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

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.

https://doi.org/10.1038/s41565-020-00782-3

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.

https://pubs.acs.org/doi/10.1021/acsnano.0c07537