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Tuning the Cavity Size and Chirality of Self-Assembling 3D DNA Crystals Chad R. Simmons, Fei Zhang, Tara MacCulloch, Nour Eddine Fahmi, Nicholas Stephanopoulos, Yan Liu, Nadrian C. Seeman, and Hao Yan J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b06485 • Publication Date (Web): 21 Jul 2017 Downloaded from http://pubs.acs.org on July 23, 2017

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Journal of the American Chemical Society

Tuning the Cavity Size and Chirality of Self-Assembling 3D DNA Crystals Chad R. Simmons,1,2 Fei Zhang,1,2 Tara MacCulloch,1,2 Noureddine Fahmi1,2 Nicholas Stephanopoulos,1,2 Yan Liu,1,2 Nadrian C. Seeman3 & Hao Yan1,2* 1

Biodesign Center for Molecular Design and Biomimetics & 2School of Molecular Sciences, Arizona State University, Tempe, Arizona 85287, USA 3

Department of Chemistry, New York University, New York, New York 10003, USA

KEYWORDS: DNA nanotechnology, self-assembled crystals, enantiomeric DNA, L-DNA, DNase resistant crystals ABSTRACT: The foundational goal of structural DNA nanotechnology—the field that uses oligonucleotides as a molecular building block for the programmable self-assembly of nanostructured systems—was to use DNA to construct three-dimensional (3D) lattices for solving macromolecular structures. The programmable nature of DNA makes it an ideal system for rationally constructing self-assembled crystals, and immobilizing guest molecules in a repeating 3D array through their specific stereospatial interactions with the scaffold. In this work, we have extended a previously described motif (4x5) by expanding the structure to a system that links four double helical layers; we use a central weaving oligonucleotide containing a sequence of four six base repeats (4x6) forming a matrix of layers that are organized and dictated by a series of Holliday junctions. In addition, we have assembled mirror image crystals (L-DNA) with the identical sequence that are completely resistant to nucleases. Bromine and selenium derivatives were obtained for the L- and D-DNA forms, respectively, allowing phase determination for both forms and solution of the resulting structures to 3.0 and 3.05 Å resolution. Both right- and left-handed forms crystallized in the trigonal space groups with mirror image three-fold helical screw axes P32 and P31 for each motif, respectively. The structures reveal a highlyorganized array of discrete and well-defined cavities that are suitable for hosting guest molecules, and allow us to dictate a priori the assembly of guest-DNA conjugates with a specified crystalline hand.

INTRODUCTION The original goal of structural DNA nanotechnology, as outlined by Seeman in 19821, was to use branched DNA junctions to construct designed DNA crystal lattices in two and three dimensions that can act as hosts for solving structures of macromolecular guest molecules. In the decades since this idea was proposed, significant advances have been made toward the rational design of macroscopic crystals for the specific purpose of organizing guest molecules such as proteins, peptides, and nanoparticles on the nanometer scale.2-8 Two key principles underlie the design of periodic self-assembled lattices: (1) the predictability of DNA secondary structure arising from Watson-Crick base pairing, allowing the preparation of both linear and branched assemblies, and (2) the ability to control programmably the association of multiple DNA molecules via stickyended cohesion.1,9-11 A fundamental motif in DNA nanotechnology is the immobile Holliday junction,12 adapted from the natural structure seen in homologous recombination but with unique asymmetric sequences to prevent branch migration.1 Crystal structures of Holliday junctions demonstrated that the two constituent duplexes adopt a roughly 60° angle with respect to one another in the presence of divalent cations.13 These stacked duplexes provide the context necessary to predict the self-assembly of 3D DNA crystals, and de-

termine the strict requirements for self-assembly in the third dimension. Because the structural details of these junctions have been studied extensively,14-17 it is now possible to construct self-assembling lattices dictated by the angles at those junctions, and the precise sticky end cohesion at either end of the DNA helices. Natural DNA containing D-sugars adopts a right-handed helical twist characterized as the B-form of DNA—in this paper, we will call this enantiomer D-DNA—however, the ability to synthesize DNA containing the L-enantiomer (L-DNA) in the laboratory provides the additional flexibility to design motifs that can assemble into the opposite chiral hand. L-DNA duplexes possess the same physical attributes as their natural counterparts, including the number of base pairs (bp) per turn, the diameter of the helix, rise per base pair, etc., with the exception of their left-handed helical twist, which results in a perfect mirror image of the corresponding D-DNA helix.18 This attribute should allow for control of the handedness of lattice packing in a self-assembled DNA crystal, thus potentially making it possible to control the lattice arrangements of guest molecules. In addition, L-DNA is resistant to digestion by nucleases, making it attractive for the design of stable crystals subject to degradation in environments compromised with DNases.19-23

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Figure 1. Topological schematic, cartoon representation, crystallization, and models of the mirror image 4x6 crystals. (a) Topological representation of the 4x6 crystal system. The motif contains three component strands that comprise the lattice. Self-assembly is mediated via 2-bp sticky-ended cohesion (shown with boxes). A “central” strand (orange) tethers each of four 21-bp duplexes by way of a sequence containing 4 repeats of a 6 base sequence (4x6). A single duplex in the ASU is shown with a gray box. Seleno- and bromo-dU positions for the D- and L-DNA, respectively, are underlined. Sequences are indicated with arrowheads for the 3’ end of each component strand. Additional details and description are described in Figure S1a and b. The orange color shown represents the D-DNA structure, however, the schematic for the L-DNA will be represented in blue throughout this report and is shown in Figure S1c and d. (b) Three-dimensional cartoon representation of the 4x6 structure. (c) Light microscope image of the 4x6 D-DNA crystals. (d) Light microscope image of the 4x6 L-DNA crystals. (e) Model of the central building “block” of the 4x6 D-DNA (orange) motif. (f) Model of the central building “block” of the 4x6 LDNA (blue) motif. (g) Stereoscopic view of the 21-bp D-DNA duplex (orange stick model) that comprises the unit cell. 2Fo-Fc map is shown contoured at σ=1.8. The model is in very good agreement with the electron density, whereby the major and minor groove, base stacking, phosphate backbone, and right-handedness of the double stranded duplex are readily observable in the map. Also shown are the neighboring helices (orange sticks) that comprise the “block” (f) 2Fo-Fc map at σ=1.8. All features are clearly observable as in (e), but with the enantiomeric mirror image in the L-DNA form and neighboring helices (blue). Scale bars: 100 µm.


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The first self-assembled DNA crystal, based on the tensegrity triangle motif, was reported in 2009 by Seeman and Mao.24 The structure revealed a periodic arrangement of cavities formed by a series of tensegrity triangles each containing three Holliday junctions with 7 base pairs between each junction, resulting in its three-fold symmetry; there were a total of 21 bp between each triangle, corresponding to two full helical turns. Recently, we determined the structure of a new crystal system inspired by the tensegrity triangle.25 Our motif was initially designed as a ‘tensegrity square,’ but this motif required the reduction of the number of inter-crossover bases from seven bases to five bases with a total of four sequence repeats instead of three (4x5). Since 4x5 bp < 3x7 bp (21 bp = 2x10.5 bp), the structure resulted in a slight under-winding relative to two full turns. Contrary to the expected ‘tensegrity square’ structure, this motif crystallized into a series of 21 base pair duplexes that were tethered together via a central strand containing a repeating sequence, yielding four attached duplex “blocks”. The crystals contained a densely-packed arrangement of continuous helical layers, with each block oriented with threefold symmetry relative to one another. However, the compact arrays lacked the uniform periodicity of void spaces desired in the initial design, so we set out to modify the crystal motif to yield a more uniform arrangement of cavities. In this work, we report a re-designed ‘tensegrity square’ system, which we call the 4x6 motif, that contains an additional base on each repeat in the central weaving strand (Figure 1a,b and S1a,b). We hypothesized that this modification would reduce the structural strain caused by a single base deficit in the central strand, which could have prevented the packing required for a strictly periodic array of cavities throughout the crystal. In addition, we sought to crystallize the left-handed enantiomer (L-DNA) of this motif in order to obtain the enantiomeric form of the naturally occurring righthanded motif (Figure S1c,d). Structures of both D-DNA and LDNA crystals were determined to 3.05 and 3.0 Å resolution, respectively. In addition to confirming the potential of rational structural design, the L-form crystals were resistant to degradation by DNase, allowing for future encapsulation and protection of guest molecules in an environment not strictly free of nucleases.

selenium and bromine derivatives (for D- and L-DNA, respectively). The motif consists of the 3 component strands self-assembled into designed crystalline arrays for each form, with a layered lattice mediated via a central strand that contains a sequence of 4 sequential repeats of 6 bases. Each structure possesses a central “block” (Figure 1c,d) of 4 two-turn duplexes linked together by the central strand, organized via a series of Holliday junctions, and arranged in a series of helical layers oriented 120° with respect to one another. Each block formed the crystalline lattice via two-base sticky end cohesion at the ends of each DNA duplex. The right- and left-handed structures crystallized in mirror image space groups P32 and P31, respectively, with the same average unit cell parameters of a = b = 69 Å, c= 56 Å, α = β= 90°, γ = 120° for each construct (Figure 1e,f). Complete data processing and refinement statistics are presented in Table 1. Each unit cell consists of 3 asymmetric units (ASU) with two 21 base pair duplexes per ASU. The basic structures were defined as a single two turn helix that was in excellent agreement with the 2Fo – Fc electron density, clearly revealing the D- and L-DNA conformers (Figure 2a,b). The major and minor grooves, phosphate backbone, and proper base-stacking at intervals of ~3.4 Å were readily observable in the maps, permitting model building and refinement of a B-DNA duplex in each crystalline hand. After refinement, the 21-bp double helices exhibited a ~30° angular “bend” which deviated from standard B-DNA, resulting in a zig-zag helix axis along each continuous layer in the lattices (Figure S3).

RESULTS AND DISCUSSION Overall Structures We crystallized (Figure 1c,d) and determined the structures of two self-assembling DNA crystal systems that are perfect mirror images of one another (Figure S2). Naturally-occurring DNA (D-DNA) adopts a right handed helical twist containing 10.5 base pairs per full turn, and can be synthesized using phosphoramidite coupling methodology.26 Its enantiomeric form (L-DNA) also can be synthesized in the laboratory using standard phosphoramidite coupling chemistry with the L-form of each nucleotide (nt), resulting in a left handed helix when pairing with its complementary strand, but otherwise displaying identical structural attributes as its D-DNA counterpart (Figure S2).17 Both the D- and L-DNA 4x6 motifs contain three strands, with lengths 24 nt (S1), 21 nt (S2), and 15 nt (S3), in a molar ratio of 1:4:4. All “native” and derivative crystals were crystallized as described in the Methods and Materials.

Figure 2. Surface rendering of the mirror image 4x6 building blocks. (a) Stereoscopic view of the central D-DNA building “block” structure that comprises the self-assembling lattice. The four tethered duplexes (semi-transparent orange surface rendering) that are mediated by the central weaving strand (purple) are shown. The right-handed pitch and four-arm junctions are clearly observable. (b) Mirror image structure (L-DNA) of (a) with the central weaving strand (red) tethering the semi-transparent left-handed DNA duplexes (blue).

The structures of both isoforms were determined to 3.05 Å and 3.0 Å resolution, each using an unbiased set of phases from



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4x6 D-DNA SADa

4x6 D-DNA Nativeb

4x6 L-DNA SADa

4x6 L-DNA Nativeb

Space group

P32

P32

P31

P31

Resolution (Å)

50 – 3.1 Å

50 - 3.05 Å

50 - 3.0 Å

50 - 3.0 Å

a, b, c (Å)

68.5 68.5 56.8

68.5 68.5 55.8

67.7 67.7 54.3

68.6 68.6 55.7

α, β, γ (°)

90 90 120

90 90 120

90 90 120

90 90 120

Rmerge

0.084(0.25)*

0.108(0.674)

0.186(0.721)

0.110(0.467)

I/σI

27.6(2.6)

23.9(1.9)

41.1(2.6)

39.0(2.6)

Completeness (%)

98.0(92.1)

89.25(57.7)

86.8(58.6)

86.1(53.7)

Redundancy

3.7(3.2)

7.6(6.7)

10.4(9.3)

10.7(9.5)

Data collection

Cell dimensions

Refinement Resolution (Å)

30 - 3.05 Å

50 - 3.0 Å

No. reflections

4710

4949

Rwork/Rfree

21.13/23.66

23.76/24.57

No. atoms

851

851

DNA

851

851

Bond lengths (Å)

0.07

.016

Bond angles (°)

0.923

1.332

R.m.s deviations

Table 1. Data collection and refinement statistics a

Data were collected at beamline 19-ID at APS

b

Data were collected at beamline 8.2.2 at ALS

*Highest resolution shell is shown in parenthesis.

and structure solution to be performed using the same procedure described above. The heavy atom positions are equivalent for each construct, and are indicated in Figure S1. Periodicity of the 4x6 cavities

The sticky ends were also clearly visualized in density, allowing for placement of the well-defined duplex. Self-assembly of each of four tethered duplexes was mediated by the 4x6 central weaving strand (Figure 2a,b), with each group of four helices serving as the fundamental building block for assembly of the lattice. Each four-arm junction was modeled and refined using the geometry and angular parameters described previously.27 Phase Determination Independent sets of crystallographic phases were determined via single-wavelength anomalous dispersion (SAD) using two different types of derivative crystals in order to determine the structure in an unbiased fashion. For the L-DNA, four bromo-dU bases were inserted into each two-turn duplex in the ASU (Figure 1a, S1a,c), and anomalous difference Fourier maps were used to determine the heavy atom substructure. Subsequent model building was carried out after determination of the bromine positions. Initial attempts to obtain adequate phasing from the 4x6 brominated DDNA derivatives were unsuccessful. As a result, seleno-dU phosphoramidites were synthesized in our laboratory with the heavy atom placed on the C5 thymine position using the procedure described by Huang and colleagues.28 The reaction protocol is outlined in Scheme 1 (SI). Quality crystals were obtained allowing phase determination

The resulting lattice from both the L- and D- crystals reveals a uniform, periodic array of discrete cavities (Figure 3a). Specifically, the array contains cavities that are roughly 3.4 x 2 x 5 nm in size, accounting for one helical turn, the width of a duplex, and with an opening that results from the layered helical arrays when viewed along the three-fold symmetry axis of roughly 5 nm across (Figure 3a,b). The estimated solvent content for these crystals was approximately 70% (compared to ~55% in the 4x5 and 6x5 crystals).25 Although the cell constants between the 4x6 and 4x5 or 6x5 structures are nearly identical, the resulting packing (P32 or P31) of the layered 4x6 lattice starkly contrasts with the packing previously observed with the 4x5 and 6x5 crystal systems, which resulted in P3221 symmetry. Analysis of the n-repeating five-base crystal structures clearly revealed a non-periodic array—with pairs of crystal layers packed with spacing between duplexes at intervals of 1 and 1.7 nm with and pores of only 2.5 nm along the three-fold axis (one half of those in the 4x6)—leading to a densely-packed array of highly constrained crystal cavities. We initially postulated that the significant exclusion of solvent in these arrays contributed to the quality of diffraction; however, even with significantly larger solvent channels, the 4x6 systems diffracted equally as well as the originally reported structure (3.1 Å) The densely packed


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Figure 3. Structural comparison of the crystal cavity periodicity between the 4x6 and 4x5 scaffolds. (a) Stereoview of the crystal packing of the 4x6 lattice. Yellow bars are indicated for measurements of cavity sizes of the scaffold. Distances were measured in PyMOL on each crystal structure to provide values that approximate the sizes of the cavities from edge to edge of neighboring helices. The 6 and 10 nm scale bars are used as references for distance. Regular periodicity of each cavity is evident. (b) 90-degree rotation with respect to (a) with a view down the three-fold symmetry axis with pores of ~5 nm in size. (c) Stereoview of the crystal packing of the 4x5 lattice24.Yellow bars are indicated for measurements of cavity sizes of the scaffold. The 6 and 10 nm scale bars are used for reference. The crystal structure revealed a non-periodic array of densely packed cavities in stark contrast with the 4x6 scaffold. (d) 90-degree rotation with respect to (c) with a view down the three-fold symmetry axis showing ~2.5 nm size pores. arrangement of the initial 4x5 design was not amenable to the eventual scaffolding of biomolecules, but the introduction of a single base between four-arm junctions in the 4x6 system allowed for an additional ~34° twist between junctions which altered the dihedral angle along the helix axis, releasing the structural “stress” initially observed in the 4x5 system, and thereby yielding a scaffold that contained well-defined cavities of identical sizes. Nuclease Resistance of the 4x6-L-DNA

Our long-term goal is to use the periodic array of cavities in each 4x6 system for the scaffolding and structural elucidation of guest molecules such as proteins. Another possible use for these crystal systems is as macromolecular sieves for scavenging targets in cellular systems, by attaching affinity groups such as peptides or aptamers to the scaffold strands. However, one obstacle towards use of DNA crystals for this potential application is the presence of nucleases in cell systems that can degrade D-DNA. To test the fidelity of crystals in solutions containing DNases, we prepared crystals of both conformers in order to compare their resistance to DNase, with the expectation that the L- enantiomer would provide a protective matrix from nuclease digestion. First, the DNase stock was tested for digestion activity on duplex DNA that did not contain sticky ends, but had the corresponding sequence from the crystallographic ASU to confirm enzymatic activity in artificial mother liquor (Figure S4). Each crystal type was then incubated in the presence and absence of DNase at 37°C, and monitored by optical microscopy over time to

probe crystal stability in the crystallization drop (Figure 4 and Figures S5-8). Both the L- and D- crystal forms alone (without addition of DNase) showed no apparent degradation of morphology as a result of the elevated temperature over the course of the experiment. Further, the addition of DNase to the mother liquor showed no immediate damage at the initial time. However, after incubation for 4 hours, we observed significant degradation of the D-DNA crystals, with near complete digestion after 6 hours. In contrast, the L-DNA crystals showed no apparent change in morphology throughout the entire time course, indicating their complete resistance to DNase (Figure 4 and Figures S4-6). A denaturing PAGE gel was also prepared at each time point shown in Figure 4 to further demonstrate the digestion of the D-DNA crystals by the nuclease (Figure S9). Even after two weeks of incubation at 37°C, all L-DNA crystals were completely intact, with no observable damage, clearly indicating that the L-form design could be well-suited as a nuclease resistant host for guests within its lattice.



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Figure 4. Crystal stability of both isoforms in the presence of nuclease. Time course of incubation of D-DNA crystals (top panels) and L-DNA crystals (bottom panels) with DNase I in a buffer containing 50 mM HEPES (pH 7.5) with 80 mM MgCl2 and 2.5 mM spermine over 24 hours to demonstrate crystal stability at 37°C. Light images of representative time points of 0, 4, 6, and 24 hours are shown. Degradation of the D-DNA crystals became apparent at 4 hours, with nearly complete dissolution at 6 hours, whereas the LDNA crystals suffered no visible damage throughout the experiment. Scale bar: 100 µm. CONCLUSION The enduring, if still evasive, goal of structural DNA nanotechnology is the precise organization of functional molecules within self-assembled DNA lattices for structural solution of biomolecules that have not yet been determined via conventional crystallization methods. The new structures described here provide a new route towards this goal, with discrete, periodic, and larger cavities well-suited for immobilization of proteins up to ~5 nm in size. In addition, the 4x6 crystals possess uninterrupted solvent channels down the threefold symmetry axis that provide access to these channels, hopefully facilitating guest diffusion throughout the scaffold. We posit that the release of structural strain that resulted in an irregular, densely packed lattice in the originally determined 4x5 structure was due to the inter-crossover distance, however, it is also possible that specified bases at each Holliday junction, and even throughout the motif, could be contributing factors to crystal packing. Efforts are underway to explore these additional possibilities. We also demonstrated the ability to engineer crystals rationally designed using L-DNA, which can serve as a nuclease-protective framework for encapsulated macromolecules. These lefthanded crystals could serve as dense, 3D scaffolds with potential to sieve and trap macromolecules from a cell lysate, binding targets using affinity groups appended to the crystals and releasing them after filtration. Future work will determine the feasibility of this strategy in vitro, followed by targeted delivery of functional or therapeutic proteins in vivo, especially in conjunction with surface functionalization with targeting ligands. The crystals could also immobilize several proteins to construct enzymatic cascades, helping build “molecular factories” within highly stable 3D frameworks. Although resistance to nucleases is an important feature of an L-DNA crystal scaffold, we will still need to explore whether the stability to digestion could lead to accumulation of these crystals in cells, potentially resulting in cytotoxicity. Nevertheless, the work here provides a method to study whether nuclease resistant L-DNA crystals, rather than slowly degradable D-DNA nanostructures, could provide a more robust route for DNA crystal materials in vivo. METHODS AND MATERIALS

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synthesizer. Following synthesis, the oligonucleotides were cleaved, deprotected, and purified by HPLC. “Native” D-DNA strands were purchased HPLC purified from Integrated DNA Technologies (Coralville, IA). Sequences and heavy atom positions are indicated in Figure S1. Crystallization of both native and modified systems for both L- and D-DNA was performed using the sitting drop vapor diffusion method using a temperature gradient of 60°C - 25°C at 0.3°C/hour in a chilling incubator (Torrey Pines Scientific, Carlsbad, CA). The mother liquor for both the native and selenium derivatized D-DNA crystals contained 50 mM cacodylate pH 6.5, 18 mM MgCl2, 0.9 mM spermine, 0.9 mM cobalt(III)hexamine, and 9% isopropanol. Native and brominated L-DNA were crystallized in a mother liquor containing 50 mM cacodylate pH 6.5, 50 mM MgCl2, 2.0 mM cobalt(III)hexamine, and 1.5 M lithium sulfate. Triangular prism-like crystals grew to a final size of ~150 x 25 x 25 μm. All crystals were harvested using cryo-loops (Hampton Research), then transferred into a drop of artificial mother liquor supplemented with 30% glycerol, incubated for ~2 minutes, and finally cryo-cooled via plunging into a liquid nitrogen bath. Data collection, processing, and structural determination Data for all crystals were collected at either the Advanced Light Source at Lawrence Berkeley National Laboratory on beamline 8.2.2 with an ADSC Q315R detector, or the Advanced Photon Source at Argonne National Laboratory at beamline 19-ID on a Pilatus 6M detector. Bromine derivatized crystals were measured at λ = 0.92 Å, and native and selenium derivatives were collected at λ = 1.0 Å. A full 360° sphere (1° oscillations) of data was collected for all crystals. All data were indexed, refined, integrated, and scaled using the HKL2000 package.29 Initial phases were determined using hkl2map,30 which contains the Shelxc/d/e programs.31,32 Initial electron density maps were calculated from the SAD phases, and revealed obvious helices with resolvable major and minor grooves. The data were then loaded into the Python-based Hierarchical Environment for Integrated Xtallography (PHENIX)33 suite to independently validate the initial substructure from hkl2map; to identify the four bromine positions in the L-DNA crystals, and the selenium atom locations in the D-DNA. Each of the positions in the ASU were identified via the Hybrid Substructure Search (HySS) and AutoSol program in the PHENIX package. The Autobuild program SOLVE was used for initial refinement and phase calculation, and the starting model was built with RESOLVE.34 Manual model building was then carried out in Coot,35 and subsequent rounds of refinement were performed using both phenix.refine and REFMAC.36,37 Anomalous difference Fourier maps from the derivative data clearly revealed the four strongest peaks for the appropriate positions at the designed heavy atom sites. A 21 base pair duplex in the ASU was treated as one rigid body during refinement, after which real space and XYZ coordinate refinement was performed against each dataset. Later, atom occupancies and temperature factors were refined followed by simulated annealing. Calculations of Rfree for each of the datasets were based upon 5% of the unique reflections. The Holliday junction connections were not made to adjacent asymmetric units until the final stages of refinement, to avoid introduction of model bias or ambiguity of the appropriate angles yielded by the structure during the initial rounds of refinement. Crystallographic statistics for all crystals, and refinement statistics of the native models are given in Table 1. The atomic coordinates and structure factors for the completed models have been deposited in the Protein Data Bank (PDB) with accession codes 5VY6 and 5VY7. All figures were generated using PyMOL.38

Crystal-DNase incubations

DNA synthesis and crystallization Synthesis and HPLC purification of “native” and brominated L-DNA oligonucleotides was performed by ChemGenes (Wilmington, MA). SelenodU synthesis was carried out as previously described.27 The reaction scheme and details can be found in the Supplementary Information Scheme 1. Selenium modified D-DNA oligonucleotides were synthesized using standard phosphoramidite coupling chemistry on an Applied Biosystems 394 DNA

To demonstrate that the DNase stock was active in the buffer used for crystal formation, blunt-ended double stranded DNA was formed from the three oligonucleotides contained in the ASU that comprise the 21 bp duplex, but without sticky ends. These strands were annealed at a 1:4:4 molar ratio (40:160:160 µM) using a temperature gradient from 95°C to 4°C for 30 minutes in 50 mM HEPES (pH 7.5) with 80 mM MgCl2 and 2.5 mM spermine at 100 µM. 10 µL of the stock was incubated at 37°C with 1 µL (2 units) of Bovine Pancreatic DNase I that was isolated from a recombinant source (New England Biolabs), for 0, 5, 10, and 15 minutes. One unit is defined as


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the amount of enzyme which will completely digest 1 µg of DNA in 10 minutes. Another 10 µL of the stock was incubated at 37°C for 15 minutes with 1 µL of DNase that had been heat-inactivated by incubating at 75°C for 10 minutes. Each sample was then diluted 10-fold with H2O and analyzed by 15% native polyacrylamide gel electrophoresis, along with a control sample with no added DNase, and a 10-base pair double stranded DNA ladder (Figure S4). DNA crystallization was performed as outlined above for both the right handed and left handed 4x6 motifs in 50 mM HEPES (pH 7.5) with 80 mM MgCl2 and 2.5 mM spermine. Crystals were imaged using a LEICA S6D microscope and application suite at time 0 min. 1 µL (2 Units) of DNase I was added to the drop, then resealed and incubated at 37°C. The crystal tray was removed and the drops were imaged again at time intervals of 10 min., 20 min., 40 min., 1 hr., 2 hr., 4 hr., 6 hr, 8 hr., and overnight. This experiment was repeated with two other crystallization buffers: 50 mM cacodylate (pH 6.0) with 10 mM MgCl2, 2.5 mM spermine, and 2.5 M NaCl and 50 mM cacodylate (pH 6.5) with 5 mM Co(NH3)6 and 2.5 mM KCl. Additional optical micrographs for these experiments can be found in the supplementary information, including the mother liquor components used for crystallization for each case. A 12% denaturing PAGE gel (Figure S5) was run at 45 mA for 1 hour on dissolved crystals, was also used to confirm the degradation of the D-DNA crystals. The crystallization drops and DNase digestion conditions were as described above. Representative crystals were removed from each drop at time 0, 4, 6, and 24 hours as shown in Figure 4, and dissolved in 10 µL of H2O, and heat incubated at 75°C to inactivate any residual DNase that might have been transferred along with the crystals into H2O, and allow for complete dissolution. The gel was stained with ethidium bromide for 10 minutes and imaged.

ASSOCIATED CONTENT This material is available free of charge via the Internet at http://pubs.acs.org.”

AUTHOR INFORMATION Corresponding Author *[email protected]

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

ACKNOWLEDGEMENT Data were collected at The Berkeley Center for Structural Biology (BCSB), and the Structural Biology Center (SBC). The BCSB (BL 8.2.2) is supported in part by the NIH, National Institute of General Medical Sciences, and the HHMI. The Advanced Light Source is supported by the U.S. DOE under DE-AC02-05CH11231. The SBC (BL 19-ID) of the Advanced Photon Source, a DOE Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357. The Yan lab was supported by grants to H.Y. and Y.L. from the NSF (nos. 1360635 and 1334109), the ARO (no. W911NF-12-1-0420), and the NIH (no. R01GM104960). H.Y. was also supported by the Presidential Strategic Initiative Fund from Arizona State University. N.S. was supported via laboratory startup funding from the Center for Molecular Design and Biomimetics at the Arizona State University Biodesign Institute. The following grants to NCS are also acknowledged: grants EFRI-1332411 and CCF-1526650 from the NSF, MURI W911NF-11-1-0024 from ARO, MURI N000140911118 from ONR and grant GBMF3849 from the Gordon and Betty Moore Foundation.

ABBREVIATIONS Nt, nucleotides; bp, basepairs; ASU, asymmetric unit; SAD, singlewavelength anomalous dispersion;

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