SM04.03.05 : Elucidating DNA Origami Assemblies on Surfaces

5:00 PM–7:00 PM Apr 3, 2018 (America - Denver)

PCC North, 300 Level, Exhibit Hall C-E

Huan Cao1 Gary Abel1 Yehan Zhang1 Tao Ye1

1, University of California, Merced, Merced, California, United States

Molecular assembly based on Watson-Crick base-pairing has enabled the construction of complex 2D and 3D DNA nanostructures to be used in widespread applications including photonics, biosensing, enzymatic catalysis, single-molecule fluorescence, and drug delivery. In particular, scaffolded DNA origami involves the cooperative binding of large numbers of short single-stranded DNA (ssDNA) helper strands to “staple” a long ssDNA into a predesigned object. However, the order of assembly and different folding pathways to assemble these DNA nanostructures still remain unclear. Recent efforts have utilized a combination of ex situ imaging techniques, i.e., transmission electron microscopy and atomic force microscopy (AFM) to observe the structures of intermediates formed at each annealing step. However, the transient intermediates may experience structural distortions and damages because these approaches require the DNA samples to be transferred from solutions and deposited onto mica surfaces for imaging. In addition, there is a lack of direct evidences as to how a particular DNA origami structure evolves from a random-coil scaffold strand.
Here, we utilized a recent DNA imaging method developed in our group to resolve the structures and kinetics of intermediates in situ along the DNA origami folding pathway. Thiolated DNA probes were inserted stochastically into the defect sites of negatively charged carboxylate-modified alkanethiol self-assembled monolayers (SAMs). These probes were then hybridized and crosslinked with double-stranded DNA precursors. Surface-tethered DNAs were then denatured to yield single-stranded scaffolds, enabling hybridization with DNA staples in the solution phase. Divalent cations were used to immobilize the surface-tethered intermediates onto the negatively charged SAMs for AFM visualization, while monovalent cation solutions enable intermediates to be released from the surface and hybridize with DNA staples. Because DNA scaffolds were tethered to surfaces, we were able to follow the evolution of each DNA origami with minimal structural distortion and damages. Using formamide to vary “effective” temperatures, we showed that the optimal folding temperature ranges not only agree with solution-phase studies, but could also be translated to surface-assisted folding of DNA origami structures. Moreover, we showed that the thiolated DNA probes act as surface seeds to nucleate different 2D origami structures, rectangles and crosses, simultaneously on the same substrate. Thus, our approach not only offers new insight into folding intermediates, but also provides a convenient method to generate single-stranded scaffolds from virtually any double-stranded DNAs, which can be mass-produced via polymerase chain reactions.