TREA

Crisscross cooperative self-assembly

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Information

  • Patent Grant
  • 12351862
  • Patent Number
    12,351,862
  • Date Filed
    Friday, January 14, 2022
    3 years ago
  • Date Issued
    Tuesday, July 8, 2025
    20 days ago
Information
Abstract
Provided herein, in some embodiments, are methods, compositions and kits for controlling nucleation and assembly of molecular nanostructures, microstructures and macrostructures.
Description
REFERENCE TO A SEQUENCE LISTING SUBMITTED AS A TEXT FILE VIA EFS-WEB

This application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Apr. 13, 2022, is named H049870616US02-SEQ-MSB and is 137600 bytes in size.


BACKGROUND

In nature, biomolecules assemble into hierarchical structures through intermolecular interactions. In synthetic biology, it is possible to rationally design biosynthetic building blocks, with hierarchical structures arising from built-in functionality at the molecular level controlling intermolecular interactions. Such biosynthetic self-assembling structures have useful applications in the field of nanotechnology, for example.


SUMMARY

Provided herein, in some embodiments, is a technology (including, for example, methods, compositions and kits) for controlling nucleation and hierarchical assembly (programmable self-assembly) of molecular structures, such as nucleic acid (e.g., DNA) and/or protein nanostructures, microstructures, and macrostructures. This technology, referred to herein as ‘crisscross cooperative assembly’ can be used to program and rapidly assemble structures that only originate from provided macromolecular ‘seeds,’ thus may be considered a ‘zero-background’ assembly method. Through the design of cooperative binding sites on individual biomolecular subunits that require simultaneous engagement with a large number of other subunits to achieve stable attachment, the system imposes an intrinsically high energetic barrier against spontaneous nucleation of structures, even in the presence of high concentrations of each individual component. Nucleation can only be triggered by providing a macromolecular ‘seed’ that resembles a pre-existing structural interface (presents multiple weak binding sites for stable capture of the next subunit). Addition of a seed that can stably capture individual subunits effectively bypasses the activation energy barrier against spontaneous nucleation to drive higher-order assembly of a microscale structure. Components can be continually added to the structures such that their growth in one-dimension, two-dimensions or three-dimensions is potentially as large as for other polymerization or crystallization processes.


Crisscross cooperative assembly, as provided herein, uses molecular (e.g., nucleic acid or protein) building blocks (FIG. 1A) that are programmed to self-assemble into crisscrossed layers (FIG. 1B). A building block, in some embodiments, may be a rod-shaped structure assembled from programmable nucleic acid hybridization interactions. As indicated above, this crisscross cooperative assembly technology uses a ‘seed’ structure from which programmable nucleic acid self-assembly begins. This seed structure is formed through irreversible interactions between a nucleating structure (FIG. 1A; ‘queen’) and a subset of building blocks (FIG. 1A; ‘drones’) that are aligned to form an initial seed layer along the nucleating structure. In the presence of a seed structure, another set of building blocks (FIG. 1A; ‘workers’) are added to the pre-existing seed layer (FIG. 1B). Binding between a sufficient number of building blocks (drones) and a nucleating structure (queen) to form a seed can trigger the addition of many additional layers of building blocks (workers), with each layer rotated by some degree (e.g., 90°) relative to adjacent layers (above and/or below).


The nucleating structure and the building blocks are engineered to interact with (e.g., bind to) each other based on a set of kinetic/nucleation energy parameters, as follows. An initial subset of building blocks (drones) should bind strongly (irreversibly/stably) to and form an aligned layer along the nucleating structure (queen). The building blocks (drones) of the initial subset should not interact with (bind to) each other. Likewise, building blocks (workers) of a subsequent subset should not interact with (bind to) each other. Further, in the absence of a nucleating structure (queen), any building block (drone) from the initial subset should have only one weak (reversible) interaction with any other building block (worker) from another subset. In the presence of a nucleating structure (queen), a single building block (drone) from an initial subset may interact with more than one building block (worker) from a subsequent subset, and a single building block (worker) from a subsequent subset may interact with more than one building block (drone) from the initial subset or another subset (‘workers’ of another subset). For example, with reference to FIG. 1B, a single building block (e.g., DNA nanorod) may bind to eight other building blocks (e.g., DNA nanorods), although the single building block binds to each of the eight building blocks only once to form two layers having a ‘crisscross’ pattern.


The single interaction between a building block (drone) from the initial subset and a building block from a subsequent subset (worker) should be weak enough such that there is an arbitrarily large entropy penalty against nucleation in the absence of a seed structure (a large number of individual workers would have to come together simultaneously). With these parameters, zero-background and minimal defects can be achieved, even at high concentrations

    • of interacting building blocks, thereby enabling rapid nucleation and assembly of nucleic acid nanostructures.


Thus, provided herein are compositions, comprising (a) a nucleating nucleic acid nanostructure, (b) a first layer of parallel elongated nucleic acid nanostructures stably bound to the nucleating nanostructure of (a), and (c) a second layer of parallel elongated nucleic acid nanostructures stably bound to the elongated nanostructures of (b) and rotated at an angle relative to the parallel elongated nanostructures of (b), wherein a single elongated nanostructure of (b) binds to multiple elongated nanostructures of (c), each through a single cooperative binding site.


In some embodiments, a single elongated nanostructure of (c) binds to multiple elongated nanostructures of (b), each through a single cooperative binding site.


Also provided herein, in some aspects, are compositions comprising: (a) nucleating nanostructures; (b) a first subset of elongated nanostructures, wherein less than 10% of the nanostructures of (b) bind to each other, and wherein the nanostructures of (b) irreversibly bind to a nucleating nanostructure of (a); and (c) a second subset of elongated nanostructures, wherein less than 10% of the nanostructures of (c) bind to each other, wherein, in the absence of a nucleating nanostructure, a nanostructure of (b) can reversibly binding to a nanostructure of (a) only at a single position on the nanostructure of (a), and wherein, in the absence of a nucleating nanostructure, a nanostructure of (a) can reversibly binding to a nanostructure of (b) only at a single position on the nanostructure of (b). See, e.g., FIGS. 1A-1B.


Also provided herein, in some embodiments, are crisscross nucleic acid nanostructures, comprising a first nanorod comprising a first plug strand and a second plug strand; a second nanorod comprising a third plug strand and a fourth plug strand, wherein the second nanorod is parallel to the first nanorod; a third nanorod comprising a fifth plug strand complementary to and bound to the first plug strand and a sixth plug strand complementary to and bound to the second plug strand; a fourth nanorod comprising a seventh plug strand complementary to and bound to the third plug strand and an eighth plug strand complementary to and bound to the fourth plug strand, wherein the third nanorod is parallel to the fourth nanorod. See, e.g., FIG. 18. A crisscross nanostructure is not limited to 4 nanorods and, in many embodiments, includes at least 4 (e.g., at least 5, 10, 15, 20, 25, 50, 100 or more) nanorods arranged in a crisscross pattern as described herein.


Thus, in some embodiments, a crisscross nucleic acid nanostructure, comprises a first plurality of nanorods parallel to each other, and a second plurality of nanorods parallel to each other, wherein the nanorods of the first plurality are bound to and perpendicular to (or are non-parallel to) the nanorods of the second plurality. See, e.g., FIG. 18.


In some embodiments, each nanorod is comprised of DNA. For example, a nanorod may be comprised of a 6-helix DNA bundle (see, e.g., Douglas S M1, Chou J J, Shih W M. DNA-nanotube-induced alignment of membrane proteins for NMR structure determination. Proc Natl Acad Sci USA. 104, 6644-6648, 2007, incorporated herein by reference).


Also provided herein, in some aspects, are crisscross nucleic acid slats, comprising: a first plurality of at least four nucleic acid strands parallel to each other, each strand of the first plurality having a length of 20-100 nucleotides (e.g., 20-30, 20-40 or 20-50 nucleotides); and a second plurality of at least four nucleic acid strands parallel to each, each strand of the second plurality having a length of 20-100 nucleotides (e.g., 20-30, 20-40 or 20-50 nucleotides), wherein the at least four nucleic acid strands of the first plurality are bound to and perpendicular to the at least four nucleic acid strands of the second plurality. See, e.g., FIGS. 21A-21B.


Also provided herein, in some aspects are crisscross nucleic acid slats, comprising: a first plurality of at least four nucleic acid strands parallel to each other, each strand of the first plurality having a length of at least 21 nucleotides; and a second plurality of at least four nucleic acid strands parallel to each, each strand of the second plurality having a length of at least 21 nucleotides, wherein the at least four nucleic acid strands of the first plurality are bound to and perpendicular to the at least four nucleic acid strands of the second plurality. See, e.g., FIGS. 21A-21B.


Further provided herein, in some aspects, are nucleic acid nanostructures comprising a nucleic acid scaffold strand folded (e.g., M13 or M13-derived) into repeating loop-like shapes (e.g., 5-15 loops, or 5, 6, 7, 8, 9 or 10 loops) secured by shorter nucleic acid staple strands, wherein the repeating loop structures are bound to at least one (e.g., at least 2, 3, 4, 5, 10, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 or more) crisscross nucleic acid slat. See, e.g., FIGS. 22, 24A, 25 and 27B.


Further still, provided herein, in some aspects, are nucleic acid nanostructures, comprising a nucleic acid scaffold strand folded into repeating loop-like shapes secured by at least two crisscross nucleic acid slats. See, e.g., FIGS. 27B and 28B.


The present disclosure also provides, in some aspects, methods of producing a crisscross nucleic acid nanostructures, comprising: combining in a reaction mixture (a) a first nanorod comprising a first plug strand and a second plug strand, (b) a second nanorod comprising a third plug strand and a fourth plug strand, wherein the second nanorod is parallel to the first nanorod, (c) a third nanorod comprising a fifth plug strand complementary to and bound to the first plug strand and a sixth plug strand complementary to and bound to the second plug strand, and (d) a fourth nanorod comprising a seventh plug strand complementary to and bound to the third plug strand and an eighth plug strand complementary to and bound to the fourth plug strand, wherein the third nanorod is parallel to the fourth nanorod; and incubating the reaction mixture under conditions (e.g., nucleic acid hybridization conditions) that result in assembly of a crisscross nucleic acid nanostructure. See, e.g., FIG. 22.


Biomolecule (analyte) detection methods are also provided herein, in some aspects. In some embodiments, a method, comprises (a) combining in a reaction mixture (i) a sample comprising a biomolecule; (ii) a nucleic acid strand capable of self-assembling into a nanostructure that comprise vertically-stacked parallel strands; (iii) a plurality of oligonucleotides, shorter than the nucleic acid strand of (ii), wherein the oligonucleotides of (iii) bind to the strand of (ii) to assemble the vertically-stacked parallel strands; (iv) two crisscross nucleic acid slats, wherein the two slats bind to the strand of (ii), and wherein each of the slats is linked to a biomolecule binding partner that specifically binds to the biomolecule in the sample; (b) incubating the reaction mixture under conditions that permit binding of the biomolecule binding partners to the biomolecule and assembly of the nanostructure into vertically-stacked parallel strands; (c) removing the plurality of oligonucleotides of (iii) from the reaction mixture of (b); (d) incubating the reaction mixture of (c) in the presence of a plurality of crisscross nucleic acid slats described herein, wherein the crisscross nucleic acid slats bind to the vertically-stacked parallel strands to form a three-dimensional barrel structure. In some embodiments, the methods further comprise imaging the three-dimensional barrel structure. See, e.g., FIGS. 28A-28B.


In some embodiments, the methods may comprise combining in a reaction mixture (e.g., with hybridization buffer) (a) a sample comprising a biomolecule and (b) a nucleic acid nanostructure comprising (i) a nucleic acid scaffold strand capable of folding into repeating loop-like shapes (e.g., 2-15 vertically-stacked loops) and (ii) two crisscross nucleic acid slats, wherein a biomolecule binding partner (e.g., an antibody) that specifically binds to the biomolecule is linked to each of the crisscross nucleic acid slats such that in the presence of the cognate biomolecule the biomolecule binding partner binds to the biomolecule and the nucleic acid nanostructure folds into repeating loop-like shapes. See, e.g., FIGS. 28A-28B.


In some embodiments, the methods further comprise combining the reaction mixture with a plurality (e.g., 2-50 or 2-100) of crisscross nucleic acid slats to form a three-dimensional barrel-like structure. See, e.g., FIG. 24B.


It should be understood that the nucleic-acid nanostructures as described herein (e.g., nanorods, slats, barrels, etc.) and variants thereof, as provided herein, may be designed, for example, using the following publicly-available tool described by Douglas S M, Marblestone A H, Teerapittayanon S, Vazquez A, Church G M, Shih W M. Rapid prototyping of 3D DNA-origami shapes with caDNAno. Nucleic Acids Res. 37, 5001-5006, 2009, incorporated herein by reference. See, also, Douglas et al. Nature, 459(7245): 414-418, 2009, incorporated herein by reference. For example, and as described elsewhere herein, it is known in the art that custom shape (e.g., megadalton-scale) DNA nanostructures may be produced using a long ‘scaffold’ strand to template the assembly of hundreds of oligonucleotide ‘staple’ strands into a planar antiparallel array of cross-linked helices. This ‘scaffolded DNA origami’ method has also been adapted to produce 3D shapes formed as pleated layers of double helices constrained to a honeycomb lattice. caDNAno, an open-source software package with a graphical user interface, may be used to aid in the design of DNA sequences for folding 3D DNA (or other nucleic acid) nanostructures. The caDNAno software is available at cadnano.org, along with example designs and video tutorials demonstrating their construction.





BRIEF DESCRIPTION OF THE DRAWINGS


FIGS. 1A-1B show an abstraction of an example of crisscross cooperative assembly system. FIG. 1A indicates the system without a nucleation site and no self-assembly. FIG. 1B indicates the system after the addition of the nucleation site and triggered spontaneous self-assembly. Growth direction is indicated by the grey arrows and shows a 1D growth in this example. Individual components are referred to as ‘queen,’ ‘drone’ and ‘worker.’ FIG. 2 shows a graph depicting the principle by which the nucleation site (queen) structure initiates higher-order structures with drones and workers by lowering the activation energy for assembly.



FIGS. 3A-3C show examples of DNA-origami crisscross assembly. FIG. 3A shows queen (Q) and drone/worker (D/W) architecture shown in cross-section (caDNAno software downloaded from cadnano.org) and in 3D representation. Each individual cylinder represents a double stranded DNA helix. FIG. 3B is a representation of 1D and 2D growth with the crisscross DNA-origami cooperative assembly. 3D growth can be achieved by creating a design that merges 1D and 2D growth. FIG. 3C shows different pathways for 2D growth.



FIG. 4 shows an example of single-stranded DNA crisscross cooperative assembly. Oligonucleotides comprising the workers and drones of the system (shown as cylinders) are nucleated by the addition of a cubic DNA-Origami queen structure with a nucleation site. Stepwise assembly is shown in illustrated magnification.



FIG. 5 shows an example of catenane crisscross cooperative assembly queen (catenane queen), useful for ultrasensitive detection. Oligonucleotides comprising the workers and drones of the system (shown as cylinders) are nucleated by the addition of a single-stranded catenane queen structure with a nucleation site. Binding sites on the structure shown to the left of the illustrated magnification indicate the nucleation for the workers/drones. Each host ring has multiple binding sites, collectively functioning as a cooperative binding site.



FIG. 6 shows a catenane queen from FIG. 5 that has been modified to serve as a biosensor. The large DNA ring has been split to incorporate and biomolecule capture site to bind a biomolecule (e.g., macromolecule) in biological samples. The presence of the biomolecule, in some embodiments may be detected in mixtures as follows: (1) A biological sample is mixed with a high concentration of the catenane queen and a biomolecule of interest binds the biomolecule capture site. (2) A chemical reaction is used to reversibly cleave the biomolecule capture site. (3) Catenane queens not bound to the target biomolecule fall apart more quickly compared to those held together by the target biomolecule. (4) Remaining catenane queens in the test mixture are re-ligated at the biomolecule capture site. (5) Drones and workers are added to the test mixture to amplify remaining queens using readily observable micrometer-scale DNA structures. This system is modular, as the biomolecule capture site may be customized to bind disease markers, including proteins or nucleic acid sequences.



FIG. 7 shows a CAD schematic of an example of base-pairing linkages between a 6 helix bundle worker and a 6 helix bundle drone to queen. A plug socket linkage design may also be used, as shown in FIGS. 18-20. The following CAD tool was used to design the structures: Douglas S M, Marblestone A H, Teerapittayanon S, Vazquez A, Church G M, Shih W M. Rapid prototyping of 3D DNA-origami shapes with caDNAno. Nucleic Acids Res. 37, 5001-5006, 2009.



FIGS. 8A-8D are schematics depicting different example ‘seed’ designs (a 6-helix bundle nanorod bound to a nucleating nanostructure) with different cooperative bind site configurations. Additional example ‘seed’ designs are shown in FIGS. 16A-16C.



FIG. 9A-9B show results from a seesaw experiment with an example of a nucleating nanostructure (queen) folded at different temperatures (A: 65-60° C.; B: 60-55° C.; C: 65-55° C.; D: 60-50° C.) at a MgCl2 concentration of 6 mM.



FIGS. 10A-10B show results from a seesaw experiment with an example of a nanostructure (drone) folded at different temperatures (A: 70-60° C.; B: 65-55° C.; C: 65-60° C.; D: 60-55° C.) at a MgCl2 concentration of 6 mM.



FIG. 11 shows a schematic depicting the assembly of an example seed structure (queen+drones). Images of the structures are also shown. Nanostructure assembly may also be carried out as shown in FIGS. 19A-19D.



FIGS. 12A-12G show a seed structure forming from the assembly of a single-stranded DNA and additional nanostructures (drones).



FIGS. 13A-13B show results from seesaw experiments with a single-stranded nucleating nanostructure (queen) at various temperatures and steps.



FIGS. 14A-14C show results demonstrating that nanostructures (workers) assemble in the presence of a nucleating nanostructure (queen) but not in the absence of a nucleating nanostructure.



FIGS. 15A-15B show results an example of nanostructures not assembling in the absence of a nucleating nanostructure (queen).



FIG. 16A shows a two-dimensional view of the gridiron queen that can bind 16 drones simultaneously in a horizontal (coordinate x in FIG. 16B) across the queen. Staples necessary to fold the scaffold into the queen are shown, and the 3′ ends of the staples may be appended with overhanging single-stranded sequences to bind drones. A three-dimensional view (FIG. 16B) shows the queen with binding sites in each drone-docking cell. A transmission electron microscope (TEM) image of the queen is shown in FIG. 16C. Lateral dimensions of the structure are approximately 72 nm×240 nm.



FIGS. 17A-17B depict drone and worker subcomponents. FIG. 17A shows drone and worker subcomponents constructed from 6-helix bundle scaffolded DNA origami to form rods that are customizable in length. The 3′ ends of staples contain overhanging single strand DNA sequences that act as plug binding handles to interact with other components. Similarly, the lowermost helix contains socket sequences (i.e. single strand DNA scaffold not complemented by a folding staple) that accept plug sequences from other components. The plugs and socket respectively are periodic and can be situated, for example, every 42 bp (˜14 nm) along the length of the component. FIG. 17B shows TEM images of test drones folded from two different scaffold sequences. The drone in the top image is ˜250 nm in length, versus the drone in the bottom image, which is ˜440 nm in length.



FIG. 18 shows a detailed view of the plug-socket binding system. The case shown in the upper panel shows the full set of 5 single-stranded plug sequences extending from the queen (the small arrow) with matching socket sites in a six-helix bundle drone. In the lower pane, the binding sequence is drawn as a series of ‘X’ to indicate that both the length and sequence of the plug and socket may be varied. The scaffold sequences are drawn in black. Note that this design (shown for a drone-queen assembly) is also used to bind drones to workers. The gridiron queen sequences, from top to bottom, correspond to SEQ ID NOs: 685 and 686. The 6 helix bundle (hb) drone sequences, from left to right, correspond to SEQ ID NOs: 687 and 688.



FIGS. 19A-19D shows how the plug-socket binding system can be used to program drones to bind to desired sites on the queen. FIG. 19A shows two 440 nm drones placed in the middle two queen cells, FIG. 19B shows one 250 nm drone placed in the middle queen cell, and FIG. 19C shows 250 nm located in every cell of the queen. The desired design is shown to the left, versus a TEM image of the assembled structure to the right. FIG. 19D shows bulk analysis of the design from FIG. 19A using agarose gel electrophoresis for one design using a 7 bp plug-socket, and another with a 10 bp plug-socket.



FIG. 20 shows the extent of free queen remaining over time as it becomes bound to a single 440 nm drone.



FIGS. 21A-21B show an example of a crisscross DNA slat-based architecture. FIG. 21A is an abstraction of the crisscross DNA slats motif (right). Light and dark strands weave and are complementary to each other at each junction. The length of each binding site is shown on the right. Each row and column amount to 21 base pairs (bp). The matrix shows the number of base pairs (bp) per binding site at each position of the abstraction and 3D rendering. FIG. 21B is a 3D rendering of the DNA slats. On the left, the top down view shows the weaving of each strand. A cross section (A-A) is shown on the right.



FIG. 22 shows the steps to DNA slats assembly. Step 1 shows DNA-origami folding of an arbitrary DNA-origami queen (a barrel queen shown as an example). Step 2 is the mixing of the crude DNA-origami queen reaction (from step 1) with DNA slats at various salt concentrations, temperatures, and DNA slat concentration.



FIG. 23A shows a flat DNA-origami queen without any DNA slats added. FIG. 23B shows a flat DNA-origami queen with the addition of DNA slats and the correct formation of a sheet, by tiling the ssDNA scaffold of the queen with DNA slats. DNA slat tiled region is indicated in light gray. Scale bars on images are 600 nm and on enlarged view 100 nm.



FIG. 24A shows a barrel DNA-origami queen without any DNA slats added. Scale bar on image is 400 nm and on enlarged view 100 nm. FIG. 24B shows a barrel DNA-origami queen with the addition of DNA slats and the correct formation of a barrel, by tiling the ssDNA scaffold of the queen with DNA slats. Scale bar indicates 50 nm. DNA slat tiled region is indicated in light gray.



FIG. 25 depicts a growth mechanism of DNA slats seeded on a queen structure. The DNA-origami queen is mixed with DNA slats to tile the ssDNA scaffold of the queen and then later extend and polymerize the growth of micron-sized structures solely through DNA slats (DNA slats may be joined end-to-end, within the same plane, through nucleotide base pairing of adjacent slats). The upper design shows a flat DNA-origami queen with the growth of three linear sheets in the horizontal direction. The lower design shows a barrel DNA-origami queen with tubular growth in the vertical direction.



FIGS. 26A-26E show three extensions of the first generation of DNA slats binding to the flat DNA-origami queen. FIG. 26A shows first generation extensions tiled with a short second generation of DNA slats, resulting in three tooth-like extensions on the queen. FIG. 26B shows first generation extensions tiled with a long second generation of DNA slats, which are terminally tiled with short third generation DNA slats. FIGS. 26C-26E show first, second, and third generations of DNA slats which are complementary to one another, resulting in extensions of linear sheet structures. FIG. 26C contains one extended first generation, FIG. 26D contains two extended first generations, and FIG. 26E contains three extended first generations. Scale bars for FIGS. 26A-26C are 100 nm and for FIGS. 26D-26E, 20 0 nm.



FIGS. 27A-27E show formation of multi-host-ring catenane systems with DNA slats in a one-pot reaction. (FIG. 27A) Formation of eight loops with M13 scaffold through staple strands. Staple (“brown”) strands fold stable DNA-Origami base and DNA slats catenate the eight loops. (FIG. 27B) 3D view of barrel queen additionally serving as multi-host-ring catenane system with high yield typical for DNA-Origami. (FIG. 27C) Abstract and 3D view of DNA slats weaving through the ssDNA M13 scaffold loops on the barrel queen. Through ligation of one side a single DNA slat catenates all eight loops. (FIG. 27D) 3D rendering of DNA slat weaving and catenating eight separate ssDNA loops. Top shows a tilted bottom view and bottom a side view. (FIG. 27E) Former technique to achieve a maximum of four-host-ring catenane system with low yield.



FIGS. 28A-28B show a barrel queen used for ultrasensitive detection. FIG. 28A shows that the biomolecule presence is connected to the DNA slats (black) holding the eight loops together. Without the biomolecule, the queen falls apart and no growth can occur, even with DNA slats present in solution. FIG. 28B shows that biomolecule presence in the reaction holds the DNA slats (black) together and provides the close proximity of ssDNA scaffold for the tube structure to nucleate and grow.



FIG. 29 shows a schematic (upper panel) of a queen with six binding sites per slat and a transmission electron microscope (TEM) image (lower panel) of the queen.



FIGS. 30A-30B show a flat DNA-origami queen nucleating a staggered DNA slats ribbon. FIG. 30A depicts a flat DNA-origami queen without the bottom right hand sheet shown in FIG. 23A. FIG. 30B is a schematic explaining how the DNA slats (moving in a diagonal direction) assemble on the ssDNA scaffold on the flat queen.



FIG. 31 shows an example of a biomolecule sensing and proofreading mechanism on DNA-Origami barrel queen. Top: Biomolecule present. (1) Biomolecule binds to antibody bridge. (2) Medium gray strand is displaced via toehold-mediated strand displacement. (3) Light gray strand binds to dark gray strands, sealing bridge. Bottom: No biomolecule present. (1) No biomolecule binds to the antibody bridge. (2) Medium gray strand is displaced via toehold-mediated strand displacement, leading to no bridge being intact and the subsequent falling apart of the barrel queen (shown in FIG. 28A).





DETAILED DESCRIPTION

Nature achieves rapid and nucleation-limited growth of cytoskeletal filaments such as actin and microtubules. This is achieved by securing each additional subunit by weak interactions to 2-3 already attached subunits at the growing end of the filament. This means that if any two monomers bind to each other in solution, they will rapidly (e.g., within milliseconds) dissociate from each other, because the single interaction is so weak. It is only after four subunits come together simultaneously—a rare event—that a stable nucleus will be formed. Therefore, untriggered spontaneous nucleation will be rare. Conversely, nucleation can be triggered by providing a macromolecular “seed” that mimics a fully formed filament end.


Rapid and nucleation-limited growth are very useful features for programmable self-assembly, however technological modification of natural filaments such as actin or microtubules has many current drawbacks: (1) there is a limited understanding of how to tune the interaction strength between subunits; (2) the level of cooperativity is relatively low (the weak interactions upon binding are spread only over 2-3 subunits), therefore the suppression of spontaneous nucleation is not as robust as it could be; and (3) growth is limited to one-dimension (filament formation).


Rapid, reversible, zero-background, triggered nucleation and growth, as provided herein, can have useful applications in nanotechnology and biotechnology, such as ultrasensitive detection, and templates for miniaturized materials.


Crisscross Cooperative Assembly


The crisscross cooperative assembly technology as provided herein is based on a concept that may apply to many self-assembling molecules, including nucleic acids and proteins. For simplicity and ease of understanding, however, reference herein primarily will address crisscross cooperative assembly in the context of nucleic acids, such as deoxyribonucleic acid (DNA). A crisscross cooperative assembly system uses three basic components: a nucleating nanostructure, an initial (first) subset of nanostructures programmed to bind to the nucleating nanostructure, and another (second) subset of nanostructures programmed to bind to the nanostructures of the initial.


An example of a crisscross cooperative assembly is provided in FIGS. 1A-1B, wherein the nucleating structure is referred to as a ‘queen,’ nanostructures of the first subset are referred to as ‘drones,’ and nanostructures of the second (and any subsequent) subset are referred to as ‘workers.’ The final structure, in this example, includes layers of aligned molecular rods, where each layer is rotated by some amount (e.g., 90 degrees) relative to the layer below and above. For example, one layer may be perpendicular to another adjacent (directly above or below) layer. In some embodiments, one layer is rotated 20, 30, 40, 50, 60, 70, 80 or 90 degrees relative to an adjacent layer (measured alone the length of a drone and/or worker nanorod, for example). Each intersection between rods on adjacent layers adds a small binding energy; any given rod intersects with a large number of rods below and above, and the net binding energy can be tuned (e.g., by adjusting the design of the binding interface, for example, the number of base pairs, or by adjusting subunit concentration, temperature, or salt concentration) to be large enough to achieve stable (irreversible) or slightly favorable (reversible) attachment as desired. Before assembly initiates, any spontaneous crossing between two rods in solution is short-lived, as the net energy is very low because there is only one interaction. Thus, a rod can be stably (or else slightly favorably (reversibly)) added to a pre-existing crisscross structure (many attachment points can immediately be realized), but a structure will not spontaneously assemble in the absence of a pre-existing one. There should be no growth unless a structural mimic of a pre-existing crisscross structure—a seed—is added to the solution.


An example protocol for a crisscross cooperative assembly system is as follows: (1) Design constitutive building blocks (queen, drones and workers) using DNA CAD tools. See, e.g., Douglas S M, Marblestone A H, Teerapittayanon S, Vazquez A, Church G M, Shih W M. Rapid prototyping of 3D DNA-origami shapes with caDNAno. Nucleic Acids Res. 37, 5001-5006, 2009, incorporated herein by reference in its entirety.


Cooperative binding site sequences and number of sites on queens are tailored to modulate the activation energy of nucleation as required. (2) Construct and purify constitutive building blocks using techniques in DNA synthesis and DNA origami. (3) Mix drones and workers in solution, and add queens to initiate growth of higher order DNA structures.


Nanostructures bind to each other through cooperative binding sites. A “cooperative binding site” is the location at which two nanostructures interact (hybridize/bind). For example, a nucleating nanostructure may be programmed with multiple nucleotide base sequences, each of which is complementary to a nucleotide base sequence of one of the nanostructures of the initial subset of nanostructures. A cooperative binding site may include plug and socket sites that include plug and socket strands. A plug strand is a nucleic acid strand (single-stranded nucleic acid) attached to a nucleic acid nanostructure, such as a nanorod. A plug strand contains a nucleotide sequence that is complementary to (and this binds to) a nucleotide sequence within a cognate socket strand. Thus, a pair of plug and socket strands include nucleotide sequences that are complementary to each other such that the plug and socket strand bind (hybridize) to each other to anchor, for example, a drone to a queen or a worker to a drone (see, e.g., FIG. 17B). In some embodiments, a queen includes multiple plug strands that direct and anchor a drone that includes multiple complementary (cognate) socket strands. Likewise, a drone may include multiple plug strands that direct and anchor a worker that includes multiple complementary socket strands.


Cooperative binding sites, e.g., plug and socket strands, may also be used to assemble nucleic acid (e.g., DNA) slats onto another nucleic acid scaffold structure in a similar manner. For example, as shown in FIG. 22, DNA slats may be appended to a nucleic acid scaffold (queen) to secure the two- or three-dimensional shape of the scaffold structure. In the example, shown in FIG. 22, DNA slats are used to secure (hold together) the barrel shape of a larger scaffold nanostructure. “Growth” of these slats along the scaffold through cooperative binding sites results in a barrel-like shape that may be visualized by microscopy, for example.


Cooperative binding sites (e.g., plug and socket sequences) are arranged on a nucleating nanostructure in a spatial configuration that facilitates binding and alignment of the initial e.g., scaffold) nanostructures. The length of a cooperative binding site may vary, depending in part on the desired strength (strong v. weak) of the intended interaction between two molecules having complementary sites. In some embodiments, a cooperative binding site has a length of 5-50 nucleotides. For example, a cooperative binding site may have a length of 5-40, 5-30, 5-20, 5-10, 5-15, 10-50, 10-40, 10-30, 10-20, 30-50, 30-40, or 40-50 nucleotides. In some embodiments, a cooperative binding site has a length of 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides. A single plug strand and/or socket strand may have a length of 5-20 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) nucleotides, for example.


The number of cooperative binding sites on a nanostructure may also vary. In some embodiments, the number of cooperative binding sites on a nanostructure is 3-1000. For example, the number of cooperative binding sites on a nanostructure may be 3-900, 3-800, 3-700, 3-600, 3-500, 3-400, 3-300, 3-200, or 3-100. In some embodiments, the number of cooperative binding sites on a nanostructure is 3-10, 3-15, 3-20, 3-25, 3-30, 3-35, 3-40, 3-45 or 3-50. In some embodiments, the number of cooperative binding sites on a nanostructure is 3-15, 3-20, 3-25, 3-30, 3-35, 3-40, 3-45 or 3-50. In some embodiments, the number of cooperative binding sites on a nanostructure is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100.


The distance between cooperative binding sites may also vary. In some embodiments, the distance between two cooperative binding sites on the same nanostructure is 20-1000 angstroms. For example, the distance between two cooperative binding sites on a nanostructures may be 20-900, 20-800, 20-700, 20-600, 20-500, 20-400, 20-300, 20-200, 20-100, 50-1000, 50-900, 50-800, 50-700, 50-600, 50-500, 50-400, 50-300, 50-200, or 50-100 angstroms. In some embodiments, the distance between two cooperative binding sites on a nanostructures is 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450 or 500 angstroms.


In some embodiments, the distance between cooperative binding sites, for example, the distance between plug strands (and/or between socket strands) may be 5 to 100 nucleotides (or nucleotide base pairs (bp)). In some embodiments, the distance between plug strands (and/or between socket strands) is 5-20, 5-25, 5-50 or 5-100 nucleotides. In some embodiments, the distance between plug strands (and/or between socket strands) is 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 nucleotides. In some embodiments, the distance between plug strands (and/or between socket strands) is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 nucleotides. In some embodiments, the distance between plug strands (and/or between socket strands) is 42+/−21 nucleotides. For example, the distance between plug strands (and/or between socket strands) may be 21, 42 or 63 nucleotides. In some embodiments, the distance between plug strands (and/or between socket strands) is 42 nucleotides.


One nucleotide unit measures 0.33 nm. Thus, in some embodiments, the distance between cooperative binding sites, for example, the distance between plug strands (and/or between socket strands) may be 5 to 35 nanometers (nm). In some embodiments, the distance between plug strands (and/or between socket strands) is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nm. In some embodiments, the distance between plug strands (and/or between socket strands) is 14+/−7 nm. For example, the distance between plug strands (and/or between socket strands) may be 7, 14 or 21 nm. In some embodiments, the distance between plug strands (and/or between socket strands) is 14 nucleotides.


In some embodiments, the distance between two cooperative binding sites on a nanostructure is evenly spaced, while in other embodiments, the distances may vary. For example, the distance between a first cooperative binding site and a second cooperative binding site may be 30 angstroms, while the distance between the second cooperative binding site and a third may be 30 angstroms, 40 angstroms or 50 angstroms.


Two or more nanostructures are considered “aligned” if they are oriented in the same direction relative to one another. For example, the 5′ ends (or 3′ ends) of the nanostructures maybe facing the same direction along its y axis. The top layer of the structure shown in FIG. 3B shows aligned nanorods bound to a nucleating nanostructure. The nanorods, in this example, are perpendicular to the nucleating nanostructure.


A nucleating nanostructure is required to initiate assembly of the first (initial) and second (and, thus, subsequent, e.g., third, fourth, fifth, etc.) subsets of nanostructures, and binding of the nanostructures in the first subset to the nucleating structure is required to initiate assembly of the nanostructures of the second subset. A “nucleating nanostructure” is any nanostructure programmed with binding sites that interacts strongly (irreversibly) with binding sites on each member of drone nanostructures of the initial subset, and aligns them for recruitment of subsequent subsets of worker nanostructures. That is, the binding sites between a nucleating nanostructure and nanostructures of the initial subset should be strong enough that the initial nanostructures bind to and align along the nucleating nanostructures and do not dissociate from the nucleating nanostructure under reaction conditions (e.g., isothermal, physiological conditions). A nucleating nanostructure may have a two-dimensional or a three-dimensional shape, for example.


Additional subsets of nanostructures may be added to the crisscross cooperative assembly system to propagate growth of the end structure (e.g., nanostructure, microstructure or macrostructure). For example, third, fourth and fifth subsets of nanostructures may be added. Binding of the nanostructures of the second subset to the first subset is required to initiate assembly of the nanostructures of the third subset; binding of the nanostructures of the third subset to the second subset is required to initiate assembly of the nanostructures of the fourth subset; and so on. The user-defined end structure may be assembled in one dimension, two dimensions (see, e.g., FIG. 3B) or three-dimensions.


Each subset of nanostructures (e.g., nanorods) should follow a specific set of binding energy parameters. More specifically, the initial subset of nanostructures (e.g., nanorods) should bind strongly (irreversibly) to and form an aligned layer (where each nanostructure is oriented in the same direction relative to one another) along the nucleating nanostructure. The nanostructures (e.g., nanorods) of the initial subset should not interact with (bind to) each other. Likewise, nanostructures (e.g., nanorods) of a subsequent subset should not interact with (bind to) each other. Further, in the absence of a nucleating structure, any nanostructure (e.g., nanorod) from the initial subset should have only one weak (reversible) interaction with any other nanostructure (e.g., nanorod) from a subsequent subset. In the presence of a nucleating structure, a single nanostructure (e.g., nanorod) from an initial subset may interact with more than one nanostructure (e.g., nanorod) from a subsequent subset, and a single nanostructure (e.g., nanorod) from a subsequent subset may interact with more than one nanostructure (e.g., nanorod) from the initial subset. For example, with reference to FIG. 1B, a single nanostructure (e.g., nanorod) may bind to eight other nanostructure (e.g., nanorod), although the single nanostructure (e.g., nanorod) binds to each of the eight nanostructure (e.g., nanorod) only once to form two layers having a ‘crisscross’ pattern.


A “strong interaction” refers to binding that is engaged more than 50% (e.g., more than 60%, 70%, 80% or 90%) of the time that the binding nanostructures are in a reaction together (the dissociation constant is lower than the concentration of the species/nanostructures in excess).


A “weak interaction”—refers to binding that is engaged less than 1% of the time that the binding nanostructures are in a reaction together (the dissociation constant is at least 100 times higher than the concentration of the species/nanostructures in excess).


A nucleating nanostructure may bind to two or more other nanostructures. In some embodiments, a nucleating nanostructure binds to 5-1000 nanostructures (e.g., DNA nanorods). For example, a nucleating nanostructure may bind to 3-900, 3-800, 3-700, 3-600, 3-500, 3-400, 3-300, 3-200, or 3-100 nanostructures. In some embodiments, a nucleating nanostructure binds to 3-10, 3-15, 3-20, 3-25, 3-30, 3-35, 3-40, 3-45 or 3-50 nanostructures. In some embodiments, a nucleating nanostructure binds to 10-15, 10-20, 10-25, 10-30, 10-35, 10-40, 10-45 or 10-50 nanostructures. In some embodiments, a nucleating nanostructure binds to 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 nanostructures (e.g., DNA nanorods).


Thus, a single subset of nanostructures (nanostructures programmed to interact with a single nucleating nanostructure) may comprise 3-900, 3-800, 3-700, 3-600, 3-500, 3-400, 3-300, 3-200, or 3-100 nanostructures. In some embodiments, a single subset of nanostructures comprises 3-10, 3-15, 3-20, 3-25, 3-30, 3-35, 3-40, 3-45 or 3-50 nanostructures. In some embodiments, a single subset of nanostructures comprises 10-15, 10-20, 10-25, 10-30, 10-35, 10-40, 10-45 or 10-50 nanostructures. In some embodiments, a single subset of nanostructures comprises 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 nanostructures (e.g., DNA nanorods).


A “subset of nanostructures” refers to a specific group of nanostructures that are similar in size (have similar dimensions) and structure/shape and are programmed to bind to either the nucleating nanostructure (the initial subset) or to a pre-existing layer formed by alignment and binding of other nanostructures that have already aligned and bound to the nucleating structure or nanostructures of another pre-existing layer.


Nanostructures within a defined subset are programmed not bind to each other. Thus, in some embodiments, less than 10% of the nanostructures of a subset bind to another nanostructure of the same subset. In some embodiments, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.2%, or less than 0.1% of the nanostructures of a subset bind to another nanostructure of the same subset. In some embodiments, none of the nanostructures of a subset bind to another nanostructure of the same subset.


With crisscross cooperative assembly, nanostructures are aligned to form multiple layers, each layer rotated by some degree relative to adjacent layers (above and below). An example of two layers rotated relative to one another is shown in FIG. 1B. The top layer of aligned nanorods is rotated 90 degrees relative to the bottom layer of aligned nanorods. The degree of rotation between two adjacent layers may vary. In some embodiments, one layer is rotated 10-90 degrees, 20-90 degrees, 30-90 degrees, 40-90 degrees, 50-90 degrees, 60-90 degrees, 70-90 degrees, or 80-90 degrees relative to an adjacent layer. In some embodiments, one layer is rotated 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90 degrees relative to an adjacent layer.


Nucleic Acid Nanostructures


A “nucleic acid nanostructure,” including a “DNA nanostructure,” refers to a nanostructure (e.g., a structure that is between 0.1 nm and 1 μm (e.g., 0.1 nm and 100 nm) in each spatial dimension, e.g., 1D, 2D or 3D) that is rationally designed to self-assemble (is programmed) into a pre-determined, defined shape that would not otherwise assemble in nature. The use of nucleic acids to build nanostructures is enabled by strict nucleotide base pairing rules (e.g., A binds to T, G binds to C, A does not bind to G or C, T does not bind to G or C), which result in portions of strands with complementary base sequences binding together to form strong, rigid structures. This allows for the rational design of nucleotide base sequences that will selectively assemble (self-assemble) to form nanostructures.


Examples of nucleic acid (e.g., DNA) nanostructures include, but are not limited to, DNA origami structures, in which a long scaffold strand (e.g., at least 500 nucleotides in length) is folded by hundreds (e.g., 100, 200, 200, 400, 500 or more) of short (e.g., less than 200, less than 100 nucleotides in length) auxiliary strands into a complex shape (Rothemund, P. W. K. Nature 440, 297-302 (2006); Douglas, S. M. et al. Nature 459, 414-418 (2009); Andersen, E. S. et al. Nature 459, 73-76 (2009); Dietz, H. et al. Science 325, 725-730 (2009); Han, D. et al. Science 332, 342-346 (2011); Liu, W et al. Angew. Chem. Int. Ed. 50, 264-267 (2011); Zhao, Z. et al. Nano Lett. 11, 2997-3002 (2011); Woo, S. & Rothemund, P. Nat. Chem. 3, 620-627 (2011); Tørring, T. et al. Chem. Soc. Rev. 40, 5636-5646 (2011)). Other more modular strategies have also been used to assemble DNA tiles (Fu, T. J. & Seeman, N. C. Biochemistry 32, 3211-3220 (1993); Winfree, E. et al. Nature 394, 539-544 (1998); Yan, H. et al. Science 301, 1882-1884 (2003); Rothemund, P. W. K. et al. PLoS Biol. 2, e424 (2004); Park, S. H. et al. Angew. Chem. Int. Ed. 45, 735-739 (2006); Schulman, R. & Winfree, E. Proc. Natl Acad. Sci. USA 104, 15236-15241 (2007); He, Y. et al. Nature 452, 198-201 (2008); Yin, P. et al. Science 321, 824-826 (2008); Sharma, J. et al. Science 323, 112-116 (2009); Zheng, J. P. et al. Nature 461, 74-77 (2009); Lin, C. et al. ChemPhysChem 7, 1641-1647 (2006)) or RNA tiles (Chworos, A. et al. Science 306, 2068-2072 (2004); Delebecque, C. J. et al. Science 333, 470-474 (2011)) into periodic (Winfree, E. et al., Nature 394, 539-544 (1998); Yan, H. et al. Science 301, 1882-1884 (2003); Chworos, A. et al. Science 306, 2068-2072 (2004); Delebecque, C. J. et al. Science 333, 470-474 (2011)) and algorithmic (Rothemund, P. W. K. et al. PLoS Biol. 2, e424 (2004)) two-dimensional lattices (Seeman, N. C. J. Theor. Biol. 99, 237-247 (1982); Park, S. H. et al. Angew. Chem. Int. Ed. 45, 735-739 (2006)), extended ribbons-(Schulman, R. & Winfree, E. Proc. Natl Acad. Sci. USA 104, 15236-15241 (2007); Yin, P. et al. Science 321, 824-826 (2008)) and tubes (Yan, H. et al. Science 301, 1882-1884 (2003); Yin, P. et al. Science 321, 824-826 (2008); Sharma, J. et al. Science 323, 112-116 (2009)), three-dimensional crystals (Zheng, J. P. et al. Nature 461, 74-77 (2009)), polyhedral (He, Y. et al. Nature 452, 198-201 (2008)) and simple finite two-dimensional shapes (Chworos, A. et al. Science 306, 2068-2072 (2004); Park, S. H. et al. Angew. Chem. Int. Ed. 45, 735-739 (2006)).


Thus, crisscross cooperative assembly building blocks (e.g., nucleating nanostructures and subsets of nanostructures) may be one of a number of nucleic acid nanostructure shapes, including, but not limited to, rods/tubes, sheets, ribbons, lattices, cubes, spheres, polyhedral, or another two-dimensional or three-dimensional shape. In some embodiments, a nanostructure has junction(s), branch(es); crossovers, and/or double-crossovers formed by nucleotide base pairing of two or more nucleic acid strands (see, e.g., Mao, C. PLoS Biology, 2(12), 2036-2038, 2004).


In some embodiments, a nucleic acid nanostructure has a handle and barrel shape, similar to that depicted in FIG. 22.


The versatile and stable nature of DNA origami enables the construction of various individual architectures that can be designed in a particular way, to facilitate to cooperative assembly of larger structures. In one example, each component is a separately folded DNA-origami structure. FIG. 3A shows an example of a DNA origami queen, drone and worker, whereby the drone and worker are of identical architecture (six helix bundle DNA nanotubes). Queen, drones and workers can then assemble in a cooperative manner to form higher order 1D, 2D and 3D structures (FIG. 3B). 3D structures are contemplated by merging 1D and 2D design principles. For example, FIG. 22 depicts a 3D queen nanostructure assembling with 2D drone/worker slats to form a barrel shape.


A nucleic acid (e.g., DNA) slat is a slat-shaped nanostructure that is composed of DNA. A slat may be an antiparallel-crossover single-stranded slat (AXSSS) comprising single strands that cross a partnering single strand only once. Also provided herein are paranemic crossover slats that include a pair of strands that cross another pair of strands.


Similar to the larger scale DNA-origami crisscross cooperative assembly, single-stranded DNA can be used to achieve cooperative assembly of higher order structures. In order to achieve this, drones and workers are replaced with oligonucleotides of various lengths (depending on the proposed architecture) that can assemble onto a DNA-origami queen nucleation site (shown in FIG. 4) or onto a single stranded DNA catenane structure shown in FIG. 5, FIG. 22 and FIGS. 24A-24B. The ring structures depicted in FIG. 5 are comprised of single-stranded DNA that has exposed binding sites for drone and worker oligonucleotides. In another example, the components are folded into a DNA origami barrel queen (FIGS. 24A-24B). The scaffold can be tiled with extended DNA slats (slats) capable of seeding further DNA slats, leading to growth of the structure. Generally, the DNA slats work in two steps: first, folding the origami queen site (for example, mixing M13 scaffold and staple strands), and second, mixing the crude DNA origami queen reaction with DNA slats, leading to growth of the structure. Varying salt concentrations, temperatures, and DNA slat concentration can alter the binding energy of the various sub-components, leading to reversible or irreversible binding, for example.


Typically, nucleic acid nanostructures do not contain coding sequences (sequences that code for a full length mRNA or protein), thus, nucleic acid nanostructures do not contain a promoter or other genetic elements that control gene/protein expression. An individual single-stranded nucleic acid (e.g., DNA strand or RNA strand without secondary structure), or an individual double-stranded nucleic acid (e.g., without secondary structure), for example, double helices found in nature or produced synthetically or recombinantly (e.g., such as a plasmid or other expression vector), are specifically excluded from the definition of a nucleic acid nanostructure.


Nanostructures, in some embodiments, have a void volume, which is the combine volume of space between nucleic acids that form a nanostructures. It should be understood that “space” includes fluid-filled space. Thus, a nanostructure in solution, have a void volume of 25% may include 75% nucleic acids and 25% reaction buffer (filling the 25% void volume of the nanostructure). In some embodiments, a nanostructure in solution, e.g., in reaction buffer, may have a void volume of at least 10% (e.g., 10-90%, 10-80%, 10-70%, 10-60%, 10-50%, 10-40%, or 10-30%), at least 20% (e.g., 20-90%, 20-80%, 20-70%, 20-60%, 20-50%, 20-40%, or 20-30%), at least 30%, (e.g., 30-90%, 30-80%, 30-70%, 30-60%, 30-50%, or 30-40%), at least 40% (e.g., 40-90%, 40-80%, 40-70%, 40-60%, or 40-50%), at least 50% (e.g., 50-90%, 50-80%, 50-70%, or 50-60%), at least 60% (e.g., 60-90%, 60-80%, or 60-70%), at least 70% (e.g., 70-90% or 70-80%), or at least 80% (e.g., 80-90%). In some embodiments, a nanostructure has a void volume of 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%.


A “nucleic acid nanorod,” including a “DNA nanorod” is a nucleic acid (e.g., DNA) nanostructure in the shape of a rod. A nanorod is a three-dimensional cylindrical shape having a length longer than its diameter. Examples of nanorods are depicted in FIGS. 1A-1B and FIGS. 3A-3B. In some embodiments, a nucleic acid nanorod comprises six helix bundles. For example, six DNA double helices may be connected to each other at two crossover sites. DNA double helices with 10.5 nucleotide pairs per turn facilitate the programming of DNA double crossover molecules to form hexagonally symmetric arrangements when the crossover points are separated by seven or fourteen nucleotide pairs (see, e.g., Mathieu F. et al. Nano Lett. 5(4), 661-664 (2005)). Other methods of assembling nucleic acid nanorods (also referred to as nanotubes) may be used (see, e.g., Feldkamp, U. et al. Angew. Chem. Int. Ed. 45(12), 1856-1876 (2006); Hariri A. et al. Nature Chemistry, 7, 295-300 (2015)).


The length and diameter of a nanorod (or other nanostructure) may vary. In some embodiments, a nanorod (or other nanostructure) has a length of 10-100 nm, or 10-500 nm. For example, a nanorod may have a length of 10-500 nm, 10-400 nm, 10-300 nm, 10-200 nm, 10-100 nm, 10-90 nm, 10-80 nm, 10-70 nm, 10-60 nm, 10-50 nm, 10-30 nm, or 10-20 nm. In some embodiments, a nanorod has a length of 100-500 nm, 200-500 nm, or 300-500 nm. In some embodiments, a nanorod has a length of 100 nm, 150 nm, 200 nm, 250 nm, 300 nm, 350 nm, 400 nm, 450 nm or 500 nm. In some embodiments, a nanorod has a length of 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 nm. In some embodiments, the length of a nanorod (or other nanostructure) is longer than 100 nm (e.g., 100-1000 nm), or shorter than 10 nm (e.g., 1-10 nm). In some embodiments, a nanorod (or other nanostructure) has a diameter of 5-90 nm. For example, a nanorod may have a diameter of 5-80 nm, 5-70 nm, 5-60 nm, 5-50 nm, 5-30 nm, 5-20 or 5-10 nm. In some embodiments, a nanorod has a diameter of 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85 or 90 nm. In some embodiments, the diameter of a nanorod is longer than 9 nm, or shorter than 5 nm. Thus, in some embodiments, a nanorod (or other nanostructure) has a circumference of 15-300 nm (C≈3.14×d).


A nucleic acid nanostructure, such as a nanorod, is considered “elongated,” if the length of the nanostructure is longer than its width/diameter (e.g., by at least 10%, 20%, 25%, 50%, 100%, or 200%).


Nucleic acid nanostructures are typically nanometer-scale structures (e.g., having lengths of 1 to 1000 nanometers). In some embodiments, however, the term “nanostructure” herein may include micrometer-scale structures (e.g., assembled from more than one nanometer-scale or micrometer-scale structure). In some embodiments, a nanostructure has a dimension (e.g., length or width/diameter) of greater than 500 nm or greater than 1000 nm. In some embodiments, a nanostructure has a dimension of 1 micrometer to 2 micrometers. In some embodiments, a nanostructure has a dimension of 10 to 500 nm, 10 to 450 nm, 10 to 400 nm, 10 to 350 nm, 10 to 300 nm, 10 to 250 nm, 10 to 200 nm, 10 to 150 nm, 10 to 100 nm, 10 to 50 nm, or 10 to 25 nm. In some embodiments, the nanostructure has a dimension of 500 to 450 nm, 500 to 400 nm, 500 to 350 nm, 500 to 300 nm, 500 to 250 nm, 500 to 200 nm, 500 to 150 nm, 500 to 100 nm, 500 to 50 nm, or 500 to 25 nm. In some embodiments, the nanostructure has a dimension of 10, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, or 500 nm.


A nucleic acid nanostructure is considered to “self-assemble.” Bottom up, self-assembly refers to the process by which molecules adopt a defined arrangement without guidance or management from an outside source. Although, it should be understood that with synthetic nucleic acid self-assembly, as provided herein, the nucleotide base sequences that guide assembly of nucleic acids are artificially designed, and the corresponding nucleic acids are accordingly synthesized by an outside source, such as one of skill in the art (using, for example, standard nucleic acid synthesis techniques). That is, one of ordinary skill in the art can ‘program’ nucleotide base sequences within a single nucleic acid strand or between two difference nucleic acid strands to selectively bind to each other in solution based on a strict set of nucleotide base pairing rules (e.g., A binds to T, G binds to C, A does not bind to G or C, T does not bind to G or C). Self-assembly may be intramolecular (folding) or intermolecular.


The nanostructures and, thus, nanostructures, microstructures and macrostructures assembled from smaller nanostructures, are “rationally designed.” A nanostructure, as discussed above, does not assemble in nature. Nucleic acid strands for use in crisscross cooperative assembly are ‘programmed’ such that among a specific population of strands, complementary nucleotide base sequences within the same strand or between two different strands bind selectively to each other to form a complex, user-defined structure, such as a rod/tube, ribbon, lattice, sheet, polyhedral, cube, sphere, or other two-dimensional or three-dimensional shape. A nanostructure may have a regular shape (sides that are all equal and interior angles that are all equal) or an irregular shape (sides and angles of any length and degree).


Methods of Crisscross Cooperative Assembly


Self-assembly of a nucleating nanostructure and subsets of nanostructures occurs, in some embodiments, in a ‘one-pot’ reaction, whereby all nucleic acid nanostructures of a crisscross cooperative assembly system are combined in a reaction buffer, and then the reaction buffer is incubated under conditions that result in self-assembly of all of the nucleic acid nanostructures.


Conditions that result in self-assembly of nucleic acid nanostructures of a crisscross cooperative assembly reaction may vary depending on the size, shape, composition and number of nucleic acid nanostructures in a particular reaction. Such conditions may be determined by one of ordinary skill in the art, for example, one who rationally designs/programs the nanostructures to self-assemble.


A crisscross cooperative assembly method may be performed at a variety of temperatures. In some embodiments, a crisscross cooperative assembly method is performed at room temperature (˜25° C.) or 37° C. A crisscross cooperative assembly method may be performed at a temperature lower than 25° C. or higher than 37° C.


The salt concentration of the reaction buffer in which a crisscross cooperative assembly reaction is performed may also vary. In some embodiments, the reaction buffer comprises MgCl2 salt at a concentration of 1 mM-10 mM (e.g., 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM or 10 mM). In some embodiments, the reaction buffer comprises NaCl at a concentration of 100 mM-500 mM (e.g., 100 mM, 200 mM, 300 mM, 400 mM or 500 mM). In some embodiments, a crisscross cooperative assembly method is performed under high-salt conditions. Thus, in some embodiments, the reaction buffer comprises MgCl2 salt at a concentration of at least 20 mM (e.g., 20-500 mM, or 20-200 mM). In some embodiments, the reaction buffer comprises NaCl at a concentration of at least 1 M (e.g., 1-2 M, 1-3 M, 1-4 M, or 1-5 M).


In any given reaction, the number of initial nanostructures (drones) exceeds the number of nucleating nanostructures (queens). Thus, in some embodiments, the ratio of nucleating nanostructure to non-nucleating nanostructure (e.g., a drone from an initial subset, or a worker from a subsequent subset) is 1:10-1:1012 (trillion). For example, the ratio of nucleating nanostructure to non-nucleating nanostructure may be 1:10-1:1000, 1:10-1:500, 1:10-1:100, 1:10-1:75, 1:10-1:50, or 1:10-1:25. In some embodiments, the ratio of nucleating nanostructure to non-nucleating nanostructure is 1:1000, 1:500, 1:100, 1:90, 1:80, 1:70, 1:60, 1:50, 1:40, 1:30, 1:20 or 1:10.


In some embodiments, a crisscross cooperative assembly reaction is incubated for 2-96 hours. For example, a crisscross cooperative assembly reaction may be incubated for 2-24 hours, 2-30 hours, 2-36 hours, 2-42 hours, 2-48 hours, 2-54 hours, 2-60 hours, 2-66 hours, 2-72 hours, 2-78 hours, 2-84 hours, 2-90 hours, or 2-96 hours. In some embodiments, a crisscross cooperative assembly reaction is incubated for 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, or 72 hours. In some embodiments, a crisscross cooperative assembly reaction is incubated for 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70 or 72 hours.


Biosensors


In some embodiments, the crisscross assembly products may be used as biosensors that are capable of detecting a selected biomolecule (analyte) using a variety of different mechanisms and the systems described herein. For example, in such systems, the presence of a biomolecule can be used to trigger crisscross assembly, which can then be detected (visualized), indicating the presence of the biomolecule.


The biomolecule may be detected using a ring system. As depicted in FIG. 6, the large DNA ring (“host ring”), single-stranded DNA, may be split to incorporate a biomolecule capture site (analyte test site) to bind macromolecules in biological samples. The DNA ring loops through and encloses a number of discrete, separate “guest” rings, which are single-stranded DNA and function as catenane queens, so that the guest rings are catenated on the host ring, similar to individual beads on a bracelet. In some embodiments, the guest rings are independently formed from separate single-stranded nucleic acids (see, e.g., FIGS. 5 and 6), while in other embodiments, the guest rings are formed from a long single nucleic acid strand assembled into multiple (e.g., vertically stacked) rings (see, e.g., FIGS. 27A and 27B). The number of guest rings can be 2, 3, 4, or 5 or more. In embodiments, each guest ring (catenane queen) comprises binding sites for drone and worker oligonucleotides and is therefore capable of crisscross assembly. In embodiments, the plurality of catenated guest rings when in close proximity forms a catenane queen comprising binding sites (e.g., plug strands) for drone and worker nucleic acids and/or structures and is thus capable of crisscross assembly. The number of binding sites per guest ring can vary, and may be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100. A biomolecule test site, located near the biomolecule capture site may also formed.


The presence of the biomolecule, in some embodiments may be detected in mixtures, such as biological samples, as follows. First, a biological sample is mixed with a high concentration of the catenane queen, allowing macromolecules of interest bind the biomolecule capture site. Then, a chemical reaction is used to reversibly cleave the biomolecule capture site. Catenane queens not bound to the target biomolecule will fall apart more quickly compared to those held together by the target biomolecule. The remaining catenane queens in the test mixture are re-ligated at the biomolecule test site. Subsequently, drones and workers are added to the test mixture to amplify remaining intact queens using readily observable micrometer-scale DNA structures. This system is modular, and the biomolecule capture site may be customized to bind disease markers, including proteins or nucleic acid sequences.


Ultraspecific biosensors can also be created by adding a biomolecule detection system to the multiple guest-ring (e.g., guest-loop) catenane systems with DNA slats, as depicted in FIGS. 27-28. In this example, a barrel-queen is used; however, other 3-dimensional shapes are also possible (e.g., sheets, blocks and dendrimers). An example of the production of a barrel queen (a rolled sheet) is described above.


An example of a DNA slat is depicted in FIGS. 21A and 21B.


Using a scaffold for DNA origami, for example an M13 scaffold and staple strands, a multiple guest-ring catenane system can be formed. For example, in FIG. 27, an eight-loop system is formed in a one-pot reaction. The number of loops (rings) can be varied, depending on the design of the system, and may be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 loops. Additional loops may be used. Unlike the system described above, the handles (elongated structures) and loops (rings) are all part of the same single-stranded DNA (e.g., M13 DNA); the handle structures are programmed to link together by specific staple strands (slats). The system is designed around the specific staple strands/slats; in the presence of biomolecule, they hold the structure together and growth can occur from the parallel loops when drones and workers are added (FIG. 28B). In the absence of biomolecule, the staple strands/slats release the structure, and no growth can occur as the queen falls apart and the binding sites are not close enough for nucleation and growth even in the presence of drones and workers (FIG. 28A). The presence of the structures can be detected using any one of the methods described above, or with any method known in the art.


DNA slats or other nucleic acids of a biosensor may be modified with one or more switchable bridges. A “switchable bridge” is a link between functional groups that forms or breaks in the presence of a particular agent (e.g., reaction agent or dissociation agent). Examples of switchable bridges include bonds formed via a “click chemistry” reaction (e.g., a between an azide and an alkyne), protein-protein binding (e.g., one or more antibodies binding to a target protein/antigen), a disulfide bond (between two thiols).


Thus, some aspects of the present disclosure provide a biosensor comprising (i) a first DNA slat comprising a first functional group (e.g., an azide or alkyne), a first binding partner (e.g., an antibody, aptamer or nanobody), and a second functional group (e.g., a thiol or nucleic acid), and (ii) a second DNA slat comprising a third functional group (e.g., a thiol or nucleic acid), a second binding partner (e.g., an antibody, aptamer or nanobody), and a fourth functional group (e.g., an azide or alkyne), wherein the first and fourth functional groups react in the presence of a reaction agent to form a link (e.g., a covalent link), wherein the first and fourth binding partners bind specifically to a biomolecule of interest to form a link (e.g., non-covalent link), and wherein the second and third functional groups form a link (e.g., a covalent link) that breaks in the presence of a dissociation agent.


In some embodiments, a biosensor comprises a first DNA slat comprising an azide, an antibody, and a thiol group, and a second DNA slat comprising an alkyne, an antibody, and a thiol group, wherein antibody of (i) and the antibody of (ii) bind specifically to a biomolecule of interest.


A “first biomolecule binding partner” and a “second biomolecule binding partner” are any molecules that bind to the same target biomolecule to form a switchable bridge linking DNA slats to each other (via a non-covalent link). In some embodiments, the first and second biomolecule binding partners are proteins or peptides. For example, the first and second biomolecule binding partners may be antibodies that bind to different epitopes of the same antigen. Thus, in some embodiments, the first and second biomolecule binding partners are antibodies (e.g., monoclonal, polyclonal, human, humanized or chimeric). In some embodiments, the first and second biomolecule binding partners are antibody fragments (e.g., Fab, F(ab′)2, Fc, scFv, or vhh). The biomolecule binding partners may also be nanobodies or aptamers. Other protein-protein binding partners may be used.


A “first functional group” and a “fourth functional group” are functional groups that react with each other to form a link (bond, such as a covalent bond or a non-covalent bond), which forms a switchable bridge linking the DNA slats to each other. In some embodiments, this bridge is formed through a click chemistry (azide-alkyne cycloaddition) reaction (e.g., V. V.


Rostovtsev, et al., Angew. Chem. Int. Ed., 2002, 41, 2596-2599; and F. Himo, et al. J. Am. Chem. Soc., 2005, 127, 210-216, each of which is incorporated herein by reference). Thus, in some embodiments, one of the first or fourth functional group is an azide, while the other of the first or fourth functional groups is an alkyne. For example, the first functional group may be azide, and the fourth functional group may be trans-cyclooctene (TCO). Other click chemistry functional groups may be used.


A “second functional group” and a “third functional group” are functional groups that react with each other to form a link (bond, such as a covalent bond or a non-covalent bond), which forms yet another switchable bridge linking the DNA slats to each other. This bridge breaks (dissociates) in the presence of a dissociation agent. A “dissociation agent” is an agent (e.g., chemical) that breaks the bond (e.g., covalent bond) between the second and third functional groups. In some embodiments, the second and third functional groups are thiol groups that react with each other to form a disulfide bridge. Thus, in some embodiments, the dissociation agent is dithiothreitol (DTT). In some embodiments, the concentration of DTT is 50 mM-200 mM. For example, the concentration of DTT may be 100 mM. Other functional groups may be used.


ADDITIONAL EMBODIMENTS

1. A composition, comprising:






    • (a) nucleating nanostructures;

    • (b) a first subset of elongated nanostructures, wherein less than 10% of the nanostructures of (b) bind to each other, and wherein the nanostructures of (b) irreversibly bind to a nucleating nanostructure of (a); and

    • (c) a second subset of elongated nanostructures, wherein less than 10% of the nanostructures of (c) bind to each other, wherein, in the absence of a nucleating nanostructure, a nanostructure of (b) can reversibly binding to a nanostructure of (a) only at a single position on the nanostructure of (a), and wherein, in the absence of a nucleating nanostructure, a nanostructure of (a) can reversibly binding to a nanostructure of (b) only at a single position on the nanostructure of (b).


      2. The composition of embodiment 1 further comprising (d) a third subset of elongated nanostructures, wherein less than 10% of the nanostructures of (d) bind to each other.


      3. The composition of embodiment 1 or 2, wherein the nanostructures of (b) are aligned in one direction and irreversibly bound to a nucleating nanostructure of (a) to form a first layer.


      4. The composition of embodiment 3, wherein the nanostructures of (c) are aligned in one direction and bound to nanostructures of the first layer to form a second layer, wherein first layer is rotated by 10 degrees to 170 degrees relative to the second layer.


      5. The composition of embodiment 4, wherein the first layer is rotated by 90 degrees relative to the second layer.


      6. The composition of embodiment 4 or 5, wherein the nanostructures of (d) are aligned in one direction and bound to nanostructures of the second layer to form a third layer, wherein second layer is rotated by 10 degrees to 170 degrees relative to the third layer.


      7. The composition of embodiment 6, wherein the first layer is rotated by 90 degrees relative to the second layer.


      8. The composition of any one of embodiments 1-7, wherein the nucleating nanostructures of (a), the nanostructures of (b), the nanostructures of (c), and/or the nanostructures of (d) are nucleic acid nanostructures.


      9. The composition of embodiment 8, wherein the nucleating nanostructures of (a), the nanostructures of (b), the nanostructures of (c), and/or the nanostructures of (d) are DNA nanostructures.


      10. The composition of embodiment 8 or 9, wherein the nucleating nanostructures of (a), the nanostructures of (b), the nanostructures of (c), and/or the nanostructures of (d) comprise a long nucleic acid strand bound to multiple nucleic acid strands that are shorter than the long nucleic acid strand.


      11. The composition of embodiment 8 or 9, wherein the nucleating nanostructures of (a), the nanostructures of (b), the nanostructures of (c), and/or the nanostructures of (d) comprise multiple nucleic acid strands, each having a length of less than 200 nm.


      12. The composition of any one of embodiments 1-6, wherein the nucleating nanostructures of (a), the nanostructures of (b), the nanostructures of (c), and/or the nanostructures of (d) are protein nanostructures.


      13. The composition of any one of embodiments 1-12, wherein the nucleating nanostructures of (a), the nanostructures of (b), the nanostructures of (c), and/or the nanostructures of (d) are rod-shaped.


      14. A method, comprising:

    • combining in reaction buffer

    • (a) nucleating nanostructures,

    • (b) a first subset of elongated nanostructures, wherein less than 10% of the nanostructures of (b) bind to each other, and wherein the nanostructures of (b) irreversibly bind to a nucleating nanostructure of (a), and

    • (c) a second subset of elongated nanostructures, wherein less than 10% of the nanostructures of (c) bind to each other,

    • wherein, in the absence of a nucleating nanostructure, a nanostructure of (b) can reversibly binding to a nanostructure of (a) only at a single position on the nanostructure of (a), and

    • wherein, in the absence of a nucleating nanostructure, a nanostructure of (a) can reversibly binding to a nanostructure of (b) only at a single position on the nanostructure of (b); and
      • incubating the reaction buffer comprising (a), (b) and (c) under conditions that result in binding of the nanostructures of (b) to the nucleating nanostructures of (a) and result in binding of the nanostructures of (c) to the nanostructures of (b) to form a hierarchical structure.


        15. The method of embodiment 14, wherein the reaction buffer further comprises (d) a third subset of elongated nanostructures, wherein less than 10% of the nanostructures of (d) bind to each other.


        16. The method of embodiment 14 or 15, wherein the nucleating nanostructures of (a), the nanostructures of (b), the nanostructures of (c), and/or the nanostructures of (d) are nucleic acid nanostructures.


        17. The method of embodiment 16, wherein the nucleating nanostructures of (a), the nanostructures of (b), the nanostructures of (c), and/or the nanostructures of (d) are DNA nanostructures.


        18. The method of embodiment 16 or 17, wherein the nucleating nanostructures of (a), the nanostructures of (b), the nanostructures of (c), and/or the nanostructures of (d) comprise a long nucleic acid strand bound to multiple nucleic acid strands that are shorter than the long nucleic acid strand.


        19. The method of embodiment 16 or 17, wherein the nucleating nanostructures of (a), the nanostructures of (b), the nanostructures of (c), and/or the nanostructures of (d) comprise multiple nucleic acid strands, each having a length of less than 200 nm.


        20. The method of any one of embodiments 14-16, wherein the nucleating nanostructures of (a), the nanostructures of (b), the nanostructures of (c), and/or the nanostructures of (d) are protein nanostructures.


        21. The method of any one of embodiments 14-20, wherein the nucleating nanostructures of (a), the nanostructures of (b), the nanostructures of (c), and/or the nanostructures of (d) are rod-shaped.


        22. A composition, comprising:

    • (a) nucleating DNA nanostructures;

    • (b) a first subset of elongated DNA nanorods, wherein less than 10% of the nanostructures of (b) bind to each other, and wherein the DNA nanorods of (b) irreversibly bind to a nucleating DNA nanostructure of (a); and

    • (c) a second subset of elongated DNA nanorods, wherein less than 10% of the DNA nanorods of (c) bind to each other,


      wherein, in the absence of a nucleating DNA nanostructure, a DNA nanorod of (b) can reversibly binding to a DNA nanorod of (a) only at a single position on the DNA nanorod of (a), and


      wherein, in the absence of a nucleating DNA nanostructure, a DNA nanorod of (a) can reversibly binding to a DNA nanorod of (b) only at a single position on the DNA nanorod of (b).





EXAMPLES
Example 1

The examples demonstrates assembly of a nucleating nanostructures (queen). The sharpest bands from a screen of nucleic acid self-assembly reactions were selected and subjected to a 2 minute incubation at 90° C. for denaturing and then an 18 hour ramp. The gel (FIG. 9A) was 2% agarose (10 μL ethidium bromide, c=10 mg/mL), and run at 60V for 240 minutes in 0.5×TBE and 11 mM MgCl2. A seesaw experiment was performed, whereby the temperature was varied between 65-60° C. (A), 60-55° C. (B), 65-55° C. (C), and 60-50° C. (D). The structures were purified using band excision of the gel, followed by 15 minutes at 16k×g FreezeNSqueeze and then stained with 2% UF following a 2 minute ddH2O post-wash. The queen folded well, and no noticeable difference was observed between conditions A through D on the seesaw experiment (FIG. 9B).


The experiment was repeated with different nanostructures that function as ‘drones’. The sharpest bands from a large screen were selected and subjected to 2 minute incubation at 90° C. for denaturing and then an 18 hour ramp. The gel (FIG. 10A) was 2% agarose (10 μL ethidium bromide, c=10 mg/mL), and run at 60V for 240 minutes in 0.5×TBE and 11 mM MgCl2. In this experiment, the temperature was varied between 70-60° C. (A), 65-55° C. (B), 65-60° C. (C), and 60-55° C. (D). The structures were purified using band excision of the gel, followed by 15 minutes at 16k×g FreezeNSqueeze and then stained with 1% UF. The drone folded well, and there was no noticeable difference between conditions A through D on the seesaw experiment (FIG. 10B).


Next, the assembly of a queen together with a drone was examined (FIG. 11). The following conditions were tested (with a 1:1:1 ratio): queen—all sites closed and both drones; queen—site 0 exposed and both drones; queen—site 1 exposed and both drones; queen—site 0/1 exposed and both drones; and queen—all sites exposed and both drones. Assembly was achieved through a 72 hour incubation period at 25° C., and the structures were purified by band excision of the gel, followed by 15 minutes at 16k×g FreezeNSqueeze followed by 2% UF staining. The samples were run on a 2% agarose gel (10 μL ethidium bromide, c=10 mg/mL), and run at 60V for 240 minutes in 0.5×TBE and 11mMM MgCl2. Approximately 10-15 ng of each structure were observed.


Example 2

A similar system was built using single-stranded DNA instead of 6 helix bundles. A schematic of the nucleating nanostructure architecture is shown in FIG. 12A. The single strands each contain binding and linker regions, including a 5 bp binding region and 3 and 5 nucleotide linker (poly T) regions. An exemplary 5 bp, 2 nucleotide linker is shown below:









[SEQ ID NO: 683)


TGCAATTTAATTCTTTTAGCATTTCAATATTTGTAGATTTGAGAATTT


CGTTTTTTTATTCA-62 mer.






The workers stack on top of drones in layers (FIGS. 12B-12G). The queen was shown to fold in both 5C or 10C steps (FIG. 12A). Folding occurred after a 2 minute 90° C. denaturing period and then an 18 hour ramp. The samples were run on a 2% agarose gel (10 μL ethidium bromide, c=10 mg/mL), and run at 60V for 240 minutes in 0.5×TBE and 11 mM MgCl2. The queen was incubated under a 50-40° C. thermal ramp, with 6 mM MgCl2. The resulting structure is shown in FIG. 13B.


Example 3

Assembly without the queen and only the workers (both short and long linkers) was examined. In this example, workers did not assemble under conditions with high salt concentration (1M NaCL, up to 15 mM MgCl2), low temperature (4° C.), and a high concentration of workers (3.125 μM). Other conditions, including 10-20 mM PEG and high salt and a high concentration of oligonucleotides were also tested. No assembly occurred. FIGS. 14A-14B show drone/workers at different concentrations successfully assembling with queens. The structures were purified with band excision, 15 minutes at 16k×g FreezeNSqueeze and 2% UF staining. Without the queens, there was no sign of assembly (FIG. 14C).


The duplex length was then increased to 8 bp and linker regions of 2 nt (v0.1) and 3 nt (v0.2) were tested. Weaving was introduced into the structure and staple strands were added to constrain the end of the scaffold loops (FIG. 15A). As seen in FIG. 16B, there was no assembly without the queen in any of the groups and under any of the salt concentration conditions.


Example 4

This Example demonstrates assembly of 6-helix bundle DNA nanorod drones into a crisscross structure via nucleation by a gridiron queen (FIGS. 19A-19D). The gridiron queen has 16 cells, each to which may bind a drone using 5 cooperative plug-socket binding sites. Shown here is site-specific binding of two 440 nm long drones (FIG. 19A), one 250 nm long drone (FIG. 19B), and sixteen 250 nm long drones (FIG. 19C). This example shows binding of drones to the gridiron queen using a 10 bp plug-socket. The agarose gel image in FIG. 19D shows that the queen in the reaction is completely bound by drones, when stoichiometric excess of drones is present. Additionally, functionality of the plug-socket binding system (FIG. 18) is shown with the TEM micrographs of these assemblies and kinetics data (FIGS. 19A-D and FIG. 20).


The gridiron queen and 6-helix bundle drones were conceptualized and then designed using the caDNAno design tool (see FIGS. 16A-16C (and data not shown) for the queen, FIGS. 17A-17B (and data not shown) for the drones). Staple sequences designed in caDNAno were ordered commercially and folded with M13 phage scaffold DNA over the following conditions: drones in 6 mM MgCl2, (90° C./2 mins, 60-50° C./18 hrs); queen in 8 mM MgCl2, ({(94° C.-86° C.) in 4° C./5 min steps}; {(85° C.-70° C.) in 1° C./5 minute steps}; {(70° C. to 40° C.) in 1° C./15 minutes steps}; {(40° C. to 25° C.) in 1° C./10 minute steps}). The scaffold for the gridiron queen was comprised of a 8634 base genome from M13 phage, and staple DNA sequences were determined by caDNAno. Binding sequences for drones were manually appended to the 3′ ends of the staple DNA to bind drones in the desired orientation. The 250 nm and 440 nm 6 hb drones were also designed in caDNAno. The scaffold DNA was comprised of either the 8064 base genome from M13 phage (for the 440 nm drone), or a custom 3825 base sequence derived from M13 phage (for the 250 nm drone). Staple DNA sequences were determined by caDNAno and purchased commercially. Scaffold sections for the sockets and plug sequences were customized to determine the orientation and final location of sub-components in assembled structures. The 5′ ends of a subset of the staples are truncated to free scaffold so that it could act as a socket to bind plugs. The 3′ ends of another subset of staples were appended with plug DNA sequences so that they could interact with other worker subcomponents.


The folded structures were separated from excess folding staples using agarose gel electrophoresis and bands containing the structure of interest were purified from the agarose gel matrix. The purified structures were placed onto carbon grids, stained with 2% uranyl formate, and analyzed by TEM to validate assembly of the correct structure (see FIG. 16C for the queen and FIG., 17B for the drones).


The purified sub-components were assembled into crisscross formation using the following conditions: 0.1 or 0.01 nM queen, 1 nM drones, 30 mM MgCl2, 45 mM Tris-borate, 1 mM EDTA, and 0.01% Tween-20; incubation at 50° Celsius for 8-24 hours. Assembly reactions were analyzed using gel electrophoresis and TEM, as shown in FIGS. 19A-19D. Kinetics of binding between the drone and queen are shown in FIG. 20.


Example 5

A similar system was built using single-stranded DNA (ssDNA) instead of 6 helix bundles, referred to as “crisscross DNA slats” (short: “DNA slats”). A schematic of the base unit is shown in FIGS. 21A-21B. The 21 nucleotide (nt) long oligonucleotide per DNA slat shown in FIGS. 21A-21B allows the 4 by 4 DNA slats array to retains the correct 10.5 bases/turn. The length of the DNA slats can be expanded by repeats of 21 nt, for example, achieving larger structures. FIG. 21A shows an abstraction of the DNA slats architecture and a matrix with the number of base pairs (bp) per binding site at each position. The alternation of 6 bp and 5 bp is used to retain the correct helicity and approximately same binding energy per DNA slat. An exemplary DNA slat strand with 16 binding sites (84 nt) is shown below:









[SEQ ID NO: 684)


TGGTTCTGGAGTTTTACTCGGGACACTTCAGCGTAATATCGGAAGCAGG


CACTTTGAAACCTATAAGTCCTGACTATTAATAAC.







FIG. 21B shows a 3D rendering from the front and cross-section of the DNA slats architecture. The strands weave over and under each other. The DNA slats can reliably tile the ssDNA overhangs (from the M13 scaffold) of different DNA-Origami queens. FIGS. 23A-24B both show examples of ssDNA scaffold being tiled upon and the addition of DNA slats. The flat DNA-Origami queen shown in FIG. 23A is folded through a 2 minute 90° C. denaturing period following an 18-hour ramp from 55° C.-50° C. with 6 mM MgCl2. The barrel DNA-Origami queen shown in FIG. 24A is folded by a 15 minute 80° C. denaturing period following an 18-hour ramp from 60° C.-25° C. with 8 mM MgCl2. The assembly process of DNA slats with the queen is shown in FIG. 22. Once the queen is folded the crude reaction queen is mixed with the DNA slats, assembly conditions can be tuned by varying the concentrations of DNA slats (100 nM-1000 nM), salt (5 mM-30 mM MgCl2 and 0-1 M NaCl), and the temperature of assembly (4° C.-55° C.). By altering the assembly conditions such as MgCl2 and the DNA slat concentration the kinetics of the assembly process can be influenced.


Example 6

This Example shows how extending the DNA slats to create more binding sites facilitates polymerization seeded on the queen. FIG. 25 shows both flat and barrel queen first tiled (as shown in FIGS. 23A-23B and FIGS. 24A-24B) and with extended DNA slats seeding the next generation of DNA slats to bind and eventually grow into micron sized structures. The queen nucleating site determines the shape of the subsequently grown structure. FIGS. 26A and 26B show the flat queen assembled in two types of terminal extensions. FIGS. 26C-26E show the flat queen with one, two, and three domain extensions. Samples shown in FIGS. 26A-26E were prepared using crude flat queen reaction (˜1 nM), DNA slats (1000 nM/strand), and 15 mM MgCl2 at 50 □ for 2 hours. FIGS. 30A-30B show a staggered design of DNA slats that bind to the flat queen, growing a ribbon like sheet. The subsequent formation of ribbons is shown in FIG. 31. Control reactions, without the flat queen, showed no assembly of the DNA slats after 18 hours of running the reaction. Samples shown in FIG. 31 were prepared using crude flat queen reaction (˜9 nM), DNA slats (7500 nM/strand), and 14 mM MgCl2 at 50□ for ˜66 hours.


Example 7

Through the use of the barrel DNA-Origami queen multiple guest-ring catenane systems can be produced in a one-pot reaction. FIGS. 27A-27C show that by folding the barrel queen, a multiple guest-ring catenane system can be achieved through the addition of DNA slats. In order to catenate the loops two DNA slats are needed. A close up view of the DNA slats weaving and catenating the ssDNA M13 scaffold loops is shown in FIG. 27C. By ligating the two DNA slats on one end, a single DNA slat is created that captures all eight loops. A 3D rendering of the purple DNA slat capturing eight loops is shown in FIG. 27D. FIG. 24 shows the addition of 64 slats, which can simply be reduced, depending on size of the size and number of guest rings. Using the barrel queen with the DNA slats achieves a high yield in a one-pot reaction. The barrel queen can subsequently be transformed into an ultrasensitive biosensor, by coupling a biomolecule detection system to the DNA slats (see, e.g., FIG. 31). Through the integration of proofreading steps, the analyte presence can be transferred into the open or closed state of the purple DNA slat. FIGS. 28A-28B show that without a biomolecule present, the queen falls apart (open DNA slats) and no nucleation of DNA slat mediated growth can occur. The presence of a biomolecule, however, keeps the DNA slats intact and holds the queen structure together, which can then trigger the growth of micron sized tubes, for example, that can subsequently be detected using low-cost optical instruments.









TABLE 1







Exemplary Gridiron Queen Staple Sequences











SEQ




ID


Sequence
Comment
NO:












ATCTGAACTCGCTACGGCGGGGGGAGCCCC
20170216_cc6hb_v3_queen, c0, h21, p0, control,
1


CGATTTAGAGCT
cyan, start 0[94], end 21[94], 42 mer






CATTGCTGATACCGTTTAGCTAACAAACATC
20170216_cc6hb_v3_queen, c0, h20, p1, control,
2


AAGAAAACAAA
cyan, start 1[95], end 20[95], 42 mer






GATACTTGCCCTCTCTGTACATAATTAATTT
20170216_cc6hb_v3_queen, c0, h19, p2, control,
3


TCCCTTAGAAT
cyan, start 0[178], end 19[94], 42 mer






GATTGGGCGTTATCAATGTTGTTTTGTCACA
20170216_cc6hb_v3_queen, c0, h18, p3, control,
4


ATCAATAGAAA
cyan, start 1[179], end 18[95], 42 mer






TCTAATGAAGACAAATCCCCACGTCACCGA
20170216_cc6hb v3_queen, c0, h17, p4, control,
5


CTTGAGCCATTT
cyan, start 0[262], end 17[94], 42 mer






AAACATCGGGTTGAGTATTATGTGGCGAGA
20170216_cc6hb_v3_queen, c1, h21, p4, control,
6


AAGGAAGGGAAG
cyan, start 1[53], end 21[136], 42 mer






CGCTGGCATTCGCATCAAAGGCGAATTATT
20170216_cc6hb v3_queen, c1, h20, p3, control,
7


CATTTCAATTAC
cyan, start 2[136], end 20[137], 42 mer






AGTTTATAAATGAGTATCAATTTAGATTAAG
20170216_cc6hb_v3_queen, c1, h19, p2, control,
8


ACGCTGAGAAG
cyan, start 1[137], end 19[136], 42 mer






TATCGACATCATTACGCATCGCAACATATA
20170216_cc6hb v3_queen, c1, h18, p1, control,
9


AAAGAAACGCAA
cyan, start 2[220], end 18[137], 42 mer






CCATGCAGACATCACGAAGGTCACCAGTAG
20170216_cc6hb_v3_queen, c1, h17, p0, control,
10


CACCATTACCAT
cyan, start 1[221], end 17[136], 42 mer






AAGATAACGCTTGTGAAAATGAGGGCGCTG
20170216_cc6hb_v3_queen, c2, h21, p0, control,
11


GCAAGTGTAGCG
cyan, start 2[94], end 21[178], 42 mer






GCTAACAGTAGGGAAACTGCGGCCTGATTG
20170216_cc6hb_v3_queen, c2, h20, p1, control,
12


CTTTGAATACCA
cyan, start 3[95], end 20[179], 42 mer






ATGGGTTCAGGATGCAGGTGAAATCATAGG
20170216_cc6hb_v3_queen, c2, h19, p2, control,
13


TCTGAGAGACTA
cyan, start 2[178], end 19[178], 42 mer






CTCGGATGGGAGTAAGCGTATGCAGTATGT
20170216_cc6hb_v3_queen, c2, h18, p3, control,
14


TAGCAAACGTAG
cyan, start 3[179], end 18[179], 42 mer






AGAGTTTCTGCGGCAGTTAATCAATGAAAC
20170216_cc6hb_v3_queen, c2, h17, p4, control,
15


CATCGATAGCAG
cyan, start 2[262], end 17[178], 42 mer






GCAATACATCAAACGCCGCGAACACCCGCC
20170216_cc6hb_v3_queen, c3, h21, p4, control,
16


GCGCTTAATGCG
cyan, start 3[53], end 21[220], 42 mer






TCAGGCACTGCGTGAAGCGGCAGTAACAGT
20170216_cc6hb_v3_queen, c3, h20, p3, control,
17


ACCTTTTACATC
cyan, start 4[136], end 20[221], 42 mer






ATCAAAACTCAACGAGCAGCGGTTGGGTTA
20170216_cc6hb_v3_queen, c3, h19, p2, control,
18


TATAACTATATG
cyan, start 3[137], end 19[220], 42 mer






AGGGTTGTCGGACTTGTGCAAGGAATACCC
20170216_cc6hb_v3_queen, c3, h18, p1, control,
19


AAAAGAACTGGC
cyan, start 4[220], end 18[221], 42 mer






AGTCCGTGAAGACGGAAACCAAATCAAGTT
20170216_cc6hb_v3_queen, c3, h17, p0, control,
20


TGCCTTTAGCGT
cyan, start 3[221], end 17[220], 42 mer






CTGGGGATTTGACGCAGACCTGGTTGCTTTG
20170216_cc6hb_v3_queen, c4, h21, p0, control,
21


ACGAGCACGTA
cyan, start 4[94], end 21[262], 42 mer






TTTTCCCAGTCACGACGTTGTGAAATTGCGT
20170216_cc6hb_v3_queen, c4, h20, p1, control,
22


AGATTTTCAGG
cyan, start 5[95], end 20[263], 42 mer






TTATCAGTAAACAGAGAGGTTTCGCAAGAC
20170216_cc6hb_v3_queen, c4, h19, p2, control,
23


AAAGAACGCGAG
cyan, start 4[178], end 19[262], 42 mer






TCAGGGATTAATGAAAGATGGAACAAAGTT
20170216_cc6hb_v3_queen, c4, h18, p3, control,
24


ACCAGAAGGAAA
cyan, start 5[179], end 18[263], 42 mer






AGTGTGGCGATCCGATAGATGCGGCATTTT
20170216_cc6hb_v3_queen, c4, h17, p4, control,
25


CGGTCATAGCCC
cyan, start 4[262], end 17[262], 42 mer






GGGGGATGTGCTGCAAGGCGAATCAGAGCG
20170216_cc6hb_v3_queen, c5, h21, p4, control,
26


GGAGCTAAACAG
cyan, start 5[53], end 21[304], 42 mer






AGCCAGCTTTCCGGCACCGCTACCTACCATA
20170216_cc6hb_v3_queen, c5, h20, p3, control,
27


TCAAAATTATT
cyan, start 6[136], end 20[305], 42 mer






CTTTATTATTCGCATTCACCCTAGTTAATTTC
20170216_cc6hb_v3_queen, c5, h19, p2, control,
28


ATCTTCTGAC
cyan, start 5[137], end 19[304], 42 mer






TTGGTGTAGATGGGCGCATCGATCTTACCG
20170216_cc6hb_v3_queen, c5, h18, p1, control,
29


AAGCCCTTTTTA
cyan, start 6[220], end 18[305], 42 mer






CAGAAATAGAAGAATTACAGCTTTCATAAT
20170216_cc6hb_v3_queen, c5, h17, p0, control,
30


CAAAATCACCGG
cyan, start 5[221], end 17[304], 42 mer






AAGCGCCATTCGCCATTCAGGAGACAGGAA
20170216_cc6hb_v3_queen, c6, h21, p0, control,
31


CGGTACGCCAGA
cyan, start 6[94], end 21[346], 42 mer






TCAGAAAAGCCCCAAAAACAGCTGATTGTT
20170216_cc6hb_v3_queen, c6, h20, p1, control,
32


TGGATTATACTT
cyan, start 7[95], end 20[347], 42 mer






GAGGGGACGACGACAGTATCGACCGACCGT
20170216_cc6hb_v3_queen, c6, h19, p2, control,
33


GTGATAAATAAG
cyan, start 6[178], end 19[346], 42 mer






TTTTTGTTAAATCAGCTCATTAGCCCAATAA
20170216_cc6hb_v3_queen, c6, h18, p3, control,
34


TAAGAGCAAGA
cyan, start 7[179], end 18[347], 42 mer






GTGGGAACAAACGGCGGATTGCGCCTCCCT
20170216_cc6hb_v3_queen, c6, h17, p4, control,
35


CAGAGCCGCCAC
cyan, start 6[262], end 17[346], 42 mer






TCGTAAAACTAGCATGTCAATATCAGTGAG
20170216_cc6hb_v3_queen, c7, h21, p4, control,
36


GCCACCGAGTAA
cyan, start 7[53], end 21[388], 42 mer






ATGATATTCAACCGTTCTAGCATATTCCTGA
20170216_cc6hb_v3_queen, c7, h20, p3, control,
37


TTATCAGATGA
cyan, start 8[136], end 20[389], 42 mer






TTAAATTGTAAACGTTAATATCGGAATCATA
20170216_cc6hb_v3_queen, c7, h19, p2, control,
38


ATTACTAGAAA
cyan, start 7[137], end 19[388], 42 mer






TATTTTAAATGCAATGCCTGATGAGCGCTAA
20170216_cc6hb_v3_queen, c7, h18, p1, control,
39


TATCAGAGAGA
cyan, start 8[220], end 18[389], 42 mer






TCAAAAATAATTCGCGTCTGGAGCCACCAC
20170216_cc6hb_v3_queen, c7, h17, p0, control,
40


CCTCAGAGCCGC
cyan, start 7[221], end 17[388], 42 mer






GGTAGCTATTTTTGAGAGATCATTAACCGTT
20170216_cc6hb_v3_queen, c8, h21, p0, control,
41


GTAGCAATACT
cyan, start 8[94], end 21[430], 42 mer






ATGGTCAATAACCTGTTTAGCTTGCGGAAC
20170216_cc6hb_v3_queen, c8, h20, p1, control,
42


AAAGAAACCACC
cyan, start 9[95], end 20[431], 42 mer






AAAAGGGTGAGAAAGGCCGGACGTTATACA
20170216_cc6hb_v3_queen, c8, h19, p2, control,
43


AATTCTTACCAG
cyan, start 8[178], end 19[430], 42 mer






AACATCCAATAAATCATACAGGGGAGAATT
20170216_cc6hb_v3_queen, c8, h18, p3, control,
44


AACTGAACACCC
cyan, start 9[179], end 18[431], 42 mer






CTTTATTTCAACGCAAGGATACGCCGCCAG
20170216_cc6hb_v3_queen, c8, h17, p4, control,
45


CATTGACAGGAG
cyan, start 8[262], end 17[430], 42 mer






CGAACGAGTAGATTTAGTTTGACTTGCCTGA
20170216_cc6hb_v3_queen, c9, h21, p4, control,
46


GTAGAAGAACT
cyan, start 9[53], end 21[472], 42 mer






CATTTTTGCGGATGGCTTAGACCGAACGTTA
20170216_cc6hb_v3_queen, c9, h20, p3, control,
47


TTAATTTTAAA
cyan, start 10[136], end 20[473], 42 mer






AGCTGAAAAGGTGGCATCAATTAGGGCTTA
20170216_cc6hb_v3_queen, c9, h19, p2, control,
48


ATTGAGAATCGC
cyan, start 9[137], end 19[472], 42 mer






AGCTTCAAAGCGAACCAGACCTTTACAGAG
20170216_cc6hb_v3_queen, c9, h18, p1, control,
49


AGAATAACATAA
cyan, start 10[220], end 18[473], 42 mer






ATTAAGCAATAAAGCCTCAGAGGCCTTGAT
20170216_cc6hb_v3_queen, c9, h17, p0, control,
50


ATTCACAAACAA
cyan, start 9[221], end 17[472], 42 mer






CTGTAGCTCAACATGTTTTAAAATATCCAGA
20170216_cc6hb_v3_queen, c10, h21, p0, control,
51


ACAATATTACC
cyan, start 10[94], end 21[514], 42 mer






GGCTTTTGCAAAAGAAGTTTTAGACTTTACA
20170216_cc6hb_v3_queen, c10, h20, p1, control,
52


AACAATTCGAC
cyan, start 11[95], end 20[515], 42 mer






AGGATTAGAGAGTACCTTTAAGTAATTTAG
20170216_cc6hb_v3_queen, c10, h19, p2, control,
53


GCAGAGGCATTT
cyan, start 10[178], end 19[514], 42 mer






AATATTCATTGAATCCCCCTCGAAACGATTT
20170216_cc6hb_v3_queen, c10, h18, p3, control,
54


TTTGTTTAACG
cyan, start 11[179], end 18[515], 42 mer






AAGAGGAAGCCCGAAAGACTTAATGGAAA
20170216_cc6hb_v3_queen, c10, h17, p4, control,
55


GCGCAGTCTCTGA
cyan, start 10[262], end 17[514], 42 mer






ACCCTCGTTTACCAGACGACGAACGCTCAT
20170216_cc6hb_v3_queen, c11, h21, p4, control,
56


GGAAATACCTAC
cyan, start 11[53], end 21[556], 42 mer






TAACGGAACAACATTATTACAAGAGCCGTC
20170216_cc6hb_v3_queen, c11, h20, p3, control,
57


AATAGATAATAC
cyan, start 12[136], end 20[557], 42 mer






ATGTTTAGACTGGATAGCGTCATAAAGTAC
20170216_cc6hb_v3_queen, c11, h19, p2, control,
58


CGACAAAAGGTA
cyan, start 11[137], end 19[556], 42 mer






TGAATTACCTTATGCGATTTTTTACAAAATA
20170216_cc6hb_v3_queen, c11, h18, p1, control,
59


AACAGCCATAT
cyan, start 12[220], end 18[557], 42 mer






AAACGAGAATGACCATAAATCCATACATGG
20170216_cc6hb_v3_queen, c11, h17, p0, control,
60


CTTTTGATGATA
cyan, start 11[221], end 17[556], 42 mer






AGATTTAGGAATACCACATTCAAATGGATT
20170216_cc6hb_v3_queen, c12, h21, p0, control,
61


ATTTACATTGGC
cyan, start 12[94], end 21[598], 42 mer






CGAGGCGCAGACGGTCAATCAGTTATCTAA
20170216_cc6hb_v3_queen, cl2,1120, p1, control,
62


AATATCTTTAGG
cyan, start 13[95], end 20[599], 42 mer






GTCAGGACGTTGGGAAGAAAAGACAATAA
20170216_cc6hb_v3_queen, c12, h19, p2, control,
63


ACAACATGTTCAG
cyan, start 12[178], end 19[598], 42 mer






AGGCTGGCTGACCTTCATCAATACCAACGC
20170216_cc6hb_v3_queen, c12, h18, p3, control,
64


TAACGAGCGTCT
cyan, start 13[179], end 18[599], 42 mer






TAAATTGGGCTTGAGATGGTTTTTTAACGGG
20170216_cc6hb_v3_queen, c12, h17, p4, control,
65


GTCAGTGCCTT
cyan, start 12[262], end 17[598], 42 mer






TGTGTCGAAATCCGCGACCTGAGTAATAAA
20170216_cc6hb_v3_queen, c13, h21, p4, control,
66


AGGGACATTCTG
cyan, start 13[53], end 21[640], 42 mer






TACGAAGGCACCAACCTAAAACTGGTCAGT
20170216_cc6hb_v3_queen, c13, h20, p3, control,
67


TGGCAAATCAAC
cyan, start 14[136], end 20[641], 42 mer






CTTTGAAAGAGGACAGATGAATATCAACAA
20170216_cc6hb_v3_queen, c13, h19, p2, control,
68


TAGATAAGTCCT
cyan, start 13[137], end 19[640], 42 mer






GTAGCAACGGCTACAGAGGCTTAGTTGCTA
20170216_cc6hb_v3_queen, c13, h18, p1, control,
69


TTTTGCACCCAG
cyan, start 14[220], end 18[641], 42 mer






GATATTCATTACCCAAATCAACAGTTAATGC
20170216_cc6hb_v3_queen, c13, h17, p0, control,
70


CCCCTGCCTAT
cyan, start 13[221], end 17[640], 42 mer






CTAAAACACTCATCTTTGACCCTGACCTGAA
20170216_cc6hb_v3_queen, c14, h21, p0, control,
71


AGCGTAAGAAT
cyan, start 14[94], end 21[682], 42 mer






CGAATAATAATTTTTTCACGTATCACCTTGC
20170216_cc6hb_v3_queen, c14, h20, p1, control,
72


TGAACCTCAAA
cyan, start 15[95], end 20[683], 42 mer






ATGAGGAAGTTTCCATTAAACATCCTAATTT
20170216_cc6hb_v3_queen, c14, h19, p2, control,
73


ACGAGCATGTA
cyan, start 14[178], end 19[682], 42 mer






TTCGAGGTGAATTTCTTAAACACCTCCCGAC
20170216_cc6hb_v3_queen, c14, h18, p3, control,
74


TTGCGGGAGGT
cyan, start 15[179], end 18[683], 42 mer






GGATCGTCACCCTCAGCAGCGACATGAAAG
20170216_cc6hb_v3_queen, c14, h17, p4, control,
75


TATTAAGAGGCT
cyan, start 14[262], end 17[682], 42 mer






TTTCAGCGGAGTGAGAATAGATGAATGGCT
20170216_cc6hb_v3_queen, c15, h21, p4, control,
76


ATTAGTCTTTAA
cyan, start 15[53], end 21[724], 42 mer






CTACAACGCCTGTAGCATTCCAGTGCCACG
20170216_cc6hb_v3_queen, c15, h20, p3, control,
77


CTGAGAGCCAGC
cyan, start 16[136], end 20[725], 42 mer






CTCCAAAAGGAGCCTTTAATTGTCTTTCCTT
20170216_cc6hb_v3_queen, c15, h19, p2, control,
78


ATCATTCCAAG
cyan, start 15[137], end 19[724], 42 mer






CAGAGCCACCACCCTCATTTTAAGGCTTATC
20170216_cc6hb_v3_queen, c15, h18, p1, contro,
79


CGGTATTCTAA
cyan, start 16[220], end 18[725], 42 mer






CCGACAATGACAACAACCATCTAGGATTAG
20170216_cc6hb_v3_queen, c15, h17, p0, control,
80


CGGGGTTTTGCT
cyan, start 15[221], end 17[724], 42 mer






TCGTAAAACTAGCATGTCAATATCAGTGAG
20170216_cc6hb_v3_queen, c7, h21, p4, 7 bp plug,
81


GCCACCGAGTAAGAAAAAC
cyan, start 7[53], end 21[388], 49 mer






ATGATATTCAACCGTTCTAGCATATTCCTGA
20170216_cc6hb_v3_queen, c7, h20, p3, 7 bp plug,
82


TTATCAGATGAAGAGTCC
cyan, start 8[136], end 20[389], 49 mer






TTAAATTGTAAACGTTAATATCGGAATCATA
20170216_cc6hb_v3_queen, c7, h19, p2, 7 bp plug,
83


ATTACTAGAAAAATAGCC
cyan, start 7[137], end 19[388], 49 mer






TATTTTAAATGCAATGCCTGATGAGCGCTAA
20170216_cc6hb_v3_queen, c7, h18, p1, 7 bp plug,
84


TATCAGAGAGAATGGTGG
cyan, start 8[220], end 18[389], 49 mer






TCAAAAATAATTCGCGTCTGGAGCCACCAC
20170216_cc6hb_v3_queen, c7, h17, p0, 7 bp plug,
85


CCTCAGAGCCGCCGGTCCA
cyan, start 7[221], end 17[388], 49 mer






GGTAGCTATTTTTGAGAGATCATTAACCGTT
20170216_cc6hb_v3_queen, c8, h21, p0, 7 bp plug,
86


GTAGCAATACTCGGTCCA
cyan, start 8[94], end 21[430], 49 mer






ATGGTCAATAACCTGTTTAGCTTGCGGAAC
20170216_cc6hb_v3_queen, c8, h20, p1, 7 bp plug,
87


AAAGAAACCACCATGGTGG
cyan, start 9[95], end 20[431], 49 mer






AAAAGGGTGAGAAAGGCCGGACGTTATACA
20170216_cc6hb_v3_queen, c8, h19, p2, 7 bp plug,
88


AATTCTTACCAGAATAGCC
cyan, start 8[178], end 19[430], 49 mer






AACATCCAATAAATCATACAGGGGAGAATT
20170216_cc6hb_v3_queen, c8, h18, p3, 7 bp plug,
89


AACTGAACACCCAGAGTCC
cyan, start 9[179], end 18[431], 49 mer






CTTTATTTCAACGCAAGGATACGCCGCCAG
20170216_cc6hb_v3_queen, c8, h17, p4, 7 bp plug,
90


CATTGACAGGAGGAAAAAC
cyan, start 8[262], end 17[430], 49 mer






TCGTAAAACTAGCATGTCAATATCAGTGAG
20170216_cc6hb_v3_queen, c7, h21, p4, 10 bp plug,
91


GCCACCGAGTAAGAAAAACCGT
cyan, start 7[53], end 21[388], 52 mer






ATGATATTCAACCGTTCTAGCATATTCCTGA
20170216_cc6hb_v3_queen, c7, h20, p3, 10 bp plug,
92


TTATCAGATGAAGAGTCCACT
cyan, start 8[136], end 20[389], 52 mer






TTAAATTGTAAACGTTAATATCGGAATCATA
20170216_cc6hb_v3_queen, c7, h19, p2, 10 bp plug,
93


ATTACTAGAAAAATAGCCCGA
cyan, start 7[137], end 19[388], 52 mer






TATTTTAAATGCAATGCCTGATGAGCGCTAA
20170216_cc6hb_v3 queen, c7, h 18, p1, 10 bp plug,
94


TATCAGAGAGAATGGTGGTTC
cyan, start 8[220], end 18[389], 52 mer






TCAAAAATAATTCGCGTCTGGAGCCACCAC
20170216_cc6hb_v3_queen, c7, h17, p0, 10 bp plug,
95


CCTCAGAGCCGCCGGTCCACGC
cyan, start 7[221], end 17[388], 52 mer






GGTAGCTATTTTTGAGAGATCATTAACCGTT
20170216_cc6hb_v3_queen, c8, h21, p0, 10 bp plug,
96


GTAGCAATACTCGGTCCACGC
cyan, start 8[94], end 21[430], 52 mer






ATGGTCAATAACCTGTTTAGCTTGCGGAAC
20170216_cc6hb_v3_queen, c8, h20, p1, 10 bp plug,
97


AAAGAAACCACCATGGTGGTTG
cyan, start 9[95], end 20[431], 52 mer






AAAAGGGTGAGAAAGGCCGGACGTTATACA
20170216_cc6hb_v3_queen, c8, h19, p2, 10 bp plug,
98


AATTCTTACCAGAATAGCCCGA
cyan, start 8[178], end 19[430], 52 mer






AACATCCAATAAATCATACAGGGGAGAATT
20170216_cc6hb_v3_queen, c8, h18, p3, 10 bp plug,
99


AACTGAACACCCAGAGTCCACT
cyan, start 9[179], end 18[431], 52 mer






CTTTATTTCAACGCAAGGATACGCCGCCAG
20170216_cc6hb_v3_queen, c8, h17, p4, 10 bp plug,
100


CATTGACAGGAGGAAAAACCGT
cyan, start 8[262], end 17[430], 52 mer






CAATATTACATAACAATCCTCCATTTGAATT
20170216_cc6hb_v3_queen, edge, na, na, na, black,
101


ACCTTTTTTAA
start 0[136], end 20[53], 42 mer






ACTGATACCGTGCAAAATTATCAAAGACAA
20170216_cc6hb_v3_queen, edge, na, na, na, black,
102


AAGGGCGACATT
start 0[220], end 18[53], 42 mer






CGTAACGATCTAAAGTTTTGTAACATCGCCA
20170216_cc6hb_v3_queen, edge, na, na, na, black,
103


TTAAAAATACC
start 16[94], end 21[766], 42 mer






GAACCCATGTACCGTAACACTCGCACTCAT
20170216_cc6hb_v3_queen, edge, na, na, na, black,
104


CGAGAACAAGCA
start 16[178],end 19[766], 42 mer






TACCGCCACCCTCAGAACCGCCGTCGAGAG
20170216_cc6hb_v3_queen, edge, na, na, na, black,
105


GGTTGATATAAG
start 16[262], end 17[766], 42 mer






TCATTAAAGGTGAATTATCACTTCTGCAATG
20170216_cc6hb_v3_queen, edge, na, na, na, black,
106


TGCGAGAAATG
start 17[53], end 0[221], 42 mer






GGGAATTAGAGCCAGCAAAATGTTTATGTA
20170216_cc6hb_v3_queen, edge, na, na, na, black,
107


GATGAAGGTATA
start 17[95], end 11262], 42 mer






CACCGTAATCAGTAGCGACAGGTTTCTTGTT
20170216_cc6hb_v3_queen, edge, na, na, na, black,
108


GTTCGCCATCC
start 17[179], end 3[262], 42 mer






CCTTATTAGCGTTTGCCATCTGCAACACAGC
20170216_cc6hb_v3_queen, edge, na, na, na, black,
109


AATAAAAATGC
start 17[263], end 5[262], 42 mer






CCTCAGAACCGCCACCCTCAGCCTTCCTGTA
20170216_cc6hb_v3_queen, edge, na, na, na, black,
110


GCCAGCTTTCA
start 17[347], end 7[262], 42 mer






GTTGAGGCAGGTCAGACGATTGCATAAAGC
20170216_cc6hb_v3_queen, edge, na, na, na, black,
111


TAAATCGGTTGT
start 17[431], end 9[262], 42 mer






ATTTACCGTTCCAGTAAGCGTAAAAATCAG
20170216_cc6hb_v3_queen, edge, na, na, na, black,
112


GTCTTTACCCTG
start 17[515], end 11[262], 42 mer






GAGTAACAGTGCCCGTATAAACGTAACAAA
20170216_cc6hb_v3_queen, edge, na, na, na, black,
113


GCTGCTCATTCA
start 17[599], end 13[262], 42 mer






GAGACTCCTCAAGAGAAGGATGCCCACGCA
20170216_cc6hb_v3_queen, edge, na, na, na, black,
114


TAACCGATATAT
start 17[683], end 15[262], 42 mer






TAGCAAGCAAATCAGATATAGCAGGGATAG
20170216_cc6hb_v3_queen, edge, na, na, na, black,
115


CAAGCCCAATAG
start 18[766] end 16[179], 42 mer






GCTTCTGTAAATCGTCGCTATAAACATATAG
20170216_cc6hb_v3_queen, edge, na, na, na, black,
116


ATGATTAAACC
start 19[53] end 0[37], 42 mer






AGTATTAACACCGCCTGCAACACAGACAGC
20170216_cc6hb_v3_queen, edge, na, na, na, black,
117


CCTCATAGTTAG
start 20[766], end 16[95], 42 mer






GCACTAAATCGGAACCCTAAATTTTGTTTTA
20170216_cc6hb_v3_queen, edge, na, na, na, black,
118


TGGAGATGATA
start 21[53], end 0[53], 42 mer






AAAGCGAAAGGAGCGGGCGCTCTGAATTTC
20170216_cc6hb_v3_queen, edge, na, na, na, black,
119


GCGTCGTCTTCA
start 21[137], end 2[53], 42 mer






CCGCTACAGGGCGCGTACTATTTTCCATGAA
20170216_cc6hb_v3_queen, edge, na, na, na, black,
120


TTGGTAACACC
start 21[221], end 4[53], 42 mer






GAGGCCGATTAAAGGGATTTTCTGCGCAAC
20170216_cc6hb_v3_queen, edge, na, na, na, black,
121


TGTTGGGAAGGG
start 21[305], end 6[53], 42 mer






AAGAGTCTGTCCATCACGCAATACAAAGGC
20170216_cc6hb_v3_queen, edge, na, na, na, black,
122


TATCAGGTCATT
start 21[389], end 8[53], 42 mer






CAAACTATCGGCCTTGCTGGTATATGCAACT
20170216_cc6hb_v3_queen, edge, na, na, na, black,
123


AAAGTACGGTG
start 21[473], end 10[53], 42 mer






ATTTTGACGCTCAATCGTCTGAACTAATGCA
20170216_cc6hb_v3_queen, edge, na, na, na, black,
124


GATACATAACG
start 21[557], end 12[53], 42 mer






GCCAACAGAGATAGAACCCTTCCCAGCGAT
20170216_cc6hb_v3_queen, edge, na, na, na, black,
125


TATACCAAGCGC
start 21[641], end 14[53], 42 mer






TGCGCGAACTGATAGCCCTAACGTCTTTCCA
20170216_cc6hb_v3_queen, edge, na, na, na, black,
126


GACGTTAGTAA
start 21[725], end 16[53], 42 mer






CAAAGGGCGAAAAACCAACAGCTGATTGCC
20170216_cc6hb_v3_queen, reference sheet, na, na,
127


CTGCGCCAGG
na, puke green, start 22[79], end 24[72], 40 mer






AGTCCACTATTAAAGAAGAGAGTTGCAGCA
20170216_cc6hb_v3_queen, reference sheet, na, na,
128


AGCAACGCGC
na, puke green, start 22[111], end 24[104], 40 mer






TAGGGTTGAGTGTTGTGCCCCAGCAGGCGA
20170216_cc6hb_v3_queen, reference sheet, na, na,
129


AAACCTGTCG
na, puke green, start 22[143], end 24[136], 40 mer






AATCCCTTATAAATCAGTTCCGAAATCGGC
20170216_cc6hb_v3_queen, reference sheet, na, na,
130


AA
na, puke green, start 22[175], end 23[175], 32 mer






GTGAGACGGGCGTCTATCA
20170216_cc6hb_v3_queen, reference sheet, na, na,
131



na, puke green, start 23[53], end 22[56], 19 mer






GTGGTTTTTGTTTCCTGTGTGAAA
20170216_cc6hb_v3_queen, reference sheet, na, na,
132



na, puke green, start 24[71], end 25[79], 24 mer






CGTATTGGTCACCGCCTGGCCCTGACGTGG
20170216_cc6hb_v3_queen, reference sheet, na, na,
133


ACTCCAACGT
na, puke green, start 24[87], end 22[80], 40 mer






GGGGAGAGATTCCACACAACATAC
20170216_cc6hb_v3_queen, reference sheet, na, na,
134



na, puke green, start 24[103], end 25[111], 24 mer






GAATCGGCCGGTCCACGCTGGTTTTCCAGTT
20170216_cc6hb_v3_queen, reference sheet, na, na,
135


TGGAACAAG
na, puke green, start 24[119], end 22[112], 40 mer






TGCCAGCTGTGTAAAGCCTGGGGT
20170216_cc6hb_v3_queen, reference sheet, na, na,
136



na, puke green, start 24[135], end 25[143], 24 mer






GTCGGGAAATCCTGTTTGATGGTGAAAGAA
20170216_cc6hb_v3_queen, reference sheet, na, na,
137


TAGCCCGAGA
na, puke green, start 24[151], end 22[144], 40 mer






GTTGCGCTCACTGCCCAACTCACATTAATTG
20170216_cc6hb_v3_queen, reference sheet, na, na,
138


C
na, puke green, start 24[175], end 25[175], 32 mer






GTCATAGCTCTTTTCACCA
20170216_cc6hb_v3_queen, reference sheet, na, na,
139



na, puke green, start 25[56], end 24[53], 19 mer






TTGTTATCCGCTCACAGCGGTTTG
20170216_cc6hb_v3_queen, reference sheet, na, na,
140



na, puke green, start 25[80], end 24[88], 24 mer






GAGCCGGAAGCATAAAGCATTAAT
20170216_cc6hb_v3_queen, reference sheet, na, na,
141



na, puke green, start 25[112], end 24[120], 24 mer






GCCTAATGAGTGAGCTGCTTTCCA
20170216_cc6hb_v3_queen, reference sheet, na, na,
142



na, puke green, start 25[144], end 24[152], 24 mer






AAAATACATACATAAAGGTGGCTATTACGG
20170216_cc6hb_v3_queen, cell not used, na, na,
143


GGTTGGAGGTCA
na, red, start 18[178], end 2[179], 42 mer






TAGCAAGGCCGGAAACGTCACCGAACAAGA
20170216_cc6hb_v3_queen, cell not used, na, na,
144


CCCGTTAGTAAC
na, red, start 17[137], end 2[221], 42 mer






CAGACTGTAGCGCGTTTTCATAACGAAGAC
20170216_cc6hb_v3_queen, cell not used, na, na,
145


GCCTGGTCGTTC
na, red, start 17[221] end 4[221] 42 mer






AACCAGAGCCACCACCGGAACACCGTAATG
20170216_cc6hb_v3_queen, cell not used, na, na,
146


GGATAGGTCACG
na, red, start 17[305], end 6[221], 42 mer






CACCAGAACCACCACCAGAGCAAAATTTTT
20170216_cc6hb_v3_queen, cell not used, na, na,
147


AGAACCCTCATA
na, red, start 17[389], end 8[221] 42 mer






ATAAATCCTCATTAAAGCCAGCAAATATCG
20170216_cc6hb_v3_queen, cell not used, na, na,
148


CGTTTTAATTCG
na, red, start 17[473], end 10[221], 42 mer






CAGGAGTGTACTGGTAATAAGTAATTTCAA
20170216_cc6hb_v3_queen, cell not used, na, na,
149


CTTTAATCATTG
na, red, start 17[557], end 12[221], 42 mer






TTCGGAACCTATTATTCTGAAAAAGACAGC
20170216_cc6hb_v3_queen, cell not used, na, na,
150


ATCGGAACGAGG
na, red, start 17[641], end 14[221], 42 mer






CAGTACCAGGCGGATAAGTGCCACCCTCAG
20170216_cc6hb_v3_queen, cell not used, na, na,
151


AACCGCCACCCT
na, red, start 17[725], end 16[221], 42 mer






ATTCATATGGTTTACCAGCGCTATCACGAGT
20170216_cc6hb_v3_queen, cell not used, na, na,
152


ACGGTGGAAAC
na, red, start 18[94], end 0[179], 42 mer






AGACACCACGGAATAAGTTTATGCAGATCC
20170216_cc6hb_v3_queen, cell not used, na, na,
153


GGTGTCTTGTCT
na, red, start 18[136], end 1[220], 42 mer






ATGATTAAGACTCCTTATTACTGCTAAACTG
20170216_cc6hb_v3_queen, cell not used, na, na,
154


GAAAGCAACGA
na, red, star[ 18 220], end 3[220], 42 mer






CCGAGGAAACGCAATAATAACGTTGCCAGG
20170216_cc6hb_v3_queen, cell not used, na, na,
155


AGGATCTGGAAC
na, red, start 18[262], end 4[179], 42 mer






AGAAAAGTAAGCAGATAGCCGCAGACATCA
20170216_cc6hb_v3_queen, cell not used, na, na,
156


TTGATTCAGCAT
na, red, start 18[304], end 5[220], 42 mer






AACAATGAAATAGCAATAGCTTAACCGTGC
20170216_cc6hb_v3_queen, cell not used, na, na,
157


ATCTGCCAGTTT
na, red, start 18[346], end 6[179], 42 mer






TAACCCACAAGAATTGAGTTATTTTAACCA
20170216_cc6hb_v3_queen, cell not used, na, na,
158


ATAGGAACGCCA
na, red, start 18[388], end 7[220], 42 mer






TGAACAAAGTCAGAGGGTAATGTAATGTGT
20170216_cc6hb_v3_queen, cell not used, na, na,
159


AGGTAAAGATTC
na, red, start 18[430], end 8[179], 42 mer






AAACAGGGAAGCGCATTAGACGCAAGGCA
20170216_cc6hb_v3_queen, cell not used, na, na,
160


AAGAATTAGCAAA
na, red, start 18[472], end 9[220], 42 mer






TCAAAAATGAAAATAGCAGCCGGAAGCAA
20170216_cc6hb_v3_queen, cell not used, na, na,
161


ACTCCAACAGGTC
na, red, start 18[514], end 10[179], 42 mer






TATTTATCCCAATCCAAATAAAAATGCTTTA
20170216_cc6hb_v3_queen, cell not used, na, na,
162


AACAGTTCAGA
na, red, start 18[556], end 11[220], 42 mer






TTCCAGAGCCTAATTTGCCAGAAGAACTGG
20170216_cc6hb_v3_queen, cell not used, na, na,
163


CTCATTATACCA
na, red, start 18[598], end 12[179], 42 mer






CTACAATTTTATCCTGAATCTGAGTAATCTT
20170216_cc6hb_v3_queen, cell not used, na, na,
164


GACAAGAACCG
na, red, start 18[640], end 13[220], 42 mer






TTTGAAGCCTTAAATCAAGATTTGAGGACT
20170216_cc6hb_v3_queen, cell not used, na, na,
165


AAAGACTTTTTC
na, red, start 18[682], end 14[179], 42 mer






GAACGCGAGGCGTTTTAGCGAAGCTTGATA
20170216_cc6hb_v3_queen, cell not used, na, na,
166


CCGATAGTTGCG
na, red, start 18[724], end 15[220], 42 mer






CCTTGAAAACATAGCGATAGCGAGTTAGAG
20170216_cc6hb_v3_queen, cell not used, na, na,
167


TCTGAGCAAAAA
na, red, start 19[95], end 1[178], 42 mer






AGTCAATAGTGAATTTATCAAGTATCTGCAT
20170216_cc6hb_v3_queen, cell not used, na, na,
168


ATGATGTCTGA
na, red, start 19[137], end 2[137], 42 mer






CCTTTTTAACCTCCGGCTTAGTGAGTATTAC
20170216_cc6hb_v3_queen, cell not used, na, na,
169


GAAGGTGTTAT
na, red, start 19[179], end 3[178], 42 mer






TAAATGCTGATGCAAATCCAACGAAGTGAG
20170216_cc6hb_v3_queen, cell not used, na, na,
170


CGAAATTAACTC
na, red, start 19[221], end 4[137], 42 mer






AAAACTTTTTCAAATATATTTTCATGCGTAT
20170216_cc6hb_v3_queen, cell not used, na, na,
171


TAACCAACAGT
na, red, start 19[263], end 5[178], 42 mer






CTAAATTTAATGGTTTGAAATGCCTCAGGA
20170216_cc6hbv3_queen, cell not used, na, na,
172


AGATCGCACTCC
na, red, start 19[305], end 6[137], 42 mer






GCGTTAAATAAGAATAAACACTTTGTTAAA
20170216_cc6hb_v3_queen, cell not used, na, na,
173


ATTCGCATTAAA
na, red, start 19[347], end 7[178], 42 mer






AAGCCTGTTTAGTATCATATGGACAGTCAA
20170216_cc6hb_v3_queen, cell not used, na, na,
174


ATCACCATCAAT
na, red, start 19[389], end 8[137], 42 mer






TATAAAGCCAACGCTCAACAGTCTACTAAT
20170216_cc6hb_v3_queen, cell not used, na, na,
175


AGTAGTAGCATT
na, red, start 19[431], end 9[178], 42 mer






CATATTTAACAACGCCAACATTTGCTCCTTT
20170216_cc6hb_v3_queen, cell not used, na, na,
176


TGATAAGAGGT
na, red, start 19[473], end 10[137], 42 mer






TCGAGCCAGTAATAAGAGAATCAATACTGC
20170216_cc6hb_v3_queen, cell not used, na, na,
177


GGAATCGTCATA
na, red, start 19[515], end 11[178], 42 mer






AAGTAATTCTGTCCAGACGACATCTACGTTA
20170216_cc6hb_v3_queen, cell not used, na, na,
178


ATAAAACGAAC
na, red, start 19[557], end 12[137], 42 mer






CTAATGCAGAACGCGCCTGTTCGGTGTACA
20170216_cc6hb_v3_queen, cell not used, na, na,
179


GACCAGGCGCAT
na, red, start 19[599], end 13[178], 42 mer






GAACAAGAAAAATAATATCCCGGGTAAAAT
20170216_cc6hb_v3_queen, cell not used, na, na,
180


ACGTAATGCCAC
na, red, start 19[641], end 14[137], 42 mer






GAAACCAATCAATAATCGGCTGTATCGGTT
20170216_cc6hb_v3_queen, cell not used, na, na,
181


TATCAGCTTGCT
na, red, start 19[683], end 15[178], 42 mer






AACGGGTATTAAACCAAGTACGAGTTTCGT
20170216_cc6hb_v3_queen, cell not used, na, na,
182


CACCAGTACAAA
na, red, start 19[725], end 16[137], 42 mer






ATTAATTACATTTAACAATTTGCACTCGCGG
20170216_cc6hb_v3_queen, cell not used, na, na,
183


GGATTTATTTT
na, red, start 20[94], end 0[95], 42 mer






CTGAGCAAAAGAAGATGATGAGAAACGAC
20170216_cc6hb_v3_queen, cell not used, na, na,
184


ATACATTGCAAGG
na, red, start 20[136], end 1[136], 42 mer






AGTTACAAAATCGCGCAGAGGAGAGTGAGA
20170216_cc6hb_v3_queen, cell not used, na, na,
185


TCGGTTTTGTAA
na, red, start 20[178], end 2[95], 42 mer






GGGAGAAACAATAACGGATTCTGTTGAGCT
20170216_cc6hb_v3_queen, cell not used, na, na,
186


TGAAACAGCAAA
na, red, start 20[220], end 3[136], 42 mer






TTTAACGTCAGATGAATATACAGAGCAGGC
20170216_cc6hb_v3_queen, cell not used, na, na,
187


AATGCATGACGA
na, red, start 20[262], end 4[95], 42 mer






TGCACGTAAAACAGAAATAAAAAAACGAC
20170216_cc6hb_v3_queen, cell not used, na, na,
188


GGCCAGTGCCAAG
na, red, start 20[304], end 5[136], 42 mer






CTGAATAATGGAAGGGTTAGATCTGGTGCC
20170216_cc6hb_v3_queen, cell not used, na, na,
189


GGAAACCAGGCA
na, red, start 20[346], end 6[95], 42 mer






TGGCAATTCATCAATATAATCGAAGATTGT
20170216_cc6hb_v3_queen, cell not used, na, na,
190


ATAAGCAAATAT
na, red, start 20[388], end 7[136], 42 mer






AGAAGGAGCGGAATTATCATCTGATAAATT
20170216_cc6hb_v3_queen, cell not used, na, na,
191


AATGCCGGAGAG
na, red, start 20[430], end 8[95], 42 mer






AGTTTGAGTAACATTATCATTTATATTTTCA
20170216_cc6hb_v3_queen, cell not used, na, na,
192


TTTGGGGCGCG
na, red, start 20[472], end 9[136], 42 mer






AACTCGTATTAAATCCTTTGCGCTTAATTGC
20170216_cc6hb_v3_queen, cell not used, na, na,
193


TGAATATAATG
na, red, start 20[514], end 10[95], 42 mer






ATTTGAGGATTTAGAAGTATTGCCAGAGGG
20170216_cc6hb_v3_queen, cell not used, na, na,
194


GGTAATAGTAAA
na, red, start 20[556], end 11[136], 42 mer






AGCACTAACAACTAATAGATTGGTAGAAAG
20170216_cc6hb_v3_queen, cell not used, na, na,
195


ATTCATCAGTTG
na, red, start 20 598], end 12[95], 42 mer






AGTTGAAAGGAATTGAGGAAGTAAGGGAA
20170216_cc6hb_v3_queen, cell not used, na, na,
196


CCGAACTGACCAA
na, red, start 20[640], end 13[136], 42 mer






TATCAAACCCTCAATCAATATCGAAAGAGG
20170216_cc6hb_v3_queen, cell not used, na, na,
197


CAAAAGAATACA
na, red, start 20[682], end 14[95], 42 mer






AGCAAATGAAAAATCTAAAGCTGAAAATCT
20170216_cc6hb_v3_queen, cell not used, na, na,
198


CCAAAAAAAAGG
na, red, start 20[724], end 15[136], 42 mer






TGACGGGGAAAGCCGGCGAACCTTACTGTT
20170216_cc6hb_v3_queen, cell not used, na, na,
199


TCTTTACATAAA
na, red, start 21[95], end 1[94], 42 mer






GTCACGCTGCGCGTAACCACCCCAGGAGAA
20170216_cc6hb_v3_queen, cell not used, na, na,
200


CGAGGATATTGC
na, red, start 21[179], end 3[94], 42 mer






TAACGTGCTTTCCTCGTTAGATTAAGTTGGG
20170216_cc6hb_v3_queen, cell not used, na, na,
201


TAACGCCAGGG
na, red, start 21[263], end 5[94], 42 mer






ATCCTGAGAAGTGTTTTTATACATATGTACC
20170216_cc6hb_v3_queen, cell not used, na, na,
202


CCGGTTGATAA
na, red, start 21[347], end 7[94], 42 mer






TCTTTGATTAGTAATAACATCACCATTAGAT
20170216_cc6hb_v3_queen, cell not used, na, na,
203


ACATTTCGCAA
na, red, start 21[431], end 9[94], 42 mer






GCCAGCCATTGCAACAGGAAAATAAAAACC
20170216_cc6hb_v3_queen, cell not used, na, na,
204


AAAATAGCGAGA
na, red, start 21[515], end 11[94], 42 mer






AGATTCACCAGTCACACGACCCTCCATGTTA
20170216_cc6hb_v3_queen, cell not used, na, na,
205


CTTAGCCGGAA
na, red, start 21[599], end 13[94], 42 mer






ACGTGGCACAGACAATATTTTAAGGAACAA
20170216_cc6hb_v3_queen, cell not used, na, na,
206


CTAAAGGAATTG
na, red, start 21[683], end 15[94], 42met






ATCTGAACTCGCTACGGCGGGGGGAGCCCC
20170407_cc6hb_v3-1_queen, c0, h21, p0, 7 bp
207


CGATTTAGAGCTCGGTCCA
plug, cyan, start 0[94], end 21[94], 49 mer






CATTGCTGATACCGTTTAGCTAACAAACATC
20170407_cc6hb_v3-1_queen, c0, h20, p0, 7 bp
208


AAGAAAAGAAAATGGTGG
plug, cyan, start 1[95], end 20195], 49 mer






GATACTTGCCCTCTCTGTACATAATTAATTT
20170407_cc6hb_v3-1_queen, c0, h19, p2, 7 bp
209


TCCCTTAGAATAATAGCC
plug, cyan, start 0[178], end 19[94], 49 mer






GATTGGGCGTTATCAATGTTGTTTTGTCACA
20170407_cc6hb_v3-1_queen, c0, h18, p3, 7 bp
210


ATCAATAGAAAAGAGTCC
plug, cyan, start 1[179], end 18[95], 49 mer






TCTAATGAAGACAAATCCCCACGTCACCGA
2017040_cc6hb_v3-1_queen, c0, h17, p4, 7 bp
211


CTTGAGCCATTTGAAAAAC
plug, cyan, start 0[262], end 17[94], 49 mer






AAACATCGGGTTGAGTATTATGTGGCGAGA
20170407_cc6hb_v3-1_queen, c1, h21, p4, 7 bp
212


AAGGAAGGGAAGGAAAAAC
plug, cyan, start 1[53], end 21[136], 49 mer






CGCTGGCATTCGCATCAAAGGCGAATTATT
20170407_cc6hb_v3-1_queen, c1, h20, p3, 7 bp
213


CATTTCAATTACAGAGTCC
plug, cyan, start 2[136], end 20[137], 49 mer






AGTTTATAAATGAGTATCAATTTAGATTAAG
20170407_cc6hb_v3-1_queen, c1, h19, p2, 7 bp
214


ACGCTGAGAAGAATAGCC
plug, cyan, start 1[137], end 19[136], 49 mer






TATCGACATCATTACGCATCGCAACATATA
20170407_cc6hb_v3-1_queen, c1, h18, p1, 7 bp
215


AAAGAAACGCAAATGGTGG
plug, cyan, start 2[220], end 18[137], 49 mer






CCATGCAGACATCACGAAGGTCACCAGTAG
20170407_cc6hb_v3-1_queen, c1, h17, p0, 7 bp
216


CACCATTACCATCGGTCCA
plug, cyan, start 1[221], end 17[136], 49 mer






AAGATAACGCTTGTGAAAATGAGGGCGCTG
20170407_cc6hb_v3-1_queen, c2, h21, p0, 7 bp
217


GCAAGTGTAGCGCGGTCCA
plug, cyan, start 2[94], end 21[178], 49 mer






GCTAACAGTAGGGAAACTGCGGCCTGATTG
20170407_cc6hb_v3-1_queen, c2, h20, p1, 7 bp
218


CTTTGAATACCAATGGTGG
plug, cyan, start 3[95], end 20[179], 49 mer






ATGGGTTCAGGATGCAGGTGAAATCATAGG
20170407_cc6hb_v3-1_queen, c2, h19, p2, 7 bp
219


TCTGAGAGACTAAATAGCC
plug, cyan, start 2[178], end 19[178], 49 mer






CTCGGATGGGAGTAAGCGTATGCAGTATGT
20170407_cc6hb_v3-1_queen, c2, h18, p3, 7 bp
220


TAGCAAACGTAGAGAGTCC
plug, cyan, start 3[179], end 18[179], 49 mer






AGAGTTTCTGCGGCAGTTAATCAATGAAAC
20170407_cc6hb_v3-1_queen, c2, h17, p4, 7 bp
221


CATCGATAGCAGGAAAAAC
plug, cyan, start 2[262], end 17[178], 49 mer






GCAATACATCAAACGCCGCGAACACCCGCC
20170407_cc6hb_v3-1_queen, c3, h21, p4, 7 bp
222


GCGCTTAATGCGGAAAAAC
plug, cyan, start 3[53], end 21[220], 49 mer






TCAGGCACTGCGTGAAGCGGCAGTAACAGT
20170407_cc6hb_v3-1_queen, c3, h20, p3, 7 bp
223


ACCTTTTACATCAGAGTCC
plug, cyan, start 4[136], end 20[221], 49 mer






ATCAAAACTCAACGAGCAGCGGTTGGGTTA
20170407_cc6hb_v3-1_queen, c3, h19, p2, 7 bp
224


TATAACTATATGAATAGCC
plug, cyan, start 3[137], end 191220], 49 mer






AGGGTTGTCGGACTTGTGCAAGGAATACCC
20170407_cc6hb_v3-1_queen, c3, h18, p1, 7 bp
225


AAAAGAACTGGCATGGTGG
plug, cyan, start 4[220], end 18[221], 49 mer






AGTCCGTGAAGACGGAAACCAAATCAAGTT
20170407_cc6hb_v3-1_queen, 3, h17, p0, 7 bp
226


TGCCTTTAGCGTCGGTCCA
plug, cyan, start 3[221], end 17[220], 49 mer






CTGGGGATTTGACGCAGACCTGGTTGCTTTG
20170407_cc6hb_v3-1_queen, c4, h21, p0, 7 bp
227


ACGAGCACGTACGGTCCA
plug, cyan, start 4[94], end 21[262], 49 mer






TTTTCCCAGTCACGACGTTGTGAAATTGCGT
20170407_cc6hb_v3-1_queen, c4, h20, p1, 7 bp
228


AGATTTTCAGGATGGTGG
plug, cyan, start 5[95], end 20[263], 49 mer






TTATCAGTAAACAGAGAGGTTTCGCAAGAC
20170407_cc6hb_v3-1_queen, c4, h19, p2, 7 bp
229


AAAGAACGCGAGAATAGCC
plug, cyan, start 4[178], end 19[262], 49 mer






TCAGGGATTAATGAAAGATGGAACAAAGTT
20170407_cc6hb_v3-1_queen, c4, h18, p3, 7 bp
230


ACCAGAAGGAAAAGAGTCC
plug, cyan, start 5[179], end 18[263], 49 mer






AGTGTGGCGATCCGATAGATGCGGCATTTT
20170407_cc6hb_v3-1_queen, c4, h17, p4, 7 bp
231


CGGTCATAGCCCGAAAAAC
plug, cyan, start 4[262], end 17[262], 49 mer






GGGGGATGTGCTGCAAGGCGAATCAGAGCG
20170407_cc6hb_v3-1_queen, c5, h21, p4, 7 bp
232


GGAGCTAAACAGGAAAAAC
plug, cyan, start 5[53], end 21[304], 49 mer






AGCCAGCTTTCCGGCACCGCTACCTACCATA
20170407_cc6hb_v3-1_queen, c5, h20, p3, 7 bp
233


TCAAAATTATTAGAGTCC
plug, cyan, start 6[136], end 20[305], 49 mer






CTTTATTATTCGCATTCACCCTAGTTAATTTC
20170407_cc6hb_v3-1_queen, c5, h19, p2, 7 bp
234


ATCTTCTGACAATAGCC
plug, cyan, start 5[137], end 19[304], 49 mer






TTGGTGTAGATGGGCGCATCGATCTTACCG
20170407_cc6hb_v3-1_queen, c5, h18, p1, 7 bp
235


AAGCCCTTTTTAATGGTGG
plug, cyan, start 6[220], end 18[305], 49 mer






CAGAAATAGAAGAATTACAGCTTTCATAAT
20170407_cc6hb_v3-1_queen, c5, h17, p0, 7 bp
236


CAAAATCACCGGCGGTCCA
plug, cyan, start 5[221], end 17[304], 49 mer






AAGCGCCATTCGCCATTCAGGAGACAGGAA
20170407_cc6hb_v3-1_queen, c6, h21, p0, 7 bp
237


CGGTACGCCAGACGGTCCA
plug, cyan, start 6[94], end 21[346], 49 mer






TCAGAAAAGCCCCAAAAACAGCTGATTGTT
20170407_cc6hb_v3-1_queen, c6, h20, p1, 7 bp
238


TGGATTATACTTATGGTGG
plug, cyan, start 7[95], end 20[347], 49 mer






GAGGGGACGACGACAGTATCGACCGACCGT
20170407_cc6hb_v3-1_queen, c6, h19, p2, 7 bp
239


GTGATAAATAAGAATAGCC
plug, cyan, start 6[178], end 19[346], 49 mer






TTTTTGTTAAATCAGCTCATTAGCCCAATAA
20170407_cc6hb_v3-1_queen, c6, h18, p3, 7 bp
240


TAAGAGCAAGAAGAGTCC
plug, cyan, start 7[179], end 18[347], 49 mer






GTGGGAACAAACGGCGGATTGCGCCTCCCT
20170407_cc6hb_v3-1_queen, c6, h17, p4, 7 bp
241


CAGAGCCGCCACGAAAAAC
plug, cyan, start 6[262], end 17[346], 49 mer






CGAACGAGTAGATTTAGTTTGACTTGCCTGA
20170407_cc6hb_v3-1_queen, c9, h21, p4, 7 bp
242


GTAGAAGAACTGAAAAAC
plug, cyan, start 9[53], end 21[472], 49 mer






CATTTTTGCGGATGGCTTAGACCGAACGTTA
20170407_cc6hb_v3-1_queen, c9, h20, p3, 7 bp
243


TTAATTTTAAAAGAGTCC
plug, cyan, start 10[36], end 20[473], 49 mer






AGCTGAAAAGGTGGCATCAATTAGGGCTTA
20170407_cc6hb_v3-1_queen, c9, h19, p2, 7 bp
244


ATTGAGAATCGCAATAGCC
plug, cyan, start 9[137], end 19[472], 49 mer






AGCTTCAAAGCGAACCAGACCTTTACAGAG
20170407_cc6hb_v3-1_queen, c9, h18, p1, 7 bp
245


AGAATAACATAAATGGTGG
plug, cyan, start 10[220], end 18[473], 49 mer






ATTAAGCAATAAAGCCTCAGAGGCCTTGAT
20170407_cc6hb_v3-1_queen, c9, h17, p0, 7 bp
246


ATTCACAAACAACGGTCCA
plug, cyan, start 9[221], end 17[472], 49 mer






CTGTAGCTCAACATGTTTTAAAATATCCAGA
20170407_cc6hb_v3-1_queen, c10, h21, p0, 7 bp
247


ACAATATTACCCGGTCCA
plug, cyan, start 10[94], end 21[514], 49 mer






GGCTTTTGCAAAAGAAGTTTTAGACTTTACA
20170407_cc6hb_v3-1_queen, c10, h20, p1, 7 bp
248


AACAATTCGACATGGTGG
plug, cyan, start 11[95], end 20[515], 49 mer






AGGATTAGAGAGTACCTTTAAGTAATTTAG
20170407_cc6hb_v3-1_queen, c10, h19, p2, 7 bp
249


GCAGAGGCATTTAATAGCC
plug, cyan, start 10[178], end 19[514], 49 mer






AATATTCATTGAATCCCCCTCGAAACGATTT
20170407_cc6hb_v3-1_queen, c10, h18, p3, 7 bp
250


TTTGTTTAACGAGAGTCC
plug, cyan, start 11[179], end 18[515], 49 mer






AAGAGGAAGCCCGAAAGACTTAATGGAAA
20170407_cc6hb_v3-1_queen, c10, h17, p4, 7 bp
251


GCGCAGTCTCTGAGAAAAAC
plug, cyan, start 10[262], end 17[514], 49 mer






ACCCTCGTTTACCAGACGACGAACGCTCAT
20170407_cc6hb_v3-1_queen, c11, h21, p4, 7 bp
252


GGAAATACCTACGAAAAAC
plug, cyan, start 11[53], end 21[556], 49 mer






TAACGGAACAACATTATTACAAGAGCCGTC
20170407_cc6hb_v3-1_queen, c11, h20, p3, 7 bp
253


AATAGATAATACAGAGTCC
plug, cyan, start 12[136], end 20[557], 49 mer






ATGTTTAGACTGGATAGCGTCATAAAGTAC
20170407_cc6hb_v3-1_queen, c11, h19, p2, 7 bp
254


CGACAAAAGGTAAATAGCC
plug, cyan, start 11[137], end 191556], 49 mer






TGAATTACCTTATGCGATTTTTTACAAAATA
20170407_cc6hb_v3-1_queen, c11, h18, p1, 7 bp
255


AACAGCCATATATGGTGG
plug, cyan, start 12[220], end 18[557], 49 mer






AAACGAGAATGACCATAAATCCATACATGG
20170407_cc6hb_v3-1_queen, c11, h17, p0, 7 bp
256


CTTTTGATGATACGGTCCA
plug, cyan, start 11[221], end 17[556], 49 mer






AGATTTAGGAATACCACATTCAAATGGATT
20170407_cc6hb_v3-1_queen, c12,h21, p0, 7 bp
257


ATTTACATTGGCCGGTCCA
plug, cyan, start 12[94], end 21[598], 49 mer






CGAGGCGCAGACGGTCAATCAGTTATCTAA
20170407_cc6hb_v3-1_queen, c12, h20, p1, 7 bp
258


AATATCTTTAGGATGGTGG
plug, cyan, start 13[95], end 20[599], 49 mer






GTCAGGACGTTGGGAAGAAAAGACAATAA
20170407_cc6hb_v3-1_queen, c12, h19, p2, 7 bp
259


ACAACATGTTCAGAATAGCC
plug, cyan, start 12[178], end 19[598], 49 mer






AGGCTGGCTGACCTTCATCAATACCAACGC
20170407_cc6hb_v3-1_queen, c12, h18, p3, 7 bp
260


TAACGAGCGTCTAGAGTCC
plug, cyan, start 13[179], end 18[599], 49 mer






TAAATTGGGCTTGAGATGGTTTTTTAACGGG
20170407_cc6hb_v3-1_queen, c12, h17, p4, 7 bp
261


GTCAGTGCCTTGAAAAAC
plug, cyan, start 12[262], end 17[598], 49 mer






TGTGTCGAAATCCGCGACCTGAGTAATAAA
20170407_cc6hb_v3-1_queen, c13, h21, p4, 7 bp
262


AGGGACATTCTGGAAAAAC
plug, cyan, start 13[53], end 21[640], 49 mer






TACGAAGGCACCAACCTAAAACTGGTCAGT
20170407_cc6hb_v3-1_queen, c13, h20, p3, 7 bp
263


TGGCAAATCAACAGAGTCC
plug, cyan, start 14[136], end 20[641], 49 mer






CTTTGAAAGAGGACAGATGAATATCAACAA
20170407_cc6hb_v3-1_queen, c13, h19, p2, 7 bp
264


TAGATAAGTCCTAATAGCC
plug, cyan, start 13[137], end 19[640], 49 mer






GTAGCAACGGCTACAGAGGCTTAGTTGCTA
20170407_cc6hb_v3-1_queen, c13, h18, p1, 7 bp
265


TTTTGCACCCAGATGGTGG
plug, cyan, start 14[220], end 18[641], 49 mer






GATATTCATTACCCAAATCAACAGTTAATGC
20170407_cc6hb_v3-1_queen, c13, h17, p0, 7 bp
266


CCCCTGCCTATCGGTCCA
plug, cyan, start 13[221], end 17[640], 49 mer






CTAAAACACTCATCTTTGACCCTGACCTGAA
20170407_cc6hb_v3-1_queen, c14, h21, p0, 7 bp
267


AGCGTAAGAATCGGTCCA
plug, cyan, start 14[94], end 21[6821, 49 mer






CGAATAATAATTTTTTCACGTATCACCTTGC
20170407_cc6hb_v3-1_queen, c14, h20, p1, 7 bp
268


TGAACCTCAAAATGGTGG
plug, cyan, start 15[95], end 20[683], 49 mer






ATGAGGAAGTTTCCATTAAACATCCTAATTT
20170407_cc6hb_v3-1_queen, c14, h19, p2, 7 bp
269


ACGAGCATGTAAATAGCC
plug, cyan, start 14[178], end 19[6821, 49 mer






TTCGAGGTGAATTTCTTAAACACCTCCCGAC
20170407_cc6hb_v3-1_queen, c14, h18, p3, 7 bp
270


TTGCGGGAGGTAGAGTCC
plug, cyan, start 15[179], end 18[6831, 49 mer






GGATCGTCACCCTCAGCAGCGACATGAAAG
20170407_cc6hb_v3-1_queen, c14, h17, p4, 7 bp
271


TATTAAGAGGCTGAAAAAC
plug, cyan, start 14[262], end 17[682], 49 mer






TTTCAGCGGAGTGAGAATAGATGAATGGCT
20170407_cc6hb_v3-1_queen, c15, h21, p4, 7 bp
272


ATTAGTCTTTAAGAAAAAC
plug, cyan, start 15[53], end 21[724], 49 mer






CTACAACGCCTGTAGCATTCCAGTGCCACG
20170407_cc6hb_v3-1_queen, c15, h20, p3, 7 bp
273


CTGAGAGCCAGCAGAGTCC
plug, cyan, start 16[136], end 20[725], 49 mer






CTCCAAAAGGAGCCTTTAATTGTCTTTCCTT
20170407_cc6hb_v3-1_queen, c15, h19, p2, 7 bp
274


ATCATTCCAAGAATAGCC
plug, cyan, start 15[137], end 19[724], 49 mer






CAGAGCCACCACCCTCATTTTAAGGCTTATC
20170407_cc6hb_v3-1_queen, c15, h18, p1, 7 bp
275


CGGTATTCTAAATGGTCG
plug, cyan, start 16[220], end 18[725], 49 mer






CCGACAATGACAACAACCATCTAGGATTAG
20170407_cc6hb_v3-1_queen, c15, h17, p0, 7 bp
276


CGGGGTTTTGCTCGGTCCA
plug, cyan, start 15[221], end 17[724], 49 mer






ATCTGAACTCGCTACGGCGGGGGGAGCCCC
20170407_cc6hb_v3-1_queen, c0, h21, p0, 10 bp
277


CGATTTAGAGCTCGGTCCACGC
plug, cyan, start 0[94], end 21[94], 52 mer






CATTGCTGATACCGTTTAGCTAACAAACATC
20170407_cc6hb_v3-1_queen, c0, h20, p1, 10 bp
278


AAGAAAACAAAATGGTGGTTC
plug, cyan, start 1[95], end 20[95], 52 mer






GATACTTGCCCTCTCTGTACATAATTAATTT
20170407_cc6hb_v3-1_queen, c0, h19, p2, 10 bp
279


TCCCTTAGAATAATAGCCCGA
plug, cyan, start 0[178], end 19[94], 52 mer






GATTGGGCGTTATCAATGTTGTTTTGTCACA
20170407_cc6hb_v3-1_queen, c0, h18, p3, 10 bp
280


ATCAATAGAAAAGAGTCCACT
plug, cyan, start 1[179], end 18[95], 52 mer






TCTAATGAAGACAAATCCCCACGTCACCGA
20170407_cc6hb_v3-1_queen, c0, h17, p4, 10 bp
281


CTTGAGCCATTTGAAAAACCGT
plug, cyan, start 0[262], end 17[94], 52 mer






AAACATCGGGTTGAGTATTATGTGGCGAGA
20170407_cc6hb_v3-1_queen, c1, h21, p4, 10 bp
282


AAGGAAGGGAAGGAAAAACCGT
plug, cyan, start 1[53], end 21[136], 52 mer






CGCTGGCATTCGCATCAAAGGCGAATTATT
20170407_cc6hb_v3-1_queen, c1, h20, p3, 10 bp
283


CATTTCAATTACAGAGTCCACT
plug, cyan, start 2[136], end 20[137], 52 mer






AGTTTATAAATGAGTATCAATTTAGATTAAG
20170407_cc6hb_v3-1_queen, c1, h19, p2, 10 bp
284


ACGCTGAGAAGAATAGCCCGA
plug, cyan, start 1[137], end 19[136], 52 mer






TATCGACATCATTACGCATCGCAACATATA
20170407_cc6hb_v3-1_queen, c1, h18, p1, 10 bp
285


AAAGAAACGCAAATGGTGGTTC
plug, cyan, start 2[220], end 18[137], 52 mer






CCATGCAGACATCACGAAGGTCACCAGTAG
20170407_cc6hb_v3-1_queen, c1, h17, p0, 10 bp
286


CACCATTACCATCGGTCCACGC
plug, cyan, start 1[221], end 17[136], 52 mer






AAGATAACGCTTGTGAAAATGAGGGCGCTG
20170407_cc6hb_v3-1_queen, c2, h21, p0, 10 bp
287


GCAAGTGTAGCGCGGTCCACGC
plug, cyan, start 2[94], end 21[178], 52 mer






GCTAACAGTAGGGAAACTGCGGCCTGATTG
20170407_cc6hb_v3-1_queen, c2, h20, p1, 10 bp
288


CTTTGAATACCAATGGTGGTTC
plug, cyan, start 3[95], end 20[179], 52 mer






ATGGGTTCAGGATGCAGGTGAAATCATAGG
20170407_cc6hb_v3-1_queen, c2, h19, p2, 10 bp
289


TCTGAGAGACTAAATAGCCCGA
plug, cyan, start 2[178], end 19[178], 52 mer






CTCGGATGGGAGTAAGCGTATGCAGTATGT
20170407_cc6hb_v3-1_queen, c2, h18, p3, 10 bp
290


TAGCAAACGTAGAGAGTCCACT
plug, cyan, start 3[179], end 18[179], 52 mer






AGAGTTTCTGCGGCAGTTAATCAATGAAAC
20170407_cc6hb_v3-1_queen, c2, h17, p4, 10 bp
291


CATCGATAGCAGGAAAAACCGT
plug, cyan, start 2[262], end 17[178], 52 mer






GCAATACATCAAACGCCGCGAACACCCGCC
20170407_cc6hb_v3-1_queen, c3, h21, p4, 10 bp
292


GCGCTTAATGCGGAAAAACCGT
plug, cyan, start 3[53], end 21[220], 52 mer






TCAGGCACTGCGTGAAGCGGCAGTAACAGT
20170407_cc6hb_v3-1_queen, c3, h20, p3, 10 bp
293


ACCTTTTACATCAGAGTCCACT
plug, cyan, start 4[136], end 20[221], 52 mer






ATCAAAACTCAACGAGCAGCGGTTGGGTTA
20170407_cc6hb_v3-1_queen, c3, h19, p2, 10 bp
294


TATAACTATATGAATAGCCCGA
plug, cyan, start 3[137], end 19[220], 52 mer






AGGGTTGTCGGACTTGTGCAAGGAATACCC
20170407_cc6hb_v3-1_queen, c3, h18, p1, 10 bp
295


AAAAGAACTGGCATGGTGGTTC
plug, cyan, start 4[220], end 18[2211, 52 mer






AGTCCGTGAAGACGGAAACCAAATCAAGTT
20170407_cc6hb_v3-1_queen, c3, h17, p0, 10 bp
296


TGCCTTTAGCGTCCGTCCACGC
plug, cyan, start 3[221], end 17[220], 52 mer






CTGGGGATTTGACGCAGACCTGGTTGCTTTG
20170407_cc6hb_v3-1_queen, c4, h21, p0, 10 bp
297


ACGAGCACGTACGGTCCACGC
plug, cyan, start 4[94], end 21[262], 52 mer






TTTTCCCAGTCACGACGTTGTGAAATTGCGT
20170407_cc6hb_v3-1_queen, c4, h20, p1, 10 bp
298


AGATTTTCAGGATGGTGGTTC
plug, cyan, start 5[95], end 20[263], 52 mer






TTATCAGTAAACAGAGAGGTTTCGCAAGAC
20170407_cc6hb_v3-1_queen, c4, h19, p2, 10 bp
299


AAAGAACGCGAGAATAGCCCGA
plug, cyan, start 4[178], end 19[262], 52 mer






TCAGGGATTAATGAAAGATGGAACAAAGTT
20170407_cc6hb_v3-1_queen, c4, h18, p3, 10 bp
300


ACCAGAAGGAAAAGAGTCCACT
plug, cyan, start 5[179], end 18[263], 52 mer






AGTGTGGCGATCCGATAGATGCGGCATTTT
20170407_cc6hb_v3-1_queen, c4, h17, p4, 10 bp
301


CGGTCATAGCCCGAAAAACCGT
plug, cyan, start 4[262], end 17[262], 52 mer






GGGGGATGTGCTCCAAGGCGAATCAGAGCG
20170407_cc6hb_v3-1_queen, c5, h21, p4, 10 bp
302


GGAGCTAAACAGGAAAAACCGT
plug, cyan, start 5[53], end 21[304], 52 mer






AGCCAGCTTTCCGGCACCGCTACCTACCATA
20170407_cc6hb_v3-1 queen, c5, h20, p3, 10 bp
303


TCAAAATTATTAGAGTCCACT
plug, cyan, start 6[136], end 20[305], 52 mer






CTTTATTATTCGCATTCACCCTAGTTAATTTC
20170407_cc6hb_v3-1_queen, c5, h19, p2, 10 bp
304


ATCTTCTGACAATAGCCCGA
plug, cyan, start 5[137], end 19[304], 52 mer






TTGGTGTAGATGGGCGCATCGATCTTACCG
20170407_cc6hb_v3-1_queen, c5, h18, p1, 10 bp
305


AAGCCCTTTTTAATGGTCGTTC
plug, cyan, start 6[220], end 18[305], 52 mer






CAGAAATAGAAGAATTACAGCTTTCATAAT
20170407_cc6hb_v3-1_queen, c5, h17, p0, 10 bp
306


CAAAATCACCGGCGGTCCACGC
plug, cyan, start 5[221], end 17[304], 52 mer






AAGCGCCATTCGCCATTCAGGAGACAGGAA
20170407_cc6hb_v3-1_queen, c6, h21, p0, 10 bp
307


CGGTACGCCAGACGGTCCACGC
plug, cyan, start 6[94], end 21[346], 52 mer






TCAGAAAAGCCCCAAAAACAGCTGATTGTT
20170407_cc6hb_v3-1_queen, c6, h20, p1, 10 bp
308


TGGATTATACTTATGGTGGTTC
plug, cyan, start 7[95], end 20[347], 52 mer






GAGGGGACGACGACAGTATCGACCGACCGT
20170407_cc6hb_v3-1_queen, c6, h19, p2, 10 bp
309


GTGATAAATAAGAATAGCCCGA
plug, cyan, start 6[178], end 19[346], 52 mer






TTTTTGTTAAATCAGCTCATTAGCCCAATAA
20170407_cc6hb_v3-1_queen, c6, h18, p3, 10 bp
310


TAAGAGCAAGAAGAGTCCACT
plug, cyan, start 7[179], end 18[347], 52 mer






GTCKJGAACAAACGGCGGATTGCGCCTCCCT
20170407_cc6hb_v3-1_queen, c6, h17, p4, 10 bp
311


CAGAGCCGCCACGAAAAACCGT
plug, cyan, start 6[262], end 17[346], 52 mer






CGAACGAGTAGATTTAGTTTGACTTGCCTGA
20170407_cc6hb_v3-1_queen, c9, h21, p4, 10 bp
312


GTAGAAGAACTGAAAAACCGT
plug, cyan, start 9[53], end 21[472], 52 mer






CATTTTTGCGGATGGCTTAGACCGAACGTTA
20170407_cc6hb_v3-1_queen, c9, h20, p3, 10 bp
313


TTAATTTTAAAAGAGTCCACT
plug, cyan, start 10[136], end 20[473], 52 mer






AGCTGAAAAGGTGGCATCAATTAGGGCTTA
20170407_cc6hb_v3-1_qucen, c9, h19, p2, 10 bp
314


ATTGAGAATCGCAATAGCCCGA
plug, cyan, start 9[137], end 19[472], 52 mer






AGCTTCAAAGCGAACCAGACCTTTACAGAG
20170407_cc6hb_v3-1_queen, c9, h18, p1, 10 bp
315


AGAATAACATAAATGGTGGTTC
plug, cyan, start 10[220], end 18[473], 52 mer






ATTAAGCAATAAAGCCTCAGAGGCCTTGAT
20170407_cc6hb_v3-1_queen, c9, h17, p0, 10 bp
316


ATTCACAAACAACGGTCCACGC
plug, cyan, start 9[221], end 17[472], 52 mer






CTGTAGCTCAACATGTTTTAAAATATCCAGA
20170407_cc6hb_v3-1_queen, c10, h21, p0, 10 bp
317


ACAATATTACCCGGTCCACGC
plug, cyan, start 10[94], end 21[514], 52 mer






GGCTTTTGCAAAAGAAGTTTTAGACTTTACA
20170407_cc6hb_v3-1_queen, c10, h20, p1, 10 bp
318


AACAATTCGACATGGTGGTTC
plug, cyan, start 11[95], end 20[515], 52 mer






AGGATTAGAGAGTACCTTTAAGTAATTTAG
20170407_cc6hb_v3-1_queen, c10, h19, p2, 10 bp
319


GCAGAGGCATTTAATAGCCCGA
plug, cyan, start 10[178], end 19[514], 52 mer






AATATTCATTGAATCCCCCTCGAAACGATTT
20170407_cc6hb_v3-1_queen, c10, h18, p3, 10 bp
320


TTTGTTTAACGAGAGTCCACT
plug, cyan, start 11[179], end 18[515], 52 mer






AAGAGGAAGCCCGAAAGACTTAATGGAAA
20170407_cc6hb_v3-1_queen, c10, h17, p4, 10 bp
321


GCGCAGTCTCTGAGAAAAACCGT
plug, cyan, start 10[262], end 17[514], 52 mer






ACCCTCGTTTACCAGACGACGAACGCTCAT
20170407_cc6hb_v3-1_queen, c11, h21, p4, 10 bp
322


GGAAATACCTACGAAAAACCGT
plug, cyan, start 11[53], end 21[556], 52 mer






TAACGGAACAACATTATTACAAGAGCCGTC
20170407_cc6hb_v3-1_queen, c11, h20, p3, 10 bp
323


AATAGATAATACAGAGTCCACT
plug, cyan, start 12[136], end 20[557], 52 mer






ATGTTTAGACTGGATAGCGTCATAAAGTAC
20170407_cc6hb_v3-1_queen, c11, h19, p2, 10 bp
324


CGACAAAAGGTAAATAGCCCGA
plug, cyan, start 11[137], end 19[556], 52 mer






TGAATTACCTTATGCGATTTTTTACAAAATA
20170407_cc6hb_v3-1_queen, c11, h18, p1, 10 bp
325


AACACCCATATATGGTGGTTC
plug, cyan, start 12[220], end 18[557], 52 mer






AAACGAGAATGACCATAAATCCATACATGG
20170407_cc6hb_v3-1_queen, c11, h17, p0, 10 bp
326


CTTTTGATGATACGGTCCACGC
plug, cyan, start 11[221], end 17[556], 52 mer






AGATTTAGGAATACCACATTCAAATGGATT
20170407_cc6hb_v3-1_queen, c12, h21, p0, 10 bp
327


ATTTACATTGGCCGGTCCACGC
plug, cyan, start 12[94], end 21[598], 52 mer






CGAGGCGCAGACGGTCAATCAGTTATCTAA
20170407_cc6hb_v3-1_queen, c12, h20, p1, 10 bp
328


AATATCTTTAGGATGGTGGTTC
plug, cyan, start 13[95], end 20[599], 52 mer






GTCAGGACGTTGGGAAGAAAAGACAATAA
20170407_cc6hb_v3-1_queen, c12, h19, p2, 10 bp
329


ACAACATGTTCAGAATAGCCCGA
plug, cyan, start 12[178], end 19[598], 52 mer






AGGCTGGCTGACCTTCATCAATACCAACGC
20170407_cc6hb_v3-1_queen, c12, h18, p3, 10 bp
330


TAACGAGCGTCTAGAGTCCACT
plug, cyan, start 13[179], end 18[599], 52 mer






TAAATTCGGCTTGAGATGGTTTTTTAACGGG
20170407_cc6hb_v3-1_queen, c12, h17, p4, 10 bp
331


GTCAGTGCCTTGAAAAACCGT
plug, cyan, start 12[262], end 17[598], 52 mer






TGTGTCGAAATCCGCGACCTGAGTAATAAA
20170407_cc6hb_v3-1_queen, c13, h21, p4, 10 bp
332


AGGGACATTCTGGAAAAACCGT
plug, cyan, start 13[53], end 21[640], 52 mer






TACGAAGGCACCAACCTAAAACTGGTCAGT
20170407_cc6hb_v3-1_queen, c13, h20, p3, 10 bp
333


TGGCAAATCAACAGAGTCCACT
plug, cyan, start 14[136], end 20[641], 52 mer






CTTTGAAAGAGGACAGATGAATATCAACAA
20170407_cc6hb_v3-1_queen, c13, h19, p2, 10 bp
334


TAGATAAGTCCTAATAGCCCGA
plug, cyan, start 13[137], end 19[640], 52 mer






GTAGCAACGGCTACAGAGGCTTAGTTGCTA
20170407_cc6hb_v3-1_queen, c13, h18, p1, 10 bp
335


TTTTGCACCCAGATGGTGGTTC
plug, cyan, start 14[220], end 18[641], 52 mer






GATATTCATTACCCAAATCAACAGTTAATGC
20170407_cc6hb_v3-1_queen, c13, h17, p0, 10 bp
336


CCCCTGCCTATCGGTCCACGC
plug, cyan, start 13[221], end 17[640], 52 mer






CTAAAACACTCATCTTTGACCCTGACCTGAA
20170407_cc6hb_v3-1_queen, c14, h21, p0, 10 bp
337


AGCGTAAGAATCGGTCCACGC
plug, cyan, start 14[94], end 21[682], 52 mer






CGAATAATAATTTTTTCACGTATCACCTTGC
20170407_cc6hb_v3-1_queen, c14, h20, p1, 10 bp
338


TGAACCTCAAAATGGTGGTTC
plug, cyan, start 15[95], end 20[683], 52 mer






ATGAGGAAGTTTCCATTAAACATCCTAATTT
20170407_cc6hb_v3-1_queen, c14, h19, p2, 10 bp
339


ACGAGCATGTAAATAGCCCGA
plug, cyan, start 14[178], end 19[682], 52 mer






TTCGAGGTGAATTTCTTAAACACCTCCCGAC
20170407_cc6hb_v3-1_queen, c14, h18, p3, 10 bp
340


TTGCGGGAGGTAGAGTCCACT
plug, cyan, start 15[179], end 18[683], 52 mer






GGATCGTCACCCTCAGCAGCGACATGAAAG
20170407_cc6hb_v3-1_queen, c14, h17, p4, 10 bp
341


TATTAAGAGGCTGAAAAACCGT
plug, cyan, start 14[262], end 17[682], 52 mer






TTTCAGCGGAGTGAGAATAGATGAATGGCT
20170407_cc6hb_v3-1_queen, c15, h21, p4, 10 bp
342


ATTAGTCTTTAAGAAAAACCGT
plug, cyan, start 15[53], end 21[724], 52 mer






CTACAACGCCTGTAGCATTCCAGTGCCACG
20170407_cc6hb_v3-1_queen, c15, h20, p3, 10 bp
343


CTGAGAGCCAGCAGAGTCCACT
plug, cyan, start 16[136], end 20[725], 52 mer






CTCCAAAAGGAGCCTTTAATTGTCTTTCCTT
20170407_cc6hb_v3-1_queen, c15, h19, p2, 10 bp
344


ATCATTCCAAGAATAGCCCGA
plug, cyan, start 15[137], end 19[724], 52 mer






CAGAGCCACCACCCTCATTTTAAGGCTTATC
20170407_cc6hb_v3-1_queen, c15, h18, p1, 10 bp
345


CGGTATTCTAAATGGTGGTTC
plug, cyan, start 16[220], end 18[725], 52 mer






CCGACAATGACAACAACCATCTAGGATTAG
20170407_cc6hb_v3-1_queen, c15, h17, p0, 10 bp
346


CGGGGTTTTGCTCGGTCCACGC
plug, cyan, start 15[221], end 17[724], 52 mer
















TABLE 2







Exemplary 250 nm Six-helix Bundle Sequences











SEQ




ID


Sequence
Comment
NO:





TCATCAACATTAAAAGAACGCGAGAAAATT
20170608 cc6hb v4-base 250 nm 6hb, grey
347


GTTAAATCAGACCGTGCAT
standard seq, start 0[41], end 4[21], 49 mer






TAATCGTAAAACTAATCTTCTGACCTAAAGC
20170608 cc6hb v4-base 250 nm 6hb, grey
348


TATTTTTGATA
standard seq, start 0[83], end 4[70], 42 mer






ATATATTTTAAATGGATAAATAAGGCGTAA
20170608 cc6hb v4-base 250 nm 6hb, grey
349


AAACATTATGTC
standard seq, start 0[125], end 4[112], 42 mer






GCGAGCTGAAAAGGTTACTAGAAAAAGCAC
20170608 cc6hb v4-base 250 nm 6hb, grey
350


GAGTAGATTTCT
standard seq, start 0[167], end 4[154], 42 mer






GGTCATTTTTGCGGTTCTTACCAGTATATTC
20170608 cc6hb v4-base 250 nm 6hb, grey
351


AAAGCGAACCC
standard seq, start 0[209], end 4[196], 42 mer






AGAAAACGAGAATGTGAGAATCGCCATATT
20170608 cc6hb v4-base 250 nm 6hb, grey
352


TAGACTGGATAG
standard seq, start 0[251], end 4[238], 42 mer






ACGCCAAAAGGAATAGAGGCATTTTCGACG
20170608 cc6hb v4-base 250 nm 6hb, grey
353


GAACAACATTAG
standard seq, start 0[293], end 4[280], 42 mer






TAGTAAATTGGGCTACAAAAGGTAAAGTAT
20170608 cc6hb v4-base 250 nm 6hb, grey
354


TCATTACCCAAG
standard seq, start 0[335], end 4[322], 42 mer






GAACGAGGCGCAGAACATGTTCAGCTAAAC
20170608 cc6hb v4-base 250 nm 6hb, grey
355


AAAGTACAACCA
standard seq, start 0[377], end 4[364], 42 mer






TTCATGAGGAAGTTGATAAGTCCTGAACTC
20170608 cc6hb v4-base 250 nm 6hb, grey
356


GTCACCCTCATT
standard seq, start 0[419], end 4[406], 42 mer






GCTTTCGAGGTGAACGAGCATGTAGAAAAT
20170608 cc6hb v4-base 250 nm 6hb, grey
357


AATAATTTTTTG
standard seq, start 0[461], end 4[448], 42 mer






TAGCGTAACGATCTTCATTCCAAGAACGCC
20170608 cc6hb v4-base 250 nm 6hb, grey
358


CATGTACCGTAA
standard seq, start 0[503], end 4[490], 42 mer






AAGTATAGCCCGGAAGAACAAGCAAGCCAC
20170608 cc6hb v4-base 250 nm 6hb, grey
359


TCCTCAAGAGCA
standard seq, start 0[545], end 4[532], 42 mer






ATACAGGAGTGTACCCGCGCCCAATAGCAA
20170608 cc6hb v4-base 250 nm 6hb, grey
360


TCCTCATTAATC
standard seq, start 0[587], end 4[574], 42 mer






CACCCTCAGAACCGGGTATTCTAAGAACTA
20170608 cc6hb v4-base 250 nm 6hb, grey
361


TTAGCGTTTGAC
standard seq, start 0[629], end 4616], 42 mer






CATTAGCAAGGCCGTGCGGGAGGTTTTGTT
20170608 cc6hb v4-base 250 nm 6hb, grey
362


AAAGGTGAATTT
standard seq, start 0[671], end 4[658], 42 mer






CAAAGACACCACGGTTGCACCCAGCTACAT
20170608 cc6hb v4-base 250 nm 6hb, grey
363


TAAGACTCCTGG
standard seq, start 0|713], end 4[700], 42 mer






AGAAACAATGAAATACGAGCGTCTTTCCGA
20170608 cc6hb v4-base 250 nm 6hb, grey
364


ATTAACTGAAAA
standard seq, start 0[755], end 4[742], 42 mer






GAACAAACGGCGGATTGACAATAATTCG
20170608 cc6hb v4-base 250 nm 6hb, grey
365



standard seq, start 1[7], end 1[34], 28 mer






CGTCTGGCCTTCCTGTCCCGGTTGATAATCA
20170608 cc6hb v4-base 250 nm 6hb, grey
366


GAAGAGTCTGG
standard seq, start 1[35], end 1[76], 42 mer






AGCAAACAAGAGAATCAGGTAAAGATTCAA
20170608 cc6hb 4-base 250 nm 6hb, grey
367


AAGTTTCAACGC
standard seq, start 1[77], end 1[118], 42 mer






AAGGATAAAAATTTTTAGTAGTAGCATTAA
20170608 cc6hb v4-base 250 nm 6hb, grey
368


CATCAATAACCT
standard seq, start 1[119], end 1[160], 42 mer






GTTTAGCTATATTTTCCTGAATATAATGCTG
20170608 cc6hb v4-base 250 nm 6hb, grey
369


TATAGAGAGTA
standard seq, start 1[161], end 1[202], 42 mer






CCTTTAATTGCTCCTTGTCTTTACCCTGACTA
20170608 cc6hb v4-base 250 nm 6hb, grey
370


TTCATTGAAT
standard seq, start 1[203], end l[244], 42 mer






CCCCCTCAAATGCTTTAACACTATCATAACC
20170608 cc6hb v4-base 250 nm 6hb, grey
371


CTTAGGAATAC
standard seq, start 1[245], end 1[286], 42 mer






CACATTCAACTAATGCCTTTAATCATTGTGA
20170608 cc6hb v4-base 250 nm 6hb, grey
372


ATTAAGGCTTG
standard seq, start 1[287], end 1[328], 42 mer






CCCTGACGAGAAACACCGAACTGACCAACT
20170608 cc6hb v4-base 250 nm 6hb, grey
373


TTGCGAAATCCG
standard seq, start 1[329], end 1[370], 42 mer






CGACCTGCTCCATGTTACGTAATGCCACTAC
20170608 cc6hb v4-base 250 nm 6hb, grey
374


GAAACGGCTAC
standard seq, start 1[371], end 1[412] 42 mer






AGAGGCTTTGAGGACTCCGATAGTTGCGCC
20170608 cc6hb v4-base 250 nm 6hb, grey
375


GACAAAGGAGCC
standard seq, start 1[413], end 1[454], 42 mer






TTTAATTGTATCGGTTAGACGTTAGTAAATG
20170608 cc6hb v4-base 250 nm 6hb, grey
376


AAACGCCTGTA
standard seq, start 1[455], end 1[496], 42 mer






GCATTCCACAGACAGCCAGGAGGTTTAGTA
20170608 cc6hb v4-base 250 nm 6hb, grey
377


CCGCCAGGCGGA
standard seq, start 1[497], end 1[538], 42 mer






TAAGTGCCGTCGAGAGGGTCAGTGCCTTGA
20170608 cc6hb v4-base 250 nm 6hb, grey
378


GTACCGTTCCAG
standard seq, start 1[539], end 1[580], 42 mer






TAAGCGTCATACATGGCCTCAGAGCCGCCA
20170608 cc6hb v4-base 250 nm 6hb, grey
379


CCAGAGCCACCA
standard seq, start 1[581], end 1[622], 42 mer






CCGGAACCGCCTCCCTCATCGATAGCAGCA
20170608 cc6hb v4-base 250 nm 6hb, grey
380


CCGTTAGAGCCA
standard seq, start 1[623], end 1[664], 42 mer






GCAAAATCACCAGTAGAATCAATAGAAAAT
20170608 cc6hb v4-base 250 nm 6hb, grey
381


TCAACATACATA
standard seq, start 1[665], end 1[706], 42 mer






AAGGTGGCAACATATAAAGCCCTTTTTAAG
20170608 cc6hb v4-base 250 nm 6hb, grey
382


AAAAGAATTGAGTTAAGCC
standard seq, start 1[707], end 1[755], 49 mer






ACAGGAAGATTGAATAGGAACGCCATCAAA
20170608 cc6hb v4-base 250 nm 6hb, grey
383


CGTAATGGGATA
standard seq, start 2[55], end 2[14], 42 mer






AGATGGGCGCATCGTACTCATTTTTTAACCT
20170608 cc6hb v4-base 250 nm 6hb, grey
384


ATAA
standard seq, start 3[14], end 3[48], 35 mer






GCAAATATTTAAATTGTAAACGTGAGATCT
20170608 cc6hb v4-base 250 nm 6hb, grey
385


ACAAAGGAATCA
standard seq, start 3[49], end 3[90], 42 mer






CCATCAATATGATATTCAACCGTACCCTGTA
20170608 cc6hb v4-base 250 nm 6hb, grey
386


ATACTTAAGAA
standard seq, start 3[91], end 3[132], 42 mer






TTAGCAAAATTAAGCAATAAAGCAGTTTGA
20170608 cc6hb v4-base 250 nm 6hb, grey
387


CCATTAGCTAAA
standard seq, start 3[133], end 3[174], 42 mer






GTACGGTGTCTGGAAGTTTCATTCAGACCGG
20170608 cc6hb v4-base 250 nm 6hb, grey
388


AAGCAACATCA
standard seq, start 3[175], end 3[216], 42 mer






AAAAGATTAAGAGGAAGCCCGAAAGCGTCC
20170608 cc6hb v4-base 250 nm 6hb, grey
389


AATACTGCAAAA
standard seq, start 3[217], end 3[258], 42 mer






TAGCGAGAGGCTTTTGCAAAAGAATTACAG
20170608 cc6hb v4-base 250 nm 6hb, grey
390


GTAGAAAGCTCA
standard seq, start 3[259], end 3[300], 42 mer






TTATACCAGTCAGGACGTTGGGAAATCAAC
20170608 cc6hb v4-base 250 nm 6hb, grey
391


GTAACAAAGACC
standard seq, start 3[301], end 3[342], 42 mer






AGGCGCATAGGCTGGCTGACCTTGGAGATT
20170608 cc6hb v4-base 250 nm 6hb, grey
392


TGTATCAGCAAA
standard seq, start 3[343], end 3[384], 42 mer






AGAATACACTAAAACACTCATCTGCAGCGA
20170608 cc6hb v4-base 250 nm 6hb, grey
393


AAGACAGATAAC
standard seq, start 3[385], end 3[426], 42 mer






CGATAETTCGGTCGCTGAGGCTTCACGTTG
20170608 cc6hb v4-base 250 nm 6hb, grey
394


AAAATCCAACT
standard seq, start 3[427], end 3[468], 42 mer






TTCAACAGTTTCAGCGGAGTGAGAACACTG
20170608 cc6hb v4-base 250 nm 6hb, grey
395


AGTTTCGGAACC
standard seq, start 3[469], end 3[510], 42 mer






GCCACCCTCAGAGCCACCACCCTAAGGATT
20170608 cc6hb 4-base 250 nm 6hb, grey
396


AGGATTAGCCCC
standard seq, start 3[551], end 3[552], 42 mer






CTGCCTATTTCGGAACCTATTATAGCCAGAA
20170608 cc6hb v4-base 250 nm 6hb, grey
397


TGGAAAGCATT
standard seq, start 3[553], end 3[594], 42 mer






GACAGGAGGTTGAGGCAGGTCAGCCATCTT
20170608 cc6hb v4-base 250 nm 6hb, grey
398


TTCATAATTGCC
standard seq, start 3[595], end 3[636], 42 mer






TTTAGCGTCAGACTGTAGCGCGTTATCACCG
20170608 cc6hb v4-base 250 nm 6hb, grey
399


TCACCGAAAGG
standard seq, start 3[637], end 3[678], 42 mer






GCGACATTCAACCGATTGAGGGATATTACG
20170608 cc6hb v4-base 250 nm 6hb, grey
400


CAGTATGTACCA
standard seq, start 3[679], end 3[720], 42 mer






GAAGGAAACCGAGGAAACGCAATCACCCTG
20170608 cc6hb v4-base 250 nm 6hb, grey
401


AACAAAGTCAGATAATATC
standard seq, start 3[721], end 2[756], 49 mer






ATATTTTGTTAAAATTCGCATTAAATTTCTTT
20170608 cc6hb v4-base 250 nm 6hb, grey
402


TTCAAATATA
standard seq, start 4[69], end 5[55], 42 mer






TAGCTGATAAATTAATGCCGGAGAGGGTAT
20170608 cc6hb v4-base 250 nm 6hb, grey
403


TTAATGGTTTGA
standard seq, start 4[111], end 5[97], 42 mer






CAGAGCATAAAGCTAAATCGGTTGTACCTA
20170608 cc6hb v4-base 250 nm 6hb, grey
404


AATAAGAATAAA
standard seq, start 4[153], end 5[139], 42 mer






ATATAACAGTTGATTCCCAATTCTGCGACTG
20170608 cc6hb v4-base 250 nm 6hb, grey
405


TTTAGTATCAT
standard seq, start 4[195], end 5[181], 42 mer






ACTTCAAATATCGCGTTTTAATTCGAGCAAG
20170608 cc6hb v4-base 250 nm 6hb, grey
406


CCAACGCTCAA
standard seq, start 4[237], end 5[223], 42 mer






TTTTGCCAGAGGGGGTAATAGTAAAATGTTT
20170608 cc6hb v4-base 250 nm 6hb, grey
407


AACAACGCCAA
standard seq, start 4[279], end 5[265], 42 mer






AAAAATCTACGTTAATAAAACGAACTAAGC
20170608 cc6hb v4-base 250 nm 6hb, grey
408


CAGTAATAAGAG
standard seq, start 4[321], end 5[307], 42 mer






TCAAGAGTAATCTTGACAAGAACCGGATAA
20170608 cc6hb v4-base 250 nm 6hb, grey
409


TTCTGTCCAGAC
standard seq, start 4[363], end 5[349], 42 mer






GACCCCCAGCGATTATACCAAGCGCGAATG
20170608 cc6hb v4-base 250 nm 6hb, grey
410


CAGAACGCGCCT
standard seq, start 4[405], end 5[391], 42 mer






CAGGGAGTTAAAGGCCGCTTTTGCGGGAAA
20170608 cc6hb v4-base 250 nm 6hb, grey
411


GAAAAATAATAT
standard seq, start 4[447], end 5[433], 42 mer






TAGAAAGGAACAACTAAAGGAATTGCGACC
20170608 cc6hb v4-base 250 nm 6hb, grey
412


AATCAATAATCG
standard seq, start 4[489], end 5[475], 42 mer






TTTTCAGGGATAGCAAGCCCAATAGGAAGG
20170608 cc6hb v4-base 250 nm 6hb, grey
413


TATTAAACCAAG
standard seq, start 4[531], end 5[517], 42 mer






TGAAACATGAAAGTATTAAGAGGCTGAGGT
20170608 cc6hb v4-base 250 nm 6hb, grey
414


TTTTATTTTCAT
standard seq, start 4[573], end 5[559], 42 mer






GATTGGCCTTGATATTCACAAACAAATAAA
20170608 cc6hb v4-base 250 nm 6hb, grey
415


GCAAATCAGATA
standard seq, start 4[615], end 5[601], 42 mer






TCATCGGCATTTTCGGTCATAGCCCCCTGCG
20170608 cc6hb v4-base 250 nm 6hb, grey
416


AGGCGTTTTAG
standard seq, start 4[657], end 5[643], 42 mer






GAAGGTAAATATTGACGGAAATTATTCAAA
20170608 cc6hb v4-base 250 nm 6hb, grey
417


GCCTTAAATCAA
standard seq, start 4[699], end 5[685], 42 mer






TAACGGAATACCCAAAAGAACTGGCATGAA
20170608 cc6hb v4-base 250 nm 6hb, grey
418


TTTTATCCTGAA
standard seq, start 4[741], end 5[727], 42 mer






CGCATTAGACGGGAAGAGCCT
20170608 cc6hb v4-base 250 nm 6hb, grey
419



standard seq, start 4[769], end 5[762], 21 mer






AAGACAATGTGAGCGAGTAACAACCCGT
20170608 cc6hb v4-base 250 nm 6hb, grey
420



standard seq, start 5[21], end 0[7], 28 mer






TTTTAGTTAATTTCGCATGTCAATCATATGT
20170608 cc6hb v4-base 250 nm 6hb, yellow
421


ACAGCCAGCTT
standard seq, start 5[56], end 0[42], 42 mer






AATACCGACCGTGTCAATGCCTGAGTAATG
20170608 cc6hb v4-base 250 nm 6hb, yellow
422


TGTGATGAACGG
standard seq, start 5[98], end 0[84], 42 mer






CACCGGAATCATAATGGCATCAATTCTACT
20170608 cc6hb v4-base 250 nm 6hb, yellow
423


AATAGAACCCTC
standard seq, start 5[140], end 0[126], 42 mer






ATGCGTTATACAAAATGGCTTAGAGCTTAAT
20170608 cc6hb v4-base 250 nm 6hb, yellow
424


TGATTTGGGGC
standard seq, start 5[182], end 0[168], 42 mer






CAGTAGGGCTTAATACCATAAATCAAAAAT
20170608 cc6hb v4-base 250 nm 6hb, yellow
425


CAGTTGATAAGA
standard seq, start 5[224], end 0[210], 42 mer






CATGTAATTTAGGCTACGAGGCATAGTAAG
20170608 cc6hb v4-base 250 nm 6hb, yellow
426


AGCAAACAGTTC
standard seq, start 5[266], end 0[252], 42 mer






AATATAAAGTACCGTGAGATGGTTTAATTTC
20170608 cc6hb v4-base 250 nm 6hb, yellow
427


AAAGATACATA
standard seq, start 5[308], end 0[294], 42 mer






GACGACAATAAACACGGTCAATCATAAGGG
20170608 cc6hb v4-base 250 nm 6hb, yellow
428


AACCAGAACGAG
standard seq, start 5[350], end 0[336], 42 mer






GTTTATCAACAATATCCATTAAACGGGTAA
20170608 cc6hb v4-base 250 nm 6hb, yellow
429


AATACTTAGCCG
standard seq, start 5[392], end 0[378], 42 mer






CCCATCCTAATTTATTTCTTAAACAGCTTGA
20170608 cc6hb v4-base 250 nm 6hb, yellow
430


TAAAAGACTTT
standard seq, start 5[434], end 0[420], 42 mer






GCTGTCTTTCCTTAAAAGTTTTGTCGTCTTTC
20170608 cc6hb v4-base 250 nm 6hb, yellow
431


CTATCAGCTT
standard seq, start 5[476], end 0[462], 42 mer






TACCGCACTCATCGATAGGTGTATCACCGTA
20170608 cc6hb v4-base 250 nm 6hb, yellow
432


CTCCTCATAGT
standard seq, start 5[518], end 0[504], 42 mer






CGTAGGAATCATTATGGTAATAAGTTTTAAC
20170608 cc6hb v4-base 250 nm 6hb, yellow
433


GGGGTTGATAT
standard seq, start 5[560], end 0[546], 42 mer






TAGAAGGCTTATCCCCACCCTCAGAGCCAC
20170608 cc6hb v4-base 250 nm 6hb, yellow
434


CACCTTTTGATG
standard seq, start 5[602], end 0[588], 42 mer






CGAACCTCCCGACTGAAACGTCACCAATGA
20170608 cc6hb v4-base 250 nm 6hb, yellow
435


AACCAGAGCCGC
standard seq, start 5[644], end 0[630], 42 mer






GATTAGTTGCTATTAATAAGTTTATTTTGTC
20170608 cc6hb v4-base 250 nm 6hb, yellow
436


ACCACCATTAC
standard seq, start 5[686], end 0[672], 42 mer






TCTTACCAACGCTAAGCAATAGCTATCTTAC
20170608 cc6hb v4-base 250 nm 6hb, yellow
437


CGAAAGAAACG
standard seq, start 5[728], end 0[714], 42 mer






CGGAGACAGTCACTATCAGGTCATTGCCTG
20170608 cc6hb v4-base 250 nm 6hb, magenta
438


AAAGCCCCAAAA
standard seq, start 2[97], end 2[56], 42 mer






ACAGGCAAGGCATTGCGGGAGAAGCCTTTA
20170608 cc6hb v4-base 250 nm 6hb, magenta
439


GGTGAGAAAGGC
standard seq, start 2[139], end 2[98], 42 mer






TTAAATATGCAAATACATTTCGCAAATGGTC
20170608 cc6hb v4-base 250 nm 6hb, magenta
440


CAATAAATCAT
standard seq, start 2[181], end 2[140], 42 mer






CAAAGCGGATTGACTCCAACAGGTCAGGAT
20170608 cc6hb v4-base 250 nm 6hb, magenta
441


GCTCAACATGTT
standard seq, start 2[223], end 2[182], 42 mer






GACGATAAAAACCGGAATCGTCATAAATAT
20170608 cc6hb v4-base 250 nm 6hb, magenta
442


TATAGTCAGAAG
standard seq, start 2[265], end 2[224], 42 mer






TTTTAAGAACTGGATTCATCAGTTGAGATTC
20170608 cc6hb v4-base 250 nm 6hb, magenta
443


GTTTACCAGAC
standard seq, start 2[307], end 2[266], 42 mer






TGAACGGTGTACAGCTGCTCATTCAGTGAAT
20170608 cc6hb v4-base 250 nm 6hb, magenta
444


ACCTTATCCGA
standard seq, start 2[349], end 2[308], 42 mer






AAAACGAAAGAGTCGCCTGATAAATTGTGT
20170608 cc6hb v4-base 250 nm 6hb, magenta
445


AAAGAGGACAGA
standard seq, start 2[391], end 2[350], 42 mer






CATCGCCCACGCCATCGGAACGAGGGTAGC
20170608 cc6hb v4-base 250 nm 6hb, magenta
446


AGGCACCAACCT
standard seq, start 2[433], end 2[392], 42 mer






GATTTTGCTAAATCCAAAAAAAAGGCTCCA
20170608 cc6hb v4-base 250 nm 6hb, magenta
447


AATGACAACAAC
standard seq, start 2[475], end 2[434], 42 mer






CCGCCACCCTCATCACCAGTACAAACTACA
2.0170608 cc6hb v4-base 250 nm 6hb, magenta
448


TTTTCTGTATGG
standard seq, start 2[517], end 2[476], 42 mer






TAAACAGTTAATGCGGGGTTTTGCTCAGTAC
20170608 cc6hb v4-base 250 nm 6hb, magenta
449


CACCCTCAGAA
standard seq, start 2[559], end 2[518], 42 mer






GAGCCGCCGCCAGCGCAGTCTCTGAATTTA
2.0170608 cc6hb v4-base 250 nm 6hb, magenta
450


ACAGTGCCCGTA
standard seq, start 2[601], end 2[560], 42 mer






ACAGAATCAAGTTCAAAATCACCGGAACCA
20170608 cc6hb v4-base 250 nm 6hb, magenta
451


GAACCACCACCA
standard seq, start 2[643], end 2|602], 42 mer






GCGCCAAAGACAACTTGAGCCATTTGGGAA
20170608 cc6hb v4-base 250 nm 6hb, magenta
452


TAATCAGTAGCG
standard seq, start 2[685], end 2[644], 42 mer






CCGAACAAAGTTTAGCAAACGTAGAAAATT
20170608 cc6hb v4-base 250 nm 6hb, magenta
453


ATGGTTTACCA
standard seq, start 2[726], end 2[686], 41 mer






AGAGAGATAACCCACAAGTAAGCAGATAG
20170608 cc6hb 4-base 250 nm 6hb, magenta
454



standard seq, start 2[755], end 2[727], 29 mer






AGAAGATGAAATTAACTAAAATATATTTGA
20170608, cc6hb v4-250 nm 16 component square,
455


AAAAGTTTTCTCGCGTTCTTTGTCTTGCGAT
default 6hb, miniseaf, node-0



TG







ATTTATCACACGGTCGGTATTTCAAACCATT
20170608, cc6hb v4-250 nm 16 component square,
456


AAATTTAGGTC
default 6hb, miniscaf, node-1






CTAGTAATTATGATTCCGGTGTTTATTCTTA
20170608, cc6hb v4-250 nm 16 component square,
457


TTTAACGCCTT
default 6hb, miniscaf, node-2






GTAAGAATTTGTATAACGCATATGATACTA
20170608, cc6hb v4-250 nm 16 component square,
458


AACAGGCTTTTT
default 6hb, miniscaf, node-3






ATTCTCAATTAAGCCCTACTGTTGAGCGTTG
20170608, cc6hb v4-250 nm 16 component square,
459


GCTTTATACTG
default 6hb, miniscaf, node-4






TGCCTCTGCCTAAATTACATGTTGGCGTTGT
20170608, cc6hb v4-250 nm 16 component square,
460


TAAATATGGCG
default 6hb, miniscaf, node-5






CTTTTGTCGGTACTTTATATTCTCTTATTACT
20170608, cc6hb v4-250 nm 16 component square,
461


GGCTCGAAAA
default 6hb, miniscaf, node-6






AACATGTTGTTTATTGTCGTCGTCTGGACAG
20170608, cc6hb v4-250 nm 16 component square,
462


AATTACTTTAC
default 6hb, miniscaf, node-7






ACTTATCTATTGTTGATAAACAGGCGCGTTC
20170608, cc6hb v4-250 nm 16 component square,
463


TGCATTAGCTG
default 6hb, miniscaf, node-8






ATGCTCGTAAATTAGGATGGGATATTATTTT
20170608, cc6hb v4-250 nm 16 component square,
464


TCTTGTTCAGG
default 6hb, miniscaf, node-9






GGAATGATAAGGAAAGACAGCCGATTATTG
20170608, cc6hb v4-250 nm 16 component square,
465


ATTGGTTTCTAC
default 6hb, miniscaf, node-10






TTGTTCTCGATGAGTGCGGTACTTGGTTTAA
20170608, cc6hb v4-250 nm 16 component square,
466


TACCCGTTCTT
default 6hb, miniscaf, node-11






GGCGCGGTAATGATTCCTACGATGAAAATA
20170608, cc6hb v4-250 nm 16 component square,
467


AAAACGGCTTGC
default 6hb, miniscaf, node-12






GAATACCGGATAAGCCTTCTATATCTGATTT
20170608, cc6hb v4-250 nm 16 component square,
468


GCTTGCTATTG
default 6hb, miniscaf, node-13






TCCCGCAAGTCGGGAGGTTCGCTAAAACGC
20170608, cc6hb v4-250 nm 16 component square,
469


CTCGCGTTCTTA
default 6hb, miniscaf, node-14






GGTGCAAAATAGCAACTAATCTTGATTTAA
20170608, cc6hb v4-250 nm 16 component square,
470


GGCTTCAAAACC
default 6hb, miniscaf, node-15






GCAAATTAGGCTCTGGAAAGACGCTCGTTA
20170608, cc6hb v4-250 nm 16 component square,
471


GCGTTGGTAAGATTCAGGATAAAATTGTAG
default 6hb, miniscaf, node-16



CTG







ATTCTCAATTAGCGTGGACCGTTGAGCGTTG
cc6hbv3_miniscaf_10s_n4
472


GCTTTATACTG







TGCCTCTGCCTGAACCACCATTTGGCGTTGT
cc6hbv3_miniscaf_10s_n5
473


TAAATATGGCG







CTTTTGTCGGTTCGGGCTATTCTCTTATTACT
cc6hbv3_miniscaf_10s_n6
474


GGCTCGAAAA







AACATGTTGTTAGTGGACTCTGTCTGGACAG
cc6hbv3_miniscaf_10s_n7
475


AATTACTTTAC







ACTTATCTATTACGGTTTTTCAGGCGCGTTC
cc6hbv3_miniscaf_10s_n8
476


TGCATTAGCTG







TAATACCATAAATCAAAAATCAGTTGATAA
cc6hbv3_yellow_term_10s_n4
477


GA







AGGCTACGAGGCATAGTAAGAGCAAACAGT
cc6hbv3_yellow_term_10s_n5
478


TC







ACCGTGAGATGGTTTAATTTCAAAGATACAT
cc6hbv3_yellow_term_10s_n6
479


A







AACACGGTCAATCATAAGGGAACCAGAACG
cc6hbv3_yellow_term_10s_n7
480


AG







AATATCCATTAAACGGGTAAAATACTTAGC
cc6hbv3_yellow_term_10s_n8
481


CG
















TABLE 3







Exemplary 440 nm Six-helix Bundle











SEQ




ID


Sequence
Comment
NO:





AACGGCATCTCCGTGAGCCTCCTCACAGAG
6hb_440nm, start 0[76], end 5[62]
482


CCTGGGGTGCCT







GGCAGCACCCATCCCTTACACTGGTGTGGTT
6hb_440nm, start 0[118], end 5[104]
483


GCGCTCACTGC







AAATCCCGTGGTCTGGTCAGCAGCAACCCC
6hb_440nm, start 0[160], end 5[146]
484


AGCTGCATTAAT







GAGCCGCCAAGCAGTTGGGCGGTTGTGTTTT
6hb_440nm, start 0[202], end 5[188]
485


GCGTATTGGGC







GGCACCGCTAAAACGACGGCCAGTGCCAAG
6hb_440nm, start 0[244], end 5[230]
486


ACGGGCAACAGC







CGCGTCTGGGCCTCAGGAAGATCGCACTAG
6hb_440nm, start 0[286], end 5[272]
487


AGTTGCAGCAAG







GGAGCAAACTTTTAACCAATAGGAACGCGA
6hb_440nm, start 0[328], end 5[314]
488


AAATCCTGTTTG







GCAAGGATATACAAAGGCTATCAGGTCATT
6hb_440nm, start 0[370], end 5[356]
489


ATAAATCAAAAG







CTGTTTAGCTAATACTTTTGCGGGAGAATCC
6hb_440nm, start 0[412], end 5[398]
490


AGTTTGGAACA







TACCTTTAAACCATTAGATACATTTCGCCAA
6hb_440nm, start 0[454], end 5[440]
491


CGTCAAAGGGC







ATCCCCCTCGAAGCAAACTCCAACAGCAC
6hb_440nm, start 0[496], end 5[482]
492


TACGTGAACCAT







ACCACATTCCAATACTGCGGAATCGTCAGT
6hb_440nm, start 0[538], end 5[524]
493


GCCGTAAAGCAC







TGCCCTGACGGTAGAAAGATTCATCAGTATT
6hb_440nm, start 0[580], end 5[566]
494


TAGAGCTTGAC







CGCGACCTGCGTAACAAAGCTGCTCATTGG
6hb_440nm, start 0[622], end 5[608]
495


AAGGGAAGAAAG







ACAGAGGCTTTGTATCATCGCCTGATAAAA
6hb_440nm, start 0[664], end 5[650]
496


GTGTAGCGGTCA







CCTTTAATTAAAGACAGCATCGGAACGAGC
6hb_440nm, start 0[706], end 5[692]
497


TTAATGCGCCGC







TAGCATTCCTGAAAATCTCCAAAAAAAACG
6hb_440nm, start 0[748], end 5[734]
498


AGCACGTATAAC







GATAAGTGCGAGTTTCGTCACCAGTACAAG
6hb_440nm, start 0[790], end 5[776]
499


CTAAACAGGAGG







AGTAAGCGTTAGGATTAGCGGGGTTTTGGT
6hb_440nm, start 0[832], end 5[818]
500


ACGCCAGAATCC







CACCGGAACAATGGAAAGCGCAGTCTCTCA
6hb_440nm, start 0[874], end 5[860]
501


CCGAGTAAAAGA







CAGCAAAATTTTCATAATCAAAATCACCTA
6hb_440nm, start 0[916], end 5[902]
502


GCAATACTTCTT







TAAAGGTGGCGTCACCGACTTGAGCCATTA
6hb_440nm, start 0[958], end 5[944]
503


GAAGAACTCAAA







ATTGAGTTAGCAGTATGTTAGCAAACGTCA
6hb_440nm, start 0[1000], end 5[986]
504


ATATTACCGCCA







ATTTGCCAGTGAGCGCTAATATCAGAGAAA
6hb_440nm, start 0[1042], end 5[1028]
505


ATACCTACATTT







TTTCATCGTTACCAACGCTAACGAGCGTTTA
6hb_440nm, start 0[1084], end 5[1070]
506


CATTGGCAGAT







CCAGACGACCGCACTCATCGAGAACAAGGG
6hb_440nm, start 0[1126], end 5[1112]
507


ACATTCTGGCCA







AATAAACACATAAAGTACCGACAAAAGGGC
6hb_440nm, start 0[1168], end 5[1154]
508


GTAAGAATACGT







ATTTATCAAACCGACCGTGTGATAAATATA
6hb_440nm, start 0[1210], end 5[1196]
509


GTCTTTAATGCG







AGATGATGATTAGATTAAGACGCTGAGATA
6hb_440nm, start 0[1252], end 5[1238]
510


AAAATACCGAAC







AGGGTTAGACGAATTATTCATTTCAATTTGA
6hb_440nm, start 0[1294], end 5[1280]
511


GGCGGTCAGTA







AGAAGTATTCTGATTGTTTGGATTATACGAG
6hb_440nm, start 0[1336], end 5[1322]
512


AGCCAGCAGCA







TCATGGTCATAGCCGTGCCTGTTCTTCGCGA
6hb_440nm, start 1[38], end 1[79]
513


GATGCCGGGTT







ACCTGCAGCCAGCTCTTTGCTCGTCATAAAG
6hb_440nm, start 1[80], end 1[121]
514


TCGGTGGTGCC







ATCCCACGCAACCAACGTCAGCGTGGTGCT
6hb_440nm, start 1[122], end 1[163]
515


AAAAAAAGCCGC







ACAGGCGGCCTTTTCTGCTCATTTGCCGCCC
6hb_440nm, start 1[164], end 1[205]
516


GGGAACGGATA







ACCTCACCGGAAACCCAGTCACGACGTTGT
6hb_440nm, start 1[206], end 1[247]
517


TCTGGTGCCGGA







AACCAGGCAAAGCGGACGACGACAGTATCG
6hb_440nm, start 1[248], end 1[289]
518


CCTTCCTGTAGC







CAGCTTTCATCAATGTTAAATCAGCTCATTA
6hb_440nm, start 1[290], end 1[331]
519


AGAGAATCGAT







GAACGGTAATCGTGCTATTTTTGAGAGATCA
6hb_440nm, start 1[332], end 1[373]
520


AAATTTTAGA







ACCCTCATATATTAAAACATTATGACCCTGT
6hb_440nm, start 1[374], end 1[415]
521


ATATTTTCATT







TGGGGCGCGAGCTCGAGTAGATTTAGTTTGT
6hb_440nm, start 1[416], end 1[457]
522


TGCTCCTTTTG







ATAAGAGGTCATTTCAAAGCGAACCAGACC
6hb_440nm, start 1[458], end 1[499]
523


AAATGCTTTAAA







CAGTTCAGAAAACTTAGACTGGATAGCGTC
6hb_440nm, start 1[500], end 1[541]
524


AACTAATGCAGA







TACATAACGCCAAGGAACAACATTATTACA
6hb_440nm, start 1[542], end 1[583]
525


GAGAAACACCAG







AACGAGTAGTAAATTCATTACCCAAATCAA
6hb_440nm, start 1[584], end 1[625]
526


CTCCATGTTACT







TAGCCGGAACGAGCAAAGTACAACGGAGAT
6hb_440am, start 1[626], end 1[667]
527


TTGAGGACTAAA







GACTTTTTCATGACGTCACCCTCAGCAGCGG
6hb_440nm, start 1[668], end 1[709]
528


TATCGGTTTAT







CAGCTTGCTTTCGTAATAATTTTTTCACGTA
6hb_440nm, start 1[710], end 1[751]
529


CAGACAGCCCT







CATAGTTAGCGTACCATGTACCGTAACACTC
6hb_440nm, start 1[752], end 1[793]
530


GTCGAGAGGGT







TGATATAAGTATACTCCTCAAGAGAAGGAT
6hb_440nm, start 1[794], end 1[835]
531


CATACATGGCTT







TTGATGATACAGGATCCTCATTAAAGCCAG
6hb_440nm, start 1[836], end 1[877]
532


CGCCTCCCTCAG







AGCCGCCACCCTCATTAGCGTTTGCCATCTC
6hb_440nm, start 1[878], end 1[919]
533


ACCAGTAGCAC







CATTACCATTAGCTAAAGGTGAATTATCACC
6hb_440nm, start 1[920], end 1[961]
534


AACATATAAAA







GAAACGCAAAGACTTAAGACTCCTTATTAC
6hb_440nm, start 1[962], end 1[1003]
535


AGCCCAATAATA







AGAGCAAGAAACACAAAGTCAGAGGGTAA
6hb_440nm, start 1[1004], end 1[1045]
536


TTTACAAAATAAA







CAGCCATATTATTAATTTTATCCTGAATCTA
6hb_440nm, start 1[1046], end 1[1087]
537


GGAATCATTAC







CGCGCCCAATAGCGGTATTAAACCAAGTAC
6hb_440nm, start 1[1088], end 1[1129]
538


GACAATAAACAA










CATGTTCAGCTAAGCCAGTAATAAGAGAAT
6hb_440nm, start 1[1130], end 1[1171]
539


CGGAATCATAAT







TACTAGAAAAAGCATTTAATGGTTTGAAAT
6hb_440nm, start 1[1172], end 1[1213]
540


AATCATAGGTCT







GAGAGACTACCTTGAAAACATAGCGATAGC
6hb_440nm, start 1[1214], end 1[1255]
541


AACAAACATCAA







GAAAACAAAATTAACAAAATCGCGCAGAG
6hb_440nm, start 1[1256], end 1[1297]
542


GACCTACCATATC







AAAATTATTTGCAAATTCATCAATATAATCA
6hb_440nm, start 1[1298], end 1[1343]
543


GACTTTACAAACAAT







TCGACAACTCTAACAACTAATCGTCAATAG
6hb_440nm, start 1[1344], end 5[1364]
544


ATAATGAACCTCAAATATC







TCTGCCAGCACGTGTTTCCTGTGTGCCGCTC
6hb_440nm, start 2[62], end 3[45]
545


AC







TGGGTAAAGGTTGGTGCCGGTGCCCCCTGC
6hb_440nm, start 2[104], end 2[63]
546


ATACCGGCGGTT







CCGGACTTGTAGAGCTTACGGCTGGAGGTG
6hb_440nm, start 2[146], end 2[105]
547


TGCGGCTCGTAA







CAAACTTAAATTAGTGATGAAGGGTAAAGT
6hb_440nm, start 2[188], end 2[147]
548


TAACGGAACGTG







CGCCAGGGTTTTCAATCGGCGAAACGTACA
6hb_440nm, start 2[230], end 2[189]
549


GAAACAGCGGAT







GCCAGTTTGAGGGCCATTCGCCATTCAGGCT
6hb_440nm, start 2[272], end 2[231]
550


AAGTTGGGTAA







GCATTAAATTTTCATTAAATGTGAGCGAGTA
6hb_440mn, start 2[314], end 2[273]
551


ACCGTGCATCT







CCGGAGAGGGTAAAAACTAGCATGTCAATC
6hb_440nm, start 2[356], end 2[315]
552


TTGTTAAAATTC







TCGGTTGTACCATTAAATGCAATGCCTGAGG
6hb_440nm, start 2[398], end 2[357]
553


ATAAATTAATG







CAATTCTGCGAAGAAAAGGTGGCATCAATT
6hb_440nm, start 2[440], end 2[399]
554


CATAAAGCTAAA







TTAATTCGAGCTTTTGCGGATGGCTTAGAGA
6hb_440nm, start 2[482], end 2[441]
555


CAGTTGATTCC







ATAGTAAAATGTGAGAATGACCATAAATCA
6hb_440nm, start 2[524], end 2[483]
556


AAATATCGCGTT







AAACGAACTAACAAGGAATTACGAGGCATA
6hb_440nm, start 2[566], end 2[525]
557


CCAGAGGGGGTA







AAGAACCGGATATTGGGCTTGAGATGGTTT
6hb_440nm, start 2[608], end 2[567]
558


TCTACGTTAATA







CCAAGCGCGAAAGCGCAGACGGTCAATCAT
6hb_440nm, start 2[650], end 2[609]
559


AGTAATCTTGAC







CTTTTGCGGGATGGAAGTTTCCATTAAACGC
6hb_440nm, start 2[692], end 2[651]
560


CAGCGATTATA







AGGAATTGCGAAAGGTGAATTTCTTAAACA
6hb_440nm, start 2[734], end 2[693]
561


AGTTAAAGGCCG







CCCAATAGGAACACGATCTAAAGTTTTGTC
6hb_440nm, start 2[776], end 2[735]
562


AGGAACAACTAA







AAGAGGCTGAGAGCCCGGAATAGGTGTATC
6hb_440nm, start 2[818], end 2[777]
563


AGGGATAGCAAG







ACAAACAAATAAAGTGTACTGGTAATAAGT
6hb_440nm, start 2[860], end 2[819]
564


CATGAAAGTATT







CATAGCCCCCTTAGAACCGCCACCCTCAGA
6hb_440nm, start 2[902], end 2[861)
565


GCCTTGATATTC







GAAATTATTCATAAGGCCGGAAACGTCACC
6hb_440nm, start 2[944], end 2[903]
566


GGCATTTTCGGT







GAACTGGCATGAACCACGGAATAAGTTTAT
6hb_440nm, start 2[986], end 2[945)
567


TAAATATTGACG







GAACACCCTGAAATGAAATAGCAATAGCTA
6hb_440nm, start 2[1028], end 2[987]
568


GAATACCCAAAA







GCACCCAGCTACTATCCCAATCCAAATAAG
6hb_440nm, start 2[1070], end 2[1029]
569


GGAGAATTAACT







ATTCCAAGAACGAAGCAAATCAGATATAGA
6hb_440nm, start 2[1112], end 2[1071]
570


AGTTGCTATTTT







AGGCATTTTCGATGCAGAACGCGCCTGTTTT
6hb_440nm, start 2[1154], end 2[1131]
571


CTTTCCTTATC







CTTCTGACCTAACTGTTTAGTATCATATGCT
6hb_440nm, start 2[1196], end 2[1155]
572


AATTTAGGCAG







CTTAGAATCCTTTTTAACCTCCGGCTTAGGA
6hb_440nm, start 2[1238], end 2[1197]
573


GTTAATTTCAT







GAATACCAAGTTATTACATTTAACAATTTCA
6hb_440nm, start 2[1280], end 2[1239]
574


ATTAATTTTCC







TCAGATGATGGCCGTAAAACAGAAATAAAG
6hb_440nm, start 2[1322], end 2[1281]
575


CCTGATTGCTTT







TCTTTAGGAGCACGTATTAAATCCTTTGCCT
6hb_440nm, start 2[1364], end 2[1323]
576


ATTCCTGATTA







AATTCCACACAAGGGCCGTTTTCACGGTCAT
6hb_440nm, start 3[46], end 3[87]
577


CAGACGATCCA







GCGCAGTGTCACCCGGGTCACTGTTGCCCTC
6hb_440nm, start 3[88], end 3[129]
578


CAGCATGAGCG







GGGTCATTGCAGGCCAGAGCACATCCTCAT
6hb_440nm, start 3[130], end 3[171]
579


AAACGATGCTGA







TTGCCGTTCCGGACGGAAAAAGAGACGCAG
6hb_440nm, start 3[172], end 3[213]
580


CGCCATGTTTAC







CAGTCCCGGAATATGTGCTGCAAGGCGATT
6hb_440nm, start 3[214], end 3[255]
581


GCGCAACTGTTG







GGAAGGGCGATCGTAGATGGGCGCATCGTA
6hb_440nm, start 3[256], end 3[297]
582


ACAACCCGTCGG







ATTCTCCGTGGGTTGTAAACGTTAATATTAT
6hb_440nm, start 3[298], end 3[339]
583


ATGTACCCCGG







TTGATAATCAGAATTCAACCGTTCTAGCTTA
6hb_440nm, start 3[340], end 3[381]
584


ATGTGTAGGTA







AAGATTCAAAAGGCAATAAAGCCTCAGAGC
6hb_440nm, start 3[382], end 3[423]
585


TACTAATAGTAG







TAGCATTAACATAAGTTTCATTCCATATACT
6hb_440nm, start 3[424], end 3[465]
586


TAATTGCTGAA







TATAATGCTGTAGAAGCCCGAAAGACTTCA
6hb_440um, start 3[466], end 3[507]
587


AAATCAGGTCTT







TACCCTGACTATTTGCAAAAGAAGTTTTGGT
6hb_440nm, start 3[508], end 3[549]
588


AAGAGCAACAC







TATCATAACCCTGACGTTGGGAAGAAAAAA
6hb_440nm, start 3[550], end 3[591]
589


ATTTCAACTTTA







ATCATTGTGAATGGCTGACCTTCATCAAGAA
6hb_440nm, start 3[592], end 3[633]
590


GGGAACCGAAC







TGACCAACTTTGACACTCATCTTTGACCCGG
6hb_440nm, start 3[634], end 3[675]
591


TAAAATACGTA







ATGCCACTACGACGCTGAGGCTTGCAGGGG
6hb_440nm, start 3[676], end 3[717]
592


CTTGATACCGAT







AGTTGCGCCGACGCGGAGTGAGAATAGAAG
6hb_440nm, start 3[718], end 3[759]
593


TCTTTCCAGACG







TTAGTAAATGAACCACCACCCTCATTTTCAC
6hb_440nm, start 3[760], end 3[801]
594


CGTACTCAGGA







GGTTTAGTACCGAACCTATTATTCTGAAATT
6hb_440nm, start 3[802], end 3[843]
595


TAACGGGGTCA







GTGCCTTGAGTAGGCAGGTCAGACGATTGG
6hb_440nm, start 3[844], end 3[885]
596


CCACCACCCTCA







GAGCCGCCACCATGTAGCGCGTTTTCATCA
6hb_440nm, start 3[886], end 3[927)
597


ATGAAACCATCG







ATAGCAGCACCGGATTGAGGGAGGGAAGGT
6hb_440nm, start 3[928], end 3[969]
598


TTGTCACAATCA







ATAGAAAATTCAGAAACGCAATAATAACGT
6hb_440nm, start 3[970], end 3[1011]
599


CTTACCGAAGCC







CTTTTTAAGAAAGGGAAGCGCATTAGACGA
6hb_440nm, start 3[1012], end 31053]
600


AACGATTTTTTG







TTTAACGTCAAAAGCCTTAAATCAAGATTA
6hb_440nm, start 3[1054], end 3[1095]
601


GGCTTATCCGGT







ATTCTAAGAACGCAATCAATAATCGGCTGA
6hb_440nm, start 3[1096], end 3[1137]
602


TCAACAATAGAT







AAGTCCTGAACATTAACAACGCCAACATGG
6hb_440nm, start 3[1138], end 3[1179]
603


TTATACAAATTC







TTACCAGTATAATTTTTCAAATATATTTTTTG
6hb_440nm, start 3[1180], end 3[1221]
604


GGTTATATAA







CTATATGTAAATTGTAAATCGTCGCTATTAT
6hb_440nm, start 3[1222], end 3[1263]
605


TTGAATTACCT







TTTTAATGGAAAAACAATAACGGATTCGA
6hb_440nm, start 3[1264], end 3[1305]
606


AATTGCGTAGAT







TTTCAGGTTTAAGAGCGGAATTATCATCACG
6hb_440nm, start 3[1306], end 3[1347]
607


AACGTTATTAA







TTTTAAAAGTTTAAAGGAATTGAGTAAAAT
6hb_440nm, start 3[1348], end 2[1365]
608


A







CGGAAGCATAAAGTGTAATTGAGGATCCCC
6hb_440nm, start 4[48], end 0[35]
609


GG







GTGCACTCTGTGGTCTCACATTAATTGCTTC
6hb_440nm, start 4[90], end 0[77]
610


AGCAAATCGTT







CACTCAATCCGCCGGGAAACCTGTCGTGGC
6hb_440nm, start 4[132], end 0[119]
611


AAGAATGCCAAC







TCCGTTTTTTCGTCGCGGGGAGAGGCGGAC
6hb_440nm, start 4[174], end 0[161]
612


ATCGACATAAAA







ATAGACTTTCTCCGTCTTTTCACCAGTGAGC
6hb_440nm, start 4[216], end 0[203]
613


TTTCAGAGGTG







CTCTTCGCTATTACCGCCTGGCCCTGAGCCA
6hb_440nm, start 4[258], end 0[245]
614


GCCAGCTTTCC







CGGATTGACCGTAATTGCCCCAGCAGGCCA
6hb_440nm, start 4[300], end 0[287]
615


TCAAAAATAATT







AAAACAGGAAGATTATCGGCAAAATCCCTT
6hb_440nm, start 4[342], end 0[329]
616


GCCTGAGAGTCT







GGCCGGAGACAGTCGGGTTGAGTGTTGTGC
6hb_440nm, start 4[384], end 0[371]
617


CTTTATTTCAAC







CATACAGGCAAGGCAAGAACGTGGACTCAA
6hb_440nm, start 4[426], end 0[413]
618


ATGGTCAATAAC







GTTTTAAATATGCACAGGGCGATGGCCCTC
6hb_440nm, start 4[468], end 0[455]
619


AGGATTAGAGAG







AAGCAAAGCGGATTTTTTTGGGGTCGAGTA
6hb_440nm, start 4[510], end 0[497]
620


AATATTCATTGA







GACGACGATAAAAAAAAGGGAGCCCCCGT
6hb_440nm, start 4[552], end 0[539]
621


GAGATTTAGGAAT







CGATTTTAAGAACTAACGTGGCGAGAAACA
6hb_440nm, start 4[594], end 0[581]
622


GTGAATAAGGCT







AGATGAACGGTGTAGCTAGGGCGCTGGCAT
6hb_440nm, start 4[636], end 0[623]
623


TGTGTCGAAATC







CCTAAAACGAAAGAACCACACCCGCCGCGG
6hb_440nm, start 4[678], end 0[665]
624


GTAGCAACGGCT







AACCATCGCCCACGTATGGTTGCTTTGAGGC
6hb_440nm, start 4[720], end 0[707]
625


TCCAAAAGGAG







TGGGATTTTGCTAAAGAATCAGAGCGGGAA
6hb_440nm, start 4[762], end 0[749]
626


CTACAACGCCTG







GAACCGCCACCCTCTTTAGACAGGAACGCT
6hb_440nm, start 4[804], end 0[791]
627


CAGTACCAGGCG







GTATAAACAGTTAAATAATCAGTGAGGCGA
6hb_440nm, start 4[846], end 0[833]
628


ATTTACCGTTCC







CCAGAGCCGCCGCCCAAATTAACCGTTGGG
6hb_440nm, start 4[888], end 0[875]
629


AACCAGAGCCAC







GCGACAGAATCAAGATCACTTGCCTGAGTT
6hb_440nm, start 4[930], end 0[917]
630


GGGAATTAGAGC







CCAGCGCCAAAGACGGTAATATCCAGAAAG
6hb_440nm, start 4[972], end 0[959]
631


AAAATACATACA







ATAGCCGAACAAAGAAAAACGCTCATGGGA
6hb_440nm, start 4[1014], end 0[1001]
632


TAACCCACAAGA







AGCAGCCTTTACAGCTGAAATGGATTATCTT
6hb_440nm, start 4[1056], end 0[1043]
633


TCCAGAGCCTA







TTAGCGAACCTCCCACCAGTAATAAAAGCA
6hb_440nm, start 4[1098], end 0[1085]
634


AGCCGTTTTTAT







ATATCCCATCCTAACTTCTGACCTGAAATAA
6hb_440nm, start 4[1140], end 0[1127]
635


AGTAATTCTGT







TCAACAGTAGGGCTTTTTGAATGGCTATAGG
6hb_440nm, start 4[1182], end 0[1169]
636


CGTTAAATAAG







AATCCAATCGCAAGTAAAACATCGCCATAG
6hb_440nm, start 4[1224], end 0[1211]
637


AGTCAATAGTGA







AAATCAATATATGTAGATAAAACAGAGGAC
6hb_440nm, start 4[1266], end 0[1253]
638


CTGAGCAAAAGA







AATATACAGTAACAAACAGTGCCACGCTTT
6hb_440nm, start 4[1308], end 0[1295]
639


CTGAATAATGGA







TATCATTTTGCGGAAGCATCACCTTGCTACA
6hb_440nm, start 4[1350], end 0[1337]
640


TTTGAGGATTT







AATGAGTGAGCTAAGCTGCGGCCAGAATGC
6hb_440nm, start 5[63], end 4[49]
641


GGCCATACGAGG







CCGCTTTCCAGTCGGGCGCGGTTGCGGTATG
6hb_440nm, start 5[105], end 4[91]
642


AGTGCCCGCCT







GAATCGGCCAACGCTCGTCGCTGGCAGCCT
6hb_440nm, start 5[147], end 4[133]
643


CCGGCGCTTTCG







GCCAGGGTGGTTTTTGGTGAAGGGATAGCT
6hb_440nm, start 5[189], end 4[175]
644


CTCCAAACGCGG







TGATTGCCCTTCACGCCAGCTGGCGAAAGG
6hb_440nm, start 5[231], end 4[217]
645


GGGTTGTGAGAG







CGGTCCACGCTGGTTGGGATAGGTCACGTT
6hb_440nm, start 5[273], end 4[259]
646


GGTGGTGCGGGC







ATGGTGGTTCCGAAGTATAAGCAAATATTT
6hb_440nm, start 5[315], end 4[301]
647


AAAAACAAACGG







AATAGCCCGAGATAAAATCACCATCAATAT
6hb_440nm, start 5[357], end 4[343]
648


GATAAAGCCCCA







AGAGTCCACTATTAAAAGAATTAGCAAAAT
6hb_440nm, start 5[399], end 4[385]
649


TAAGGTGAGAAA







GAAAAACCGTCTATACTAAAGTACGGTGTC
6hb_440nm, start 5[441], end 4[427]
650


TGGCCAATAAAT







CACCCAAATCAAGTGCATCAAAAAGATTAA
6hb_440nm, start 5[483], end 4[469]
651


GAGGCTCAACAT







TAAATCGGAACCCTCCAAAATAGCGAGAGG
6hb_440nm, start 5[525], end 4[511]
652


CTTTATAGTCAG







GGGGAAAGCCGGCGGGCTCATTATACCAGT
6hb_440nm, start 5[567], end 4[553]
653


CAGCGTTTACCA







CGAAAGGAGCGGGCCAGACCAGGCGCATA
6hb_440nm, start 5[609], end 4[595]
654


GGCTTACCTTATG







CGCTGCGCGTAACCGGCAAAAGAATACACT
6hb_440nm, start 5[651], end 4[637]
655


AAAAAAGAGGAC







TACAGGGCGCGTACCATAACCGATATATTC
6hb_440nm, start 5[693], end 4[679]
656


GGTAGGCACCAA







GTGCTTTCCTCGTTACAACTTTCAACAGTTT
6hb_440nm, start 5[735], end 4[721]
657


CAAATGACAAC







CCGATTAAAGGGATAGAACCGCCACCCTCA
6hb_440nm, start 5[777], end 4[763]
658


GAGTTTTCTGTA







TGAGAAGTGTTTTTGCCCCCTGCCTATTTC
6hb_440nm, start 5[819], end 4[805]
659


GGCCACCCTCA







GTCTGTCCATCACGAGCATTGACAGGAGGT
6hb_440nm, start 5[861], end 4[847]
660


TGAACAGTGCCC







TGATTAGTAATAACTTTGCCTTTAGCGTCAG
6hb_440nm, start 5[903], end 4[889]
661


ACGAACCACCA







CTATCGGCCTTGCTAAAAGGGCGACATTCA
6hb_440nm, start 5[945], end 4[931]
662


ACCTAATCAGTA







GCCATTGCAACAGGTTACCAGAAGGAAACC
6hb_440nm, start 5[987], end 4[973]
663


GAGTATGGTTTA







TGACGCTCAATCGTAGAGAATAACATAAAA
6hb_440nm, start 5[1029], end 4[1015]
664


ACAAGTAAGCAG







TCACCAGTCACACGGACTTGCGGGAGGTTT
6hb_440nm, start 5[1071], end 4[1057]
665


TGAAATGAAAAT







ACAGAGATAGAACCTTTACGAGCATGTAGA
6hb_440nm, start 5[1113], end 4[1099]
666


AACCGAGGCGTT







GGCACAGACAATATTAATTGAGAATCGCCA
6hb_440nm, start 5[1155], end 4[1141]
667


TATAGAAAAATA







CGAACTGATAGCCCACAAAGAACGCGAGAA
6hb_440nm, start 5[1197], end 4[1183]
668


AACAGCCAACGC







GAACCACCAGCAGAGAGTGAATAACCTTGC
6hb_440nm, start 5[1239], end 4[1225)
669


TTCGCTGATGCA







TTAACACCGCCTGCGTACCTTTTACATCGGG
6hb_440nm, start 5[1281], end 4[1267]
670


AGACAGTACAT







AATGAAAAATCTAAACAAAGAAACCACCAG
6hb_440nm, start 5[1323], end 4[1309]
671


AAGCGTCAGATG







AAACCCTCAATCAAGTTGGCAAATCAACAG
6hb_440nm, start 5[1365], end 4[1351]
672


TTGGAGTAACAT







CGCTGGTTGGGATAGGTCACGTTGGTGGTG
6hb_440nm, 7 bp socket end distal to
673


CGGGC
queen, start 5[280], end 4[259)






TTCCGAAGTATAAGCAAATATTTAAAAACA
6hb_440nm, 7 bp socket end distal to
674


AACGG
queen, start 5[322], end 41301)






CGAGATAAAATCACCATCAATATGATAAAG
6hb_440nm, 7 bp socket end distal to
675


CCCCA
queen, start 5[364], end 4[343]






ACTATTAAAAGAATTAGCAAAATTAAGGTG
6hb_440nm, 7 bp socket end distal to
676


AGAAA
queen, start 5[406], end 4[385]






CGTCTATACTAAAGTACGGTGTCTGGCCAAT
6hb_440nm, 7 bp socket end distal to
677


AAAT
queen, start 5[448],end4[427]






TGGTTGGGATAGGTCACGTTGGTGGTGCGG
6hb_440nm, 10 bp socket end distal to
678


GC
queen, start 5[283], end 4[259]






CGAAGTATAAGCAAATATTTAAAAACAAAC
6hb_440nm, 10 bp socket end distal to
679


GG
queen, start 5[325],end4[301]






GATAAAATCACCATCAATATGATAAAGCCC
6hb_440nm, 10 bp socket end distal to
680


CA
queen, start 5[367], end 4[343]






ATTAAAAGAATTAGCAAAATTAAGGTGAGA
6hb_440nm, 10 bp socket end distal to
681


AA
queen, start 5[409], end 4[385]






CTATACTAAAGTACGGTGTCTGGCCAATAA
6hb_440nm, 10 bp socket end distal to
682


AT
queen, start 5[451],end 4[427]









All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.


The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.” It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.


In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of” and “consisting essentially of” shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

Claims
  • 1. A crisscross nucleic acid nanostructure, comprising: (a) a first plurality of deoxyribonucleic acid (DNA) nanorods aligned parallel to each other; and(b) a second plurality of DNA nanorods aligned parallel to each other,wherein the DNA nanorods of the first plurality are bound to and perpendicular to the DNA nanorods of the second plurality, a single DNA nanorod of (b) binds to multiple DNA nanorods of (a), each through a single cooperative binding site, and single DNA nanorod of (a) binds to multiple DNA nanorods of (b), each through a single cooperative binding site.
  • 2. The crisscross nucleic acid nanostructure of claim 1, wherein the DNA nanorods of the first plurality have a length of 10-500 nm and/or the DNA nanorods of the second plurality have a length of 10-500 nm.
  • 3. The crisscross nucleic acid nanostructure of claim 1, wherein the DNA nanorods of the first plurality comprise a 6-helix DNA bundle and/or the DNA nanorods of the second plurality comprise a 6-helix DNA bundle.
  • 4. The crisscross nucleic acid nanostructure of claim 1, wherein the first plurality comprises at least 4 DNA nanorods and/or the second plurality comprises at least 4 DNA nanorods.
  • 5. The crisscross nucleic acid nanostructure of claim 1, wherein the first plurality comprises at least 10 DNA nanorods and/or the second plurality comprises at least 10 DNA nanorods.
  • 6. The crisscross nucleic acid nanostructure of claim 1, wherein the first plurality comprises at least 25 DNA nanorods and/or the second plurality comprises at least 25 DNA nanorods.
  • 7. The crisscross nucleic acid nanostructure of claim 1, wherein the first plurality comprises at least 50 DNA nanorods and/or the second plurality comprises at least 50 DNA nanorods.
  • 8. The crisscross nucleic acid nanostructure of claim 1, wherein the cooperative binding site has a length of 5-50 nucleotides.
  • 9. The crisscross nucleic acid nanostructure of claim 1, wherein the nanostructure comprises 3-1000 cooperative binding sites.
  • 10. The crisscross nucleic acid nanostructure of claim 9, wherein the distance between each of the cooperative binding sites is 20-1000 angstroms.
  • 11. A crisscross nucleic acid slat, comprising: a first plurality of at least four DNA strands parallel to each other, each strand of the first plurality having a length of at least 21 nucleotides; anda second plurality of at least four DNA strands parallel to each, each strand of the second plurality having a length of at least 21 nucleotides, wherein the at least four DNA strands of the first plurality are bound to and perpendicular to the at least four DNA strands of the second plurality.
RELATED APPLICATION

This application is a continuation of U.S. application Ser. No. 16/322,787, filed Feb. 1, 2019, which is a national stage filing under 35 U.S.C. § 371 of international application number PCT/US2017/045013, filed Aug. 2, 2017, which claims the benefit under 35 U.S.C. § 119(e) of U.S. provisional Application No. 62/370,098, filed Aug. 2, 2016, each of which is incorporated by reference herein in its entirety.

FEDERALLY SPONSORED RESEARCH

This invention was made with government support under 1435964 awarded by the Office of Naval Research. The government has certain rights in the invention.

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Related Publications (1)
Number Date Country
20220403453 A1 Dec 2022 US
Provisional Applications (1)
Number Date Country
62370098 Aug 2016 US
Continuations (1)
Number Date Country
Parent 16322787 US
Child 17576550 US