Constitutional Dynamic Networks Research Articles

Spatially Localized Entropy-Driven Evolution of Nucleic Acid-Based Constitutional Dynamic Networks for Intracellular Imaging and Spatiotemporal Programmable Gene Therapy.

The primer-guided entropy-driven high-throughput evolution of the DNA-based constitutional dynamic network, CDN, is introduced. The entropy gain associated with the process provides a catalytic principle for the amplified emergence of the CDN. The concept is applied to develop a programmable, spatially localized DNA circuit for effective in vitro and in vivo theranostic, gene-regulated treatment of cancer cells. The localized circuit consists of a DNA tetrahedron core modified at its corners with four tethers that include encoded base sequences exhibiting the capacity to emerge and assemble into a [2 × 2] CDN. Two of the tethers are caged by a pair of siRNA subunits, blocking the circuit into a mute, dynamically inactive configuration. In the presence of miRNA-21 as primer, the siRNA subunits are displaced, resulting in amplified release of the siRNAs silencing the HIF-1α mRNA and fast dynamic reconfiguration of the tethers into a CDN. The resulting CDN is, however, engineered to be dynamically reconfigured by miRNA-155 into an equilibrated mixture enriched with a DNAzyme component, catalyzing the cleavage of EGR-1 mRNA. The DNA tetrahedron nanostructure stimulates enhanced permeation into cancer cells. The miRNA-triggered entropy-driven reconfiguration of the spatially localized circuit leads to the programmable, cooperative bis-gene-silencing of HIF-1α and EGR-1 mRNAs, resulting in the effective and selective apoptosis of breast cancer cells and effective inhibition of tumors in tumor bearing mice.

Triple Adaptation of Constitutional Dynamic Networks of Imines in Response to Micellar Agents: Internal Uptake-Interfacial Localization-Shape Transition.

Understanding the behavior of complex chemical reaction networks and how environmental conditions can modulate their organization as well as the associated outcomes may take advantage of the design of related artificial systems. Microenvironments with defined boundaries are of particular interest for their unique properties and prebiotic significance. Dynamic covalent libraries (DCvLs) and their underlying constitutional dynamic networks (CDNs) have been shown to be appropriate for studying adaptation to several processes, including compartmentalization. However, microcompartments (e.g., micelles) provide specific environments for the selective protection from interfering reactions such as hydrolysis and an enhanced chemical promiscuity due to the interface, governing different processes of network modulation. Different interactions between the micelles and the library constituents lead to dynamic sensing, resulting in different expressions of the network through pattern generation. The constituents integrated into the micelles are protected from hydrolysis and hence preferentially expressed in the network composition at the cost of constitutionally linked members. In the present work, micellar integration was observed for two processes: internal uptake based on hydrophobic forces and interfacial localization relying on attractive electrostatic interactions. The latter drives a complex triple adaptation of the network with feedback on the shape of the self-assembled entity. Our results demonstrate how microcompartments can enforce the expression of constituents of CDNs by reducing the hydrolysis of uptaken members, unravelling processes that govern the response of reactions networks. Such studies open the way toward using DCvLs and CDNs to understand the emergence of complexity within reaction networks by their interactions with microenvironments.

Phototriggered Equilibrated and Transient Orthogonally Operating Constitutional Dynamic Networks Guiding Biocatalytic Cascades.

The photochemical deprotection of structurally engineered o-nitrobenzylphosphate-caged hairpin nucleic acids is introduced as a versatile method to evolve constitutional dynamic networks, CDNs. The photogenerated CDNs, in the presence of fuel strands, interact with auxiliary CDNs, resulting in their dynamically equilibrated reconfiguration. By modification of the constituents associated with the auxiliary CDNs with glucose oxidase (GOx)/horseradish peroxidase (HRP) or the lactate dehydrogenase (LDH)/nicotinamide adenine dinucleotide (NAD+) cofactor, the photogenerated CDN drives the orthogonal operation upregulated/downregulated operation of the GOx/HRP and LDH/NAD+ biocatalytic cascade in the conjugate mixture of auxiliary CDNs. Also, the photogenerated CDN was applied to control the reconfiguration of coupled CDNs, leading to upregulated/downregulated formation of the antithrombin aptamer units, resulting in the dictated inhibition of thrombin activity (fibrinogen coagulation). Moreover, a reaction module consisting of GOx/HRP-modified o-nitrobenzyl phosphate-caged DNA hairpins, photoresponsive caged auxiliary duplexes, and nickase leads upon irradiation to the emergence of a transient, dissipative CDN activating in the presence of two alternate auxiliary triggers, achieving transient operation of up- and downregulated GOx/HRP biocatalytic cascades.

Dynamic DNA Networks-Guided Directional and Orthogonal Transient Biocatalytic Cascades.

DNA frameworks, consisting of constitutional dynamic networks (CDNs) undergoing fuel-driven reconfiguration, are coupled to a dissipative reaction module that triggers the reconfigured CDNs into a transient intermediate CDNs recovering the parent CDN state. Biocatalytic cascades consisting of the glucose oxidase (GOx)/horseradish peroxidase (HRP) couple or the lactate dehydrogenase (LDH)/nicotinamide adenine dinucleotide (NAD+) couple are tethered to the constituents of two different CDNs, allowing the CDNs-guided operation of the spatially confined GOx/HRP or LDH/NAD+ biocatalytic cascades. By applying two different fuel triggers, the directional transient CDN-guided upregulation/downregulation of the two biocatalytic cascades are demonstrated. By mixing the GOx/HRP-biocatalyst-modified CDN with the LDH/NAD+-biocatalyst-functionalized CDN, a composite CDN is assembled. Triggering the composite CDN with two different fuel strands results in orthogonal transient upregulation of the GOx/HRP cascade and transient downregulation of the LDH/NAD+ cascade or vice versa. The transient CDNs-guided biocatalytic cascades are computationally simulated by kinetic models, and the computational analyses allow the prediction of the performance of transient biocatalytic cascades under different auxiliary conditions. The concept of orthogonally triggered temporal, transient, biocatalytic cascades by means of CDN frameworks is applied to design an orthogonally operating CDN for the temporal upregulated or downregulated transient thrombin-induced coagulation of fibrinogen to fibrin.

Front Cover: Behavior of Constitutional Dynamic Networks: Competition, Selection, Self‐sorting in Cryptate Systems (ChemistryEurope 1/2023)

Front Cover: Behavior of Constitutional Dynamic Networks: Competition, Selection, Self‐sorting in Cryptate Systems (ChemistryEurope 1/2023)

Cascaded, Feedback-Driven, and Spatially Localized Emergence of Constitutional Dynamic Networks Driven by Enzyme-Free Catalytic DNA Circuits.

The enzyme-free catalytic hairpin assembly (CHA) process is introduced as a functional reaction module for guided, high-throughput, emergence, and evolution of constitutional dynamic networks, CDNs, from a set of nucleic acids. The process is applied to assemble networks of variable complexities, functionalities, and spatial confinement, and the systems provide possible mechanistic pathways for the evolution of dynamic networks under prebiotic conditions. Subjecting a set of four or six structurally engineered hairpins to a promoter P1 leads to the CHA-guided emergence of a [2 × 2] CDN or the evolution of a [3 × 3] CDN, respectively. Reacting of a set of branched three-arm DNA-hairpin-functionalized junctions to the promoter strand activates the CHA-induced emergence of a three-dimensional (3D) CDN framework emulating native gene regulatory networks. In addition, activation of a two-layer CHA cascade circuit or a cross-catalytic CHA circuit and cascaded driving feedback-driven evolution of CDNs are demonstrated. Also, subjecting a four-hairpin-modified DNA tetrahedron nanostructure to an auxiliary promoter strand simulates the evolution of a dynamically equilibrated DNA tetrahedron-based CDN that undergoes secondary fueled dynamic reconfiguration. Finally, the effective permeation of DNA tetrahedron structures into cells is utilized to integrate the four-hairpin-functionalized tetrahedron reaction module into cells. The spatially localized miRNA-triggered CHA evolution and reconfiguration of CDNs allowed the logic-gated imaging of intracellular RNAs. Beyond the bioanalytical applications of the systems, the study introduces possible mechanistic pathways for the evolution of functional networks under prebiotic conditions.

Behavior of Constitutional Dynamic Networks: Competition, Selection, Self‐sorting in Cryptate Systems

AbstractUnderstanding dynamic systems is a crucial step toward the design of complex matter. Here, we aim to study the behavior of Constitutional Dynamic Networks (CDNs) in conditions of dynamic competition, taking cryptands and metal cations as a test bed. The CDNs of cryptates were analyzed by NMR spectroscopy. The experimental results were complemented by extensive numerical simulations, based on a large amount of thermodynamic and kinetic data available in the literature for cryptates. Although the CDN′s output is a result of the interplay between the individual stability constants of the complexes in a mixture, the overall effect may be governed by only one – the most thermodynamically stable member of a network. Significantly, these findings indicate that an increase in complexity (multiplicity and connectivity) of a system may, in conditions of dynamic competition, result in “simplexity”, i. e. a simplification of the output of the system.

Dynamic Catalysis Guided by Nucleic Acid Networks and DNA Nanostructures.

Nucleic acid networks conjugated to native enzymes and supramolecular DNA nanostructures modified with enzymes or DNAzymes act as functional reaction modules for guiding dynamic catalytic transformations. These systems are exemplified with the assembly of constitutional dynamic networks (CDNs) composed of nucleic acid-functionalized enzymes, as constituents, undergoing triggered structural reconfiguration, leading to dynamically switched biocatalytic cascades. By coupling two nucleic acid/enzyme networks, the intercommunicated feedback-driven dynamic biocatalytic operation of the system is demonstrated. In addition, the tailoring of a nucleic acid/enzyme reaction network driving a dissipative, transient, biocatalytic cascade is introduced as a model system for out-of-equilibrium dynamically modulated biocatalytic transformation in nature. Also, supramolecular nucleic acid machines or DNA nanostructures, modified with DNAzyme or enzyme constituents, act as functional reaction modules driving temporal dynamic catalysis. The design of dynamic supramolecular machines is exemplified with the introduction of an interlocked two-ring catenane device that is dynamically reversibly switched between two states operating two different DNAzymes, and with the tailoring of a DNA-tweezers device functionalized with enzyme/DNAzyme constituents that guides the dynamic ON/OFF operation of a biocatalytic cascade by opening and closing the molecular device. In addition, DNA origami nanostructures provide functional scaffolds for the programmed positioning of enzymes or DNAzyme for the switchable operation of catalytic transformations. This is introduced by the tailored functionalization of the edges of origami tiles with nucleic acids guiding the switchable formation of DNAzyme catalysts through the dimerization/separation of the tiles. In addition, the programmed deposition of two-enzyme/cofactor constituents on the origami raft allowed the dynamic photochemical activation of the cofactor-mediated biocatalytic cascade on the spatially biocatalytic assembly on the scaffold. Furthermore, photoinduced "mechanical" switchable and reversible unlocking and closing of nanoholes in the origami frameworks allow the "ON" and "OFF" operation of DNAzyme units in the nanoholes, confined environments. The future challenges and potential applications of dynamic nucleic acid/enzyme and DNAzyme conjugates are discussed in the conclusion paragraph.

Autonomous Chemistry Enabling Environment-Adaptive Electrochemical Energy Storage Devices

Autonomous Chemistry Enabling Environment-Adaptive Electrochemical Energy Storage Devices

Transient Out‐of‐Equilibrium Nucleic Acid‐Based Dissipative Networks and Their Applications

AbstractDynamic, dissipative reactions play important roles in biological processes, and emulating such biological processes by artificial circuits and networks is a challenging topic in the area of Systems Chemistry. The present article summarizes efforts to apply the information encoded in the base sequence of nucleic acids to assemble transient, dissipative DNA‐based networks mimicking natural processes. The design principles of the transient nucleic acid‐based systems are introduced. Triggered reaction moduli lead to a transient intermediary constituent recovered to the parent moduli by enzymes, DNAzyme, or light as control units are described. Transient DNA‐based circuitries of enhanced complexities revealing temporal gated or cascaded transformations are demonstrated. Different applications of dynamically triggered transient circuits are introduced, including the temporal aggregation and deaggregation of plasmonic nanoparticles or semiconductor quantum dots and the control over their optical properties, the transient release and uptake of loads by aptamer scaffolds, and the transient reconfiguration of constitutional dynamic networks and the control over emergent catalytic functions. The future perspectives of dissipative nucleic acid‐based networks and their potential future applications are discussed.

Tetrahedron-Based Constitutional Dynamic Network for COVID-19 or Other Coronaviruses Diagnostics and Its Logic Gate Applications.

Considering the large-scale outbreak of the coronavirus, it is essential to develop a versatile sensing system for different coronaviruses diagnostics, such as COVID-19, severe acute respiratory syndrome-related coronavirus (SARS-CoV), and bat SARS-like coronavirus (Bat-SL-CoVZC45). In this work, a tetrahedron-based constitutional dynamic network was built as the sensing platform for coronavirus detection. Four different DNA probes were used to construct the tetrahedron structure. DNAzyme and the fluorophore modified substrate strand were used to generate different fluorescence signals, which can be used to distinguish different coronaviruses. The coronavirus biosensor shows a high sensitivity for COVID-19, Bat-SL-CoVZC45, and SARS-CoV detection, with detection limits of 2.5, 3.1, and 2.9 fM, respectively. Also, the platform is robust, and the possible interference from clinical samples was negligible. Using different coronaviruses as inputs, we have fabricated several concatenated logic gates, such as "AND-OR", "INHIBIT-AND", "AND-AND-AND", and "AND-INHIBIT". Importantly, our logic system can also be used to identify SARS-CoV-2 Delta and Lambda variants in the logic operations. Due to the unique advantages of high sensitivity and selectivity, multiple logic biocomputing capabilities, and multireadout mode, this flexible sensing system provides a versatile sensing strategy for intelligent diagnostics of different coronaviruses with low false-negative rates.

Integration of photocatalytic and dark-operating catalytic biomimetic transformations through DNA-based constitutional dynamic networks

Nucleic acid-based constitutional dynamic networks (CDNs) have recently emerged as versatile tools to control a variety of catalytic processes. A key challenge in the application of these systems is achieving intercommunication between different CDNs to mimic the complex interlinked networks found in cellular biology. In particular, the possibility to interface photochemical ‘energy-harvesting’ processes with dark-operating ‘metabolic’ processes, in a similar way to plants, represents an up to now unexplored yet enticing research direction. The present study introduces two CDNs that allow the intercommunication of photocatalytic and dark-operating catalytic functions mediated by environmental components that facilitate the dynamic coupling of the networks. The dynamic feedback-driven intercommunication of the networks is accomplished via information transfer between the two CDNs effected by hairpin fuel strands in the environment of the system, leading to the coupling of the photochemical and dark-operating modules.

DNA Assembly-Based Stimuli-Responsive Systems.

Stimuli‐responsive designs with exogenous stimuli enable remote and reversible control of DNA nanostructures, which break many limitations of static nanostructures and inspired development of dynamic DNA nanotechnology. Moreover, the introduction of various types of organic molecules, polymers, chemical bonds, and chemical reactions with stimuli‐responsive properties development has greatly expand the application scope of dynamic DNA nanotechnology. Here, DNA assembly‐based stimuli‐responsive systems are reviewed, with the focus on response units and mechanisms that depend on different exogenous stimuli (DNA strand, pH, light, temperature, electricity, metal ions, etc.), and their applications in fields of nanofabrication (DNA architectures, hybrid architectures, nanomachines, and constitutional dynamic networks) and biomedical research (biosensing, bioimaging, therapeutics, and theranostics) are discussed. Finally, the opportunities and challenges for DNA assembly‐based stimuli‐responsive systems are overviewed and discussed.

DNA-based constitutional dynamic networks as functional modules for logic gates and computing circuit operations.

A nucleic acid-based constitutional dynamic network (CDN) is introduced as a single computational module that, in the presence of different sets of inputs, operates a variety of logic gates including a half adder, 2 : 1 multiplexer and 1 : 2 demultiplexer, a ternary multiplication matrix and a cascaded logic circuit. The CDN-based computational module leads to four logically equivalent outputs for each of the logic operations. Beyond the significance of the four logically equivalent outputs in establishing reliable and robust readout signals of the computational module, each of the outputs may be fanned out, in the presence of different inputs, to a set of different logic circuits. In addition, the ability to intercommunicate constitutional dynamic networks (CDNs) and to construct DNA-based CDNs of higher complexity provides versatile means to design computing circuits of enhanced complexity.

Dictated Emergence of Nucleic Acid-Based Constitutional Dynamic Networks by DNA Replication Machineries.

The emergence of nucleic acid-based constitutional dynamic networks, CDNs, from a pool of nucleic acids is a key process for the understanding and modality of the evolution of biological networks. We present a versatile method that applies a library of nucleic acids coupled to biocatalytic DNA machineries as functional modules for the emergence of CDNs of diverse composition, complexity, and structural diversity. A set of four DNA template/blocker scaffolds coupled to the polymerase/dNTP replication machinery leads, in the presence of a primer, P1, to the gated replication of the scaffolds and to the displacement of four components that reconfigure into a [2 × 2] CDN. Using six template/blocker scaffolds and the polymerase/dNTPs, the P1-guided emergence of a [3 × 3] CDN is demonstrated. In addition, by further engineering the template/blocker scaffolds, the hierarchical control over the composition of the P1-guided emergence of [3 × 3] CDNs is accomplished. Also, sequence-engineered template/blocker scaffolds, coupled to the polymerase/dNTP machinery, lead, in the presence of two primers P1 and/or P2, to the selective emergence of two different [2 × 2] CDNs or to a [3 × 3] CDN. Also, a set of six appropriately engineered template/blocker scaffolds, coupled to the polymerase/dNTP machinery, leads to the emergence of a CDN composed of four equilibrated DNA tetrahedra constituents. Finally, by further sequence engineering of the set of template/blocker scaffolds and their coupling to a nicking/polymerization/dNTP replication machinery, the amplified high-throughput emergence of CDNs is demonstrated.

Nucleic Acid Based Constitutional Dynamic Networks: From Basic Principles to Applications.

Inspired by nature where intracellular dynamic interactions between DNA, RNA and proteins processed within complex networks leading to programmed reaction patterns, extensive research efforts are directed to mimic these processes by chemical means, "Systems Chemistry". The present perspective introduces nucleic acids as functional modules to construct constitutional dynamic networks, CDNs, mimicking natural networks. The base sequences comprising nucleic acids provide a rich "tool box" to assemble signal-triggered reconfigurable CDNs revealing adaptive and hierarchically adaptive properties, intercommunication between CDNs, and feedback-driven reaction pathways similar to natural systems. Pathways for the evolution of CDNs and the formation of networks of enhanced complexities are discussed. Different applications of constitutional dynamic networks are introduced including programmed catalysis, CDN-guided optical and catalytic functions of nanoparticle aggregates, and CDN-dictated stiffness and self-healing functions of hydrogels. Future perspectives of the field in designing dissipative transient CDNs, CDNs-guided transcription/translation synthesis of selective proteins, and the challenging integration of CDNs into cell-like containments aiming to assemble "artificial cells" are addressed.

Constitutional Dynamic Networks-Guided Synthesis of Programmed "Genes", Transcription of mRNAs, and Translation of Proteins.

Inspired by nature, where dynamic networks control the levels of gene expression and the activities of transcribed/translated proteins, we introduce nucleic acid-based constitutional dynamic networks (CDNs) as functional modules mimicking native circuits by demonstrating CDNs-guided programmed synthesis of genes, controlled transcription of RNAs, and dictated transcription/translation synthesis of proteins. An auxiliary CDN consisting of four dynamically equilibrated constituents AA', AB', BA', and BB' is orthogonally triggered by two different inputs yielding two different compositionally reconfigured CDNs. Subjecting the parent auxiliary CDN to two hairpins, HA and HB, and two templates TA and TB and a nicking/replication machinery leads to the cleavage of the hairpins and to the activation of the nicking/replication machineries that synthesize two "genes", e.g., the histidine-dependent DNAzyme g1 and the Zn2+-ion-dependent DNAzyme g2. The triggered orthogonal reconfiguration of the parent CDN to the respective CDNs leads to the programmed preferred CDN-guided synthesis of g1 or g2. Similarly, the triggered reconfigured CDNs are subjected to two hairpins HC and HD, the templates I'/I and J'/J, and the RNA polymerase (RNAp)/NTPs machinery. While the cleavage of the hairpins by the constituents associated with the parent CDN leads to the transcription of the broccoli aptamer recognizing the DFHBI ligand and of the aptamer recognizing the malachite green (MG) ligand, the orthogonally triggered CDNs lead to the CDNs-guided enhanced transcription of either the DFHBI aptamer or the MG aptamer. In addition, subjecting the triggered reconfigured CDNs to predesigned hairpins HE and HF, the templates M'/M and N'/N, the RNAp/NTPs machinery, and the cell-free ribosome t-RNA machinery leads to the CDNs-guided transcription/translation of the green fluorescence protein (GFP) or red fluorescence protein (RFP).

Controlling biocatalytic cascades with enzyme–DNA dynamic networks

Cellular transformations, such as gene expression or temporal protein activities, are controlled by complex stimuli-responsive network circuitries regulated by enzymes, metabolites or transcription factors. Inspired by nature, extensive research efforts are directed to mimic these processes by in vitro chemical systems. Here we report on the assembly of constitutional dynamic networks (CDNs), composed of nucleic acid–enzyme conjugates, that act as modules for triggered, network-driven, biocatalytic cascades and for the intercommunication of network-guided biocatalytic cascades. Two CDNs were assembled—one network includes a constituent module functionalized with glucose oxidase and horseradish peroxidase in spatially close positions, and the second CDN includes a constituent module modified at sterically intimate positions with nicotinamide adenine dinucleotide and alcohol dehydrogenase. Biocatalytic cascades proceed in the two networks and, on the triggered reconfiguration of the CDNs, controlled and switchable biocatalytic cascades in the CDNs are demonstrated. The two CDNs are coupled, and the triggered feedback-driven intercommunication of the networks is realized. Extensive research efforts in systems chemistry are directed to the development of in vitro systems that mimic complex natural networks. Now, stimuli-responsive nucleic acid-based networks conjugated to biocatalysts for the triggered and orthogonal control over biocatalytic cascades are reported.

Dissipative Constitutional Dynamic Networks for Tunable Transient Responses and Catalytic Functions.

Following the significance of dissipative, out-of-equilibrium biological processes controlling living systems, we introduce nucleic acid-based dissipative constitutional dynamic networks (CDNs) that exhibit tunable transient composition changes of the networks dictated by auxiliary fuel strands. CDN "X" composed of four equilibrated nucleic acid constituents, AA', AB', BA', and BB', and the accompanying "dormant" structures T1L1 and T2L2 and nicking enzyme Nt.BbvCI, undergoes dissipative orthogonal transitions to CDN "Y" and back or to CDN "Z" and back. In the presence of the fuel strand L1' or L2', the displacement of the respective "dormant" structure releases the trigger T1 or T2 that activates the reconfiguration of CDN "X" to CDN "Y" or CDN "X" to CDN "Z". The generated duplex L1L1' or L2L2' is designed to be nicked by Nt.BbvCI, leading to the regeneration of L1 or L2 that rebinds to T1 or T2, resulting in the dissipative cyclic recovery of CDN "X". Kinetic simulations of the dissipative processes allow us to predict the dissipative behavior of the systems under different auxiliary conditions. Subjecting CDN "X" to altering sets of the fuel strands L1' and L2' yields programmed reconfiguration patterns of dissipative reaction cycles. By engineering functional nucleic acid tethers on the constituents and the triggering strands, orthogonal dissipative emerging catalytic transformations dictated by the dissipative CDNs are demonstrated.

Functional Constitutional Dynamic Networks Revealing Evolutionary Reproduction/Variation/Selection Principles.

Within the broad research efforts to engineer chemical pathways to yield high-throughput evolutionary synthesis of genes and their screening for dictated functionalities, we introduce the evolution of nucleic-acid-based constitutional dynamic networks (CDNs) that follow reproduction/variation/selection principles. These fundamental principles are demonstrated by assembling a library of nucleic-acid strands and hairpins as functional modules for evolving networks. Primary T1-initiated selection of components from the library assembles a parent CDN X, where the evolved constituents exhibit catalytic properties to cleave the hairpins in the library. Cleavage of the hairpins yields fragments, which reproduces T1 to replicate CDN X, whereas the other fragments T2 and T3 select other components to evolve two other CDNs, Y and Z (variation). By applying appropriate counter triggers, we demonstrate the guided selection of networks from the evolved CDNs. By integrating additional hairpin substrates into the system, CDN-dictated emergent catalytic transformations are accomplished. The study provides pathways to construct evolutionary dynamic networks revealing enhanced gated and cascaded functions.