Abstract
Investigations into the refolding of DNA origami leads to the creation of reconstructable nanostructures and deepens our understanding of the sustainability of life. Here, we report the refolding of the DNA origami structure inside a micron-sized compartment. In our experiments, conventional DNA origami and truss-type DNA origami were annealed and purified to remove the excess staples in a test tube. The DNA origami was then encapsulated inside of a micron-sized compartment of water-in-oil droplets, composed of neutral surfactants. The re-annealing process was then performed to initiate refolding in the compartment. The resulting 100-nm-sized DNA nanostructures were observed using atomic force microscopy (AFM), and the qualities of their structures were evaluated based on their shape. We found that the refolding of the DNA origami structure was favored inside the droplets compared with refolding in bulk solution. The refolded structures were able to fold even under “quick” one-minute annealing conditions. In addition, the smaller droplets (average diameter: 1.2 µm) appeared to be more advantageous for the refolding of the origamis than larger droplets. These results are expected to contribute to understanding the principles of life phenomena based on multimolecular polymer self-assembly in a micron-sized compartment, and for the production and maintenance of artificially designed molecules.
Highlights
All living organisms maintain life through the synthesis and function of a vast number of biopolymers
Two types of planar DNA origami were designed, conventional DNA origami with normal crossover branching [9] and truss DNA origami [18,19], and their structures were confirmed by imaging using atomic force microscopy (AFM)
Two types of re-annealing profiles were used after 5 min at 70 ◦ C: a “Normal_45min” profile, performed at −1 ◦ C/min, from 70 ◦ C to 25 ◦ C; and a “Quick_1min” profile, performed at −50 ◦ C/min. We collected these DNA samples by centrifugation, and after polyethylene glycol (PEG) purification, we evaluated the morphology of the DNA origami structures using AFM observation (Figures S5–S17)
Summary
All living organisms maintain life through the synthesis and function of a vast number of biopolymers. Most elongated polymers function in their folded states; polymers can often become unfolded—and dysfunctional—due to environmental changes, such as pH, salt concentration, hydrophobic balance, and cell cycle. The living cell inevitably employs a refolding mechanism to recover from the unfolded and/or misfolded state of the polymer. The self-refolding of proteins [1], molecular chaperones [2] that refold denatured or misfolded proteins, and chromatin re-modelling complexes [3,4] that reproduce the chromatin structure of genomic DNA, have been identified. In the field of nanoengineering, the folding of artificially designed DNA has been a rapidly emerging field [5,6,7,8]. DNA origami is one of the major structures in DNA nanotechnology
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