Materials
Staple strands for the DNA origami seeds were purchased from Sigma-Aldrich (desalt purification). SST strands for the SST sublattices were purchased from Sangon Biotech (PAGE purification). The sequences of all strands can be found in Supplementary Tables 1–6. Scaffold DNA strands (p7560, CS3-L and CS4) were purchased from Tilibit Nanosystems. All DNA strands were stored at −20 °C after being dissolved in ultrapure water. Other chemicals were purchased from Sigma-Aldrich. Carbon-coated TEM grids were purchased from Ted Pella. Quantifoil grids were purchased from Quantifoil Micro Tools GmbH. AFM tips were purchased from Bruker.
Preparation of DNA origami seeds
DNA origami seeds were designed using caDNAno software. Seed S is a DNA origami bundle consisting of 48 helices (8-helix × 8-helix rim, 4-helix × 4-helix pore) arranged in a square array (Fig. 1b(i)). Seed H is a DNA origami bundle composed of 54 helices (12-helix × 6-helix rim, 6-helix × 3-helix pore) arranged in a honeycomb array (Fig. 1b(ii)). The hollow seed design facilitates the accurate identification of the seed within a DNA moiré superlattice for microscopy characterizations. The twisted geometries of the DNA origami seeds and the twist angles of the Z2 (or Z4) segments were simulated using the SNUPI program28. The simulation parameters are provided in Supplementary Fig. 3. Position vectors on the seeds were calculated on the basis of coordinates extracted from the SNUPI simulation output files. To assemble the DNA origami seeds, staple strands were mixed with scaffold strands in a molar ratio of 10:1 in TE-MgCl2 buffer (10 mM Tris, 1 mM EDTA and 20 mM MgCl2, pH 7.8). The mixture was then annealed in a PCR thermocycler using the following protocol: 65 °C for 20 min; from 60 °C to 40 °C at 40 min per degree Celsius; and from 40 °C to 25 °C at 15 min per degree Celsius.
Agarose gel analysis and sample purification
The DNA origami seeds were electrophoresed on a 1.0 % agarose gel containing 1× GelRed in 0.5× TBE buffer with 11 mM Mg2+ for 3 h at 80 V with ice cooling. The target band was excised from the gel and squeezed between two glass slides. The concentration of the purified DNA origami seeds was determined using ultraviolet–visible absorption spectroscopy. All purified DNA origami seeds were stored at 4 °C before further use.
Growth of unseeded DNA sublattice monolayers
The square and honeycomb lattices are created using 32-nt SSTs with four 8-nt domains and 36-nt SSTs with four 9-nt domains, respectively. The kagome lattice is formed using two 32-nt SST variants: one adopts a U-shape, as in the square lattice, while the other is linear, comprising three domains (8 nt, 16 nt and 8 nt). To form the lattice monolayers, the SST strands were mixed in an equimolar stoichiometric ratio from a 200 µM stock in TE buffer (10 mM Tris and 1 mM EDTA, pH 7.8) supplemented with 40 mM MgCl2. The mixture was then annealed in a PCR thermocycler using the following protocol: 65 °C for 20 min; 48 °C to 47 °C at 12 h per degree Celsius for the honeycomb lattice; 44 °C to 43 °C at 12 h per degree Celsius for the kagome lattice; and 39 °C to 38 °C at 12 h per degree Celsius for the square lattice.
Growth of seeded monolayers, bilayers and trilayers
The SST strands (1.2 µM), purified DNA origami seeds (0.1–1.1 nM) and MgCl2 (final concentration of 40 mM) were separately preheated for 5 min at proper temperatures (honeycomb, 50 °C; kagome, 48 °C; square, 45 °C). They were then mixed quickly to avoid temperature fluctuations and incubated for 5 min. Finally, seeded growth proceeded at the designated nucleation and growth temperatures.
Dynamic light scattering measurements of homogeneous nucleation kinetics
Dynamic light scattering experiments were conducted using Zetasizer Pro (Malvern Instruments). The SST strands were pipetted into a cuvette, and the size of individual SST strands was measured. To obtain their nucleation kinetics, the SST strands and 40 mM MgCl2 were preheated for 5 min and then mixed. Measurements were taken immediately on the mixture at 15-s intervals.
Optical microscopy imaging
A 1 cm × 1 cm silicon wafer was cleaned sequentially with Milli-Q water, acetone and ethanol. It was then dried using nitrogen gas. Afterwards, the wafer was treated with plasma cleaning (30 mA, 5 min) to ensure a hydrophilic surface. Tenfold diluted DNA sublattices or superlattices were absorbed for 1 h onto the freshly prepared silicon wafer. Then,75% (vol/vol), 90% (vol/vol) and 100% (vol/vol) ethanol was sequentially used to wash the surface after absorption. Finally, the wafer was dried using nitrogen gas.
SEM imaging
Tenfold diluted DNA sublattices or superlattices were absorbed for 1 h onto the freshly prepared 1 cm × 1 cm silicon wafer. The wafer was then stained for 30 s using a 2% aqueous uranyl formate solution containing 25 mM NaOH. Then, 75% (vol/vol), 90% (vol/vol) and 100% (vol/vol) ethanol was sequentially used to wash the surface after absorption. Finally, the wafer was dried using nitrogen gas. SEM imaging was performed using Raith eLine Plus.
AFM imaging
The silicon wafers with stained DNA samples were directly used for AFM imaging with a ScanAsyst-air tip in air mode.
TEM imaging
Five microlitres of purified DNA origami seeds (1 nM) or tenfold diluted DNA sublattices or superlattices were absorbed for 10 min onto a glow discharged, carbon-coated TEM grid. The grids were then stained for 10 s using a 2% aqueous uranyl formate solution containing 25 mM NaOH. TEM imaging was performed using Phillip CM 200 TEM operated at 200 kV. STEM images were performed using JEOL JEM-ARM200F operated at 60 kV, equipped with a cold field-emission gun and a probe Cs corrector (DCOR, CEOS GmbH).
Cryo-EM characterization
Three microlitres of DNA lattices were pipetted onto glow-discharged Quantifoil grids. The sample was plunge-frozen using a Vitrobot Mark IV at 20 °C and humidity of 90%, with a wait time of 0 s, blot time of 6 s, blot force of −1 and drain time of 0 s. Imaging was performed using Talos Arctica (Thermo Fisher Scientific) operated at 200 kV, equipped with a Falcon III detector. Class-average cryo-EM images were obtained using the EMAN2 software package, version 2.99.47.
All-atom molecular dynamics simulations
Molecular dynamics simulations were performed using the Gromacs2021 software package. The AMBER99sb force field was used to describe the interactions within the system. The simulation box was constructed using Gromacs, with all components of the system being properly positioned according to the requirements. The box was then filled with water, utilizing the TIP3P water model. Mg2+ and Cl− were added to neutralize the system, ensuring that it was overall charge neutral. Before the production run, the system underwent energy minimization followed by a 100-ps equilibration simulation to allow proper relaxation. The production molecular dynamics simulation used the leapfrog algorithm to integrate Newton’s equations of motion, with an integration time step of 0.002 ps and a total of 50,000,000 steps, resulting in a total simulation time of 100 ns. The V-rescale thermostat was used to maintain the simulation temperature at 318.15 K, and the Parrinello–Rahman barostat was applied to keep the pressure constant at 1.0 bar. The Verlet scheme was used for neighbour searching. The cut-off radius for Coulombic interactions was set to 1.2 nm, with the long-range electrostatic interactions being corrected using the particle mesh Ewald method.
Statistics and reproducibility
No statistical method was used to predetermine sample size. No data were excluded from the analyses. The experiments were not randomized. The investigators were not blinded to allocation during experiments and outcome assessment.
