Analysis of Tertiary Structural Features of Branched DNA Nanostructures with Partially Common Sequences Using Small-Angle X-ray Scattering.

We demonstrated that polypod-like structured DNA, composed of 3-8 kinds of oligodeoxynucleotides (ODNs), exhibit accelerated cellular uptake depending on their structural properties. The biological activities, including immunostimulation and unwanted adverse effects of ODNs depend on their sequences. Therefore, as the number of different types of ODNs within a polypod-like structured DNA increases, the possible risks and concerns regarding its future clinical applications also increase. To minimize this risk and to explore the relationship between structural properties and cellular uptake, we designed Tet(id12), a tetrapod-like structured DNA with 12 identical palindrome sequences in the center of ODNs, and compared it with other tetrapod-like structures without palindrome sequences, symmetric Tet(sym) and asymmetric Tet(asym12). The thermal stability analysis of Tet(id12) revealed a two-step dissociation process. Polyacrylamide gel electrophoresis, small-angle X-ray scattering profile, and the Guinier plot also revealed that the conformation of Tet(id12) was close to that of Tet(sym). No significant differences were observed between Tet(id12) and others in terms of cellular uptake. These results indicate that ODNs with palindrome sequences can be used to design polypod-like structured DNAs without significant changes to their physicochemical and biological properties, although their thermal stability is somewhat different from the ones without palindrome sequences.