Self-replication of nucleic acid origami tiles

Abstract

The present invention provides a method for self-replication of multimers of nucleic acid origami tiles by exponentially amplifying the multimer from initial seeds of monomeric units of nucleic acid origami tiles and also provides for the selective exponential amplification of a designated multimer, such as with specific properties or characteristics, over one or more competing multimers in the presence of a mixture of monomers for each of the possible multimers. The selection of the designated multimer based on an environmental change allows the designated multimer to outgrow all competing multimers.

Claims

1. A method for exponential self-replication of nucleic acid origami tiles, comprising: (i) providing a set of monomers of seed nucleic acid origami tiles, monomers of first generation daughter nucleic acid origami tiles and monomers of second generation daughter nucleic acid origami tiles, each monomer having a long scaffold strand that is folded by a plurality of short staple strands into an origami tile structure having a plurality of horizontal edges and a plurality of faces, with a plurality of sticky cohesive ends protruding from one or more horizontal edges of the tiles and from one or more faces of the tiles; (ii) forming a multimer from monomers of seed nucleic acid origami tiles by cohesion of complementary horizontal sticky cohesive ends between the edge of one monomer to the edge of another monomer; (iii) mixing the resulting multimer of seed tiles with monomers of first generation daughter (1G) tiles to allow the monomers to anneal to each other by horizontal sticky end cohesion between edges of adjacent 1G monomeric tiles, as enhanced by the 1G monomers first annealing to the multimer of seed tiles by vertical sticky end cohesion between sticky cohesive ends protruding from the faces of adjacent seed and 1G tiles, to form a stacked multimer of seed and 1G tiles; (iv) covalently linking the 1G tiles which are in sticky end cohesion with each other in the stacked multimer; (v) heating to denature the horizontal sticky end cohesion between monomers of seed tiles and the vertical sticky end cohesion between monomers of seed tiles and 1G tiles to separate the heat resistant covalently linked 1G tiles as a multimer of 1G tiles; (vi) mixing the multimer of covalently linked 1G tiles with monomers of second generation daughter (2G) tiles to allow the monomers to anneal to each other by horizontal sticky end cohesion between edges of adjacent 2G monomeric tiles, as enhanced by the 2G monomers first annealing to the multimer of covalently linked 1G tiles by vertical sticky end cohesion between sticky cohesive ends protruding from the faces of adjacent 1G and 2G tiles, to form a stacked multimer of 1G and 2G tiles; (vii) covalently linking the 2G tiles which are in sticky end cohesion with each other in the stacked multimer; (viii) heating to denature the vertical sticky end cohesion between the multimer of 1G tiles and the multimer of 2G tiles in the stacked multimer to separate the multimers of covalently linked 1G tiles and covalently linked 2G tiles; (ix) incubating the separated multimers with monomers of 1G tiles and 2G tiles to allow the monomers of 1G and 2G tiles to anneal respectively to other 1G and 2G monomers by horizontal sticky end cohesion between edges of adjacent monomers tiles and to a multimer of covalently linked 1G or 2G tiles by vertical sticky end cohesion between sticky cohesive ends protruding from the faces of adjacent 1G and 2G tiles to form stacked multimers of 1G and 2G tiles; (x) for 1G and 2G tiles not already covalently linked, covalently linking 1G tiles in horizontal sticky end cohesion to each other and covalently link 2G tiles in horizontal sticky end cohesion to each other in the stacked multimers; (xi) heating to denature the vertical sticky end cohesion between multimers of covalently linked 1G tiles and multimers of covalently linked 2G tiles; (xii) repeating steps (ix)-(xi) one or more times to self-replicate and exponentially amplify multimers of nucleic acid origami tiles.

2. The method of claim 1, wherein the nucleic acid origami tiles are DNA origami tiles.

3. The method of claim 1, wherein, in step (ix) or in one of the repetitions thereof in (xii), additional amounts of monomers of 1G and 2G tiles are added and mixed with the separated multimers of 1G and 2G tiles.

4. The method of claim 1, wherein the monomers of seed, 1G and 2G tiles are substantially two dimensional with a top and bottom face/surface.

5. The method of claim 4, wherein the monomers are rectangular.

6. The method of claim 1, wherein a hairpin formed from a nucleic acid strand protrudes perpendicularly from a face of the monomer.

7. The method of claim 6, wherein a pendant molecule or moiety is attached to the hairpin.

8. The method of claim 1, wherein the covalently linked 1G and 2G tiles are from photo-crosslinking.

9. The method of claim 8, wherein the photo-crosslinking is with a 3-cyanovinylcarbazole nucleoside.

10. The method of claim 1, further comprising monomers of one or more different sets of seed, 1G and 2G daughter tiles are provided in the same self-replication mixture to selectively amplify a nucleic acid multimer over competing monomers and multimers from the other set(s).

11. The method of claim 10, wherein, for each of the different set(s) of tiles, the monomers each have a sticky cohesive end, which is necessary for forming a multimer, labeled with a different near-infrared dye that produces light-activated local heat generation at a specific wavelength that is different from those of the dyes on competing monomers and multimers of the other sets of tiles.

12. The method of claim 11, further comprising irradiating the tiles at one or more different wavelengths to effect light-activated local heat generation, thereby suppressing sticky end cohesion of competing monomers labeled with dyes that are light-activated at the one or more wavelengths to selectively amplify a multimer in which sticky end cohesion is not suppressed.

13. The method of claim 1, wherein: each monomer is substantially two dimensional with a top and bottom face and at least three horizontal edges; at least one edge of each monomer has a plurality of protruding single stranded nucleic acid ends that serve as sticky ends for annealing to complementary single stranded nucleic acid ends of another monomer so as to form adjacent monomers joined by sticky end cohesion; and the top or bottom face of each monomer has a plurality of single stranded nucleic acid ends that protrude perpendicularly from the plane of the substantially two dimensional monomer (monomeric tile); and the monomers of seed tiles differ from monomers of 1G tiles in the nature of the plurality of single stranded nucleic acid ends that serve as sticky ends for annealing to complementary nucleic acid ends of another monomer.

14. The method of claim 13, wherein: in step (ii), the multimer of seed tiles is formed in a plane with a top and bottom face from the monomers of seed tiles through the annealing of a plurality of sticky ends with nucleic acid sequence complementarity at the adjacent edges of monomeric seed tiles; and the plurality of single stranded nucleic acid ends that protrude perpendicularly from the top or bottom face of each monomeric seed tile are on the same face of the multimer of seed tiles; in step (iii), the formed multimer of seed tiles is mixed with monomers of 1G tiles and the monomers of 1G tiles are allowed to anneal to each other and to the multimer of seed tiles to form a stacked multimer of seed and 1G tiles that serves as a first recognition complex for self-replication, wherein: the annealing of monomers of 1G tiles to each other through sequence complementarity between the plurality of protruding single stranded ends at the edges of adjacent monomeric 1G tiles forms a multimer of 1G tiles in a plane with a top and bottom face; the plurality of single stranded nucleic acid ends protruding perpendicularly from the plane of the substantially two dimensional monomeric 1G tiles are on the same face of the multimer of 1G tiles; and the annealing of monomers of 1G tiles to the monomeric seed tiles in the multimer of seed tiles is through sequence complementarity between the plurality of single stranded nucleic acid ends that protrude perpendicularly from the planes of the monomeric daughter tiles and the monomeric seed tiles so as to form the stacked multimer in which the plane of the multimer of seed tiles is parallel to the plane of the multimer of 1G tiles and joined thereto by sticky end cohesion; in step (iv), at least two of the plurality of annealed sticky ends between adjacent monomers in the multimer of daughter tiles forming the first recognition complex are allowed to react to covalently link with complementary strands in the at least two annealed sticky ends so as to be resistant to heat denaturation at a melting temperature (Tm) which denatures the sticky end cohesion between the monomers of seed tiles and monomers of 1G tiles; in step (v), the sticky end cohesion between monomers of seed tiles and monomers of 1G tiles is denatured by heating at the Tm so as to separate the multimer of 1G tiles, which is resistant to heat denaturation, from the multimer of seed tiles to release the multimers of the first recognition complex; in step (vi), the heat resistant multimer of 1G tiles are allowed to anneal with monomers of 2G tiles and the monomers of 2G tiles are allowed to anneal to each other to form a second stacked multimer of 1G and 2G tiles that serves as a second recognition complex for self-replication, wherein: monomers of 2G tiles are annealed to each other through sequence complementarity between the plurality of protruding single stranded ends at the edges of adjacent monomeric 2G tiles to form a multimer of 2G tiles in a plane with a top and bottom face; the plurality of single stranded nucleic acid ends protruding perpendicularly from the plane of the substantially two dimensional monomeric 2G tiles are on the same face of the multimer of 2G tiles; and the monomers of 2G tiles are annealed to the monomeric 1G tiles in the heat resistant multimer of 1G tiles through sequence complementarity between the plurality of single stranded nucleic acid ends that protrude perpendicularly from the planes of the monomeric 2G tiles and the monomeric 1G tiles so as to form the second stacked multimer in which the plane of the heat resistant multimer of 1G tiles is parallel to the plane of the multimer of 2G tiles and joined thereto by sticky end cohesion; in step (vii), at least two of the plurality of annealed sticky ends between adjacent monomers in the multimer of 2G tiles forming the second recognition complex are allowed to react to covalently link the complementary strands in the at least two annealed sticky ends together so as to be resistant to heat denaturation at the Tm which denatures the sticky end cohesion between the monomers of the 1G tiles and monomers of 2G tiles; in step (viii), the sticky end cohesion between the heat resistant multimer of 1G tiles in one plane and the heat resistant multimer of 2G tiles in a second parallel plane is denatured by heating at the Tm to separate the heat resistant multimer of 2G tiles from the heat resistant multimer of 1G tiles to release the heat resistant multimers of 1G and 2G tiles from the second recognition complex; in step (ix), the heat resistant multimer of 1G tiles and the heat resistant multimer of 2G tiles obtained from step (viii) are mixed with monomers of 1G tiles and 2G tiles with monomers of 1G tiles being allowed to anneal to each other and to the heat resistant multimer of 2G tiles and monomers of 2G tiles being allowed to anneal to each other and to heat resistant multimers of 1G tiles, both forming the second stacked multimer that serves as the second recognition complex, wherein the monomers of 1G and 2G tiles are annealed to corresponding monomers of 1G and 2G tiles through sequence complementarity between the plurality of protruding single stranded ends at the edges of adjacent monomeric 1G or 2G tiles to form a multimer of 1G tiles and a multimer of 2G tiles, both of which have a plane with a top and bottom face; the plurality of single stranded nucleic acid ends protruding perpendicularly from the plane of the substantially two dimensional monomeric 1G tiles and the plane of the substantially two dimensional monomeric 2G tiles are on the same face of the multimer of 1G tiles and the multimer of 2G tiles, respectively; and the monomers of 1G or 2G tiles are annealed respectively to the monomeric 2G or 1G tiles in the multimer of 2G or 1G tiles through sequence complementarity between the plurality of single stranded nucleic acid ends that protrude perpendicularly from the planes of the monomeric 1G tiles and the monomeric 2G tiles so as to form the second stacked multimer in which the plane of the multimer of 2G tiles is parallel to the plane of the multimer of 1G tiles and joined thereto by sticky end cohesion; (x) at least two of the plurality of annealed sticky ends between adjacent monomers in the multimer of 1G tiles and in the multimer of 2G tiles in the second recognition complex are allowed to react to covalently link the complementary strands in the at least two annealed sticky ends together so as to be resistant to heat denaturation at the Tm which denatures the vertical sticky end cohesion between monomers of 1G tiles and monomers of 2G tiles; (xi) the sticky end cohesion between the heat resistant multimer of 1G tiles in one plane and the heat resistant multimer of 2G tiles in a second parallel plane is denatured by heating at the Tm to separate the heat resistant multimers and release them from the second recognition complex; (xii) repeating steps (ix)-(xi) one or more times to self-replicate and exponentially amplify multimers of 1G and 2G tiles.

15. The method of claim 13, wherein: each monomer is a rectangle with four horizontal edges; each monomer has eight single stranded nucleic acid ends that protrude perpendicularly from the same face of the substantially two dimensional rectangular tile for vertical sticky end cohesion to the complementary single stranded nucleic acid ends of another monomeric tile; each monomer of seed tiles has eight horizontal single stranded nucleic acid ends at one edge of the substantially two dimensional rectangular seed tile for horizontal sticky end cohesion to the complementary single stranded nucleic acid ends of another monomeric seed tile; and each monomer of 1G and 2G tiles has six horizontal single stranded nucleic acid ends at each of two opposing edges of the substantially two dimensional rectangular 1G or 2G tile for horizontal sticky end cohesion to the complementary single stranded nucleic acid ends of, respectively, another monomeric 1G or 2G tile, with four of the six horizontal single stranded nucleic acid ends on one edge having a 3-cyanovinylcarbazole nucleoside for photo-crosslinking to the complementary single stranded nucleic acid ends of an adjacent monomer.

16. The method of claim 4, wherein the monomers are planar.

17. The method of claim 1, wherein the monomers are planar DNA origami tiles having a scaffold strand in the range of 5-10 kb in size.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIGS. 1A-1C schematically illustrate DNA origami tile design and self-replication cycling. FIG. 1ASeed and later generation tiles design, (Seed and first-generation tile (A-tile) design, comprising three domains: (1) DNA hairpin structures to create a topographic feature that can be detected by AFM (seed and second-generation tiles labeled with T and first generation tiles labeled with A); (2) vertical sticky ends, which pair with the successive-generation tiles; (3) both T and A have the same horizontal sticky ends as linkers (dotted with .sup.CNVK and dashed) to connect other tiles in the same generation, right side sticky ends complementary to left side. FIG. 1BPhoto-cross-linking with .sup.CNVK Photo-cross-linking reagent in a DNA duplex. FIG. 1CSelf-replication cycling of the dimer seed system, including TT seed formation with special horizontal complementary sticky ends, recognition and hybridization of daughter tiles to seeds with vertical bonds, formation of new-generation dimers using horizontal bonds and the .sup.CNVK photo-cross-linking reaction and separation of the two successive generations by heating the system to 46 C. The left-side cycles include seed tiles and the right-side cycles do not.

(2) FIGS. 2A-2C show self-replication of DNA seeds. FIG. 2ASchematic illustrations and AFM images of a (i) DNA dimer seed TT, (ii) a first generation tile (A-tile) and (iii) a double-layer complex of seed and two first-generation tiles. FIG. 2BThe amplification of dimers (including seeds and later-generations) increased exponentially before leveling off as the supply of substrates was exhausted. Curves with square (and solid line), circles and triangles represent self-replication cycling containing seeds, first-generation and second-generation tiles with ratios of 1:1024:1022, 1:32:30 and 1:16:14, respectively. The curve with squares and a dashed line is the theoretical curve for exponential growth. The dimer amplification factor was calculated at the end of each cycle. FIG. 2CAFM images of self-replication cycles for the dimer system: (i) cycle 0, (ii) cycle 8 and (iii) zoomed-in image of dimers TT and AA in cycle 8. The initial ratio of seed, first-generation tile and second-generation tile is 1:32:30. Scale bars in (i) and (ii) are 500 nm. The scale bar in (iii) is 100 nm.

(3) FIGS. 3A-3C show amplification of a serial transfer experiment. FIG. 3AA self-replication amplification curve that was obtained by six successive replications, allowing approximately 14-fold amplification before transferring 8% of the mixture to the next replication tube, which contained a fresh supply of later-generation monomers. FIG. 3BA non-denaturing agarose gel demonstrating and quantifying the amplification of dimers in replication cycles 8-12, after a transfer experiment. Lane 1 contains a 1 kb DNA marker. Lanes 2 and 3 contain first-generation tile A and dimer seed TT, respectively. Lanes 4-8 contain the mixtures from replication cycles 8 to 12. The intensity of the band representing dimer tiles increases as cycling occurs. The gel was run at 48 C. Plots of dimer amplification versus cycle (cycles 8 to 12) obtained from nondenaturing gel and AFM imaging show that the two curves almost overlap, indicating that the AFM quantification is consistent with the gel quantification (data not shown). FIG. 3CAFM images of the mixtures in cycles 17-20 (scale bars: 500 nm).

(4) FIGS. 4A-4G show self-replication enabling selection. FIG. 4ADesign of first-generation and second-generation tiles (H and I) modified with near infrared (NIR) dyes (IR dyes). IR Dye 800 molecules (dotted circles) and IR Dye 700 molecules (hatched circles) were attached to a monomer I and a monomer H, respectively. (i) For first-generation tiles, four of the dyes are located in the vertical direction and four others are on helper strands in the horizontal direction. CNV-containing strands are shown as dotted lines. (ii) Four IR dyes are all attached to solid line helper strands in the vertical direction for seeds or second-generation tiles. FIG. 4BAFM images of the two competing species seeds II and HH. FIG. 4CReplication selection using different wavelength lasers as environmental factors is indicated schematically. Using the 685-nm laser diode, the species II significantly replicated to produce copies of II. The species HH amplifies faster when the wavelength is changed to 785 nm. FIG. 4DYields of daughter-generation formation under 685-nm or 785-nm laser irradiation. The ratio of template and monomer was 1:2 in all replication vessels. FIG. 4EThe proportion between species HH and II under various environmental conditions. All solid lines and all dashed lines represent fractional results of dimer HH and dimer II, separately. Solid and dashed curves with square data points were measured without laser irradiation, showing the fraction of HH or II is 50% and indicating the replication of both seeds. Solid and dashed curves with diamond data points represent the data obtained under 785-nm laser exposure. The fraction of dimer HH reached above 94%. Dimer II became the dominant species under 685-laser exposure (triangle dashed line), while species HH decreased to less than 10% of all dimer tiles (triangle solid line). Solid and dashed curves with circle data points show the dominant group can be switched after environmental conditions change (laser wavelength changed from 685 nm to 785 nm after the first 2 cycles). FIG. 4FAFM images of purified dimers (from the replication mixtures), HH and II, under 685-nm laser irradiation. FIG. 4GAFM images of purified dimers (from the replication mixtures), HH and II, under 785-nm laser irradiation. Scale bars in FIGS. 4F and 4G are 500 nm.

(5) FIGS. 5A-5D show overlapping quadrants of the rectangular DNA origami base structure in its entirety without any modification (SEQ ID NOs:1-216; FIG. 5A upper left quadrant; FIG. 5B upper right quadrant; FIG. 5C lower left quadrant; and FIG. 5D lower right quadrant) that overlay together at the overlapping portions to form the entire rectangular DNA origami base structure.

(6) FIG. 6 schematically illustrates the structure of the DNA hairpin involved in producing letters (SEQ ID NO:345).

(7) FIG. 7 shows sequences of horizontal sticky ends for first-generation (FG) tiles and second-generation (SG) tiles. Numbers on each generation tile are the label numbers of staple strands modified with horizontal sticky ends in the origami.

(8) FIGS. 8A-8C show AFM images of the single origami tiles. FIG. 8Aimage of origami with T pattern (tapping mode in buffer). FIG. 8Bimage of origami with the A pattern (tapping in buffer). (Scale bars in FIGS. 8A and 8B: 250 nm.) FIG. 8Cimage of the vertical dimer of the origami tile T and A by vertical sticky-end pairing (tapping in air at 4 C., scale bar in FIG. 8C: 500 nm).

(9) FIGS. 9A and 9B are nondenaturing agarose gel showing formation of complex of dimer DNA origami seed/first-generation tiles and daughter generation. FIG. 9AAssembly of dimer DNA origami seed and two first-generation tiles (M: 1000 nt pair marker; Lane 1: DNA first-generation tile A; Lane 2: dimer DNA origami seed TT). Lane 3 and Lane 4 show the cohesion of dimer DNA origami seed and two first-generation tiles after and before photo-cross-linking reaction, respectively. The gel was run at 10 C. FIG. 9BSeparation of seed and daughter tiles (Lane M: 1000 nt pairs DNA marker; Lane 3: DNA first-generation tile A; Lane 4: dimer DNA origami seed TT). The band representing the complex of DNA seed and first-generation tile disappears, leading to successful separation. Lane 1 presents separation of seed and photo-cross-linked daughter. There is no lower band (monomer), which indicates the successful formation of daughter generation by template photo-cross-linking reaction. Lane 2 was loaded with the complex of seed and first-generation tile without UV irradiation. The lower band represents the monomer tile A, showing no cross-linking between adjacent monomer to form daughter generation AA. The gel was run at 48 C.

(10) FIGS. 10A-10D show AFM images of the dimer origami tiles. FIG. 10Adimer seed with TT pattern. FIG. 10BExtra relatively high resolution images of dimer seed TT. There is one line on each side of the T pattern, which corresponds to topography of vertical sticky ends. FIGS. 10C and 10Dvertical dimer of seed TT and first-generation tiles A by vertical sticky-end pairing. Two first-generation tiles were bound to one seed. Scale bar in FIG. 10A is 500 nm. Scale bars in FIGS. 10B-10D are 250 nm.

(11) FIG. 11 shows DNA origami dimer percentage (yield) during replication cycling. Dimer percentage is calculated from the equation PD=ND/(ND+NM/2), where PD is dimer percentage, and ND and NM are the amounts of dimers and monomers, respectively (shown in Table 6). Solid curves with triangles, circles and squares represent the result obtained with various initial ratios of seeds, A-tiles and T-tiles (triangles: 1:16:14, circles: 1:32:30 and squares: 1:1024:1022). The theoretical curves of exponential growth are shown as dashed lines. The replication of dimers (including seeds and later-generations) increased exponentially before leveling off as the supply of substrates became exhausted. The system without seeds is presented as a control, drawn as a solid curve with diamonds, which affirms that seeds triggered the exponential self-replication.

(12) FIG. 12 shows AFM images of dimer replication cycling (ratio of seeds, A-tiles and T-tiles is 1:1024:1022). The statistics for amplification is calculated from several AFM images in each cycle. The scale bars are 500 nm.

(13) FIG. 13 is a graph showing the heat generation profile of tubes containing buffer or IR dye-modified oligonucleotides when irradiated with IR lasers for 30 minutes. Solid curves were measured under 685-nm laser exposure, while dashed curves were obtained with 785-nm laser. The concentrations of the oligonucleotides (19 M) are controlled to be the same as the local concentration of the dyes in double-layer origami dimers. The lasers and the samples, when operating, were enclosed in a box to prevent external emission of the NIR light and to avoid air circulation that would affect the temperature of the samples. The starting temperature was 3.8 C. The temperature of the solution was measured with a thermocouple for the 30-minute laser exposure. The solution containing oligonucleotides modified by IR Dye 700 produced a significantly higher peak temperature increase of 4 C. than those under 685-nm laser irradiation. The IR Dye 800-modified oligonucleotide solution generated the highest peak temperature increase.

(14) FIGS. 14A-14D show AFM images of replication selection cycling. FIG. 14AAFM images of each 2 self-replication cycles under 685-nm laser irradiation, showing the fraction of species II increase as cycling. FIG. 14BAFM images of each 2 self-replication cycles under 785-nm laser irradiation, showing the fraction of species HH increase as cycling. FIG. 14CAFM images of each 2 self-replication cycles with laser switch (laser was changed from 685 nm to 785 nm after two replication cycles), indicating that the minor species HH became dominant when the environment was switched. FIG. 14DAFM images of each 2 self-replication cycles without laser irradiation, showing that the fraction of HH or II maintains 50% and indicating the replication of both seeds. The scale bars are 500 nm.

DETAILED DESCRIPTION OF THE INVENTION

(15) The present invention is directed to the self-replication of nucleic acid origami tiles that have flat surfaces with single-stranded nucleic acid protruding both from the edges (horizontally) and in one direction roughly perpendicular to one of the flat surfaces itself. Given a mother seed of multiple tiles (multimer), a replica can be made of that seed by complementary pairing with the perpendicular strands. The arrangement of individual multiple daughter tiles (first generation tiles and planar like the seed) is covalently fused (e.g., by photo-crosslinking of the horizontal strands that pair with each other) when they assemble in order on the seed. The mother and daughter structures can be separated by mild heating, so that there are now twice as many seeds in the solution. Both mother and daughter can then serve as seeds for the next generation. There is no apparent limit to the amplification, given sufficient daughter tiles. In addition, the daughter tiles from a pool of different mother seeds can be selected for amplification or not. As a non-limiting working example of selection, different dyes have been attached to various daughters. When the solution is illuminated with laser light so that one of the dyes absorbs, that daughter tile is locally heated, so that it cannot be replicated. The daughter that contains a different dye and that does not absorb replicates, and the species with those daughters outstrips the other product (competing multimer), so that after numerous generations, its population is hugely greater than the absorbing species. In this manner, the present invention can be used to amplify and select a given multimer that has specific characteristics or properties from among a population of different multimers.

(16) The experiments in the Example hereinbelow present a preferred embodiment of the present invention in which two dimensional rectangular DNA origami tiles replicate a seed pattern, doubling the copies in each cycle in an exponential fashion. The replication process is driven by diurnal-like cycles of temperature and UV illumination, leading to an amplification of more than 7 million in 24 cycles. This system is also used to demonstrate that two similarly-growing sub-populations with incorporated dyes have growth rates that can be controlled by applying specific environmental stimuli. Appropriately colored light heats the system only in the vicinity of the dyes, enabling the non-absorbing progeny to replicate preferentially, and thereby to take over the system. This addressable selectivity of different constituents in the same solution should be adapted readily to the selection and evolution of multi-component nanoscopic-microscopic self-replicating materials.

(17) The present method for self-replication of nucleic acid origami tiles, includes:

(18) (i) providing a set of monomers of seed, first generation daughter and second generation daughter nucleic acid origami tiles, each monomer having a plurality of horizontal edges and a plurality of faces, with a plurality of sticky cohesive ends protruding from one or more horizontal edges of the tiles and from a one or more faces of the tiles;

(19) (ii) forming a multimer from monomers of seed nucleic acid origami tiles by cohesion of complementary horizontal sticky cohesive ends between edges of adjacent monomers;

(20) (iii) mixing the resulting multimer of seed tiles with monomers of first generation daughter (1G) tiles to allow the monomers to anneal to each other by horizontal sticky end cohesion between edges of adjacent 1G monomeric tiles and to the multimer of seed tiles by sticky end cohesion between sticky cohesive ends protruding from the faces of adjacent seed and 1G tiles to form a stacked multimer of seed and 1G tiles;

(21) (iv) covalently linking 1G tiles in sticky end cohesion with each other in the stacked multimer;

(22) (v) heating to denature the horizontal sticky end cohesion between monomers of seed tiles and the vertical sticky end cohesion between monomers of seed tiles and 1G tiles to separate the heat resistant covalently linked 1G tiles as a multimer of 1G tiles;

(23) (vi) mixing the multimer of covalently linked 1G tiles with monomers of second generation daughter (2G) tiles to allow the monomers to anneal to each other by horizontal sticky end cohesion between edges of adjacent 2G monomeric tiles and to the multimer of covalently linked 1G tiles by sticky end cohesion between sticky cohesive ends protruding from the faces of adjacent 1G and 2G tiles to form a stacked multimer of 1G and 2G tiles;

(24) (vii) covalently linking 2G tiles in sticky end cohesion with each other in the stacked multimer;

(25) (viii) heating to denature the vertical sticky end cohesion between the multimer of 1G tiles and the multimer of 2G tiles in the stacked multimer to separate the multimers of covalently linked 1G tiles and covalently linked 2G tiles;

(26) (ix) incubating the separated multimers with monomers of 1G tiles and 2G tiles to allow the monomers of 1G and 2G tiles to anneal respectively to other 1G and 2G monomers by horizontal sticky end cohesion between edges of adjacent monomers tiles and to a multimer of covalently linked 1G or 2G tiles by vertical sticky end cohesion between sticky cohesive ends protruding from the faces of adjacent 1G and 2G tiles to form stacked multimers of 1G and 2G tiles;

(27) (x) for 1G and 2G tiles not already covalently linked, covalently linking 1G tiles in horizontal sticky end cohesion to each other and covalently link 2G tiles in horizontal sticky end cohesion to each other in the stacked multimers;

(28) (xi) heating to denature the vertical sticky end cohesion between multimers of covalently linked 1G tiles and multimers of covalently linked 2G tiles;

(29) (xii) repeating/cycling steps (ix)-(xi) one or more times to self-replicate and amplify multimers of nucleic acid origami tiles.

(30) Origami tiles composed of nucleic acids are well known in the art since the publication of Rothemund (2006). As used herein, a nucleic acid origami tile is formed into a particular pattern (e.g., polygons and polygonal lattices) using a scaffold strand (most typically, but not necessarily, a large scaffold strand such as a M13 single-stranded form (7250 nucleotide genome)) by staple strands, e.g., 200-250 staple strands with the M13 genomic DNA as the scaffold strand. Preferably, the origami tile is a DNA origami tile. The nucleic acid origami tile can be designed and the sequences generated by, for example, a software program caDNAno 1 (Douglas et al., 2009).

(31) While the nucleic acid origami tile may be two or three dimensional, it is preferred that the nucleic acid origami tile is substantially two dimensional, i.e., where the third dimension is very small or negligible compared to the other two dimensions, such as one having a planar appearance (length and breadth but no depth). Top and bottom surfaces of the plane are also referred to herein as faces or sides ending at the edges, which are the outer boundaries of the planar structure. Accordingly, a substantially two dimensional origami tile has two surfaces (a top and a bottom surface) with a plurality of edges that form the outer boundaries of the surfaces, e.g., four edges in a rectangular two dimensional origami tile. Preferably, the origami tile is rectangular.

(32) The size of a nucleic acid origami tile mainly depends on the length of the scaffold strand used. Most often used and preferred is a scaffold strand in the 5-10 kb range, such as an M13 genomic DNA strand. It has been reported that a scaffold strand of 26 kb in length has been used successfully (Zhang et al., 2012).

(33) The staple strands used together with the scaffold to form the origami tile into a particular pattern may have other features, like hairpins visible in the AFM or dyes like the ones used in the selection process disclosed below, or just stick ends, or perhaps nothing beyond the complementarity to the scaffold.

(34) In the present invention, a nucleic acid origami tile is used as a single monomeric unit (monomer) for the purpose of forming multimers of nucleic acid origami tiles that can self-replicate. The joining/linking of monomers into a multimer is through sticky end cohesion, which involves single stranded nucleic acid ends (cohesive sticky ends) on a monomer that have sequence complementarity with the corresponding single stranded nucleic acid ends on another monomer so as to anneal the single stranded nucleic acid ends together by cohesion. The sticky ends on the monomers are single-stranded nucleic acid protruding both from the edges (horizontally; herein designated has horizontal sticky ends) and in one direction roughly perpendicular to one of the flat surfaces itself (herein designated as vertical sticky ends). When multimers are formed by horizontal sticky end cohesion between sticky ends on the edges of monomers and there is vertical sticky end cohesion between multimers, then this is termed stacked multimers, where in the case of substantially two dimensional monomers and multimers, a stacked multimer is one where two monomers form two parallel planes, one stacked over the other.

(35) There are a plurality of horizontal and vertical sticky cohesive ends on at least one edge and on one face of nucleic acid monomeric tile. As a preferred embodiment, there are eight horizontal sticky cohesive ends on one edge and eight vertical sticky cohesive ends on one face of the seed tile. In this same preferred embodiment, there are six horizontal sticky cohesive ends on one edge and eight vertical sticky cohesive ends on one face of the first generation (1G) and second generation (2G) tiles. Preferably, the design of the sticky cohesive ends allows the horizontal sticky cohesive ends of the seed to remain stably annealed at a high temperature such as at least 50 C. (e.g., the melting temperature Tm of the horizontal sticky cohesive ends on the seed tiles are at least 55 C.), whereas the vertical sticky cohesive ends have a Tm that is at least 15 C. lower. This allows, for instance, the horizontal sticky end cohesion between seed monomers to be stable at a temperature of 55 C. whereas the denaturation of the vertical sticky end cohesion between different generation of multimers (e.g., between seed and 1G, between 1G and 2G) is complete at 40 C. The sticky cohesive ends on daughter 1G and 2G tiles are also shorter than those ends on seed tiles so that daughter cannot form multimers of daughter tiles without the aid of templates in the temperature range of cycling in the present method, such as 4 C. to 50 C. It is only when monomers of daughter 1G tiles are immobilized on a multimer of seed tiles acting as template that adjacent monomers of daughter 1G tiles are subject to a greatly increased local concentration of daughter 1G tiles, thereby leading to formation of stable horizontal sticky end cohesion between adjacent monomers of daughter tiles.

(36) The covalent linking of adjacent 1G or 2G monomers in a 1G or 2G multimer is preferably by photo-crosslinking. Such photo-crosslinking is well known and available in the art. Non-limiting examples includes photo-crosslinking with a nucleotide functionalized with 3-cyanovinylcarbazole (CNV), psoralen or cinnamate molecules. A nucleotide functionalized with such a molecule can react when exposed to ultraviolet light to form a covalent bond with the base on the opposing sticky cohesive end to which it was initially annealed by hydrogen bonding.

(37) As will be appreciated by those of skill in the art from studying the embodiment in the Example shown in FIGS. 1a and 1c that, for purposes of being able to visualize successive generations by atomic force microscopy (AFM), the seed and later generation tiles are arbitrarily provided with differences in raised projections on the surface so as to be distinguishable using AFM. Furthermore, there are slight differences in the sequences of the sticky cohesive ends in order to allow monomers of the same generation to join to each other by horizontal sticky end cohesion and for monomers and multimers of different generations (e.g., seed with 1G, 1G with 2G) to join to each other by vertical sticky end cohesion to ultimately form the stacked multimers. Nevertheless, for practical purposes, it is the particular sequence and structure of the multimeric origami tile itself along with pendant molecules or moieties that are disposed on its surface (as opposed to the sequences of the sticky cohesive ends that are merely to facilitate the joining of monomers and multimers) that are considered to be replicated in the present self-replication process.

(38) The present method of self-replication of a multimer of nucleic acid origami tiles can also be used to selectively amplify one or more different multimers from a pool of various monomers of different sets of seed, 1G and 2G daughter tiles so they outgrow their competitors (other competing multimers). For selective amplication, the monomers each have a sticky cohesive end, which is necessary for forming a multimer, labeled with a different dye (e.g., near-infrared) that produces light-activated local heat generation at a specific wavelength that is different from those of the dyes on competing monomers and multimers of the other sets of tiles. The tiles are irradiated at one or more different wavelengths to effect light-activated local heat generation in the vicinity of a particular dye or dyes, thereby suppressing sticky end cohesion of competing monomers labeled with dyes that are light-activated/absorbing at the one or more wavelengths to selectively amplify a multimer in which sticky end cohesion is not suppressed. Thus, the non-absorbing progeny replicates preferentially, and thereby takes over the system.

(39) The term nucleic acid as used herein is any polymeric system containing a sequence of nucleotides, where individual nucleotides are capable of forming highly specific paired interaction with other nucleotides, i.e., they form weak bonds with some nucleotides, vanishingly weak bonds with others, or they may even repel some other nucleotides. The interactions or bonds between nucleotides must be weak enough to be broken individually but strong enough that the cooperative action of a few neighbors forms a stable hybrid. A feature of the nucleotides is that they are capable of interacting with a nucleotide opposing them to form a specifically paired attraction. In natural DNA and RNA, hydrogen bonding forms this interaction. Non-limiting examples of nucleic acids include DNA, RNA, Peptide Nucleic Acid (PNA), and Locked Nucleic Action (LNA). A review of some nucleic acid variations, including derivatized/modified bases and other unusual bases, is presented in Freier et al. (1997).

(40) It should also be appreciated that the term nucleic acid refers to both DNA and RNA and hybrids of the two. The structure need not resemble anything which can theoretically be made from nature. For example, one or more strands may contain PNA or other backbone molecules (Lukeman et al., 2004). A particular oligonucleotide or polynucleotide strand may employ bases other than the standard five, adenine, cytosine, guanine, thymine and uracil. Derivatized (e.g., methylated) and other unusual bases such as iso-guanine, iso-cytosine, amino-adenine, K, X, , (Piccirilli et al., 1990), inosine and other derivatives of purine and pyrimidine may be used. A preferable feature in the selection of the bases is that they be capable of interacting with a base opposing them to form a specifically paired attraction. In natural DNA and RNA, hydrogen bonding forms this interaction. However, opposite ion charges, hydrophobic interactions and van der Waals forces may also be acceptable forms of interaction. These interactions expand the choices over naturally occurring bases to give a wider assortment of physical properties.

(41) Within a particular strand, the heterocyclic base may be entirely missing from the sugar moiety. This may be particularly desirable where the strands bend, form a junction, or where one desires fewer forces holding the strands together.

(42) A particular strand need not have a single contiguous ribose-phosphate or deoxyribose-phosphate backbone. It could be a peptide nucleic acid with a peptide backbone. One may employ a simple inorganic or organic moiety or polymeric spacer between segments of polynucleotide. Spacers such as polyethylene, polyvinyl polymers, polypropylene, polyethylene glycol, polystyrene, polypeptides (enzymes, antibodies, etc.) peptide nucleic acids (PNA), polysaccharides (starches, cellulose, etc.) silicones, silanes and copolymers, etc., may be employed. An example of such a hybrid structure is dodecadiol having phophoramidite at one end. This structure has been inserted covalently instead of four T nucleotides to form a hairpin loop in a fashion similar to the nucleotides it replaces. See Mitchel J. Doktycz, Ph.D. Thesis (1991), University of Illinois, Chicago.

(43) In nature and in the field of molecular biology, double stranded DNA generally occurs in the B form. However, for the purposes of this invention, it may be desirable for DNA or other double stranded polynucleotide to exist in the A, C, D or Z form. Various bases, derivations and modifications may be used to stabilize the structure in the A, C, D or Z form as well.

(44) A nanoparticle (e.g., gold (Au) or CdSe) and/or a pendant molecule can be attached to the surface of a monomeric tile. The pendant molecule can be small molecules, catalysts, enzymes, peptides and other proteins, i.e., ribosomes, (Niemeyer et al., 1994). Such attachment is not limited to direct attachment to the surface of the tile itself but may be through hairpins protruding from the surface of the tile or via attachment to oligonucleotides that bind to the surface of the tile or to hairpins or single stranded protrusions from the surface of the tile.

(45) The self-replication of the nucleic acid origami tiles provides a way of producing multimers of the same monomeric unit or a combination of different monomeric units with greater ease by exponential amplification. Origami tiles have found utility in controlled release of drugs (Zhao et al., 2012; Cho et al., 2014), and there is a use for exponential amplification as a means of producing origami tiles carrying a drug.

(46) Having now generally described the invention, the same will be more readily understood through reference to the following example which is provided by way of illustration and is not intended to be limiting of the present invention.

EXAMPLE

(47) Materials and Methods

(48) DNA Strand Design, Synthesis, and Purification.

(49) The DNA origami was designed and the sequences were generated by program caDNAno 1 (Douglas et al., 2009). DNA sequences of sticky ends were generated using the program Uniquimer (Wei et al., 2007). Single-stranded M13mp18 DNA genome was purchased from Bayou Biolabs. 3-Cyanovinylcarbazole phosphoramidite (.sup.CNVK) was purchased from Glen Research. IRDye 700 and IRDye 800 phosphoriamidites were purchased from LI-COR Biosciences. The DNA strands with modifications (.sup.CNVK or IR dyes) were synthesized on an Applied Biosystems 394 DNA synthesizer. Other DNA strands were purchased from Integrated DNA Technology, Inc. (idtdna.com). All the sticky-end-containing strands were purified using denaturing PAGE gel electrophoresis. The designed origami tiles are all based on using the genomic M13mp18 DNA (SEQ ID NO:344) as a scaffold to which 216 staple strands (Table 1; SEQ ID NO:1-216) anneal to form the origami base tile structure without any modifications (no hairpin structures and sticky ends). This origami base structure that is used for all the different types of origami tile structures used in this Example is shown in FIGS. 5A-5D. The origami base structure is shown split into four quadrants (FIGS. 5A-5D) with sufficient overlap between adjacent quadrants so that the entire origami structure can be determined and more easily visualized for clarity. The extra four T shown at both the 5 and 3 ends of staple strands 9, 10, 27, 28, 45, 46, 63, 64, 81, 82, 99, 100, 116, 117, 134, 135, 152, 153, 170, 171, 188, 189, 204 and 205 in Table 1 but not shown in FIGS. 5A-5D, are designed merely to prevent origami tiles from aggregating or binding to each other due to non-specific stacking interactions in the helical direction.

(50) TABLE-US-00001 TABLE1 SequencesofRegularOrigamiStapleStrands Number Sequence SEQIDNO: 1 TTCTTTGATTAGTAATTATCGGCCTTGCTGGTACACGACC SEQIDNO:1 2 GCAAATTATTACCGCCAGCCATTGATGGATTA SEQIDNO:2 3 GAGGCCACCATGGAAATACCTTTCCAGTCGGG SEQIDNO:3 4 CCAGAATCCGTGCCAGCTGCATTAAGCTAACT SEQIDNO:4 5 GAAAAACCGCGGGGAGAGGCGGTTTAAAGTGT SEQIDNO:5 6 ATTAAAGAGGGTGGTTTTTCTTTTCACAATTC SEQIDNO:6 7 AGTGTTGTAACAGCTGATTGCCCTTAGCTGTT SEQIDNO:7 8 ATAAATCAAGAGAGTTGCAGCAAGGGGTACCG SEQIDNO:8 9 TTTTATCCTGTTTGATGGTGGCCCCAGCAGGCGAAATTTT SEQIDNO:9 10 TTTTGTAGAAGAACTCAAACAACATCACTTGCCTGATTTT SEQIDNO:10 11 AAAACGCTCGAGTAAAAGAGTCTGTCCATCAC SEQIDNO:11 12 AAACCTGTCTGAGAAGTGTTTTTATAATCAGT SEQIDNO:12 13 GCCAACGCGTCTATCAAGACAGGAACGGTACG SEQIDNO:13 14 GGGCGCCAACGTGGACTCCAACGTCAAAGGGC SEQIDNO:14 15 AGACGGGCTCCAGTTTGGAACAAGAGTCCACT SEQIDNO:15 16 TGGCCCTGAAAGAATAGCCCGAGATAGGGTTG SEQIDNO:16 17 GCTGGTTTGTTCCGAAATCGGCAAAATCCCTT SEQIDNO:17 18 AGTAATAATTCTGACCTGAAAGCGAACTAATA SEQIDNO:18 19 CACCAGTCAATATCCAGAACAATAACCGTTGTAGCAATAC SEQIDNO:19 20 TTTACATTAGACAATATTTTTGAAAGGTTATC SEQIDNO:20 21 CGCTCACTGCCCGCTACATTTTGAATGCGCGA SEQIDNO:21 22 CACATTAATTAAAAATACCGAACGAAATATCA SEQIDNO:22 23 AAAGCCTGTAAAACAGAGGTGAGGGAAAAATC SEQIDNO:23 24 CACACAACCGCCTGCAACAGCCAGCTGGCGAA SEQIDNO:24 25 TCCTGTGTGTGCTGCAAGGCGATTTGGGAAGG SEQIDNO:25 26 AGCTCGAAGGGTTTTCCCAGTCACAGCGCCAT SEQIDNO:26 27 TTTTGCATGCCTGCAGGTCGGGCCAGTGCCAAGCTTTTTT SEQIDNO:27 28 TTTTAACAGAGATAGAACCCAAGGGACATTCTGGCCTTTT SEQIDNO:28 29 AGTCTTTACGCTCAATCGTCTGAACAACAGGA SEQIDNO:29 30 ACTGATAGTTGGCAAATCAACAGTTTAAAAGT SEQIDNO:30 31 GCAGAAGAGGGTGCCTAATGAGTGATGAATCG SEQIDNO:31 32 ATTAACACATACGAGCCGGAAGCATGCGTATT SEQIDNO:32 33 AGGGGGATGAAATTGTTATCCGCTCACCAGTG SEQIDNO:33 34 TAACGCCATTCGTAATCATGGTCATCACCGCC SEQIDNO:34 35 AAAACGACACTCTAGAGGATCCCCCGGTCCAC SEQIDNO:35 36 GATTAGAGAGTATTAGACTTTACAAATAATGG SEQIDNO:36 37 GCACTAACTAAGAATACGTGGCACGGCAGATT SEQIDNO:37 38 TAAAATATGTATTAAATCCTTTGCATATAATC SEQIDNO:38 39 CTGGTCAGCCCTAAAACATCGCCATTGCGTTG SEQIDNO:39 40 AACCCTCAAACAAAGAAACGAGCGAGTAACAA SEQIDNO:40 41 TAAAGCATATTCTCCGTGGGAACAGGCCTTCC SEQIDNO:41 42 TCTTCGCTATTACGTGCCACGCTGTAATGGGA SEQIDNO:42 43 GCGATCGGGCATCGTAACCGTGCA SEQIDNO:43 44 TCGCCATTGACGACGACAGTATCGGTAAACGT SEQIDNO:44 45 TTTTCGGCACCGCTTCTGGTACTCCAGCCAGCTTTCTTTT SEQIDNO:45 46 TTTTCATTTGAGGATTTAGACCGTCAATAGATAATATTTT SEQIDNO:46 47 TATTAATTTGAAAGGAATTGAGGATGGCTATT SEQIDNO:47 48 TTGAGTAAATTCCTGATTATCAGACCTTTTAC SEQIDNO:48 49 CCCGTCGGCACCTTGCTGAACCTCAACCACCA SEQIDNO:49 50 ATTGACCGAGAGCCAGCAGCAAATCGGTCAGT SEQIDNO:50 51 TAGGTCACAATAGGAACGCCATCATGAGCAAA SEQIDNO:51 52 TTTGAGGGCAGGCTGCGCAACTGTAAGTTGGG SEQIDNO:52 53 AAGATCGCGCCGGAAACCAGGCAAGACGTTGT SEQIDNO:53 54 AAGGGTTAAACAGAAATAAAGAAAAATCATAG SEQIDNO:54 55 TACTTCTGAACAATTCGACAACTCCTTTAGGA SEQIDNO:55 56 CTGATTGTGTTTAACGTCAGATGAACGCTGAG SEQIDNO:56 57 ATCATCATCATTATCATTTTGCGGATCAATAT SEQIDNO:57 58 CAACATTAAATGTCACCAGAAGGAGCCTGATT SEQIDNO:58 59 TGTAGCCACGCGCAGAGGCGAATTAATATATG SEQIDNO:59 60 TTTTAACCGTTGGTGTAGATGGGCTGCGGGCC SEQIDNO:60 61 AAATTTTTGTTAAATCGAAAACAAAATTGAACGGTAATCG SEQIDNO:61 62 TAATATTTGCATGTCAATCATATGTCATTGCC SEQIDNO:62 63 TTTTACAGGAAGATTGTATACAGAAAAGCCCCAAAATTTT SEQIDNO:63 64 TTTTAATTATTTGCACGTAAGAACCTACCATATCAATTTT SEQIDNO:64 65 TAACAGTATGATGGCAATTCATCACCGAACGT SEQIDNO:65 66 ATCGGGAGTCCTTGAAAACATAGCTTTCAAAT SEQIDNO:66 67 GCTTTGAAAATCGTCGCTATTAATAGCCTTTA SEQIDNO:67 68 CAATTACCAAAATAATTCGCGTCTAACGGCGG SEQIDNO:68 69 AGAAGATGTTACCTTTTTTAATGGGAGTAATG SEQIDNO:69 70 TAAAACTATGTTAAAATTCGCATTTCTGCCAG SEQIDNO:70 71 TTGATAATAGCAAATATTTAAATTGCCTCAGG SEQIDNO:71 72 GTCTGAGATTATATAACTATATGTATAAACAC SEQIDNO:72 73 TTTATCAATTGCGTAGATTTTCAGTTGGATTA SEQIDNO:73 74 AAGAGTCACCAATCGCAAGACAAACGACCGTG SEQIDNO:74 75 CCTTAGAAAAACAATAACGGATTCGCGGAATT SEQIDNO:75 76 CTTCTGTATACCAAGTTACAAAATGCTTTCAT SEQIDNO:76 77 TGAGTGAATAGAACCCTCATATATAAGCCTCA SEQIDNO:77 78 CATTTGAAATGAAACAAACATCAAAGCTCATT SEQIDNO:78 79 ACAAGAGAATCGATAATTACATTTAGAAAGGC SEQIDNO:79 80 TGAGAGTCTATGATATTCAACCGTGAGCTGAA SEQIDNO:80 81 TTTTCTATTTTTGAGAGATCATGCCGGAGAGGGTAGTTTT SEQIDNO:81 82 TTTTTCCGGCTTAGGTTGGGGACTACCTTTTTAACCTTTT SEQIDNO:82 83 GAAAACTTGATAGCTTAGATTAAGATATACAG SEQIDNO:83 84 ATATTTTAGTTAATTTGCGGGAGATAATTTTC SEQIDNO:84 85 TTTCAACGCCAAAAACATTATGACCAGAGGCA SEQIDNO:85 86 CAATGCCTAAACAGTACATAAATCATTCATTT SEQIDNO:86 87 TGTAGGTAGCAAGGCAAAGAATTATCCAGACG SEQIDNO:87 88 CGGAGACAGTAGTAGCATTAACATTTCCATAT SEQIDNO:88 89 ATAAATTATACAAAGGCTATCAGGTACCCCGG SEQIDNO:89 90 CGGAATCATGCGTTATACAAATTCTATTTTCATCGTAGGA SEQIDNO:90 91 AAATAAGAAAATGCTGATGCAAATATAGTGAA SEQIDNO:91 92 TGATAAATAACGCTCAACAGTAGGACCGCACT SEQIDNO:92 93 CCTAAATTCCATATTTAACAACGCTTATCATT SEQIDNO:93 94 CGGTTGTACAAGGATAAAAATTTTTAACCTTG SEQIDNO:94 95 GAGCATAAATAAAGTACCGACAAAAAAAATAA SEQIDNO:95 96 TCATACAGAAGATTCAAAAGGGTGAACAATTT SEQIDNO:96 97 TACTAATAGTCAAATCACCATCAATGGAGCAA SEQIDNO:97 98 AAGGTGGCACGAGTAGATTTAGTTTCAACATG SEQIDNO:98 99 TTTTAACCTGTTTAGCTATATTCGCAAATGGTCAATTTTT SEQIDNO:99 100 TTTTCCTGTTTAGTATCATATAATTACTAGAAAAAGTTTT SEQIDNO:100 101 GAGAATCGTAATGGTTTGAAATACGAACGCGA SEQIDNO:101 102 AATTTAGGCCTGTAATACTTTTCATCTTCTGA SEQIDNO:102 103 TTTTCGAGGTAGAAACCAATCAATGTCAGAAG SEQIDNO:103 104 TAATTCTGGCAAAATTAAGCAATATTTAAATG SEQIDNO:104 105 ACGACAATTGTTTATCAACAATAGGTTTTAAT SEQIDNO:105 106 AACAGTTGGTGTCTGGAAGTTTCAGGAAGCAA SEQIDNO:106 107 AGATACATTTTTCATTTGGGGCGCTCTAGCTG SEQIDNO:107 108 GCCGTTTTTTACCAGTATAAAGCCAAGGCGTT SEQIDNO:108 109 CATCGAGATAAACAGTTCAGAAAAATCGTCAT SEQIDNO:109 110 CCAAGAACTCAAAAATCAGGTCTTTGTTTAGA SEQIDNO:110 111 ACGAGCATCCAGTAATAAGAGAATAGCTAAAT SEQIDNO:111 112 TATCCCATTAAGAGGAAGCCCGAAATAAAAAC SEQIDNO:112 113 AACGCGCCAAACAACATGTTCAGCCCAATAAA SEQIDNO:113 114 AAAGTACGATTCCCAATTCTGCGAATCAATTC SEQIDNO:114 115 TTTTAAATGTACCTTTAATTGCTCAATACCAC SEQIDNO:115 116 TTTTTAGAGCTTAATTGCTGATTTTTGCGGATGGCTTTTT SEQIDNO:116 117 TTTTATAGCAAGCAAATCAGATCATTACCGCGCCCATTTT SEQIDNO:117 118 ATATAGAAGGCTTATCCGGTACTCAAATGCTTACAAGCAA SEQIDNO:118 119 ACCATAAAGGGTATTAAACCAAGTGCTTAATT SEQIDNO:119 120 CTATTATAAATCGGCTGTCTTTCCCAACATGT SEQIDNO:120 121 CAAAGCGGAAGAAGTTTTGCCAGACCAGTTAC SEQIDNO:121 122 AATATCGCATAAGTCCTGAACAAGAGGTAAAG SEQIDNO:122 123 TCGAGCTTACACTATCATAACCCTTAATCATT SEQIDNO:123 124 ACTCCAACGCCAAAAGGAATTACGGAACTGGC SEQIDNO:124 125 AAGAGGTCAATATAATGCTGTAGCTGACCATT SEQIDNO:125 126 AGGCGTTTAGCCTTAAATCAAGATGGTAATTG SEQIDNO:126 127 AAATATTCCCCAGCTACAATTTTAGAATTAAC SEQIDNO:127 128 CTGGATAGCGCTAACGAGCGTCTTAACATAAA SEQIDNO:128 129 TTTTGCAAATTGCATCAAAAAGATCCTAATTT SEQIDNO:129 130 CAAAATAGCCCAATCCAAAGAGATGGTTTAAT SEQIDNO:130 131 TAAGAGCACAAAGCGAACCAGACCTAATGCAG SEQIDNO:131 132 TACATAACAGGTCAGGATTAGAGAATGCAACT SEQIDNO:132 133 ATTCAACTGAAGAAAAATCTACGTAACCGGAT SEQIDNO:133 134 TTTTCAGGTAGAAAGATTCACGGAACAACATTATTATTTT SEQIDNO:134 135 TTTTTTGCGGGAGGTTTTGATAGCGAACCTCCCGACTTTT SEQIDNO:135 136 ATTTTGCAATTGAATCCCCTTCTAAGAACGCG SEQIDNO:136 137 CTTACCAACGTCCAATACTGCGGACGAGAATG SEQIDNO:137 138 CTAATTTGGGGGGTAATAGTAAAATACCCTGA SEQIDNO:138 139 AAAATAAAAAAATGAAAATAGCAGCGCGAAAC SEQIDNO:139 140 TTCAACTTCGTTTACCAGACGACGAGACTTCA SEQIDNO:140 141 GTGAATTATGACGAGAAACACCAGTGCTCCAT SEQIDNO:141 142 TCATTATAAAAGCTGCTCATTCAGGACGGTCA SEQIDNO:142 143 CGAACTAATCAGTTGAGATTTAGGCTTTTGAT SEQIDNO:143 144 AGCGCTAAAGCCCAATAATAAGAGAACGCAAT SEQIDNO:144 145 TGAACACCAGCAATAGCTATCTTAGCCGAACA SEQIDNO:145 146 AACAGGGAACTCATCTTTGACCCCAAGAATAC SEQIDNO:146 147 TAACGTCACAGCCATATTATTTATCGAGAGGC SEQIDNO:147 148 GTAAATTGGGCTTTAAGAAACGATTCGCCTGA SEQIDNO:148 149 GCTTGCCCCCTTATGCGATTTTAAAGGCATAG SEQIDNO:149 150 AACGTAACCCAGTCAGGACGTTGGAATGCAGA SEQIDNO:150 151 ATTCATTAACTTTGAAAGAGGACAGGGATCGT SEQIDNO:151 152 TTTTGGCTGACCTTCATCAAACCAGGCGCATAGGCTTTTT SEQIDNO:152 153 TTTTCACAAGAATTGAGTTATATCAGAGAGATAACCTTTT SEQIDNO:153 154 AATGAAATCTGAACAAAGTCAGAGTAGTTGCT SEQIDNO:154 155 CTTTTTAAAGCGCATTAGACGGGATCCTGAAT SEQIDNO:155 156 ATACCAAGCCTTTACAGAGAGAATTCCAGAGC SEQIDNO:156 157 AAAGTACAACGAAGGCACCAACCTGTCACAAT SEQIDNO:157 158 TAAATTGTTCCATTAAACGGGTAACAGCGCCA SEQIDNO:158 159 GTTACTTATTGAGGACTAAAGACTGATTGAGG SEQIDNO:159 160 ATCATAAGTCGGAACGAGGGTAGCATTATTCA SEQIDNO:160 161 GTGTACAGGAGTAATCTTGACAAGTAATAAAA SEQIDNO:161 162 AATAACGGCTTATTACGCAGTATGGAGCCACC SEQIDNO:162 163 AAGTTACCATACATACATAAAGGTCCATCTTT SEQIDNO:163 164 ACTAAAACAACGCAAAGACACCACATTTTCGG SEQIDNO:164 165 ATGCCACTACGGAGATTTGTATCATTTTTGTT SEQIDNO:165 166 AGGAAGTTGTCGAAATCCGCGACCAACGAGTA SEQIDNO:166 167 CAGAGGCTGCCGGAACGAGGCGCATGAATAAG SEQIDNO:167 168 AGACAGCAGGAACCGAACTGACCACCCAAATC SEQIDNO:168 169 CACCCTCACGACTTGAGCCAACCATCGCCCAC SEQIDNO:169 170 TTTTGAGGCTTGCAGGGAGTGATATATTCGGTCGCTTTTT SEQIDNO:170 171 TTTTGGCATGATTAAGACTCAATACCCAAAAGAACTTTTT SEQIDNO:171 172 CGTAGAAAAGAAGGAAACCGAGGACAAGAAAC SEQIDNO:172 173 ATAAAAGAGAAAAGTAAGCAGATACCGAAGCC SEQIDNO:173 174 TTTATTTTAAAACGAAAGAGGCAACAGCGATT SEQIDNO:174 175 CAATAGAATTAGCGTCAGACTGTAGTATGGGA SEQIDNO:175 176 AAGACAAACGTAATCAGTAGCGACTTCAGCGG SEQIDNO:176 177 GAGGGAAGAACGTCACCAATGAAAAAGGAATT SEQIDNO:177 178 TTAAAGGTCCAGTAGCACCATTACAAAATCTC SEQIDNO:178 179 GCATAACCTAAAGGCCGCTTTTGCGATGAACG SEQIDNO:179 180 ACCGGAACCCACCCTCAGAGCCACGAGGTTGA SEQIDNO:180 181 TCATAATCACCAGAACCACCGTAACGATCTAA SEQIDNO:181 182 TCATAGCCCGTCTTTCCAGACGTTACGCCTGTAGCATTCC SEQIDNO:182 183 GTTTGCCTAATTCATATGGTTTACAATACGTA SEQIDNO:183 184 AGCAGCACAGGGCGACATTCAACCTTTTCATG SEQIDNO:184 185 AGGCCGGAGTAAATATTGACGGAAAACGGCTA SEQIDNO:185 186 CAAAATCAGAATTATCACCGTCACGCAGCGAA SEQIDNO:186 187 CGACAATGACAACATTTGGGAATTCTTTAATT SEQIDNO:187 188 TTTTACAGCTTGATACCGATGAGGTGAATTTCTTAATTTT SEQIDNO:188 189 TTTTGCCACCCTCAGAACCGCGCCTCCCTCAGAGCCTTTT SEQIDNO:189 190 GAGCCGCCAAAATCACCGGAACCATTAGCAAA SEQIDNO:190 191 AGTTTTGTCCCTTATTAGCGTTTGGGCAACAT SEQIDNO:191 192 AATTTTCTGCGCGTTTTCATCGGCGGAATAAG SEQIDNO:192 193 TTTTGCTAAACACTGAGTTTCGTCAATAAGTT SEQIDNO:193 194 AGTGAGAAGATAGCAAGCCCAATAACAGTGCC SEQIDNO:194 195 GCGAATAACCACCCTCAGAGCCACCCTATTTCGGAACCTA SEQIDNO:195 196 CAAAAAAACGCCACCCTCAGAACCGCCACCCT SEQIDNO:196 197 GTATCGGTAGGTGTATCACCGTACGGATTAGG SEQIDNO:197 198 GGCAGGTCAATCCTCATTAAAGCCAGAATGGA SEQIDNO:198 199 ACAGACAGCCCTCATAGTTAGCACCAGAGCCGTCTCTGAA SEQIDNO:199 TTTACCGT 200 TGTACCGTAACAACTTTCAACAGTAGAATCAA SEQIDNO:200 201 TTTTCAGGTAGAAAGGAACAACTACCATCGAT SEQIDNO:201 202 CAGAACCGTAATTTTTTCACGTTGCATTAGCA SEQIDNO:202 203 TTTAGTACAGGCTCCAAAAGGAGCAGAGCCAG SEQIDNO:203 204 CCCGGAATTTATCAGCTTGCTTTCAGTTGCGC SEQIDNO:204 205 TTTTTCGAGAGGGTTGATATAGGCGGATAAGTGCCGTTTT SEQIDNO:205 206 TTTTTATTCACAAACAAATAAGACGATTGGCCTTGATTTT SEQIDNO:206 207 AAGCGCAGCCGCCAGCATTGACAGCACCCTCA SEQIDNO:207 208 TCCAGTAAGCGTCATA SEQIDNO:208 209 CATGGCTTTTGATGAT SEQIDNO:209 210 ACAGGAGTGTACTGGTACCAGTACAAACTACAAGTAAATG SEQIDNO:210 211 TTAACGGGGTCAGTGCCTTGAGTAGGAACCCA SEQIDNO:211 212 CGTATAAACAGTTAATGCCCCCTGCACCCTCA SEQIDNO:212 213 TTATTCTGAAACATGA SEQIDNO:213 214 AAGTATTAAGAGGCTG SEQIDNO:214 215 AGACTCCTCAAGAGAATCAGGAGG SEQIDNO:215 216 ATTAGCGGGGTTTTGCTCAGTACCAAGTATAG SEQIDNO:216

(51) To label origami tiles for identification by AFM, DNA hairpins are added to increase the height of a desired location on the origami tile as shown in FIG. 6. The sequences of the hairpin modified staple strands involved in producing the letter T on the origami tile are shown in Table 2 below, where the nucleotides forming the hairpins are shown in lower case.

(52) TABLE-US-00002 TABLE2 SequencesofStapleStrandsInvolvedinProducingtheLetterT Label Sequence SEQIDNO: T-039 atgaatccattggattcatcaagtgctttttagcacttgtcgagattCTGG SEQIDNO:217 TCAGCCCTAAAACATCGCCATTGCGTTG T-040-1 AACCCTCAAACAAAGA SEQIDNO:218 T-040 atgaatcatttggattcatcaagtgctttttagcacttgtcgagattAACGA SEQIDNO:219 GCGAGTAACAA T-041-1 TAAAGCATATTCTCCG SEQIDNO:220 T-041 atgaatccattggattcatcaagtgctttttagcacttgtcgagattTGGGAA SEQIDNO:221 CAGGCCTTCCTGTAGCCACGCGCAGA T-042 atgaatcatttggattcatcaagtgctttttagcacttgtcgagattTCTTCG SEQIDNO:222 CTATTACGTGCCACGCTGTAATGGGA T-049-1 CCCGTCGGCACCTTGC SEQIDNO:223 T-049 atgaatcatttggattcatcaagtgctttttagcacttgtcgagattTGAACCTCAACCACCA SEQIDNO:224 T-050-1 ATTGACCGAGAGCCAG SEQIDNO:225 T-050 atgaatccattggattcatcaagtgctattagcacttgtcgagattCAGCAAATCGGTCAGT SEQIDNO:226 T-051 atgaatcatttggattcatcaagtgctttttagcacttgtcgagattTAGGTCACAA SEQIDNO:227 TAGGAACGCCATCATGAGCAAA T-059 atgaatcatttggattcatcaagtgctttttagcacttgtcgagattGGCGAATTAATAT SEQIDNO:228 ATGTGAGTGAATAGAACCC T-077 atgaatcatttggattcatcaagtgctttttagcacttgtcgagattTCATATATAAGCCT SEQIDNO:229 CAGAGCATAAATAAAGTA T-095 atgaatcatttggattcatcaagtgctttttagcacttgtcgagattCCGACAAAAAAAA SEQIDNO:230 TAATATCCCATTAAGAGGA T-112 atgaatcatttggattcatcaagtgctttttagcacttgtcgagattAGCCCGAAATAAA SEQIDNO:231 AACCAAAATAGCCCAATCC T-130 atgaatcatttggattcatcaagtgctttttagcacttgtcgagattAAAGAGATGGTTTAAT SEQIDNO:232 T-158 atgaatcatttggattcatcaagtgctttttagcacttgtcgagattTAAATTGTTCCAT SEQIDNO:233 TAAACGGGTAACAGCGCCA T-176 atgaatcatttggattcatcaagtgctttttagcacttgtcgagattAAGACAAACGTAATC SEQIDNO:234 AGTAGCGACTTCAGCGG

(53) For an I pattern origami tile, the staple strands involved in producing the letter T in Table 2 above were used except that staple strands T-039, T-040, T-040-1, T-042, T-049, T-049-1, T-050, T-050-1 and T-051 are replaced with the regular staple strands with the same label numbers as shown in Table 1.

(54) The sequences of the hairpin-modified staple strands involved in producing the letter A on the origami tile are shown in Table 3 below, where the nucleotides forming the hairpins are shown in lowercase.

(55) TABLE-US-00003 TABLE3 SequencesofStapleStrandsInvolvedinProducingtheLetterA Label Sequence SEQIDNO: A-023-1 AAAGCCTGTAAAACAG SEQIDNO:235 A-023 atgaatccttttggattcatcaagtgctttttagcacttgtcgagattAGGTGAGGGAAAAATC SEQIDNO:236 A-040-1 AACCCTCAAACAAAGA SEQIDNO:237 A-040 atgaatccttttggattcatcaagtgctttttagcacttgtcgagattAACGAGCGAGTAACAA SEQIDNO:238 A-049-1 CCCGTCGGCACCTTGC SEQIDNO:239 A-049 atgaatccttttggattcatcaagtgctttttagcacttgtcgagattTGAACCTCAACCACCA SEQIDNO:240 A-050-1 ATTGACCGAGAGCCAG SEQIDNO:241 A-050 atgaatccttttggattcatcaagtgctttttagcacttgtcgagattCAGCAAATCGGTCAGT SEQIDNO:242 A-051 atgaatccttttggattcatcaagtgctttttagcacttgtcgagattTAGGTCACAATAGGAACGCCATC SEQIDNO:243 ATGAGCAAA A-057 atgaatccttttggattcatcaagtgctttttagcacttgtcgagattATCATCATCATTATCATTTTGCGG SEQIDNO:244 ATCAATAT A-060 atgaatccttttggattcatcaagtgctttttagcacttgtcgagattTTTTAACCGTTGGTGTAGATGGG SEQIDNO:245 CTGCGGGCC A-075 atgaatccttttggattcatcaagtgctttttagcacttgtcgagattCCTTAGAAAAACAATAACGGATT SEQIDNO:246 CGCGGAATT A-078 atgaatccttttggattcatcaagtgctttttagcacttgtcgagattCATTTGAAATGAAACAAACATCA SEQIDNO:247 AAGCTCATT A-096 atgaatccttttggattcatcaagtgctttttagcacttgtcgagattTCATACAGAAGATTCAAAAGGGT SEQIDNO:248 GAACAATTT A-102 atgaatccttttggattcatcaagtgctttttagcacttgtcgagattACTTTTCATCTTCTGA SEQIDNO:249 A-111 atgaatccttttggattcatcaagtgctttttagcacttgtcgagattACGAGCATCCAGTAATAAGAGAA SEQIDNO:250 TAGCTAAAT A-112-1 TATCCCATTAAGAGGA SEQIDNO:251 A-112 atgaatccttttggattcatcaagtgctttttagcacttgtcgagattAGCCCGAAATAAAAAC SEQIDNO:252 A-113 atgaatccttttggattcatcaagtgctttttagcacttgtcgagattAACGCGCCAAACAACATGTTCAG SEQIDNO:253 CCCAATAAA A-120 atgaatccttttggattcatcaagtgctttttagcacttgtcgagattGTCTTTCCCAACATGTAATTTAGG SEQIDNO:254 CCTGTAAT A-121 atgaatccttttggattcatcaagtgctttttagcacttgtcgagattCAAAGCGGAAGAAGTTTTGCCAG SEQIDNO:255 ACCAGTTAC A-122-1 AATATCGCATAAGTCC SEQIDNO:256 A-122 atgaatccttttggattcatcaagtgctttttagcacttgtcgagattTGAACAAGAGGTAAAG SEQIDNO:257 A-123 atgaatccttttggattcatcaagtgctttttagcacttgtcgagattTCGAGCTTACACTATCATAACCCT SEQIDNO:258 TAATCATT A-131 atgaatccttttggattcatcaagtgctttttagcacttgtcgagattTAAGAGCACAAAGCGAACCAGA SEQIDNO:259 CCTAATGCAG A-138 atgaatccttttggattcatcaagtgctttttagcacttgtcgagattTAGTAAAATACCCTGACTATTAT SEQIDNO:260 AAATCGGCT A-149 atgaatccttttggattcatcaagtgctttttagcacttgtcgagattGCTTGCCCCCTTATGCGATTTTAA SEQIDNO:261 AGGCATAG A-156-1 ATACCAAGCCTTTACA SEQIDNO:262 A-156 atgaatccttttggattcatcaagtgctttttagcacttgtcgagattGAGAGAATTCCAGAGCCTAATTT SEQIDNO:263 GGGGGGTAA

(56) As for the locations of the hairpin-modified staple strands for the H pattern origami tile, the staple strands involved in producing the letter A were used, except that staple strands with hairpin sequences A-023, A-023-1, A-040, A-040-1, A-049, A-049-1, A-050, A-050-1 and A-050 were replaced with the regular staple strands with the same label numbers as shown in Table 1.

(57) In order to connect two tiles together to form dimer seeds, some of the staple strands were modified to have sticky ends that protrude from the sides of the origami tile in the direction of the helices. The sequences of such modified staple strands are shown in Table 4 below, wherein the sticky end sequences are shown in lowercase.

(58) TABLE-US-00004 TABLE4 Sequencesofhorizontalstickyendsfordimerseed Dimer-R-045 TTTTCGGCACCGCTTCTGGTACTCCAGCCAGCTTTCtgtcgtggtca SEQIDNO:264 Dimer-R-063 TTTTACAGGAAGATTGTATACAGAAAAGCCCCAAAAgcgcttcaata SEQIDNO:265 Dimer-R-081 TTTTCTATTTTTGAGAGATCATGCCGGAGAGGGTAGcgcattcactt SEQIDNO:266 Dimer-R-099 TTTTAACCTGTTTAGCTATATTCGCAAATGGTCAATtgggtcttcct SEQIDNO:267 Dimer-R-116 TTTTTAGAGCTTAATTGCTGATTTTTGCGGATGGCTttattggcgtt SEQIDNO:268 Dimer-R-134 TTTTCAGGTAGAAAGATTCACGGAACAACATTATTAggcttgttcga SEQIDNO:269 Dimer-R-152 TTTTGGCTGACCTTCATCAAACCAGGCGCATAGGCTagtttccgtgc SEQIDNO:270 Dimer-R-170 TTTTGAGGCTTGCAGGGAGTGATATATTCGGTCGCTaaccgagtatc SEQIDNO:271 Dimer-L-046 TTTTCATTTGAGGATTTAGACCGTCAATAGATAATAtgaccacgaca SEQIDNO:272 Dimer-L-064 TTTTAATTATTTGCACGTAAGAACCTACCATATCAAtattgaagcgc SEQIDNO:273 Dimer-L-082 TTTTTCCGGCTTAGGTTGGGGACTACCTTTTTAACCaagtgaatgcg SEQIDNO:274 Dimer-L-100 TTTTCCTGTTTAGTATCATATAATTACTAGAAAAAGaggaagaccca SEQIDNO:275 Dimer-L-117 TTTTATAGCAAGCAAATCAGATCATTACCGCGCCCAaacgccaataa SEQIDNO:276 Dimer-L-135 TTTTTTGCGGGAGGTTTTGATAGCGAACCTCCCGACtcgaacaagcc SEQIDNO:277 Dimer-L-153 TTTTCACAAGAATTGAGTTATATCAGAGAGATAACCgcacggaaact SEQIDNO:278 Dimer-L-171 TTTTGGCATGATTAAGACTCAATACCCAAAAGAACTgatactcggtt SEQIDNO:279

(59) There are two groups of vertical sticky-end pairs, with each vertical sticky end protruding perpendicularly from the surface/face (top or bottom) of an origami tile. For each set, strand 038 on the seed tile is complementary to strand 044 on the first generation tile (seed strand 038 to FG-044) and so on. One set was used on one unit tile (monomer) of seed in the dimer system. In order to ensure that the vertical sticky ends face up from the origami tile surface/face, complementary strands containing poly A sticky ends (black lines; see also Table 5 below), which would bind to the poly T segments of the vertical sticky ends. The sequences of the vertical sticky ends for the dimer seeds and first generation tiles are shown in Table 5 below.

(60) TABLE-US-00005 TABLE5 Sequencesofverticalstickyendsfordimerseedsandfirstgenerationtiles SEQIDNO: ComplementarystrandstothepolyTsegmentinverticalstickyends 9A-056 AAAAAAAAACTGATTGTGTTTAACGTCAGATGAACGCTGAG SEQIDNO:280 9A-092 AAAAAAAAATGATAAATAACGCTCAACAGTAGGACCGCACT SEQIDNO:281 9A-127 AAAAAAAAAAAATATTCCCCAGCTACAATTTTAGAATTAAC SEQIDNO:282 9A-163 AAAAAAAAAAAGTTACCATACATACATAAAGGTCCATCTTT SEQIDNO:283 9A-062 AAAAAAAAATAATATTTGCATGTCAATCATATGTCATTGCC SEQIDNO:284 9A-098 AAAAAAAAAAAGGTGGCACGAGTAGATTTAGTTTCAACATG SEQIDNO:285 9A-133 AAAAAAAAAATTCAACTGAAGAAAAATCTACGTAACCGGAT SEQIDNO:286 9A-169 AAAAAAAAACACCCTCACGACTTGAGCCAACCATCGCCCAC SEQIDNO:287 Verticalstickyends(twosets) 1-Seed-038 TAAAATATGTATTAAATCCTTTGCATATAATCtttttttttatgagacgg SEQIDNO:288 1-Seed-044 TCGCCATTGACGACGACAGTATCGGTAAACGTtttttttttgtaggcagt SEQIDNO:289 1-Seed-074 AAGAGTCACCAATCGCAAGACAAACGACCGTGtttttttttcgtgttcag SEQIDNO:290 1-Seed-080 TGAGAGTCTATGATATTCAACCGTGAGCTGAAtttttttttcgtatgtgc SEQIDNO:291 1-Seed-109 CATCGAGATAAACAGTTCAGAAAAATCGTCATtttttttttcagcgttag SEQIDNO:292 1-Seed-115 TTTTAAATGTACCTTTAATTGCTCAATACCACtttttttttcttggttcg SEQIDNO:293 1-Seed-145 TGAACACCAGCAATAGCTATCTTAGCCGAACAtttttttttccattccga SEQIDNO:294 1-Seed-151 ATTCATTAACTTTGAAAGAGGACAGGGATCGTtttttttttggagagtcc SEQIDNO:295 1-FG-038 TAAAATATGTATTAAATCCTTTGCATATAATCtttttttttactgcctac SEQIDNO:296 1-FG-044 TCGCCATTGACGACGACAGTATCGGTAAACGTtttttttttccgtctcat SEQIDNO:297 1-FG-074 AAGAGTCACCAATCGCAAGACAAACGACCGTGtttttttttgcacatacg SEQIDNO:298 1-FG-080 TGAGAGTCTATGATATTCAACCGTGAGCTGAAtttttttttctgaacacg SEQIDNO:299 1-FG-109 CATCGAGATAAACAGTTCAGAAAAATCGTCATtttttttttcgaaccaag SEQIDNO:300 1-FG-115 TTTTAAATGTACCTTTAATTGCTCAATACCACtttttttttctaacgctg SEQIDNO:301 1-FG-145 TGAACACCAGCAATAGCTATCTTAGCCGAACAtttttttttggactctcc SEQIDNO:302 1-FG-151 ATTCATTAACTTTGAAAGAGGACAGGGATCGTttttttttttcggaatgg SEQIDNO:303 2-Seed-038 TAAAATATGTATTAAATCCTTTGCATATAATCttttttttttatgcaccc SEQIDNO:304 2-Seed-044 TCGCCATTGACGACGACAGTATCGGTAAACGTtttttttttatcgagtgc SEQIDNO:305 2-Seed-074 AAGAGTCACCAATCGCAAGACAAACGACCGTGtttttttttacctgggtc SEQIDNO:306 2-Seed-080 TGAGAGTCTATGATATTCAACCGTGAGCTGAAtttttttttggaaagtcg SEQIDNO:307 2-Seed-109 CATCGAGATAAACAGTTCAGAAAAATCGTCATttttttttttgcttcacg SEQIDNO:308 2-Seed-115 TTTTAAATGTACCTTTAATTGCTCAATACCACtttttttttagctgttgt SEQIDNO:309 2-Seed-145 TGAACACCAGCAATAGCTATCTTAGCCGAACAtttttttttcctcttgcc SEQIDNO:310 2-Seed-151 ATTCATTAACTTTGAAAGAGGACAGGGATCGTtttttttttgagcgattc SEQIDNO:311 2-FG-038 TAAAATATGTATTAAATCCTTTGCATATAATCtttttttttgcactcgat SEQIDNO:312 2-FG-044 TCGCCATTGACGACGACAGTATCGGTAAACGTtttttttttgggtgcata SEQIDNO:313 2-FG-074 AAGAGTCACCAATCGCAAGACAAACGACCGTGtttttttttcgactttcc SEQIDNO:314 2-FG-080 TGAGAGTCTATGATATTCAACCGTGAGCTGAAtttttttttgacccaggt SEQIDNO:315 2-FG-109 CATCGAGATAAACAGTTCAGAAAAATCGTCATtttttttttacaacagct SEQIDNO:316 2-FG-115 TTTTAAATGTACCTTTAATTGCTCAATACCACtttttttttcgtgaagca SEQIDNO:317 2-FG-145 TGAACACCAGCAATAGCTATCTTAGCCGAACAtttttttttgaatcgctc SEQIDNO:318 2-FG-151 ATTCATTAACTTTGAAAGAGGACAGGGATCGTtttttttttggcaagagg SEQIDNO:319

(61) There are six sticky ends on both sides (at the ends of the DNA helices) of the first generation and second generation tiles, as shown in FIG. 7 of these six sticky ends on a side, four of them contain 3-cyanounylcarbazole nucleosides (.sup.CNVK), labeled as X in FIG. 7, for photo-crosslinking. Under UV irradiation at a wavelength of 366 nm, UV-sensitive .sup.CNVK nucleotides can form a covalent bond with thymine bases diagonally opposite on the other strand in the cohesion between a complementary pair of sticky ends from two adjacent tiles. Two conventional sticky end pairs on both sides of each tile are added to strengthen the binding between the two tiles and to improve the photo-crosslinking yield.

(62) Formation of Individual DNA Origami Tiles.

(63) The mixture of staple strands, sticky-ended strands and M13mp18 DNA genome was diluted using 1TAE/Mg.sup.2+ buffer (40 mM Tris-HCl, pH 8.0, 20 mM acetic acid, 2.5 mM EDTA and 12.5 mM magnesium acetate). The final concentration of M13mp18 DNA genome in the solution was 10 nM, and the molar ratio of the M13mp18 DNA genome to each staple strand was 1:10. The sample was cooled from 90 C. to 16 C. on a thermocycling machine over 2 hrs. The origami tiles were purified using Amicon Ultra 0.5 mL centrifugal filters (100K, Millipore). At the same time, the buffer was exchanged to become 1TAE/Mg.sup.2+ buffer (28 mM magnesium acetate).

(64) Formation of Self-Replication Seeds.

(65) Individual DNA origami tiles (two for dimer seed, four for tetramer seed) were mixed stoichiometrically. The solution was slowly annealed from 61 C. to 24 C. with a ramp of 0.7 C./h, and then cooled further to 4 C. with a ramp of 3 C./h in an incubator.

(66) Self-Replication Cycling.

(67) (1) First-generation tiles and second-generation tiles were prepared by the method above. (2) Seeds, first-generation tiles, and second-generation tiles, with specific concentration ratio (for example, seed:first-generation tile:second-generation tile=1:32:30 in dimer system), were mixed well. (3) The mixture was annealed at 50 C. for 20 min, cooled from 50 C. to 24 C. at a rate of 1.0 C./h and from 24 C. to 4 C. at a rate of 3.0 C./h using an incubator. (4) The solution was exposed to 360 nm ultraviolet lamp (UVP, Model XX-15L, 15 W) at 10 C. for 2-3 hrs. (5) 2 L of the solution was taken out of (4) for AFM imaging. The remaining solution underwent steps (3) to (5) for further self-replication cycles.

(68) Amplification of DNA Origami Dimer by a Serial Transfer Experiment.

(69) After 4 cycles of replication (when half of the monomers were consumed and a leveling off began), 8% of the material from the reaction mixture was transferred to the next replication tube, which contained a fresh supply of later-generation monomers, with an initial ratio of dimer, first-generation and second-generation tiles to be around 1:32:30 after each transfer.

(70) Self-Replication Selection Cycling.

(71) (1) Two seeds (HH and II) and their first- and second-generation tiles (H and I) were prepared by the same method of formation as self-replication seeds. (2) All the tiles with specific ratio (seed:first-generation:second generation=1:8:6) were mixed well. (3) The mixture was annealed at 50 C. for 20 min, cooled from 50 C. to 24 C. at a rate of 1.0 C./h and from 24 C. to 4 C. at a rate of 3.0 C./h using an incubator. (4) The solution was first exposed to the laser diode with the wavelength of 685 nm (Thorlabs HL6750MG, 50 mW, for dominance of II) or 785 nm (Thorlabs L785P090, 90 mW, for dominance of HH) at 4 C. for 20 min. The control sample without laser exposure skipped this step. The solution was exposed to the laser diode and ultraviolet lamp at 4 C. for 50 min. (5) A 2 L of the solution was taken out of (4) for AFM imaging. The rest of the solution underwent the steps from (3) to (5) for further self-replication cycles. (6) After each two cycles, the first- and second-generation tiles of the dominate products were added into the solution to keep the initial ratio (dominant dimer:corresponding first-generation tiles:corresponding second-generation tiles=1:7:7). The first- and second-generation tiles of the other species were also added to keep the equal amount of both species (H and I).

(72) AFM Imaging.

(73) Most of the AFM imaging was performed in tapping-mode in buffer, except for the imaging of double-layer complexes of the seed and the first generation. (1) Tapping-mode in Buffer: A 3-5 L of diluted DNA sample (preheated at 46 C. for 1 h) was spotted on freshly cleaved mica (Ted pella, Inc.) and was left for 1 min to be absorbed. A 30 L 1TAE/Mg.sup.2+ buffer (28 mM magnesium acetate) was added to both the mica surface and the liquid cell. (2) Tapping-mode in air: A 5-7 L of diluted DNA sample was spotted on freshly cleaved mica (Ted pella, Inc.) at 4 C. and was left for 3 min to be absorbed. The mica was washed with 3 drops of ddH.sub.2O three times, and excess water was removed by blotting the mica with a filter paper. The mica was then blown dry using compressed air. All AFM imaging was performed on a NanoScope IV MultiMode SPM (Digital Instruments) with silicon tips (Veeco, Inc.).

(74) Results

(75) The present inventors have centered their efforts on a general process that autonomously replicates seeds fabricated from individual units into copies with the same shape and information that will continue to replicate for numerous further generations. The goal here is to make devices and materials that can grow exponentially and evolve to have specific desired properties in response to selective pressures. The basic idea of the present process is (i) to have each unit in the seed bind specifically to a complementary unit from a bath with a temperature dependent bond at low temperature, (ii) to covalently link the assembled daughter units using UV-photoactivated bonding and then (iii) separate seeds from daughters by heating. Both seeds and daughters serve as templates for the further progeny, doubling the population with each generation. A realization of such a system is shown in FIG. 1A, there are two complementary tiles made from rectangular DNA origami constructs (Rothemund, 2006). On one side the tiles are decorated by DNA hairpins that form a T or an A to be read by atomic force microscopy (AFM). On the flip sides are eight vertical sticky ends (red strands) that are complementary, T tiles to A tiles, and pair these tiles in the vertical direction. They encode the information for recognition and guarantee information transfer from a seed to later generations. The sticky ends are supported vertically by hybridizing their lower nine nucleotides with eight strands to form double helices. A second set of sticky ends (drawn in dotted and dashed lines) is attached to both ends of the origami tiles horizontally, in the direction of the origami tile's DNA double helices. These can bind to other tiles to form patterns of the origami tiles, TT, TA, ATA. etc. There are six horizontal sticky ends on each side of each tile, of which four contain the photo-cross-linkable nucleotide (FIG. 1B) 3-cyanovinylcarbazole nucleoside (.sup.CNVK) (Yoshimura et al., 2008). All hairpins, sticky ends and supports are extensions of the staple strands that hold the origami together.

(76) FIG. 1C shows a schematic of self-replication cycling for the DNA origami pattern, using a dimer DNA seed. To begin, the seed TT is constructed from two T-tiles using sticky-end cohesion. The T-tiles of the seed have different groups of horizontal sticky ends with high melting temperature, no .sup.CNVK and no external bonds. An AFM image of the seed is shown in FIG. 2A, demonstrating that the dimer seed forms as designed with high yield (see FIG. 10A for zoomed-out images). Seeds as initiators are added to a pool of A-tile and T-tile monomers. First-generation tiles (A-tiles, AFM image shown in FIG. 2A, zoom-out images shown in FIG. 13B) and seeds with complementary vertical cohesive sequences are paired together to form double-layer origami dimers (FIG. 2A and FIGS. 10C-10D), analogous to the DNA base pairing of adenine and thymine (5-ApA-3 with 5-TpT-3). Two free monomer tiles in solution cannot bind to each other, in the temperature range of 4 C.50 C., during the replication cycle, owing to the low melting temperature of the horizontal sticky ends. When immobilized on the seed by vertical binding, adjacent DNA origami tiles are present at a greatly enhanced local concentration, leading to the formation of six sticky end pairs .sup.CNVK will between adjacent DNA tiles. Upon 366-nm UV irradiation, sticky ends containing bond covalently to the thymine base diagonally opposite in the complementary strand. The same DNA sequences of horizontal sticky ends are used in the A-tile or the T-tile, except for the seed, to ensure successful replication.

(77) To quantify the amplification, N, of dimers, each generation was sampled using AFM images to measure the percentage of dimers, P.sub.n, compared to the initial percentage of seeds, P.sub.0, with N=P.sub.n/P.sub.0 (see Table 6 below).

(78) TABLE-US-00006 TABLE 6 Statistics for replication of dimer DNA origami tile calculated from AFM images Seed (TT):First-generation tile (A):Second-generation tile (T) 1:16:14 1:32:30 1:1024:1022 Cycle N Monomer Dimer Monomer Dimer Monomer Dimer 1 1346 100 707 24 2 1000 154 899 62 1414 3 3 977 404 732 109 1036 5 4 647 373 504 212 1020 10 5 635 467 542 372 1177 21 6 437 439 466 401 983 34 7 449 559 1812 142 8 346 567 2760 391 9 1524 554 10 1340 701 11 995 667 12 984 766 13 698 746 14 531 918

(79) FIG. 2C(i) shows an AFM image of the initial state of the pool containing dimer TT-seeds, A-tiles and T-tiles in a ratio of 1:32:30. After 8 cycles of self-replication, the dimers become dominant products in the pool by consuming monomers of A-tiles and T-tiles to produce further-generation AA and TT dimers (FIG. 2C(ii)). FIG. 2B shows the exponential amplification from analysis of more than 700 tiles in each cycle of self-replication (generation). For an initial ratio of TT-seed:A-tile:Ttile=1:1024:1022 (solid curve with squares; AFM images of cycles 2-14 are shown in FIG. 12), the exponential amplification is more than a 480-fold amplification after 9 cycles (amplification of 1.986/cycle). All amplification curves level off when about half of the monomers are consumed. The yield of the replicated generations with precisely encoded information TT or AA is estimated to be more than 99% (more than 100 dimers sampled in zoomed-in AFM images; no AT or TA dimers were found). However, as the cycle number increases, some trimer tiles are found with an extremely low yield (<1%). To demonstrate that seeds trigger self-replication, a control has been performed in which the solution contains only monomers of A-tiles and T-tiles; fewer than 14% dimers are detected after 20 replication cycles (FIG. 11).

(80) Exponential growth was allowed to proceed indefinitely in a serial transfer experiment wherein a portion of a half-reacted mixture is transferred to a new pool that contains fresh later-generation monomers of A-tiles and T-tiles. The system with a ratio of 1:32:30 was used. Six successive replications were carried out in this manner, each 4 cycles in duration (when half of the monomers were consumed and a leveling off began; FIG. 3A) and then 8% of the material was transferred from one reaction solution to the next. The initial solution contained TT seeds, but all subsequent replication pools contained only those dimers TT or AA that were transferred over. The initial concentration ratio of dimers, A-tiles and T-tiles was maintained around 1:32:30 after each transfer. FIG. 3C shows the products of a randomly chosen replication unit of 4 cycles in duration, cycles 17-20, which verify that the percentage of dimers grows as the cycle number increases. Exponential growth was maintained throughout 24 cycles, with an overall amplification (1.93/cycle) of greater than 7,000,000-fold for dimers TT and AA as shown in FIG. 3 (see Table 7 below for statistics of amplification of dimers through a serial transfer experiment).

(81) TABLE-US-00007 TABLE 7 Statistics for amplification of dimer by a serial transfer experiment Cycle Dimer Ampli- N M D Percentage fication 0 0.03125 1 1 707 24 0.0635 2.03 2 899 62 0.121 3.87 3 732 109 0.230 7.36 4 504 212 0.457 14.6 4 (T1) 1698 30 0.0341 1.09 5 1199 39 0.0611 1.79 6 1286 94 0.128 3.75 7 1409 228 0.245 7.19 8 1144 507 0.470 13.8 8 (T2) 1588 27 0.0329 1.05 9 1536 61 0.0736 2.24 10 2081 147 0.124 3.77 11 2249 385 0.255 7.76 12 939 391 0.454 13.8 12 (T3) 2227 38 0.0330 1.06 13 1016 35 0.0645 1.95 14 1454 104 0.125 3.79 15 1680 265 0.240 7.27 16 617 268 0.465 14.1 16 (T4) 2290 40 0.0338 1.08 17 2441 89 0.0680 2.01 18 1056 71 0.118 3.51 19 1039 150 0.224 6.64 20 970 408 0.457 13.5 20 (T5) 1686 26 0.0299 0.957 21 2424 82 0.0634 2.12 22 2015 143 0.124 4.15 23 1326 213 0.243 8.13 24 875 318 0.421 14.1

(82) Nondenaturing agarose gel electrophoresis was also applied to examine the self-replication products from cycle 8 to cycle 12 (FIG. 3B). The intensity of the upper bands, representing dimers, increases exponentially from cycle 8 to cycle 12 (lane 4 to lane 8); while, the intensity of the lower bands, monomers, decreases. The plot of the dimer amplification versus cycle quantified from the gel is consistent with the plot obtained from AFM images.

(83) A selection experiment was performed to demonstrate that the present DNA origami system is capable of evolution. DNA origami dimers were subjected to an environment change that affected their ability to replicate. Here, DNA origami dimers labeled with HH and II were used as competing species. In contrast to the complementary system used above, dimer HH can only replicate HH, and II replicates II. Near-infrared dyes that have light-activated heat generation properties were used to generate the environmental change. IR Dye 700 (hatched circles in FIG. 4A) and IR Dye 800 (dotted circles in FIG. 4A), have been chosen and attached to two origami tiles HH and II (FIG. 4B), respectively (see Table 8 below for formation of dimers HH and II).

(84) TABLE-US-00008 TABLE8 SequencesofIRdye-modifiedstrandsforself-replicationselection First-generationtileH IR700-9A-092 aAAAAAAAATGATAAATAACGCTCA SEQIDNO:320 ACAGTAGGACCGCACT IR700-9A-127 aAAAAAAAAAAATATTCCCCAGCTA SEQIDNO:321 CAATTTTAGAATTAAC IR700-9A-098 aAAAAAAAAAAGGTGGCACGAGTA SEQIDNO:322 GATTTAGTTTCAACATG IR700-9A-133 aAAAAAAAAATTCAACTGAAGAAA SEQIDNO:323 AATCTACGTAACCGGAT IR700-063 tAGGTTTTACAGGAAGATTGTATAC SEQIDNO:324 AGAAAAGCCCCAAAATTTT IR700-064 aCCTATTTAATTATTTGCACGTAAGA SEQIDNO:325 ACCTACCATATCAATTTT IR700-152 aGTCTTTTGGCTGACCTTCATCAAAC SEQIDNO:326 CAGGCGCATAGGCTTTTT IR700-153 aGACTTTTCACAAGAATTGAGTTAT SEQIDNO:327 ATCAGAGAGATAACCTTTT Seedorsecond-generation IR700-9A-092 aAAAAAAAATGATAAATAACGCTCA SEQIDNO:328 tileH ACAGTAGGACCGCACT IR700-9A-127 aAAAAAAAAAAATATTCCCCAGCTA SEQIDNO:329 CAATTTTAGAATTAAC IR700-9A-098 aAAAAAAAAAAGGTGGCACGAGTA SEQIDNO:330 GATTTAGTTTCAACATG IR700-9A-133 aAAAAAAAAATTCAACTGAAGAAA SEQIDNO:331 AATCTACGTAACCGGAT First-generationtileI IR800-9A-092 aAAAAAAAATGATAAATAACGCTCA SEQIDNO:332 ACAGTAGGACCGCACT IR800-9A-127 aAAAAAAAAAAATATTCCCCAGCTA SEQIDNO:333 CAATTTTAGAATTAAC IR800-9A-098 aAAAAAAAAAAGGTGGCACGAGTA SEQIDNO:334 GATTTAGTTTCAACATG IR800-9A-133 aAAAAAAAAATTCAACTGAAGAAA SEQIDNO:335 AATCTACGTAACCGGAT IR800-063 tAGGTTTTACAGGAAGATTGTATAC SEQIDNO:336 AGAAAAGCCCCAAAATTTT IR800-064 aCCTATTTAATTATTTGCACGTAAGA SEQIDNO:337 ACCTACCATATCAATTTT IR800-152 aGTCTTTTGGCTGACCTTCATCAAAC SEQIDNO:338 CAGGCGCATAGGCTTTTT IR800-153 aGACTTTTCACAAGAATTGAGTTAT SEQIDNO:339 ATCAGAGAGATAACCTTTT Seedorsecond-generation IR800-9A-092 aAAAAAAAATGATAAATAACGCTCA SEQIDNO:340 tileI ACAGTAGGACCGCACT IR800-9A-127 aAAAAAAAAAAATATTCCCCAGCTA SEQIDNO:341 CAATTTTAGAATTAAC IR800-9A-098 aAAAAAAAAAAGGTGGCACGAGTA SEQIDNO:342 GATTTAGTTTCAACATG IR800-9A-133 aAAAAAAAAATTCAACTGAAGAAA SEQIDNO:343 AATCTACGTAACCGGAT

(85) The heat generation profiles of the oligonucleotides modified with IR Dye 700 or IR Dye 800 at their 5 ends were measured during 30 minutes of laser irradiation. The concentrations of the oligonucleotides modified with dyes are controlled to be similar to the local concentration of the dyes in double-layer origami dimers. IR Dye 700- and IR Dye 800-modified oligonucleotides produced a temperature increase of about 5 C. under 685-nm laser and 785-nm laser irradiation, respectively (FIG. 13). The effect of light/heat on the formation of first-generation dimers from a template:monomer ratio of 1:2 is shown in FIG. 4D. Formation of species HH was barely affected by 785-nm laser diode, but 685-nm laser irradiation caused a significant reduction in yield (see Table 9 below for statistics).

(86) TABLE-US-00009 TABLE 9 Statistics for formation yields of daughter generations under 685-nm or 785-nm laser irradiation. Monomer Dimer Dimer Formation Yield HH-785 108 181 0.702 HH-685 475 277 0.0387 II-785 323 168 0.143 II-685 257 319 0.597

(87) The opposite results were obtained in the formation of species II. The two IR dyes indeed produce a replication selection where an environmental change results from laser color change. In the selection system, the initial concentrations of seeds HH and II were the same and the ratio of seeds:monomers=1:16 for both I-tiles and H-tiles. After vertical recognition and cooling, successive generations of NIR dye modified tiles were exposed to laser irradiation for 20 minutes before UV exposure. The IR dyes absorbed energy and locally heated the tiles of the corresponding wavelength. The vertical binding efficiency of successive generations and concomitantly the horizontal photo-cross-linking yield of later generations decreases, due to the local temperature increase. The temperature increase decays inversely with distance from absorbing tiles; thus, if the average temperature rise of a tile containing a dye is 10 C., a tile 1 micron away is heated less than 1 C. The replication yield of the absorbing DNA origami dimers was observed to be reduced. As illustrated in FIG. 4C, species II amplifies under 685-nm light, while species HH amplifies under 785-nm light. So as to prevent degradation of heat production, fresh NIR dye-modified monomers were added into the replication mixture after each 2 cycles, thereby keeping a ratio of 1:16 between dimer and monomer. The result of the self-replication selection under different environments is shown in FIG. 4E. The proportion between dimer HH and II was quantified from AFM images using the simple relationships, D.sub.II=1D.sub.HH, where D.sub.HH and D.sub.II are the fractions of dimers HH and dimers II, respectively (FIGS. 14A-14D and Table 10 below).

(88) TABLE-US-00010 TABLE 10 S Statistics for self-replication selection Control Selection with Selection with Selection with (Without laser) 785-nm laser 685-nm laser laser switch Cycle Monomer HH II Monomer HH II Monomer HH II Monomer HH II 0 529 19 19 529 19 19 529 19 19 529 19 19 2 319 51 52 765 95 34 308 20 42 308 20 42 4 300 51 58 964 129 16 823 23 117 1526 67 54 6 316 59 64 2013 251 15 1584 19 185 560 49 16

(89) After 6 cycles, species HH became dominant under 785-nm laser irradiation, representing more than 94% of the total dimers (solid diamond curve in FIG. 4E). In contrast, dimer II became the preferred species, 90%, under 685-nm laser irradiation (dashed triangle curve). When no laser was applied (control system, no environment change, curves with square as datapoints), the ratio of dimer HH and II remained constant around 1:1, confirming that both species underwent replication amplification at similar rates. It was also demonstrated that minor species can become dominant if the environment changes favorably (the laser was changed from 685 nm to 785 nm after two replication cycles). The curves with circles as datapoints in FIG. 4E show that the dimers II with a higher fraction in the first two cycles turned into minor species after the laser was switched to 785 nm; meanwhile, the dimers HH became dominant. After the dimer purification from the replication mixtures, AFM images of cycles 2 and 6 (each 2 self-replication cycles) under 685-nm or 785-nm laser exposure are presented in FIGS. 4F-4G. The results indicate that artificial evolution promoting one component over another has indeed been achieved using the wavelength of light as the selection factor.

(90) The replication/selection system the present inventors have demonstrated depends upon the catalysis of progeny formation by a covalently crosslinked parent. It is related to the purification/selection system used by Adleman when he amplified the correct start and end points of a Hamiltonian path search using PCR (Adleman, 1994). The process demonstrated in this Example should be readily adapted to other systems that need to optimize the properties of materials. A multi-component seed is introduced into a bath of the individual components, it templates the assembly of complementary (or similar) components by specific reversible recognition, which can be chemical or physical (Sacanna et al., 2010); a subsequent step fixes the daughter configuration permanently and the daughter is then separated from the template. The material with the desired properties is distinguished from other members in the pool by enabling it to replicate. Cycling produces exponential growth and environmental changes can affect growth rates, leading to selective evolution. The present demonstration with DNA origami tiles may prove particularly useful since origami tiles can be attached to many nano- and micron-scale components; these components can then be assembled and replicated into devices that are selectively evolved and optimized for different purposes.

(91) Having now fully described this invention, it will be appreciated by those skilled in the art that the same can be performed within a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation.

(92) While this invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications. This application is intended to cover any variations, uses, or adaptations of the inventions following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth as follows in the scope of the appended claims.

(93) All references cited herein, including journal articles or abstracts, published or corresponding U.S. or foreign patent applications, issued U.S. or foreign patents, or any other references, are entirely incorporated by reference herein, including all data, tables, figures, and text presented in the cited references. Additionally, the entire contents of the references cited within the references cited herein are also entirely incorporated by references.

(94) Reference to known method steps, conventional methods steps, known methods or conventional methods is not in any way an admission that any aspect, description or embodiment of the present invention is disclosed, taught or suggested in the relevant art.

(95) The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying knowledge within the skill of the art (including the contents of the references cited herein), readily modify and/or adapt for various applications such specific embodiments, without undue experimentation, without departing from the general concept of the present invention. Therefore, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein. It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance presented herein, in combination with the knowledge of one of ordinary skill in the art.

REFERENCES

(96) Adleman, L. M., Molecular computation of solutions to combinatorial problems, Science 266, 1021-1024 (1994). Cho, Y. et al., Controlled release of an anti-cancer drug from DNA structured nano-films. Scientific Reports 4:4078 (2014), doi:10.1038/srep04078. Douglas, S. M., Marblestone A. H., Terrapittayanon, S., Vasquez, A., Church, G. M., Shih, W. M., Rapid prototyping of 3D DNA-origami shapes with caDNAno. Nucl. Acids Res. 37, 5001-5006 (2009). Eckardt, L. H., Naumann, K., Pankau, W. M., Rein, M., Schweitzer, M., Windhab, N., von Kiedrowski, G., Chemical copying of connectivity, Nature 420, 286-286 (2002). Ellington, A. D., Szostak, J. W., In Vitro selection of RNA molecules that bind to specific ligands, Nature 346 818-822 (1990). Lee, D. H., Severin, K., Yokobayashi, Y. & Ghadiri, M. R. Emergence of symbiosis in peptide self-replication through a peptide hypercyclic network. Nature 390, 591-594 (1997). Leunissen, M. E., Dreyfus, R., Sha, R., Wang, T., Seeman, N. C., Pine, D., Chaikin, P. M., Towards self-replicating materials of DNA-functionalized colloids. Soft Matter 5, 2422-2430 (2009). Lin, C. Rinker, S., Wang, X., Liu, Y, Seeman, N.C., H. Yan, H., In Vivo Cloning of Artificial DNA Nanostructures, Proc. Nat. Acad. Sci. (USA) 105, 17626-17631 (2008). Lincoln, T. A. & Joyce, G. F. Self-sustained replication of an RNA enzyme. Science 323, 1229-1232 (2009). Rothemund, P. W. K., Folding DNA to create nanoscale shapes and patterns Nature, 440, 297-302, 2006. Sacanna, S., Irvine, W. T. M., Chaikin, P. M. & Pine, D. J. Lock and key colloids. Nature 464, 575-578 (2010). Schulman, R. & Winfree, E. Synthesis of crystals with a programmable kinetic barrier to nucleation. Proc. Natl Acad. Sci. USA 104, 15236-15241 (2007). Tuerk, C., Gold, L., Systematic evolution of ligands by exponential enrichment, Science 249, 505-510 (1990). Wang, T., Sha, R., Dreyfus, R., Leunissen, M. E., Maass, C., Pine, D., Chaikin, P. M., Seeman, N.C., Self-replication of information-bearing nanoscale patterns, Nature, 478, 225-228, (2011). Wei, B., Wang, Z. & Mi, Y., Uniquimer: Software of de novo DNA sequence generation for DNA self-assemblyAn introduction and the related applications in DNA self-assembly J. Comp. & Theor. Nanosci. 4, 133-141 (2007). Wintner, E. A., Conn, M. M. & Rebek, J. Jr. Studies in molecular replication. Acc. Chem. Res. 27, 198-203 (1994). Yoshimura, R. & Fujimoto, K. Ultrafast reversible photo-cross-linking reaction: toward in situ DNA manipulation. Org. Lett. 10, 3227-3230 (2008). Zhang, H. et al., Folding super-sized DNA origami with scaffold strands from long-range PCR Chem. Commun. 48:6405-6407 (2012). Zhao, Y. et al., DNA origami delivery system for cancer therapy with tunable release properties. ACSNANO 6(10):8684-8691 (2012).