Efficient amplification of self-gelling polypod-like structured DNA by rolling circle amplification and enzymatic digestion

Abstract

The application of DNA as a functional material such as DNA hydrogel has attracted much attention. Despite an increasing interest, the high cost of DNA synthesis is a limiting factor for its utilization. To reduce the cost, we report here a highly efficient amplification technique for polypod-like structured DNA (polypodna) with adhesive ends that spontaneously forms DNA hydrogel. Two types of polypodna with three (tripodna) and four (tetrapodna) pods were selected, and a template oligodeoxynucleotide, containing a tandem sequence of a looped tripodna or tetrapodna, respectively, along with restriction enzyme (TspRI) sites, was designed. The template was circularized using T4 DNA ligase, and amplified by rolling circle amplification (RCA). The RCA product was highly viscous and resistant to restriction digestion. Observation under an electron microscope revealed microflower-like structures. These structures were composed of long DNA and magnesium pyrophosphate, and their treatment with EDTA followed by restriction digestion with TspRI resulted in numerous copies of polypodna with adhesive ends, which formed a DNA hydrogel. Thus, we believe this technique provides a new approach to produce DNA nanostructures, and helps in expanding their practical applications.

Self-assembly of hierarchically ordered structures in DNA nanotube systems

“…The formation of complex, mesoscopic, condensed aggregates consisting of repeating DNA motifs generated by PCR reactions has been previously addressed, for instance, in the frame of medical applications [32][33][34][35][36]. Other studies have focused on mesoscopic crystallization phases of short, non-hybridizing DNA oligomers through either end-to-end stacking or crowding effects [37][38][39].…”

Droplet Microfluidics Approach for Single-DNA Molecule Amplification and Condensation into DNA-Magnesium-Pyrophosphate Particles

“…The fraction of RNA per single particle was found to be around 20 wt %, which we anticipate to be of a similar level for the DNA-Mg-PP i particles produced in this work. Others have also shown the generation of various macromolecular nucleic acid structures that are likely to be mediated through multiple ion bridges coordinated by magnesium ions and pyrophosphate [ 41 , 42 , 43 , 44 , 45 ]. Given our results and previous reports, it is appealing to think that under certain environmental conditions, the inorganic pyrophosphate and divalent ions (magnesium, calcium, etc.)…”

DNA Nanostructure as an Efficient Drug Delivery Platform for Immunotherapy

“…Besides dendrimers, Polypod-like nanostructure referred to another kind of branched nanoassembiles consist of structural body "trunk" together with many "legs". Investigators have reported various types of polypod-like DNA nanostructure, including polypodna (Yata et al, 2015), DNA nanocentipede. Their structural advantages are their long backbone and numerous branch structures.…”

“…Polypodna itself could serve as an immunostimulatory agent. Y-shaped polypodna could induce great amounts of cytokines TNF-a and IL-6 than normal native double-stranded DNA (Yata et al, 2015). Y L -DNA (ligated Y-shaped DNA) also exhibits TLR9-mediated activation of DCs and macrophages, as revealed by promoted expression of the immune-relevant molecules (Yang et al, 2019).…”