Communication and Cross-Regulation Between Multiple Concatenated Enzymatic Reaction Networks

Nature connects multiple fuel-driven chemical/enzymatic reaction networks (CRNs/ERNs) via cross-regulation to hierarchically control biofunctions for a tailored adaption in complex sensory landscapes. In contrast, emerging artificial fuel-driven systems most-ly focus on a single CRN and their implementation to direct self-assembly or material responses. In this work, we introduce a facile example of communication and cross-regulation among multiple DNA-based ERNs regulated by a concatenated RNA transcription regulator. For this purpose, we run two fuel-driven DNA-based ERNs by concurrent NAD+-fueled ligation and restriction via endo-nucleases (REases) in parallel. ERN one allows for the dynamic steady-state formation of the promoter sequence for T7 RNA poly-merase, which activates RNA transcription. The produced RNA regulator can repress or promote the second ERN via RNA-mediated strand displacement. Furthermore, adding RNase H to degrade the produced RNA can restart the reaction or tune the lag time of two ERNs, giving rise to a repression-recovery and promotion-stop processes. We believe that concatenation of multiple CRNs provides a basis for the design of more elaborate autonomous regulatory mechanisms in systems chemistry and synthetic biology.

Memory, switches, and logic gates through bistability in chemically fueled crystals

The ability to store information in chemical reaction networks is essential for evolution, calculations, and, more generally, for the complex behavior, we associate with life. In biology, cellular memory is regulated through transcriptional states that are bistable, i.e., a state that can either be on or off and can be flipped from one to another through a transient signal. Such memory circuits have been realized synthetically through the rewiring of genetic systems in vivo or through the rational design of reaction networks based on DNA and highly evolved enzymes in vitro. Completely bottom-up analogs based on small molecules are rare and hard to design and thus represent a challenge for systems chemistry. In this work, we show that bistability can be designed from an extremely simple non-equilibrium reaction cycle that is coupled to crystallization. The crystals exert the necessary feedback on the reaction cycle required for the bistability resulting in an on-state with assemblies and an off-state without. We can switch the state on and off, such that each state represents volatile memory that can be stored in continuously stirred tank reactors indefinitely despite the fact that molecules are turned over on a minute-timescale. We showcase the system’s abilities by creating a matrix display that can store images and by performing Boolean logic by coupling several switches together.

Can prebiotic reaction systems survive in the wild? An interference chemistry approach

The origin of life occurred by a series of prebiotic reaction pathways (collectively a system) hosted in one or more geochemical environments (together forming an origin of life scenario). State-of-the-art prebiotic chemistry links together reactions to create systems, intended to be more representative of the diverse chemical pathways that may have proceeded on early Earth. By practical necessity, prebiotic systems chemistry must be investigated under simplified conditions in comparison to likely natural environments. The mismatch in complexity between lab and environment poses a challenge: how to build systems chemistry that is robust not only in the idealised conditions of a lab, but also under natural levels of environmental stress? Here, we propose and formalise a conceptual framework for such work: interference chemistry. We define interference chemistry as the interaction between prebiotic systems chemistry and the environmental scenarios proposed to host it. Natural environments in which prebiotic chemistry could have occurred are messy, containing many spectator ions, mineral phases, and spatially and temporally variable physical processes, e.g., wet/dry cycles. Each of these environmental variables may interfere either constructively or destructively with prebiotic pathways, respectively aiding or inhibiting their efficacy. Exploring interference chemistry for a reaction system will point towards favoured or disfavoured regions of environmental parameter space. To do so, innovation is needed in both the investigation of early planetary environmental conditions, and the continued incorporation of these constraints into experimental systems chemistry. We argue that interference chemistry provides a compelling way to assess combinations of system and environment, leading the way to increasingly prebiotically plausible scenarios for the origin of life on Earth.

Ultrafast and Selective Labeling of Endogenous Proteins Using Affinity-based Benzotriazole Chemistry

Chemical modification of proteins is enormously useful for characterizing protein function in complex biological systems and for drug development. Selective labeling of native or endogenous proteins is challenging owing to the existence of distinct functional groups in proteins and in living systems. Chemistry for rapid and selective labeling of proteins remains in high demand. Here we have developed novel affinity labeling probes using benzotriazole (BTA) chemistry. We showed that affinity-based BTA probes selectively and covalently label a lysine residue in the vicinity of the ligand binding site of a target protein with a reaction half-time of 28-42 s. The reaction rate constant is comparable to the fastest biorthogonal chemistry. This approach was used to selectively label different cytosolic and membrane proteins in vitro and in live cells. BTA chemistry could be widely useful for labeling of native/endogenous proteins, target identification and development of covalent inhibitors.

Regulating the dynamic folding of a DNA hairpin at the expense of a small, molecular fuel

Molecular machines, such as ATPases or motor proteins, couple the catalysis of a chemical reaction, most commonly hydrolysis of nucleotide triphosphates, to their conformational change. In essence, they continuously convert a chemical fuel to drive their motion. An outstanding goal of nanotechnology remains to synthesize a nanomachine with similar functions, precision, and speed. The field of DNA nan- otechnology has given rise to the engineering precision required for such a device. Simultaneously, the field of systems chemistry developed fast chemical reaction cycles that convert fuel to change the function of molecules. In this work, we thus combined a fast, chemical reaction cycle with the precision of DNA nanotechnology to yield kinetic control over the conformational state of a DNA hairpin. Future work on such systems will result in fast and precise DNA nanodevices.

Spatial localisation meets biomolecular networks

Spatial organisation through localisation/compartmentalisation of species is a ubiquitous but poorly understood feature of cellular biomolecular networks. Current technologies in systems and synthetic biology (spatial proteomics, imaging, synthetic compartmentalisation) necessitate a systematic approach to elucidating the interplay of networks and spatial organisation. We develop a systems framework towards this end and focus on the effect of spatial localisation of network components revealing its multiple facets: (i) As a key distinct regulator of network behaviour, and an enabler of new network capabilities (ii) As a potent new regulator of pattern formation and self-organisation (iii) As an often hidden factor impacting inference of temporal networks from data (iv) As an engineering tool for rewiring networks and network/circuit design. These insights, transparently arising from the most basic considerations of networks and spatial organisation, have broad relevance in natural and engineered biology and in related areas such as cell-free systems, systems chemistry and bionanotechnology.

Preparation of ZIF@ADH/NAD-MSN/LDH Core Shell Nanocomposites for the Enhancement of Coenzyme Catalyzed Double Enzyme Cascade

The field of enzyme cascades in limited microscale or nanoscale environments has undergone a quick growth and attracted increasing interests in the field of rapid development of systems chemistry. In this study, alcohol dehydrogenase (ADH), lactate dehydrogenase (LDH), and mesoporous silica nanoparticles (MSN) immobilized nicotinamide adenine dinucleotide (NAD+) were successfully immobilized on the zeolitic imidazolate frameworks (ZIFs). This immobilized product was named ZIF@ADH/NAD-MSN/LDH, and the effect of the multi-enzyme cascade was studied by measuring the catalytic synthesis of lactic acid. The loading efficiency of the enzyme in the in-situ co-immobilization method reached 92.65%. The synthesis rate of lactic acid was increased to 70.10%, which was about 2.82 times that of the free enzyme under the optimal conditions (40 °C, pH = 8). Additionally, ZIF@ADH/NAD-MSN/LDH had experimental stability (71.67% relative activity after four experiments) and storage stability (93.45% relative activity after three weeks of storage at 4 °C; 76.89% relative activity after incubation in acetonitrile-aqueous solution for 1 h; 27.42% relative activity after incubation in 15% N, N-Dimethylformamide (DMF) solution for 1 h). In summary, in this paper, the cyclic regeneration of coenzymes was achieved, and the reaction efficiency of the multi-enzyme biocatalytic cascade was improved due to the reduction of substrate diffusion.