Methods 47 (2009) 168–176
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Methods journal homepage: www.elsevier.com/locate/ymeth
Review Article
Crystallographic studies of DNA and RNA Blaine H.M. Mooers * Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, 975 NE 10th Street, BRC 466, Oklahoma City, OK 73104, USA
a r t i c l e
i n f o
Article history: Accepted 10 September 2008 Available online 10 October 2008 Keywords: Nucleic acid crystallography DNA crystallography RNA crystallography DNA structure RNA structure Crystallization constructs DNA structural biology RNA structural biology
a b s t r a c t Our knowledge of nucleic acid structure grew rapidly over the past decade with the determination to high resolution of larger structures of great biological significance. Advances in sample preparation, crystallization techniques, cryocrystallography, access to synchrotron radiation, and crystallographic software continue to accelerate the structure determination of nucleic acids. Crystallographic studies of DNA and RNA molecules share many considerations that we outline here. The application of crystallography to RNA is illustrated with the structure determination of the CUG repeat that is linked to type I myotonic dystrophy. Ó 2008 Elsevier Inc. All rights reserved.
1. Introduction Since the model of the DNA double helix proposed by Watson and Crick in 1953, the crystal structures of nucleic acids have provided valuable insights into their biological functions. The fiber diffraction method used by Rosalind Franklin and others has been largely eclipsed by single-crystal methods. The application of single-crystal crystallography to polynucleotides was delayed by difficulties in making milligram quantities of material suitable for growing diffraction quality crystals. The first crystal structures of an RNA (tRNA) were reported in 1974 [1,2] and of a DNA oligonucleotide was reported in 1979 [3], almost two decades after the first crystal structure of a protein. In the past two decades, nucleic acid structure determinations accelerated because of three developments: better methods of making material for crystallization, the widespread adoption of cryocrystallography, and improved access to synchrotron radiation. There are several thousand crystal structures of nucleic acids when protein complexes are included. RNA crystallography, particularly, has enjoyed increased attention with the publication of several structures longer than 100 nucleotides [4–7]. Crystallography provides precise molecular structures. Mediumresolution structures (2.0 Å) have an uncertainty in atomic position of 0.1–0.2 Å, and atomic-resolution structures (better than 1.2 Å) have an uncertainty in atomic position of 0.02–0.04 Å. Precise atomic coordinates allow for the determination of precise molecular parameters. Molecular parameters of biological interest include * Fax: +1 405 271 3910. E-mail address:
[email protected] 1046-2023/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.ymeth.2008.09.006
hydrogen bonding geometry, torsion angles, conformational parameters, and molecular surfaces. In addition, crystal structures provide insight into molecular flexibility and reveal hydration patterns, ion binding, and nucleic acid–ligand interactions. In macromolecular crystallography, studies of nucleic acids have a number of unique features: (1) unwanted variation in polynucleotide chain length, (2) a refolding step after purification, (3) frequent use of polyamines in crystallization, (4) insensitivity of crystallization to changes in pH, (5) vulnerability to acid and alkaline hydrolysis, (6) limited crystal packing contacts, (7) errors in the crystal packing of double helices, (8) challenges in making heavy atom derivatives, (9) use of covalently incorporated halogen atoms for phasing experiments, (10) extremely strong diffraction spots from double-helical structures, (11) molecular symmetry confounding molecular replacement searches, and (12) specialized methods for conformational analysis. We address these issues in the context of recent advances in methodology. The crystallographic study of nucleic acid–protein complexes and nucleic acid fibers are not covered (see reviews elsewhere [8]). For related reviews in this series, see [8–11]. To illustrate the use of some of the methods presented, we discuss the structure determination of a RNA double helix formed by CUG triplet repeats.
2. Nucleic acid crystallization 2.1. Basic principles A crystal of a nucleic acid is a three-dimensional array of nucleic acids held together by non-covalent interactions. As in crystals of
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proteins, the large spaces between nucleic acids are occupied by semi-ordered solvent molecules near the surface of the nucleic acid and by disordered solvent molecules in the bulk solvent several angstroms away from the surface of the nucleic acid. Parts of the nucleic acid far from contact points with other macromolecules may have high levels of flexibility and may adopt several conformational states. These disordered parts of the structure may be invisible in the electron density map because the electron density map gives a view of the structure averaged over the several million unit cells in a typical crystal. A minor variant of a nucleic acid with a known crystal structure may be crystallized with similar conditions if the variant does not change its conformation or its intermolecular contacts. The reproducible nature of the crystallization experiment also enables the crystallization of molecules with halogen atoms (e.g., bromine or iodine) incorporated for phasing by anomalous scattering or by isomorphous replacement [12]. On the other hand, the experimental conditions for crystallization of a nucleic acid that differs in length and conformation from known crystal structures are difficult to predict accurately. Many factors influence the crystallization of nucleic acids: (1) chain length heterogeneity, (2) conformational heterogeneity, (3) loop flexibility, (4) molecular shape (i.e., helical form), (5) ion binding sites, (6) ligand binding sites, and (7) intermolecular contact sites. Nucleic acids have few sites for making intermolecular contacts for four reasons. First, the nucleotide side chains (i.e., the nitrogenous bases), with their rich array of hydrogen bond donors and acceptors, are often largely buried by base stacking and rarely form intermolecular contacts. Second, the close contacts between the backbones of neighboring helices are limited to geometries that allow a multivalent cation to bridge two or three negatively charged backbones. Third, double-stranded helices with a regular repeating conformation such as the dinucleotide repeat of Z-DNA may crystallize with errors in crystal packing (i.e., statistical disorder, twinning, and so on). Fourth, contacts between molecules are largely limited to the packing of the ends of helices on top of other helices or in the grooves of neighboring helices [13]. 2.2. Constructs for crystallization The limitations to nucleic acid crystallization may be exploited in the design of constructs to enhance the success at crystallization (for reviews, see [14–19]). Double-stranded oligonucleotides are designed with blunt ends or staggered ends to promote the formation of a semi-continuous helix in the crystal lattice by the stacking of helices end-on-end [20]. The tendency to crystallize in semicontinuous helices seems to be greater with oligonucleotides one helical turn in length or longer. Oligonucleotides shorter than one helical turn may crystallize with both ends stacked end-onend in a semi-continuous helix [21] (Fig. 1A), with both ends of the duplex packing into the grooves of neighboring duplexes [22] (Fig. 1B), or with one end of the duplex packing in the groove of a neighboring duplex and one end stacking on the end of another neighboring duplex [23] (Fig. 1C). Staggered ends do not always hybridize to form a semi-continuous helix with the duplexes stacked above and below (Fig. 2A) [24]. For example, one overhanging nucleotide at one end of a double-stranded helix may remain stacked while the overhanging nucleotide at the other end of the helix may flip out of the helix and join another helix in a neighboring column of stacked helices [25] (Fig. 2B). Alternatively, the overhanging nucleotide may project into a groove of a neighboring duplex in a semi-continuous stack of duplexes and form a base triplet [26,27] (Fig. 2C). Sometimes the overhanging nucleotide is not visible in the electron density map because of static disorder [28].
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Double-stranded helices for crystallization should be designed to avoid end-on-end disorder. The two ends of a duplex may present identical contact sites to the crystal lattice. A double helix composed of two strands with different sequences (a heterodimer or heteroduplex) may enter the same site in a crystal lattice in either of two orientations (i.e., pointed up or pointed down) with similar probability. The resulting electron density map will show two overlapped base pairs where one is expected. End-on-end disorder is avoided by using self-complementary sequences, duplicating the sequence in an inverted repeat (explained below), or making the double helix asymmetric by adding different overhangs to the ends of the double helix. Self-complementary sequences avoid the need to mix two strands in a 1:1 molar ratio. A crystallization solution with two different strands may contain three double-stranded molecular species: the intended heterodimer and two unintended homodimers. One of the homodimers may be favored for crystallization over the heterodimer [29]. If the sequence motif of biological interest is not self-complimentary, a fusion may be made between the sequence of interest and its reverse complement [30]. The resulting inverted repeat will hybridize with itself to give two copies of the domain of interest in a head-to-head orientation. A spacer sequence placed in the middle of the duplex may modulate potential crystal packing interactions by varying the total length of the oligonucleotide and the relative orientations of the two domains [31]. Double helices longer than one helical turn are vulnerable to translational disorder because of non-unique side-by-side packing of neighboring stacks of helices in the crystal lattice [32,33]. The lattice may be a blend of two or more lattices related by a translation along the helical axis. Many attempts to crystallize RNA hairpins using relatively short oligonucleotides with inverted repeat sequences have failed because hairpins exist in equilibrium with double-stranded duplexes. The high concentration of nucleic acid required for crystallization drives the equilibrium towards duplexes despite the formation of several non-canonical base pairs (i.e., mismatched base pairs) in the spacer between the repeats designed to be part of the hairpin’s loop [34]. The crystallization of hairpins often requires their stabilization by a RNA-binding protein. The sequence and length of a single-stranded RNA are varied to find a suitable construct for the crystallization. The constructs are tested for biochemical activity, which is especially important with ribozymes. The biologically active RNAs are then screened for their ability to crystallize. Sometimes, nonessential regions are deleted, or just the biologically active part of the nucleic acid is targeted for crystallization. For example, the short double-helical segment formed by the acceptor arm of tRNA is sufficient for recognition by tRNA synthetases and has been crystallized to study the structure of the acceptor arm at very high resolution [35]. The most likely folds of the minimized sequence may be assessed in silico with RNA folding programs and native polyacrylamide gel electrophoresis. The thermodynamic stability of the construct may be measured by UVmonitored thermal melting in solutions with different ionic strength. Sequence alignments of homologous RNAs may be used to ensure that the original structure is conserved in the construct used for crystallization. Where applicable, the secondary structure should be supported by biochemical data and the 50 and 30 ends of the motif should be well defined. Nonessential regions may also be replaced with motifs that promote intermolecular interactions. The best-known example is the GNRA tetraloop and its cognate receptor, which is an asymmetric internal loop in a double-helical segment [36]. This pair of motifs was used to grow higher quality crystals of the hepatitis delta virus (HDV) ribozyme [36].
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Fig. 1. Crystal packing motifs of blunt-ended double-stranded nucleic acids. (A) End-on-end stacking by B-DNA decamers [21]. (B) End in minor groove packing of a A-DNA decamer [22]. (C) Both end-on-end stacking and end-in-minor groove packing of a DNA/RNA hybrid decamer [23]. Prepared with PyMOL (Warren L. DeLano ‘‘The PyMOL Molecular Graphics System.” DeLano Scientific LLC, San Carlos, CA, USA. http://www.pymol.org).
Fig. 2. Crystal packing motifs of double-stranded nucleic acids with staggered-ends. The thin bars represent bases and the dashed lines represent intermolecular hydrogen bonds. (A) A RNA octamer with two-base overhangs on each end that remains stacked and part of a semi-continuous helix [24]. (B) A Z-DNA heptamer that has one 50 terminal nucleotide that remains stacked while the other 50 -terminal nucleotide flips out of the helix and formed a base-pair with a neighboring duplex [25]. (C) A B-DNA decamer with two-base overhangs that form base triples in the major grooves of neighboring decamers [27]. One overhang projects into the major groove of a stacked neighbor (blue), and the other overhang projects into the major groove of a duplex (red) in a nearby stack of duplexes. Prepared with PyMOL (Warren L. DeLano ‘‘The PyMOL Molecular Graphics System.” DeLano Scientific LLC, San Carlos, CA, USA. http://www.pymol.org).
Another approach is to use a RNA-binding protein to provide a high density of potential lattice contacts. The protein’s binding site is incorporated into a nonessential region of the RNA, and the RNA–protein complex is crystallized. For example, the use of the U1A protein as a crystallization chaperone [37] dramatically improved the crystals of the HDV and hairpin ribozymes [38,39]. The use of selenomethione labeled U1A protein enabled structure determination by multiwavelength anomalous diffraction (MAD) [37]. Nonessential regions may also be used to introduce known heavy atom binding sites to promote heavy atom binding and
thus allow the use of heavy atom methods to obtain experimental crystallographic phases. For example, hexamine binding sites have been engineered using GU wobble base pairs [40]. GU wobble base pairs present an electronegative surface on the major groove side of the base-pair that favors the binding of metals including cobalt hexamine and iridium hexamine [40,41]. The flanking base pairs influence hexamine binding because the hexamines will not bind to all GU base pairs [40]. Several short sequences that bind cobalt hexamine have been identified [40]. For example, the two self-complementary tetranucleotides GUGC and GGUC have motif I [42] (GU wobble base-pair followed
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by a YG base-pair where Y represents U or C) and are known to bind cobalt hexamine [40]. As in protein crystallography, crystal quality may be improved by using nucleic acids with higher thermal stability. Thermophiles are natural sources of such molecules. If the RNA is universally abundant, homologs may sometimes be purified directly from a thermophile (e.g., the ribosome 30-subunit from the bacteria Thermus thermophilus [43]). Alternatively, in vitro selection may be used to find more stable variants. For example, in vitro selection found a single base deletion in the P4–P6 domain of the Tetrahymena ribozyme that improved diffraction from 2.8 to 2.24 Å [44], and a mutant of the full-length group II intron improved diffraction from 5.0 to 3.8 Å [45]. 2.3. Precautions in handling DNA and RNA The preparation and handling of nucleic acids require the conscientious use of sterile technique to avoid destruction of the nucleic acids by nucleases. Greater precautions need to be taken in the handling of RNA to keep it free from RNase. Standard procedures include wearing clean latex gloves, baking glassware and electrophoresis plates at 200 °C for 2 h, using DEPC treated water, and filtering all solutions with 0.02 lm filters. Purified DNA and RNA may be stored in lyophilized form at 20 °C or in solution at 4 °C. RNA should be stored in buffers with a pH between 6 and 7 to avoid alkaline hydrolysis at higher pH and acid hydrolysis at lower pH. 2.4. Production of milligram quantities As in protein crystallography, milligram quantities of homogeneous DNA or RNA with greater than 95% purity are required to first find optimal crystallization conditions and then to grow large numbers of diffraction quality crystals to screen for cryoconditions and heavy atom soaking. Milligram quantities of DNA and RNA oligonucleotides (<60 nucleotides) may be made by chemical synthesis and purified by HPLC or PAGE gel electrophoresis. Repeats of long DNA sequences (hundreds of nucleotides) may be amplified by PCR, released by restriction enzyme digestion, purified by PAGE gel electrophoresis and used in structural studies of large DNA– protein complexes involving long DNAs (e.g., the nucleosome) [46]. RNA may also be made by run-off transcription in vitro, over-expression in E. coli as a fusion with tRNA [47], purified directly from a natural source (e.g., tRNAs [48,49] and the bacterial ribosome subunits [10], or 4) ligating two shorter RNAs using T4 ligase. The last approach enables the incorporation of a synthetic oligonucleotide bearing a modified nucleotide containing a halogen atom for crystallographic phasing or a substituent group for ribozyme inhibition. The large-scale production of RNA by in vitro transcription is the principal method of making larger RNAs. The DNA template typically contains a phage T7 promoter sequence and the RNA gene [50]. The DNA sequence of the first three nucleotides from the start of transcription influences the subsequent yield, and a limited number of sequences are known to lead to high yields of RNA transcripts. In addition, phage T7 RNA polymerase tends to terminate transcription prematurely by several nucleotides and to add several nucleotides beyond the end of the DNA template. The correct length is often impossible to select on a denaturing PAGE gel. The sequence restraints at the 50 -end may be avoided by inserting a hammerhead ribozyme that self-cleaves at its 30 -end and at the 50 -end of the RNA of interest. There are no sequence restraints in the target RNA on the nucleotides 30 to the cleavage site, so the hammerhead ribozyme is preferred for generating the 50 -terminus of the desired RNA. The hammerhead ribozyme requires that several residues in its interior complement several nucleotides in
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the target sequence, so it must be redesigned for each new RNA gene. The heterogeneity at the 30 -end of the RNA of interest may be eliminated by incorporating a cis-acting ribozyme 30 to the RNA of interest [51] or by adding a trans-acting ribozyme [52,53]. The 30 cis-acting hammerhead ribozyme requires the dinucleotide UX (where X is any nucleotide except G) on the 50 side of the cleavage site, the HDV ribozyme requires that a G not be on the 50 side of the cleavage site, and the Neurospora Varkud satellite RNA ribozyme requires that a C not be in this position. Trans-acting ribozymes need a sequence of 7–11 nucleotides that complements the cleavage site at the 30 -end of the target RNA [52,53]. The trans-acting ribozyme must be redesigned for each target RNA and must differ significantly in size from the target RNA to enable separation during purification. If the ribozyme has strong interactions with the target RNA, the ribozyme fusion may misfold and not cleave. Misfolding may be anticipated by predicting the fold of the fusion with a secondary structure prediction program such as Mfold [54]. Such programs also enable testing whether a base-pair reversal will break an undesired interaction. The interfering interaction between the ribozyme and target RNA may be broken by heating the RNA to 55–60 °C and cooling slowly to room temperature several times. Recently, two affinity chromatography methods have been reported that use a RNA-binding protein attached to a column to capture transcripts with a RNA affinity tag on the 30 end [55,56]. The transcript has the RNA of interest followed by a self-cleaving ribozyme and a pair of stable hairpins that serve as the affinity tag. The desired RNA is released from the column after the addition of a ligand that binds to the ribozyme and induces self-cleavage. These methods allow RNA purification under native conditions in a couple of days. 2.5. Renaturation DNAs and RNAs purified under denaturing conditions need to be properly refolded prior to crystallization. Refolding conditions are empirically determined. Refolding success may be assessed by testing the biochemical activity of ribozymes or by measuring the binding affinity of an interacting nucleic acid or protein partner. The nucleic acid is resuspended in nuclease-free water or a buffer such as 10 mM sodium cacodylate near neutral pH. Cacodylate is a popular buffer for nucleic acids because it contains arsenic that inhibits microbial growth; however, it must be handled safely. Initial refolding buffers for RNA should have a slightly acidic pH and only monovalent cations because divalent cations promote cleavage at high temperature. The nucleic acid is unfolded by heating for 1–5 min at a high temperature (60–95 °C) and then refolded by either slow cooling (1 h or overnight) to room temperature to promote duplex formation or flash cooling by transfer to ice water to promote hairpin formation. Divalent cations (e.g., 5–20 mM MgCl2) may be added after the temperature has dropped below about 40 °C. 2.6. Crystallization strategies Both the hanging drop and the sitting drop techniques are used to crystallize nucleic acids by vapor diffusion. Hanging drops are often set up using Linbro or VDX plates with 24-wells. The crystallization drop (2–16 ll in volume) is assembled by mixing the nucleic acid stock solution with the reservoir solution in a 1:1 ratio on a siliconized glass cover slip that is suspended over a 500– 1000 ll reservoir and sealed with silicon vacuum grease or petroleum jelly. Sitting drops have a larger volume range (2–50 ll) and are easier to assemble independently of the reservoir solution. The reservoir solution may consist of only the precipitating agent while
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the sitting drop may contain the precipitating agent, various additives, the buffer, and the nucleic acid. High initial concentrations of the precipitating agent 2-methyl-2,4-pentanediol (MPD) in the crystallization drop may inhibit the crystallization of some DNA duplexes. Often, the drop is set up with MPD at an initial concentration that is 5–10% of the concentration in the reservoir. The lower initial concentration of precipitating agent reduces crystal nuclei and lengthens the time required to reach equilibrium with the reservoir thereby promoting the growth of larger crystals. During screening experiments, daily inspection of crystallization drops in the first week identifies crystals that appear and then quickly dissolve or degrade. Crystals that grow too quickly often have growth defects that compromise the quality of the subsequent diffraction data. Early detection of rapid crystal growth is useful for revising the crystallization conditions to slow crystal growth. During crystallization experiments to optimize crystal quality and size, crystallization trays are left undisturbed for a week or more to avoid interrupting crystal growth and thus compromising the final crystal size and quality. Crystallization robots may be used to reduce pipetting errors and conserve material by using nanoliter-scale drops. The high throughput of crystallization experiments leads to a rapid accumulation of trays experiments that need to be monitored. Imaging robots automate the monitoring of crystal growth. A number of crystallization screens have been designed for the crystallization of either a broad range of nucleic acids or a subset of nucleic acids (Table 1). Nucleic acid crystallization solutions frequently differ from the solutions in commercial protein crystallization screens by containing MPD, cobalt hexamine, or spermine. Cobalt hexamine and spermine interact with nucleic acids directly and are often found in electron density maps. MPD is a precipitating agent that is handled under a chemical hood because it is a volatile neurotoxin. Small molecular weight PEGs (200, 300, and 400) are effective substitutes for MPD. Each kind of nucleic acid requires different crystallization agents. For example, 10–80 mM of MgCl2 is often required for RNA crystallization, and the presence of 100–300 mM of a monovalent cation (e.g., KCl) may profoundly improve the size and quality of RNA crystals [50]. Knowledge from the literature of crystallization conditions for molecules similar to the target molecule may save time and effort. Generally, several crystallization screens (Table 1) are used to find crystallization leads because some screens will yield a few poor leads while other screens will yield many good leads. If there are no promising leads after using many screens, it is more effective to modify the construct for crystallization rather than pursue
Table 1 Nucleic acid sparse-matrix crystallization screens Screen
Commercial source
No. of solutions
Target
Natrix [17]
Hampton research
48
Nucleix [17] (expanded version of Natrix Screen) DNA [111] RNA [112] Nucleic Acid Mini [113] Doudna [114] Golden I [115] Golden II [50] G-rich DNA [116] Kundrot RNA [117]
Qiagen
96
Ribozymes DNA oligos RNA oligos RNA–protein DNA–protein Same as Natrix
Sigma–Aldrich Sigma–Aldrich Hampton research None None None None None
48 48 24 44 48 24 24 72
DNAzymes RNA oligos RNA oligos Ribozymes Ribozymes Ribozymes DNA quartets RNA oligos Ribozymes
poor crystallization leads. The best leads are explored in subsequent two-factor optimization experiments. Finally, the optimized condition is replicated in 24–96 crystallization drops to produce many (>100) large crystals for room temperature X-ray data collection, cryocondition optimization, and heavy atom derivatization. 2.7. Heavy atom incorporation Heavy atoms are added to crystals to help find the phases needed to calculate the initial electron density maps for a new structure. There are several approaches to introducing heavy atoms including synthetic incorporation, co-crystallization, and soaking. Heavy atoms may be incorporated into the polynucleotide by chemical synthesis with modified nucleotides in which a natural atom has been replaced with a heavier atom such as bromine, iodine, or selenium. The heavy atom site is fully occupied, and its location in the structure is known a priori, which is an advantage when using Patterson methods to locate heavy atoms. The synthetic incorporation of heavy atoms is practical with short oligonucleotides (<35 nucleotides) and with long RNAs made from two pieces where one piece contains a modified nucleotide [57]. The modified RNA may simply be added in trans or, if necessary, ligated with T4 RNA ligase [8]. When bromine or iodine atoms are attached to the C5 carbon atom in the rings of uracil or cytosine [58], the halogen atoms project into the major groove, reduce the solubility of the nucleic acid, and promote too much crystal nucleation, so halogenated oligonucleotides often require modified crystallization conditions. The modified nucleotide may also introduce a conformational change [59–63] or a change in crystal packing that makes the derivative non-isomorphous with the wild-type nucleic acid. Not all modified oligonucleotides will crystallize, so a series of oligonucleotides with the modified site in different positions may be tested. Oligonucleotides with modified nucleotides at two or more sites are even less soluble. Halogenated nucleotides are light sensitive, so crystallization experiments are stored in the dark. Halogen atoms are vulnerable to eventual radiolysis during X-ray data collection [64], so the X-ray dose is considered while planning data collection. Selenium atoms may be incorporated into synthetic oligonucleotides by replacing phosphorous atom [65], the oxygen atom in the 20 position [66–70] or in the 50 bridging backbone, or at the thymine 4 position [71]. Oligonucleotides with these latter modifications often crystallize more readily and give higher quality diffraction data than halogenated oligonucleotides. Finally, hexamine binding sites and cesium binding sites may be engineered into nonessential segments of helices [40]. Cationic forms of alkali metals, transitions metals, lanthanides, and other heavy atoms may also be introduced into crystals by soaking existing crystals or by inclusion in the initial crystallization drop. The later method is known as co-crystallization. Anionic forms of heavy atom compounds have not been exploited widely since nucleic acids are thought to be efficient cation binders. Nevertheless, nucleotide amino, imino, and hydroxyl groups may create specific anion binding sites [72]. The preparation of heavy atom derivatives of nucleic acids is often difficult because many heavy atom compounds do not bind well enough to be useful [15,50,73]. The heavy atoms must be added without introducing large changes in the crystal lattice for a derivatized crystal to be useful in the method of isomorphous replacement. The structural adjustments are likely to be restricted by crystal packing. Hysteresis may delay the onset of changes in the crystal lattice during soaks with heavy atoms for 2 h, so soaks lasting less than 2 h may minimize changes in the lattice while allowing enough time for binding by heavy atoms [74]. Covalent bonding to a nucleic acid by a heavy atom compound may be checked by MALDI-TOF mass spectrome-
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try [74] or by native PAGE gel electrophoresis [75]. These same methods may also be used to test for the presence of bound heavy atoms in a crystal after washing away excess crystallization solution and then dissolving the crystal in water. Success at heavy atom derivatization by crystal soaking is reported more often than success by co-crystallization, perhaps because co-crystallization experiments require large amounts of precious materials and the labor of additional crystallization experiments. The nucleic acid may also change its structure during co-crystallization to accommodate the binding of the heavy atom. These changes may be large if the heavy atom is replacing a tightly bound magnesium in the native structure and if the heavy atom is binding with a different geometry. These structural changes often lead to precipitation of the nucleic acid or otherwise prevent crystallization. If the co-crystal has a heavy atom with a strong anomalous signal and if it diffracts well, then an anomalous scattering experiment is possible with one crystal, and the lack of isomorphism is no longer a serious limitation. Ideally, the co-crystallization solution contains a cryogenic precipitating agent to ease the subsequent step of cryoprotection. Generally, multivalent cations heavier than magnesium (e.g., Co2+, Zn2+, Mn2+, Ba2+, Tb2+, Pb3+, and Sm3+) are used to replace hydrated magnesium bound as Mg[(H2O)6]+2 to specific sites such as the Hoogsteen face of guanines in the major groove, non-Watson– Crick base pairs, and bulged residues [40]. The hexamines of cobalt, iridium. osmium, ruthenium, and rhodium may replace magnesium cations with octahedral coordination [58]. Nevertheless, cobalt hexamine may lose two amines and form covalent bonds with the N7 nitrogen atoms of two guanines to give Co[(NH3)4]+3, so cobalt hexamine is not a perfect mimetic for Mg[(H2O)6]+2 [41]. Monovalent cations heavier than sodium or potassium (Rb+, Cs+, and Tl+) may replace sodium and potassium if these cations have localized binding sites [76]. The ‘‘Metals in RNA” database (MeNRA, http://merna.lbl.gov/) [77] may be used to identify potential metal binding sites and to select heavy atoms to be added by soaking crystals or by co-crystallization.
As found for protein crystals, about half of the unit cell volume of nucleic acid crystals is occupied by disordered solvent molecules and the X-ray diffraction is weak at high resolution. Other aspects of diffraction from nucleic acid crystals, however, may surprise experienced protein crystallographers. First, crystals of oligonucleotides generally give unit cells with at least one short dimension (<40 Å) that lead to a relatively small number of unique X-ray reflections in at least one direction. A rotation of the crystal by 1° will give a small number of spots. Rotation angles of 2.5–5° are often more efficient. Second, the very strong reflections associated with base stacking have counts that exceed the dynamic range of the detector, especially when using synchrotron radiation. Saturation of the detector may be avoided in a second pass with the beam intensity attenuated, and perhaps with shorter exposure time and with the detector moved farther back from the crystal. For successful scaling of the intensities from the two passes, the exposure of the low intensity pass should not be less than a tenth of the exposure of the high intensity pass. In cases of strongly diffracting crystals, three passes may be required. Third, nucleic acids have smaller partial specific volume (0.5 cm3/g) than proteins (0.73 cm3/g) because of base stacking [79,80]. The smaller partial specific volume must be taken into account when calculating the Matthews coefficient (VM) and percent solvent of a putative unit cell to determine the potential number of nucleic acid molecules in the asymmetric unit [81]. A useful online calculator may be found at http://www.ruppweb.org/Mattprob/ [79].
3. Diffraction studies
3.4. Neutron diffraction studies of nucleic acids
3.1. Crystal evaluation
Hydrogen atoms have a small X-ray scattering form factor relative to the heavier carbon, nitrogen, oxygen, and phosphorous atoms found in nucleic acids, so hydrogen atoms are almost invisible in electron density maps. On the other hand, hydrogen and deuterium atoms have scattering lengths (the analog of scattering factors in X-ray scattering) that are comparable to heavier atoms, so hydrogen and deuterium atoms show up strongly in neutron density maps and may reveal the orientation of well-ordered water molecules. A study of a B-DNA decamer found the spine of hydration in the minor groove to have a more intricate network of hydrogen bonding than inferred from an earlier X-ray crystal structure [82]. One study of a Z-DNA hexanucleotide offered new insights into the acidic nature of the hydrogen atom on the C8 carbon atom of guanine, the hydration patterns in the major and minor grooves, and the coordination of metal ions by both nucleic acid and solvent hydrogen atoms [83]. The prerequisites for neutron diffraction studies include deuterated material and very large crystals. Hydrogen atoms contribute to a continuous background scattering and should be replaced with deuterium atoms by transfer of the sample to D2O or by making deuterated nucleic acid or by doing both. Crystals of very large volume (0.5–4 mm3) are needed because neutrons scatter weakly compared to X-rays. The neutron diffraction data are often collected at room temperature over several weeks. Protein crystallography beam-lines are available at several neutron scattering labs (e.g., Japan Atomic Energy Research Institute, Los Alamos Neutron Science Center, Institut Laue-Langevin, and soon the Spallation Neutron Source).
Diffraction quality crystals are tested for the presence of nucleic acid by staining with Coomassie blue or ethidium bromide or by dissolving the crystal and checking the contents by PAGE or MALDI-TOF. The unit cell dimensions, resolution limit, and mosaic spread are determined by collecting initial X-ray diffraction data at room temperature. The room temperature values are used to evaluate changes induced by cryoprotection. 3.2. Cryoprotection of crystals The cryoprotection of nucleic acid crystals follows the same principles as the cryoprotection of protein crystals. A quantity of cryoprotectant is added to prevent the formation of ice during the rapid cooling of the crystal to 66–100 K; often a slightly higher concentration of cryoprotectant than the minimum required for cryoprotection will lead to better diffraction [78]. The cryoprotectant is either a nearly saturated solution of a cryogenic salt (e.g., LiSO4 or sodium malonate) or a high concentration (25–40%) of an alcohol, a sugar, or a small molecular weight PEG. Often crystals are stabilized by transfers through a series of drops with progressively higher concentrations of cryoprotectant. Alternatively, the concentration of a volatile cryoprotectant like MPD in the drop may be raised by incremental additions to the reservoir. The increments have to be small (2.5%) to avoid nucleating the growth of new crystals on the surfaces of existing crystals. These additions sometimes promote
further crystal growth. Crystals that are able to resist shearing apart in viscous solutions may be isolated from their mother liquor in the petroleum product paratone N, or in one of the oils that are less viscous, and then frozen directly. The discovery of adequate cryoconditions may require weeks of trial-and-error. 3.3. X-ray data collection
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3.5. Structure determination New structures are generally best determined with a method that uses heavy atom derivatives since structures determined by molecular replacement are prone to serious mistakes. Structure determination by multiple isomorphous replacement (MIR) or MAD may be done in a semi-automated fashion with a comprehensive crystallographic software package (e.g., PHENIX [84], CCP4 [85], SHARP [86], and SHELX [87]). A successful phasing experiment requires a signal that is above the noise of the anomalous differences or the isomorphous differences. A strong signal requires one or more well-ordered heavy atoms. The anomalous signal of the phosphorous atoms in the backbone is weak and may be enhanced by collecting highly redundant data. The signal has been successfully used for phasing with ultrahigh resolution X-ray data (<1.0 Å) [88]. Many short, double-helical DNA and RNA structures have been solved by molecular replacement. Idealized models are used to search for the position and orientation of the molecule in the unit cell using the X-ray data truncated at low resolution (3–4 Å) to compensate for the discrepancies between the model and the actual structure. High levels of molecular symmetry in doublestranded DNA and RNA may lead to close similarity between the true solution and the nearest false solution. In this case, many trial models must be inspected manually and tested by trial refinement before the correct solution is found. Recently, idealized known secondary-structure fragments were used in an iterative molecular replacement procedure to solve the structure of a 142-nucleotide L1 ligase ribozyme heterodimer [89]. The placed fragments were used to obtain an estimated phase set that was further modified by solvent flattening. The resulting electron density map was readily interpretable. The iterative molecular replacement approach took advantage of the modular nature of large RNA structures. Fragments of two nucleotides and three phosphates may also be placed by molecular replacement searches to build up the final structure [90]. The molecular symmetry of double-helical structures is so high that planar densities associated with base-pair stacking appear in Patterson maps made with the native X-ray data. The spacing of these densities reveals the helical rise, and the number of planes reveals the helical repeat. The orientation of the densities suggests the orientation of the helical axis in the unit cell. The positions of crystallographic symmetry axes also limit the location of the double helix. A true ab initio structure determination using the native diffraction data alone and probability theory (direct methods) is sometimes possible when the X-ray data extend to atomic resolution [91] and the structure has less than 2000 atoms. When heavy atoms are present, the size limit may be pushed beyond 2000 atoms [92]. Alternatively, Patterson superimposition methods may find heavy atoms with only the derivative X-ray diffraction data (no native data required). The positions of the heavy atoms are then used to find the lighter atoms [93]. 4. Structure analysis 4.1. Conformational analysis The analysis of a nucleic acid structure may be broken down into describing the helical structure, the backbone torsion angles, the molecular surface, the electrostatic surface, and the hydration pattern. The double helices of DNA and RNA are described with helical parameters at the local base step level and globally [94]. Different methods of helical analysis may lead to conflicting interpretations in the analysis of the same structure. A standard reference frame was agreed upon that gives local helical parameters that are inde-
pendent of the method used [95]. Traditionally, the basic unit used in the analysis of backbone conformation was the nucleotide, but an alternative unit that includes both the N and N + 1 phosphates revealed the rotameric nature of the RNA backbone [96]. Common hydration patterns of similar structures are illuminated by the clever use of Fourier synthesis [97]. The full non-linear Poisson–Boltzmann equation is required for accurate calculation of the electrostatic surface of nucleic acids due to the high charge density of polynucleotide backbones. This equation is in the programs QNIFFT [98] and the Adaptive Poisson–Boltzmann Solver (APBS) [99]. The APBS program also includes the size-modified Poisson– Boltzmann equation that takes into account ion-size [100]. 4.2. Figure preparation Most molecular graphics programs will correctly display crystal structures of nucleic acids as line, ball-and-stick, and molecular surface models. A smaller number of programs can make ribbon cartoons of the nucleic acids suitable for publication (e.g., Pymol as extended by Nuccyl (Jovine, L. nuccyl (2003) http://www.biosci.ki.se/groups/ljo/software/nuccyl.html), Chimera [101], DRAWNA [102], Ribbons [103], and Curves [104]).
5. Application to the structure determination of CUG triplet repeats Expansions (>50) of the CTG repeats found in the 30 untranslated region of the human dystrophin myotonin protein kinase gene cause type I myotonic dystrophy by a RNA gain-of-function mechanism [105]. The corresponding CUG repeats form hairpin loops that attract several RNA-binding proteins, including the splicing factor muscleblind [106]. The riboprotein complex remains sequestered in the nucleus, and the depletion of the splicing factor pool may alter the splicing of genes relevant to the clinical features of type 1 myotonic dystrophy [107]. We made a synthetic RNA with six CUG repeats to serve as a model of the stem of the stem-loop formed by CUG repeats. The RNA was purified to single nucleotide resolution on 2 mm thick, 40 mm long, 10% polyacrylamide (19:1) gels containing 6 M urea at 55 °C. The RNA was visualized by UV shadowing, excised, eluted, ethanol precipitated, suspended in dd(H2O), and desalted with size-exclusion chromatography. The RNA concentration was adjusted to 0.35 mM in a solution of 50 mM MOPS (pH 7.0) and 300 mM NaCl. The RNA was annealed by heating to 95 °C for 5 min and by cooling slowly to room temperature. Crystals grew in 1–2 weeks at room temperature by vapor diffusion using the hanging drop method. The well solution contained 50 mM MOPS (pH 7.0), 300 mM NaCl, 20 mM MgCl2, and 40% MPD. Isomorphous crystals were grown of RNA with brominated uridine in the fifth position from the 50 -end or iodinated uridine in the second position. RNAs with modified uridines in several other positions did not give good crystals. Crystals were frozen directly from the crystallization drop because of the high percentage of MPD. Native data were collected at SSRL BL9-1 to resolution of 1.58 Å. The strong reflections with a resolution of 3 Å saturated the CCD detector, so a second run of images was collected with the beam attenuated 10-fold. These data were used in molecular replacement trials using the program EPMR [108] and using an idealized model of A-form RNA. Solutions were found with acceptable packing and unexplained continuous electron density between the ends of the duplexes. We resorted to heavy atom methods to solve the phase problem. Three-wavelength MAD data were collected at ALS BL 8.2.2. The program SHELX [87] was used to find the positions of the bromine atoms. Four heavy atom sites were found in the asymmetric unit,
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whereas two sites were expected. The four sites were explained by the presence of two superimposed RNA double helices with one helix shifted relative to the second by a translation that corresponds to the length of one helical repeat (11 base pairs). This arrangement of the helices was validated by difference Fourier maps made using the X-ray data from the native, brominated, and iodinated structures. The structure was refined using REFMAC [109]. The final structure differed little from ideal A-form RNA despite the presence of six UU mismatches. The stacking of the guanines and cytosines in the G-C base steps dominated the structure and prevented narrowing of the helix at the UU base pairs. Each pair of Us was too far apart to share two direct hydrogen bonds. These unusual UU mismatches may be important in protein recognition and binding. 6. Concluding remarks The field of nucleic acid structure needs a structural genomics approach focused on RNA [110]. Several groups are developing techniques that enhance the throughput of RNA structures in the structural biology pipeline. Some of these techniques will be transferred to DNA crystallography. Over the next decade, the number, size, and complexity of nucleic acid structures determined by crystallography will continue to grow. Acknowledgments I thank Drs. J. Andrew Berglund and Sandra Greive of the University of Oregon, Zach Porterfield of OUHSC for valuable comments on this manuscript. This work was supported by an OHRS award (HR08-138) from the Oklahoma Center for the Advancement of Science and Technology. References [1] S.H. Kim, F.L. Suddath, G.J. Quigley, A. McPherson, J.L. Sussman, A.H.-J. Wang, N.C. Seeman, A. Rich, Science 185 (1974) 435–440. [2] J.D. Robertus, J.E. Ladner, J.T. Finch, D. Rhodes, R.S. Brown, B.F.C. Clark, A. Klug, Nature 250 (1974) 546–551. [3] A.H.-J. Wang, G.J. Quigley, F.J. Kolpak, J.L. Crawford, J.H. van Boom, G. van der Marel, A. Rich, Nature 282 (1979) 680–686. [4] J.H. Cate, A.R. Gooding, E. Podell, K. Zhou, B.L. Golden, C.E. Kundrot, T.R. Cech, J.A. Doudna, Science 273 (1996) 1678–1685. [5] N. Ban, P. Nissen, J. Hansen, P.B. Moore, T.A. Steitz, Science 289 (2000) 905– 920. [6] B.S. Schuwirth, M.A. Borovinskaya, C.W. Hau, W. Zhang, A. Vila-Sanjurjo, J.M. Holton, J.H. Doudna Cate, Science 310 (2005) 827–833. [7] N. Toor, K.S. Keating, S.D. Taylor, A.M. Pyle, Science 320 (2008) 77–82. [8] A. Ke, J.A. Doudna, Methods 34 (2004) 408–414. [9] J.H.D. Cate, Methods 25 (2001) 303–308. [10] M. Gluehmann, R. Zarivach, A. Bashan, J. Harms, F. Schluenzen, H. Bartels, I. Agmon, G. Rosenblum, M. Pioletti, T. Auerbach, H. Avila, H.A.S. Hansen, F. Franceschi, A. Yonath, Methods 25 (2001) 292–302. [11] N.H. Campbell, G.N. Parkinson, Methods 43 (2007) 252–263. [12] R. Wing, H. Drew, T. Takano, C. Broka, S. Tanaka, K. Itakura, R.E. Dickerson, Nature 287 (1980) 755–758. [13] S.R. Holbrook, S.H. Kim, Biopolymers 44 (1997) 3–21. [14] W.G. Scott, J.B. Murray, Methods Enzymol. 317 (2000) 180–198. [15] J.H. Cate, J.A. Doudna, Methods Enzymol. 317 (2000) 169–180. [16] J.E. Wedekind, D.B. McKay, Methods Enzymol. 317 (2000) 149–168. [17] W.G. Scott, J.T. Finch, R. Grenfell, J. Fogg, T. Smith, M.J. Gait, A. Klug, J. Mol. Biol. 250 (1995) 327–332. [18] P.J. Paukstelis, J. Nowakowski, J.J. Birktoft, N.C. Seeman, Chem. Biol. 11 (2004) 1119–1126. [19] N.C. Seeman, Biochemistry 42 (2003) 7259–7269. [20] J.E. Wedekind, D.B. McKay, Nat. Struct. Biol. 6 (1999) 261–268. [21] B.F. Eichman, J.M. Vargason, B.H.M. Mooers, P.S. Ho, Proc. Natl. Acad. Sci. USA 97 (2000) 3971–3976. [22] Y.-G. Gao, H. Robinson, A.H.-J. Wang, Eur. J. Biochem. 261 (1999) 413–420. [23] N.C. Horton, B.C. Finzel, J. Mol. Biol. 264 (1996) 521–533. [24] R. Biswas, M. Sundaralingam, J. Mol. Biol. 270 (1997) 511–519. [25] B.H.M. Mooers, B.F. Eichman, P.S. Ho, J. Mol. Biol. 269 (1997) 796–810. [26] L. Van Meervelt, D. Vlieghe, A. Dautant, B. Gallois, G. Précigoux, O. Kennard, Nature 374 (1995) 742–744.
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