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UNIT 12.9DNA Origami: Synthesis andSelf-Assembly

Arivazhagan Rajendran,1,2 Masayuki Endo,2,3 and Hiroshi Sugiyama1,2,3

1Department of Chemistry, Graduate School of Science, Kyoto University, Kyoto, Japan2CREST, Japan Science and Technology Corporation (JST), Tokyo, Japan3Institute for Integrated Cell-Material Sciences (iCeMS), Kyoto University, Kyoto, Japan

ABSTRACT

DNA origami is an emerging technology for designing defined two- and three-dimensional (2D and 3D) DNA nanostructures. Here, we report an introductory practicalguide with step-by-step experimental details for the design and synthesis of origamistructures, and their size expansion in 1D and 2D space by means of self-assembly. Curr.Protoc. Nucleic Acid Chem. 48:12.9.1-12.9.18. C© 2012 by John Wiley & Sons, Inc.

Keywords: DNA origami � designed nanospace � self-assembly �

DNA nanotechnology � atomic force microscopy

INTRODUCTION

This unit contains an introductory practical guide with step-by-step protocols for thedesign and synthesis of DNA origami structures, and their size expansion in 1D and2D space by means of self-assembly. DNA nanotechnology was used to prepare theprogrammed 2D arrays of DNA and RNA with a size of about 10 to 20 nm (Chworoset al., 2004; Park et al., 2006). Though such a small structure was used for variouspurposes, their applications were limited and strategies for the construction of definedlarger assemblies are indeed required. The size of the nanostructures was scaled upby the recent development of the “scaffolded DNA origami” method (Fig. 12.9.1),which drastically improved the size of the nanostructure about 10-fold (∼100 × 80nm; Rothemund, 2006). This method requires a special design of the structure of interestand the design principles of the origami structures and their self-assembly are describedin the first method (see Basic Protocol 1). The synthesis, purification, characterization,and storage of origami structures are described in the second method (see Basic Protocol2). For practical applications, the sizes of the origami structures need to be scaled upfurther. For instance, conventional photolithography techniques require a size domain of1 μm. The bottom-up approach we have developed with the 1D and 2D self-assembly oforigami structures further improved the size (Endo et al., 2010; Rajendran et al., 2011a).The additional design strategies for the self-assembly are explained in the first method(see Basic Protocol 1). The experimental procedures for the 1D self-assembly are given inthe third method (see Basic Protocol 3), and the final method, as given in Basic Protocol4, describes the 2D self-assembly of multiple origami structures. An overview of recentprogress in DNA origami technology can be found in UNIT 12.8.

BASICPROTOCOL 1

STRUCTURAL DESIGN OF DNA ORIGAMI AND ADDITIONAL DESIGNSTRATEGIES FOR 1D AND 2D SELF-ASSEMBLY

In the original report (Rothemund, 2006), the design of the origami structure was per-formed in five steps, the first two by hand and the last three aided by computer. Alterna-tively, these five steps can be done either manually or using a semiautomated computerprogram. Shih et al. developed an open-source software package called “caDNAno” for

Current Protocols in Nucleic Acid Chemistry 12.9.1-12.9.18, March 2012Published online March 2012 in Wiley Online Library (wileyonlinelibrary.com).DOI: 10.1002/0471142700.nc1209s48Copyright C© 2012 John Wiley & Sons, Inc.

NucleicAcid-BasedMicroarrays andNanostructures

12.9.1

Supplement 48

DNA Origami:Synthesis andSelf-Assembly

12.9.2

Supplement 48 Current Protocols in Nucleic Acid Chemistry

Figure 12.9.1 The schematic drawing of the structures prepared using the DNA origami method and their AFMimages. The top row represents the folding paths of the scaffold. Only the raster fill patterns of the scaffold areshown and the staples are not included here. The middle row represents the diagrams after the addition of staples,showing the bend of helices at crossovers where helices touch and away from crossovers where helices bendapart. AFM images of the representative schemes are given in the bottom row. AFM images correspond to a sizeof 165 × 165 nm.

the design of 2D and 3D origami structures (Douglas et al., 2009a). Using this software,all five steps of the origami design can be performed. Moreover, it is developed by follow-ing the basic rules of the origami design and, thus, the design is relatively easy even fornon-experts, reduces the labor time, and is less prone to error. It can be downloaded fromhttp://www.cadnano.org. The alternative option for the computer design is the “SARSE”program, which can be downloaded from http://www.cdna.dk/origami. Since thousandsof base pairs are involved in this method, it is complex and error prone. Thus, it issuggested to use a computer program rather than the manual design. Moreover, thereare several basic principles involved in the origami design, which should be followed inorder to maintain the B-DNA structure and planarity. Hence, irrespective of the mode ofdesign, it is important to understand the following principles.

Step 1: Building a geometrical model of the origami structure1. Build a geometric model of DNA origami structure that approximates the desired

shape (Fig. 12.9.2A).

2. Fill the shape from top to bottom with an even number of parallel double helices.

3. Cut the helices to fit the shape in sequential pairs and have them be an integer numberof turns in length.

4. Incorporate a periodic array of crossovers. Design the crossovers in such a waythat they designate positions at which strands running along one helix switch to anadjacent helix and continue there. This arrangement is important to hold the helicestogether.

5. One may use any odd number of half-turns between the crossovers, though the typicalone is three half-turns.


12.9.3

Current Protocols in Nucleic Acid Chemistry Supplement 48

Figure 12.9.2 Design of a DNA origami structure. (A) The schematic design of a shape (red) approximated byparallel double helices joined by periodic crossovers (blue). (B) A scaffold strand (black) runs through every helixand forms more crossovers (red). (C) As first designed, most staples bind two helices and are 16-mers. Arrowspoint to nicks that can be sealed to create longer strands. The yellow diamond indicates a position at which sta-ples may be cut and resealed to bridge the seam. (D) A finished design after merges and rearrangements of thestaples along the seam. Most staples are 32-mers spanning three helices. For the color version of the figure go tohttp://www.currentprotocols.com/protocol/nc1209.

Step 2: Folding of the scaffold strand6. Fold a single long or circular scaffold strand back and forth in a raster fill pattern.

This will cause the scaffold to consist of one of the two strands in every helix presentin the entire origami structure (Fig. 12.9.2B). The other one will be the staple strand.

7. Raster filling of the scaffold will lead to scaffold crossovers and these can be formedonly at those positions where the DNA twist places it at a tangent point betweenhelices.

8. The distance between successive scaffold crossovers must be an odd number ofhalf-turns when the raster progresses from one helix to another and onto a third.

9. It must be an even number of half-turns between the crossovers when the scaffoldreverses the direction vertically and returns to a previously visited helix, i.e., thecrossovers between the same two helices but on the opposite ends of the raster.


12.9.4


Step 3: The design of a set of staple strands (Fig. 12.9.2C)10. Input the above designed lists of DNA lengths and offsets in units of half-turns

along with the sequence information of the scaffold to a computer program(called multishapes.m; it may be downloaded from http://www.dna.caltech.edu/SupplementaryMaterial/).

11. Use an integer number of bases between periodic crossovers (e.g., 16 bp for 1.5turns).

Though 16 base pairs per 1.5 turns were used in the original origami design, the typicalB-DNA parameter of 15.75 base pairs per 1.5 turns can also be imposed by omitting abase in every 6 helical turns. For example, using 16 base pairs in the first three 1.5 turnsand 15 base pairs in the fourth 1.5 turns will lead to 63 base pairs per 6 turns. This canbe done by using the skip base option in the caDNAno program.

12. The program will design a set of staple strands based on the Watson-Crick comple-ments of the scaffold.

13. Note that the crossovers are antiparallel, as the staples reverse direction at thesecrossovers. In addition, helices are likely to bend gently to meet at crossovers, thus,only a single phosphate from each backbone occurs in the gap and there is no unpairedbase at the inter-helix gap.

Step 4: Minimizing strain14. Arrange the periodic crossovers with glide symmetry, i.e., the minor groove should

face alternating directions in alternating columns of periodic crossovers. This ar-rangement will balance the strain caused by representing 1.5 turns with 16 bp.

15. The scaffold crossovers can be left without balancing in this way.

16. Calculate the twist of the scaffold crossovers, change their position, and recomputethe sequence of the staples.

17. Along seams and some edges, the minor groove angle of 150◦ places scaffoldcrossovers in tension with adjacent periodic crossovers and these can be left as theyare.

Step 5: Defining the staples with larger binding domain18. There is a nick in the backbone where two staples meet and these nicks occur on

both top and bottom faces of the helices.

19. Merge pairs of adjacent staples across nicks to yield fewer, longer staples(Fig. 12.9.2D).

20. Additional pattern of breaks and merges may be imposed to yield staples that crossthe seam. This will strengthen the seam.

21. Different choices of the merges can be used.

Additional design strategies for the programmed 1D assembly of jigsaw puzzle(JP)-shaped origami (additionally required for Basic Protocol 3)For the self-assembly of DNA origami structures, we have designed the jigsaw puzzle–shaped origami (Fig. 12.9.3). We believe that the jigsaw shape could provide shape-fittingsteric hindrance, so that the specificity can be achieved among the monomer units.

22. Shape-fitting of the side-edges for selective connection and exclusion of undesiredpairing of DNA JPs: This can be achieved by introducing a tenon and mortise at thesides of the origami, so that a tenon can only be attached to a mortise and vice versa(Fig. 12.9.3).


12.9.5


~ 100 nm

self-assembly

~ 8

0 nm

teno

n

mor

tise

CA

B

Figure 12.9.3 The design of a DNA origami “jigsaw piece” structure and its self-assembly. (A) Structure of thedesigned DNA jigsaw piece with a tenon and mortise. (B) Scheme of the self-assembly of the DNA jigsaw piecemonomer. (C) The detailed connection around the tenon and mortise between neighboring DNA jigsaw piecemonomers. Black and colored DNA sequences denote the M13 and the staple strands, respectively. Blue andgreen arrow staples represent the left- and right-side edges of the DNA jigsaw piece monomer, respectively. Redarrows represent the connecting staples that bridge the neighboring tiles. For the color version of the figure go tohttp://www.currentprotocols.com/protocol/nc1209.

23. Hybridization of the DNA strands at the connection sites using part of the staplestrands: This can be achieved by base pairing part of the connecting strand with theM13 in a JP, leaving an overhang region of eight bases. During self-assembly, theoverhang region can be hybridized directly to the M13 in the neighboring JP and,thus, this strand bridges neighboring tiles through specific connections.

24. Nonspecific π-stacking interactions of the side-edges enable the formation of stablecomplexes and connection of the side-edges. This π-stacking interactions can beutilized by performing the self-assembly along the helical axis rather than the helicalside.

Additional design strategies for the programmed 2D assembly of JPs (additionallyrequired for Basic Protocol 4)The 2D self-assembly of origami structures can also be carried out using the jigsaw-shaped origami structures. In this case, we have altered our initial structure and preparednine different origami puzzles to form a 3 × 3 structure.

25. Design nine different JPs, each of which should be a 24-helix tile with the size of∼100 × 80 nm (Fig. 12.9.4).

26. Place the sequence-programmed connection sites, a tenon, and corresponding mor-tise, to allow assembly along the helical axis (x-direction) with the adjacent JP viathese adhesive connections.

27. Place each of the two single-stranded overhangs at the tenon and the mortise.


12.9.6


teno

n

mor

tise

poolpool

single-stranded overhang

L

I

J

CA

B

JP-1 JP-2 JP-3

JP-4 JP-5 JP-6

JP-7 JP-8 JP-9

~ 100 nm (288 bp) x

y

~ 8

0 nm

(24

dupl

exes

)

Figure 12.9.4 (A) A model structure of the designed DNA JP having a mortise and a tenon (JP-5).(B) Scheme of the JP-5 represented as a matrix of blocks. One block represents four 32-mer duplexes.(C) Scheme of the nine monomer JPs showing the position of the tenon, the mortise, and the hairpin mark-ers along with their AFM images. Pink blocks at the bottom of each JP represent the loops. Circles denote theset of four individual hairpin markers. AFM images correspond to a size of 200 × 200 nm. For the color versionof the figure go to http://www.currentprotocols.com/protocol/nc1209.

28. Keep two single-stranded overhangs at the sides of each monomer to make thebinding stronger along the helical axis.

29. Place nine protruding single-stranded overhangs at the bottom side for the first rowJPs, the top side for the last row, and both sides for the middle row that base pairwith the M13mp18 viral DNA in the neighboring JP to facilitate assembly along thehelical side (y-direction).

30. In addition to the single-stranded overhangs, create jigsaw shapes at the top andbottom sides of each origami structure to promote the assembly.

31. Differentiate the individual monomers by changing the position of the tenon and themortise and also the sequences at the bottom.

32. Introduce two loops at the bottom of each JP and make each sequence differentbetween the monomers at the bottom side by adjusting the sequences in the loop.This arrangement is important for the monomers to assemble exclusively with theirrespective partners. Note that these loops are duplexes containing double-crossoversformed by the portion of the viral DNA with their complementary staple strands.


12.9.7


33. For the identification of each monomer, introduce a set of hairpin DNA strands (eachset contains four individual hairpins) as markers that were adjacent to the tenon andthe mortise.

Typical hairpin sequence for the pixel enhancement in atomic force microscopy (AFM) im-age is 5′-TCCTCTTTTGAGGAACAAGTTTTCTTGT-3′, if the structures are characterizedusing AFM.

34. To avoid the intertile-stacking interactions among the same JP monomer, introducetetra-thymidine (T4) units at both of the side edges of each monomer JP.

The number of connecting strands/single-stranded overhangs can be varied. A reducednumber of strands may lower the binding efficiency and more strands may increase therisk of nonspecific interactions; both may contribute to the lower yield of the assembledproduct.

BASICPROTOCOL 2

SYNTHESIS OF DNA ORIGAMI

Like an organic synthesis, the synthesis of DNA origami involves the following foursteps: (1) synthesis, (2) purification, (3) characterization of the formed structure, and (4)lyophilization and storage.

The origami structures can be characterized without purification due to the fact that thestaple strands are small and may not have a visible feature when characterized by aparticular technique (e.g., AFM). Moreover, when compared to the origami structures,these staples have less binding affinity to the mica surface. However, specific experimentsmay require the purified structures.

The origami structures can be characterized using microscopic techniques, such as trans-mission electron microscopy (TEM) and AFM. TEM is especially useful for the structuralcharacterization of 3D origami structures and AFM is powerful for the analysis of 2Dstructures.

Materials

Required set of staple strands based on the origami design (see Basic Protocol 1)M13mp18 single-stranded DNA (New England Biolabs, cat. no, N4040S)10× origami buffer (see recipe)Deionized water by a Milli-Q system (≥ 18.0 M� cm specific resistance;

Millipore)Sephacryl S-300 (High resolution; GE Healthcare, cat. no., 17-0599-01)Sephacryl S-300 solution (see recipe)

PCR tubesThermal cyclerAutomatic shakerMicro Bio-Spin chromatography columns (Bio-Rad, cat. no. 732-6204)1.5-mL microcentrifuge tubesVortex mixerMicrocentrifugeMica plate (e.g., 1.5-mm plate; Nano Live Vision, RIBM)AFM instrument–80◦C freezerLyophilizer

NOTE: The use of a particular brand chemical, reagent, or material throughout thisprotocol is purely the authors’ choice. In fact, any brand can be used with the same orsimilar grade.


12.9.8


Synthesize the origami structures1. After the structural design, as described in Basic Protocol 1, extract the staple

sequences from the software and purchase them from the company.

The staple length of 32-mer will be a better option in terms of length and stability. Thesingle-stranded viral genome (M13mp18), which serves as a scaffold, can be obtainedfrom New England Biolabs.

It is not necessary to use the M13mp18 circular DNA for any origami synthesis. Inprinciple, any available long/circular single-stranded DNA can be used based on thedesign and purpose. This viral genome has 7249 bases and thus can be used to prepare anorigami structure with the size of roughly 100 nm in diameter. Gel-purified staple strandsmay be sufficient and HPLC purification may not be required, as the origami method ishighly sequence-specific and not sensitive to the purity of the DNA.

2. Mix a portion of each staple in PCR tubes to make a single solution of the staplestrands with the desired stock concentration (typically a final concentration of 0.2 μMof each staple).

3. Prepare a stock solution of the origami buffer.

Typically, 200 mM Tris·Cl (pH 7.6) containing 100 mM MgCl2 and 10 mM EDTA (all arein the 10× stock concentration).

4. Mix the staples (5 μL of 0.2 μM stock), M13 (2.5 μL of 0.1-μM stock), and origamibuffer (2.5 μL of 10× stock). Adjust the final volume to 25 μL using Milli-Q water.

5. Shake the solution gently (or vortex and centrifuge) to ensure the homogeneity ofthe solution.

6. Anneal the solution from 85◦ to 15◦C at a rate of −1◦C/min using a thermal cycler(in the original report, it was 95◦ to 20◦C in <2 hr).

Purify7. Remove the excess staples from the origami solution using the gel filtration method.

Sephacryl S-300 usually contains 20% ethanol as a preservative. Remove the ethanoland resuspend the sephacryl S-300 gel in ∼40 mL of the desired buffer (usually in1× origami buffer; i.e., 20 mM Tris·Cl containing 10 mM MgCl2 and 1 mM EDTA)and keep it shaking overnight (∼128 rpm) in an automatic shaker.

8. Pack the chromatographic column with sephacryl S-300 as follows:

a. Poke a small hole in the bottom of a chromatographic column and place it into aclean 1.5-mL microcentrifuge tube.

b. Add ∼500 μL sephacryl S-300 solution and centrifuge the column for 3 min at1000 × g, 15◦C. Discard the buffer solution collected in the microcentrifuge tubeand repeat the steps (i.e., adding of the sephacryl solution, centrifugation, anddiscarding of the buffer collected in the microcentrifuge tube) until the column ispacked up to a little below its neck (∼1.5 cm in height).

c. Finally, centrifuge again under similar conditions but without adding sephacryl toensure that the column is sufficiently free from excess buffer.

9. Exchange the microcentrifuge tube with a new one. Without disturbing the gelbed, carefully apply the origami sample directly onto the top center of the gel bed.Centrifuge the column for 3 min at 1000 × g, 15◦C.

10. Collect the staples-free, purified origami sample in the microcentrifuge tube.

Characterize the structure by AFM11. Adjust the concentration of the origami structure to 1 nM with 1× origami buffer.


12.9.9


2520 0051mn 0981 1125 nm

Figure 12.9.5 AFM images of the rectangular DNA origami JP (left) and frame structure (right) lyophilized andredissolved in Milli-Q water. The image sizes are given in each image.

12. Adsorb/apply the sample onto a freshly cleaved mica plate (∼2 μL of 1 nM samplefor ∼1.5-mm mica plate in the case of Nano Live Vision, RIBM) for 5 min at roomtemperature.

13. Remove excess sample by gently washing the mica surface three to five times usingthe 1× origami buffer that was used for the preparation of origami.

14. Perform scanning using the tapping mode in the same buffer (i.e., 1× origami buffer).

The surface of mica is negatively charged and thus Mg2+ in the buffer is important for theorigami binding on the mica surface. The origami easily attaches on the mica surface andno surface modification (such as chemical cross-linking or surface coating) is needed.The sample volume can be varied depending on the size of the mica plate. For less robustbiological samples, such as DNA origami, AFM observation in the dynamic tappingmode may be suitable, as the tip-sample interaction forces are minimized in this mode.The scanning may also be performed in air.

Lyophilize and store (Fig. 12.9.5)15. Freeze the origami structures for about 1 hr at –80◦C.

16. Make a hole on the top of the container (PCR tube) to prevent the sample fromspreading out during the lyophilization.

17. Lyophilize the frozen sample overnight under high vacuum using a lyophilizer.

18. Store up to a few weeks in a −30◦C freezer and resuspend in Milli-Q water, whenrequired.

BASICPROTOCOL 3

1D SELF-ASSEMBLY OF ORIGAMI STRUCTURES

The size of the origami structures prepared with the M13 scaffold is ∼100 × 80 nm, whichis still smaller in area, and strategies for the construction of defined larger assemblies arelimited. A critical challenge facing the further development of DNA origami technologyis scaling up the size of origami structures. The size of the origami was first expanded byRothemund with the assembly of triangular origami into a hexagon, with a yield of <2%(Rothemund, 2006). Later, 3D heterotrimers in the shape of a wireframe icosahedronwere reported with no information about the yield of the assembled structures (Douglaset al., 2009b). We have recently developed a new method to scale up DNA origamiusing jigsaw pieces (JPs) and successfully prepared a unidirectional DNA assembly(Endo et al., 2010). Our method utilizes the sequence information of the DNA for the


12.9.10


self-assembly, i.e., the origami structures can be connected by using single-strandedconnecting strands. It requires no additional materials or modified sequences. Thus, itresults in the assembled product fully made of DNA without any modification, chemicals,or biochemicals. Herein, we present the protocol of the self-assembly-based 1D sizeexpansion of the origami structures. The basic rules for the origami design and preparationare similar as those mentioned for the monomer origami. However, in addition to thementioned rules, there are some additional design principles that should be followed inorder to promote the self-assembly, as given in Basic Protocol 1.

For the controlled self-assembly of the origami JPs, we divided the self-assembly processinto two sets of steps by employing different annealing procedures:

i. Formation of the individual DNA JP monomer via fast annealing.ii. Self-assembly of the DNA piece monomer into a programmed 1D DNA assembly

via slow annealing.

In the methods described below, the connecting strands were placed within the JP bypartly hybridizing them to the M13 that leaves single-stranded overhangs of 8 basesin each strand. In an alternative procedure (not described in this unit), the connectingstaples can be added during the self-assembly. In the latter procedure, the concentrationof the connecting strands can be varied. In method 1 described below, the position of thetenon and mortise can be moved to any row along the helical axis; however, both shouldbe in the same row for their connection during the assembly (Fig. 12.9.6A). Further,the self-assembly can be performed using a single monomer JP (method 1) or multiplemonomers (method 2).

Additional Materials (also see Basic Protocol 2)

AFM instrument

Method 1: Self-assembly of single DNA origami JP monomer (Fig. 12.9.6)

1a. Design the origami monomers based on the basic principles and additional rules, asmentioned in the Basic Protocol 1. Place the tenon and mortise in the same row, buton either of the sides (Fig. 12.9.3).

2a. Insert a T4 loop at all the corner staples, except on the tenon and mortise.

This will reduce the π -stacking interaction at the corners of the monomers, therebyreducing nonspecific interactions between them. However, it cannot remove the π -stackingcompletely.

3a. Use hairpin-modified sequences near the tenon and mortise to recognize their positionin the AFM image.

4a. Mix the staples and connecting strand solutions (5 μL of 0.2 μM stock), M13 (2.5 μLof 0.1 μM stock), and origami buffer (2.5 μL of 10× stock). Adjust the final volume to25 μL with Milli-Q water. Vortex gently for a few seconds and centrifuge the solutionfor a few seconds at a mild force, room temperature, to ensure the homogeneity ofthe solution.

5a. Anneal the solution from 85◦ to 25◦C at a rate of –2◦C/min (termed fast annealing).

6a. Purify the origami to remove excess staples and connecting strands by following theprocedure given in Basic Protocol 2 (see steps 8 to 10).

7a. After purification, carry out the self-assembly by slow annealing from 50◦ to 15◦Cat a rate of –0.05◦C/min.

8a. Characterize the self-assembled product using AFM (Fig. 12.9.6C) by following theprocedure listed in Basic Protocol 2 (see steps 12 to 14).


12.9.11


A

B

C

A B C D

A

375 375 nm 375 375 nm 375 375 nm 375 375 nm

1200 900 nm 1000 750 nm 1000 750 nm 1000 750 nm

DCB

DCBA

Figure 12.9.6 DNA JP monomers and self-assembled structures. (A) Schematic drawings of the monomersA-D. Each JP differs by their relative positions of the tenon and mortise. Pink blocks represent hairpin DNAmarkers for the identification of the tenon and mortise in the AFM image. (B) AFM images of the DNA jigsawpiece monomers A, B, C, and D after fast annealing from 85◦ to 25◦C at a rate of −2◦C/min. (C) Oligomerizationof the single DNA jigsaw piece monomer by self-assembly from 50◦ to 15◦C at a rate of –0.05◦C/min. For thecolor version of the figure go to http://www.currentprotocols.com/protocol/nc1209.

Method 2: Self-assembly of different DNA piece monomers (Fig. 12.9.7)

The 1D self-assembly can also be performed by using different JP monomers that differfrom each other by the position of the tenon and mortise.

1b. Design the JP monomers (in the present case five different JPs) by changing theposition of the tenon and mortise, as shown in Figure 12.9.7.

In principle, the tenon and mortise can be placed anywhere in the side edge. However, atenon in one JP and a mortise in its neighbor should be placed in the same row so thatthey can be attached during the self-assembly.

2b. Insert a T4 loop at all the corner staples except on the tenon and mortise (see step2a).

3b. Use hairpin-modified sequences near the tenon and mortise to recognize their positionin the AFM image.

4b. In this design, the staple sequences near the tenon and mortise differ between JPs,while other positions have identical sequences in all JPs. Thus, during the preparationof the staples solutions, mix the common sequences in one set and the others in asecond set. Then, prepare the final staple solutions of each monomer separately.

5b. Mix the components of the origami and prepare individual monomers separately byfast annealing (85◦ to 25◦C at a rate of –2◦C/min). Adopt the same concentrationsas listed in the previous procedure above (see step 4a).


12.9.12


A

CB

H E F G I

H E F G I

image size of all monomers: 375 375 nm

self-assembly

750 750 nm

HE

FG

I

H E F G I

Figure 12.9.7 Self-assembly of five different DNA JP monomers. (A) Scheme of the five different monomersand their self-assembly. (B) AFM images of the DNA jigsaw piece monomers after fast annealing. (C) AFM imageof the self-assembled pentamer after slow annealing.

6b. After fast annealing, remove excess staples by gel filtration (see Basic Protocol 2,steps 8 to 10).

7b. Mix an equimolar concentration of each purified monomer and perform slow an-nealing (50◦ to 15◦C at a rate of –0.05◦C/min).

8b. After the second step of annealing, the self-assembled product can be characterizedusing AFM (Fig. 12.9.7C) under liquid or in air (see Basic Protocol 2, steps 12 to14).

Both, the higher concentration of DNA JP monomers and slower annealing speed tend toincrease the yield of the target assembly. The self-assembly of prefunctionalized origamistructures can also be carried out using this method. For example, the hairpin-modifiedstaples can be used to write alphabets on the DNA origami surface. These alphabet-containing monomers can be self-assembled to display words in nanoscale. The nanopar-ticles, proteins, and other functional components containing origami may also be self-assembled by following this procedure.

BASICPROTOCOL 4

2D SELF-ASSEMBLY OF MULTIPLE ORIGAMI STRUCTURES

Though the 1D self-assembly was successfully carried out, the 2D construction of origamitiles is critical for the development of robust materials based on DNA. The 2D scale-upof the origami structure was recently initiated using small DNA tiles with a size of 16 ×17 nm as folding staples (Zhao et al., 2010). Apart from this example, there is no reportfor the preparation of defined larger 2D origami sheet-like structures, and hence thedevelopment of new methods with added advantages is urgently required. We recentlydemonstrated a new route for the 2D extension of DNA origami using multiple JPs byprogrammed self-assembly, the spontaneous association of components into organized2D structures using noncovalent interactions (Rajendran et al., 2011a). This methodexplains how nine different jigsaw-shaped origami structures can be self-assembled in2D space to create a 3 × 3 structure. In principle, this method can be extended for theprogrammed self-assembly in 2D space reaching the size domain of few micrometers.


12.9.13


Though the self-assembly can be performed in several ways, there are three major waysone can consider:

i. Two-step assembly: Preparation of monomers in the first step and the self-assemblyin the second step.

ii. Three-step assembly: Preparation of the monomers in the first step, self-assemblyof trimers along helical axis (x-direction) in the second step, and, in the final step,self-assembly of pre-assembled trimers.

iii. Alternative three-step assembly: Preparation of the monomers, assembly of trimersalong the helical side (y-direction), and final assembly of pre-assembled trimers inthe last step.

Though the first two methods successfully yielded the final product, these methods fail toproduce the final structure with reasonable yield. The maximum yield obtained by thesemethods is ∼10%. This low yield in the first method is logical because in a reaction withmore components (in the present case there are nine components), the low yield would beexpected. In the second method, the number of components in every step would be fewerand thus a better yield is expected. However, a lower yield was observed and it might bedue to the single-stranded overhangs that were introduced to facilitate the self-assemblyalong the helical side. These single-stranded overhangs could possibly display undesirednonspecific interactions with other JPs or the trimer assembly and lead to aggregation.Thus, these single-stranded overhangs may not be effectively available for the assemblyalong the helical side, and hence, the yield of the final product was low. Therefore,the single-stranded overhangs at the bottom side need to be protected before the 2Dself-assembly. This can be done by hybridizing the single-stranded overhangs with theircomplementary bases in the neighboring JP. In other words, the trimer formation alongthe helical side (vertical assembly along y-direction) can protect the single-strandedoverhangs during the trimer formation, and then the 2D self-assembly can be furthercarried out as denoted by the third method. Note that the number of single-strandedoverhangs is more at the top and bottom (each side 9) of each JP when compared to thecorners (each corner 4). For the origami design and materials, see Basic Protocols 1 and2, respectively.

2D assembly in three steps1. Design and prepare nine different origami JPs using the procedures described in

Basic Protocols 1 and 2 (Fig. 12.9.4C).

A typical concentration of 0.01 μM of M13, 0.04 μM of staples, and 1× buffer conditionwill be sufficient. A final volume of 25 μL would suffice.

2. After the monomer preparation, remove excess staples by gel filtration of eachorigami JPs separately (see Basic Protocol 2, steps 8 to 10).

3. Mix the monomers in each column in equimolar ratio and make three differentsolutions.

4. Anneal them from 50◦ to 15◦C at a rate of –0.05◦C/min.

5. After trimer assembly, characterize the trimer structures using AFM (Fig. 12.9.8A;see Basic Protocol 2, steps 12 to 14).

6. Mix the pre-assembled trimers in equimolar ratio and perform the final step ofassembly by adopting the same annealing condition.

7. Characterize the final structure using AFM (Fig. 12.9.8B-C).


12.9.14


Figure 12.9.8 (A) Schemes and AFM images of the trimers assembled along the helical side. (B) Schematicdrawing of the desired final structure of the self-assembly. (C) AFM image of the self-assembled structure preparedfrom the pre-assembled trimers. The bright spots in the image represent the loops at the bottom of each monomer.Light spots represent the hairpin markers, which are adjacent to the tenons and the mortises. The image sizesare shown below each image.

REAGENTS AND SOLUTIONSUse deionized, distilled water in all recipes and protocol steps. For common stock solutions, seeAPPENDIX 2A; for suppliers, see SUPPLIERS APPENDIX.

Origami buffer, 10×Dissolve Tris·Cl, pH 7.6 (APPENDIX 2A; 200 mM), MgCl2 (100 mM), and EDTA (10mM) in Milli-Q water.

Here, final concentrations for the 10× stock are given. A total volume of 25 mL would bemore than sufficient.

Sephacryl S-300 solution

Prepare the solution as follows:

a. Add 25 mL sephacryl S-300 and 25 mL Milli-Q water into a 50-mL plastic tube.

b. Shake the solution vigorously until a homogeneous solution is obtained andcentrifuge the tube for 1 min at 1000 × g, room temperature.

c. Discard the supernatant, which contains water and ethanol.continued


12.9.15


d. Add ∼40 mL Milli-Q water and repeat the abovementioned steps (b) and (c)three to four times.

e. Repeat step (d) three to four times by replacing Milli-Q with ∼40 mL of 1×origami buffer (containing 20 mM Tris·Cl, 10 mM MgCl2, and 1 mM EDTA;see recipe for 10× origami buffer).

f. Finally, resuspend the gel in ∼40 mL of 1× origami buffer and keep shakingovernight in an automatic shaker at ∼128 rpm, room temperature.

g. After the overnight shaking, use the solution for the gel filtration to remove theexcess staple strands from the origami solution.

h. Store the solution for several months at 5◦C.

COMMENTARY

Background Information

Structural DNA nanotechnologyDNA molecules are not merely associated

with genetics and the carrying of information.They have been used as excellent constructionunits in structural DNA nanotechnology due totheir unique structural motifs, robust physic-ochemical properties, and self-assembling na-ture. The field of DNA nanotechnology waspioneered by Seeman, who laid a theoret-ical framework for the use of DNA as ananoscale building material (Seeman, 1982).Subsequently, DNA was used in the prepara-tion of increasingly complex shapes including2D arrays with 8 to 16 unique positions andless than 20 nm spacing (Chworos et al., 2004;Park et al., 2006), and 3D shapes, such as acube (Chen and Seeman, 1991) and truncatedoctahedron (Zhang and Seeman, 1994). Thesestructures were decorated by spatially posi-tioning the nanoparticles (Sharma et al., 2006)and functional molecules (Liu et al., 2005;Williams et al., 2007) with sub-nanometer pre-cision aiming the construction of functional-ized materials. They have also been used fora variety of applications such as molecularmechanics and synthetic chemistry and biol-ogy (Seeman, 2003; Feldkamp and Niemeyer,2006; Endo and Sugiyama, 2009). Most ofthese studies were focused on the use of flexi-ble and linear DNA as templates. Nevertheless,these structures offer relatively small area,which is not sufficient for diverse applications,and a larger assembly with a size of a few mi-crometers is required for the preparation ofpractical devices. For example, conventionalphotolithography techniques require a size do-main of 1 μm. In addition to the larger surfacearea, it is desirable to use more rigid DNAnanostructures rather than the flexible one.To circumvent these obstacles, an importantmilestone for the advance of DNA nanotech-nology was recently demonstrated by Rothe-

mund who developed a versatile and simplemethod of self-assembly, the so called “scaf-folded DNA origami” (Rothemund, 2006).Using this technique, the preparation of de-fined larger assemblies of almost any arbi-trary shape (Fig. 12.9.1) in 2D space with thesize ∼100 nm in diameter can be carried out.As the name implies, a single-stranded viralgenome (M13mp18) serves as a scaffold inthis method. Hundreds of predesigned shortoligomers hybridize with the scaffold strandthrough complementary base pairing to formmany branched junctions between adjacent he-lices. A “one-pot” nanomolar-scale synthesisyields one hundred trillion origami tiles witha yield of nearly 100%. This method has beensuccessfully utilized for the preparation of var-ious 2D (Endo et al., 2011a; Liu et al., 2011;Rajendran et al., 2011a) and later 3D assem-blies (Andersen et al., 2009; Douglas et al.,2009b ; Endo et al., 2009, 2011b) of custom-designed shapes. One of the most salient fea-tures of the origami is that each position ofthe structure has a precise address by meansof sequence codes of the staples. The numberof staples defines the number of such uniquepositions on the origami surface, and thusthese structures possess over 200 positions.Thus, each staple strand can serve as an at-tachment point for different kinds of nanoob-jects, making the method suitable for a widerange of applications. These structures havebeen used for several applications includingthe self-assembly of nanoparticles (Ding etal., 2010; Pal et al., 2010), proteins (Chhabraet al., 2007; Kuzuya et al., 2009), and variousfunctional components (Stephanopoulos et al.,2010; Maune et al., 2010) into deliberately de-signed patterns.

Bottom-up approachIn the early history of DNA nanotech-

nology, DNA was used for the preparationof the 2D array with less than 20 unique


12.9.16


addressable positions (Chworos et al., 2004;Park et al., 2006). Though such a small struc-ture was used for various purposes, their appli-cations were limited due to the limited numberof functionalities that can be incorporated intosuch a structure. The addressable positionswere scaled up by the recent development ofthe “scaffolded DNA origami” method, whichdrastically improved the size of the nanos-tructure about 10-fold (over 200 unique posi-tions; Rothemund, 2006). The bottom-up ap-proach we have developed with the 1D and2D self-assembly of origami structures fur-ther improved the addressable positions, lead-ing to ∼2000 such specific addressable regionson the self-assembled structures (Endo et al.,2010; Rajendran et al., 2011a).

Purification, storage, and stabilityAs explained before, the origami structures

can be purified using a gel filtration method.Alternatively, they can be purified by gel elec-trophoresis. Due to the smaller size of thestaples and the scaffold strand, they migratefaster in the gel, while the rigid and tightlypacked origami structures migrate slowly, asthey are higher in molecular weight. Theself-assembled structures are difficult to pu-rify by either of these methods, and a pu-rification method should be developed. Theorigami structures can be lyophilized andstored under freezing conditions. When re-quired, they can be redissolved and used. Weanticipate that the self-assembled structurescan also be lyophilized and stored for futureuse. It has been demonstrated recently thatthe DNA origami retain their structure and re-main folded against nuclease digestion (Castroet al., 2011) and in the presence of cell lysate(Mei et al., 2011), making them suitable forbiological studies. However, these structuresare thermally stable only up to ∼55◦C. Thus,strategies for the improvement of the thermalstability of origami structures are indeed re-quired. We have recently developed a methodto induce the thermal stability of the origamibased on the photo-cross-linking. After irre-versible cross-linking, the origami structureswere found to be stable over 85◦C (Rajendranet al., 2011b).

ApplicationsThe origami structures were successfully

used for the nanopatterning of gold nanoparti-cles, proteins, virus capsids, and various func-tional components into a deliberately designedpattern. They can also act as templates for thegrowth of nanowires, aid in the structural de-

termination of proteins, and provide new plat-forms for the genomic applications (Douglaset al., 2007). They have been used for thesingle-molecular analysis of various chemi-cal and biochemical reactions and functions(Rajendran et al., 2012). Due to their resistanceagainst enzymes and cell lysate, it is possible touse these structures at the cellular level. As wehave proved, these structures can also be usedfor the higher temperature applications. Al-though the self-assembled structures have notyet been used, we believe that these structureswill be very useful for various bio and nanoapplications. In addition, this bottom-up ap-proach can be combined with top-down tech-nologies, such as the lithographic technique,to realize the functionalized materials (Hunget al., 2010).

DrawbacksPrimary methods for the size expansion

(1 μm or larger) of the origami structures arelimited. However, a larger structure may be re-quired to increase the addressability even morefor the patterning of multiple functionalities si-multaneously. Self-assembly can achieve thistask efficiently; however, it may be subjectedto kinetic and thermodynamic limitations. Liuet al. described one example of the preparationof a larger 2D origami structure with net-likeshape (Liu et al., 2011), whereas strategies forthe construction of sheet-like structures withdefined size, controllable growth, and higheryield are expected.

Critical Parameters andTroubleshooting

Since thousands of base pairs are involvedin the origami method, it is complex and er-ror prone. Thus, it is recommended to use acomputer program for the design of origamistructures and staple strands rather than themanual design. The PCR tubes and pipet tipsshould be enzyme-free and autoclaved beforethe experiments. Prevent the evaporation of thesolvent and drying up of the sample during theannealing. This can be done by maintainingthe lid temperature of the thermal cycler hotterthan the sample. Sephacryl S-300 solution isnot homogeneous and needs vigorous shakingbefore use. The exclusion limit of sephacrylS-300 is 118 bp and sequences shorter thanthe exclusion limit will be retained by the gel.Since the origami method uses 32-mer sta-ples in general, sephacryl S-300 is suitable. Ifrequired, sephacryl S-200, S-400, S-500, andS-1000 can be used for the exclusion limit of30, 271, 1078, and 20,000 bp, respectively.


12.9.17


This gel filtration column may be used fora sample volume between 20 to 75 μL. Re-covery may reduce with volumes <20 μL or>75 μL. The centrifugation temperature of15◦C may be required to ensure the foldedstructure of origami. In case of 1D and 2D self-assembly, the self-assembly will not be suc-cessful in the absence of connecting strands.Failure to remove the excess staples before theself-assembly may lead to a decrease in theproduct yield. Wrong orientation of the tenonand mortise (e.g., tenon and mortise kept atdifferent rows) will yield no assembled prod-uct due to the shape-fitting factor and ineffi-cient π -stacking. The best annealing condi-tion for the self-assembly lies in the range–0.2 to –0.05◦C/min, and a slow annealingrate, such as –0.01◦C/min, may lead to aggre-gation. The melting point of the origami struc-ture is ∼55◦C and the self-assembly shouldbe performed below this temperature. Anyhigher temperature will lead to a nonspecificand damaged structures.

Anticipated ResultsBasic Protocol 1 gives a deep understanding

of the design strategies of monomer origami,as well as their self-assembly in 1D and2D space. In Basic Protocol 2, a “one-pot”nanomolar-scale synthesis yields one hundredtrillion monomer origami tiles with the yieldof nearly 100%. Regarding the size of theorigami structures, ∼100 × 80 nm can beobtained, though the dimensions can be var-ied (within this size limit) by the design. The1D self-assembly may result in the final struc-ture with the yield of ∼20% and more than10 monomer units can be assembled with thelength of >1 μm (using method 1 of Basic Pro-tocol 3, self-assembly of single DNA origamiJP monomer). In case of the self-assembly ofdifferent DNA piece monomers (method 2 ofBasic Protocol 3), the yield of the final struc-ture could be ∼20% and the length of the prod-uct is ∼600 nm, as only five JP monomers wereassembled. The yield of the 2D self-assembledproduct, as described in the Basic Protocol 4,could be ∼35% and its dimension is ∼365 ×260 nm.

Time ConsiderationsThe design of an origami structure using a

computer program can be done in 2 to 4 hr.Synthesis and purification of the staples mayrequire less than 10 days if it is purchased froma reputable company. The origami can be pre-pared in 1 to 2 hr. One-step self-assembly canbe accomplished in ∼6 hr and two-step assem-

bly in ∼12 hr. Purification of the origami struc-ture by gel filtration can be done in 30 min.Lyophilization of the origami samples may re-quire an overnight. The time required for AFManalysis of a solution is ∼30 min.

AcknowledgmentsThe authors express their sincere thanks for

the CREST grant from the Japan Science andTechnology Corporation (JST), grants for theWPI program (iCeMS, Kyoto University), andfor the global COE program from the Min-istry of Education, Culture, Sports, Scienceand Technology (MEXT), Japan.

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