DNA Hydrogels
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0950-D09-03
DNA Hydrogels Soong Ho Um, Nokyoung Park, and Dan Luo Biological and Environmental Engineering, Cornell University, Riley-Robb Hall, Wing dr., Ithaca, NY, 14853 ABSTRACT DNA hydrogel was constructed by the cross-connection of the branched DNA molecules through enzymatic catalytic reaction. Three different types of branched DNA, X-, Y-, T-shaped DNA were tested as both building block and crosslinker. Swelling degree and degradation of the DNA hydrogel strongly relied on the initial concentration and types of DNA building blocks: XDNA gel with highest concentration (0.2 mM) showed the highest swelling degree (approximately 700%) and the strongest resistance to degradation over other hydrogels. DNA hydrogel was explored as new controlled drug delivery, cell encapsulation matrix, and even novel protein production system. INTRODUCTION DNA has been recognized as genetic information storage. To date, owing to its unique chemical and physical properties such as flexibility and programmability, DNA material as new generic material source have been employed to construct bottom-up and self-assembled materials with specific functionality. For example, Seeman and his colleagues have created a branched DNA which is artificially manufactured and applied to diverse research areas[1-3]. However, such DNA materials have been restricted as a template for making material complexes in small scale but not in large scale. Here we report bulk DNA hydrogel which is entirely composed of DNA[4]. It is expected to achieve a practical application of DNA material into a variety of medical related research. EXPERIMENT The protocol for synthesizing DNA building blocks were described on our previous papers in more detail[5-7]. DNA hydrogel was formed by ligating DNA building block (50 nmol) via T4 DNA ligase (30 Weiss units) provided from Promega. DNA gel degradation was evaluated by measuring their daily DNA mass loss in the presence of various media. For drug release assay, the released drugs in the supernatants were periodically replaced with fresh PBS and then incubated in the buffer at 37 oC with shaking. They were evaluated using insulin specific ELISA kit (ALPCO diagnostics) and measuring CPT absorbance. To image cell encapsulation in DNA hydrogel, CHO cells were pre-stained with CellTrackerTM Red CMTPX Probes (Molecular Probes) for long-term tracing of living cells. For gelation, the stained cells were mixed with X-DNA building blocks (0.2 mM) and 3 Weiss units of T4 DNA ligase. Biocompatibility of DNA hydrogel was evaluated using CellTiter 96® Aqueous Non-radioactive Cell Proliferation Assay Kits (Promega). For a DNA gel based protein production, a gene coding Renilla luciferase (RL) was cloned into the expression vector, pIVEX 1.3 WG (Roche Applied Science) and used as a target gene source for protein producing DNA hydrogel (P-gel). The P-gel expressed a target protein using wheat germ based in vitro protein production system (RTS 100 WG, Roche Applied Science). The expressed Renilla luciferase was assessed using Renilla luci
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