Abstract

DNA hydrogels have unique properties, including sequence programmability, precise molecular recognition, stimuli-responsiveness, biocompatibility and biodegradability, that have enabled their use in diverse applications ranging from material science to biomedicine. Here, we describe a rolling circle amplification (RCA)-based synthesis of 3D DNA hydrogels with rationally programmed sequences and tunable physical, chemical and biological properties. RCA is a simple and highly efficient isothermal enzymatic amplification strategy to synthesize ultralong single-stranded DNA that benefits from mild reaction conditions, and stability and efficiency in complex biological environments. Other available methods for synthesis of DNA hydrogels include hybridization chain reactions, which need a large amount of hairpin strands to produce DNA chains, and PCR, which requires temperature cycling. In contrast, the RCA process is conducted at a constant temperature and requires a small amount of circular DNA template. In this protocol, the polymerase phi29 catalyzes the elongation and displacement of DNA chains to amplify DNA, which subsequently forms a 3D hydrogel network via various cross-linking strategies, including entanglement of DNA chains, multi-primed chain amplification, hybridization between DNA chains, and hybridization with functional moieties. We also describe how to use the protocol for isolation of bone marrow mesenchymal stem cells and cell delivery. The whole protocol takes ~2 d to complete, including hydrogel synthesis and applications in cell isolation and cell delivery.