Custom-Size, Functional, and Durable DNA Origami with Design-Specific Scaffolds

Abstract

DNA origami nano-objects are usually designed around generic single-stranded "scaffolds". Many properties of the target object are determined by details of those generic scaffold sequences. Here, we enable designers to fully specify the target structure not only in terms of desired 3D shape but also in terms of the sequences used. To this end, we built design tools to construct scaffold sequences de novo based on strand diagrams, and we developed scalable production methods for creating design-specific scaffold strands with fully user-defined sequences. We used 17 custom scaffolds having different lengths and sequence properties to study the influence of sequence redundancy and sequence composition on multilayer DNA origami assembly and to realize efficient one-pot assembly of multiscaffold DNA origami objects. Furthermore, as examples for functionalized scaffolds, we created a scaffold that enables direct, covalent cross-linking of DNA origami via UV irradiation, and we built DNAzyme-containing scaffolds that allow postfolding DNA origami domain separation.

Keywords: DNA nanotechnology; DNA origami; nanostructures; phagemid; self-assembly.

Figure 1

Design-specific scaffold sequences in minimum-constraint vectors for making fully user-defined DNA origami. (A) Schematic diagram of input for the scaffold smith used for creating custom scaffold sequences: exemplary caDNAno design diagram with scaffold strand indicated in blue and staple strands in multiple colors (I), user-specific constraints (II), and weighting factors for a stochastic base distribution (III). (B) Illustration of scaffold production with helper-plasmid system using phagemids with a split-ori approach (top) and a modified split-ori approach where the backbone sequence is flanked by self-cleaving DNAzymes (bottom). Zn²⁺ addition leads to excision of the backbone and linearization. Black, constant parts for each type of scaffold; gray, user-definable parts; light green, backbone present only in the double-stranded plasmid and not in the single-stranded product; red, self-cleaving DNAzymes.

Figure 2

Influence of base composition and sequence redundancy of custom scaffolds on DNA origami self-assembly. Blue indicates M13-based scaffolds; orange, magenta, red, cyan, and green indicate custom scaffolds. (A) Schematic representations of six different 42-helix bundles folded using the six different scaffolds. SC1, M13-based scaffold; SC2, reduced backbone phagemid scaffold with CpG-free de Bruijn insert sequence; SC3, conventional phagemid with high duplicity fragment and de Bruijn insert sequence; SC4, conventional phagemid with de Bruijn insert sequence; SC5 and SC6, split-ori based scaffold with de Bruijn sequence; L, length; GC, GC content of the corresponding scaffold. (B) Electrophoretic mobility analysis of self-assembly reactions of the 42-helix bundles shown in (A) at different temperatures and salt concentrations. SC, scaffold reference; C50 and C20, assembly reactions containing 50 nM (C50) or 20 nM (C20) scaffold, 200 nM staples, and 20 mM MgCl₂ that were subjected to an annealing ramp from 60 to 44 °C (1 h per °C); temperature screen, assembly mixtures as in C50 but subjected to annealing ramps covering the temperature intervals indicated above each lane (1 h per °C); magnesium screen, assembly reactions containing 50 nM scaffold, 200 nM staples, and MgCl₂ concentrations between 5 mM (M5) and 30 mM (M30). P, pocket; F, folded 42-helix bundle. All samples were loaded onto the gel at an approximate scaffold concentration of 20 nM. All temperature ramps contained an initial denaturation step at 65 °C for 15 min. Laser scanned fluorescent images of the electrophoretic analysis were autoleveled. (C) Statistics of sequence duplicates of different scaffold variants as a function of fragment length. Colors as in (A). (D) Experimentally observed optimal folding temperature intervals of the 42-helix bundles plotted against total NN energy of corresponding scaffold variant. Total NN energy was calculated using nearest-neighbor free energy parameters, ignoring edge effects. Dots in red indicate upper, and dots in blue indicate lower limit of the highest folding temperature interval where the sample appeared fully folded. Solid lines represent linear fits.

Figure 3

DNA origami objects with sizes ranging between 1024 bp (633 kDa) and 37800 bp (23.4 MDa) can be assembled using mini-scaffolds or in one-pot assembly reactions containing multiple scaffolds. Blue indicates M13-based scaffolds; orange, green, cyan, and red indicate custom scaffolds. (A) Schematic representation of a circular DNA single strand (top left) that, in the presence of Zn²⁺, cleaves itself to yield four copies of a short, linear scaffold (top right) that can subsequently be used to assemble a small DNA origami object (bottom). (B) Schematic representation (top) and average TEM images of 13-helix bundle (13hb) variants assembled from linear mini-scaffolds comprising 1024 (I), 1536 (II), or 2048 bases (III). Scale bar: 20 nm. (C) Electrophoretic mobility analysis of mini-scaffolds and 13-helix bundle variants described in (B). (D) Schematic representations, single TEM images, and average TEM images (from top to bottom) of a 42-helix bundle assembled with five scaffolds in one-pot reactions. Scale bar: 50 nm. (E) Schematic representations, single TEM images, and average TEM images (from top to bottom) of an improved 42-helix bundle design with five interlocked scaffolds. Scale bar: 50 nm. (F) Electrophoretic mobility analysis of the two 42-helix bundle versions shown in (D,E). (G) Schematic representation (top), average TEM images with corresponding model views (left), and gel electrophoretic analysis (right) of a 126-helix bundle (126hb) assembled with two interlocked scaffolds. Scale bar: 50 nm. (H) Overlay of a cryo-EM density map fragment and the corresponding scaffold routing diagram. Blue and orange paths indicate the two orthogonal scaffolds. Laser scanned fluorescent images of the electrophoretic analyses were autoleveled. P, pocket; sta, staples.

Figure 4

Self-cleaving DNA origami. (A) Schematic representations of circular scaffolds containing two self-excising DNAzyme cassettes (top left) that can be cleaved into two linear scaffolds (bottom left) or assembled into a switch object (top right). Individual switch arms (bottom right) can be obtained by cleavage of assembled switch objects or assembly using cleaved linear scaffolds. (B) Electrophoretic analysis of reaction kinetics of scaffold cleavage. Controls: cleaved scaffold (lane 1), undigested sample (lane 2), and switch arms assembled separately (lane 7) using cleaved scaffold. (C) Field-of-view TEM images of uncleaved (left) and cleaved (right) switch objects. (D) Electrophoretic analysis of cleavage reactions containing unpurified (lanes 1 and 5) and PEG-purified (lanes 2–4, 6–8) switch objects at 1.4, 4, 10, or 20 mM MgCl₂. Laser scanned fluorescent images of the electrophoretic analysis were autoleveled, and the highlighted region was autoleveled individually. P, pocket; U, undigested species; D, digested species. Scale bar: 100 nm.

Figure 5

UV point-welding of DNA origami with a custom scaffold. (A) Section of a multilayer DNA origami strand diagram with a customized scaffold featuring AA motifs every 8 base pairs, which results in adjacent Thymidines in separate staple strands that may be UV-cross-linked. Blue lines, scaffold strand; gray lines, staple strands. (B) Schematic representation (left) and average TEM images of the pointer object assembled with the welding scaffold. Average images of the pointer as obtained in the presence of 30 mM MgCl₂ before irradiation (I), after irradiation for 2 h at 310 nm (II) in the presence of 30 mM MgCl₂, and after irradiation for 2 h at 310 nm and 48 h long incubation in low ionic strength phosphate-buffered saline (PBS) at 40 °C (III). (C) Electrophoretic analysis of nonirradiated and irradiated pointer objects incubated over time in PBS at 40 °C. L, 1kB Ladder; NI, not irradiated; RT, room temperature; P, pocket; F, folded species; sta, staples. Scale bar: 50 nm.