Chemical reservoir computation in a self-organizing reaction network.

Chemical reaction networks, such as those found in metabolism and signalling pathways, enable cells to process information from their environment1,2. Current approaches to molecular information processing and computation typically pursue digital computation models and require extensive molecular-level engineering3. Despite considerable advances, these approaches have not reached the level of information processing capabilities seen in living systems. Here we report on the discovery and implementation of a chemical reservoir computer based on the formose reaction4. We demonstrate how this complex, self-organizing chemical reaction network can perform several nonlinear classification tasks in parallel, predict the dynamics of other complex systems and achieve time-series forecasting. This in chemico information processing system provides proof of principle for the emergent computational capabilities of complex chemical reaction networks, paving the way for a new class of biomimetic information processing systems.

A catalytically active oscillator made from small organic molecules.

Oscillatory systems regulate many biological processes, including key cellular functions such as metabolism and cell division, as well as larger-scale processes such as circadian rhythm and heartbeat1-4. Abiotic chemical oscillations, discovered originally in inorganic systems5,6, inspired the development of various synthetic oscillators for application as autonomous time-keeping systems in analytical chemistry, materials chemistry and the biomedical field7-17. Expanding their role beyond that of a pacemaker by having synthetic chemical oscillators periodically drive a secondary function would turn them into significantly more powerful tools. However, this is not trivial because the participation of components of the oscillator in the secondary function might jeopardize its time-keeping ability. We now report a small molecule oscillator that can catalyse an independent chemical reaction in situ without impairing its oscillating properties. In a flow system, the concentration of the catalytically active product of the oscillator shows sustained oscillations and the catalysed reaction is accelerated only during concentration peaks. Augmentation of synthetic oscillators with periodic catalytic action allows the construction of complex systems that, in the future, may benefit applications in automated synthesis, systems and polymerization chemistry and periodic drug delivery.

Exploring Emergent Properties in Enzymatic Reaction Networks: Design and Control of Dynamic Functional Systems.

The intricate and complex features of enzymatic reaction networks (ERNs) play a key role in the emergence and sustenance of life. Constructing such networks in vitro enables stepwise build up in complexity and introduces the opportunity to control enzymatic activity using physicochemical stimuli. Rational design and modulation of network motifs enable the engineering of artificial systems with emergent functionalities. Such functional systems are useful for a variety of reasons such as creating new-to-nature dynamic materials, producing value-added chemicals, constructing metabolic modules for synthetic cells, and even enabling molecular computation. In this review, we offer insights into the chemical characteristics of ERNs while also delving into their potential applications and associated challenges.

How Droplets Can Accelerate Reactions─Coacervate Protocells as Catalytic Microcompartments.

ConspectusCoacervates are droplets formed by liquid-liquid phase separation (LLPS) and are often used as model protocells-primitive cell-like compartments that could have aided the emergence of life. Their continued presence as membraneless organelles in modern cells gives further credit to their relevance. The local physicochemical environment inside coacervates is distinctly different from the surrounding dilute solution and offers an interesting microenvironment for prebiotic reactions. Coacervates can selectively take up reactants and enhance their effective concentration, stabilize products, destabilize reactants and lower transition states, and can therefore play a similar role as micellar catalysts in providing rate enhancement and selectivity in reaction outcome. Rate enhancement and selectivity must have been essential for the origins of life by enabling chemical reactions to occur at appreciable rates and overcoming competition from hydrolysis.In this Accounts, we dissect the mechanisms by which coacervate protocells can accelerate reactions and provide selectivity. These mechanisms can similarly be exploited by membraneless organelles to control cellular processes. First, coacervates can affect the local concentration of reactants and accelerate reactions by copartitioning of reactants or exclusion of a product or inhibitor. Second, the local environment inside the coacervate can change the energy landscape for reactions taking place inside the droplets. The coacervate is more apolar than the surrounding solution and often rich in charged moieties, which can affect the stability of reactants, transition states and products. The crowded nature of the droplets can favor complexation of large molecules such as ribozymes. Their locally different proton and water activity can facilitate reactions involving a (de)protonation step, condensation reactions and reactions that are sensitive to hydrolysis. Not only the coacervate core, but also the surface can accelerate reactions and provides an interesting site for chemical reactions with gradients in pH, water activity and charge. The coacervate is often rich in catalytic amino acids and can localize catalysts like divalent metal ions, leading to further rate enhancement inside the droplets. Lastly, these coacervate properties can favor certain reaction pathways, and thereby give selectivity over the reaction outcome.These mechanisms are further illustrated with a case study on ribozyme reactions inside coacervates, for which there is a fine balance between concentration and reactivity that can be tuned by the coacervate composition. Furthermore, coacervates can both catalyze ribozyme reactions and provide product selectivity, demonstrating that coacervates could have functioned as enzyme-like catalytic microcompartments at the origins of life.

Role of the extracellular matrix in regulating stem cell fate.

The field of stem cells and regenerative medicine offers considerable promise as a means of delivering new treatments for a wide range of diseases. In order to maximize the effectiveness of cell-based therapies - whether stimulating expansion of endogenous cells or transplanting cells into patients - it is essential to understand the environmental (niche) signals that regulate stem cell behaviour. One of those signals is from the extracellular matrix (ECM). New technologies have offered insights into how stem cells sense signals from the ECM and how they respond to these signals at the molecular level, which ultimately regulate their fate.

Integrated Single-Cell (Phospho-)Protein and RNA Detection Uncovers Phenotypic Characteristics and Active Signal Transduction of Human Antibody-Secreting Cells.

Single-cell technologies are currently widely applied to obtain a deeper understanding of the phenotype of single-cells in heterogenous mixtures. However, integrated multilayer approaches including simultaneous detection of mRNA, protein expression, and intracellular phospho-proteins are still challenging. Here, we combined an adapted method to in vitro-differentiate peripheral B-cells into antibody-secreting cells (ASCs) (i.e., plasmablasts and plasma cells) with integrated multi-omic single-cell sequencing technologies to detect and quantify immunoglobulin subclass-specific surface markers, transcriptional profiles, and signaling transduction pathway components. Using a common set of surface proteins, we integrated two multimodal datasets to combine mRNA, protein expression, and phospho-protein detection in one integrated dataset. Next, we tested whether ASCs that only seem to differ in its ability to secrete different IgM, IgA, or IgG antibodies exhibit other differences that characterize these different ASCs. Our approach detected differential expression of plasmablast and plasma cell markers, homing receptors, and TNF receptors. In addition, differential sensitivity was observed for the different cytokine stimulations that were applied during in vitro differentiation. For example, IgM ASCs were more sensitive to IL-15, while IgG ASC responded more to IL-6 and IFN addition. Furthermore, tonic BCR activity was detected in IgA and IgM ASCs, while IgG ASC exhibited active BCR-independent SYK activity and NF-κB and mTOR signaling. We confirmed these findings using flow cytometry and small molecules inhibitors, demonstrating the importance of SYK, NF-κB, and mTOR activity for plasmablast/plasma cell differentiation/survival and/or IgG secretion. Taken together, our integrated multi-omics approach allowed high-resolution phenotypic characterization of single cells in a heterogenous sample of in vitro-differentiated human ASCs. Our strategy is expected to further our understanding of human ASCs in healthy and diseased samples and provide a valuable tool to identify novel biomarkers and potential drug targets.

A Prebiotic Precursor to Life's Phosphate Transfer System with an ATP Analog and Histidyl Peptide Organocatalysts.

Biochemistry is dependent upon enzyme catalysts accelerating key reactions. At the origin of life, prebiotic chemistry must have incorporated catalytic reactions. While this would have yielded much needed amplification of certain reaction products, it would come at the possible cost of rapidly depleting the high energy molecules that acted as chemical fuels. Biochemistry solves this problem by combining kinetically stable and thermodynamically activated molecules (e.g., ATP) with enzyme catalysts. Here, we demonstrate a prebiotic phosphate transfer system involving an ATP analog (imidazole phosphate) and histidyl peptides, which function as organocatalytic enzyme analogs. We demonstrate that histidyl peptides catalyze phosphorylations via a phosphorylated histidyl intermediate. We integrate these histidyl-catalyzed phosphorylations into a complete prebiotic scenario whereby inorganic phosphate is incorporated into organic compounds though physicochemical wet-dry cycles. Our work demonstrates a plausible system for the catalyzed production of phosphorylated compounds on the early Earth and how organocatalytic peptides, as enzyme precursors, could have played an important role in this.

Division in synthetic cells.

The bottom-up construction of a living cell using non-living materials represents a grand challenge in science and technology. Reproduction of cells into similar offspring is key to life, and therefore, building a synthetic cell that can autonomously divide is one of the most fundamental tasks that need to be achieved in bottom-up synthetic biology. In this review, we summarize the strategies of inducing synthetic division by using physical, chemical, and biological stimuli, and highlight the future challenges to the construction of autonomous synthetic cell division.

Direct Analysis of Complex Reaction Mixtures: Formose Reaction.

Complex reaction mixtures, like those postulated on early Earth, present an analytical challenge because of the number of components, their similarity, and vastly different concentrations. Interpreting the reaction networks is typically based on simplified or partial data, limiting our insight. We present a new approach based on online monitoring of reaction mixtures formed by the formose reaction by ion-mobility-separation mass-spectrometry. Monitoring the reaction mixtures led to large data sets that we analyzed by non-negative matrix factorization, thereby identifying ion-signal groups capturing the time evolution of the network. The groups comprised ≈300 major ion signals corresponding to sugar-calcium complexes formed during the formose reaction. Multivariate analysis of the kinetic profiles of these complexes provided an overview of the interconnected kinetic processes in the solution, highlighting different pathways for sugar growth and the effects of different initiators on the initial kinetics. Reconstructing the network's topology further, we revealed so far unnoticed fast retro-aldol reaction of ketoses, which significantly affects the initial reaction dynamics. We also detected the onset of sugar-backbone branching for C6  sugars and cyclization reactions starting for C5  sugars. This top-down analytical approach opens a new way to analyze complex dynamic mixtures online with unprecedented coverage and time resolution.

Dynamic Environmental Conditions Affect the Composition of a Model Prebiotic Reaction Network.

Prebiotic environments are dynamic, containing a range of periodic and aperiodic variations in reaction conditions. However, the impact of the temporal dynamics of environmental conditions upon prebiotic chemical reaction networks has not been investigated. Here, we demonstrate how the magnitude and rate of temporal fluctuations of the catalysts Ca2+ and hydroxide control the product distributions of the formose reaction. Surprisingly, the product compositions of the formose reaction under dynamic conditions deviate significantly from those under steady state conditions. We attribute these compositional changes to the non-uniform propagation of fluctuations through the network, thereby shaping reaction outcomes. An examination of temporal concentration patterns showed that collections of compounds responded collectively to perturbations, indicating that key gating reactions branching from the Breslow cycle may be important responsive features of the formose reaction. Our findings show how the compositions of prebiotic reaction networks were shaped by sequential environmental events, illustrating the necessity for considering the temporal traits of prebiotic environments that supported the origin of life.

Tuning Material States and Functionalities of G-Quadruplex-Modulated RNA-Peptide Condensates.

RNA encodes sequence- and structure-dependent interactions to modulate the assembly and properties of biomolecular condensates. RNA G-quadruplexes (rG4s) formed by guanine-rich sequences can trigger the formation of liquid- or solid-like condensates that are involved in many aberrant phase transitions. However, exactly how rG4 motifs modulate different phase transitions and impart distinct material properties to condensates is unclear. Here, using RNA oligonucleotides and cationic peptides as model systems, we show that RNA-peptide condensates exhibit tunability in material properties over a wide spectrum via interactions arising from rG4 folding/unfolding kinetics. rG4-containing oligonucleotides formed strong pairwise attraction with peptides and tended to form solid-like condensates, while their less-structured non-G4 mutants formed liquid-like droplets. We find that the coupling between rG4 dissociation and RNA-peptide complex coacervation triggers solid-to-liquid transition of condensates prior to the complete unfolding of rG4s. This coupling points to a mechanism that material states of rG4-modulated condensates can be finely tuned from solid-like to liquid-like by the addition of less-structured RNA oligonucleotides, which have weak but dominant binding with peptides. We further show that the tunable material states of condensates can enhance RNA aptamer compartmentalization and RNA cleavage reactions. Our results suggest that condensates with complex properties can emerge from subtle changes in RNA oligonucleotides, contributing ways to treat dysfunctional condensates in diseases and insights into prebiotic compartmentalization.

Structure-Property Relationships Governing Membrane-Penetrating Behaviour of Complex Coacervates.

Complex coacervates are phase-separated liquid droplets composed of oppositely charged multivalent molecules. The unique material properties of the complex coacervate interior favours the sequestration of biomolecules and facilitates reactions. Recently, it is shown that coacervates can be used for direct cytosolic delivery of sequestered biomolecules in living cells. Here, it is studied that the physical properties required for complex coacervates composed of oligo-arginine and RNA to cross phospholipid bilayers and enter liposomes penetration depends on two main parameters: the difference in ζ-potential between the complex coacervates and the liposomes, and the partitioning coefficient (Kp ) of lipids into the complex coacervates. Following these guidelines, a range of complex coacervates is found that is able to penetrate the membrane of living cells, thus paving the way for further development of coacervates as delivery vehicles of therapeutic agents.

Computing Arithmetic Functions Using Immobilised Enzymatic Reaction Networks.

Living systems use enzymatic reaction networks to process biochemical information and make decisions in response to external or internal stimuli. Herein, we present a modular and reusable platform for molecular information processing using enzymes immobilised in hydrogel beads and compartmentalised in a continuous stirred tank reactor. We demonstrate how this setup allows us to perform simple arithmetic operations, such as addition, subtraction and multiplication, using various concentrations of substrates or inhibitors as inputs and the production of a fluorescent molecule as the readout.

Endocytosis of Coacervates into Liposomes.

Recent studies have shown that the interactions between condensates and biological membranes are of functional importance. Here, we study how the interaction between complex coacervates and liposomes as model systems can lead to wetting, membrane deformation, and endocytosis. Depending on the interaction strength between coacervates and liposomes, the wetting behavior ranged from nonwetting to engulfment (endocytosis) and complete wetting. Endocytosis of coacervates was found to be a general phenomenon: coacervates made from a wide range of components could be taken up by liposomes. A simple theory taking into account surface energies and coacervate sizes can explain the observed morphologies. Our findings can help to better understand condensate-membrane interactions in cellular systems and provide new avenues for intracellular delivery using coacervates.

A Bayesian Approach to Extracting Kinetic Information from Artificial Enzymatic Networks.

In order to create artificial enzymatic networks capable of increasingly complex behavior, an improved methodology in understanding and controlling the kinetics of these networks is needed. Here, we introduce a Bayesian analysis method allowing for the accurate inference of enzyme kinetic parameters and determination of most likely reaction mechanisms, by combining data from different experiments and network topologies in a single probabilistic analysis framework. This Bayesian approach explicitly allows us to continuously improve our parameter estimates and behavior predictions by iteratively adding new data to our models, while automatically taking into account uncertainties introduced by the experimental setups or the chemical processes in general. We demonstrate the potential of this approach by characterizing systems of enzymes compartmentalized in beads inside flow reactors. The methods we introduce here provide a new approach to the design of increasingly complex artificial enzymatic networks, making the design of such networks more efficient, and robust against the accumulation of experimental errors.

Photoswitchable Molecular Communication between Programmable DNA-Based Artificial Membraneless Organelles.

Spatiotemporal organization of distinct biological processes in cytomimetic compartments is a crucial step towards engineering functional artificial cells. Mimicking controlled bi-directional molecular communication inside artificial cells remains a considerable challenge. Here we present photoswitchable molecular transport between programmable membraneless organelle-like DNA coacervates in a synthetic microcompartment. We use droplet microfluidics to fabricate membraneless non-fusing DNA coacervates by liquid-liquid phase separation in a water-in-oil droplet, and employ the interior DNA coacervates as artificial organelles to imitate intracellular communication via photo-regulated uni- and bi-directional transfer of biomolecules. Our results highlight a promising new route to assembly of multicompartment artificial cells with functional networks.

Reversible Photoswitchable Inhibitors Generate Ultrasensitivity in Out-of-Equilibrium Enzymatic Reactions.

Ultrasensitivity is a ubiquitous emergent property of biochemical reaction networks. The design and construction of synthetic reaction networks exhibiting ultrasensitivity has been challenging, but would greatly expand the potential properties of life-like materials. Herein, we exploit a general and modular strategy to reversibly regulate the activity of enzymes using light and show how ultrasensitivity arises in simple out-of-equilibrium enzymatic systems upon incorporation of reversible photoswitchable inhibitors (PIs). Utilizing a chromophore/warhead strategy, PIs of the protease α-chymotrypsin were synthesized, which led to the discovery of inhibitors with large differences in inhibition constants (Ki) for the different photoisomers. A microfluidic flow setup was used to study enzymatic reactions under out-of-equilibrium conditions by continuous addition and removal of reagents. Upon irradiation of the continuously stirred tank reactor with different light pulse sequences, i.e., varying the pulse duration or frequency of UV and blue light irradiation, reversible switching between photoisomers resulted in ultrasensitive responses in enzymatic activity as well as frequency filtering of input signals. This general and modular strategy enables reversible and tunable control over the kinetic rates of individual enzyme-catalyzed reactions and makes a programmable linkage of enzymes to a wide range of network topologies feasible.

Dynamic Environments as a Tool to Preserve Desired Output in a Chemical Reaction Network.

Current efforts to design functional molecular systems have overlooked the importance of coupling out-of-equilibrium behaviour with changes in the environment. Here, the authors use an oscillating reaction network and demonstrate that the application of environmental forcing, in the form of periodic changes in temperature and in the inflow of the concentration of one of the network components, removes the dependency of the periodicity of this network on temperature or flow rates and enforces a stable periodicity across a wide range of conditions. Coupling a system to a dynamic environment can thus be used as a simple tool to regulate the output of a network. In addition, the authors show that coupling can also induce an increase in behavioural complexity to include quasi-periodic oscillations.

Robustness, Entrainment, and Hybridization in Dissipative Molecular Networks, and the Origin of Life.

How simple chemical reactions self-assembled into complex, robust networks at the origin of life is unknown. This general problem-self-assembly of dissipative molecular networks-is also important in understanding the growth of complexity from simplicity in molecular and biomolecular systems. Here, we describe how heterogeneity in the composition of a small network of oscillatory organic reactions can sustain (rather than stop) these oscillations, when homogeneity in their composition does not. Specifically, multiple reactants in an amide-forming network sustain oscillation when the environment (here, the space velocity) changes, while homogeneous networks-those with fewer reactants-do not. Remarkably, a mixture of two reactants of different structure-neither of which produces oscillations individually-oscillates when combined. These results demonstrate that molecular heterogeneity present in mixtures of reactants can promote rather than suppress complex behaviors.

Branched DNA Architectures Produced by PCR-Based Assembly as Gene Compartments for Cell-Free Gene-Expression Reactions.

The physical distance between genes plays important roles in controlling gene expression reactions in vivo. Herein, we report the design and synthesis of a branched gene architecture in which three transcription units are integrated into one framework through assembly based on the polymerase chain reaction (PCR), together with the exploitation of these constructs as "gene compartments" for cell-free gene expression reactions, probing the impact of this physical environment on gene transcription and translation. We find that the branched gene system enhances gene expression yields, in particular at low concentrations of DNA and RNA polymerase (RNAP); furthermore, in a crowded microenvironment that mimics the intracellular microenvironment, gene expression from branched genes maintains a relatively high level. We propose that the branched gene assembly forms a membrane-free gene compartment that resembles the nucleoid of prokaryotes and enables RNAP to shuttle more efficiently between neighboring transcription units, thus enhancing gene expression efficiency. Our branched DNA architecture provides a valuable platform for studying the influence of "cellular" physical environments on biochemical reactions in simplified cell-free systems.