Synthetic Systems Research Articles

Guide-specific loss of efficiency and off-target reduction with Cas9 variants.

High-fidelity clustered regularly interspaced palindromic repeats (CRISPR)-associated protein 9 (Cas9) variants have been developed to reduce the off-target effects of CRISPR systems at a cost of efficiency loss. To systematically evaluate the efficiency and off-target tolerance of Cas9 variants in complex with different single guide RNAs (sgRNAs), we applied high-throughput viability screens and a synthetic paired sgRNA-target system to assess thousands of sgRNAs in combination with two high-fidelity Cas9 variants HiFi and LZ3. Comparing these variants against wild-type SpCas9, we found that ∼20% of sgRNAs are associated with a significant loss of efficiency when complexed with either HiFi or LZ3. The loss of efficiency is dependent on the sequence context in the seed region of sgRNAs, as well as at positions 15-18 in the non-seed region that interacts with the REC3 domain of Cas9, suggesting that the variant-specific mutations in the REC3 domain account for the loss of efficiency. We also observed various degrees of sequence-dependent off-target reduction when different sgRNAs are used in combination with the variants. Given these observations, we developed GuideVar, a transfer learning-based computational framework for the prediction of on-target efficiency and off-target effects with high-fidelity variants. GuideVar facilitates the prioritization of sgRNAs in the applications with HiFi and LZ3, as demonstrated by the improvement of signal-to-noise ratios in high-throughput viability screens using these high-fidelity variants.

A synthetic cell-free 36-enzyme reaction system for vitamin B12 production

Adenosylcobalamin (AdoCbl), a biologically active form of vitamin B12 (coenzyme B12), is one of the most complex metal-containing natural compounds and an essential vitamin for animals. However, AdoCbl can only be de novo synthesized by prokaryotes, and its industrial manufacturing to date was limited to bacterial fermentation. Here, we report a method for the synthesis of AdoCbl based on a cell-free reaction system performing a cascade of catalytic reactions from 5-aminolevulinic acid (5-ALA), an inexpensive compound. More than 30 biocatalytic reactions are integrated and optimized to achieve the complete cell-free synthesis of AdoCbl, after overcoming feedback inhibition, the complicated detection, instability of intermediate products, as well as imbalance and competition of cofactors. In the end, this cell-free system produces 417.41 μg/L and 5.78 mg/L of AdoCbl using 5-ALA and the purified intermediate product hydrogenobyrate as substrates, respectively. The strategies of coordinating synthetic modules of complex cell-free system describe here will be generally useful for developing cell-free platforms to produce complex natural compounds with long and complicated biosynthetic pathways.

Evolving a mitigation of the stress response pathway to change the basic chemistry of life

Despite billions of years of evolution, there have been only minor changes in the number and types of proteinogenic amino acids and the standard genetic code with codon assignments across the three domains of life. The rigidity of the genetic code sets it apart from other aspects of organismal evolution, giving rise to key questions about its origins and the constraints it places on innovation in translation. Through adaptive laboratory evolution (ALE) in Escherichia coli, we aimed to replace tryptophan (Trp) in the genetic code with an analogue L-β-(thieno[3,2-b]pyrrolyl)alanine ([3,2]Tpa). This required Escherichia coli to recruit thienopyrrole instead of indole and allowed reassignment of UGG codons. Crossing the stress response system emerged as a major obstacle for ancestral growth in the presence of [3,2]Tp and Trp limitation. During ALE, a pivotal innovation was the deactivation of the master regulon RpoS, which allowed growth solely in the presence of [3,2]Tp in minimal medium. Notably, knocking out the rpoS gene in the ancestral strain also facilitated growth on [3,2]Tp. Our findings suggest that regulatory constraints, not just a rigid translation mechanism, guard Life’s canonical amino acid repertoire. This knowledge will not only facilitate the design of more effective synthetic amino acid incorporation systems but may also shed light on a general biological mechanism trapping organismal configurations in a status quo.

From autocatalysis to survival of the fittest in self-reproducing lipid systems.

Studying autocatalysis - in which molecules catalyse their own formation - might help to explain the emergence of chemical systems that exhibit traits normally associated with biology. When coupled to other processes, autocatalysis can lead to complex systems-level behaviour in apparently simple mixtures. Lipids are an important class of chemicals that appear simple in isolation, but collectively show complex supramolecular and mesoscale dynamics. Here we discuss autocatalytic lipids as a source of extraordinary behaviour such as primitive chemical evolution, chemotaxis, temporally controllable materials and even as supramolecular catalysts for continuous synthesis. We survey the literature since the first examples of lipid autocatalysis and highlight state-of-the-art synthetic systems that emulate life, displaying behaviour such as metabolism and homeostasis, with special consideration for generating structural complexity and out-of-equilibrium models of life. Autocatalytic lipid systems have enormous potential for building complexity from simple components, and connections between physical effects and molecular reactivity are only just beginning to be discovered.

Targeted and Equally Distributed Delivery of mRNA to Organs with Pentaerythritol-Based One-Component Ionizable Amphiphilic Janus Dendrimers.

Delivery of nucleic acids with viral and synthetic vectors has pioneered genetic nanomedicine. Four-component lipid nanoparticles (LNPs) consisting of ionizable lipids, phospholipids, cholesterol, and PEG-conjugated lipids, assembled by microfluidic or T-tube, are the benchmark synthetic vector for delivery of mRNA. One-component multifunctional sequence-defined ionizable amphiphilic Janus dendrimer (IAJD) delivery systems for mRNA were developed by us to complement LNPs. IAJDs consist of multifunctional hydrophilic low-generation dendrons or minidendrons conjugated to hydrophobic dendrons. They were inspired by amphiphilic Janus dendrimers and glycodendrimers. IAJDs coassemble with mRNA into predictable-size vesicles, named dendrimersome nanoparticles (DNPs), by simple injection in acetate buffer, rather than by the complex technology required by LNPs. Assembly of DNPs by simple injection together with sequence design in the hydrophilic and hydrophobic modules of IAJDs endowed rapid screening to access discovery. Molecular design principles for targeted delivery were elaborated when the branching points of IAJDs were constructed from symmetrically and nonsymmetrically substituted plant phenolic acids interconnected by pentaerythritol (PE). Here, we report the first library containing simplified IAJDs constructed in only three steps from symmetrically trialkylated PE in the hydrophobic domain and four different piperazine-based ionizable amines in the hydrophilic part. Rapid coassembly with mRNA and in vivo screening led to the discovery of the two most active IAJDs targeting the spleen, liver, and lymph nodes, one predominantly to the spleen and liver and six delivering equally to the spleen, liver, lung, and lymph nodes. These IAJDs represent the simplest synthetic vectors and the first viral or synthetic system delivering equally to multiple organs.

Seeing the Membrane from Both Sides Now: Lipid Asymmetry and Its Strange Consequences.

Almost all biomembranes are constructed as lipid bilayers and, in almost all of these, the two opposing monolayers (leaflets) have distinct lipid compositions. This lipid asymmetry arises through the concerted action of a suite of energy-dependent enzymes that maintain living bilayers in a far-from-equilibrium steady-state. Recent discoveries reveal that lipid compositional asymmetry imparts biophysical asymmetries and that this dualistic organization may have major consequences for cellular physiology. Importantly, while transbilayer asymmetry appears to be an essential, near-ubiquitous characteristic of biological membranes, it has been challenging to reproduce in reconstituted or synthetic systems. Although recent methodological developments have overcome some critical challenges, it remains difficult to extrapolate results from available models to biological systems. Concurrently, there are few experimental approaches for targeted, controlled manipulation of lipid asymmetry in living cells. Thus, the biophysical and functional consequences of membrane asymmetry remain almost wholly unexplored. This perspective summarizes the current state of knowledge and highlights emerging themes that are beginning to make inroads into the fundamental question of why life tends toward asymmetry in its bilayers.

Image restoration for optical synthetic aperture system via variational physics-informed network

Optical synthetic aperture with homogeneous circular sub-mirrors greatly improves the spatial resolution of space telescopes; however, the discrete and sparse characteristics of the sub-mirrors reduce the mid-frequency modulation transfer function (MTF), resulting in blurred images being obtained. In this paper, a method combining variational physics-informed with deep learning is presented, which shows blind image restoration without complex priors. The constraint effect of traditional maximum a posterior (MAP) framework is removed by variational inference framework, which is embedded into Variational Physics-informed Network (VPIN) to optimize neural network training. Residual dense blocks (RDBs) construction is contributed to image feature extraction. Networks with SSIM-corrected loss functions can be trained at the feature level to help with convergence. When SNR = 30 dB, the PSNR of Golay-6 remote sensing test set increases from 20.16 dB to 23.90 dB, SSIM is from 0.610 to 0.842, and MS-SSIM is from 0.930 to 0.955.

Immunogenic cell death (ICD) enhancers-Drugs that enhance the perception of ICD by dendritic cells.

The search for immunostimulatory drugs applicable to cancer immunotherapy may profit from target-agnostic methods in which agents are screened for their functional impact on immune cells cultured in vitro without any preconceived idea on their mode of action. We have built a synthetic mini-immune system in which stressed and dying cancer cells (derived from standardized cell lines) are confronted with dendritic cells (DCs, derived from immortalized precursors) and CD8+ T-cell hybridoma cells expressing a defined T-cell receptor. Using this system, we can identify three types of immunostimulatory drugs: (i) pharmacological agents that stimulate immunogenic cell death (ICD) of malignant cells; (ii) drugs that act on DCs to enhance their response to ICD; and (iii) drugs that act on T cells to increase their effector function. Here, we focus on strategies to develop drugs that enhance the perception of ICD by DCs and to which we refer as "ICD enhancers." We discuss examples of ICD enhancers, including ligands of pattern recognition receptors (exemplified by TLR3 ligands that correct the deficient function of DCs lacking FPR1) and immunometabolic modifiers (exemplified by hexokinase-2 inhibitors), as well as methods for target deconvolution applicable to the mechanistic characterization of ICD enhancers.

Population-level antagonism between FGF and BMP signaling steers mesoderm differentiation in embryonic stem cells.

The mesodermal precursor populations for different internal organ systems are specified during gastrulation by the combined activity of extracellular signaling systems such as BMP, Wnt, Nodal, and FGF. The BMP, Wnt and Nodal signaling requirements for the differentiation of specific mesoderm subtypes in mammals have been mapped in detail, but how FGF shapes mesodermal cell type diversity is not precisely known. It is also not clear how FGF signaling integrates with the activity of other signaling systems involved in mesoderm differentiation. Here, we address these questions by analyzing the effects of targeted signaling manipulations in differentiating stem cell populations with single cell resolution. We identify opposing functions of BMP and FGF, and map FGF-dependent and -independent mesodermal lineages. Stimulation with exogenous FGF boosts the expression of endogenous Fgfs while repressing Bmp ligand genes. This positive autoregulation of FGF signaling, coupled to the repression of BMP signaling, may contribute to the specification of reproducible and coherent cohorts of cells with the same identity via a community effect, both in the embryo and in synthetic embryo-like systems.

DNA droplets for intelligent and dynamical artificial cells: from the viewpoint of computation and non-equilibrium systems.

Living systems are molecular assemblies whose dynamics are maintained by non-equilibrium chemical reactions. To date, artificial cells have been studied from such physical and chemical viewpoints. This review briefly gives a perspective on using DNA droplets in constructing artificial cells. A DNA droplet is a coacervate composed of DNA nanostructures, a novel category of synthetic DNA self-assembled systems. The DNA droplets have programmability in physical properties based on DNA base sequence design. The aspect of DNA as an information molecule allows physical and chemical control of nanostructure formation, molecular assembly and molecular reactions through the design of DNA base pairing. As a result, the construction of artificial cells equipped with non-equilibrium behaviours such as dynamical motions, phase separations, molecular sensing and computation using chemical energy is becoming possible. This review mainly focuses on such dynamical DNA droplets for artificial cell research in terms of computation and non-equilibrium chemical reactions.

Carotenoid ornaments and the spandrels of physiology: a critique of theory to explain condition dependency.

Even as numerous studies have documented that the red and yellow coloration resulting from the deposition of carotenoids serves as an honest signal of condition, the evolution of condition dependency is contentious. The resource trade-off hypothesis proposes that condition-dependent honest signalling relies on a trade-off of resources between ornamental display and body maintenance. By this model, condition dependency can evolve through selection for a re-allocation of resources to promote ornament expression. By contrast, the index hypothesis proposes that selection focuses mate choice on carotenoid coloration that is inherently condition dependent because production of such coloration is inexorably tied to vital cellular processes. These hypotheses for the origins of condition dependency make strongly contrasting and testable predictions about ornamental traits. To assess these two models, we review the mechanisms of production of carotenoids, patterns of condition dependency involving different classes of carotenoids, and patterns of behavioural responses to carotenoid coloration. We review evidence that traits can be condition dependent without the influence of sexual selection and that novel traits can show condition-dependent expression as soon as they appear in a population, without the possibility of sexual selection. We conclude by highlighting new opportunities for studying condition-dependent signalling made possible by genetic manipulation and expression of ornamental traits in synthetic biological systems.

Inter-Vesicle Signal Transduction Using a Photo-Responsive Zinc Ionophore.

Transmission of chemical information between cells and across lipid bilayer membranes is of profound significance in many biological processes. The design of synthetic signalling systems is a critical step towards preparing artificial cells with collective behaviour. Here, we report the first example of a synthetic inter-vesicle signalling system, in which diffusible chemical signals trigger transmembrane ion transport in a manner reminiscent of signalling pathways in biology. The system is derived from novel ortho-nitrobenzyl and BODIPY photo-caged ZnII transporters, in which cation transport is triggered by photo-decaging with UV or red light, respectively. This decaging reaction can be used to trigger the release of the cationophores from a small population of sender vesicles. This in turn triggers the transport of ions across the membrane of a larger population of receiver vesicles, but not across the sender vesicle membrane, leading to overall inter-vesicle signal transduction and amplification.

A novel star polymer for regulating fluid loss in oil-based mud under high temperature conditions

Oil-Based Mud (OBM) is a highly effective drilling fluid system for challenging wellbore conditions such as water-sensitive and high-temperature drilling operations. To minimize filtration loss and achieve thin filter cake, fluid loss control additives are essential. This study introduces a star polymer as an alternative to traditional additives like gilsonite or linear polymer. The star polymer was synthesized using Reversible Addition Fragmentation Chain Transfer polymerization (RAFT) with hydrophilic and lipophilic monomers and crosslinkers. Mud properties such as rheology, fluid loss, mud cake thickness, and emulsion stability were evaluated for OBM and compared with commercially available fluid loss control additives at simulated downhole pressure and temperatures. Spectroscopy and thermogravimetric analysis were used to determine the chemical architecture and thermal stability of the star polymer. The results showed that the star polymer demonstrated thermal stability up to 225 °C, resulting in a small amount of fluid loss volume and ultra-thin filter cake for OBM systems with mud weight at 10.5 ppg and 13.4 ppg. Furthermore, the star polymer improved emulsion stability without significantly affecting plastic viscosity (PV), unlike commercially available fluid loss control additives. The results were consistent for both diesel and synthetic oil-based mud systems.

Inter‐Vesicle Signal Transduction Using a Photo‐Responsive Zinc Ionophore

AbstractTransmission of chemical information between cells and across lipid bilayer membranes is of profound significance in many biological processes. The design of synthetic signalling systems is a critical step towards preparing artificial cells with collective behaviour. Here, we report the first example of a synthetic inter‐vesicle signalling system, in which diffusible chemical signals trigger transmembrane ion transport in a manner reminiscent of signalling pathways in biology. The system is derived from novel ortho‐nitrobenzyl and BODIPY photo‐caged ZnII transporters, in which cation transport is triggered by photo‐decaging with UV or red light, respectively. This decaging reaction can be used to trigger the release of the cationophores from a small population of sender vesicles. This in turn triggers the transport of ions across the membrane of a larger population of receiver vesicles, but not across the sender vesicle membrane, leading to overall inter‐vesicle signal transduction and amplification.

Capture of small clusters by ligand-solvent interaction.

Clusters are considered to become increasingly significant for elaborating the nanocrystal's formation mechanism. However, capturing the clusters with high chemical potential is challenging because of the lack of effective strategies. In this work, the key role of ligand-solvent interaction has been revealed for the stabilization of clusters in silver telluride synthesis. The Flory interaction coefficient that comprehensively regards the temperature and dispersion, polarity, and hydrogen bonding of the solvent has been used to evaluate the ligand-solvent interaction and thus assist in the design of synthetic systems. Small silver telluride clusters have been successfully captured, and the composition of the smallest cluster is determined as Ag7Te8(SCy)2 (SCy represents the ligand). This work provides new insights into the design of cluster/nanocrystal synthesis systems and paves the way to revealing the mechanism of precursor-cluster-nanocrystal conversion.

Method to probe the pronounced growth of correlation lengths in active glass-forming liquids using an elongated probe.

The growth of correlation lengths in equilibrium glass-forming liquids near the glass transition is considered a critical finding in the quest to understand the physics of glass formation. These understandings helped us understand various dynamical phenomena observed in supercooled liquids. It is known that at least two different length scales exist; one is of thermodynamic origin, while the other is dynamical in nature. Recent observations of glassy dynamics in biological and synthetic systems where the external or internal driving source controls the dynamics, apart from the usual thermal noise, lead to the emergence of the field of active glassy matter. A question of whether the physics of glass formation in these active systems is also accompanied by growing dynamic and static lengths is indeed timely. In this article, we probe the growth of dynamic and static lengths in a model active glass system using rod-like elongated probe particles, an experimentally viable method. We show that the dynamic and static lengths in these nonequilibrium systems grow much more rapidly than their passive counterparts. We then offer an understanding of the violation of the Stokes-Einstein relation and Stokes-Einstein-Debye relation using these lengths via a scaling theory.

High Performance of Gas Sensor Based on Novel Cu-Based Binary Composite Phase-Modified ZnO

Novel Cu-based binary composite phases were synthesized with an octadecylamine (ODA) synthetic system. HR-TEM was used to characterize the binary composite phase indicating that the Cu-based binary composite phase was a mixed phase of two oxides. ZnO gas sensing films decorated with Cu-based binary composite phase were parallel fabricated. The gas sensing performance to nitric oxides (NO, N2O, NO[Formula: see text] was studied. Cu–Mo–O binary composite phase-modified ZnO was screened out with high sensitivity and selectivity to NO. The gas sensing response to 0.5 ppm NO was 128.1. The detection limit of Cu–Mo–O binary composite phase-modified ZnO to NO was less than 10 ppb. Cu–Mo–O binary composite phase-modified ZnO also shows good selectivity to NO even using NO2 as interference gas. It is of great importance for the detection of NO.

A framework for synthetic power system dynamics.

We present a modular framework for generating synthetic power grids that consider the heterogeneity of real power grid dynamics but remain simple and tractable. This enables the generation of large sets of synthetic grids for a wide range of applications. For the first time, our synthetic model also includes the major drivers of fluctuations on short-time scales and a set of validators that ensure the resulting system dynamics are plausible. The synthetic grids generated are robust and show good synchronization under all evaluated scenarios, as should be expected for realistic power grids. A software package that includes an efficient Julia implementation of the framework is released as a companion to the paper.

Configuration-independent thermal invariants under flow reversal in thin vascular systems.

Modulating temperature fields is indispensable for advancing modern technologies: space probes, electronic packing, and implantable medical devices, to name a few. Bio-inspired thermal regulation achieved via fluid flow within a network of embedded vesicles is notably desirable for slender synthetic material systems. This far-reaching study-availing theory, numerics, and experiments-reveals a counter-intuitive yet fundamental property of vascular-based fluid-flow-engendered thermal regulation. For such thin systems, the mean surface temperature and the outlet temperature-consequently, the heat extracted by the flowing fluid (coolant)-are invariant under flow reversal (i.e. swapping the inlet and outlet). Despite markedly different temperature fields under flow reversal, our newfound invariance-a discovery-holds for anisotropic thermal conductivity, any inlet and ambient temperatures, transient and steady-state responses, irregular domains, and arbitrary internal vascular topologies, including those with branching. The reported configuration-independent result benefits thermal regulation designers. For instance, the flexibility in the coolant's inlet location eases coordination challenges between electronics and various delivery systems in microfluidic devices without compromising performance (e.g. soft implantable coolers for pain management). Last but not least, the invariance offers an innovative way to verify computer codes, especially when analytical solutions are unavailable for intricate domain and vascular configurations.

Super-swelling behavior of stacked lipid bilayer systems

Bilayer systems comprising lipid mixtures are the most well-studied model of biological membranes. While the plasma membrane of the cell is a single bilayer, many intra- and extra-cellular biomembranes comprise stacks of bilayers. Most bilayer stacks in nature are periodic, maintaining a precise water layer separation between bilayers. That equilibrium water separation is governed by multiple inter-bilayer forces and is highly responsive. Biomembranes re-configure inter-bilayer spacing in response to temperature, composition, or mass transport cues. In synthetic bilayer systems for applications in cosmetics or topical treatments, control of the hydration level is a critical design handle. Herein we investigate a binary lipid system that leverages key inter-bilayer forces leading to unprecedented levels of aqueous swelling while maintaining a coherent multilamellar form. We found that combining cationic lipids with bicontinuous cubic phase-forming lipids (lipids with positive Gaussian modulus), results in the stabilization of multilamellar phases against repulsive steric forces that typically lead to bilayer delamination at high degrees of swelling. Using ultra-small-angle X-ray scattering alongside confocal laser scanning microscopy, we characterized various super-swelled states of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and glycerol monooleate (GMO) lipids, as well as other analogous systems, at varied concentration and molar ratios. Through these experiments we established swelling profiles of various binary lipid systems that were near-linear with decreasing lipid volume fraction, showing maximum swelling with periodicity well above 200 nanometers.Graphical abstractConfocal fluorescence micrograph of super-swelled multilamellar structures in 90GMOD sample at 25 mM concentration. Inset plot shows intensity profile of orange line, with pink triangles indicating maxima.