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International Journal of Bioprinting DNA-functionalized hyaluronic acid bioink

2.1.2. Assembly induced enzymatically and arrangement (Figure 2d and e). Hydrogen bonds,
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Enzymes also play a pivotal role as essential molecular rather than covalent bonds, provide major support to the
agents in facilitating the assembly of DNA hydrogels. For HCR-mediated network in DNA gelation systems, which
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example, by using Taq polymerase, coupled with a pair of is endowed with a less rigid but dynamic structure. This
thermostable Y-shaped DNA building blocks, thermostable method employs HCR between single-stranded DNA
DNA hydrogels could be synthesized via polymerase molecules with hairpin structures or initiator sequences to
chain reaction (PCR) (Figure 2c). Firstly, the DNA was form long-chain polymers, which crosslink with polymer
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crosslinked with psoralen and extended into a dumbbell chains to create a 3D network structure. The advantages
shape. Subsequently, these oligonucleotides formed of this method encompass precise control over hydrogel
covalently interstranded, branched DNA nanostructures, patterns, shapes, responsiveness, and the ability to
highlighting the crucial role of polymerase enzymes in the introduce various functional elements, but this method is
efficient production of robust DNA hydrogels. The two tied a time-consuming hydrogel preparation process, which
Y-structured DNA units underwent psoralen treatment, involves multiple reaction steps and purification, and it may
resulting in crosslinked structures. These crosslinked be susceptible to impurities or non-specific hybridization.
entities served as modular primers for PCR. Moreover,
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the utilization of T4 ligase-mediated reactions enables the 2.2. DNA as functional units
effective crosslinking of branched X-, Y-, and T-shaped Beyond their role as crosslinking agents, DNA-
DNAs, resulting in the formation of an interconnected functionalized hydrogels can also incorporate
gel system. The resulting DNA hydrogel leverages this reconfigurable DNA architectures and targeted DNA
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50,51
enzyme-driven, ligase-mediated crosslinking mechanism aptamers as responsive and recognition elements, such
under physiological conditions, showcasing its potential as G-quadruplexes (G4), i-motifs, triplex nucleic acids,
plasmid, and aptamers, which facilitate the intelligent
for applications such as controlled substance encapsulation. and functional regulation of hydrogels. By incorporating
Different swelling profiles of DNA hydrogels can be different functional elements, DNA hydrogels can achieve
achieved by adjusting the initial concentration and types structural regulation, molecular recognition, and functional
of building blocks. This method employs reactions of display under different environmental conditions.
enzymes such as ligases or polymerases to connect DNA
molecules with different end sequences or branched 2.2.1. G-quadruplex
structures to form a 3D network structure. The advantages G-quadruplex and i-motif are both crosslinking modes
of this method lie in its performance under physiological induced by the secondary structure of DNA, depending
conditions and the ability to adjust crosslinking density on specific base pairing and structural conformation.
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and mechanical strength by selecting different enzyme G-quadruplex is a four-stranded structure formed by
types and concentrations. However, the drawbacks of this guanine (G) bases of DNA. By regulating ion concentration
method include the complexity of hydrogel preparation and pH value, the physical and chemical properties of
process, which necessitates precise control of enzyme hydrogels can be controlled. Lu et al. explored a novel DNA
activity and reaction time, and the susceptibility to factors hydrogel synthesized with acrylamide and G-rich nucleic
such as enzyme residues or inhibitors. acid. In the presence of K ions, the nucleic acid crosslinkers
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formed G-quadruplex structures, enabling reversible
2.1.3. Assembly based on hybridization chain reaction hydrogel formation. Furthermore, the authors incorporated
Hybridization chain reaction (HCR) is a unique, isothermal hemin into the G-quadruplex hydrogel, endowing it with
DNA strand displacement process involving two hairpin catalytic activity similar to horseradish peroxidase. By
structures. It facilitates the formation of cross-opening modulating K ions, krypto[2.2.2], temperature, and pH
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polymer nucleic acid strands, acting as a versatile strategy as stimulus factors, the authors could regulate the phase
to fabricate responsive hydrogels endowed with tunable transitions of the hydrogel between solution, hydrogel,
characteristics. For example, DNA initiators, similar to and solid states as well as the conductivity and spectral
catalysts in chemical synthesis or seeds in crystal growth, properties of polyaniline (Figure 3a).
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were employed to achieve precise temporal and spatial
control in gelation process. These initiators direct 2.2.2. i-motif
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3D self-assembly and clamped hybridization, yielding i-motif is a special structure formed by the pairing of
hydrogels with enhanced mechanical properties. Moreover, protonated cytosine and guanine under acidic conditions.
employing printed, surface-confined DNA initiators, these It can regulate the structural changes of hydrogels
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hydrogels can be patterned into two-dimensional shapes (Figure 3b). For example, the incorporation of i-motif
without external constraints to allow for the generation of structures at the sticky ends of the DNA strands facilitates
hydrogels with precisely defined geometry, composition, the formation of DNA hydrogels that respond rapidly to pH

Volume 10 Issue 2 (2024) 30 doi: 10.36922/ijb.1814