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DNA-Driven Assembly of Bidisperse, Micron-Sized Colloids Valeria T. Milam,†,‡ Amy L. Hiddessen,†,‡ John C. Crocker,†,‡ David J. Graves,† and Daniel A. Hammer*,†,‡,§ Department of Chemical and Biomolecular Engineering, Institute for Medicine and Engineering, and Department of Bioengineering, University of Pennsylvania, Philadelphia, Pennsylvania 19104 Received March 3, 2003. In Final Form: July 11, 2003 Oligonucleotides are unique chemical moieties that can serve as a useful assembly tool. Unlike most bioadhesion molecules that bind together with high affinity, the attraction between complementary DNA strands can vary greatly depending on strand characteristics (e.g., length and sequence choice) and solution conditions (e.g., ionic strength and temperature). We have studied DNA-mediated assembly of micronsized, bidisperse mixtures using primarily optical and confocal microscopy. To increase hybridization efficiency between complementary strands, DNA sequences were designed to have a low self-affinity that minimizes intrastrand loop and hairpin formations. Single strands of biotinylated DNA were tethered to NeutrAvidin-coated 1.10 and 1.87 micron beads. The resulting oligonucleotide density on the large bead surface was quantified using flow cytometry. In binary mixtures, we found we could vary the degree of binding between complementary beads depending on the number of matching pairs and the ionic strength of the solution. We have also observed a variety of colloidal structures such as chains of alternating large and small particles by exploring additional experimental variables such as particle number ratio and volume fraction.
Introduction Numerous studies have investigated the phase behavior and structure of colloidal suspensions in which interactions between particles are nonspecific. Nonspecific colloidal forces such as van der Waals and electrostatic forces arise from the bulk material properties of suspensions. Crosslinking of biological complexes such as carbohydrateselectin, avidin-biotin, or antigen-antibody bonds, on the other hand, originates from specific recognition between particular binding sites. The range and magnitude of nonspecific forces cause subtle variations in the fractal nature1 and mechanical strength2,3 of a colloidal gel comprised of attractive particles or in the lattice spacing between particles in a colloidal crystal.4 Monodisperse hard-sphere suspensions exhibit coexisting fluid and crystal phases at 0.494 < φ < 0.545.5 For 0.545 < φ < 0.58, only the crystalline phase exists. The net increase in free volume as the particles pack tightly into arrays makes this ordering transition an entropically favorable process. Size ratio and composition effects provide bidisperse systems with even richer phase behavior. In bidisperse suspensions of high size asymmetry, depletion effects6-9 * To whom correspondence should be addressed. Current address: Department of Bioengineering, University of Pennsylvania, 120 Hayden Hall, 3320 Smith Walk, Philadelphia, PA 19104. Phone: 215-573-6761. Fax: 215-573-2071. E-mail: hammer@ seas.upenn.edu. † Department of Chemical and Biomolecular Engineering. ‡ Institute for Medicine and Engineering. § Department of Bioengineering. (1) Martin, J. E.; Wilcoxon, J. P. Phys. Rev. A 1989, 39, 252-258. (2) Rueb, C. J.; Zukoski, C. F. J. Rheol. 1997, 41, 197-218. (3) Guo, J. J.; Lewis, J. A. J. Am. Ceram. Soc. 1999, 82, 2345-2358. (4) Matsuoka, H.; Harada, T.; Kago, K.; Yamaoka, H. Langmuir 1996, 12, 5588-5594. (5) Pusey, P. N.; van Megen, W. Nature 1986, 320, 340-342. (6) Asakura, S.; Oosawa, F. J. Polym. Sci. 1958, 33, 183-192. (7) Vrij, A. Pure Appl. Chem. 1976, 48, 471-483. (8) Walz, J. Y.; Sharma, A. J. Colloid Interface Sci. 1994, 168, 485496. (9) Chatterjee, A. P.; Schweizer, K. S. J. Chem. Phys. 1998, 109, 10464-10476.
arising from dilute additions of small particles10 or free polymer11 can expand the fluid-crystal coexistence region of the large, hard spheres. In addition to coexisting phases such as fluid-crystal, binary systems with low size ratios can also arrange into superlattice crystal structures such as AB13 and AB2.12-18 As in the monodisperse case, colloidal crystallization in these binary colloidal suspensions is an entropically driven process that depends on the suspension composition. Attractive specific forces, on the other hand, could potentially drive colloidal crystallization at much lower volume fractions through recognition-mediated assembly. In addition, specific forces can potentially construct unique particle structures that are otherwise entropically unfavorable. Thus, great potential for novel materials can be realized using a system of colloids functionalized with biological adhesion molecules. Biomolecular recognition between antibody-antigen19 or biotin-streptavidin20,21 groups has been used to link monodisperse nanoparticles. The strong affinities between these bioconjugates resulted in irreversible aggregation of particles. The use of bid(10) Imhof, A.; Dhont, J. K. G. Phys. Rev. Lett. 1995, 75, 1662-1665. (11) Ilett, S. M.; Orrock, A.; Poon, W. C. K.; Pusey, P. N. Phys. Rev. E 1995, 51, 1344-1352. (12) Murray, M. J.; Sanders, J. V. Philos. Mag. A 1980, 42, 721-740. (13) Yoshimura, S.; Hachisu, S. Prog. Colloid Polym. Sci. 1983, 68, 59-70. (14) Bartlett, P.; Ottewill, R. H.; Pusey, P. N. Phys. Rev. Lett. 1992, 68, 3801-3804. (15) Eldridge, M. D.; Madden, P. A.; Frenkel, D. Nature 1993, 365, 35-37. (16) Hunt, N.; Jardine, R.; Bartlett, P. Phys. Rev. E 2000, 62, 900913. (17) Schofield, A. B. Phys. Rev. E 2001, 64, 051403. (18) Christianson, R. J.; Gasser, U.; Bailey, A. E.; Prasad, V.; Manley, S.; Segre, P. N.; Cipelletti, L.; Weitz, D. A.; Schofield, A. B.; Pusey, P. N.; Doherty, M. P.; Sankaran, S.; Jankovsky, A. L.; Shiley, B.; Bowen, J.; Dendorfer, K.; Eggers, J.; Koudelka, J.; Kurta, C.; Lorik, T. In preparation. (19) Shenton, W.; Davis, S. A.; Mann, S. Adv. Mater. 1999, 11, 449452. (20) Connolly, S.; Fitzmaurice, D. Adv. Mater. 1999, 11, 1202-1205. (21) Mann, S.; Shenton, W.; Li, M.; Connolly, S.; Fitzmaurice, D. Adv. Mater. 2000, 12, 147-150.
10.1021/la034376c CCC: $25.00 © 2003 American Chemical Society Published on Web 10/25/2003
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isperse biocolloids further extends the fabrication flexibility and range of material possibilities. Motivated by previous work with weakly adhesive particles serving as model cells,22,23 Hiddessen et al.24 functionalized bidisperse particles with the same carbohydrate-selectin moieties involved in transient adhesion of leukocyte cells to blood vessels. Variation in the number ratio of heterogeneous particles resulted in a variety of structures including colloidal micelles, chains, and rings. Interestingly, linkages between particles in these structures were reversible depending on the calcium concentration. Recently, reversibility has also been observed in this binary system between colloids with low surface coverage of these weakly attractive bioconjugates.25 Fifty years following Crick and Watson’s seminal paper,26 deoxyribonucleic acid is being investigated as an assembly tool by several groups. Helical formation resulting from molecular recognition between complementary DNA strands has made this macromolecule alone viable for constructing interesting objects such as tiles,27 for templating nanowires,28 and for building nanomachines such as tweezers29 and switches.30 Unlike most bioadhesion molecules such as biotin-avidin which bind together with high affinity, the attraction between complementary strands can vary greatly depending on solution conditions and strand characteristics. Though individual strands of DNA are highly charged due to its phosphate backbone, counterions from sufficient salt additions mediate an effective attraction between oligonucleotides causing hybridization via hydrogen bonding.31 In Watson-Crick base pairs, three hydrogen bonds impart greater stability in guanine-cytosine (G-C) matches than the two hydrogen bonds in adenine-thymine (A-T) matches.32 Thus, increasing the G-C content raises the melting temperature at which complementary strands dissociate. Increasing the number of base matches between strands also increases helical stability; however, flexible single strands can form loops or hairpins and compromise interstrand hybridization efficiency.33,34 DNA-mediated colloidal assembly offers a unique tool for fine-tuning the magnitude and range of interparticle forces depending on the solution conditions (e.g., ionic strength and temperature) and strand characteristics (e.g., sequence choice and number of matching base pairs). The effect of the interaction potential on theoretical phase behavior for monodisperse DNA-covered beads has been addressed in simulations by Tkachenko.35 Similar to results of previous depletion studies,10,11 Tkachenko found (22) Brunk, D. K.; Goetz, D. J.; Hammer, D. A. Biophys. J. 1996, 71, 2902-2907. (23) Hammer, D. A.; Discher, D. E. Annu. Rev. Mater. Res. 2001, 31, 387-404. (24) Hiddessen, A. L.; Rodgers, S. D.; Weitz, D. A.; Hammer, D. A. Langmuir 2000, 16, 9744-9753. (25) Hiddessen, A. L.; Weitz, D. A.; Hammer, D. A. Submitted. (26) Watson, J. D.; Crick, F. H. C. Nature 1953, 171, 737-738. (27) Winfree, E.; Liu, F.; Wenzler, L. A.; Seeman, N. C. Nature 1998, 394, 539-544. (28) Braun, E.; Eichen, Y.; Sivan, U.; Ben-Yoseph, G. Nature 1998, 391, 775-778. (29) Yurke, B.; Turberfield, A. J.; Mills, A. P., Jr.; Simmel, F. C.; Neumann, J. L. Nature 2000, 406, 605-608. (30) Mao, C.; Sun, W.; Shen, Z.; Seeman, N. C. Nature 1999, 397, 144-146. (31) Gelbart, W. M.; Bruinsma, R. J.; Pincus, P. A.; Parsegian, V. A. Phys. Today 2000, 53, 38-44. (32) Adams, R. L. P.; Knowler, J. T.; Leader, D. P. The Biochemistry of the Nucleic Acids, 11th ed.; Chapman & Hall: London, 1992. (33) SantaLucia, J., Jr. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 14601465. (34) SantaLucia, J., Jr.; Zuker, M.; Bommarito, A.; Irani, R. J. Unpublished results, 1999. (35) Tkachenko, A. V. Phys. Rev. Lett. 2002, 89, 148303.
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colloidal crystallization favorable in the presence of weakly attractive interparticle forces. Thus, DNA interactions should be tunable to the precise potential to promote crystal formation. In addition to the ability to finely control the degree of attraction, the association between hybridized strands is reversible upon heating above its melting temperature. The ability to break and reform DNA linkages may be an important tool for annealing kinetically trapped structures.27,36,37 Experimental work in this area has focused on monodisperse nanoparticle systems. Alivisatos et al.38 formed dimers and trimers of gold nanocrystals using a single DNA strand template. Mirkin’s group39 has successfully used oligonucleotide-functionalized 13-nm gold particles to form larger clusters. Recently, this group expanded these studies to include binary nanoparticle systems.40 Soto et al.41 reported DNAmediated adhesion between bidisperse, submicron particles. Structural characterization of all these systems, however, was limited to transmission electron microscopy of dried samples. To facilitate structural characterization of suspensions without having to account for particle consolidation or aggregation arising from drying effects,3,42 we have explored DNA-mediated assemblies of bidisperse, micronsized particles in suspension using optical and confocal microscopy. The use of bidisperse particles allowed us to easily distinguish nonspecific attractions (which would cause both homogeneous and heterogeneous aggregation) from specific, DNA-mediated attractions (resulting in heterogeneous aggregation only). The lack of homogeneous aggregation in all cases indicates that the attractive forces giving rise to heterogeneous aggregation are due to hybridization between tethered oligonucleotides on opposing particle surfaces. Our size choice also facilitated visual monitoring of particle aggregation dynamics to assess the effects of strand and solution conditions on the degree of DNA-mediated attractions between heterogeneous beads. We found that both particle aggregation rates and the calculated Tm (melting temperature) decreased as either the number of matching base pairs or salt concentration was lowered. Experimental Methods Oligonucleotide Design. Biotinylated and dye-tagged DNA strands were purchased from Biosource (Camarillo, CA). The sequence designs are shown schematically in Figure 1. The sequence choice for the hybridization segment of the design was based on a custom-developed Monte Carlo minimization algorithm of an ad hoc scoring function. The scoring function considered binding a given sequence with translated versions of itself and its complement, identifying each contiguous segment of Watson-Crick base pairing. Each of these segments increased the score by an amount exponential to the segment length. Segments which had one or two internal mismatches were counted but increased the score somewhat less. The desired full length Watson-Crick pairing of the sequence and its complement was explicitly not counted toward the score. Thus, this routine sought to minimize sequence repetition and oligonucleotide secondary structure by minimizing the length and number of (36) Mirkin, C. A.; Letsinger, R. L.; Mucic, R. C.; Storhoff, J. J. Nature 1996, 382, 607-609. (37) Mao, C.; Sun, W.; Seeman, N. C. Nature 1997, 386, 137-138. (38) Alivisatos, A. P.; Johnsson, K. P.; Peng, X.; Wilson, T. E.; Loweth, C. J.; Bruchez, M. P., Jr.; Schultz, P. G. Nature 1996, 382, 609-611. (39) Park, S.-J.; Lazarides, A. A.; Mirkin, C. A.; Letsinger, R. L. Angew. Chem., Int. Ed. 2001, 40, 2909-2912. (40) Mucic, R. C.; Storhoff, J. J.; Mirkin, C. A.; Letsinger, R. L. J. Am. Chem. Soc. 1998, 120, 12674-12675. (41) Soto, C. M.; Srinivasan, A.; Ratna, B. R. J. Am. Chem. Soc. 2002, 124, 8508-8509. (42) Deegan, R. D.; Bakajin, O.; Dupont, T. F.; Huber, G.; Nagel, S. R.; Witten, T. A. Nature 1997, 389, 827-829.
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Figure 1. Schematic representation of the hybridization configuration linking complementary DNA strands on opposing particle surfaces. The base sequences for each matching pair of strands are given below the schematic. Identical a strands were conjugated to small beads of 1.10 µm diameter, and identical b strands were conjugated to large beads of 1.87 µm diameter. Each strand has a biotin group (identified by the letter B) at the 5′ end. Hybridized segments are underlined, while the remaining bases closest to the particle surface act as spacers. Dye-labeled target strand sequences for fluorescence scans via flow cytometry are identified as Cy5 strand series. Table 1. Calculated Melting Temperaturesa calculated Tm (°C)
number of base pair matches
200 mM NaCl
50 mM NaCl
20 mM NaCl
12 10 8
54 48 22
44 38 12
37 31 5
a Calculations (ref 45) for hybridized segments of sequences shown in Figure 1 are based on thermodynamic properties of oligonucleotide solutions at 0.4 mM. This molarity value is based on the ratio of the surface concentration of tethered DNA (determined via cytometry analysis) to the “shell” volume of the DNA layer fully extended from the particle surface.
nonintentional Watson-Crick base pairings between sequences within the same strand and between two complementary strands. The resulting “self-melt” temperatures of all strands were very low (Tself-melt , 0 °C), making intrastrand loops and hairpin configurations unfavorable. Throughout the remainder of this paper, complementary strands are designated by the letters a and b followed by a number signifying the total number of bases. The first 6-8 bases near the bead surface serve as a spacer arm to make hybridization of the last 8-12 bases more favorable. Cy5-labeled DNA from Biosource (see Figure 1) served as target strands for flow cytometry measurements. Calculated melting temperatures for the hybridization segment of each strand are provided in Table 1. Bead Preparation. Yellow-green fluorescent 1.10 µm diameter beads (Molecular Probes, Eugene, OR) and nonfluorescent 1.87 µm diameter beads (Bangs Laboratories, Fishers, IN) served as the small and large colloidal species. The particle diameters and particle size ratio of 0.59 closely match those of previous studies in which entropically driven superlattice formation was observed.14 Both populations of polystyrene beads were modified with NeutrAvidin surface functionalities by the manufacturers. Conjugation and hybridization (pH 7.0-7.3, 20-200 mM NaCl) buffers contained 1 wt % bovine serum albumin. NeutrAvidin particles were incubated with the desired biotinylated oligonucleotide sequence following the manufacturer’s protocol.43 Following incubation, beads were heated to 50 °C in TTE buffer (43) TechNote 302; Bangs Laboratories, Inc.: Fishers, IN, 2002.
to remove any unstable NeutAvidin-biotin couplings and then washed two times in 200 mM NaCl hybridization buffer. To prepare dispersed, stock suspensions of each bead type, aggregates formed during the conjugation steps were separated from singlets via centrifugation. To determine the bead number density in the stock solutions, 8-10 aliquots of 0.02 µL were pipetted from each stock suspension onto a coverslip. Beads were counted from each dried droplet to determine the average bead number density. The amount of liquid in each stock suspension was then modified through buffer additions to adjust the total bead volume fraction to 10-4. Beads with grafted DNA strands will be subsequently identified in the text by the bead diameter and grafted sequence (see Figure 1). Small NeutrAvidin beads were functionalized with biotinylated a18, a17, or a16 oligonucleotide strands (designated hereafter as 1.10µm-a18, 1.10µma17, and 1.10µm-a16, respectively). Large beads were functionalized with b18, b17, or b16 (designated hereafter as 1.87µmb18, 1.87µm-b17, and 1.87µm-b16, respectively). Flow Cytometry. The presence and activity of immobilized DNA strands (b18, b17, or b16) on large, nonfluorescent beads were quantified with dye-labeled target strands using flow cytometry. Cy5-labeled DNA strands were incubated with DNAtethered, nonfluorescent beads for approximately 3 h in 200 mM NaCl hybridization buffer. Following incubation, beads were washed four times in 200 mM NaCl hybridization buffer. Cytometry experiments were run on a Becton Dickinson FACScan flow cytometer (Becton Dickinson, San Jose, CA). Cytometry samples consisted of (a) DNA-tethered beads alone, (b) tethered beads incubated with target strands (Cy5-b12) identical in sequence to the hybridization segment of the immobilized probe strands, and (c) tethered beads incubated with target strands (Cy5-a12) complementary to the immobilized probe strands. The number of hybridized strands was determined using calibration curves derived from Quantum Phycoerithrin-Cy5 microbead standards purchased from Bangs Laboratories, Inc. These calibration curves were constructed using the reported molecules of equivalent soluble fluorochrome intensity (MESF) for these bead standards as a linear function of the mean fluorescence peak channels (i.e., fluorescence intensity). From these calibration curves, the corresponding MESF can then be derived from the peak fluorescence intensity for DNA-tethered beads exposed to complementary dye-labeled target strands. This MESF value corresponds to the number of target strands hybridized to probe strands on the bead surface. These values are divided by the bead surface area and reported as the density of hybridized strands. Preparation and Imaging of Suspensions. Bidisperse suspensions of the desired composition were prepared by vortexing volumetric quantities of stock suspensions together. Approximately 20 µL of the suspension mixture was loaded in a microchamber consisting of two coverslips separated by a Parafilm spacer. For dimer formation studies, suspensions were prepared with an initial total volume fraction (φt,0) of 10-5 and with a particle number ratio (Nsmall/Nlarge) of 1:1. All other kinetic and structural studies were completed using suspensions with an initial total volume fraction of 10-4 and with a particle number ratio of 1:1, 4:1, 8:1, or 50:1. All suspensions were examined via optical microscopy (Nikon Diaphot 200, Tokyo, Japan) using a plan achromat 10× or 100× (oil-immersion) objective and a black and white CCD camera (Cohu Inc., San Diego, CA). Using phase contrast, static images were captured and analyzed using Imaq software (National Instruments, Austin, TX). The time-dependent structural evolution of suspensions was studied by periodically imaging (10×) suspensions 10 min to 24 h after loading into the microchambers. After 2-5 days, micrographs of selected suspensions were taken at 100× to reveal more detail on the local particle arrangement within the clusters. Confocal images of the mixtures were taken using a Bio-Rad 2000 KR-3MP (Hercules, CA) system equipped with a Nikon TE 300 fluorescent microscope.
Results and Discussion Oligonucleotide Density on Nonfluorescent Beads. Figure 2 shows the fluorescence histogram of (bead) counts as a function of fluorescence intensity resulting from flow
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Figure 2. Fluorescence histograms obtained from flow cytometry of 1.87 µm diameter NeutrAvidin particles functionalized with DNA (b sequence series) and exposed to various dye-labeled target strands. Results are presented in a histogram of (bead) counts as a function of fluorescence intensity (FL-4). Negative controls consisted of DNA-tethered particles (1.87µmb18) (a) alone (neg-1) to measure bead autofluorescence and (b) exposed to noncomplementary Cy5-labeled target DNA strands (neg-2) to check for nonspecific association of free target DNA with either the particle surface or with the immobilized probe strands. A significant shift in fluorescence intensity was observed only in the case of 1.87 µm diameter NeutrAvidin particles functionalized with DNA probe strands b16, b17, or b18 (c) exposed to complementary Cy5-labeled target DNA strands (pos-16, pos-17, and pos-18 respectively). The target strand sequences were Cy5-b12 (3′-Cy5-TAC ATA GTT CCA5′) for the neg-2 case and Cy5-a12 (5′-ATG TAT CAA GGTCy5-3′) for pos-16, pos-17, and pos-18 (sequence order presentation of target strands chosen for facile comparison to b16, b17, and b18).
cytometry measurements of DNA-functionalized beads incubated with various target strands. A reference peak (neg-1) of DNA-tethered beads alone (1.87µm-b18) accounts for autofluorescence of unlabeled particles. The fluorescence intensity does not change for 1.87µm-b18 beads incubated with noncomplementary target strands (neg-2). These noncomplementary target strands (Cy5b12, see Figure 1) have hybridization sequences that are identical to the hybridization sequence of the DNAtethered strand (b18). The closely matched fluorescence intensities of neg-1 and neg-2 suggest both negligible hybridization between homotypic probe and target strands as well as negligible nonspecific association of target strands to the bead surface. Identical fluorescence peak values (not shown) were observed for the negative controls using 1.87µm-b17 and 1.87µm-b16 alone and incubated with noncomplementary target strands (Cy5-b12). Compared to neg-1 and neg-2, a significant increase in fluorescence intensity, as evidenced by a rightward shift in peak values, was observed for tethered beads exposed to complementary target strands. Specifically, relative increases in fluorescence intensities were observed for pos-16 (1.87µm-b16 + Cy5-a12), pos-17 (1.87µm-b17 + Cy5-a12), and pos-18 (1.87µm-b18 + Cy5-a12). The increase in fluorescence intensity in these cases suggests both successful DNA functionalization of beads and hybridization activity between immobilized probe strands and complementary target strands in solution. The peak fluorescence values correspond to an average of 648, 1684, and 2418 hybridized strands/µm2 of bead surface area for pos-16, pos-17, and pos-18, respectively.
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Figure 3. Phase contrast micrograph (20×) of the colloidal fluid phase observed in mixtures of 1.10 and 1.87 µm beads functionalized with identical oligonucleotide strands b18. The inset at 100× shows more clearly that particles remain disperse with negligible aggregation. Micrographs were taken 1 day after mixing the heterogeneous beads together in 100 mM NaCl hybridization buffer.
The measured density of hybridized strands for pos-18 is a reasonable estimate based on the manufacturer’s reported density of active sites on the beads (7110 sites/ µm2). The coupling of the “probe” strand on the bead surface depends only on the relatively irreversible (Ka ) 1015 M-1) biotin-NeutrAvidin bond44 and not on strand characteristics such as sequence length. Thus, it is reasonable to conclude that the steady decline in the number of hybridized strands with decreasing segment overlap is likely due to the decreasing hybridization affinity between target and immobilized probe strands and not to any reduction in the surface density of tethered strands. The decrease in the calculated Tm for matching strand hybridization shown in Table 1 is also indicative of decreasing hybridization affinity as segment length shortens. Evidence of Negligible Colloidal Assembly in the Absence of Specific Forces. Several experiments were performed in order to confirm that colloidal assembly was unfavorable in the absence of DNA hybridization between opposing heterogeneous surfaces. To allow ample time for possible particle aggregation, suspensions were imaged 24 h after mixing. Our first negative control consisted of mixing small and large beads conjugated with identical DNA strands at 100 mM NaCl. As shown in Figure 3, negligible aggregation occurred. Similar results were observed at 200 mM NaCl (data not shown). This negligible affinity between beads with identical strands concurs with cytometry results that also indicated negligible association between immobilized probe strands and target strands with equivalent sequence segments. An additional negative control for assembly experiments consisted of mixing bare, small NeutrAvidin beads with DNA-tethered, large beads (1.87µm-b18). The lack of colloidal aggregation in this control (not shown) suggests a negligible affinity between grafted DNA sequences and NeutrAvidin moieties (44) Hiller, Y.; Gershoni, J. M.; Bayer, E. A.; Wilchek, M. Biochem. J. 1987, 248, 167-171. (45) Breslauer, K. J.; Frank, R.; Blocker, H.; Marky, L. A. Proc. Natl. Acad. Sci. U.S.A. 1986, 83, 3746-3750.
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Figure 4. Phase contrast micrographs (20×) of colloidal gels formed 4 h after mixing binary suspensions (φt,0 ) 10-4) comprised of 1.10 and 1.87 µm beads with complementary strands of varying overlapping segment lengths and ionic strengths. Mixtures of small and large beads were functionalized with a18 and b18 respectively (12 base pair overlap) at (a) 200 mM and (b) 50 mM NaCl, with a17 and b17 respectively (10 base pair overlap) at (c) 200 mM and (d) 50 mM NaCl, and with a16 and b16 respectively (8 base pair overlap) at (e) 200 mM and (f) 50 mM NaCl.
on opposing particle surfaces. In both sets of negative controls, the absence of complementary DNA strands makes both homotypic and heterotypic clustering between particles unfavorable. Thus, nonspecific attractions such as van der Waals forces do not appear to induce particle aggregation or assembly. Our final negative control consisted of mixtures of 1.10µm-a18 and 1.87µm-b18 in 20 mM NaCl hybridization buffer. Since significant ionic screening of the charged DNA backbone by counterions is prerequisite for hydrogen bonding between matching base groups, hybridization is generally unfavorable at low electrolyte concentrations. This diminished affinity between complementary strands as salt concentration decreases is evident from the lowering of Tm values shown in Table 1. Even for the longest sequences investigated here in which tethered strands on small and large beads consisted of 12 base pair matches, aggregation did not occur at 20 mM NaCl. The lack of interparticle attractions between heterogeneous beads suggests hybridization between complementary tethered strands is unfavorable due to strand characteristics, solution conditions, or both. Interestingly, the estimated melting temperature of 37 °C (see Table 1) suggests hybridization may be favorable for these same strands in 20 mM NaCl solution. However, these calculations are based on the thermodynamic properties of oligonucleotide solutions. By immobilizing the DNA on a surface, we have restricted its hybridization activity to the contact zone between particle surfaces. Ignoring possible surface roughness, we estimate the DNA tethers extend approximately 10 nm from the particle surface. This range for attractive interactions stemming from hybridization may be insufficient to compete with longer range repulsive electrostatic interactions in which the Debye or “screening” length, 1/κ, is 2.1 nm at 20 mM NaCl. Thus, electrosteric repulsions between particles may not allow the close proximity between particle surfaces necessary for hybridization. At this point, however, we cannot offer more detailed confirmation of the repulsive nature between particles without electrophoretic data and appropriate electrosteric modeling, both of which are currently lacking. Evidence of Colloidal Assembly in the Presence of Specific Forces. We examined mixtures in which
conditions (i.e., complementary strands and moderate or high ionic strength) were favorable for DNA-mediated colloidal assembly or aggregation. To isolate the effects of the variables of interest, the particle number ratio (Nsmall/Nlarge of 1:1) and total bead volume fraction (φt,0 ) 10-4) were the same for all suspension mixtures. Thus, we concurrently examined the effects of sequence overlap length and ionic strength on the overall rates and extent of binary, colloidal clustering as shown in the micrographs of Figure 4. At 200 mM NaCl (1/κ ) 0.7 nm), all suspensions aggregated 4 h after mixing with (a) 12, (c) 10, or (e) 8 base pair matches between tethered oligonucleotides. Micrographs detailing the time-dependent structural evolution of these suspensions are available in the Supporting Information (see Figure 1S). In each case, small clusters of several particles formed within 10 min after mixing. Clustering was extensive in all suspensions within about 2 h. After 24 h, the mixture in the 8 base pair case consisted of mostly aggregates and some singlet particles. For the 10 and 12 base pair cases, aggregation was nearly complete with only a few particles remaining dispersed after 24 h. Overall, the degree of aggregation appears to increase slightly with hybridization sequence length. The calculated solution Tm for these three mixtures at 200 mM NaCl ranged from 22 to 54 °C, indicating hybridization is favorable for the experimental room-temperature conditions. Thus, it appears that, unlike the mixture at 20 mM NaCl, sufficient charge screening occurs at this higher electrolyte concentration to allow hybridizaton between functionalized particle surfaces. At a lower ionic strength of 50 mM NaCl (1/κ ) 1.4 nm), small clusters formed within 10 min following mixing of the small and large beads (see the Supporting Information). For the shortest segment overlap of 8 base pairs (1.10µm-a16 + 1.87µm-b16), the extent of aggregation, however, did not change with time, even after 24 h. In this case, the Tm estimate of 12 °C suggests that these conditions are unfavorable for solution hybridization. Thus, in this case DNA-mediated attractions between these beads are too weak to induce colloidal assembly. Compared to the mixtures in 200 mM NaCl solution, overall assembly was more gradual at this lower salt concentration for mixtures with 10 and 12 base pair matches. In addition, the extent of aggregation diminished
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Figure 5. Phase contrast micrographs (100×) showing reversible dimer formation 2 h after mixing for colloidal suspensions comprised of 1.10 and 1.87 µm beads functionalized with a18 and b18 oligonucleotide strands, respectively, in 50 mM NaCl buffer (φt,0 ) 10-5). The boxed region shows both heterogeneous and homogeneous dimers (a) forming and (b) breaking apart seconds later.
Figure 6. Phase contrast (a-c) and confocal (d-f) micrographs (100× and 60×, respectively) of colloidal structures in binary suspensions comprised of varying number ratios (Nsmall/Nlarge) of fluorescent 1.10 µm and nonfluorescent 1.87 µm beads functionalized with a18 and b18 oligonucleotide strands, respectively, in 50 mM NaCl buffer. Primary clusters are comprised of (a,d) chains and occasional close-packed structures (indicated by arrows) of alternating small and large particles for Nsmall/Nlarge of 1:1, (b,e) colloidal satellites with a large particle surrounded and often bridged together by an incomplete shell of small particles for Nsmall/Nlarge of 4:1, and (c,f) colloidal satellites with a large particle surrounded and less frequently bridged together by a complete or nearly complete shell of small particles for Nsmall/Nlarge of 8:1 as indicated by arrows in the phase contrast micrographs.
at this lower salt concentration as the number of base pair matches decreased from (b) 12 to (f) 8. This reduction in cluster size as the hybridization segment decreases is more apparent at 50 mM NaCl than at 200 mM NaCl. As the electrolyte concentration is lowered, the repulsive range of electrosteric interactions between DNA-covered particles increases and competes with the short-range attraction arising from hybridization. Overall, the degree of aggregation between DNAtethered particles decreases with either decreasing number of overlapping base pairs or salt concentration. Since control experiments show that nonspecific interactions are negligible, the affinity between heterogeneous particles must arise from DNA hybridization between beads. As overlapping segments shorten or solution ionic strength is reduced, the decrease in the calculated solution Tm indicated hybridization between complementary strands becomes less favorable. In each mixture studied, particle assembly occurred only for grafted oligonucleotides with melting temperatures that equaled or exceeded the experimental room temperature. However, a relatively high Tm is not necessarily sufficient to induce assembly
at low salt concentration as observed in the case of 1.10µma18 + 1.87µm-b18 mixtures at 20 mM NaCl. We further examined the case of 1.10µm-a18 and 1.87µm-b18 mixtures (12 base pair overlap) at 50 mM NaCl in a more dilute suspension (φt,0 ) 10-5) in order to monitor dimer formation. This electrolyte concentration was chosen because particle assembly appears complete after 24 h. The aggregation kinetics, however, are slower in 50 mM NaCl solution than in 200 mM NaCl. For 1.10µm-a18 + 1.87µm-b18 mixtures, the specific, attractive forces still dominate over repulsive electrosterics but are weaker in magnitude and range at 50 mM than at 200 mM NaCl. Such weaker attractions may ultimately favor more tightly packed particle arrangements or even crystalline structures35 over fractal clusters. As shown in Figure 5, homogeneous and heterogeneous particles collide in panel a and then break apart seconds later in panel b. As shown in control experiments and in the high-resolution micrographs of Figure 6, repulsive forces between like surfaces continue to dominate their interaction potential and prevent nonspecific adhesive bonds from forming between homogeneous beads. Though aggregation kinetics
DNA-Driven Assembly of Colloids
are slower in 50 mM NaCl solution than in 200 mM NaCl solution, DNA hybridization between heterogeneous beads ultimately favors colloidal assembly as shown earlier in Figure 4b. We expanded our investigation of this suspension system with weaker, specific attractions (50 mM NaCl, 12 base pair overlap) to include compositional effects on assembly structure. As shown in the next section, we observed a variety of structures such as chains and colloidal satellites depending on the number ratio of small to large particles. Effect of Number Ratio on Colloidal Assembly. Holding the hybridization chemistry constant (50 mM NaCl, 12 base pair overlap), we varied the number ratio of small to large particles to study the effect of number ratio on the structure of the suspension. We used both 100× phase contrast and 60× confocal microscopy to study assembly of the materials. Confocal microscopy was useful for investigating the particle arrangement since the small beads are fluorescent. The number ratio of small to large particles (Nsmall/Nlarge) was varied from 1:1 to 50:1, but the total microsphere volume fraction was the same (φt,0 ) 10-4) in each case. In each case aggregates formed, but the primary clusters varied in the number of linked, heterogeneous particles. For Nsmall/Nlarge of 1:1, chains of alternating large and small particles (indicated by an arrow in the phase contrast micrograph) were observed as shown in Figure 6a,d. These chains generally either formed dangling ends several particles long or served as connections between more compact primary clusters. Occasionally, these clusters consisted of close-packed particle arrangements, indicated by a second, lower arrow in Figure 6a. Chainlike structures were also observed at Nsmall/Nlarge of 4:1 as shown in Figure 6b,e. However, more of the small colloidal species act as a single particle “branch” rather than as a bridging link to a neighboring large particle. As the number ratio increased to 8:1 (see Figure 6c,f), large particles appear surrounded by a shell of small particles. This shell was generally an incomplete monolayer of small particles; however, the overall aggregate structure was that of a colloidal micelle or satellite. The incomplete micelles were easier to structurally characterize via confocal images shown in panel f where the fluorescent small particles form a shell around a single, nonfluorescent bead. Some of the incomplete colloidal micelles remained as individual heteroaggregates, but most shared small particle bridges to form particle “chords” with a larger aspect ratio than was seen in chains (one particle in diameter) for the 1:1 mixture. At the highest number ratio investigated of 50:1 (not shown), the shell of these colloidal micelles was generally a nearly saturated monolayer of small particles. Many of these heteroag-
Langmuir, Vol. 19, No. 24, 2003 10323
gregates remained as individual colloidal micelles, but many still appeared as linked clusters. Due to the excess of small particles at this composition, many of these colloids remained as dispersed, unassociated singlets. Conclusions We observed the ability to tune the degree of attractive interactions between DNA-functionalized beads to control rates of colloidal assembly and the particle arrangements in suspension by varying the hybridization segment length, ionic strength, and composition of suspension mixtures. Overall, the hybridization affinity between complementary oligonucleotide strands increases with either the number of base pair matches or salt concentration. In addition, at a high salt concentration (200 mM NaCl) the degree of aggregation appears less sensitive to the number of base pair matches than at a lower salt concentration (50 mM NaCl). At the lower ionic strength, electrosteric repulsions compete with hybridization affinity between beads. Correspondingly, the Tm values, number of hybridized surface strands from cytometry measurements, and propensity for particle assembly also decreased with decreasing hybridization affinity. An elevated Tm (i.e., Tm g 22-25 °C) is required but not necessarily sufficient for DNA-mediated particle assembly if long-range electrosteric repulsions are not sufficiently screened to allow short-range hydrogen bonding between complementary tethered strands. Future work will focus on systems with weakly attractive DNA-mediated assembly. We plan to continue to “tune” the degree of attraction by controlling the number density of tethered strands available for hybridization between beads. For this future study, we plan to titrate the surface density of DNA active in particle bonding by adding soluble, complementary strands to occupy a percentage of the tethered strands. Lastly, we plan to study annealing effects on the structural evolution of these suspensions. Acknowledgment. We gratefully acknowledge financial support from the NASA-NAG3-2417 and the NSF MRSEC DMR00-79909 and BES-0314265. We also thank C. Pletcher and G. Gray-Board for helpful technical suggestions and Y. Zhang and A. Kim for valuable discussions. Supporting Information Available: Phase contrast micrographs of the time-dependent structural evolution of colloidal gels. This material is available free of charge via the Internet at http://pubs.acs.org. LA034376C
