Litters of self-replicating origami cross-tilesRebecca Zhuoa,b,1, Feng Zhoua,b,1,2, Xiaojin Hea,b,1, Ruojie Shab, Nadrian C. Seemanb,2, and Paul M. Chaikina,2

aCenter for Soft Matter Research, New York University, New York, NY 10003; and bDepartment of Chemistry, New York University, New York, NY 10003

Edited by Jack W. Szostak, Massachusetts General Hospital, Boston, MA, and approved December 20, 2018 (received for review July 26, 2018)

Self-replication and exponential growth are ubiquitous in naturebut until recently there were few examples of artificial self-replication. Often replication is a templated process where aparent produces a single offspring, doubling the population ineach generation. Many species however produce more than oneoffspring at a time, enabling faster population growth and higherprobability of species perpetuation. We have made a system ofcross-shaped origami tiles that yields a number of offspring, fourto eight or more, depending on the concentration of monomerunits to be assembled. The parent dimer template serves as a seedto crystallize a one-dimensional crystal, a ladder. The ladder rungsare then UV–cross-linked and the offspring are then released byheating, to yield a litter of autonomous daughters. In the comple-ment study, we also optimize the growth conditions to speed upthe process and yield a 103 increase in the growth rate for thesingle-offspring replication system. Self-replication and exponen-tial growth of autonomous motifs is useful for fundamental stud-ies of selection and evolution as well as for materials design,fabrication, and directed evolution. Methods that increase thegrowth rate, the primary evolutionary drive, not only speed upexperiments but provide additional mechanisms for evolving ma-terials toward desired functionalities.

self-replication | exponential growth | cross-tile DNA origami | 1D crystal |multiple offspring

The interest in self-replication covers many areas, from thesearch for the origin of life to processes of use in materials

science and mass production of nanostructures. Typically repli-cation uses a copying process which gives a single copy and anamplification of 2 per copying event. Recent interest in artificialself-replication has led to systems and proposals which produceexponential growth of molecules (1–12), information (5, 13, 14),vesicles (15), fibers (16), and DNA constructs (17–20). In pre-vious work (20), a self-replication system was developed usingrectangular DNA origami (21) rafts and an exponential ampli-fication, approximately doubling each cycle. In the present pa-per, we adapt this technique to two systems and expand it to yieldeither more daughters per cycle, i.e., litters, or a shorter cycletime, both providing ways of enhancing the number of offspringper unit time. The litter-producing technique is a synthesis of tworeplication schemes: crystal growth that replicates the in-formation content of DNA tiles but not the separate individualparental motif (13), and templated replication (20) which copiesboth the physical construct and the information content ofthe parent.

Results and DiscussionThe basic replication scheme remains the same as in ref. 20. Abath contains monomer DNA origami tiles functionalized withDNA single strands (sticky ends) for horizontal and verticalbinding to other complementary monomer tiles. For the workdescribed here, “horizontal” and “vertical” refer to directions inthe plane of the cross-shaped origami tile. A seed dimer, ahorizontally bonded set of monomers, is introduced into thebath. On cooling the system, the dimer vertically binds and holdsin place two complementary monomers. While the system is coldthe bound monomers bind horizontally. Four positions on thehorizontal sticky ends are composed of the 3-cyanovinvylcarbazole

nucleoside (cnvK) (20, 22) which, when UV activated, can cross-link to a thymine nucleotide on the complementary sticky end,hence covalently linking monomers into daughter dimers. Uponheating the system, the vertical bonds are released and the seed/parent dimer and the daughter dimers are released. The daughterdimers can then act as parents for the next generation, ideallydoubling the number of parent dimers each cycle/generation.The demonstration of replication and exponential growth in

ref. 20 was done with a rectangular origami molecule, essentiallya raft of 12 double-stranded helices laced together with “staplestrands.” It is known that interorigami binding is best accom-plished along the axes of the double helices. Therefore, for therectangular rafts we utilized the short edges of the rectangle forhorizontal bonds and the top and bottom faces for vertical bonds.In the present work we make use of “cross-tile” origami (23) withdouble helices along both perpendicular directions of the cross. Inthis configuration we can have the more stable bonding along theouter four edges of the cross. This also leaves the two faces foradditional binding for other uses and more flexible designs.We first use our cross-tile system to repeat our previous self

replication of a seed dimer with an amplification factor of 2. Theprocess is illustrated in Fig. 1. The seed dimer (red tile in Fig.1A) consists of two individual monomers connected by a set of11-bp horizontal sticky ends. The melting temperature of theseed dimer is high enough that it remains intact as the templatethroughout the thermal annealing cycle. The set of six verticalsticky ends on each cross-tile of the seed dimer (Fig. 1A, α) al-lows it to bind to two next-generation tiles. The first-generation(FG) and second-generation (SG) tiles, shown in blue and greenin Fig. 1A, contain vertical sticky ends, where α′ on FG is com-plementary to α on both SG and the seed dimer. These monomer

Significance

In nature, self-replication and exponential growth are com-mon. Here we introduce a system of artificial self-replicatorsbased on DNA origami cross-tile motifs. Unlike previous sys-tems based on making a single copy of a parent template, thecross-tile systems, like many natural species, can produce “lit-ters” of offspring per generation. The cross-tiles enable aladder-like structure to crystallize from the template on cool-ing. UV exposure covalently binds the assembled componentsof the offspring and heating releases the autonomous daugh-ters from the parent, up to 10 offspring per cycle. The enhancedgrowth rate speeds up experimental studies and provides anevolutionary advantage for selection.

Author contributions: R.Z., F.Z., X.H., R.S., N.C.S., and P.M.C. designed research; R.Z., F.Z.,and R.S. performed research; R.Z., F.Z., X.H., R.S., N.C.S., and P.M.C. analyzed data; andR.Z., F.Z., X.H., R.S., N.C.S., and P.M.C. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Published under the PNAS license.1R.Z., F.Z., and X.H. contributed equally to this work.2To whom correspondence may be addressed. Email: zfedward1988@gmail.com, ned.seeman@nyu.edu, or chaikin@nyu.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1812793116/-/DCSupplemental.

Published online January 23, 2019.

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tiles have a set of unique horizontal sticky ends: β and its com-plement β′ on FG, and γ and its complement γ′ on SG. There aresix horizontal sticky ends in each set on both the left and rightside of each tile. Four of the horizontal sticky ends contain CNVK.The CNVK units are positioned diagonally across from thymine onthe complementary set, such that when the sample is subjected toUV light, the daughters are linked together covalently (Fig. 1B)(20, 23). When the seed dimer tiles capture two FG tiles by ver-tical sticky-end hybridization at a low temperature, a tetramerstructure is formed. The FG tiles are then held close together,which increases the local concentration and results in their hori-zontal base pairing. In this sense the seed acts as a catalyst forbinding the monomers to form the daughter dimer. The mono-mers in suspension are at a low concentration so that they willnot bind to each other in the thermal annealing temperaturerange. Upon UV exposure, the hybridized horizontal stickyends on the FG tiles are cross-linked to form dimers. Afterheating, the seeds and daughters separate, and the new FGdimers can now act as templates to produce later-generation di-mers. The later-generation dimers will follow the same replicationcycling, ideally doubling the number of dimers each cycle. Avaluable feature of the cross-tiles is that they contain a thickenedportion that appears as an “equal sign,” visible on the atomic forcemicroscopy (AFM), thereby constituting an orientational label foreach tile. The equal signs are visible as dark stippled areas in Fig.1A and in other figures.

To implement this approach experimentally, the seed dimer,daughter, and tetramer tiles were formed and characterized us-ing AFM, as shown in Fig. 2A. Next, we used a thermal annealingprotocol (Materials and Methods) to form the tetramer structurebetween seed dimers and FG tiles. A distinct tetramer band on anondenaturing agarose gel run at 18 °C, which is the temperaturethat UV photo–cross-linking is carried out, suggests the stabilityof the tetramer structure (Fig. 2B, lane 3). Furthermore, the FGtile in lane 2 shows a single monomer band, demonstrating thatthere is no horizontal binding between the monomers. The tet-ramer structure can be separated vertically by heat (SI Appendix,Fig. S3). Fluorescence resonance energy transfer (FRET) resultsindicate that the melting temperature of the vertical sticky endsis around 35 °C (SI Appendix, Fig. S5).We conducted the exponential growth experiments by using

three different initial ratios (1:8:6, 1:16:14, and 1:32:30) betweenthe seed dimer, FG monomers, and SG monomers, respectively.Ideally for the one-sided design the ratio seed:FG:SG is 1:2M:2M-2,producing 2M dimers for M cycles, but typically we put in enoughmonomers for one additional cycle. The number of cycles that wererun for each ratio was determined by when, theoretically, half of themonomers would be consumed. To quantify the amplification fac-tor, we divided the fraction of dimers in each cycle by the initialfraction of seed dimmers:

N  = fn=f0.

N: amplification factor;

fn: fraction of dimers after n cycle of self-replication;

f0: fraction of dimers before self-replication;

fn = number of dimers/(number of dimers + number ofmonomers/2).

For ratios 1:8:6 and 1:16:14, the amount of dimers andmonomers was counted from AFM images (SI Appendix, Figs. S6and S7). To calculate the dimer fraction, the total number ofdimers was multiplied by two and then divided by the totalnumber of monomers. At least 1,000 tiles were counted per cyclefor both ratios. For ratio 1:32:30, the dimer percentage for eachcycle was quantified by comparing the intensity of upper gelbands (dimer, trimer) to the total intensity of the bands in theentire lane (Fig. 2C). The fraction of monomers/dimers is de-termined by the integration of the gel intensity plot using ImageJas discussed and illustrated in SI Appendix, Fig. S4. The plot inFig. 2D shows the amplification factor over the course of self-replication cycling for the different ratios, with each ratio averaging∼1.7 per cycle. Trimers begin to appear after the first cycle becauseit is then possible for the seed dimer to hybridize with both a singledaughter monomer and a cross-linked daughter dimer.After demonstrating the success of this cross-tile self-replication

system, we proceeded to enhance the replication efficiency. Be-cause the dimer tiles can only pick up two daughter tiles on onevertical side, the theoretical maximum amplification rate per cycleis 2. To exceed this limit, we extended the other vertical edge withthe same set of vertical sticky ends on the tiles, as shown in Fig.3A. In this ladder design, the seed dimer and SG tiles now havethe vertical sticky-end set α extending from both the top andbottom, while the FG tile has vertical sticky-end set α′. Whenmixed together, they can hybridize to form railroad-track–likestructures of a variable length. The seed dimer, which serves asthe parent template in the initial stage, is absent from this sche-matic to emphasize that the new dimers formed from self-replication can serve as parents in the subsequent cycles. Suc-cessful formation of the ladder structure after hybridization andUV cross-linking was confirmed via AFM (Fig. 3B). Furthermore,to confirm that no replication occurs without the initial seed

Fig. 1. Self-replication of cross-shaped DNA origami tiles. (A) Schematics ofseed dimer (red), FG monomer (blue), and SG monomer (green). The seeddimer (S) contains a set of six vertical sticky ends that extend from the top ofeach tile (α), and is held together by six 11-bp complementary horizontalsticky ends (blue lines on seed tile). Both FG and SG monomers have a uniqueset of six horizontal sticky ends on each side of the tile (β and its complementβ′ for FG, and γ and its complement γ′ for SG), where there is a CNVK unit oneach of the four strands (pink lines). The FG monomer has a set of six verticalsticky ends that extends from the bottom, α′, which are complementary to α.The SG monomer has an identical set of vertical sticky ends to the seed, α. (B)Schematic of FG dimer. When subject to UV light, the CNVK unit can photo–cross-link with thymine on the complementary strand to form a covalentbond. (C) Self-replication cycling. The seed dimer hybridizes with two next-generation monomers (FG) vertically to form a tetramer. After UV irradiation,the two next-generation monomers attached to the seed are chemically cross-linked via CNVK photo–cross-linking reaction. By heating the system to 48 °C,the tetramer will separate into the seed dimer and the FG dimer. The cross-linked dimers can then act as seeds to generate further generations.

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dimer, a sample containing only FG and SG monomer tiles wassubject to replication cycling. The results show a faint dimer bandon a nondenaturing agarose gel (SI Appendix, Fig. S10), which issubstantially lower than when the seed dimer is used (SI Appendix,Fig. S13).To start the replication using the ladder design, we used two

population pools: the starting ratio was 1:32:30 and 1:64:62 be-tween seed: FG:SG, respectively. A serial transfer was conductedafter each cycle, where a portion of the sample was transferred toa fresh pool of daughter tiles to dilute the dimer population to itsinitial percentage. This ensures that there are sufficient mono-mers for replication. A total of six cycles were run for each ratio,and the amplification was quantified by both AFM counting (SIAppendix, Figs. S11–S12) and agarose gel electrophoresis (SIAppendix, Fig. S13). For the two-sided ladder design, the am-plification factor should be proportional to the ratio of mono-mers to seeds. The results from gel quantification are plotted inFig. 3C, showing that the amplification factor per cycle averages∼4 for the ratio of 1:32:30 and ∼8 for the ratio of 1:64:62. Withthe ladder design, the amplification factor is no longer limited to2, and leads to a system that achieves a much greater amplifi-cation factor per cycle. The plot in Fig. 3D shows the overallamplification factor versus the cycle number, obtained from gelquantification and AFM counting data. After six cycles, the ratioof 1:32:30 (green, blue lines) results in an ∼2,700-fold overallamplification, while the ratio of 1:64:62 (red, purple lines) resultsin an ∼270,000-fold overall amplification.

In addition to the ladder design that exceeds the replicationlimit of 2 for each temperature–UV cycle, we also performedexperiments to optimize yield by decreasing the annealing ramptime to accomplish more cycles within the same amount of time.The previous slow-annealing method (20) used a long annealingtime, about 30 h, to assure “complete” specific self-assembly ofthe tetramer structure (parent dimer + two daughter mono-mers). However, subsequent experiments reported here showthat the tetramer structure can be formed within a 40-min iso-thermal annealing period after heating the seed dimer and FGtiles at 48 °C for 30 min (Fig. 4A). Since the tetramer structure isthe platform for self-replication, the kinetics of the binding be-tween vertical sticky ends to form the tetramer structure and thepairing between the horizontal sticky ends for cross-linking areessential. FRET results show that the binding of vertical stickyends occurs rapidly (Fig. 5 A–C): 71% of the vertical sticky endsbind after 5 min of isothermal annealing at 16 °C, and 90% ofvertical sticky-ended binding is completed after 40 min of iso-thermal annealing, compared with the slow-annealing method.Vertical sticky-end binding saturates at ∼91% after 40 min, in-dicating that the majority of vertical binding for tetramer struc-ture formation is completed within 40 min. Furthermore, theFRET results of horizontal sticky-end binding show similar ki-netics through isothermal annealing (Fig. 5 D–F). As indicatedby the FRET signal, binding increases rapidly at the beginning,and reaches a plateau of 97% after 40 min, indicating that 40 minof isothermal annealing is sufficient to form the tetramer struc-ture for self-replication.

Fig. 2. Self-replication of cross-shaped DNA origami seeds. (A) AFM images of seed dimers, FG monomers, and seed-FG tetramers with their respective schematicdrawings are shown from left to right. (Inset) The AFM image of seed dimers shows the orientation of equal sign in the target dimer structure. (Scale bars, 200nm.) (B) A nondenaturing agarose gel (0.8%) shows the formation of the tetramer between a seed dimer and two FG monomers (lane 3). Lane 1 contains dimerseed, and lane 2 contains FG monomer. The gel was run at 18 °C. (C) A nondenaturing agarose gel showing the amplification and quantification of dimers in fourcycles, with a starting ratio of 1:32:30 between seed:FG:SG. Lane 1 and lane 2 contain dimer seed and FG monomer, respectively. Lanes 3–5 contain the mixturesfrom cycles 0, 2 and 4, respectively. The intensity of the band showing dimers increases as the cycling proceeds. The gel was run at 48 °C. (D) Plot showing the totalamplification factor at each cycle for different ratios between seed:FG:SG. The green, blue, and red curves represent the replication cycling containing seeds, FGtiles, and SG tiles with the ratios of 1:8:6, 1:16:14, and 1:32:30, respectively. The green and blue curves were obtained by evaluating AFM images, and the red curvewas collected from quantification of the gel shown in C. Each curve shows an average replication rate of ∼1.7 per cycle.

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Upon UV exposure for 20 min, a systematic study shows thatthe replication rate increases from 1.28 to 1.50 for the prolongedisothermal annealing time from 10 to 40 min; the data werecollected from gel quantification (Fig. 4B). Then, a total of fiveself-replication cycles, by a series transfer after each cycle, wererun using this fast-annealing process of 40-min isothermal anneal-ing, and the amplification was quantified by agarose gel electro-phoresis (Fig. 4 C and D). The replication rate of each cycle wasconsistent, indicating this is a reproducible procedure, and the av-erage replication rate was 1.51. Although we obtained a smallerreplication rate per cycle compared with the current slow-annealingmethod, we were able to accomplish 20 cycles within the sameamount of time. In this manner, the fast-annealing method results inan ∼4,400-fold overall amplification in the time it takes for one cycleof the current method.

ConclusionsWe have introduced two systems for artificial self-replication andexponential growth. Both use the same basic process as in ourprevious work: a reversible temperature cycle and an irreversibleUV cross-linking step at low temperature, which fixes the config-uration of the daughters as template on their parents. The systemshave several advantages. The basic origami tiles can be function-alized with DNA sticky ends on all four edges and on top andbottom faces, allowing templating in many different configurations.However, what we have focused on here is a significant speed-up inyield per unit time. This was accomplished either by increasing the

number of offspring per cycle from ∼2 to ∼8 or by decreasing thetime needed for a cycle. In principle we could optimize both.Exponential growth is a primary driver for evolution. A species

with a higher net growth rate can take over a population. For ex-ample, we start with two systems: one, species “A,”which doubles percycle and another, “B,” which increases eightfold per cycle but only atlow pH. If we only seed A, then only A will grow exponentially.However, if an environmental change, say pH, is introduced so thatthere is a small probability that an A dimer will lead to a B seed, thenB will quickly take over the system as long as the pH remains low.Similar population changes might be done with light using differentUV cross-linking molecules or optical quenchers. Competition alongwith selection may be incorporated by placing the cross-linkingstrands in solution rather than on the origami tiles themselves.The basic processing idea used in litter self-replication is to

assemble a structure by specific reversible hybridization of DNAfunctionalized components and then to link a subset of the DNAcontacts covalently. Heating then releases the covalently linkedmotifs from their scaffold which itself disassembles. This generalprocess may be useful for other replication and assembly schemes.

Materials and MethodsDNA Synthesis and Purification. Single-stranded M13mp18 DNA genome waspurchased from Bayou Biolabs. CNVK was purchased from Glen Research. TheDNA strands containing the CNVK molecule were synthesized on an AppliedBiosystems 394 DNA synthesizer. The remaining DNA strands were purchasedfrom Integrated DNA Technology, Inc. The sticky-end strands were purifiedusing denaturing PAGE.

Fig. 3. Ladder self-replication of cross-shaped DNA origami tiles using a serial transfer. (A) Schematic of the ladder self-replication. The seed dimer, FG (blue)and SG (green) monomer contain their respective vertical sticky-end set on both the top and bottom of each tile, detailed in SI Appendix, Fig. S1. The seed tileand SG monomer will have set α, and the FG monomer will have set α′, as labeled in Fig. 1A. The parent, as a cross-linked dimer, can hybridize with thedaughter tiles in both vertical directions to form a ladder. When exposed to UV light, a cross-linked ladder is formed. After heating to 48 °C, the ladderseparates vertically into individual cross-linked FG and SG dimers. These can then serve as parents as the cycle repeats. The seed dimer, not shown in thisschematic, can act as a parent in the same way. (B) AFM images of the cross-linked ladder. (Scale bars, 200 nm.) These are taken from green-bar (1:32:30)experiments. (C) Column chart showing the amplification factors after each transfer and cycle of self-replication for both the ratio of 1:32:30 between seed:FG:SG (green) and the ratio of 1:64:62 (purple) from gel quantification. At transfer 0 cycle 1, the dimer percentage amplifies by ∼fourfold for the ratio of1:32:30 and ∼sixfold for the ratio of 1:64:62, and the chart shows this repeated cycling until cycle 6. (D) Overall amplification of a serial transfer experiment.Overall amplification curves were obtained by six transfer cycles, resulting in an ∼2,700-fold amplification for the sample of the ratio 1:32:30 (green curveobtained from gel quantification, blue curve obtained from evaluating AFM images), and 270,000-fold amplification for the sample of the ratio of 1:64:62(purple curve obtained from gel quantification, red curve obtained from evaluating AFM images).

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Formation of Individual DNA Origami Tiles. Staple strands and M13mp18 DNAgenome were mixed together in 1X TAE/Mg2+ buffer (40 mM Tris·HCl, 20 nMacetic acid, 2.5 mM EDTA, 12.5 mM magnesium acetate, pH 8.0). The molarratio M13mp18 DNA genome:staple strand was 1:6, and the final concen-tration of the mixture was 10 nM. The mixture was heated to 70 °C for

20 min and cooled to 22 °C at a rate of −7 °C/h in a thermocycling incubator.The resulting origami tiles were purified in 100K Millipore Amicon Ultra0.5-mL centrifugal filters using 1X TAE/Mg2+ (16 mMmagnesium acetate) forbuffer exchange.

Formation of Seed Dimer. The individual left and right sides of the seed dimerorigami tiles were mixed in a 1:1 ratio. The mixture was heated to 53 °C for20 min, and cooled to 22 °C at a rate of −0.7 °C/h.

Self-Replication Cycling. (i) Seed dimers, FG tiles, and SG tiles were mixedtogether in a specified concentration ratio (i.e., seed dimer: FG:SG = 1:8:6).(ii) The solution was heated to 50 °C for 20 min, cooled to 22 °C at a rate of−1 °C/h, and further cooled to 18 °C at a rate of −3 °C/h. (iii) The sample wasplaced in an 18 °C water bath and exposed to 366-nm UV light (UVP, modelXX-15L, 15W). (iv) 5 μL of sample was taken out for AFM imaging. (v) Stepsii–iv were repeated with the same sample for further cycles.

Self-Replication Cycling Using a Ladder Design with Serial Transfers. (i) Seeddimers, FG tiles, and SG tiles were mixed together with either a ratio of1:32:30 or 1:64:62 between seed dimer to FG and SG tiles. (ii) The solutionwas heated to 50 °C for 20 min, cooled to 22 °C at a rate of −1 °C/h, andfurther cooled to 18 °C at a rate of −3 °C/h. (iii) The sample was placed in an18 °C water bath and exposed to 366-nm UV light (model XX-15L, 15W;UVP). (iv) For both AFM imaging and nondenaturing 0.8% agarose gelelectrophoresis, 10 μL of sample was taken out. (v) The sample was analyzedunder AFM to determine the resulting amplified seed dimer amount. (vi) Anadditional 10 μL of sample was transferred to a new tube, and the seeddimer was diluted to its initial concentration ratio by adding FG and SG tiles.(vii) Steps ii–vi were repeated with the same sample until five transfers werecompleted.

AFM Imaging.AFM imagingwas performed in tappingmode in air for the self-replication cycling, and in buffer for the images of the cross-linked ladder. (i)Tapping in air: 2–5 μL of a diluted sample was deposited on a clean micasurface (Ted Pella, Inc.) for 1 min. Unless specified, the sample and mica wereincubated at 48 °C for 30 min and the deposition was conducted at 48 °C.

Fig. 5. Kinetic study of self-assembly of seed and FG during the fast-annealing process through FRET. (A) Schematics of formation of cy3–cy5 vertical pairthrough hybridization between seed–cy3 (donor) and FG–cy5 (acceptor). (B) Fluorescence spectra of seed–cy3 (donor alone) and seed-FG tetramer (cy3–cy5vertical pair). (C) Fraction of the cy3–cy5 vertical pair formed through the hybridization between the seed–cy3 and FG–cy5 over the first 60 min afterannealing at 16 °C. (D) Schematics of formation of cy3–cy5 horizontal pair through hybridization between cy5-FG–cy3. (E) Fluorescence spectra of cy5-FG–cy3(donor–acceptor separated) and seed-FG tetramer (cy3–cy5 horizontal pair). (F) Fraction of the cy3–cy5 horizontal pair formed through the hybridizationbetween the cy5-FG–cy3 over the first 60 min after annealing at 16 °C.

0.5

1

1.5

2

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T0 T1 T2 T3 T4

Dimer % before crosslinking

Dimer % after crosslinking

replication rate

500 nm

A B

C D

1.1

1.2

1.3

1.4

1.5

1.6

0 10 20 30 40 50

Rep

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Annealing time (min)

Dim

er %

Re plicat io n rate

T0C0 T0C1 T1C0 T1C1 T2C0 T2C1 T3C0 T3C1 T4C0 T4C1

Fig. 4. Self-replication of cross-shaped DNA origami using fast-annealingprocess. (A) AFM image of formation of seed-FG tetramer through 40-minfast annealing process. (B) Replication rate of DNA origami corresponded tothe annealing time from 10 to 40 min, obtained from gel quantification. (C)Gel image of five-cycle fast-annealing self-replication of DNA origami usinga serial transfer. T0C0 in the image represents transfer 0 cycle 0. The gel wasrun at 48 °C (D) Plot showing the change of the dimer percentage andreplication rate of each cycle of fast-annealing self-replication, obtainedfrom gel quantification of C.

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The mica was then washed with 50–70 μL of ddH2O three times, using filterpaper to absorb the water. The mica was subsequently dried using com-pressed air. (ii) Tapping in buffer: 2–5 μL of diluted sample (∼0.5 nM) wasdeposited on a clean mica surface for 1 min. Then, 30 μL of buffer was addedon the mica surface, and an additional 30 μL of buffer was added on theliquid cell. Tapping in air and buffer modes was performed on both theNanoscope IV Multimode SPM and the Nansocope V Multimode 8 scanningprobe microscope (PeakForce QNM Software, ScanAsyst-HR accessory). Sili-con tips (Veeco, Inc.) were used for the Nanoscope IV Multimode SPM, andsilicon nitride tips (ScanAsyst-Fluid, ScanAsyst-Air; Bruker Nano, Inc.) wereused for the Nanoscope V Multimode 8 SPM.

Fast-Annealing Self-Replication. Each fast-annealing self-replication cycleconsists of three steps: (i) heating at 48 °C for 30 min to break vertical sticky-ends binding; (ii) fast isothermal annealing at 16 °C for 40 min to formtetramer structure; and (iii) UV cross-linking conducted with a 365-nm UVLED (M365LP1c, power intensity is 24 mW/mm2; Thorlabs Inc) at 18 °C for20 min. The concentration of the FG tiles was maintained as 6.4 nM, and theconcentration of seed and SG tiles was prepared according to the relativeratio. Fresh FG and SG tiles were replenished after each cycle.

FRET Experiment. FRET experiment was conducted using Horiba PTI Quan-taMaster 400 Fluorescent Spectrometer. Cy3 (donor) and Cy5 (acceptor) dyewas modified on the vertical and horizontal sticky ends to make seed-cy3(donor), FG-cy5 (acceptor), and cy3-FG-cy5 (donor acceptor). The seed con-centration was 3.2 nM and the FG and SG tiles were 6.4 nM throughout theFRET experiment. For the thermodynamic study of DNA binding (SI Appen-dix, Fig. S5), the sample was ramped from 20 °C to 48 °C, then back to 20 °Cfor three cycles. The ramping rate was 3 min/°C, and the fluorescent signalwas measured at each degree. For the dynamic study of fast-annealing ex-periment (Fig. 5), the sample was heated at 48 °C for 30 min and transferredto the fluorimeter at 20 °C to start recording the fluorescent signal imme-diately. We first measured the fluorescent signal of donor alone (ID), then

the donor–acceptor pair (IDA) subsequently. The FRET efficiency (E) wascalculated as follows:

E= 1−IDAID

.

Then the fraction of sticky-ends DNA binding (f) was

f =E− Eð0%Þ

Eð100%Þ− Eð0%Þ,

where E(0%) indicates the FRET efficiency obtained at 48 °C where there isno sticky-end DNA binding. E(100%) indicates the FRET efficiency obtainedat 20 °C after slow annealing where all of the available sticky-end DNA arehybridized.

ACKNOWLEDGMENTS. This research has been primarily supported byDepartment of Energy (DOE) DE-SC0007991 (to P.M.C., N.C.S., R.S., R.Z.,F.Z., and X.H.) for initiation, design, analysis, and imaging. P.M.C. and X.H.acknowledge support from Center for Bio-Inspired Energy Sciences, an En-ergy Frontier Research Center funded by the DOE, Office of Sciences, BasicEnergy Sciences, under Award DE-SC0000989, for initiation, DNA sequencedesign, preparation, and characterization of confocal microscopy. R.S.,N.C.S., and X.H. acknowledge partial support from NSF Emerging Frontiersin Research and Innovation (EFRI)-1332411 and Division of Computing andCommunication Foundations (CCF)-1526650 for laboratory supplies, underAward Division of Materials Research (DMR) DMR-1420073 for synthesisand characterization of the DNA origami. R.S. and N.C.S. acknowledge Mul-tidisciplinary University Research Initiatives (MURI) W911NF-11-1-0024 from theArmy Research Office, MURI N000140911118 from the Office of Naval Researchfor partial salary support. R.S. and N.C.S. acknowledge partial support fromRGP0010/2017 from Human Frontiers Science Program, R.S. and N.C.S. ac-knowledge partial support from DOE DE-SC0007991 for DNA synthesis andpartial salary support. The authors are grateful for shared facilities providedthrough the Materials Research Science and Engineering Centers program ofthe NSF under Award DMR-1420073.

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