Biocompatible DNA hydrogel composed of minimized Takumi-shaped DNA nanostructure exhibits sustained retention after in vivo administration

Our previous studies showed that DNA hydrogels containing unmethylated CpG motifs effectively induced antigen-specific immune responses when combined with the appropriate antigens. A potential drawback of existing DNA hydrogels for further applications is the need for many oligodeoxynucleotide (ODN) types. Therefore, in this study, we attempted to optimize and minimize the nanostructured DNA units for DNA hydrogels to reduce the preparation cost, design difficulty, and possible risk of sequence-dependent off-target effects, and prepare DNA hydrogels with sustained retention ability. A Takumi-shaped unit with a stem and four flanking cohesive parts was constructed using one type of ODN with a palindromic sequence. A DNA hydrogel was prepared by mixing two Takumi-shaped units with complementary cohesive parts. The required length of the cohesive part was first examined using ODNs with 14 or 18 bases of stem length. Electrophoresis, melting temperature measurements, and viscoelastic analysis showed that the properties of the cohesive part determined the hydrogel properties. ODNs with a cohesive part consisting of GC-rich 5'-ccgcaagacg-3' efficiently formed a hydrogel with sustained retention in mice after administration. Several ODNs with optimized cohesive sequences and different stem lengths and sequences were designed. Analyses showed that a stem of 10 bases or longer was required for efficient hydrogel formation, and ODNs with a 12-base stem part exhibited the most prolonged retention after subcutaneous injection into mice. Thus, the present study demonstrated the requirements of minimal DNA units for preparing DNA hydrogels with sustained retention ability.