A Novel Enzymatic Polymerization Based Synthesis Strategy For DNA Hydrogel And Its Application

Functional hydrogel has become an important material for biological and medical applications as its close resemblance of their physical and mechanical properties to that of biological tissues. Over the past decade, hydrogel entirely formed by DNA has attracted substantial interests for its variety of applications, including biosensors, drug delivery and release, cell-free protein producing and single cell enveloping and releasing owing to its excellent biocompatibility, biodegradability, and easy to functionalization. However, the reported preparation methods of DNA hydrogel are still rare, and in previous methods, a large amount of DNA were required to prepare the DNA hydrogel, even reached to sub-millimolar level, greatly increasing the cost of forming gels, which limited the development of DNA hydrogel. Herin, based on a template-independent DNA polymerase, terminal deoxynucleotidyl transferase (TdT), two-dimension or three-dimensional as building blocks was utilized to develop a novel, facile, and cost-efficient synthesis strategy to prepare pure DNA hydrogel, which can be applied in encapsulation and release of protein, conctruction of enzyme cascads, and protein enrichment.1. Based on DNA polymerization by TdT, we have firstly demonstrated a novel synthesis strategy for DNA hydrogel. A branched DNA structure, X-DNA, was used as the primer, it contains four arms with exposed 3'-OH ends which allows polymerization to extend in four directions via TdT, generating X-shaped DNA motifs with poly A and poly T tails in dATP and dTTP pool respectively. Consequently, the hydrogel was obtained through hybridization between poly T and poly A tails. In this system, TdT polymerization would significantly reduce the required amount of original DNA motifs, and the hybridization of poly A and poly T tails would enhance the mechanical strength of gel. In addition, scheme of the DNA hydrogel formation was investigated, and we find that linear DNA without cross-linking points as the building blocks and the branched DNA extended by unpaired dNTP, like dATP and dCTP, failed to form gel. The results indicated that the successful formation of DNA hydrogel actually depends on hybridization of elongated branched structures. Then rheological measurement, SEM, and AFM were used to characterize the mechanical property, surface morphology and the inner structure of the hydrogel. And our hydrogel can be useful for controlled encapsulation and release of bio-macromolecules due to its enzymatic responsive properties.2. Based on this novel DNA hydrogel synthesis strategy, a functional DNAzyme hydrogel was designed, only with addition of DNAzyme sequences to the building blocks. This DNAzyme gel exhibits enhanced peroxidase activity after binding with hemin, which could produce colorimetric signal. Based on this phenomenon, we developed bienzyme and trienzyme cascades through combining GOx and ?-Gal with the output capable for naked-eye observation. Our hydrogel not only serve as scaffold but also act as a catalytic unit in the cascade. The efficient cascade reaction provides a potential method for glucose/lactose detection. Importantly, the cascade reaction in DNA hydrogel is a promising modular platform for constructing other multiple enzyme or enzyme/DNAzyme hybrid system in the future.3. A three-dimensional DNA structure, DNA nanotube, was utilized as building blocks to develop a DNA hydrogel with more stable and stronger mechanical properties. In this study, DNA nanotube contains several exposed 3'-OH ends which allows polymerization to extend, generating DNA nanotubes with poly A and poly T tails in the side chains. Consequently, the hydrogel was obtained through hybridization between the side chains. Rheological measurement proved the formation of a true gel. In addition, we investigated the application of the gel. The results showed it can be used for proteins and enzymes enrichment with less effects on their activity. Hence, the hydrogel may also be applied for construction of multiple enzyme system.