RESEARCH:

Stabilization and structural changes of DNA origami by ligation:

  The low thermal stability of DNA nanostructures is the major drawback in their practical applications. Most of the DNA nanotubes/tiles and the DNA origami structures melt below 60◦C due to the presence of discontinuities in the phosphate backbone (i.e., nicks) of the staple strands. In molecular biology, enzymatic ligation is commonly used to seal the nicks in the duplex DNA. However, in DNA nanotechnology, the ligation procedures are neither optimized for the DNA origami nor routinely applied to link the nicks in it. Here, we report a detailed analysis and optimization of the conditions for the enzymatic ligation of the staple strands in four types of 2D square lattice DNA origami. Our results indicated that the ligation takes overnight, efficient at 37◦C rather than the usual 16◦C or room temperature, and typically requires much higher concentration of T4 DNA ligase. Under the optimized conditions, up to 10 staples ligation with a maximum ligation efficiency of 55% was achieved. Also, the ligation is found to increase the thermal stability of the origami as low as 5◦C to as high as 20◦C, depending on the structure. Further, our studies indicated that the ligation of the staple strands influences the globular structure/planarity of the DNA origami, and the origami is more compact when the staples are ligated. The globular structure of the native and ligated origami was also found to be altered dynamically and progressively upon ethidium bromide intercalation in a concentration-dependent manner.

Single molecular analysis using DNA origami:

During the last two decades, scientists have developed various methods that allow the detection and manipulation of single molecules, which have also been called “in singulo” approaches. Fundamental understanding of biochemical reactions, folding of biomolecules, and the screening of drugs were achieved by using these methods. Singlemolecule analysis was also performed in the field of DNA nanotechnology, mainly by using atomic force microscopy. However, until recently, the approaches used commonly in nanotechnology adopted structures with a dimension of 10–20 nm, which is not suitable for many applications. The recent development of scaffolded DNA origami by Rothemund made it possible for the construction of larger defined assemblies. One of the most salient features of the origami method is the precise addressability of the structures formed: Each staple can serve as an attachment point for different kinds of nanoobjects. Thus, the method is suitable for the precise positioning of various functionalities and for the single-molecule analysis of many chemical and biochemical processes. 

Four-stranded DNA structures:

i-Motif: We now present the first example in which triplet repeat DNAs adopt the i-motif structure at neutral pH by molecular crowding. Crowding stabilized the i-motif and the pK_a of N3 of cytosine was raised in such a microenvironment. Molecular crowding is known to accelerate the formation of the multi-stranded i-motif while the triplet repeats adopt the single-strand structur

G-quadurplex: We have investigated the new folding pathways of human telomeric type-1 and type-2 G-quadruplex conformations via the intermediate hairpin and triplex structures. The stabilization energies calculated by ab initio method evidenced the formation of hairpin structure with Hoogsteen GG base pairs. Further calculations revealed that the G-triplet is more stable than the hairpin conformation and equally stable when compared to the G-tetrad. This indicated the possibility of the triplex intermediate. The overall folding is facilitated by the K⁺ association in each step as it decreases the electrostatic repulsion. The K⁺ binding site was identified by the molecular dynamic simulations. We then focused on the syn/anti arrangement and found that the anti conformation of deoxyguanosine is more stable than the syn conformation which indicated that the folding would increase the number of anti conformations. The K⁺ binding to a hairpin near the second lateral TTA loop was found to be preferable, considering entropic effects. The stacking of G-tetrads with the same conformation (anti/anti or syn/syn) is more stable than the mixed stacking (anti/syn and vice versa). These results suggest the formation of type-1 and type-2 G-quadruplex structures with the possibility of the hairpin and triplex intermediates.

Programmed self-assembly of DNA origami:

The DNA origami structures were size expanded in two-dimensional (2D) area by using multiple origami structures, named “2D DNA jigsaw pieces”. We designed and prepared nine different jigsaw pieces and tried to obtain a 3´3 assembly. The proof of concept was obtained by performing the assembly in four different ways. Among them, the stepwise self-assembly from the three vertical trimer assemblies gave the target with 35% yield. Finally, the surfaces of jigsaw pieces were decorated with hairpin DNAs to display the letters of the alphabet, and the self-assembled structure displayed the word “DNA JIG SAW” in nanoscale. In other approach, we have carried out the 2D assembly using a connector that has connection sites at all four corners. By utilizing this four-way connector, five and eight origami monomers were assembled to form cruciate and hollow square structures, respectively. These methods can be extended to create self-assembled modules carrying various functional molecules for practical applications.