Introduction

III. Two-Dimensional Crystallization of DNA Origami Checkerboards via Cation-

III.1. Introduction

DNA allows programming of nanoscale shapes and patterns¹, whose resolution cannot yet be met with current top-down micro/nanofabrication methods. Transferring self-assembled DNA nanostructures onto substrates is a promising approach towards patterning surfaces with nanoscale resolution for potential technological applications. However, depositing large-scale self-assembled DNA nanostructures on surfaces in a reliable way is challenging because the sample transfer process from solution to surface can cause breakage or distortion of the product structures.

Assembling DNA nanostructures directly on substrate surfaces can eliminate the sample transfer process. There have been studies where small DNA motifs were allowed to self-assemble on substrate surfaces to form large lattices^77-79, but technologically more interesting systems would be at a scale large enough to be reached by the lithographic regime, which was proved to be able to direct the orientation of DNA origami nanostructures on substrates⁸⁰. At the same time, the use of DNA origami allows higher complexity and access to unique addressability in each structural unit.

Here, we report large-scale self-assembly of DNA origami into two-dimensional checkerboard

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pattern crystals based on surface diffusion, where the diffusion of DNA origami occurs on the substrate surface and is dynamically controlled by changing the cationic condition of the system.

Unlike previous surface-based self-assembly studies of DNA nanostructures^77-79, our protocol can operate at an isothermal condition without the need of a thermal annealing process, which gives a further practical advantage. The bonds between origami were mediated by blunt ends at four corners that are capable of connecting through stacking interactions for the following two reasons:

first, to allow potential rearrangements during the surface assembly process via a putative sliding mechanism (as opposed to using sticky ends which may require unwinding and rewinding of the strands), and second, to achieve symmetry between the corners (each corner would be able to form the same kind of bond regardless of the orientation). We designed and tested origami that have a defined number (N ∈ {3, 5, 7}) of blunt ends at each of the four corners (Figure III-1).

Figure III-1. Schematic of origami design used for this study and the surface diffusion protocol.

For the two-dimensional assembly, (1) we first deposit origami on a mica surface, then (2) change the buffer condition to allow them to diffuse around within the confined space near the surface, and then (3) change the buffer condition again to fix them back on the surface (Figure III- 1). For the first step of depositing origami on a mica surface, we use a buffer with divalent cation Mg²⁺, the condition under which origami are formed and typically imaged by AFM. For allowing origami to diffuse around near the surface, we exchange the buffer to a solution that contains mostly the monovalent cation Na⁺. Monovalent cations weaken the binding of DNA onto mica surfaces^81,82 and have often been observed to release DNA previously adsorbed onto mica^83,84. At this step we also apply heat (40°C, with ~4 hr incubation), but it turns out that heat is not an essential factor although it facilitates the process. Then, in the next step, we exchange the buffer back to a divalent cation condition, Ni²⁺ instead of Mg²⁺ at this time, to stop diffusion and “freeze” the product structures onto the surface: Ni²⁺ is known to bind DNA onto mica more strongly than Mg²⁺ because

Step (1) Step (2) Step (3)

in Mg²⁺ + Na⁺

+ heat (optional) + Ni²⁺

N ∈ {3, 5, 7}

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Ni²⁺, as a transition metal ion, can form more stable coordination complexes with surrounding negatively charged ligands^85,86. It has been shown that Ni²⁺ ions enable a permanent binding of DNA to mica even at high Na⁺ concentration⁸³.

Our protocol is based on the previous findings in the literature that the surface mobility change and reversible binding of long duplex DNA on mica can be achieved by changing the cationic conditions of the system, such as the concentration of a divalent cation (Zn²⁺)⁸⁷ and the relative concentrations of divalent and monovalent cations^83,84. Mica is a layered mineral with a negative surface charge, and DNA is a negatively charged polymer. Thus, divalent cations are thought to mediate the binding between DNA and mica by bridging the negative charges on the DNA and the mica81-83,86,88. Such direct binding of DNA to mica can be considered a short-range attraction⁸², which induces strong adsorption of DNA onto mica and practically allows stable imaging of DNA by AFM. The surface diffusion phenomenon, during which our origami rearrange to form checkerboard crystals, is considered to be governed by a long-range interaction⁸²: though the monovalent cation mostly neutralizes the charges on the DNA and the mica, weakening the binding interaction, the divalent cation pre-existing and pre-adsorbed on the mica surface effectively inverts the charge of the mica surface and prevents the DNA from completely leaving the surface⁸². In other words, the addition of Na⁺ into the Mg²⁺ solution drives the system to the regime of a non-adsorbing⁸⁴ but long-range-attractive⁸² interaction between the DNA origami and the mica surface. Adsorbed Mg²⁺ is believed to persist on the mica surface during step (2) for two reasons. First, when exchanging the buffer, the original Mg²⁺ buffer is not removed, but only diluted. We leave ~5ul, to which we add 40 ul of the Na⁺ solution. Second, Mg ions have higher valence than Na ions which would exert a stronger electrostatic attraction to the negatively charged centers on mica, and the ionic radius of Mg²⁺ (~0.65 Å) allows the ions to “fit” into cavities near the negatively charged centers in the atomic structure of mica, further ensuring stronger binding (the cavities are compatible with ions with radii <~0.74 Å; Na⁺ has a ~1 Å radius)⁸⁶.

We discuss in detail the factors that allow the surface diffusion and two-dimensional crystallization of DNA origami rectangles. We also examine why the crystals would take the checkerboard form, rather than a linear chain form. We explore the effects of the strength of the stacking interaction between corners and of the concentration of Mg²⁺ during the surface diffusion step. Finally, we present a limitation of our approach, which is that the crystal size is limited by the irregularity of mica surfaces.

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