Enhancing efficiency and control in DNA hydrogel synthesis: A dual rolling circle amplification approach and parameter optimization study

DNA hydrogels are a focus of current research in the realm of cutting-edge functional biomaterials. Rolling circle amplification (RCA) technology has surfaced as a flexible approach for producing functional DNA hydrogels in a one-pot manner, owing to its effectiveness and ease of use. This study introduces a simple dual RCA approach for producing DNA hydrogels. Our research delves into the impacts of different reaction parameters, such as reaction buffer, ethylenediaminetetraacetic acid (EDTA) concentration, base pair types and ratios, circular DNA template concentration, and RCA temperature, on the yield, morphology, and rheological characteristics of the resulting DNA hydrogels. Our findings demonstrate that DNA hydrogels prepared with TE buffer containing 1 mM EDTA, higher concentrations of circular DNA templates, and an RCA reaction temperature of 30 degrees C exhibited improved morphological characteristics and favorable mechanical properties. Furthermore, while G-C base pairs significantly enhance the mechanical properties of the hydrogel at lower concentrations, their excessive presence may negatively impact both the formation and mechanical integrity of the hydrogel. The findings from this study are crucial for boosting the efficiency and cost-effectiveness of DNA hydrogel synthesis, enhancing their quality, and broadening their range of applications.