Abstract

Rolling circle amplification (RCA) is a widely used method for the synthesis of DNA nanoparticles and macro-hydrogels. Several strategies, including oscillation-promoted entanglement of DNA chains, multi-round chain amplification, hybridization between DNA chains, and hybridization with functional moieties, were applied to synthesize DNA macro-hydrogels; alternatively, flower-like nanoparticles were also produced. Here we report a straightforward yet effective method to manipulate the morphology of RCA products from nanoparticles to 3D hydrogels using an additional cold treatment step of the circular DNA template prior to elongation using phi29 DNA polymerase. This process induces a minor aggregation of the circular DNA template, significantly enhancing the entanglement of DNA chains in subsequent steps. Compared to contemporary synthesis methods for RCA-based macro-hydrogels, our technique provides milder reaction conditions, shorter reaction time, and a more straightforward system. Notably, our method eliminates the need for oscillation during amplification and requires only a single round of RCA with a single type of circular DNA, thereby simplifying the synthesis process.