Isothermal assembly of DNA nanostructures

Abstract

DNA nanostructures are typically assembled using a thermal annealing protocol by heating the DNA mixture to high temperatures and then cooling it down to a lower temperature. Recent efforts have shown the assembly of DNA nanostructures by incubation at constant temperatures in a process called isothermal assembly. DNA motifs, polyhedra, lattices, and other nanostructures based on single-stranded tiles and the DNA origami strategy have all been constructed using the isothermal assembly process. Several additives such as denaturing agents, cationic amino acids, and natural products aid in the isothermal process at room temperature and physiological temperature. This review focusses on the developments in isothermal assembly of DNA nanostructures, key takeaways from recent studies, and the advantages and limitations of isothermal assembly in the broader context of DNA nanotechnology.

Graphical Abstract

DNA nanostructures can be assembled by incubation at constant temperatures in a process called isothermal assembly. Several additives such as denaturing agents, cationic amino acids and natural products aid in the isothermal process at room temperature and physiological temperature.

1. Introduction

The programmability of DNA has allowed the construction of nanometer to micrometer-scale structures.¹ Some key features that make DNA a useful nanoscale construction material are its inherent nanoscale dimensions and its persistence length of ~50 nm (150 base pairs). Branched DNA junctions with multiple helical arms, in combination with sticky end cohesion, further allow the creation of larger objects and lattices.² Nanostructures built using DNA include multi-stranded, multi-helical DNA motifs,^3,4 objects such as polyhedra,^5,6 and one-, two-, and three-dimensional arrays.^7–9 Larger structures have been constructed using the DNA origami method, where a single-stranded DNA scaffold is folded into arbitrary shapes using short complementary staple strands.¹⁰ In other methods, multiple single-stranded tiles with specific recognition domains can be designed to associate to form 2D and 3D brick-like structures.^11,12 DNA nanostructures can also be dynamic, where they respond to external stimuli and reconfigure into different conformations. Some examples include DNA nanostructures that undergo structural transformation in response to other nucleic acids, enzymes, pH, temperature, light, and aptamer–antigen interactions.^13,14 These custom-designed nanostructures can be further functionalized with guest molecules or stimuli-responsive linkers for applications in diagnostics,^15,16 structural biology,^17,18 data storage,^19,20 enhancing catalytic reactions^21,22 and drug delivery (Fig. 1).^23,24

Fig. 1.

DNA nanotechnology. (a) Features of DNA useful in nanoscale construction. (b) Examples of DNA nanostructures. (c) Stimuli-responsive DNA nanostructures. (d) Applications of DNA nanostructures. Image adapted from ref. 25 with permission. Copyright 2021, Springer Nature.

2. Thermal annealing and isothermal assembly

Historically, the design and construction of DNA nanostructures involved the optimization of specific assembly protocols. Primary among these was the necessity to denature any secondary structures associated with the component strands of a DNA nanostructure, and then to provide suitable assembly temperatures for the strands to hybridize to each other according to the design. The “traditional” route to assembling DNA nanostructures typically involves a thermal annealing protocol in which the DNA strands are heated in a specific buffer to a high temperature (90–95 °C) and cooled slowly at specific rates to avoid kinetic traps and ensure proper sequence-specific DNA hybridization.^26,27 For basic DNA motifs, such as branched junctions and small multi-stranded motifs, the hybridization process is typically achieved by an ~2-hour-long thermal annealing protocol.^28–30 For larger structures involving multi-helical domains, this protocol has been optimized to range from a few hours to 2 days.^3,31 In the DNA origami strategy, the complexity of the structures dictated the annealing temperatures and times, with flat, 2D structures well-formed by annealing for 1–2 h, while more complex 3D shapes have required very precisely controlled temperature gradients over multiple days.^10,32,33 While thermal annealing is a well-established method, assembly at constant moderate temperatures (isothermal assembly) is an alternate strategy for DNA nanostructure construction (Fig. 2). There are a few contexts in which isothermal assembly could prove advantageous over thermal annealing: (1) compatibility with different counter ions and solution conditions, (2) prevention of DNA degradation at high temperatures, (3) analysis of DNA folding processes, and (4) encapsulation of temperature-sensitive guest molecules.

Fig. 2.

Assembly of DNA nanostructures. The thermal annealing protocol involves heating of DNA strands to high temperatures, followed by cooling down to lower temperatures. In the isothermal strategy, component DNA strands are incubated at constant moderate temperatures.

Recent studies have explored the assembly of DNA nanostructures in counter ions other than magnesium. The use of other counter ions instead of, or in combination with, Mg²⁺ is useful to enhance the biostability of DNA nanostructures,²⁹ increase assembly yields,³⁴ and control the optical properties of DNA-nanoparticle complexes.³⁵ In our own work, we showed successful assembly of DNA motifs and origami triangles in Ca²⁺, Ba²⁺, Na⁺, K⁺, and Li⁺ using a thermal annealing protocol. However, some ions are not compatible with high temperatures. For example, thermal annealing from high temperatures affected the assembly of DNA nanostructures in Ni²⁺, possibly due to degradation or truncation of the DNA at high temperatures.^36,37 In contrast, incubation of the DNA strands in Ni²⁺-containing buffer isothermally at temperatures <45 °C showed successful assembly, indicating that the use of moderate constant temperatures provides better assembly in these ions. Relatedly, temperature-dependent depurination and strand cleavage of single-stranded DNA might occur during the thermal annealing process,³⁸ resulting in lower yields of the structures, necessitating assembly at lower temperatures.

In DNA nanostructure assembly, the annealing method is optimized based on the folding efficiency and the highest assembly yield. However, the high temperatures used to denature the component DNA strands can also be an impediment to using DNA nanostructures as scaffolds to host temperature-sensitive guest molecules. In such cases, the annealing protocols have been amenable to changes for the incorporation of guest molecules, where the DNA nanostructure assembly is started at a high temperature and the guests (e.g.: proteins or peptides) are added at an intermediate temperature, followed by cooling down to a lower temperature. In other cases, the DNA nanostructure is first thermally annealed, and the temperature-sensitive guests are attached later at a lower temperature, resulting in a functionalized construct.^15,39,40 Assembly of DNA nanostructures at moderate constant temperatures allows the creation of guest-functionalized nanostructures in one step, as shown in recent examples of DNA origami structures hosting proteins.^41,42 Another aspect of isothermal assembly is the ease of monitoring of the assembly process.^43,44 Several studies have looked at the details of the assembly process and the kinetics of the folding, but it is challenging to monitor the interactions under a temperature gradient. Many techniques used to study DNA folding processes are not compatible with thermal annealing protocols. This includes bulk methods such as isothermal titration calorimetry (ITC) and single-molecule methods such as optical and magnetic tweezers. Studies into optimal isothermal assembly of different types of DNA nanostructures and motifs will allow us to use these techniques to understand the equilibrium assembly mechanisms underlying the formation of the structures.

Some of the parameters involved in isothermal assembly are similar to thermal annealing. For example, in some isothermal methods, the initial denaturation of component strands has been provided by a heat shock before incubation at constant temperatures^45,46 or the addition of a denaturing agent.^42,47 Similar to how thermal annealing protocols vary from a few hours to days based on the complexity of the nanostructures, assembly at constant temperatures, especially for complex DNA origami structures, requires several days for assembly at 25 °C.⁴¹ There has been considerable effort recently to achieve isothermal assembly of DNA nanostructures, specifically at room temperature or physiological temperature. These strategies involve specific design modifications, solution supplements, and optimization of temperatures and times depending on the structural complexity of the DNA nanostructure being assembled (Table 1). Collectively, these studies provide a resource for choosing ideal assembly conditions that are both suited for the type of DNA nanostructure as well as the application they are meant for.

Table 1.

Examples of DNA nanostructures assembled isothermally

Structure	Assembly temperature	Additives	Ref.
DNA motifs and arrays
DNA nanoprism	37 °C	Spermidine	48
DNA nanotube	22 °C, 37 °C, or 45 °C	Arginine, lysine	49
4-way junction	4 °C, 20 °C, 37 °C, or 50 °C	Ca2+	50
Double crossover motifs	4 °C, 20 °C, 37 °C, or 50 °C	Ca2+, Mg2+, Sr2+, Ni2+, Na+, K+, Li+	50
3-helix motif	4 °C, 20 °C, 37 °C, or 50 °C	Ca2+	50
4-helix motif	4 °C, 20 °C, 37 °C, or 50 °C	Ca2+	50
DNA and RNA cubes	37 °C	Mg2+	51
Square lattice	25 °C	Na+	41
3D DNA tensegrity triangle crystals	50 °C	Ca2+	50
Anti-junction array	51.3 °C	Mg2+	52
Structure based on single stranded tiles
2D rectangular structures	15–69 °C	Mg2+, PEG-8000	53
Rectangular brick	25 °C	Na+	41
gPNA nanofibers	45–70 °C	75% DMSO	54
DNA nanotubes	21 °C	6 M urea	55
2D shapes	20–23 °C	30% formamide	42
DNA origami
Plate	55 °C	Mg2+	45
Rectangle	50 °C	Mg2+	45
Gear	49 °C	Mg2+	45
Triangle	25 °C	Na+	41
Rectangle	25 °C	Na+	41
Smiley face	25 °C	Na+	41
Toblerone bar	25 °C	Na+	41
Rectangle	Room temperature	Formamide	47
Rectangle	Room temperature	Urea	47
Nanotube	Room temperature	Formamide	47
Triangle	20–23 °C	Formamide	42
Rectangle	37 °C	Betaine	56
Rectangle	37 °C	Arginine	49
Rectangle	20 °C	Glycholine	57

3. Examples of isothermally assembled DNA nanostructures

3.1. DNA objects and lattices

DNA polyhedra are some of the most used nanostructures in the field of DNA nanotechnology.^58,59 There are several designs for such constructions including one-pot assembly and hierarchical assembly of DNA polyhedra such as a DNA tetrahedron.^31,60 As an example of isothermal assembly, Qian and coworkers constructed a DNA nanoprism in the presence of spermidine (Fig. 3a).⁴⁸ Spermidine is a naturally abundant polyamine present in ribosomal fractions and living tissues.⁶¹ The three positive charges in spermidine help bring the negatively charged DNA together efficiently. They designed two triangular structures with sticky ends on the vertices that connect together to form a prism with edge lengths of ~7 nm. They successfully assembled the nanoprism isothermally at 37 °C in solutions containing 100 μM spermidine. At higher temperatures (45 °C), the DNA nanoprisms were partially disassociated to DNA triangles, whereas at lower temperatures (4 and 22 °C), the assembled products were either single-stranded component DNA oligonucleotides or aggregates. Isothermal assembly of DNA nanoprisms was unaffected by changes in pH, with similar assembly yields in solutions of pH 6 to 10. Compared to conventional Mg²⁺-assembled DNA nanostructures, the spermidine-DNA nanoprism complex showed higher nuclease resistance, with 40% of the structures remaining intact in 10% fetal bovine serum over 48 h, while the structures assembled in Mg²⁺ were fully degraded. The higher nuclease resistance may be due to the multivalent DNA-spermidine interaction that partially inhibits DNase activity toward the DNA nanostructure. Towards biological applications, the spermidine-assembled structures were shown to have a higher cellular uptake efficacy in multiple cancerous cell lines and were used as a drug delivery system for mTOR siRNA as a lung cancer therapeutic. The same group used cationic amino acids for isothermal assembly of DNA nanotubes (Fig. 3b).⁴⁹ DNA nanotubes of length 13.8 nm and diameter of 6 nm were assembled from three DNA strands by incubation with arginine at 4 °C, 22 °C, 37 °C, or 45 °C for 3 h. The yield of DNA nanotubes at 4 °C was relatively lower than that at other temperatures, probably due to the slow kinetics at lower temperatures. Further, DNA nanotubes could be synthesized with high yields from pH 6 to 8. They also demonstrated lysine-induced DNA nanotube assembly at constant temperatures of 22 °C, 37 °C and 45 °C. The wide pH range and isothermal self-assembly ability suggest that assembly supplements such as spermidine and arginine are useful additives for robust assembly of DNA nanostructures.

Fig. 3.

DNA motifs and objects assembled at constant temperatures. (a) A DNA nanoprism assembled in the presence of spermidine.⁴⁸ (b) A DNA tube assembled in the presence of cationic amino acids such as arginine.⁴⁹ (c) DNA motifs isothermally assembled in different counter ions.⁵⁰ (d) DNA and RNA cubes assembled by enzymatic treatment of DNA/RNA hybrid duplexes.⁵¹ (e) Assembly of 2D nanogrid from DNA four-point-star motifs in Na⁺-containing buffer. AFM image reproduced from ref. 41 (open access). Copyright 2023, Springer Nature. (f) Isothermal assembly of the DNA tensegrity triangle motif in Ca²⁺ and further self-assembly into 3D DNA crystals. Image adapted from ref. 50 (open access). Copyright 2025, The American Association for the Advancement of Science. (g) DNA arrays isothermally assembled in a Mg²⁺-containing buffer.⁵² The representation of the antijunctions is simplified to show assembly from two strands. The actual assembly involves multiple component strands. AFM image reproduced from ref. 52 with permission. Copyright 2017, The American Association for the Advancement of Science.

The effect of different counter ions on the self-assembly of DNA nanostructures has also been studied.^29,62 In our recent work, we tested the assembly of a double crossover (DX) DNA motif in counter ions such as Li⁺, Na⁺, K⁺, Mg²⁺, Ca²⁺, Sr²⁺, Ni²⁺, Cu²⁺, Ag⁺, Zn²⁺, and Pb²⁺ at constant assembly temperatures of 4 °C, 20 °C, 37 °C and 50 °C (Fig. 3c).⁵⁰ Assembly was successful in Na⁺, Li⁺, K⁺, Ca²⁺, Mg²⁺, and Sr²⁺, with higher assembly yields at higher temperatures and in higher ion concentrations in most cases. For monovalent ions, the highest assembly yields were observed at 10 mM or 25 mM ion concentration at 37 °C. Ions such as Pb²⁺, Zn²⁺, Cu²⁺, and Ag⁺ showed degradation or aggregation of the DNA strands or structure. These ions are known to destabilize DNA duplexes⁶³ and cause DNA cleavage,^36,64 and have been known to affect the assembly of DNA nanostructures when using a thermal annealing process.²⁹ One advantage of the isothermal assembly process was the successful assembly of the DX motif in Ni²⁺ at temperatures below 45 °C, otherwise unattainable using a thermal annealing protocol. We also showed successful isothermal assembly of a 4-arm DNA junction, a 3-helix motif, and a 4-helix motif in solutions containing Ca²⁺.

In addition to DNA motifs, other types of DNA nanostructures have also been assembled isothermally. Jerala and colleagues designed DNA knots that folded efficiently on slow annealing, but some designs also folded on rapid temperature quenching or dilution from a chemical denaturant.⁶⁵ For a folding-optimized design of a single-chain DNA square pyramid, assembly yields were similar for slow annealing and rapid quenching of the denatured DNA mixture on ice or liquid nitrogen. This method, however, was not efficient for a multi-stranded DNA square pyramid, indicating the advantage of using single-chain designs for DNA knots for rapid folding compared to multi-stranded designs. In another strategy, the DNA mixture was initially denatured in 8 M urea or 70% formamide, followed by dilution into 1 × TAE-Mg²⁺. Again, assembly yields were similar for the slow annealed and quenched samples for the optimized DNA tetrahedra design. However, the experimental procedure in this study first annealed the structure using slow anneal, denatured it by exposing to 95 °C for 5 min, followed by temperature quenching. Thus, it’s unclear whether DNA strands can be taken as is, followed by temperature quenching for proper folding of the DNA knots.

Afonin and coworkers developed an enzyme-triggered isothermal assembly process to create a cube structure (Fig. 3d).⁵¹ They started with RNA/DNA hybrid duplexes with the individual set of DNA and RNA component strands corresponding to DNA and RNA cubes respectively. On the addition of DNase I, the DNA strands in the hybrid duplexes are digested, leaving only the RNA component strands that then isothermally assemble at 37 °C to form an RNA cube. On the other hand, addition of RNase H selectively degrades only the RNA strands in the DNA/RNA hybrid duplexes, leaving the DNA strands to isothermally assemble into DNA nanocubes. The DNA cubes were formed minutes after RNase H addition, whereas the RNA cubes formed only after 30 min after DNase addition. Further, DNA cube formation by RNase H addition worked well at 37 °C or 4 °C, whereas DNase-triggered RNA cube formation was only successful at 37 °C. Structures created by this nuclease-driven process retained their immunostimulatory functionality similar to those assembled by the traditional annealing process.

Larger arrays composed of DNA motifs have also been created isothermally. The selection of studies discussed here has used isothermally assembled starting units or assembled entire arrays isothermally. This is in contrast to existing studies where pre-annealed DNA motifs can be used for hierarchical assembly of arrays and higher-order structures at constant temperatures.^5,66 Franco and coworkers created DNA nanotubes based on DX motifs.⁶⁷ The component strands were isothermally assembled into DX motifs in Na⁺-containing buffer and eventually into nanotubes that were tens of micrometers in length. Baigl and colleagues assembled a self-repeating square lattice using 4-point-star motifs that are composed of nine DNA strands (Fig. 3e).⁴¹ They demonstrated successful assembly of the lattice at 25 °C (with a 24 h incubation time) by replacing Mg²⁺ with Na⁺ in the buffer. 2D lattices with extended dimensions were successfully obtained in buffer containing 100–150 mM NaCl. In our recent work, we isothermally assembled tensegrity triangle DNA motifs in Ca²⁺-containing buffers at 50 °C and used them for creating 3D DNA crystals (Fig. 3f).⁵⁰ 3D crystals obtained from isothermally assembled motifs had a rhombohedral crystal habit similar to the crystals produced using a thermal annealing protocol⁹ or those assembled by incubating thermally annealed tensegrity triangle motifs at room temperature for crystallization.^9,68 Another example of an array created isothermally is a 42-bp DNA relay array based on anti-junctions developed by Ke and coworkers (Fig. 3g).⁵² The array contained 11 units by 4 units, and the optimal assembly temperature for the best yield was 51.3 °C in a Mg²⁺-containing buffer. They improved the assembly yields by modifying the design of the motifs to contain single-stranded poly-T extensions around the boundary of the array, a strategy that also reduced aggregation in the assembly process.

3.2. DNA bricks assembled from single-stranded tiles

In addition to finite DNA objects and infinite arrays, DNA nanostructures have also been constructed isothermally using single-stranded tiles (SSTs). SSTs were first reported by Yin and coworkers for building 2D and 3D micrometer-scale structures.^11,12 Each motif is a 42-base oligonucleotide composed of 4 consecutive modular domains. Complementary domains from two strands bind to form a tile. The tiles connect to each other via single-stranded overhangs to form a larger 2D or 3D brick array. They further showed two types of assemblies. The motifs without linkers form 2D rectangles composed of 10 parallel helices (with lengths ranging from 108 to 252 nt) connected by periodic single-stranded crossovers. Motifs with poly-T linkers form flexible rectangular, fish-net patterns containing short segments of DNA helices connected by single-stranded linkers at all junctions (Fig. 4a).

Fig. 4.

Single-stranded tile-based DNA nanostructures assembled isothermally. (a) Single stranded DNA tiles with and without poly-T linkers used to create 2D arrays. AFM image reproduced from ref. 53 with permission. Copyright 2013, American Chemical Society. (b) Assembly of 2D arrays in Na⁺ containing buffer.⁴¹ AFM image reproduced from ref. 41 (open access). Copyright 2023, Springer Nature. (c) Assembly of 9-helix structure in glycholine. AFM image reproduced from ref. 57 with permission. Copyright 2015, John Wiley and Sons. (d) Assembly of 2D arrays at room temperature using 30% formamide. AFM image reproduced from ref. 42 with permission. Copyright 2013, John Wiley and Sons.

Yin and colleagues extended their work to create the DNA bricks from SSTs isothermally.⁵³ They used U-shaped motifs⁶⁹ to form 2D rectangular structures and modified the domain length of the SSTs from 8 to 21 nt to achieve isothermal assembly from 15–69 °C over a 12 h incubation period (Fig. 4a). They found that higher assembly temperatures were required for structures with longer domains. By introducing poly-T linkers between the domains, the assembly temperature was reduced, allowing isothermal assembly at the physiological temperature of 37 °C. With these modified designs, isothermal assembly was also successful in a pH range of 7 to 9, under Mg²⁺ concentrations as low as 4 mM. Addition of molecular crowding agents such as PEG-8000 aided in the assembly of these structures under low ionic conditions but affected assembly when tested at higher ionic concentrations. They also showed that the assembly temperature can be modulated by changing the binding energy of strand-to-strand interactions in several ways, such as altering the GC content of binding domains or disrupting the continuity of complementary segments.

The isothermal assembly of DNA bricks from SSTs has also been shown in Na⁺-containing buffers (Fig. 4b). Specifically, Baigl and colleagues assembled a rectangular DNA brick consisting of 97 SSTs in 100–150 mM Na⁺ by incubating the strands at 25 °C for 24 h.⁴¹ In a variation on the SST-based assembly, Taylor and coworkers developed SST motifs with (l)-serine γPNA.⁵⁴ They tested the isothermal assembly of γPNA fibers at temperatures ranging from 45–70 °C in 75% dimethyl sulfoxide (DMSO). They observed the formation of nanofibers >2 μm in length at assembly temperatures ranging from 55 °C to 65 °C over a 2 h incubation period. Temperatures lower and higher than these did not yield nanofibers. Use of other solution conditions has also enabled the isothermal assembly of DNA nanostructures. Hud and coworkers constructed a 9-helix triangle with 87% yield by incubation at 20 °C for 24 h in 75% glycholine (a water-free solvent composed of a 4 : 1 molar ratio of glycerol and choline chloride).⁵⁷

In another work, Song and coworkers used SSTs to create DNA nanotubes isothermally at 21 °C.⁵⁵ A key requirement for isothermal assembly of the DNA nanotubes was the presence of 6 M urea, which acts as a substitute for higher temperature.⁷⁰ No assembly was observed under conditions without urea. In a similar strategy using denaturing agents, Gothelf and coworkers demonstrated the isothermal assembly of a variety of structures from SSTs by using 30% formamide as a denaturing agent in the assembly process (Fig. 4c).⁴² Formamide lowers the DNA melting temperature linearly by ~0.6 °C per % formamide in the buffer depending on the GC content, helix conformation, and state of hydration of DNA.^71–73 For their test structures – a six helix bundle and a rectangle – the melting temperatures for the scaffold-staple interactions were calculated to be between 40 and 60 °C. Addition of 25–60% formamide reduces this temperature range to room temperature (20–23 °C), allowing assembly by the use of formamide instead of a temperature gradient. Further, formamide eliminates the need for protector strands in SST assembly due to the prevention of aggregation during the assembly process. This was in contrast to the traditional annealing method that caused aggregation when the protector strands were left out from the mixture. Overall, the addition of reagents such as formamide lowers DNA melting temperatures by functioning as a denaturing agent⁷⁴ and accelerates the rate of hybridization.⁷⁵

3.3. DNA origami

Dietz and colleagues assembled a variety of DNA origami objects at constant temperatures by analyzing the folding and unfolding rates of these structures at different temperatures (Fig. 5a).⁴⁵ Their model structures were a DNA plate, a DNA rectangle and a DNA gear that folded at 55 °C (in 15 min), at 50 °C (in 30 min), and 49 °C (in 40 min), respectively. This isothermal folding process, however, included a denaturing step at 65 °C for 15 min to remove any secondary structures in the DNA template strand. For a 2D rectangular DNA origami, folding was achieved in 5 minutes, and isothermal assembly also avoided aggregates and side products compared to an annealing protocol. Similarly, Weck and Heuer-Jungemann recently created arbitrary finite super-structures using DNA origami monomers that were constructed by denaturing the scaffold and staples for 5 min at 65 °C followed by isothermal folding at 50.5 °C for 3 h.⁴⁶

Fig. 5.

DNA origami assembly at constant temperatures. (a) Assembly of DNA origami structures in Mg²⁺-containing buffers. TEM images reproduced from ref. 45 with permission. Copyright 2012, The American Association for the Advancement of Science. (b) Assembly of DNA origami structures in Na⁺-containing buffers. AFM images reproduced from ref. 41 (open access). Copyright 2023, Springer Nature. (c) Filling of pores in DNA origami frames over different time periods. AFM images reproduced from ref. 44 with permission. Copyright 2013, John Wiley and Sons. (d) Completion of DNA origami assembly at room temperature. AFM images reproduced from ref. 43 with permission. Copyright 2019, American Chemical Society. (e) DNA origami assembly at room temperature using a formamide gradient. AFM and TEM images reproduced from ref. 47 with permission. Copyright 2008, American Chemical Society. (f) Completion of assembly of partially formed DNA origami structures and assembly of full DNA origami structures in formamide. AFM images reproduced from ref. 42 with permission. Copyright 2013, John Wiley and Sons. (g) Assembly of DNA origami aided by arginine. AFM images reproduced from ref. 49 (open access). Copyright 2021, Elsevier. (h) Assembly of DNA origami structure in glycholine. AFM and TEM images reproduced from ref. 57 with permission. Copyright 2015, John Wiley and Sons.

Baigl and colleagues demonstrated the assembly of DNA origami triangles at 25 °C in Na⁺ containing buffers (Fig. 5b).⁴¹ The optimal assembly conditions were 100 mM Na⁺ for proper folding at 25–40 °C and 150 mM Na⁺ for assembly in the 15–55 °C range. This was in contrast to Mg²⁺ containing buffers that did not yield any properly folded origami structures, possibly due to the formation of kinetically trapped structures for which the magnesium-stabilized base pairing would require a higher thermal energy to allow structure reconfigurability.⁷⁶ For Ca²⁺ containing buffer, assembly was observed only when incubated at a higher constant temperature of 55–60 °C. Assembly at 25 °C in Na⁺ was also shown for 2D rectangles and smiley face DNA origami structures. Further, they also demonstrated the isothermal assembly of protein-functionalized DNA origami by incubating biotinylated staples, M13 scaffold and streptavidin in a one-pot assembly at 25 °C over 24 h. Most of the bound proteins were found at the positions prescribed by biotinylated staples. They used this isothermal strategy for 3D origami shapes such as a multilayer square-lattice pattern of 24 helix bundles forming a triangular structure with dimensions 57 nm × 71 nm × 10 nm (similar to a Toblerone bar). For the 3D shapes, the requirement of Na⁺ was higher (100–200 mM) for successful assembly, and the yields were comparatively lower for isothermal assembly at 25 °C or 37 °C for 48 h compared to traditional annealing protocol over 41 h. Zhang and coworkers used a similar Na⁺-containing buffer to isothermally assemble DNA origami structures with parallel and antiparallel crossovers at 35 °C.⁷⁷

Isothermal assembly strategies have allowed the monitoring of DNA origami formation and the optimization of several different factors such as temperature, staple concentration, and ionic content. Dong and coworkers constructed 2D DNA origami rectangles with small (20.4 × 30 nm) or large (40.8 nm × 42 nm) pores in the middle of the origami to study the isothermal assembly kinetics in the pores of the DNA origami structures.⁴⁴ Using high-speed AFM, they observed that the pores were completely filled in ~6 min at 60 °C, but the assembly yields were lower for the large pore (Fig. 5c). For the same experiment at room temperature, filling up of the small pore took 1 h while the larger pore took 2 h, in contrast to just a few minutes at 60 °C. They found that the staple-scaffold hybridization rate at 60 °C was 10 times that of room temperature, and the structures assembled at 60 °C contained fewer defects and were more mechanically stable compared to those assembled at room temperature. Further, the pores were not entirely filled when assembled at room temperature. The low assembly yield at room temperature was improved by increasing the scaffold-to-staple ratio to 1 : 50. The same group continued this kinetic study with a 2D rectangle DNA origami (Fig. 5d).⁴³ Six different patterns with and without “seam” strands were designed to systematically investigate the pattern of seeding growth on 2D origami at room temperature. They observed the completion of the corner or one half of the DNA rectangle in ~120 min. However, when the seam strands were missing, the assembly of complete origami structures did not occur. Similarly, Li and colleagues pre-formed a DNA origami triangle by thermal annealing of the scaffold and a sub-set of staple strands, followed by isothermal completion of the full triangle assembly at 25 °C on the addition of the remaining staple strands.⁷⁸

The concept of using denaturing agents such as formamide to lower DNA melting temperatures has also been used in the assembly of DNA origami nanostructures at constant temperatures. Simmel and colleagues assembled DNA origami structures by mixing the component DNA strands in solutions containing a larger amount of formamide, followed by dilution or buffer exchange into solutions with lower amounts of formamide.⁴⁷ This was achieved either by the continuous pumping in of buffer without formamide or by sequential dialysis into solutions containing lower amounts of formamide. They started with 85% formamide in the solution and reduced the formamide to 1% for the assembly of a 2D DNA rectangle and 11% for a 3D DNA nanotube (Fig. 5e). For the 2D rectangular structures, assembly times were similar (~1 h) for both thermal annealing and isothermal procedures. For DNA nanotubes made from six-helix bundles, isothermal assembly at room temperature required overnight assembly using the dialysis-based method, whereas thermal annealing yielded folded structures using a 2 h protocol. A similar protocol using 8 M or 16 M urea instead of formamide also yielded 2D DNA rectangles using an isothermal protocol.

In another example of using denaturing agents for DNA nanostructure self-assembly, Gothelf and coworkers used formamide as a substitute for high temperature and demonstrated the assembly of DNA origami structures at 20–23 °C (Fig. 5f).⁴² The optimal concentration of formamide was found to be 40% in a solution containing 1 × TAE and 12.5 mM Mg²⁺. They demonstrated both stepwise as well as full assembly of DNA origami triangles. For the stepwise assembly, they first annealed the structure with only one or two edges of the triangle, followed by the addition of the remaining staples and incubation at room temperature (20–23 °C). Both pathways yielded high assembly yields of 85% for the full triangle, over 16 h. For the full triangle assembly at room temperature, the yields were ~60%. They demonstrated the assembly of other 2D DNA origami structures, including a DNA rectangle, a cross-shaped tile, and a nanotube. Solutions containing 40% formamide were optimal for 2D structures, whereas 30% formamide yielded the highest assembly yields for the 3D nanotube, possibly due to the shorter seeding segments (14 bp versus 16 bp) and unit segments (7 bp versus 8 bp) in the staple patterns of the 3D nanostructure design. Further, they also demonstrated a one-step formation of streptavidin-patterned DNA origami structures, indicating that isothermal assembly processes can overcome the limitation of annealing where the guest molecules have to be added separately at a lower temperature. They also used this strategy to construct a structure that combined DNA origami and SSTs, creating a DNA rectangle with an 8-helix ribbon at room temperature. In these cases, the use of formamide and isothermal assembly also seemed to reduce the aggregation of the structures due to base stacking.

There have also been efforts to assemble DNA origami structures by avoiding denaturing conditions. Fritzsche and colleagues demonstrated the isothermal assembly of DNA origami rectangles in temperatures ranging from 43–57 °C.⁵⁶ The assembly efficiency increased from 43 °C to 50 °C, and the highest assembly yield (80–85%) was observed between 50 °C and 55 °C. The effect of temperature and yield here was attributed to staples with higher melting temperatures binding to regions with similar complementary sequences but not the specific designed spot. Further, they supplemented the solution with betaine, a compound known to improve the amplification of genes in PCR reactions by reducing the formation of secondary structure caused by GC-rich regions.⁷⁹ Addition of 1 M betaine to the DNA mixture and incubation at 37 °C for 24 h yielded DNA origami structures with 25% yield whereas no folding was observed in solutions without betaine. By increasing the concentration of betaine to 3.3 M, room temperature assembly was also achievable in 1 h. In another work, Qian and colleagues achieved isothermal assembly of a DNA origami sheet at 37 °C by adding arginine to the solution (Fig. 5g).⁴⁹ Assembly was not proper at lower temperatures of 4 °C and 22 °C. Additionally, DNA origami sheets could be assembled from pH 6 to 9, with its highest yield at pH 7.

Hud and colleagues isothermally assembled a DNA origami rectangle by incubating the DNA strands in glycholine instead of Mg²⁺-containing buffer followed by dehydration under high vacuum for ≥12 h (Fig. 5h).⁵⁷ In fully anhydrous conditions, the assembly required denaturing at 70 °C for 20 min followed by incubation at 20 °C for up to six days when a high yield of fully formed rectangles was achieved (93% yield). The long duration required for complete folding in glycholine at room temperature was attributed to the slow DNA diffusion and reorganization in a viscous solvent such as glycholine. When using 90% glycholine to reduce the solution viscosity, assembly of the rectangle was achieved in 3 h when incubated at 20 °C, with a 92% yield, and a 6-helix-bundle was folded with an 84% yield by incubation at 25 °C for 48 h in 75% glycholine.

4. Outlook and discussion

Isothermal assembly strategies provide information on the kinetics of assembly processes and have the potential to simplify the assembly process of DNA nanostructures functionalized with heat-sensitive guest molecules while reducing the resources needed for assembly, such as a thermal cycler. This is important as it helps to make DNA nanotechnology accessible to labs with fewer resources and may help the undergraduate education of DNA nanotechnology.^80–82 A general takeaway from isothermal assembly strategies is that the assembly yield and kinetics of assembly are lower for more complex, 3D nanostructures at lower assembly temperatures. The intricacy of folding in inter- vs. intramolecular DNA complexes makes generalizing a universal folding protocol at constant temperatures challenging. Modifications to the sequence or structure design can help address lower assembly yields at room temperature, such as the addition of poly-T linkers in SST-based DNA nanostructures.⁵³ For larger structures such as DNA origami, the sequence, length and the proposed position of the staple strands in the nanostructure design also play a role in achieving high assembly yields isothermally. Addition of excess of staple strands during isothermal DNA origami construction has helped in higher folding efficiency,⁴⁴ possibly due to mishybridized staples bring displaced by the correct strands.¹⁰ The theoretical range for the melting temperatures of staple strands used in a 2D rectangle-shaped origami are between 53–83 °C.⁵⁶ In most origami designs, the staple strands have the same length, and thus the different melting temperatures of the staple-scaffold interactions are a result of the different GC content of the staples or because of base stacking effects between the scaffold-bound staples.⁸³ One should note that the overall GC content of different DNA origami structures formed from the same scaffold would be the same since the sequence of the entire structure is dictated by the commonly used M13 scaffold. Thus, more precise design of staples sequences and the complexity of the structure will aid in low-temperature assembly. Isothermal assembly of DNA nanostructure can also be further improved by combining these strategies with toe-hold-mediated strand displacement cascades⁸⁴ for triggered isothermal assembly.^85,86

The assembly yields of the structures play important roles in their applications. For example, the immunomodulatory properties of well-folded structures is different from that of misfolded structures and excess component strands.^87,88 Thus assembly yield and purification of nanostructures before use in an application is a crucial step in both thermal annealing and isothermal assembly techniques. The use of supplements such as denaturing agents or assembly-aiding reagents also help optimize isothermal assembly of DNA nanostructures. These additives expand the range of temperatures and the solution conditions in which these structures can be assembled and have been used to understand the folding and unfolding processes of DNA motifs.⁸⁹ Addition of denaturing agents substitutes the need for high temperature to reduce the secondary structure of DNA origami scaffold or component DNA strands that may interfere with the correct formation of the designed structure at low temperatures. However, the sequence of the DNA nanostructure might still play a prominent role in the efficiency of isothermal assembly. For instance, the use of a denaturing agent worked well for DNA origami rectangles and nanotubes but not for nanotubes designed using a single self-complementary sequence.⁴⁷ Depending on the intended application of the DNA nanostructure, these additives can also be purified out by buffer exchange into a physiological buffer in the case of a biological application. These challenges are also addressed by the continued development of several DNA nanostructure purification methods.^90,91

When using other solvents for isothermal assembly, it is important to consider how they affect the structure of DNA. For example, the typical B-form DNA present in aqueous solutions is used in the design of DNA nanostructures. DNA duplexes maintain a B-form helical structure in glycholine (that has been used in DNA nanostructure self-assembly) but not in other anhydrous deep eutectic solvents such as reline.⁵⁷ Once a solvent is found to enable DNA nanostructure assembly, tuning the solvent composition allows rapid folding of the DNA nanostructure, such as the addition of water to glycholine providing relatively fast folding of a 3D DNA origami structure and a 2D SST system under isothermal conditions.⁵⁷ The use of anhydrous solvents further allows the interfacing of isothermal assembly strategies for materials science applications on wafers and metallic surfaces. In comparison to aqueous solutions, deep eutectic solvents have been shown to have enhanced electrodeposition of metallic and semiconducting materials, improved inorganic nanoparticle shape control and stability, and low volatility.^92,93 While denaturing agents and anhydrous solvents are not compatible with biological applications, the assembled structures can be transferred to aqueous buffers to provide DNA structures with greater stability as well as improved compatibility with enzymes.^47,57 Further, solvents such as hydrated ionic liquids (e.g.: choline dihydrogen phosphate) have been shown to both enhance the biostability and functionality of nucleic acid structures,⁹⁴ enabling the use of such solvents in biological applications.

Isothermal assembly strategies also allow the interfacing of DNA nanostructures with biomolecular processes. For example, DNA methylation can be used as a stimulus to trigger the autonomous operation of DNA machines and be useful for the analysis of methyltransferase activity and screening of methyltransferase inhibitors.⁹⁵ Such isothermal assembly processes allow the continuous autonomous behavior of many engineered systems,¹⁴ the creation of self-replicating synthetic DNA systems that previously needed adjustment of ambient temperatures,⁹⁶ and the in vivo cloning of artificial DNA structures that requires physiological temperature.⁹⁷ There are already examples of using enzymes to initiate isothermal assembly using inert components.⁵¹ The advantage of such triggered assembly from inert components is to enable storage and transport without cold chain requirements, where the inert components are stable at storage temperature of 40–50 °C and can be readily converted to the DNA nanostructure isothermally after treatment with corresponding nucleases. For triggered isothermal assembly in a wider range of constant temperatures, one could use photoresponsive linkers, where inert strands could be activated by light, leading to the assembly of nanostructures. These inert complexes can also be based on DNA structures that reconfigure based on structure-based affinity, such as in the conversion of a topologically left-handed and parallel switchback DNA to a right-handed antiparallel conventional duplex.^98,99 Operating temperatures of 37 °C for such reactions has a significant advantage over thermal annealing in creating such assemblies in the cell, similar to co-transcriptional folding shown for RNA nanostructures.¹⁰⁰ Isothermal assembly strategies also allow scaling up of these processes by being compatible with microfluidics for in situ assembly on the addition of components. The strategy of assembling DNA nanostructures isothermally thus opens up new routes to create a variety of nanostructures for different applications and complements existing assembly strategies in DNA nanotechnology.

Data availability

All the data supporting this article are included in the manuscript.