Self-Assembly of Frayed Wires and Frayed-Wire Networks

NANO LETTERS

Self-Assembly of Frayed Wires and Frayed-Wire Networks: Nanoconstruction with Multistranded DNA

2002 Vol. 2, No. 4 269-274

Michael A. Batalia,†,| Ekaterina Protozanova,‡,§ Robert B. Macgregor, Jr.,‡ and Dorothy A. Erie*,† Department of Chemistry, UniVersity of North Carolina at Chapel Hill, CB#3290, Chapel Hill, North Carolina 27599, and Department of Pharmaceutical Sciences, Faculty of Pharmacy, UniVersity of Toronto, 19 Russell Street, Toronto ON M5S 2S2, Canada Received November 20, 2001; Revised Manuscript Received January 18, 2002

ABSTRACT New types of self-assembled 2-D and 3-D DNA networks are presented and characterized using scanning force microscopy. The building blocks of these networks are guanine-rich DNA sequences, d(A15G15) and biotinylated-d(A15G15). These sequences self-assemble into high molecular weight structures, frayed wires, that are composed of a multistranded guanine core with single-stranded adenosine tracts emanating from the core at ∼6 nm intervals. Avidin peroxidase was attached to the biotinylated frayed wires with retention of activity, demonstrating that these structures can be used as scaffolds to support multiple copies of non-DNA molecules. Finally, the frayed wires were linked together with CT15 or T15 to form large 2- and 3-dimensional networks.

The nascent field of nanotechnology provides an exciting view of the future. The ultimate goal of this field is engineering and construction at the atomic scale. The possible uses for nanodevices range from electrical circuits to computers to biological devices and sensors. The “bottomup” approach to nanotechnology involves a combination of chemistry, molecular biology, atomic force manipulation, and materials science for constructing nanometer-scale structures, components, and devices.1 In such an approach, self-assembly of the molecules (or components) is highly desirable, because it can permit the formation of large networks with relative ease. DNA is an excellent molecule for the formation of macromolecular networks because it is easy to synthesize, has a high specificity of interaction, and is conformationally flexible. The complementary base-paring properties of DNA have been used to make 2-dimensional crystals2-6 and prototypes of DNA computers7,8 and electric circuits.9 DNA, however, forms not only two-stranded secondary structures, but also three-, four-, and greater than four-stranded struc* Corresponding author. E-mail: [email protected]. Phone: (919) 962-6370. Fax: (919) 966-3675. † University of North Carolina at Chapel Hill. ‡ University of Toronto. § Present Address: Boston University, Boston, MA 02215. | Present address: Office of Technology Transfer, Campus Box 8210, 2401 Research Dr., North Carolina Sate University, Raleigh, NC 276958210. 10.1021/nl015672h CCC: $22.00 Published on Web 02/22/2002

© 2002 American Chemical Society

tures. In this work, we take advantage of these diverse properties of DNA. We present scanning force microscope (SFM) images of a new type of self-assembled 2- and 3-dimensional DNA networks and demonstrate that enzymes can be attached to these networks and still maintain their activity. The building blocks of these networks are guaninerich sequences, such as d(A15G15), which will assemble into high molecular weight multistranded structures known as frayed wires.10 These assemblies are highly thermostable structures containing a multistranded guanine core with single-stranded DNA emanating form the core.11 The singlestranded “arms” allow frayed wires to be linked together with a complementary DNA strand to form networks. Methods. The frayed wires were assembled as described previously.10 Briefly, lyophilized oligonucleotides were diluted to a concentration of 1 µM in a buffer composed of Tris-borate pH 8.0 with 5 mM MgCl2. Oligonucleotides d(A15G15) were assembled into frayed wires by heating to approximately 100 °C and then allowing the sample to cool to 4 °C overnight. Prior to scanning force microscopy, samples were briefly centrifuged at 10 000 g (approximately 5 s) to remove insoluble aggregates. The centrifuged samples were deposited onto freshly cleaved mica, rinsed several times with deionized water, blown dry with N2(g), and imaged in air under environmental control. Imaging was

(a)

Figure 1. (a) SFM image of frayed wires (1 µm × 1 µm). Frayed wires are observed to be mostly linear structures with heights of approximately 2 nm and lengths ranging from 5 to 200 nm. This height is consistent with the frayed wire cores containing four or more strands of DNA. (The measured height of double- and triple-stranded DNA when imaged in air is typically 0.5 to 1 nm12,13,33). (b) Highresolution image of a single frayed wire. Inset: section analysis showing the height along the long axis of the frayed wire. Most of the frayed wires exhibit striations along the short axis, independent of their orientation relative to the scan axis. In some cases, these striations are perpendicular to the short axis and in other cases, they are inclined toward the long axis. Statistical analysis of a large subset of frayed wires indicates that their average height (indicated by the gray arrows) is 2.1 ( 0.1 nm and the period between striations is 6.7 nm ( 1.1 nm (indicated by black arrows). (c) Structural model of the frayed wires. This model represents one possible conformation of the frayed wires. A portion of the A-tracts is depicted as unstructured in this model based on circular dichroism (CD) studies.11,16 The CD studies indicate that while the A-tracts are important for concatenation, they are free to pair with complementary oligonucleotides.

performed with a Nanoscope IIIa instrument (Digital Instruments, Santa Barbara, CA) using tapping mode in air. Nanosensor Pointprobe noncontact/tapping mode sensors (Molecular Imaging, Inc., Phoenix, AR) with spring constants of 48 N/m and resonance frequencies of 190 kHz were used for all imaging. A Phenomenex Biosep-SEC 3000 size-exclusion column mounted on a Waters HPLC was used for all chromatographic runs. The column was equilibrated with the annealing buffer and calibrated using molecular weight standards (BioRad gel filtration standards). Avidin peroxidase was purchased from Sigma Chemical and purified over the column. The purified avidin peroxidase eluted from the column at 12.5 min. Limiting amounts of purified avidin peroxidase were added to either the unbiotinylated or the biotinylated frayed wires, and these mixtures were separated over the column. For activity assays, void-volume fractions were collected from each of the chromatographic runs shown in Figure 3a. Prior to the activity assay, the samples were concentrated in Centricon microconcentrators. The sample concentration was measured by UV absorption at 264 nm. Aliquots of each complex were added to an indicator buffer composed of 100 270

mM sodium citrate, 0.06% hydrogen peroxide, and 1% of a saturated solution of 2,2′-azino-bis(3-ethylbenzthiazole-6sulfonic acid (ABTS), and the absorption at 595 nm was monitored to follow activity (assay adapted from Sigma’s standard assay). Results and Discussion. DNA frayed wires are selfassembling structures originating from spontaneous association of oligodeoxyribonucleotides possessing long tracks of guanines. The assembly of frayed wires simply requires heating the appropriate guanine-rich DNA oligonucleotides to 100 °C and allowing them to cool slowly. SFM was used to visualize the structures formed using this method. As can be seen from inspection of Figure 1a, d(A15G15) selfassembles into a heterogeneous population of mostly linear molecules. The average height of the aggregated structures is 2.1 ( 0.1 nm, which is approximately three times the measured height of double-stranded DNA12,13 (Figure 1b), suggesting a structure containing at least four DNA strands. These results are similar to those reported for “G-wires”, an aggregated form of d(G4T4G4).14,15 Examination of multiple images from different experiments revealed that self-assembly of the frayed wire population is robust. Particle shape analysis was performed on several hundred molecules from Nano Lett., Vol. 2, No. 4, 2002

multiple experiments using the NIH Image program (developed at the National Institutes of Health, 1999). The length distribution was observed to be a normal population that ranged from 5 to ∼200 nm with a mode of 20 nm. Frayed wire area varies linearly with length, indicating a constant diameter for the assemblies. While most of the frayed wires are linear, many have irregularities, such as 90° bends and disordered ends. Analysis of high-resolution images reveals striations perpendicular to the major axis with a periodicity of 6.7 ( 1.1 nm (Figure 1b). We suggest that the striations, or peaks, correspond to the A-tracts of the oligonucleotide building blocks. Consistent with this suggestion, solution experiments have demonstrated that the non-guanine portions of the oligonucleotides in frayed wires are single stranded.11,16 Taken together, these results lead us to propose a structural model in which the core is formed by association of multiple strands of guanine residues and the 5′ to 3′ concatanation of the cores is mediated via interactions with the “singlestranded” A-tracts. (Figure 1c). The structures can be likened to a barbed wire, with the A-tracts forming the barb. In this model, the separation of A-tracts would be fifteen G-residues. Any staggering of the oligonucleotide strands relative to one another would result in the A-tracts being closer together. Based on our SFM data, the rise per guanine would be 0.45 nm (6.7 nm/15 G-residues), if all the molecules were perfectly aligned and oriented in an end-to-end fashion with respect to each other. This rise is larger than that found in crystal structures of tetraplex DNA (0.34 nm).17 This larger rise presumably arises from differences between guanineguanine interactions of frayed wires and tetraplex DNA,18 or from gaps between concatamers (Figure 1c). While d(A15G15) forms discrete species that can be resolved on native and denaturing electrophoresis gels,10 d(AG15) was found to form only high molecular weight structures that were too large to be resolved by electrophoresis. SFM revealed that these molecules form a gel-like meshwork of aggregated oligonucleotides. Surface analysis of these guanine nucleotide gels showed that they are very rough with heights between 1.0 and 1.4 nm and a large surface area (data not shown). The height and morphological characteristics of these gels are not consistent with the formation of frayed wires. This result in conjunction with other solution studies10,11,16 indicates that the non-guanine portion of the oligonucleotide must be a critical length to form frayed wires. Although the non-guanine portion of the oligonucleotides appears to be essential for the formation and solubility of frayed wires, addition of oligonucleotides that are partially complementary to d(A15G15), such as d(CT15) and d(C7T15), does not disrupt the formation or stability of frayed wires.10 In fact, it was found that the addition of these oligonucleotides shifted the distribution of species to higher molecular weights.10,19 We have used SFM to characterize the frayedwire structures formed in the presence of d(CT15) and d(T15). In experiments with the complementary strand d(CT15), complexes were formed by two methods. The first method consisted of annealing the d(A15G15) and d(CT15) together Nano Lett., Vol. 2, No. 4, 2002

from 100 °C to 4 °C. The second method consisted of annealing d(CT15) to preformed frayed wires at 10 °C for 3 h or overnight. Addition of either d(T15) or d(CT15) resulted in assembly of frayed wire networks (Figure 2a). Preformed frayed wires that were incubated with d(CT15) at 10 °C formed networks containing frayed wires of varying lengths (data not shown), undoubtedly because the starting frayed wires have a broad distribution of lengths. This method was not as effective in generating complicated networks relative to annealing frayed wires in the presence of d(CT15). In the latter case, the frayed wires were observed to be uniform 20-25 nm repeats (Figure 2b and 2c). Connections between frayed wires were found to be oriented mostly along the long axis of the molecules (Figure 2b). These connections, presumably duplex or triplex DNA (Figure 2d), are sufficiently flexible to allow multiple conformations of the assemblies when the number of connections is small, such as at the ends of the frayed wires. As the number of connections increases between frayed wires, the networks preferentially form runs of parallel frayed wires (Figure 2b). Networks can also form mixed parallel/perpendicular arrays. Density between 0.6 and 1.2 nm in height was consistently observed between frayed wires and was spaced at approximately 6-nm intervals (Figure 2e). These results are consistent with the frayed wires being linked together via double or triple-stranded helices (Figure 2d) and support our proposal that the peaks observed in the frayed wires (Figure 1b) are the A-tracts. Finally, frayed wire networks are not limited to two dimensions. Networks two and three layers thick were observed in the same samples as single monolayer networks (Figures 2a and 2f), demonstrating that it is possible to form 3-dimensional networks of frayed wires. These 3-dimensional networks were observed to have irregular shapes, yet many molecules had similar features within the population. Significantly, these large 3-dimensional networks were observed only in images of samples that contained both d(A15G15) and d(CT15) and were never observed in samples containing only d(A15G15). From our data, it is evident that frayed wires can selfassemble to form large 2- and 3-dimensional networks; however, for these networks to be useful in biotechnology, we need to be able to attach non-DNA molecules such as enzymes or fluorescent probes to them. To assess this possibility, we formed frayed wires with 5′-biotinylated d(A15G15), 5′-biotin-d(A15G15), because the biotin group can be used to link a host of molecules or other structures via a streptavidin link.20 SFM images revealed that biotinylated frayed wires are very similar to the frayed wire structures shown in Figure 1 (data not shown). To test whether biotinylated frayed wires could be used as scaffolding for enzymes or other molecules, we investigated whether avidin peroxidase could be linked to biotinylated frayed wires and still maintain its enzymatic activity. To this end, we purified commercial avidin peroxidase (Sigma) on a HPLC Biosep-SEC (size-exclusion) column with a molecular weight cutoff of 250 000 Daltons. An aliquot was collected at the center of the distribution, which eluted from the column at approximately 12.5 min (data not 271

Figure 2. SFM images of frayed wire networks. (a) 2 µm image of frayed-wire networks formed by the addition of the partially complementary oligonucleotide d(CT15) to d(A15G15) in the annealing reaction. (b) 1 µm SFM image showing parallel arrays of frayed wires. Presumably, noncovalent bonds form between the arms and the complementary d(CT15) strands to join the frayed wires. (c) Surface plot showing details of frayed wire networks. The frayed wires are very close to each other but exhibit quite a bit of flexibility. (d) Three models showing potential duplex and triplex connections between frayed wires. The frayed wires are represented by the cylinders and the possible pairings between d(CT15) and the A-tracts are shown as lines. Left: Frayed wires connected by a TAA triple strand in which the AT forms a Watson-Crick base pair and the second A forms an AA reverse Hoogsteen base pair in the major groove.34 Middle and right: Frayed wires are connected by standard B-DNA with a single d(CT15) pairing with two A-tracts. In the middle panel, d(CT15) folds back to make an antiparallel helix with the bottom A-tract. In the left panel, the d(CT15) is “linear” and the bottom A-tract folds back on itself to make an antiparallel helix. (e) Section analysis of a single frayed-wire network. A density with a height of approximately 0.9 nm is observed between frayed wires. This density is consistent with AFM measurements of duplex and triplex DNA structures.12,13,33 In addition, this particular structure allows another measurement of the periodicity of the frayed-wire arms (Figure 1b). The frayed wires are spaced approximately every 6 nm on alternate sides of the central frayed wire to which they are attached. (f) High-resolution image and section analysis of 3-dimensional networks of frayed wires. The inset shows that the three particles analyzed differ in height by integral numbers of layers. Heights are consistent with 3-dimensional networks of one (black line), two (red line), and three (green line) layers. Notably, these large structures are observed only if the frayed wires are incubated d(CT15) or dT15. The structures seen in the images shown in this figure are typical of those seen in multiple depositions. 272

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Figure 3. (a) Chromatograms of solutions of avidin peroxidase with unbiotinylated frayed wires (top panel) and with biotinylated frayed wires (bottom panel). The chromatogram (top panel) of the unbiotinylated frayed (first peak; void volume) wires and avidin peroxidase (second smaller peak) shows that very little of the avidin peroxidase shifts to higher molecular weights. In contrast, the chromatogram of the biotinylated frayed wires with avidin peroxidase (bottom panel) shows a complete shift of the avidin-peroxidase peak to the void volume. (b) Activity assays of void-volume fractions that were collected from the chromatographic runs shown in (a). Absorption at 595 nm is plotted as a function of time to monitor activity. Curve A is substrate alone, curves B and C are substrate with the void volume peaks from the chromatogram in the top panel (unbiotinylated) and the bottom panel (biotinylated), respectively. The activity of the void volume peak from the biotinylated frayed wires (bottom panel in a) is significantly greater than that from the unbiotinylated frayed wires (top panel in a).

to the unbiotinylated frayed wires, as expected. In contrast, the chromatogram of the biotinylated frayed wires shows a complete shift of the avidin peroxidase peak into the void volume. Fractions of the void volumes from these chromatographic runs were collected and tested for enzymatic activity. Inspection of Figure 3b shows that the void-volume peak of the biotinylated frayed wires exhibits a high level of enzymatic activity, while that of the unbiotinylated frayed wires shows very little activity. The activity associated with unbiotinylated frayed wires is attributed to free avidin peroxidase that cannot be completely separated from the frayed wires because there is some overlap of the two chromatographic peaks. These results demonstrate that it is possible to use frayed wires as a scaffold upon which enzymes or other molecules of interest can be attached. SFM images of the biotinylated frayed wires conjugated to the avidin-peroxidase show bent and cross-linked frayed wires (data not shown). Advanced molecular manufacturing will require robust, malleable building blocks.20 To reach this goal, numerous systems have already been developed including dendrimers, rotaxanes, and carbon nanotubes.21-31 We have demonstrated that guanine-rich DNA oligomers can self-assemble into regular, stable, extreme molecular weight species that can be functionalized and conjugated to form 2- and 3-dimensional “active” structures. Such structures should be a powerful tool in biotechnology. For example, because many copies of a non-DNA molecule can be attached to a single frayed wire or a frayed-wire network, they could be used to amplify extracellular signals via antennae-like arrays, generate multienzyme complexes, and potentially link cells together. Finally, this system is especially attractive due to the low cost of materials, ease of synthesis, and proliferation of scanning probe microscopes and nanomanipulators. Numerous novel structures could be obtained by varying the oligomer length and composition, in addition to controlling the annealing conditions. Acknowledgment. We thank Linda Spremulli, Matt Redinbo, and Charlie Goss for helpful input. M.A.B. was supported by the UNC Lineberger Comprehensive Cancer Center Postodoctoral Fellowship and the Department of Defense Army Breast Cancer Research Fellowship. E.P. was supported as a PMAC/MRC Scholar. This work was supported by NIH grants GM54136 and ES09895 (D.A.E.) and the Natural Science and Engineering Council of Canada and Glaxo Wellcome Canada (R.B.M.). References

shown). On the same column, unbiotinylated frayed wires and biotinylated frayed wires eluted in the void volume (Figure 3a). Equal molar amounts of purified avidin peroxidase were added to aliquots of frayed wires and biotinylated frayed wires, and the resulting complexes were passed over the column referenced above (Figure 3a). For the sample with standard (unbiotinylated) frayed wires, the chromatogram shows two peaks, one corresponding to the frayed wires and one corresponding to avidin peroxidase. This result indicates that most of the avidin peroxidase does not bind Nano Lett., Vol. 2, No. 4, 2002

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