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Zhong et al. Chem Synth 2023;3:27 https://dx.doi.org/10.20517/cs.2023.15 Page 13 of 25

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Figure 7. Schematic design of communication and cross-regulation system integrating NAD -fueled dissipative RNA transcription sub-
network and toehold-mediated strand displacement reactions sub-networks. This figure is quoted with permission from Sun et al. [127] .

functional systems. Relevant applications include a synthetic biochemical clock in a cell-like environment,
dynamic patterns, molecular motion, production timer, and more [129,133] . In addition, Schaffter et al. reported
[131]
bistable networks using genelets as building blocks . The standard bistable network consists of two
reciprocally-inhibited genelets. Two DNA activators are designed to control the transcription of the genelet,
respectively. When one DNA activator binds to its corresponding promoter domain to activate the
transcription of the genelet, the newly transcribed RNA signal represses the transcription of another genelet
by replacing its DNA activator from the promoter domain of the second genelet. Moreover, RNA bound to
DNA is degraded by RNase H, freeing DNA activators. It displaces typical bistable dynamic behaviors,
which means only one genelet can be activated at a time. The resulting bistable network can change its states
in response to different environmental signals and coordinates the temporal regulation of state-specific
downstream signal dynamics.

Furthermore, Schaffter et al. develop a hierarchical engineering strategy for building more complex network
systems, as shown in Figure 8 . At the fundamental level of reactive nodes, they created the hairpin clamp
[87]
(HPC5) genelet toolbox with three states (OFF/ON/BLK) to improve the performance of genelet nodes.
When the T7 RNAP promoter sequence of a genelet is incomplete, transcription is almost non-existent
(defined as OFF state). When a DNA activator completes the promoter domain, transcription is switched to
ON. Transcription disappears when a DNA blocker replaces the DNA activator (BLK). Genelets can also
regulate one another through upstream transcription, either by activating or blocking downstream genelets
to trigger state transitions. A series of modularized, interchangeable nodes with similar performance and
minimum crosstalk form a standardized library. Engineering these standard nodes into feedforward and
feedback loops creates functional modules. For example, these nodes are used to design three incoherent
feedforward loop modules that produce pulses of genelet activation via upstream transcription. Finally,
multiple incoherent feedforward loop modules are coordinated into a composite network that generates
sequential pulsed signals with tunable pulse shapes and delay time. Beyond that, these elements are also
used to engineer a switchable tristable network, which includes three mutually repressive bistable sub-
networks. And integrating incoherent feedforward loop modules into a switchable multistable network