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Page 12 of 25 Zhong et al. Chem Synth 2023;3:27 https://dx.doi.org/10.20517/cs.2023.15

Complex far-from-equilibrium signal dynamic behaviors
Integrating numerous sub-networks with different composites and functionality is a promising approach to
constructing complex dynamic networks with enhanced functionalities. The synthetic signaling dynamic
network is entering a new stage with the development of the integrating strategy. Walther et al. designed a
set of toehold-mediated strand displacement reactions as signal conversion modules [32,108,109,127] . Integrating
the ATP-fueled network as a transient signal generator and strand displacement reactions as signal
conversion modules assembles the ATP-driven dissipative networks with self-resetting and cascaded
[108]
behavior . This work paves the way for network-guided regulation of the downstream system. Coupling
ATP-fueled dissipative networks to DNA assemblies allows temporary control of DNA nanostructures.
ATP-powered DNA ligation induces the transient DNA strand displacement process for activating the self-
assembly of DNA nanotubes and restriction-induced backward reactions trigger the degradation of the
[32]
resulting DNA nanotubes . More interestingly, the reciprocal feedback reaction pathway can be designed
to concatenate two sub-networks to form a communicating system for an ATP-driven automaton .
[127]
Recently, Sun et al. reported a signal communication and cross-regulation system based on dissipative DNA
[127]
reaction networks, as depicted in Figure 7 . The system includes one signal-sender network by fuel-driven
RNA transcription and two signal-receiver networks by ligation-digestion strand displacement reaction
networks. In the signal-sender network, A transcriptional template is designed to generate an RNA signal,
where the activation and deactivation of RNA transcription are controlled by the fuel-driven ligation/
digestion reaction guided by the formation and dissociation of the T7 promoter domain. Instead of the T4
DNA ligase, E. coli DNA ligase is used to ligate DNA fragments with 3'-OH and 5'-phosphate ends with β-
+
nicotinamide adenine dinucleotide (NAD ) fuel. At the same time, BsaI allows the enzymatic digestion of
+
the resulting DNA template. The signal-receiver network includes two NAD -fueled dissipative sub-
networks undergoing similar ligation-digestion reactions. The transient RNA signal from the sender
network enters the receiver network for signal regulation. The signal systems display four types of signal
output patterns, repression, promotion, repression-recovery, and promotion-stop, regulated by the
variations of different DNA sequence designs and enzymes involved in the system. For example, in the
repression process, RNA is designed to interact with the reactive monomer via toehold-mediated strand-
displacement reactions, which inhibits the ligation of two reactive monomers into dimer structures, partially
shutting down the receiver network and repressing signal outputs. By adding RNase H to degrade the RNA
simultaneously, RNA in the form of a non-reactive monomer is digesting to reactivate the NAD -fueled
+
dissipative sub-network in the receiver network. Thus, the signal pattern transforms from a repression form
into a repression-recovery form.

As analogous to the signal dynamic in cellular gene regulatory networks, a synthetic RNA transcription
machinery based on T7 RNA polymerase and RNase H, genelet, was designed to produce and degrade RNA
signals for establishing a signal turnover process. It provides a powerful tool for assembling in vitro
transcriptional regulatory networks, such as oscillating and bistable networks, for complex signaling
dynamics [128-132] . For example, the delay-negative feedback oscillator and the three-repressor ring oscillator
[128]
were constructed based on modular RNA transcription machines . These networks consist of 2 or 3
switchable transcriptional templates. Each transcriptional template contains an incomplete promoter site for
T7 RNA polymerase. When a DNA activator strand completes the promoter site, nucleotide triphosphates
(NTPs) fuel the transcription of an RNA signal that can regulate the transcription of the target genelet by
controlling the availability of the corresponding DNA activator through the strand displacement process.
Therefore, regulatory models, such as negative feedback loops, can be programmed by designing the nucleic
acid sequences of transcriptional templates. In addition to RNA signal production by activated
transcription, RNA signals are also degraded by RNase H to maintain the continuous dynamic of the
system. The resulting oscillating network can generate periodic RNA signals for regulating downstream