Page 116 - Read Online

Page 10 of 25 Zhong et al. Chem Synth 2023;3:27 https://dx.doi.org/10.20517/cs.2023.15

Furthermore, a dissipative three-layer cascaded network is designed based on auto-inhibition processes of
2+
[105]
Mg -ion-dependent DNAzymes . Three coupled sub-networks are constructed to execute a cascaded
2+
signal process to control the transient formation and depletion of three different Mg -ion-dependent
2+
DNAzymes. For example, the sub-network I consists of the toehold-functionalized duplex AA', Mg ions,
and corresponding ribonuclease-containing hairpin fuel H . Subjecting the sub-network I to the fuel T
1
1
results in the formation of Mg -ion-dependent DNAzyme AT . Hairpin fuel H acts as the substrate of
2+
1
1
DNAzyme AT to be cleaved into two fragmented strands, H and H . The resulting strand H can displace
1
1b
1a
1a
the strand T from AT to generate the waste strand H T , leading to the disassembly of the Mg -ion-
2+
1 1
1
1
dependent DNAzyme AT for the re-formation of AA'. And the other fragmented strand, H , is used as the
1b
1
cascaded signal to unlock the fuel strand T from the coupler ST for operating the next-layer sub-network.
2
2
Two additional dissipative sub-networks, II and III, are designed following a similar design principle. Sub-
network II consists of the duplex BB' and hairpin fuel H . Upon the release of fuel T , the transient operation
2
2
of DNAzyme BT is activated, and the cleavage of H yields functional fragments H and H . H interacts
2
2a
2
2a
2b
with T to dissipate DNAzyme BT in sub-network II. H is a cascaded signal to trigger the release of fuel
2
2
2b
strand T that activates the transient operation of sub-network II to generate molecular signals H and C'.
3
3b
These signals can be used as regulatory signals for the downstream functional system, such as polymerase/
nicking machinery and the rolling circle polymerization machinery for complex transient biocatalytic
transformations.
Transient signals by enzyme-catalyzed reactions
Nucleic acids are involved in various biochemical reactions, providing infinite possibilities for constructing
complex reaction networks . Different enzyme-catalyzed DNA reactions were already used to build
[51]
dissipative networks for transient signals [32,106-107,109,118-121] . For example, RNA strands are fuel molecules to
drive dissipative systems. And RNase H, as the fuel-consuming catalysis, decomposes RNA strands in RNA/
DNA heteroduplexes for the dissipation of fuels [106,118-121] . By integrating this dissipative mechanism and
DNA-based strand displacement reactions, the variations of RNA strands, such as base sequences and
concentrations, are transduced to different transient molecular signals, including DNA strands , small
[106]
bioactive molecules, ATP, cocaine , and even the structural information of DNA structures [120,121] . The
[118]
RNase H-catalyzed dissipative strand displacement reaction also allows us to control the delay time of
[122]
molecular signals, which is vital to program time-dependent biological activities . Figure 6 illustrates that
the nucleic acid-based timing network contains three blocker-inhibited strand displacement reaction
pathways . Three-type blocker strands, RNA blocker, DNA blocker containing the modified base 8-oxo-
[122]
7,8 dihydroguanine (G -blocker), and DNA blocker containing four deoxyuridine mutations (uracil-
oxo
blocker) are designed to inhibit respective toehold-mediated strand displacement reactions by binding to
the toehold domains of the target DNA strands. And RNase H, formamidopyrimidine DNA glycosylase
(Fpg), and uracil-DNA glycosylase (UDG) decompose the corresponding blocker strands and unseal strand
displacement reactions, respectively. The kinetics of the blocker enzymatic degradation thus determines
when the blocker-inhibited strand displacement reactions start. By controlling the concentrations of the
blocker strands and the enzymes used in the network, the network enables the orthogonal modulate of the
delay times of different signals for potential regulation of downstream biological activities.
Adenosine triphosphate (ATP), one of the most important energy materials in living systems, is essential for
many enzyme-catalyzed biological reactions [123-125] . In the past few years, Walther et al. introduced ATP as a
fuel molecule to design a series of nucleic acid-based dynamic networks through enzymatic DNA
reactions [32,107-109,115,126] . For example, they reported T4 DNA ligase and an antagonistic restriction enzyme,
BamHI, driving a nucleic acid-based dissipative network . T4 DNA ligase can ligate DNA oligomer
[126]
substrates encoded with complementary sticky ends at both sides with the consumption of ATP. The