Reading List: Rats on the latest celebrity diet

Caloric Restriction Reprograms the Single-Cell Transcriptional Landscape of Rattus Norvegicus Aging

https://www.sciencedirect.com/science/article/pii/S0092867420301525

 

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

https://advances.sciencemag.org/content/advances/6/21/eaaz8344.full.pdf

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

https://advances.sciencemag.org/content/6/4/eaay5952/tab-article-info

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

https://doi.org/10.1103/PhysRevLett.123.110603

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

https://www.nature.com/articles/s41565-020-0699-0

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

https://www.biorxiv.org/content/10.1101/2020.06.05.136820v1?rss=1

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 

https://ieeexplore.ieee.org/document/9030122

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

https://arxiv.org/abs/1912.06786

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.

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

Design of hidden thermodynamic driving for non-equilibrium systems via mismatch elimination during DNA strand displacement

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). 

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.

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

https://www.nature.com/articles/s41467-019-13517-3

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

https://www.ncbi.nlm.nih.gov/pubmed/28065574

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

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.

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

https://pubs.acs.org/doi/10.1021/acssynbio.9b00508

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.

Kinetic Proofreading and the limits of thermodynamic uncertainty: a review. Jenny Poulton

This paper attempts to link together two important concepts in theoretical biophysics: kinetic proofreading and the thermodynamic uncertainty relation. It analyses both in the context of copying sequence information in a polymer, but feels like it confuses more than it clarifies. The paper does make some clear statements about the predictability of travelling through a copying network. However, it fails to link this quantity to either speed or accuracy.  It further fails to make the case for the intrinsic use of this predictability, in systems such of this.

It is worth taking a moment to briefly discuss the ideas of kinetic proofreading and the thermodynamic uncertainty relation separately, before we attempt to link the together. Kinetic proofreading was first posited separately by Hopfield (1974) and Ninio (1975). It is a method by which biochemical copying systems, such as RNA translation, can improve accuracy by spending extra energy. It is also an excellent example of the motivation [TO1] behind using simple theoretical models to describe systems; while kinetic proofreading was initially posited as a completely theoretical idea, it was widely adopted by the biological community as it gave good agreement with real biological results.

In general, simple copying systems approximate to a system as shown in figure 1. A copy polymer is growing on a template polymer, connected only by its final monomer. A new monomer, of either a matching or non-matching type will bind to the template polymer. It will then polymerise into the chain, and the previous final link between copy and template will break.

Now in a copying system, the most obvious question to ask is about accuracy, how well does the copy match the template, and how does the system discriminate? Discrimination comes in the very first step. Because non-matching monomers are more weakly bound to the template polymer, they fall off more quickly than matching monomers. Thus, if the rates are carefully tuned, the system can polymerise the monomer into the copy polymer chain fast enough that matching monomers are unlikely to fall off before incorporation, but non-matching ones will fall off. Thus the system can generate accuracy.

Kinetic proofreading adds an extra energy driven step. Instead of the system polymerising the monomer into the chain directly, the system first has to spend energy activating the monomer, and only then can the monomer be incorporated into the chain. As long as the activation step is driven energetically towards activation, either through a chemical gradient or more directly, then this effectively gives the incorrect monomer two opportunities to fall off rather than be incorporated; in some limits squaring the discrimination term. Thus you can pay extra energy to increase accuracy.

Now before I move onto the thermodynamic uncertainty relation, I’m going to take a moment to stress that in a kinetic proofreading system, one is usually considering the error, ie. how alike the copy polymer and the template polymer are. This is not the same as the uncertainty in the thermodynamic uncertainty relation.

Figure 1; Left: a simple three step copying reaction in which a monomer binds to the template, is polymerised into the chain and the previous final monomer detaches. Right: the system with an additional proofreading step. The system must be energetically driven to activate the monomer, at which point it has a second opportunity to fall off before incorporation.

So what is the thermodynamic uncertainty relation? The thermodynamic uncertainty relation considers a stochastic process, often visualised by a network of states which outline progression through a process. In the case of biological copying, the process is that of adding a monomer to the end of a growing polymer as shown in figure 2. However, it could equally be the process of a molecular walker taking a step along a track. The thermodynamic uncertainty relation tracks the uncertainty in the net number of times a particular thing  happens . For example, the number of times a system undergoes a specific transition. In a case of a molecular walker  could be the uncertainty in how far the walker had walked with the net number of forward steps being . In the case of a copy process   could be the uncertainty in the net number of correct things the system has added (ie how many times the system has gone round the upper right loop in figure 2) or it could be the uncertainty the net number of incorrect things that have been added (upper left loop). Crucially  would not automatically give you a relationship between the number of right and wrong things added. The form of the uncertainty relationship  tells us that we can again spend energy to reduce the uncertainty; here  is the energy cost of the process per unit time multiplied by the time.

So while both relationships have a form of uncertainty, and this uncertainty can be reduced in both cases by paying energy, the two uncertainties; the error  and the uncertainty , are not immediately related to each other and should not be conflated.

Now the aspect of kinetic proofreading which is most straightforward to link to the thermodynamic uncertainty is not the error but the speed. Banerjee et al discusses how for many simple copying processes such as those found in T7 DNAP enzymes acting on DNA and TRNA selection in E. coli ribosomes, in general systems are willing to tolerate a certain amount of error in order to maximise speed. While the thermodynamic uncertainty relationship doesn’t directly measure speed, it does characterise the uncertainty in progress, ie how reliable said speed is. Someone with a stronger molecular biology background than I might be able to convince me that predictability in copying speed is important, sadly the paper fails to do so.

Figure 2 The left hand side represents the network for adding a monomer in DNA related actions, the right hand side represents that for RNA related actions. In both the green “reduced system” at the bottom shows the path for adding a new monomer and extending the chain whereas the blue cycle adds and removes a monomer through kinetic proofreading. Reactions a and b are gradually turned off later in the paper.

The paper focusses on the number of times the system above goes round the green cycles in figure 2, with this being   .  The thermodynamic uncertainty variable  is defined relative to this. They define a lower bound on  by considering the reduced cycle corresponding to that system which contains only the green cycle. For this cycle which is unicyclic the uncertainty is well defined and here labelled; . They thus define a quantity  which defines the thermodynamic uncertainty relative to the minimum uncertainty and compare this quantity for a number of different systems. However it should be clear that it is not true that  means lower error, it merely states that it the net number of times the system goes round that particular cycle becomes more predictable. They compare this variable for a series of biological systems, with two subtly different networks shown in figure 2. These are Err ribosome (right) WT ribosome (right), Acc ribosome (right) and T7 polymerase (left). Recall that  . They present these results and suggest that a lower  means that the system is capable of better trading off error and speed, although this isn’t necessarily persuasively explained.

In the next section the authors ask how turning off two of the reactions, which reduce accessibility to parts of the network, changes . They relate this to error  and , a time constant which quantifies the time taken to go round the cycle.

Removing reaction a) as labelled in figure 2 prevents the system from attempting to add incorrect monomers, and removes access to the whole left hand side of the cycle. Removing reaction b) prevents the system from removing correct monomers via kinetic proofreading, but does not change the overall topology of the network in the way removing a) does.

The authors state that in both cases  is reduced when the reactions are removed. This would seem self-evident. They also state that energy cost for faster speeds is minimized when reactions are removed, because you don’t spend extra energy being pushed around futile cycles.

The authors point out that in case a) Q decouples from  as the reaction is removed, but the network retains it’s dependence on  in case b). They then explore how  depends on Q. In both cases they find that as the reactions are removed,  decouples from Q. This tells us that in the accurate limit where only the reduced cycle happens, Q is not related to . So the measurement of Q seems like a curious choice when it is decoupled from both  and  in the accurate limit.

The thermodynamic uncertainty relation seems like a tool unsuited to answer the important questions about accurate copying. While there may be good reasons to perform this analysis, the authors have failed to provide them.

A full house of papers combining DNA with other biomolecules to stabilise, direct and monitor assembly

Coating and Stabilization of Liposomes by Clathrin-Inspired DNA Self-Assembly 
doi:10.1021/acsnano.9b09453

Assembling a DNA triskelion array layer on liposomes stabilized the membrane while keeping the fluidic nature of the lipid molecules. The vesicle did not rupture on the mica surface an nor was it dissolved by adding triton-X 100 detergent.


Peptide Assembly Directed and Quantified Using Megadalton DNA Nanostructures 
https://doi.org/10.1021/acsnano.9b04251

Sequence-structure relationships are sufficiently well understood for alpha-helical polypeptides to enable bottom-up design of simple alpha helix complexes. Two halves of a heterodimeric peptide were each tethered to large DNA nanostructures. The DNA nanostructures could be clearly differentiated with TEM. The peptide sequences were intentionally designed with a hydrophobic seam along which two alpha-helices could form a bond, bringing together two DNA nanostructures. To prevent DNA sticking to the peptide complexes, the sequences of peptides were chosen to be charge neutral at pH 7. Multiple peptide halves can be attached to each DNA nanostructure, altering the valency of the alpha helix bonding interaction. The authors measure the Kd disassociation constant for the peptide interactions using CD melting and by counting samples using TEM. This semi-quantitative technique is a first step toward the creation of complex rationally-designed peptide-oligonucleotide nanostructures but is not the most promising route toward measuring peptide interactions.


Directed Energy Transfer through DNA-Templated J-Aggregates
https://pubs.acs.org/doi/10.1021/acs.bioconjchem.9b00043

DNA photonic wires are DNA duplexes labelled with fluorophores that are able to transfer optical excitation through long distances by FRET interactions. In Nature, optical excitation transference is usually achieved by dye clusters templated over polymeric chains, such as proteins. Cyanine dyes are able to produce such clusters by stacking their aromatic groups together.

In the present paper, the authors template the formation of dye clusters, out of the fluorophore pseudoisocyanine, over a DNA duplex of poly(A)-poly(T).The DNA scaffold is able to produce a continuous cyanine aggregate across 48 base pairs. The optical transfer of the continuous cyanine cluster is compared with a DNA scaffold that produces a gap in the cluster. A single base pair gap in the cluster results in a sensible decrease of the optical transference efficiency, remarking the importance of the continuous templating.