair overhang with a photocatalysis modification onto the final product of catalysed hairpin assembly, the response to the presence of an initiating strand was further amplified, resulting in quadratic amplification.


Nucleobase-Templated Polymerization: Copying the Chain Length and Polydispersity of Living Polymers into Conjugated Polymers. 
https://doi.org/10.1021/ja809613n

In the absence of a template, step polymerisation processes often offer little control over the average average length and width of the distribution of polymers produced. The average molecular weight and the spread of the molecular weight distribution of an ensemble of polymers have significant effects on the macroscopic properties, such as viscosity, of the polymer bulk.

Living systems use templates to direct the synthesis of polymers. The template functions as a guide for information transfer, but also fixes the polymer length and narrows the length distribution.
In this work, a thymine block template polymer was used to direct the synthesis of another polymer. To achieve a narrow polymer length distribution, the templates must also have a narrow length distribution. The template was created by 'living polymerisation'. Living polymerisation is a catch-all for polymerisation processes in which termination is prohibited and the initiation rate is much faster than the elongation rate, leading to a smaller variance in polymer length.

Once the templates were assembled, they could be used to grow templated polymers. After the templated polymers were elongated, they were non-autonomously separated from the template, which did not cause the polymers to fragment. The distribution of templated polymer lengths had an average close that of the templates and a narrow spread. By contrast, polymerisation with incompatible templates and in the absence of templates resulted in short polymers with broad length distributions.

This is experimental confirmation that templates, regardless of informational content, are unsurprisingly effective in narrowing and controlling polymer length distributions.


De novo design of protein logic gates
https://science.sciencemag.org/content/368/6486/78

In the present work, the lab of David Baker demonstrate that they can design and create different alpha helical bundle motifs with tunable binding affinities. These motifs  bind orthogonally only to  programmed domains. With these domains, incorporated into transcription factors via fusion proteins, the authors are able to implement the six basic Boolean Logic Gate functions in genetic circuits that work independently of the type of host cell.


DNA origami cryptography for secure communication

Biomolecular cryptography exploits theromdynamically controlled biomolecular interactions instead of typical computational schemes for the same level of encryption. This paper suggests a DNA origami-based encryption method with a key size of 700 bits (for comparison, typical RSA key length is 1024 bits to ensure day-to-day web browsing security).

Alice wants to pass a secret message to Bob. Alice converts the message to a spot pattern (based on binary conversion of alphabets in the message and their positions). A custom DNA scaffold sequence is routed through a defined geometry covering this spot pattern. M-strands (biotinylated message strands), corresponding to the spot patterns, are hybridized onto the scaffold strand.
The scaffold is now passed onto Bob. Bob holds the staples to fold the DNA origami structures to reveal the biotin patterns. He then uses streptavidin to make the biotin patterns recognizable and obtain the hidden secret message. The security is maintained by unpredictability of the sequence, length and folding of the scaffold strand.


A blueprint for a synthetic genetic feedback controller to reprogram cell fate

The paper considers the problem of controlling cell phenotypes by manipulating the concentration of transcription factors in the underlying gene-regulatory networks. To this end, a fast-slow/high-gain feedback controller is developed, consisting of fast production and degradation of the transcription factors, which, for suitable multi-stable (multi-phenotypic) gene-regulatory networks, destroys all but one stable equilibrium and achieves desired uni-stability (uni-phenotype). The controller is mathematically justified at the deterministic level using suitable perturbation methods. Furthermore, an experimental implementation of the controller is also proposed, based on an intracellular integration of suitable synthetic genes which can be controlled by inducible promoters. 


Nicking-Assisted Reactant Recycle To Implement Entropy-Driven DNA Circuit

Molecular circuits implemented using nucleic acid nanotechnology typically produce double-stranded waste complexes when they run. In this work, the authors propose that these waste complexes can be reconverted into active reaction-ready multi-stranded "gates" through the action of a nicking enzyme that cleaves the backbone of one of the fuel strands. This approach means that, in the simplest of settings, only a supply of single-stranded molecules (rather than harder-to-produce gate complexes) is required to sustain circuit function.

Although impressive, these circuits show a fairly high level of unwanted leak reactions. Moreover, the recycling of waste does not occur indefinitely, and complex cascaded circuits cannot be produced due to sequence constraints. The article really emphasizes the need for in situ production of nucleic acid complexes.


The Protection Role of Magnesium Ions on Coupled Transcription and Translation in Lyophilized Cell-Free System 

The storage of a cell-free protein synthesis platform usually involves lyophilization that decreases or even inactivates transcription/translation machinery due to conformational damage of the involved enzymes. The authors proposed that two-metal-ion regulation by magnesium provides protection and regulation of the enzymes and they are essential to preserving the activity of the cell-free protein synthesis systems. This work has important implications for maximizing protein yields in cell-free systems.