Yawen Liu,Zhaozhu Zheng,He Gong,Meng Liu,Shaozhe Guo,Gang Li,Xiaoqin Wang,David L. Kaplan
The structure of DNA is susceptible to alterations at high temperature and on changing pH, irradiation and exposure to DNase. Options to protect and preserve DNA during storage are important for applications in genetic diagnosis, identity authentication, drug development and bioresearch. In the present study, the stability of total DNA purified from human dermal fibroblast cells, as well as that of plasmid DNA, was studied in silk protein materials. The DNA/silk mixtures were stabilized on filter paper (silk/DNA + filter) or filter paper pre-coated with silk and treated with methanol (silk/DNA + PT-filter) as a route to practical utility. After air-drying and water extraction, 50–70% of the DNA and silk could be retrieved and showed a single band on electrophoretic gels. 6% silk/DNA + PT-filter samples provided improved stability in comparison with 3% silk/DNA + filter samples and DNA + filter samples for DNA preservation, with ∼40% of the band intensity remaining at 37 °C after 40 days and ∼10% after exposure to UV light for 10 hours. Quantitative analysis using the PicoGreen assay confirmed the results. The use of Tris/borate/EDTA (TBE) buffer enhanced the preservation and/or extraction of the DNA. The DNA extracted after storage maintained integrity and function based on serving as a functional template for PCR amplification of the gene for zinc finger protein 750 (ZNF750) and for transgene expression of red fluorescence protein (dsRed) in HEK293 cells. The high molecular weight and high content of a crystalline beta-sheet structure formed on the coated surfaces likely accounted for the preservation effects observed for the silk/DNA + PT-filter samples. Although similar preservation effects were also obtained for lyophilized silk/DNA samples, the rapid and simple processing available with the silk–DNA–filter membrane system makes it appealing for future applications.
DNA-based biomaterials, including polyelectrolyte complex (PEC) hydrogels composed of DNA and other molecules, have received considerable attention due to their programmability and good biocompatibility. However, the cost for DNA synthesis is fairly high, especially for applications where a large quantity of DNA is needed. Construction of biomaterials using biomass-derived DNA is a feasible strategy to address this challenge. In this work, we fabricated and characterized PEC hydrogels with or without covalent cross-linking using biomass-derived DNA and chitosan (CS). Characterization results revealed detailed microstructures and mechanical properties of these two hydrogels, suggesting their potential application in DNA preservation and protein immobilization. We next developed a versatile DNA preservation strategy combining DNA-CS PEC hydrogel-based DNA encapsulation and chitosanase-mediated DNA release, which successfully maintained the integrity and functionality of various types of DNA, i.e., double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), plasmid DNA, and genomic DNA, during the treatment of DNase I and hydrogen peroxide (H2O2). Remarkably, this method also demonstrated good efficiency in protecting DNA containing a broadly used unnatural base pair, dNaM-dTPT3, against the attack of silver ion. Additionally, we developed a convenient and reliable strategy for protein immobilization with the covalently cross-linked PEC hydrogel. This strategy demonstrated good performance in the reusability of the immobilized enzyme, evidenced by 56.4% product yield of the initial cycle of usage achieved by the immobilized horseradish peroxidase (HRP) in the tenth cycle of usage. This work not only broadens the application scope of biomass-derived polymers, but also provides feasible strategies for the sustainable and cost-effective preservation and immobilization of biological macromolecules.
危险品化学品经营许可证（带存储）