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Zhong et al. Chem Synth 2023;3:27 https://dx.doi.org/10.20517/cs.2023.15 Page 5 of 25

Figure 2. (A) Positive feedback-driven regulation of nucleic acid-based CDN R; (B) Intercommunication and mutual regulation between
[75,79]
two nucleic acid-based CDNs S and T. This figure is quoted with permission from Yue et al. . CDN: constitutional dynamic network.
beyond simulating natural networks.

Contrary to the feedback loop, the feedforward loop consists of an input signal (X) that regulates an
intermediate signal (Y), while both X and Y regulate an output signal (Z) [72,76] . Haley et al. proposed using
internal base-pairing mismatches within DNA duplex substrates to control their strand displacement
reaction rates, as shown in Figure 3 . The mismatch position-dependent strand displacement reactions are
[77]
demonstrated through experiment and simulation. During the strand displacement process, the mismatch
position of the DNA duplex substrate can be chosen rationally to provide a hidden thermodynamic drive.
The strand displacement reaction rates are highest when the mismatch is eliminated early but not
immediately. And a mismatch positioned close to the toehold domain of the DNA duplex substrate
increases the reaction rate by approximately two orders of magnitude.

In contrast, a mismatch placed far from the toehold domain of the DNA duplex substrate has a minimal
effect. Based on the mismatch position-dependent strand displacement reactions, a feedforward DNA
reaction network is designed and constructed as a simple pulse-generating device. The network includes
two competitive toehold-mediated strand displacement reactions with an asymmetrically placed mismatch
in the duplex substrate OT, 18 bases from the 5' toehold and three from the 3' toehold. The displacement of
strand T from OT using 3' invading strand I is kinetically favored. The reaction between OT and 5'
3
invading strand I is thermodynamically favored as it is more excess than I , and both eliminate the initially
5
3
present mismatch. By subjecting a four-fold excess of I and a two-fold excess of I to a solution of duplex
3
5
substrate OT, the kinetic-driven reaction of I in displacing strand O is activated, as the mismatch position
3
of OT speeds up this process. This reaction is controlled by the competitive invader strand I , where the
5
product duplex I T is displaced by the thermodynamically favored strand I to control the output through
5
3
repression. One of the emerging properties of such feedforward networks is their transient response to
[78]
persistent stimuli, characterized as a pulse generator where the system returns to its original steady state .
Communication
[79]
Feedback pathways are also constructed between two CDNs to assemble intercommunicating networks .
Figure 2B depicts the signal intercommunication between two CDNs, S and T . The equilibrated CDN S
[79]
and CDN T include four constituents, EE′, EF′, FE′, FF′ and constituents GG′, GH′, HG′, HH′, respectively.