Article pubs.acs.org/JACS
Tuning the Cavity Size and Chirality of Self-Assembling 3D DNA Crystals Chad R. Simmons,†,‡ Fei Zhang,†,‡ Tara MacCulloch,†,‡ Noureddine Fahmi,†,‡ Nicholas Stephanopoulos,†,‡ Yan Liu,†,‡ Nadrian C. Seeman,§ and Hao Yan*,†,‡ †
Biodesign Center for Molecular Design and Biomimetics and ‡School of Molecular Sciences, Arizona State University, Tempe, Arizona 85287, United States § Department of Chemistry, New York University, New York, New York 10003, United States S Supporting Information *
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 systemswas 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 (4 × 5) 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 (4 × 6), 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 3-fold helical screw axes P32 and P31 for each motif, respectively. The structures reveal a highly organized 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.
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INTRODUCTION The original goal of structural DNA nanotechnology, as outlined by Seeman in 1982,1 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 © 2017 American Chemical Society
self-assembly of 3D DNA crystals and determine 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 DNAin this paper, we will call this enantiomer D-DNAhowever, 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 lefthanded 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 Received: June 21, 2017 Published: July 21, 2017 11254
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Figure 1. Topological schematic, cartoon representation, crystallization, and models of the mirror image 4 × 6 crystals. (a) Topological representation of the 4 × 6 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 (4 × 6). 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 4 × 6 structure. (c) Light microscope image of the 4 × 6 D-DNA crystals. (d) Light microscope image of the 4 × 6 L-DNA crystals. (e) Model of the central building “block” of the 4 × 6 D-DNA (orange) motif. (f) Model of the central building “block” of the 4 × 6 L-DNA (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”. (h) 2Fo − Fc map at σ = 1.8. All features are clearly observable as in (g), but with the enantiomeric mirror image in the L-DNA form and neighboring helices (blue). Scale bars: 100 μm.
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
resistant to digestion by nucleases, making it attractive for the design of stable crystals subject to degradation in environments compromised with DNases.19−23 11255
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Journal of the American Chemical Society Table 1. Data Collection and Refinement Statistics 4 × 6 D-DNA SADa Data Collection space group resolution (Å) cell dimensions a, b, c (Å) α, β, γ (deg) Rmerge I/σI completeness (%) redundancy Refinement resolution (Å) no. reflns Rwork/Rfree no. atoms DNA rms deviations bond lengths (Å) bond angles (deg) a
4 × 6 D-DNA nativeb
4 × 6 L-DNA SADa
4 × 6 L-DNA nativeb
P32 50−3.1
P32 50−3.05
P31 50−3.0
P31 50−3.0
68.5, 68.5, 56.8 90, 90, 120 0.084(0.25)c 27.6(2.6) 98.0(92.1) 3.7(3.2)
68.5, 68.5, 55.8 90, 90, 120 0.108(0.674) 23.9(1.9) 89.25(57.7) 7.6(6.7)
67.7, 67.7, 54.3 90, 90, 120 0.186(0.721) 41.1(2.6) 86.8(58.6) 10.4(9.3)
68.6, 68.6, 55.7 90, 90, 120 0.110(0.467) 39.0(2.6) 86.1(53.7) 10.7(9.5)
30−3.05 4710 21.13/23.66 851 851
50−3.0 4949 23.76/24.57 851 851
0.07 0.923
0.016 1.332
Data were collected at beamline 19-ID at APS. bData were collected at beamline 8.2.2 at ALS. cHighest resolution shell is shown in parentheses.
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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 to its D-DNA counterpart (Figure S2).17 Both the D- and L-DNA 4 × 6 motifs contain three strands, with lengths of 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. The structures of both isoforms were determined to 3.05 and 3.0 Å resolution, each using an unbiased set of phases from selenium and bromine derivatives (for D - and L-DNA, respectively). The motif consists of the three 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 1e,f) 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 1g,h). 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-
series of tensegrity triangles each containing three Holliday junctions with 7 base pairs between each junction, resulting in its 3-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 intercrossover bases from seven bases to five bases with a total of four sequence repeats instead of three (4 × 5). Since 4 × 5 bp < 3 × 7 bp (21 bp = 2 × 10.5 bp), the structure resulted in a slight underwinding 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 3-fold 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 redesigned “tensegrity square” system, which we call the 4 × 6 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 (LDNA) of this motif in order to obtain the enantiomeric form of the naturally occurring right-handed motif (Figure S1c,d). Structures of both D-DNA and L-DNA 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. 11256
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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 twoturn duplex in the ASU (Figures 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 4 × 6 brominated D-DNA 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 (Supporting Information). Quality crystals were obtained allowing phase determination 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 4 × 6 Cavities. 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 4 × 2 × 5 nm in size, resulting from an opening between layers from junctions that are separated by two full helical turns, and with an opening that results from the layered helical arrays when viewed along the 3-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 4 × 5 and 6 × 5 crystals).25 Although the cell
Figure 2. Surface rendering of the mirror image 4 × 6 building blocks. (a) Stereoscopic view of the central D-DNA building “block” structure that comprises the self-assembling lattice. The four tethered duplexes (semitransparent 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 (LDNA) of (a) with the central weaving strand (red) tethering the semitransparent left-handed DNA duplexes (blue).
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 zigzag helix axis along each continuous layer in the lattices (Figure S3). 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 4 × 6 central weaving
Figure 3. Structural comparison of the crystal cavity periodicity between the 4 × 6 and 4 × 5 scaffolds. (a) Stereoview of the crystal packing of the 4 × 6 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 3-fold symmetry axis with pores of ∼5 nm in size. (c) Stereoview of the crystal packing of the 4 × 5 lattice.24 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 nonperiodic array of densely packed cavities in stark contrast with the 4 × 6 scaffold. (d) 90-degree rotation with respect to (c) with a view down the 3-fold symmetry axis showing ∼2.5 nm size pores. 11257
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Journal of the American Chemical Society constants between the 4 × 6 and 4 × 5 or 6 × 5 structures are nearly identical, the resulting packing (P32 or P31) of the layered 4 × 6 lattice starkly contrasts with the packing previously observed with the 4 × 5 and 6 × 5 crystal systems, which resulted in P3221 symmetry. Analysis of the n-repeating five-base crystal structures clearly revealed a nonperiodic arraywith 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 3-fold axis (one-half of those in the 4 × 6)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 4 × 6 systems diffracted equally as well as the originally reported structure (3.1 Å). The densely packed arrangement of the initial 4 × 5 design was not amenable to the eventual scaffolding of biomolecules, but the introduction of a single base between four-arm junctions in the 4 × 6 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 4 × 5 system and thereby yielding a scaffold that contained welldefined cavities of identical sizes. Nuclease Resistance of the 4 × 6-L-DNA. Our long-term goal is to use the periodic array of cavities in each 4 × 6 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 toward use of DNA crystals for this potential application is the presence of nucleases in cell systems, which 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 h, we observed significant degradation of the DDNA crystals, with near complete digestion after 6 h. 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 2 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 wellsuited as a nuclease-resistant host for guests within its lattice.
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 h to demonstrate crystal stability at 37 °C. Light images of representative time points of 0, 4, 6, and 24 h are shown. Degradation of the D-DNA crystals became apparent at 4 h, with nearly complete dissolution at 6 h, whereas the L-DNA crystals suffered no visible damage throughout the experiment. Scale bar: 100 μm.
biomolecules that have not yet been determined via conventional crystallization methods. The new structures described here provide a new route toward this goal, with discrete, periodic, and larger cavities well-suited for immobilization of proteins up to ∼5 nm in size. In addition, the 4 × 6 crystals possess uninterrupted solvent channels down the 3-fold 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 4 × 5 structure was due to the intercrossover 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 under way 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 left-handed 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.
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METHODS AND MATERIALS
DNA Synthesis and Crystallization. Synthesis and HPLC purification of “native” and brominated L-DNA oligonucleotides was performed by ChemGenes (Wilmington, MA, USA). Seleno-dU synthesis was carried out as previously described.27 The reaction scheme and details can be found in the Supporting Information Scheme 1. Selenium-modified D-DNA oligonucleotides were synthesized using
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 11258
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digest 1 μg of DNA in 10 min. Another 10 μL of the stock was incubated at 37 °C for 15 min with 1 μL of DNase that had been heat-inactivated by incubating at 75 °C for 10 min. 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 10base pair double-stranded DNA ladder (Figure S4). DNA crystallization was performed as outlined above for both the right-handed and left-handed 4 × 6 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. A 1 μL (2 units) amount 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 h, 2 h, 4 h, 6 h, 8 h, 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 Supporting 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 h on dissolved crystals and 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 h as shown in Figure 4, 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 min and imaged.
standard phosphoramidite coupling chemistry on an Applied Biosystems 394 DNA 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, USA). 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 60−25 °C at 0.3 °C/h in a chilling incubator (Torrey Pines Scientific, Carlsbad, CA, USA). The mother liquor for both the native and seleniumderivatized 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% 2propanol. 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 × 25 × 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 min, 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 and 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 data set. Later, atom occupancies and temperature factors were refined followed by simulated annealing. Calculations of Rfree for each of the data sets 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. 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 constitute 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 to 4 °C for 30 min in 50 mM HEPES (pH 7.5) with 80 mM MgCl2 and 2.5 mM spermine at 100 μM. A 10 μL amount 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 min. One unit is defined as the amount of enzyme that will completely
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/jacs.7b06485.
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Additional information (PDF)
AUTHOR INFORMATION
Corresponding Author
*
[email protected] ORCID
Hao Yan: 0000-0001-7397-9852 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS 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 DEAC02-05CH11231. The SBC (BL 19-ID) of the Advanced Photon Source, a DOE Office of Science User Facility, is 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-12-10420), 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 11259
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(37) Collaborative Computational Project, Number 4. The CCP4 suite: Programs for protein crystallography. Acta Crystallogr., Sect. D: Biol. Crystallogr. 1994, 50, 76010.1107/S0907444994003112. (38) DeLano, W. L. The PYMOL Molecular Graphics System; DeLano Scientific: San Carlos, CA, 2002.
ONR, and grant GBMF3849 from the Gordon and Betty Moore Foundation.
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DOI: 10.1021/jacs.7b06485 J. Am. Chem. Soc. 2017, 139, 11254−11260
