Ken Sachenbacher,Ali Khoshouei,Maximilian Nicolas Honemann,Wouter Engelen,Elija Feigl,Hendrik Dietz
Triple-Stranded DNA As a Structural Element in DNA Origami
DNA origami,DNA triplex,Cryo EM,pH switching,nanodevices
Molecular self-assembly with DNA origami offers an attractive route to fabricate arbitrary three-dimensional nanostructures. In DNA origami, B-form double-helical DNA domains (dsDNA) are commonly linked with covalent phosphodiester strand crossovers to build up three-dimensional objects. To expand the palette of structural motifs in DNA origami, here we describe hybrid duplex–triplex DNA motifs as pH-dependent building blocks in DNA origami. We investigate design rules for incorporating triplex forming oligonucleotides and noncanonical duplex–triplex crossovers in multilayer DNA origami objects. We use single-particle cryoelectron microscopy to elucidate the structural basis of triplex domains and of duplex–triplex crossovers. We find that duplex–triplex crossovers can complement and fully replace the canonical duplex–duplex crossovers within DNA origami objects, for example, to increase the crossover density for potentially greater rigidity and reduced interhelical spacing, and to create connections at sites where conventional crossovers may be undesirable. We also show the pH-induced formation of a DNA origami object stabilized entirely by triplex-mediated strand crossovers.
Triple-Stranded DNA As a Structural Element in DNA Origami

Molecular self-assembly with DNA origami offers an attractive route to fabricate arbitrary three-dimensional nanostructures. In DNA origami, B-form double-helical DNA domains (dsDNA) are commonly linked with covalent phosphodiester strand crossovers to build up three-dimensional objects. To expand the palette of structural motifs in DNA origami, here we describe hybrid duplex–triplex DNA motifs as pH-dependent building blocks in DNA origami. We investigate design rules for incorporating triplex forming oligonucleotides and noncanonical duplex–triplex crossovers in multilayer DNA origami objects. We use single-particle cryoelectron microscopy to elucidate the structural basis of triplex domains and of duplex–triplex crossovers. We find that duplex–triplex crossovers can complement and fully replace the canonical duplex–duplex crossovers within DNA origami objects, for example, to increase the crossover density for potentially greater rigidity and reduced interhelical spacing, and to create connections at sites where conventional crossovers may be undesirable. We also show the pH-induced formation of a DNA origami object stabilized entirely by triplex-mediated strand crossovers.

Programmable self-assembly with DNA origami enables constructing nanoscale objects with arbitrary shapes. The main building blocks used in DNA origami include B-form double-helical DNA domains (dsDNA), DNA strand crossovers, and single-stranded DNA motifs. Besides B-form dsDNA, which forms through canonical Watson–Crick base-pairing, there exist a multitude of other noncanonical DNA motifs. One of these noncanonical motifs is triple-stranded DNA (tsDNA, or triplex DNA). tsDNA forms through parallel or antiparallel Hoogsteen base pairing of a third strand, termed triplex-forming oligonucleotide (TFO), to the major groove of a dsDNA domain. The thermodynamic stability of the triplex depends, among other factors, on the density of TAT or CGC triplets and differs from the thermodynamic stability of dsDNA. By decreasing the amount of TAT triplets contained in the triplex, the pH at which the triplex unfolds can be lowered. This is because the formation of a CGC triplet requires the protonation of N3 of the cytosine in the TFO, therefore CGC triplets only form at acidic pH. Since the stability of parallel Hoogsteen TAT and CGC triplet motifs in triplex DNA is pH sensitive, changes in pH may be utilized to switch between dsDNA and tsDNA secondary structures, for example, to control the conformation of DNA origami. In this work, we investigate potential uses of triplex DNA as a structural element in DNA origami. There are several avenues for how triplex DNA could potentially be utilized in DNA origami objects (Figure 1). For example, triplex motifs may be used to install noncanonical duplex–triplex crossovers, to locally reinforce the connectivity in a DNA origami object or to replace a conventional duplex–duplex crossover (Figure 1b). DNA origami objects with increased overall crossover density would be expected to be more rigid, whereas the conformations of objects or domains that are solely stabilized by triplex crossovers may by controlled by pH. To test for these possibilities, we investigated the formation of intermolecular parallel triplexes in DNA origami test cuboids, varying the sequence composition with different ratios of TAT and CGC triplets. The cuboids were multilayer DNA origami with 24 and 44 helices packed in a square lattice and were folded from 1032- and 2873-bases-long custom-sequence scaffold strands, respectively (Supplementary Figures 5, 6, 10, 11, 17, and 18). We used the smaller cuboid to investigate the binding of triplex forming oligonucleotides (TFOs) to dsDNA regions termed triplex target sites (TTSs) contained deep within the object. To enable read-out, we attached Cy3 dyes to the 3′ termini of one of the staple strands in the cuboid proximal to a TTS, and we attached Cy5 dyes to the 5′ ends of TFOs. We analyzed these samples using agarose electrophoretic mobility as a function of pH. At pH 5, we observed a band in the TFO-sensitive Cy5 channel that overlapped with the band reflecting folded DNA origami objects (Figure 2A). This band was absent at pH 8. We conclude that at pH 5 the TFOs successfully associated with the TTS. As a negative control, we used TFO sequences featuring a low percentage of both TAT and CGC triplets, which in turn leads to a low propensity for triplex formation. Here, we did not observe any signal in the Cy5-channel that overlapped with the DNA origami band seen in the Cy3 channel at any of the pH conditions tested, indicating that binding did not occur, as expected for these sequence motifs (Figure 2b). We used the larger cuboid to investigate duplex–triplex crossover formation (Figure 2d–g). To this end, we designed staple oligonucleotides with ssDNA extensions that can function as TFO domains. These TFO domains are designed to form triplex domains (again denoted as triplex target sites, TTS) with a sequence motif on a neighboring double helix. To form these triplexes, the molecules must thus create a noncanonical duplex–triplex crossover. We used fluorescence resonance energy transfer (FRET) dye pairs as reporters for the formation of these triplexes. To this end, we attached a Cy3 dye to the 5 prime end of one of the staples oligonucleotides in the neighboring helix featuring the TTS and a Cy5 dye was attached to the 5 prime end of the TFO-forming ssDNA extension, respectively, such that FRET can occur in case of successful formation of the triplex crossovers. To use FRET as a reporter for triplex formation in gel electrophoresis, we divided the intensity of the sample band in the channel with Cy3 excitation and Cy5 emission by the intensity of the Cy3 channel (Figure 2c). We expect that if FRET takes place, the intensity of the DNA origami band in the Cy3 channel will decrease because, within the object, energy is transferred to the Cy5 dye while the intensity of the channel with Cy3 excitation and Cy5 emission will increase. This leads to a higher ratio between the two intensities when triplex formation has taken place. Indeed, at pH 5, our gel electrophoresis experiments reflected this expected behavior for triplex ssDNA extension sequences with a high percentage of both TAT and CGC triplets (Figure 2d,e). We conclude that the duplex–triplex crossovers formed as designed. This was true for duplex–triplex crossovers designed proximal to sites with a pre-existing canonical duplex–duplex crossover (Figure 2e) but also for duplex–triplex crossovers at sites distal to canonical crossovers (Figure 2d). At pH 8, no triplex formation took place independent of sequence. We also investigated the formation of duplex–triplex crossovers by measuring FRET in solution (Figure 2f,g). Titration curves in the range from pH 5 to pH 8 show increasing FRET intensity for triplexes featuring a high percentage of both TAT and CGC triplets. Triplex ssDNA extensions having a low percentage of both TAT and CGC had constant, low FRET intensity under the pH conditions tested, indicating that these triplexes did not form. Both the duplex–triplex crossover proximal to a site with a pre-existing canonical duplex–duplex crossover as well as the duplex–triplex crossover at a site distal to canonical crossovers have a TAT percentage of 43%. The pKa values determined by our measurements are 6.67 ± 0.04 and 6.53 ± 0.05, respectively. These values agree with previous findings in which triplexes with a TAT percentage of 50% or less have a pKa of 6.5. We used single-particle cryoelectron microscopy to determine the structures of triplex domains and of triplex-mediated interhelical connections within multilayer DNA origami. We used the larger cuboid as one test object for these purposes. We chose sequence stretches with a high percentage of both TAT and CGC triplets and determined 3D cryo EM electron density maps with and without adding the TFO (Supplementary Figures 22–24, 27). The resulting maps had sufficient resolution to discern helical details of all helices in the cuboid. The triplex domains were clearly visible as extra density when tracking the major groove of the host duplex TTS (Figure 3a). This extra density was absent in the cryo EM structure determined for the control object lacking the TFO. Another site in the DNA origami object featured a designed triplex-mediated interhelical connection (Figure 3b, right). This triplex-mediated parallel strand crossover can be seen in the cryo EM map as a clear link between the two neighboring helices. The resolution was sufficient to also appreciate structural differences between the triplex-mediated crossover versus the conventional duplex–duplex crossovers in the control object that did not have the TFO (Figure 3b, left). For example, while the conventional crossover is in the same plane as the helical axis, the triplex-mediated crossover is localized out-of-plane (Figure 3b). This is because the triplex-mediated crossover results from the binding of the ssDNA extension from one of the helices reaching out over the major groove of the neighboring helix. We note that this triplex-mediated crossover also included two thymidines as spacers. To investigate the structural consequences of a high density of direct, spacer-free triplex-mediated crossovers in the context of a multilayer DNA origami object, we designed another object termed “rod”. The rod was designed to fold from a 1032-nt-long custom-sequence scaffold strand (Supplementary Figures 2, 8, 14, 21, and 27) and is comprised of 10 helices arranged in a honeycomb lattice (modified design from ref (22)). The object features 11 staple oligonucleotides with terminal ssDNA overhangs. These overhangs featured TFO sequences that can form triplex domains at pH 5 with sequence motifs located on neighboring helices. As a control, we also prepared a variant of the rod object with staples that lacked the TFO-forming extensions (Supplementary Figures 1, 7, 13, 20, and 27). Otherwise, the internal strand routing of both objects was identical. We experimentally determined 3D electron density maps of both objects using cryo EM single-particle analysis at pH 5, which is the condition where the TFO-mediated crossovers are expected to form (Figure 4a). The resulting 3D maps had sufficient resolution to discern helical details including groove signatures and crossovers practically throughout the entire object. Therefore, we were able to directly compare the structures of the triplex-mediated crossover regions with the same regions in the object’s version that lacked the TFO motifs. The structures of four distinct duplex–triplex crossovers as seen in the rod object are shown in Figure 4b–e, together with the same region as determined for the rod control lacking TFO motifs. Two additional duplex–triplex crossover regions are shown in Supplementary Figure 25. Regions in which triplex formation either did not take place or could not be resolved are shown in Supplementary Figure 26. Several of the triplex-mediated crossovers that we installed in the rod were located near conventional duplex–duplex staple crossovers (Figure 4b,d, and e). These triplex-mediated crossovers thus increase the overall crossover density. The TFO strands can be clearly discerned in the 3D map as extra density that tracks the major groove of the neighboring helix (Figure 4b–e). One of the triplex mediated crossovers was located at the helical periphery edge of the rod object, without other nearby staple crossovers (Figure 4c). Here, the two neighboring helices are being bent toward each other, which suggests that the triplex-mediated crossover can also bear some load. To further analyze the effect of a high density of duplex–triplex crossovers in DNA origami, we generated pseudoatomic models of the duplex regions of the origami object (Supplementary Figure 28). We used these models to determine the distances between the C1 (base-sugar connector) carbon atoms of staple bases of neighboring helices, at the location of duplex–triplex crossovers (reinforced rod) and the corresponding positions in the rod (in which these crossovers are missing). The analysis showed that the distances between C1 carbon atoms at duplex–triplex crossover locations either stayed the same or decreased relative to the corresponding distances in the control rod object lacking triplex crossovers. Triplex-mediated crossovers enable designing objects that can undergo pH induced folding. As a simple proof-of-concept demonstration, we designed a small rod-like-shaped DNA origami object that folds from a 1032-nt-long custom-sequence scaffold strand which we termed a pH-switching rod (Supplementary Figures 3, 9, 15, and 16). The object comprises 10 helices arranged in a honeycomb lattice. In the strand diagram of the pH-switching rod, we replaced all conventional duplex–duplex staple crossovers by duplex–triplex crossovers. The folding of the object then takes place in two steps. In the first step, the scaffold strand is mixed with the staple strands, and the folding mixture is then subjected to a thermal annealing ramp (Figure 5a). This leads to an object that can be considered as a double-helical ring that is pinched together at five sites by scaffold strand connections. There are also 26 single-stranded TFO overhangs with sequences that have a propensity to form triplexes. We designed these ssDNA TFO overhangs to bind to specific TTSs sequence motifs located at distal sites in the pinched ring object. In the second step, we acidify the reaction mixture, which leads to emergence of the actual rod-like shape of the object through formation of the designed triplex-mediated strand crossovers. We investigated the shape of the object after thermal annealing of the staples and after acidification by direct imaging using negative-staining TEM (Figure 5b). The TEM images show unstructured objects after thermal annealing which convert into rod-shaped particles after acidification. We also used FRET measurements to probe triplex formation exemplarily at one of the 26 triplex sites contained in the design. A titration curve from pH 5.2 to pH 7 showed that as pH decreases, the FRET intensity increased (Figure 5d), which indicates formation of the triplex crossover as described above. Investigating the FRET intensity of the same triplex across multiple cycles of folding/unfolding showed that the process is reversible when pH is elevated and is cyclical for at least three rounds of folding/unfolding. These findings are corroborated by images we acquired using negative-staining TEM for each cycle. TEM images show unstructured objects after pH is elevated and rod-like objects after acidification. To quantify the efficiency of folding after each acidification, we determined the fraction of folded vs unfolded particles per condition from TEM data (Supplementary Figure 19). The folding yield was in the range of 40% to 50% after each folding cycle. As a negative control, we designed a variant of the pH-switching rod (Supplementary Figure 4) featuring TFO sequences which would lead to unstable triplexes (by having low percentage of both TAT and CGC triplets). Agarose gel electrophoresis (Figure 5c) showed that at pH 8 the sample band containing the pH-switching rod, after the thermal annealing step, had lower electrophoretic mobility than the negative control with the weak TFO sequences. This may be due to the differences in the handle sequences that may mediate some additional internal compaction in the negative control sample. Importantly, at pH 5, the electrophoretic mobility of the pH-switching rod became greater than those of the control with weak TFO motifs. This finding suggests that the pH-switching rod folded into a compact conformation, whereas the negative control with the unstable TFO motifs remained in the less structured, pinched-plasmid conformation. Taken together, the TEM image data, the FRET measurements, and the gel-electrophoretic experiments all consistently show that the pH-folding/unfolding of the rod by triplex-mediated crossovers functioned as designed and that it is reversible. Furthermore, both the fraction of folded vs unfolded particles calculated from 2D classification as well as the FRET measurements indicate that the folding efficiency of the DNA origami objects did not deteriorate over three cycles of folding/unfolding. We also investigated the stability of the pH-switching rod and the rod in low-salt environments (Supplementary Figure 12). The rod DNA origami object with conventional crossovers was already quite insensitive to low ionic strength conditions, and the triplexes did not alter noticeably the stability in low-salt environments.

In conclusion, we successfully implemented triplex domains and additional triplex-mediated crossovers in DNA origami. We analyzed the formation of this duplex–triplex crossover via fluorescence and FRET measurements and determined structures through single-particle cryo-EM. Finally, we designed and studied the folding of a DNA origami object that can undergo pH induced folding mediated by triplex-crossovers. Triplex domains and triplex mediated crossovers can increase the mass density of DNA origami, reduce interhelical spacing, and potentially improve rigidity by having more bonds per unit volume. Triplex-mediated crossovers also create additional opportunities for DNA origami designers to fabricate dynamical structures whose conformations can be switched by pH. Objects are conceivable where segments can fold and unfold, as well as objects which can fold and unfold in stages, for example, by tuning the TAT percentage in the ssDNA handles functioning as TFOs. Our study provides proof-of-concept methods and experiments and validated design solutions that researchers can now adapt for their applications.

All origami objects were designed with the caDNAno software. Adobe Illustrator was used to highlight duplex–triplex crossover locations.

All folding mixtures were composed of a single stranded scaffold strand at 50 nM (see table below), staple strands at 200 nM (oligonucleotides purchased from IDT and biomers), buffer solution, and MgCl2. The folding buffer contained 5 mM TRIS, 1 mM EDTA, 5 mM NaCl, and 20 mM MgCl2 for the pH-switching rod, 25 mM MgCl2 for the rod, and 30 mM MgCl2 for the reinforced rod, small cuboid and large cuboid (1xFOB20, 1xFOB25, and 1xFOB30, respectively). The mixtures were subjected to thermal annealing ramps using a Tetrad thermal cycler by Bio Rad. All thermal annealing ramps first heat up to 65 °C for 15 min in order to ensure that all potential secondary structures, that the scaffold or staple strands could have formed, are unfolded. This is followed by thermal ramps which vary between objects. Rod, reinforced rod, pH-switching rod, and small cuboid: 60 to 44 °C for 1 h/1 °C. Large cuboid with TFO and large cuboid with duplex–triplex crossover: 56 to 53 °C for 5 h/1 °C. Large cuboid: 56 to 53 °C for 10 h/1 °C. Afterward, all folding mixtures were kept at 20 °C until the samples were used for experiments. Optimal folding conditions were acquired through initial folding screens.

Dialysis tubes (D - Tube Dialyzer Mini, with a molecular weight cutoff (MWCO) of 12–14 kDa) were used to fold/unfold the pH-switching rod using a pH gradient. For the folding step, the dialysis tube was first filled with 200 μL of 1xFOB5 buffer solution and then placed inside a beaker filled with 500 mL of 1xBis-Tris8 buffer (5 mM Bis-Tris, 1 mM EDTA, 5 mM NaCl, and 8 mM MgCl2) with 42 mM MgAC at pH 7.1, standing on a magnetic stirrer. The dialysis tube is stirred for 6 min, after which the buffer is pipetted out and 200 μL of sample are pipetted into the tube. The beaker of 1xBis-Tris8 buffer at pH 7.1 is connected to a beaker filled with 1500 mL of 1xMES8 buffer (5 mM MES, 5 mM NaCl, and 8 mM MgCl2) with 42 mM MgAC at pH 5.1 through a peristaltic pump from Ismatec, which pumps buffer from the low pH buffer reservoir to the high pH buffer reservoir at a constant rate of 1 mL/min (6 mL/min for the titration curve). For the unfolding step, the dialysis tube was then placed inside a beaker filled with 500 mL of 1xMES8 buffer (5 mM MES, 5 mM NaCl, and 8 mM MgCl2) with 42 mM MgAC at pH 5.5, standing on a magnetic stirrer. The beaker of 1xMES8 buffer at pH 5.5 is again connected to a beaker filled with 1500 mL of 0.05xTBE8 buffer (5 mM Tris-Borat, 0.1 mM EDTA, and 8 mM MgCl2) with 42 mM MgAC at pH 8.6 through the peristaltic pump, which pumps buffer from the high pH buffer reservoir to the low pH buffer reservoir at a constant rate of 1 mL/min. For the titration curve, every ΔpH = 0.2, 10 μL of sample at 25 nM was pipetted out and diluted with 40 μL of buffer solution at the same pH as the sample (these samples at ΔpH = 0.2 increments were analyzed as described in Fluorescence and FRET Measurements). For the reversibility/cyclicality experiments, after every folding/unfolding step, 10 μL of sample at 25 nM was pipetted out and diluted with 40 μL of buffer solution at the same pH as the sample (these samples were analyzed as described in Fluorescence and FRET Measurements). Furthermore, 5 μL of sample at 25 nM was pipetted out for TEM analysis.

For Cryo-EM sample preparation (except for the rod object), PEG (polyethylene glycol) purification was used. Here, the sample is concentrated, and the excess staples are removed from the solution. The PEG solution contained 30% of PEG 8000 (15% for the large cuboid), 5 mM Tris, 1 mM EDTA, and 500 mM NaCl. A total of 800 μL of PEG was mixed with 800 μL of sample (reinforced rod sample: MgCl2 conc. of 30 mM; large cuboid, large cuboid with TFO, and large cuboid with duplex–triplex crossover: MgCl2 conc. Of 100 mM) and centrifuged inside of a microcentrifuge for 25 min at 12–20 °C and 20.000–21.000 rcf. Afterward, the supernatant is discarded, and the pellet is diluted in 100 μL of 1xMES5 buffer at pH 5 (large cuboid with duplex–triplex crossover and reinforced rod) or folding buffer 1xFOB5 (large cuboid and large cuboid with TFO). The solution is then placed in a lab shaker and equilibrated for 3 h at 30 °C and 450 rcf and equilibrated overnight at 20 °C and 450 rcf (reinforced rod) or just equilibrated overnight at 30 °C and 450 rcf for all other objects. After PEG purification, the 1xFOB5 buffer of the large cuboid with the TFO sample was exchanged to 1xMES5 buffer at pH 5 using Amicon Ultra 0.5 mL Ultracel filters with a molecular weight cutoff (MWCO) of 50 kDa (ultrafiltration). Here, the filter was washed for 10 min at 10 k rcf; then the sample was concentrated in three centrifugation steps (each 10 min at 10 k rcf). The sample was then brought to a concentration of 500 nM and a MgCl2 conc. of 5.5 mM. Then, the sample was incubated together with the TFO overnight, before it was further concentrated using ultrafiltration as described above. All other samples were also further concentrated after PEG purification, via ultrafiltration (large cuboid, large cuboid with TFO, large cuboid with duplex–triplex crossover, MWCO of 50 kDa, or reinforced rod, 30 kDa). The rod object was not PEG purified. Instead, it was concentrated, and the excess staples were removed from the solution via ultrafiltration using large (15 mL) filters and then further concentrated using ultrafiltration as all other cryo-EM samples described above. For TFO binding experiments (small cuboid), ultrafiltration (MWCO of 30 kDa and centrifugation steps of 15 min) was used for buffer exchange (1xFOB5 buffer at pH 8 to 1xMES5 buffer at pH 5) and concentration. The sample was then brought to a concentration of 50 nM and a MgCl2 concentration of 5–40 mM. Then, the sample was incubated together with the TFO (in a 1:10 ratio) for 3 h, before being pipetted onto the gel. For FRET analysis via gel electrophoresis of the large cuboid with duplex–triplex crossover, ultrafiltration (MWCO of 50 kDa and centrifugation steps of 10 min) was used for buffer exchange (1xFOB5 buffer at pH 8 to 1xMES5 buffer at pH 5) and concentration. The sample was then brought to a concentration of 20 nM, before being pipetted onto the gel. For FRET analysis via plate reader of the large cuboid with duplex–triplex crossover, gel purification and ultrafiltration (MWCO of 50 kDa and centrifugation steps of 10 min) was used. Afterward, the sample was then brought to a concentration of 100 nM and a MgCl2 concentration of 5 mM. A total of 2.5 μL of sample was then added to 47.5 μL of buffers (1xMES5 with 0.5 M of NaCl, 1xBis-Tris5 with 0.5 M of NaCl,and 1xTris5 with 0.5 M of NaCl) at varying pH values. The sample was then brought to 20 mM MgCl2, leading to a final sample concentration of 5 nM and 20 mM MgCl2. For TEM imaging, FRET analysis via plate reader and gel analysis of the pH-switching rod, ultrafiltration (MWCO of 10 kDa and centrifugation steps for 15 min) was used to concentrate the sample (except for TEM images of the unfolded pH-switching rod, here the sample was PEG and filter purified).

The folding quality under different folding conditions, as well as triplex formation, was investigated using agarose gel electrophoresis. With an agarose percentage of 2% and an MgCl2 concentration between 5 and 20 mM. Samples were loaded onto the gel at a concentration of 25–50 nM. Gels were stained with ethidium bromide (EtBr) if no fluorescent dyes were attached to any of the TFOs or origami objects. If dyes were attached, the gels were post stained in an EtBr bath. For gels at low pH (run time: 60–75 min, voltage: 70–90 V, and pH 5.0–5.1), the gels were placed in a water bath and cooled with ice, and the buffer was exchanged every 15 min or cooled using an hc series chiller, hc 130a by HAILEA, and the buffer was circulated by an EHEIM universal pump. For gels at high pH (run time, 90 min; voltage, 90 V; and pH 8.4) the gels were placed in a water bath and cooled with ice (for MgCl2 concentrations above 5.5 mM). The loading dye contained 0.1% bromphenol blue, 15% Ficoll 400, and for gels at high pH, 5 mM Tris-HCl, and for gels at low pH, 5 mM MES buffer. After gel electrophoresis, gels were scanned with a Typhoon 9500 FLA laser scanner by GE Healthcare at a resolution of 50 μm/pixel. The acquired images were then processed with Adobe Illustrator.

Fluorescence measurements were carried out to validate the binding of triplex forming oligonucleotides (TFOs) containing cyanine 5 (Cy5) dyes to dsDNA regions (in the DNA origami object) containing cyanine 3 (Cy3) dyes. These dsDNA regions are called triplex target sites (TTSs). Förster resonance energy transfer (FRET) measurements were done to validate triplex formation and analyze at which pH the formation takes place. Cy3 and Cy5 dyes were used as donor–acceptor pairs. For this, a TTS in one dsDNA helix of the origami was modified with a Cy3 dye, and an ssDNA extension (functioning as a TFO) in the neighboring helix was modified with a Cy5 dye. FRET experiments were carried out through gel electrophoresis and using a plate reader. In the gel electrophoresis experiments, the gels were scanned with a Typhoon 9500 FLA laser scanner in the Cy3, Cy5, and FRET (Cy3–Cy5) channel. For the second set of FRET experiments, a CLARIOstar Plus plate reader by BMG LABTECH was used. Samples were pipetted into a BRAND 384 well plate with a transparent bottom and then placed into the CLARIOstar Plus plate reader. Subsequently, FRET was then measured by exciting Cy3 at a wavelength of 510 nm and then measuring emission in the range of 540–700 nm.

Custom sequence scaffolds were prepared as described previously. The scaffold strands were purified from double transformed E. coli cells and stored in 1xTE (10 mM Tris 1 mM EDTA pH 8.0) at a concentration of 300 nM for the 2873 or 600 nM for the 1033 variant. The 1033 base scaffold was produced by Zn2+ digestion of a precursor ssDNA that contained four identical copies, separated by the DNAzyme cassettes described in ref (29). The 1032 base scaffold was produced by Zn2+ digestion of a circular ssDNA precursor containing one copy of the scaffold flanked by two DNAzymes and a small backbone that contained the packaging signal and the f1 Ori. The Plasmid map for the 1032 base scaffold (Supplementary Figure 29), ssDNA sequences of all scaffolds, and the restraints file for the 1032 base scaffold (Supplementary Notes) can be found in the Supporting Information.

A De Bruijn sequence of order (n = 2, k = 15) was used as the basis for the creation of triplex motifs, with subsequences with five or more repetitive bases being excluded. The Scaffold Smith software, published in ref (20), was used for the construction of the scaffold sequence. The restraints file can be found in the Supporting Information. A “Pseudo-DB” order 7 with a maximum of five duplicates was chosen with a base step weight of 1 for all combinations. The resulting sequence was ordered from Genewiz and cloned into a scaffold production backbone. The resulting plasmid was used for scaffold production as described above.

FCF400-CU carbon coated copper grids by Electron Microscopy Sciences were first glow discharged (45 s and 35 mA) using an EMS K100X plasma cleaner by Electron Microscopy Sciences. Five microliters of DNA origami sample was then pipetted onto the grid, incubated for 30 s to 2 min, and then blotted away using filter paper. Then, 5 μL of 2% aqueous uranyl formate (UFO) solution containing 25 mM of NaOH was pipetted onto the grid and immediately absorbed using filter paper. Afterward, 20 μL of UFO solution was applied onto the grid, incubated for 30–40 s, and once again removed by filter paper. Before using the UFO solution for negative stain TEM, it was vortexed for a few seconds and then centrifuged for 2.5 min at 20 k rcf. For the 2D classes of the pH-switching rod, the UFO solution did not contain 25 mM NaOH. Afterward, the grids were air-dried for 5 min or blow-dried with a hair dryer for 30 s if the humidity was too high. Two TEMs were used for direct imaging of the origami objects on the grids: the CM-100 (Phillips) with an acceleration voltage of 100 kV and integrated electron detector AMT 4 Megapixel CCD and the FEI Technai-120 with an acceleration voltage of 120 kV and integrated electron detector TemCam-F416 (4 k × 4 k). For acquiring 2D class averages, the FEI Technai-120 was used. Imaging software used were AMT 600 and SerialEM for the CM-100 and Tecnai-120, respectively. The Relion software was used for 2D classification.

As in negative stain TEM, all cryo-EM grids were first glow discharged (90 s and 50 mA) using an EMS K100X plasma cleaner from Electron Microscopy Sciences. Different grids were used for different origami samples (large cuboid with duplex–triplex crossover and large cuboid with TFO: Quantifoil grids, 1.2/1.3 300 mesh; large cuboid, C-flat grids; CF-1.2/1.3–2Cu; rod and reinforced rod, C-flat grids, CF-1.2/1.3–4Cu). After glow discharging, 5 μL of origami sample at 5 mM MgCl2 concentration was applied to the grid inside of a FEI Vitrobot Mark IV (large cuboid: blot time, 3 s; blot force, −1 and 20 °C; large cuboid with TFO: blot time, 3 s; blot force, 25 and 4 °C; large cuboid with duplex–triplex crossover: blot time, 1 s; blot force, −1 and 4 °C; rod and reinforced rod: blot time, 3 s; blot force, 15 and 4 °C; for all: drain time, 0 s and 100% humidity). The grid was then plunge-frozen in liquid ethane and subsequently stored in liquid nitrogen. A Titan Krios G2 (FEI), with an acceleration voltage of 300 kV and integrated electron detector (Falcon III 4k direct electron detector), was used for all Cryo-EM imaging. Further details on Cryo-EM grid preparation and imaging conditions can be found in the Supporting Information (Supplementary Table 1).

First, the micrographs acquired during cryo-EM were imported into the Relion software, which was used for data processing. The 2D micrograph movies were then motion corrected using MotionCor2 and contrast transfer function estimated using CTFFIND4. Particles were then picked from the micrographs using Cryolo. The picked particles were then extracted from the micrographs. This was followed by reference-free 2D classification. The best 2D classes were selected by eye and used for further data processing. An initial model is then created using the particles from the selected 2D classes or a model is imported. This is followed by one to two (rod) rounds of 3D classification and again selection through visual inspection, of the best 3D classes. Then, the particles from the selected 3D classes were subjected to 3D autorefinement, followed by postprocessing. Afterward, the particles were polished and another round of 3D-auto refinement as well as postprocessing was done. For the large cuboid and the large cuboid with duplex–triplex crossover, Relion multibody refinement was carried out. The 3D reconstruction was split into parts using the eraser tool in UCSF Chimera.

Pseudoatomic models were generated using the “dnaFit” model construction package. During its atomic level “shrink-wrap” procedure, the electron density of the cryo-EM data biases a structural model generated with ENRG-MD toward a position of best overall alignment. All pseudoatomic models are based on the same topology to ensure comparability of their topologies; triplex strands are not included. To quantify the helical structure, a customized version of the “FitViewer” Tool,https://github.com/elija-feigl/FitViewer, was used to select bases in accordance with the cadnano design diagram. The positional information on individual atom sites on the pseudoatomic model was used to measure atom–atom distances and compute the helical traces of the structures. Helical traces were generated using the helical midpoints as defined by the standard reference frame for nucleic acids.