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Self-Assembled DNA hydrogel based on enzymatically-polymerized DNA for protein encapsulation and enzyme/DNAzyme hybrid cascade reaction Binbin Xiang, Kaiyu He, Rong Zhu, Zhuoliang Liu, Shu Zeng, Yan Huang, Zhou Nie, and Shouzhuo Yao ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b03572 • Publication Date (Web): 16 Aug 2016 Downloaded from http://pubs.acs.org on August 19, 2016

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ACS Applied Materials & Interfaces

Self-Assembled DNA Hydrogel Based on Enzymatically-polymerized DNA for Protein Encapsulation and Enzyme/DNAzyme Hybrid Cascade Reaction Binbin Xiang, Kaiyu He, Rong Zhu, Zhuoliang Liu, Shu Zeng, Yan Huang, Zhou Nie*, Shouzhuo Yao

State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha, 410082, P. R. China.

KEYWORDS: DNA hydrogel, self-assembly, protein encapsulation, multiple-enzyme system, cascade reaction

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ABSTRACT DNA hydrogel is a promising biomaterial for biological and medical applications with its native biocompatibility and biodegradability. Herein, we provided a novel, versatile, and cost-effective approach for self-assembly of DNA hydrogel using the enzymatically-polymerized DNA building blocks. The X-shaped DNA motif was elongated by terminal deoxynucleotidyl transferase (TdT) to form the building blocks, and the hybridization between dual building blocks via their complementary TdT-polymerized DNA tails leads to gel formation. TdT polymerization dramatically reduced the required amount of original DNA motifs, and the hybridization-mediated crosslinking of building blocks endows the gel with high mechanical strength. The DNA hydrogel can be applied for encapsulation and controllable release of protein cargos, for instance green fluorescent protein (GFP), due to its enzymatic responsive properties. Moreover, this versatile strategy was extended to construct a functional DNAzyme hydrogel, by integrating the peroxidase-mimicking DNAzyme into DNA motifs. Furthermore, a hybrid cascade enzymatic reaction system was constructed by co-encapsulating glucose oxidase (GOx) and β-galactosidase (β-Gal) into DNAzyme hydrogel. This efficient cascade reaction provides not only a potential method for glucose/lactose detection by naked eye, but also a promising modular platform for constructing multiple enzyme or enzyme/DNAzyme hybrid system.


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1. Introduction Functional hydrogel has become an important material for biological and medical applications due to its close resemblance in physical and mechanical properties to that of biological tissues.1 Over the past decade, hydrogel entirely formed by DNA has attracted substantial interests for its variety of applications, including biosensors,2 drug delivery and release,3 cell-free protein producing,4 and single cell enveloping and releasing5 owing to its sequence-directed self-assembly, biocompatibility, biodegradability, and mechanical stability. However, the reported preparation methods of DNA hydrogel are still rare. For example, the first pure DNA hydrogel was prepared by the assembly of branched DNA motifs through enzymatic ligation.6 Similarly, a fast pH-switchable and a designable thermal, enzymatic responsive DNA hydrogel have been developed.7, 8 While in previous methods, a large amount of DNA were required to prepare the DNA hydrogel, even reached to sub-millimolar level,9 greatly increasing the cost of forming gels, which limited the development of DNA hydrogel. There were two strategies applicable for solving this problem. The first strategy is grafting nucleic acids to hydrophilic polymers such as polyacrylamide chain, and the formation of hydrogel is based on the interaction of DNA.10-13 However, this method required multi-step modification for DNA−polymer hybrids, which was extremely labor intensive. In the other approach, DNA chains were elongated via Φ29 DNA polymerase through rolling-circle amplification (RCA), then formed a physical hydrogel14 without hybridization whose mechanical strength was relatively weak (the storage modulus, G’ 8 mM). It's worth mentioning that the general range of blood glucose concentration for healthy person is about 3–8 mM.28 We tried the proposed glucose assay in serum, and the results indicated that the serum sample with 4.53 mM blood glucose shows the color change comparable to the standard colorimetric sample with 4 mM glucose (Figure S13). The results implied that the bienzyme cascade in our hydrogel provided a potentially qualitative and semiquantitative analysis for blood glucose. In addition, the selectivity of this multiple enzyme system to discriminate glucose from its analogues was also investigated (Figure S14). There was almost no


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color change for fructose, lactose, and sucrose recorded by digital camera which showed the high selectivity of this method for the detection of glucose.

Figure 4. (a) Schematic activation of the GOx/DNAzyme bienzyme cascade (b) The color becomes greener as the concentration of glucose increased. Glucose is the main product of β-Gal-catalyzed hydrolysis reaction. Therefore a trienzyme system could be easy to construct. As shown in Figure 5a, the first enzyme β-Gal loaded in the gel catalyzed hydrolysis of lactose to glucose. Then the oxidation of glucose by GOx produced hydrogen peroxide that can be catalyzed by DNAzyme hydrogel to oxidize ABTS2-. The activation of the trienzyme cascade is shown in Figure 5b. Upon addition of lactose, green color product was observed and its amount increased with the increasing concentration of lactose, which provided a potential method to qualitatively and semiquantitatively detect lactose.



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Figure 5. (a) Schematic activation of the β-Gal/GOx/DNAzyme trienzyme (b) The color becomes greener as the concentration of lactose increased. 4. Conclusions In summary, we have developed a novel, facile, and cost-efficient synthesis strategy to prepare pure DNA hydrogel based on TdT polymerization. The elongation of X-shaped DNA motifs by TdT polymerization was exploited for preparing the hydrogel, which greatly reduced the originally required amount of DNA motifs. Second, based on the mechanical strength and enzymatic responsive properties, the gel can be useful for controllable encapsulation and release of bio-macromolecules, such as ScGFP used in this paper. Finally, Because of the versatility of our design principle, our proposed strategy was extended to construct functional hydrogel, such as DNAzyme gel, 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 combining GOx and β-Gal with the output capable for naked-eye observation. Our hydrogel did 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


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hydrogel is a promising modular platform for constructing other multiple enzyme or enzyme/DNAzyme hybrid system in the future.

ASSOCIATED CONTENT Supporting Information. Additional information including additional experimental section and extensive figures as noted in text is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. Fax: +86-731-88821848. Tel: +86-731-88821626. Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENT This work was financially supported by the National Natural Science Foundation of China (Nos. 21575038, 21235002, and 21305037), the Foundation for Innovative Research Groups of NSFC (Grant 21521063), Young Top-notch Talent for Ten



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Thousand Talent Program, the Natural Science Foundation of Hunan Province (No. 2015JJ1005).

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