DNA Coiled Coil Superstructures in Oligonucleotide Crystals

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DNA Coiled Coil Superstructures in Oligonucleotide Crystals Daniela De Luchi, Lourdes Urpí, Juan A. Subirana,* and Lourdes Campos* Departament d’Enginyeria Química, Universitat Politecnica de Catalunya, Diagonal 647, E-08028 Barcelona, Spain ABSTRACT: We present the structure of coiled coils found in two oligonucleotide DNA crystals. The sequences d(ATATATATAT) and d(CGATATATAT) are used. In the first case several coiled coils with different structures are found. In the second case a single structure is present. The results are compared with other coiled coil structures. They might be used in the development of new nanomaterials.

1. INTRODUCTION Process systems engineering aims to produce mathematical models to cope with uncertainties in complex systems. The model is later translated into adequate software. Data are introduced and operating decisions are then taken. The methodology is basically very similar to what is done in macromolecular crystallography, the subject matter of this paper: experimental data that contain errors and uncertainties are used. They are analyzed by different mathematical models, which depend on the method of data collection. The result is a molecular model, instead of operating decisions. The methodological steps are similar in both cases. As an example, we may compare an early paper of Puigjaner and Subirana in the field of X-ray scattering1 with a recent analysis of supply chains by Puigjaner et al.2 In this paper we present the structure of new coiled coils that are found in some DNA oligonucleotide crystals. They are compared with previous data on similar structures. Coiled coils have a strong potential for the development of new nanomaterials. Since the pioneering work of Seeman,3 the self-assembly properties of DNA through specific base pairing have been used to build a large number of nanostructures with different shapes.4-7 By the controlled deletion and insertion of individual bases, DNA twisted and curved bundles have also been built.8 In some cases other components may be incorporated into DNA in order to assemble complex nanomaterials.9,10 A parallel field of endeavor has been the study of helical superstructures built from different polymers,11-13 including protein coiled coils.14,15 In our laboratory we have recently investigated the formation of coiled coils in oligonucleotide crystals.16-19 Such coiled coils have not yet been used in the preparation of nanostructures, although they offer the potential to build helical springs at the molecular level. However, in one case we have found that microscopic helical crystals can be built.19 To further understand under what conditions such coiled coils are formed, in this paper we report for the first time the structure of two oligonucleotide decamers. Depending on the experimental conditions they form coiled coils with different geometries. We further compare their structure with previous reports on related compounds.

diffusion in hanging drops in 50 mM cacodylate buffers, pH 6.5, with spermine (1-4 mM) and MPD (2-methyl-2,4-pentanediol, 20-40%) as precipitants. To optimize conditions, various monovalent cations were added to the drops. Many trials were carried out, and similar results were obtained with different ions. Very often birefringent needles with blunt ends were obtained, which indicated poor quality of the crystals. Attempts to improve their quality were carried out by narrowing down the conditions used for crystallization. Diffraction data were collected at the BM16 Spanish line at the ESRF, Grenoble, France. Depending on the resolution, the rotation angle for each image was varied between 2° and 15°. As the angle of rotation is increased, more data on the diffraction appear in the image, but different diffraction spots may overlap. When possible, the collected data were processed automatically with either DENZO or HKL2000.20 However, in many cases only a limited number of spots were collected, which is due to several factors: low resolution, high mosaicity, intrinsic symmetry of the DNA duplexes, eventual presence of a small pseudocell, etc. Furthermore, in most cases the unit cell is highly asymmetric, with a very large c parameter. The combination of all these factors prevented automatic processing. In such cases the parameters of the true unit cell were determined manually with the help of MOSFLM.21 For the purpose of the results reported here, there are only a few parameters of interest which can be adequately calculated manually: the size of the unit cell; length, orientation and distance among duplexes inside the unit cell. In the rotation diagrams that we have collected there is always a region in which the c axis of the unit cell is approximately parallel to the spindle axis of the goniometer. Then the c axis is approximately vertical in oscillation patterns, such as those shown in Figures 5 and 7. From them it is immediately clear that the oligonucleotide duplexes are inclined toward the c direction of the unit cell since the strong stacking reflection appears as a sharp streak and is oriented at both sides of the meridional direction. The c value of the unit cell was determined from the distance between closely spaced spots, such as those visible in the different Special Issue: Puigjaner Issue

2. EXPERIMENTAL DETAILS Oligonucleotides were prepared by the phosphoramidite method and purified by HPLC. Crystals were obtained by vapor r 2010 American Chemical Society

Received: June 21, 2010 Accepted: September 20, 2010 Published: October 14, 2010 5218

dx.doi.org/10.1021/ie101328m | Ind. Eng. Chem. Res. 2011, 50, 5218–5224

Industrial & Engineering Chemistry Research frames in Figures 5 and 7. In the case of AT5, form II, only sharp streaks were visible. From their spacing the value of c could also be determined. Further details are given in the Results section. Simulations and drawings were prepared with the CERIUS2 program (Accelrys Inc., San Diego, CA).

3. RESULTS 3.1. Structure of Individual Duplexes. Two oligonucleotide sequences have been studied in the present paper: d(ATATATATAT), abbreviated AT5; d(CGATATATAT), abbreviated CGAT4.

Figure 1. Sequence and organization of the oligonucleotide decamers used in the present study. Individual AT5 molecules pair in opposite directions. They may form standard duplexes as shown in AT5 (Form I). Consecutive duplex molecules are stacked and form a continuous helical structure. In the case of AT5 (Form II), the molecules pair in a random staggered fashion, with a variable displacement of individual molecules, as indicated by arrows. In the case of AT5 (Form III) and CGAT4, only a unique staggered organization is found.

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These oligonucleotides have a self-complementary sequence and form DNA duplexes as shown schematically in Figure 1. In the case of CGAT4 the duplexes have sticky ends. As a result, they form a continuous DNA duplex with periodic interruptions every ten bases. The AT5 case is more complicated since individual molecules may pair in a staggered fashion as shown in Figure 1. In Form I each oligonucleotide pairs with an identical molecule so that individual duplexes of ten base pairs are formed. They are stacked in order to build a pseudocontinuous helix. This structure has been found in the presence of pentamidine and will be discussed below. In Form III individual molecules are displaced two bases with respect to its partner so that a continuous structure similar to CGAT4 is formed. In Form II random mutual displacements are present so that a mixture of similar structures coexist in the crystal. The basic duplex structure of the two decamers used in this study is shown in Figure 2. A standard DNA structure with Watson-Crick base pairs is presented. However, distortions of this basic structure are found in the actual coiled coils, as will be apparent below. Also, in some cases a partial transition to Hoogsteen base pairing may occur.22 3.2. Geometry of Coiled Coils. The simplest arrangement of a continuous stack of oligonucleotide duplexes is the HASO structure (helical arrangement of stacked oligonucleotides). It has been described in detail elsewhere.23 An example is shown in Figure 3. Each duplex rotates an angle Ω with respect to its neighbor so that a helical stack is generated. A HASO structure such as that shown in Figure 3 may also be considered a coiled coil with radius 0. The geometry of the coiled coil can be approximated as a series of rods of length l, which represent the axis of each duplex, as is schematically indicated in Figure 4. Neighbor rods form an angle θ among themselves and an angle β with the vertical direction parallel to the axis of the coil. In the projection each rod spans an angle R. As we will see below, from the experimental diffraction patterns we can determine the angle β, the pitch of the coiled coil, and the number N of duplexes in one turn of the coil. From

Figure 2. Molecular models of AT5 (Form I), AT5 (Form III), and CGAT4. Adenine in red, thymine in blue, guanine in green, and cytosine in yellow. 5219

dx.doi.org/10.1021/ie101328m |Ind. Eng. Chem. Res. 2011, 50, 5218–5224

Industrial & Engineering Chemistry Research

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Figure 4. Geometrical model of coiled coils of radius R. Each rod of length l represents the axis of one individual duplex. They are inclined an angle β with respect to the vertical axis of the coiled coil. Each duplex spans an angle R on projection. Consecutive duplexes are related by a kink angle θ and torsion τ.

Figure 3. Model of the HASO structure. Identical duplexes (shown in different colors) are stacked. Each duplex is rotated an angle Ω with respect to its neighbor. This rotation is equivalent to the angle τ in the coiled coils.

simple trigonometric calculations we can determine all the geometrical parameters of the coiled coil: l cos β ¼ p=N

ð1Þ

R ¼ 360=N

ð2Þ

R ¼ l sin β=2 sinðR=2Þ

ð3Þ

However, the molecular interactions which determine the formation of a coiled coil structure are the kink angle θ between neighbor duplexes and the torsion angle τ between consecutive duplexes also represented in Figure 4. They can be calculated as cosðθ=2Þ ¼ sin β sinðR=2Þ

ð4Þ

ð5Þ cos τ ¼ 2½cosðR=2Þ=sinðθ=2Þ2 - 1 De Luchi has described in detail18 the derivation of these equations. Both angles θ and τ can be calculated from the experimental results under the assumption that the rods are not intrinsically bent and that both ends of each duplex have an

identical geometry. As we will see below, these two assumptions are only approximate and may not apply in some cases. In the limiting case of the HASO structure, β = 0° and θ = 180°. The obvious result is that R = 0 and τ = R. A further limitation of these calculations is that it is not possible to directly determine if the coil will be right-handed or left-handed, which depends on whether the torsion angle τ is positive or negative. Additional hypothesis must be made as we will show below. 3.3. Structure of CGAT4. Diffraction patterns of this oligonucleotide are shown in Figure 5. The stacking reflections are clearly inclined at an angle β of about 25°-28° in different experiments, with 22-23 duplexes per helical turn of the coiled coil. The structural parameters are given in Table 1. The diffraction pattern shows a high crystallinity, and no horizontal streaks are present, which might indicate screw disorder, as had been previously observed in AT616 and CGAT5.17 Further examples of screw disorder will be shown below for the case of AT5. However, the resolution is limited to about 6-7 Å, probably due to the high hydration of the structure (see below) and the large value of the c parameter of the unit cell. As a result, it was not possible to obtain results at the atomic level; only its overall organization could be determined. The related coiled coil formed by CGAT5 was determined to be right-handed.17 In that case the value of τ was found to be 57°. CGAT4 is shorter, with two A 3 T pairs less. The average value of a DNA helical step is about 36°. Therefore, the value of τ is expected to be τ = 57°-72° = -15°. On the other hand, its absolute value can be calculated from eq 5 to be 13.8°, which is close to the expected value. We can therefore conclude that in CGAT4 the parameter τ is negative and the coiled coil is lefthanded. Thus, the change from CGAT5 to CGAT4 results in a different chirality of the coiled coil. This is not surprising since another decamer, AT5 in the presence of pentamidine, also shows unequivocally a left-handed coiled coil.22 The dimensions of the unit cell of CGAT4 and its symmetry features indicate that there are two coiled coils in the unit cell. The volume occupied by a base pair in the unit cell can be calculated to be 2300 Å3, a value much higher than what is found in the usual oligonucleotide crystals, which is about 1400 Å3. This observation indicates that the crystals are highly hydrated. Inspection of the patterns shown in Figure 5 demonstrates that, in the direction of the c* axis, only reflections with an even index are present. This feature indicates that there is a screw axis 5220



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Figure 5. An example of the diffraction patterns obtained from CGAT4 crystals. The axis of the crystal in reciprocal space are indicated by a*, b*, and c*. On the left is shown a 15° oscillation pattern. The inclination of the broad stacking reflection is clearly apparent. On the right are shown two 5° oscillation patterns of the central region which approximately correspond to the a*c* and a*b* diffraction planes. In the meridional region the (0 0 46) reflection is indicated. Only spots with l = even number appear in this region. Note the closer spacing of spots in other regions of the pattern (empty arrow).

Table 1. Structural Parameters of Coiled Coilsa N

β

22-23 23-24

25°-28° 25°

47 52

crystalline crystalline

P1

14-19

8°-24°

10-40

screw disorder

P6522

12

42.5

crystalline

oligonucleotide

unit cell (Å)

space group

CGAT4 AT5 3 Form III AT5 3 Form II AT5 Form Ib

a = 30.5; b = 49.4; c = 646/661 a = 29.0; b = 49.2; c = 679

P21221 P21221

a = b = 28.8; c = 467/550 a = b = 27.8; c = 312

R (Å)

comments

N = number of duplexes in one turn of the coiled coil. β = inclination of the coiled coil. R = radius of the coiled coil. b Form I is found in the presence of pentamidine.22 a

in this direction. This observation, as well as the distribution of intensities in the 001 plane, justifies our assignment of the P21221 space group. Another feature of the diffraction pattern is the absence of reflections in the c* direction, in the region around (0 0 l) = (0 0 23). The latter spacing corresponds to the projection of one duplex on the axis of the coiled coil. Therefore, it appears that the repeat unit in this direction corresponds to one-half of a duplex. This observation indicates that the two coiled coils in the unit cell are displaced by one-half duplex, as shown in Figure 6. This

regular arrangement of neighbor coils is probably due to ionmediated interactions among them. 3.4. Structure of AT5. Crystals of this oligonucleotide have a high tendency for screw disorder. Only in some cases a crystalline structure was found, as in Form III, which is practically isomorphous with CGAT4. The diffraction pattern is presented in Figure 7a. The crystallographic parameters, given in Table 1, are practically identical in both cases. In the general case (Form II), the diffraction patterns showed a high degree of screw disorder, as is apparent in Figure 7b,c. They are similar to the previous 5221



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Figure 6. A group of neighbor duplexes of CGAT4. They are displaced by one-half of their length in the vertical direction. As a result, the 00l reflections are visible only when l = 46n and neighbor l values, where n is a whole number. Each individual duplex is presented in a different color.

Figure 7. Diffraction patterns obtained from AT5. In (a) is shown a rotation image (15°) of Form III; it is practically isomorphous with CGAT4. The other two patterns correspond to Form II. A 15° rotation image is shown in (b). A 2° rotation image is presented in (c): it shows a detailed view of the central part of the diffraction pattern. In this particular sample the coiled coil has 19 duplexes per turn.

results we obtained with AT6.16 However, in the present case, the dimensions of the coiled coil vary significantly in different samples, within the limits indicated in Table 1. Apparently, individual samples present a variable degree of staggering, as indicated by the arrows in Figure 1. A particular case is the complex of AT5 with the pentamidine drug (Form I), which we have reported elsewhere.22 It forms a left-handed coiled coil with 12 molecules per turn. The pentamidine molecule sits in the minor groove and forms cross-links with neighbor molecules in the crystal. As a result, the structure is highly crystalline. The two ends of each individual duplex have a different structure so that the general formulas given in section 3.1 cannot be applied in this case.

4. OVERVIEW AND DISCUSSION The results we have presented together with previous studies16-18 show that alternating AT sequences, either alone

or with CG sticky ends, have a tendency to form coiled coils. Oligonucleotides with this composition and with different lengths also show this tendency.18 Thus, the coiled coil structure, usually associated with proteins, is also available to DNA oligonucleotides. No coiled coil has been described in oligonucleotides with a mixed sequence,24 with the exception of a few particular cases,25 which we will describe below. Alternating AT sequences may show both Hoogsteen26 and Watson-Crick base pairs, which demonstrates that they are very versatile. The AT5 coiled coil in the presence of pentamidine22 presents a mixture of both kinds of base pairs, whereas CGAT5 has the AT5 segment in the Hoogsteen conformation.17 The shorter sequence CGAT2 forms a complex set of helical structures in which the AT segment forms Watson-Crick base pairs (work in progress). Thus, the tendency to form coiled coils by alternating AT sequences may be attributed to the versatility of these sequences, which may adopt different structures in different environments. 5222



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Figure 8. Examples of different coiled coils as described in the text. The radius R and pitch p are indicated in each case (Å).

The presence of a CG sticky end is not a requirement since we have found coiled coils in its absence. In fact, a mixed sequence oligonucleotide with a CG sticky end does not form coiled coil structures.27 Coiled coils with very different geometries can be obtained from a variety of oligonucleotides. As a conclusion, we present in Figure 8 a few examples. The radius and pitch of each coiled coil are given in the figure: a A HASO structure obtained from octamer AAATATTT with 11 duplexes per turn.28 Each duplex is shown in a different color. b A unique case of a coiled coil25 formed by CGCTAGCG. In this case the ends of the duplexes interact externally, without continuous stacking, as is found in many other oligonucleotide crystals.29 In this structure and in the following case (c), the terminal CG base pairs are shown in green and the rest in red. c The CGAT5 structure.17 d The CGAT4 structure described in this paper, with two coiled coils in the unit cell. The unit cell is indicated by blue dashed lines. e The AT5-pentamidine coiled coil22 (Form I). f A coiled coil formed by a mixed sequence icosamer duplex in the presence of protein.30 In this case the coiled coil is due to bends in the central part of the oligonucleotide duplex induced by a specific protein. This example illustrates that new geometries may be discovered when further protein/ DNA complexes are studied. The nucleosome, for example, may also be considered a coiled coil with a very different geometry,31 although in this case a single DNA molecule forms a superhelix. g A simplified view of a group of coiled coils in a crystal. They pack like a bundle of spaghetti.

’ AUTHOR INFORMATION Corresponding Author

*Tel.: 34 þ 934016688. E-mail: [email protected]; [email protected].

’ ACKNOWLEDGMENT We are very thankful to Ms. Trinidad Sanmartín for her valuable and careful collaboration in the preparation of many crystals used in this study. We thank the staff of the BM16 Spanish beamline at the ESRF (Grenoble) for assistance in data collection. We are also thankful to Drs. Valentina Tereshko, Isabel Uson, and Ignacio Fita for help and advice throughout this work. It has been supported by grants BFU2006-04035 and BFU2009-10380 from the Ministerio de Ciencia y Tecnología and 2001 SGR 00250 and 2009 SGR 1208 from the Generalitat de Catalunya. ’ REFERENCES (1) Puigjaner, L. C.; Subirana, J. A. Low-Angle X-ray Scattering by Disordered and Partially Ordered Helical Systems. J. Appl. Crystallogr. 1974, 7, 169. (2) Puigjaner, L.; Laínez, J. M.; Rodrigo, C. Tracking the Dynamics of the Supply Chain for Enhanced Production Sustainability. Ind. Eng. Chem. Res. 2009, 48, 9556. (3) Seeman, N. C. At the Crossroads of Chemistry, Biology, and Materials: Structural DNA Nanotecnology. Chem. Biol. 2003, 10, 1151. (4) Lin, C.; Liu, Y.; Yan, H. Designer DNA Nanoarchitectures. Biochemistry 2009, 48, 1664. (5) Douglas, S. M.; Dietz, H.; Liedl, T.; H€ogberg, B.; Graf, F.; Shih, W. M. Self-assembly of DNA into nanoscale three-dimensional shapes. Nature 2009, 459, 414. 5223


Industrial & Engineering Chemistry Research (6) Wang, R.; Liu, W.; Seeman, N. C. Prototyping Nanorod Control: A DNA Helix Sheathed within a DNA Six-Helix Bundle. Chem. Biol. 2009, 16, 862. (7) Zheng, J.; Birktoft, J. J.; Chen, J.; Wang, T.; Sha, R.; Constantinou, P. E.; Ginell, S. L.; Mao, C.; Seeman, N. C. From molecular to macroscopic via the rational design of a self-assembled 3D DNA crystal. Nature 2009, 461, 74. (8) Dietz, H.; Douglas, S. M.; Shih, W. M. Folding DNA into Twisted and Curved Nanoscale Shapes. Science 2009, 325, 725. (9) Aldaye, F. A.; Palmer, A. L.; Sleiman, H. F. Assembling Materials with DNA as the Guide. Science 2008, 321, 1795. (10) Sharma, J.; Chhabra, R.; Cheng, A.; Brownell, J.; Liu, Y.; Yan, H. Control of Self.Assembly of DNA Tubules Through Integration of Gold Nanoparticles. Science 2009, 323, 112. (11) Yashima, E.; Maeda, K.; Furusho, Y. Single- and DoubleStranded Helical Polymers: Synthesis, Structures and Functions. Acc. Chem. Res. 2008, 41, 1166. (12) Pokroy, B.; Kang, S. H.; Mahadevan, L.; Aizenberg, J. SelfOrganization of a Mesoscale Bristle into Ordered Hierarchical Helical Assemblies. Science 2009, 323, 237. (13) Lee, C. C.; Grenier, C.; Meijer, E. W.; Schenning, A. P. H. J. Preparation and characterization of helical self-assembled nanofibers. Chem. Soc. Rev. 2009, 38, 671. (14) Dong, H.; Paramonov, S. E.; Hartgerink, J. D. Self-Assembly of R-Helical Coiled Coil Nanofibers. J. Am. Chem. Soc. 2008, 130, 13691. (15) Testa, O. D.; Moutevelis, E.; Woolfson, D. N. CCþ: a relational database of coiled-coil structures. Nucleic Acids Res. 2009, 37, 315. (16) Campos, J. L.; Urpí, L.; Sanmartín, T.; Gouyette, C.; Subirana, J. A. DNA coiled coils. Proc. Natl. Acad. Sci. USA 2005, 102, 3663. (17) De Luchi, D.; Tereshko, V.; Gouyette, C.; Subirana, J. A. Structure of the DNA coiled-coil formed by d(CGATATATATAT). ChemBioChem 2006, 7, 585. (18) De Luchi, D.Ph.D. Thesis. Available at http://www.tdx.cat/ TDX-1106108-105434, 2008. (19) Subirana, J. A.; Creixell, M.; Baldini, R.; Campos, J. L. Macroscopic DNA Helices. Biomacromolecules 2008, 9, 6–8. (20) Otwinowski, Z.; Minor, W. Processing of X-Ray diffraction data collected in oscillation mode. Methods Enzymol. 1997, 276, 307. (21) Leslie, A. G. W. Recent changes to the Mosflm package for processing film and image plate data. Joint CCP4 þ ESF-EAMCB Newsletter Protein Crystallogr. 1992, 26. (22) Moreno, T.; Pous, J.; Subirana, J. A.; Campos, J. L. Coiled-coil conformation of a pentamidine-DNA complex. Acta Crystallogr. 2010, 66D, 25. (23) Campos, L.; Valls, N.; Urpí, L.; Gouyette, C.; Sanmartín, T.; Richter, M.; Alechaga, E.; Santaolalla, A.; Baldini, R.; Creixell, M.; Ciurans, R.; Skokan, P.; Pous, J.; Subirana, J. A. Overview of the structure of all-AT oligonucleotides: organization in helices and packing interactions. Biophys. J. 2006, 91, 892. (24) Nucleic Acid Database: http://ndbserver.rutgers.edu. (25) Tereshko, V.; Urpí, L.; Malinina, L.; Huynh-Dinh, T.; Subirana, J. A. Structure of the B-DNA Oligomers d(CGCTAGCG) and d(CGCTCTAGAGCG) in New Crystal Forms. Biochemistry 1996, 35, 11589. (26) Abrescia, N. G. A.; Thompson, A.; Huynh-Dinh, T.; Subirana, J. A. Crystal structure of an antiparallel DNA fragment with Hoogsteen base pairing. Proc. Natl. Acad. Sci. USA 2002, 99, 2806. (27) Qiu, H.; Dewan, J. C.; Seeman, N. C. A DNA Decamer with a Sticky End: The Crystal Structure of d-CGACGATCGT. J. Mol. Biol. 1997, 267, 881. (28) Valls, N.; Richter, M.; Subirana, J. A. Structure of a DNA duplex with all-AT base pairs. Acta Crystallogr. 2005, D61, 1587. (29) Dickerson, R. E.; Goodsell, D. S.; Kopka, M. L.; Pjura, P. E. The effect of crystal packing on oligonucleotide double helix structure. J. Biomol. Struct. Dyn. 1987, 5, 557. (30) Schumacher, M. A.; Glover, T. C.; Brzoska, A. J.; Jensen, S. O.; Dunham, T. D.; Skurray, R. A.; Firth, N. Segrosome structure revealed by a complex of ParR with centromere DNA. Nature 2007, 450, 1268.

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(31) Luger, K.; M€ader, A. W.; Richmond, R. K.; Sargent, D. F.; Richmond, T. J. Crystal structure of the nucleosome core particle at 2.8Å resolution. Nature 1997, 389, 251.

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DNA Coiled Coil Superstructures in Oligonucleotide Crystals

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