Effect of Macromolecular Crowding on DNA:Au Nanoparticle

Polymer molecular weight was less important than polymer chemistry and weight percent in solution. ..... (50) Laurent, T. C.; Preston, B. N.; Carlsson...
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Effect of Macromolecular Crowding on DNA:Au Nanoparticle Bioconjugate Assembly Glenn P. Goodrich,† Marcus R. Helfrich,† Jennifer J. Overberg, and Christine D. Keating* Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania 16802 Received June 24, 2004. In Final Form: August 26, 2004 DNA:Au nanosphere bioconjugates have applications in biosensing and in the bottom-up assembly of materials. These bioconjugates can be selectively assembled into three-dimensional aggregates upon addition of complementary DNA oligonucleotides and can be dissociated by heating above a melting transition temperature at which the DNA duplexes are denatured. Herein we describe the impact of polymeric solutes on the thermal denaturation behavior of DNA:Au nanoparticle bioconjugate assemblies. Polymeric solutes can dramatically impact biochemical reactions via macromolecular crowding. Poly(ethylene glycol)s (PEGs) and dextrans of varying molecular weights were used as crowding reagents. While both PEG and dextran increased the stability of DNA:Au aggregates, melting transition temperatures in the presence of PEG were impacted more significantly. Polymer molecular weight was less important than polymer chemistry and weight percent in solution. For a high (15%) weight percent of PEG, aggregation was observed even in the absence of complementary oligonucleotides. These results underscore the importance of polymer chemistry in addition to physical volume exclusion in macromolecular crowding and point to the importance of understanding these effects when designing biorecognition-based nanoparticle assembly schemes in complex matrixes (i.e., any involving polymeric solutes).

Introduction DNA:Au nanoparticle bioconjugates are promising building blocks for nanoscale materials and have found numerous applications in bioanalysis.1-8 In each of these applications, selective hybridization of Au nanoparticlebound DNA oligonucleotides to complementary strands in solution, on planar surfaces, or bound to other nanoparticles is required. Typically such hybridization reactions are carried out in aqueous buffer solutions, where DNA hybridization thermodynamics,9 and recently DNA: Au bioconjugate hybridization thermodynamics,10 are well understood. In recent years, there has been a growing appreciation of the impact physical forces such as volume exclusion can exert on biological systems. Often biomolecules exhibit higher activities in vivo than are observed in vitro.11-15 This result has been explained by “macro* To whom correspondence should be addressed. E-mail: [email protected]. † These authors contributed equally. (1) Taton, T. A.; Mirkin, C. A.; Letsinger, R. L. Science 2000, 289, 1757-1760. (2) Cao, Y. C.; Jin, R.; Mirkin, C. A. Science 2002, 297, 1536-1540. (3) Park, S.-J.; Taton, A. L.; Mirkin, C. A. Science 2002, 295, 15031506. (4) Nam, J.-M.; Thaxton, C. S.; Mirkin, C. A. Science 2003, 301, 18841886. (5) He, L.; Musick, M. D.; Nicewarner, S. R.; Salinas, F. G.; Benkovic, S. J.; Natan, M. J.; Keating, C. D. J. Am. Chem. Soc. 2000, 122, 90719077. (6) Reichert, J.; Csaki, A.; Kohler, J. M.; Fritzsche, W. Anal. Chem. 2000, 72, 6025-6029. (7) Cobbe, S.; Connolly, S.; Ryan, D.; Nagle, L.; Eritja, R.; Fitzmaurice, D. J. Phys. Chem. B 2003, 107, 470-477. (8) Niemeyer, C. M.; Ceyhan, B.; Gao, S.; Chi, L.; Peschel, S.; Simon, U. Colloid Polym. Sci. 2001, 279, 68-72. (9) Bloomfield, V. A.; Crothers, D. M.; Tinoco, I., Jr. Nucleic Acids: Structures, Properties and Functions; University Science Books: Sausalito, CA, 2000. (10) Jin, R.; Wu, G.; Li, Z.; Mirkin, C. A.; Schatz, G. C. J. Am. Chem. Soc. 2003, 125, 1643-1654. (11) Ellis, R. J. Trends Biochem. Sci. 2001, 26, 597-604. (12) Al-Habori, M. Int. J. Biochem. Cell Biol. 2001, 33, 844-864. (13) Minton, A. P. J. Biol. Chem. 2001, 276, 10577-10580.

molecular crowding” in cytoplasm due to the high weight percent of intracellular macromolecules (up to 30% in some cases).15 Macromolecules such as proteins and nucleic acids can act as volume excluders, raising the effective concentration of other (bio)macromolecules in the solution.16 Polymer-induced changes in the hydration of macromolecules can also occur, leading to effects beyond those expected for volume exclusion alone.17-19 The impact of macromolecular crowding on specific biological processes such as DNA condensation in bacteria,20 DNA recircularization,21 protein aggregation and folding,22-25 and DNA duplex/triplex formation18,19,26,27 have been reported. Noninteracting polymers such as poly(ethylene glycol) (PEG) are commonly used as crowding reagents. Large differences in activity and hybridization are often observed for proteins and nucleic acids in PEG solution as compared with buffer alone. For example, DNA ligases isolated from rat liver nuclei catalyze blunt end ligation in 7% PEG 6000 and intracellularly, but not in dilute aqueous buffer.28 (14) Johansson, H. O.; Brooks, D. E.; Haynes, C. A. Int. Rev. Cytol. 2000, 192, 155-170. (15) Fulton, A. B. Cell 1982, 30, 345-347. (16) Asakura, S.; Oosawa, F. J. Polym. Sci. 1958, 33, 183-192. (17) Parsegian, V. A.; Rand, R. P.; Rau, D. C. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 3987-3992. (18) Goobes, R.; Kahana, N.; Cohen, O.; Minsky, A. Biochemistry 2003, 42, 2431-2440. (19) Spink, C. H.; Chaires, J. B. Biochemistry 1999, 38, 496-508. (20) Zimmerman, S. B.; Murphy, L. D. FEBS Lett. 1996, 390, 245248. (21) Louie, D.; Serwer, P. J. Mol. Biol. 1994, 242, 547-558. (22) Qu, Y. X.; Bolen, D. W. Biophys. J. 2002, 82, 1443. (23) Minton, A. P. Curr. Opin. Struct. Biol. 2000, 10, 34-39. (24) van den Berg, B.; Ellis, R. J.; Dobson, C. M. EMBO J. 1999, 18, 6927-6933. (25) Martin, J. Biochemistry 2002, 41, 5050-5055. (26) Spink, C. H.; Chaires, J. B. J. Am. Chem. Soc. 1995, 117, 1288712888. (27) Goobes, R.; Minsky, A. J. Am. Chem. Soc. 2001, 123, 1269212693. (28) Zimmerman, S. B.; Pheiffer, B. H. Proc. Natl. Acad. Sci. U.S.A. 1983, 80, 5852-5856.

10.1021/la048434l CCC: $27.50 © 2004 American Chemical Society Published on Web 10/12/2004

Macromolecular Crowding Effect on DNA:Au Assembly

DNA condensation can be induced in PEG solution,29 as can cytoskeletal protein polymerization.30 The effect of volume exclusion on colloidal solutions has also been studied.31-34 Yodh and co-workers reported that particles of different sizes phase separate due to volume exclusion.31 In this experiment, the different sized colloidal particles are shown to crowd each other, and the degree of phase separation and crystallization can be controlled by varying the ratio of particle diameters. Yodh et al. have also described using volume exclusion by colloidal particles32 and polystyrene33 to selectively confine larger colloidal particles near patterned surfaces. It is clear that polymer solutions can dramatically impact the solution behavior of both biomacromolecules and inorganic colloids. To the best of our knowledge, however, there have been no reports on how macromolecular crowding impacts biomolecule-directed nanoparticle assembly. Hybridization between DNA:Au nanoparticle bioconjugates is the best understood biorecognition driven nanoparticle assembly system10,35,36 and as such was chosen as the experimental system here. Biospecific DNA hybridization driven assembly of DNA: Au bioconjugates in solution and at planar surfaces is a critical aspect of many sensitive and selective biosensing strategies,1-6,37-39 and also has relevance to nanomaterials assembly.8,40,41 Typically these experiments are performed in buffer solutions that contain salts and oligonucleotides but no larger polymeric solutes. As these technologies move forward, there may be applications requiring use in more complex matrixes, such as within living cells or biological fluids for biosensors or in functional polymeric matrixes for electronic device assembly. We are particularly interested in DNA-directed nanowire assembly at the aqueous-aqueous interface of polymer-containing aqueous two-phase systems. For any of these applications in polymer-containing solutions, it will be critical to understand the impact of macromolecular crowding on biospecific particle assembly. Volume exclusion raises the thermodynamic activity (i.e., the effective concentration) of macromolecules in nonideal solutions.42 Given the dependence of DNA hybridization on DNA concentration, polymeric solutes might be expected to impact the stability of DNA duplexes and, thus, of DNA/nanoparticle aggregates. Differences in the DNA duplex stability are exploited to distinguish between fully complementary and mismatched sequences;37,38 thus, changes in sensitivity to mismatches is one potential concern in “crowded” solutions. It is even (29) Lerman, L. S. Proc. Natl. Acad. Sci. U.S.A. 1971, 68, 18861890. (30) Cuneo, P.; Magri, E.; Verzola, A.; Grazi, E. Biochem. J. 1992, 281, 507-512. (31) Kaplan, P. D.; Rouke, J. L.; Yodh, A. G.; Pine, D. J. Phys. Rev. Lett. 1994, 72, 582-585. (32) Dinsmore, A. D.; Yodh, A. G. Langmuir 1999, 15, 314-316. (33) Lin, K. H.; Crocker, J. C.; Prasad, V.; Schofield, A.; Weitz, D. A.; Lubensky, T. C.; Yodh, A. G. Phys. Rev. Lett. 2000, 85, 1770-1773. (34) Verma, R.; Crocker, J. C.; Lubensky, T. C.; Yodh, A. G. Phys. Rev. Lett. 1998, 81, 4004-4007. (35) Mirkin, C. A. Inorg. Chem. 2000, 39, 2258-2272. (36) Storhoff, J. J.; Lazarides, A. A.; Mucic, R. C.; Mirkin, C. A.; Letsinger, R. L.; Schatz, G. C. J. Am. Chem. Soc. 2000, 122, 4640-4650. (37) Elghanian, R.; Storhoff, J. J.; Mucic, R. C.; Letsinger, R. L.; Mirkin, C. A. Science 1997, 277, 1078-1081. (38) Storhoff, J. J.; Elghanian, R.; Mucic, R. C.; Mirkin, C. A.; Letsinger, R. L. J. Am. Chem. Soc. 1998, 120, 1959-1964. (39) Maxwell, D. J.; Taylor, J. R.; Nie, S. J. Am. Chem. Soc. 2002, 124, 9606-9612. (40) Zanchet, D.; Micheel, C. M.; Parak, W. J.; Gerion, D.; Williams, S. C.; Alivisatos, A. P. J. Phys. Chem. B 2002, 106, 11758-11763. (41) Taton, T. A.; Mucic, R. C.; Mirkin, C. A.; Letsinger, R. L. J. Am. Chem. Soc. 2000, 122, 6305-6306. (42) Ellis, J. R. Curr. Opin. Struct. Biol. 2001, 11, 114-119.

Langmuir, Vol. 20, No. 23, 2004 10247 Scheme 1. Schematic Representation of the Bioassembly Process Investigated in This Worka

a First, two sets of DNA:Au conjugates (A and B) are prepared by chemically modifying the surface of the nanoparticle with single-stranded thiolated DNA (12 base pairs in length). These bioconjugates can be selectively aggregated when mixed in the presence of a 24-base-pair linker strand that is half complementary to the DNA on particle A and half to that on particle B. This process can be reversed by heating above the melting transition temperature.

possible that given a high enough polymer concentration, the crowding pressure would be such that noncomplementary DNA sequences would be able to hybridize or that nanoparticles would irreversibly aggregate. To rationally design DNA hybridization based assembly systems in polymers it is necessary to study the effects of these polymers on DNA:Au nanoparticle conjugates. Herein we describe the effects of two polymeric volume excluders, PEG and dextran, on the thermodynamics of DNA:Au nanoparticle assembly. Scheme 1 illustrates the experimental system, in which DNA-coated Au nanospheres are aggregated by hybridization to a linking oligonucleotide. To determine the practical implications of assembly in polymer solutions, the effect on the aggregate Tm was studied as a function of polymer concentration and polymer molecular weight. The results of the nanoparticle-based experiments were compared to DNA duplex melts in the absence of Au nanoparticles, to determine the effect of immobilizing the DNA onto nanometer-sized metal particles. In nearly all cases, increasing the polymer content in the solution resulted in increased DNA duplex stability and, thus, increased aggregate stability; however, we find significant differences in the impact of PEG versus dextran polymers. At high PEG weight percents, aggregation of DNA:Au bioconjugates was observed in the absence of linking DNA strands, an effect that could be avoided by decreasing the solution ionic strength. Experimental Section Materials. All water used in these experiments was purified to >18 MΩ, using a Barnstead Nanopure filtration system. All chemicals were used as received except where noted. HAuCl4‚ 3H2O was purchased from Acros Organics, DL-dithiothreitol (DTT), sodium citrate trihydrate, NaCl, and sodium phosphate, monobasic, were purchased from Sigma. Sodium phosphate‚ dibasic was purchased from EM Science, concentrated HCl and HNO3 were obtained from J. T. Baker, NAP-5 columns were purchased from Amersham Pharmacia. The DNA oligonucleotides used were purchased from Integrated DNA Technologies

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Langmuir, Vol. 20, No. 23, 2004 Table 1. DNA Sequences Used in This Work

sequence description Au conjugate sequence 1 Au conjugate sequence 2 Au conjugate linker complement to linker

sequence (5′ to 3′) TCT CAA CTC GTA-(C3H6-SH) (SH-C6H12)-CGC ATT CAG GAT TAC GAG TTG AGA ATC CTG AAT GCG CGC ATT CAG GAT TCT CAA CTC GTA

(listed in Table 1). Dextrans (average molecular weights: MW 144 kDa, lot no. 101K1251; 10.2 kDa, lot no. 101K1115; and 480 kDa, lot no. 32K1454) were purchased from Sigma. Dextran with molecular weight 1000 Da was purchased from Sigma-Aldrich. PEG 12 000 MW (lot no. 396070) and 35 000 MW (lot no. 419735) were purchased from Fluka Chemica, and PEG 8000 (lot no. 20K0211) and PEG 600 MW (lot no. 41K0261) were purchased from Sigma. Instrumentation. UV-Vis Spectroscopy. All UV-vis spectra were obtained using a Hewlett-Packard 8453A diode-array spectrophotometer with 1-nm resolution and a 1-s integration time. Spectra were collected using the ChemStation UV-vis software. All spectra were taken using 10-µL quartz cuvettes (Starna Cells). Transmission Electron Microscopy (TEM). A JEOL 1200 EXII transmission electron microscope was used to obtain TEM micrographs. Images were taken using a GATAN Bioscan 792 digital imaging system using the GATAN Digital Microscopy software package. DNA:Au Nanoparticle Bioconjugate Preparation. Au colloid was prepared by citrate reduction of HAuCl4 as previously reported.36,43 The resulting Au colloids had an absorbance maximum at 518 nm. The mean diameter of the Au colloids was determined to be 12 ( 1.2 nm by TEM, and the particle concentration was determined to be 1.58 × 10-8 M using atomic absorption spectroscopy. Au colloid was stored in a clean brown glass bottle until use. Thiol-modified DNA oligonucleotides were purchased from IDT, Inc., as disulfides. Before use, the disulfide was cleaved by dissolving the lyophilized oligonucleotide in 300 µL of 100 mM DTT at room temperature for 30 min. The DTT and the short organic thiol cleaved from the oligonucleotide were removed from the solution by elution down a NAP-5 Sephadex column using 600 µL of 18 MΩ H2O. The concentration of the purified oligonucleotides was determined by UV-vis spectroscopy using the A260 peak and the extinction coefficient specific for the strand of DNA. Purified oligonucleotides were stored at -20 °C until use. Au:DNA conjugates were prepared using a slightly modified version of the procedure described by Mirkin and co-workers.44-46 In general, 50 µL of 100 µM thiolated oligonucleotide was added to 950 µL of Au colloid in a 1.7-mL eppendorf tube. The tubes were placed in a 37 °C heat block for 30 min. The conjugate solutions were then brought to a concentration of 0.1 M NaCl/10 mM phosphate by the addition of 350 µL of 18 MΩ H2O followed by 150 µL of 1 M NaCl/100 mM phosphate buffer (pH 7). The conjugates were then allowed to “age” in the buffer solution at 37 °C for at least 1.5 h. After aging, excess oligo was removed by centrifugation. The conjugates were spun down twice using a Biofuge Pico centrifuge (Heraeus) at ∼12 300g for 35 min. After each centrifugation, the supernatant was removed, and the soft pellet containing the Au particles was resuspended in 0.3 M NaCl/10 mM phosphate buffer pH 7. The conjugate concentration was determined by UV-vis spectroscopy using the extinction coefficient (1.94 × 108 M-1 cm-1) for the Au particles.47 (43) Grabar, K. C.; Freeman, R. G.; Hommer, M. B.; Natan, M. J. Anal. Chem. 1995, 67, 735-743. (44) Grabar, K. C.; Allison, K. J.; Baker, B. E.; Bright, R. M.; Brown, K. R.; Freeman, R. G.; Fox, A. P.; Keating, C. D.; Musick, M. D.; Natan, M. J. Langmuir 1996, 12, 2353-2361. (45) Nicewarner-Pen˜a, S. R.; Raina, S.; Goodrich, G. P.; Fedoroff, N. V.; Keating, C. D. J. Am. Chem. Soc. 2002, 124, 7314-7323. (46) Mirkin, C. A.; Letsinger, R. L.; Mucic, R. C.; Storhoff, J. J. Nature 1996, 382, 607-609.

Goodrich et al. DNA:Au Nanoparticle Bioconjugate Hybridization. DNA:Au conjugate hybridizations followed the 3-oligonucleotide system described by Mirkin and co-workers.45,46 Twelve-base DNA sequences attached to Au nanoparticles were linked together upon hybridization to a 24-base linking sequence complementary to both of them. In general, 150 µL of each conjugate to be used was added to 300 µL of hybridization buffer in a 1.5-mL eppendorf tube. Hybridization was initiated by the addition of 0.05 nmol of a cross-linking oligo. The hybridization temperature was controlled by placing the eppendorf tubes in a constant temperature heat block (VWR). All hybridizations were allowed to proceed overnight. DNA Hybridization. Equal volumes of two complementary DNA 24-mers (100 µM stock solutions in 0.3 M NaCl, 10 mM phosphate buffer pH 7) were added to a 1.7-mL eppendorf tube and heated to 70 °C for 10 min. The DNA solution was allowed to cool to room temperature and was left at room temperature to hybridize for at least 5 h. Melting Analysis. Determination of the melting temperature, Tm, for the bioconjugate aggregates was performed using the thermal denaturation mode of the UV Chemstation software for the HP 8453A spectrophotometer equipped with a HewlettPackard 89090A Peltier temperature control unit. To conserve DNA for use in multiple melting experiments, the melt transitions for the nanoparticle aggregates were carried out using a 100-µL quartz cuvette (Starna Cells). To reduce solution evaporation during the thermal denaturation, a small amount of mineral oil was added to the top of the solution and the cuvette was capped. In some cases, evaporation led to large standard deviations in the melting transition temperature determined. In this case, samples were analyzed using a resealable quartz cuvette (4-mL volume) with a micro-stir bar (Starna Cells). For the DNA:Au conjugate melt analysis, all thermal denaturation curves were taken using 1 °C intervals, with a hold time of 5 min. For the DNA:DNA duplex melts, the thermal denaturation plots were taken using 1 °C intervals with a hold time of 2 min. The Tm of the system was determined from the first derivative of the resulting melt curve. Determination of Percent DNA:Au Conjugate Aggregated. Upon aggregation, the DNA:Au conjugates sediment to the bottom of the cuvette. Thus, aggregation was followed by measuring the fraction of free DNA:Au conjugates remaining in suspension. Specifically, 75 µL of each of the DNA:Au conjugates prepared for use in the thermal denaturation experiments were added to 850 µL of buffered polymer solution with the appropriate amount of NaCl already dissolved. The conjugates were mixed thoroughly and allowed to sit overnight. The plasmon absorbance at 520 nm was measured for each of the samples, and the percent aggregation was calculated using the equation 100 - [(A520,sample/A520,no salt) × 100].

Results and Discussion The melting transition temperature (Tm) is frequently used as a measure of the stability of a DNA duplex. For free DNA in solution, the Tm is defined as the temperature at which 50% of the DNA duplex has melted.9 Experimentally, Tm’s are determined by observing the absorbance at 260 nm as the solution temperature is increased. Upon DNA duplex dissociation, this absorbance increases dramatically. Plotting the first derivative of the absorbance versus temperature yields the Tm. DNA:Au nanoparticle bioconjugate assemblies are also characterized by a melting transition that can be determined by either measuring the increase in the absorbance at 260 nm or the shift in the surface plasmon resonance of the gold colloid (the wavelength of which is dependent on the particle size and composition). Indeed, Mirkin and coworkers have shown that the melting transitions are sharper for bioconjugates than for DNA alone.37 This (47) This was determined experimentally using Beers law, with the nanoparticle concentration based on the particle size and number of Au atoms per milliliter.

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Figure 3. Optical extinction spectra for DNA:Au conjugates in 0 (a), 10 (b), 15 (c), and 20% (d) PEG 12 kDa without a complementary linker strand.

Figure 1. Thermal denaturation plot (A) and derivative plot (B) for DNA:Au nanoparticle conjugates prepared in the absence (solid line), and in the presence (dotted line) of 5% PEG 8 kDa.

Figure 2. Effect of increasing concentration of PEG 8kDa, PEG 12 kDa, and PEG 35 kDa on Tm for DNA:Au nanoparticle conjugate aggregates. Lines represent the linear least-squares fit for each data set.

phenomenon has been explained recently by Schatz, and co-workers as a cooperative phenomenon.10 To determine the effect of polymeric solutes on Tm for DNA:nanoparticle assemblies, thermal denaturation experiments were performed in solutions containing increasing weight percents of PEG. A marked increase in the melting transition temperature for DNA:Au conjugates aggregated due to specific DNA hybridization is observed in the presence of 5 wt % PEG 12 kDa (Figure 1). Figure 2 compares the effect of 35, 12, and 8 kDa PEGs on DNA:Au aggregates Tm as a function of the polymer weight percent. As a result of the small volumes of sample used in these experiments and resulting difficulties with evaporation and poor mixing, relatively large variations in Tm are observed for repeated measurements. Nonetheless, clear trends in Tm are observed. The average Tm for the DNA:Au aggregates increases as the weight percent of PEG increases in the solution for all of the molecular weights tested and does not appear to be sensitive to polymer molecular weight. Although the intercept for each MW of PEG is different due to batch-to-batch variations in DNA:Au conjugate preparation,48 the slope of the lines remains very similar, which supports the hypothesis that the increase in Tm is dependent primarily on the weight percent of the polymer and not on the molecular weight or molarity. This behavior differs from previously reported data for free DNA duplex melting in solution with low

molecular weight PEGs (e.g., 200-6000 kDa) where the molecular weight of the polymer was found to affect the Tm strongly.49 This effect may be the result of the large PEG molecular weights used in our experiments. Given the marked increase in thermodynamic stability for DNA:Au assemblies observed with increasing PEG concentration (Figures 1 and 2), it was important to determine what effect polymeric solutes would have on assembly specificity. To determine whether high PEG concentrations alone could drive nonspecific particle aggregation, the same experiments were repeated in the absence of linking oligonucleotide using 0, 10, 15, and 20% PEG 12kDa. Figure 3 shows the extinction spectra for a mixture of the two DNA:Au conjugates used in these experiments. Because no linking strand of DNA was added to the mixture, no DNA-induced aggregation should take place. A sharp plasmon absorption around 520 nm can be seen for the particles alone in solution in the absence of polymer. This curve broadens as the concentration of polymer is increased to 10 and 15% PEG 12 kDa, indicating aggregation. These data demonstrate that high weight percents of PEG are sufficient to drive the association of DNA:Au nanoparticle bioconjugates even in the absence of selective DNA hybridization. While there are numerous literature examples of macromolecular crowding facilitating association of weakly interacting biomacromolecules,29 this demonstration of DNA:Au nanoparticle assembly in the absence of any complementary linking DNA strand illustrates the potential significance of crowding in biomolecule-directed nanoparticle assembly experiments. Polymer-driven bioconjugate association in the absence of selective biorecognition would clearly be disastrous in biosensing or deterministic assembly applications. Fortunately, it is possible to offset the effects of macromolecular crowding in polymeric solutions by increasing electrostatic repulsive forces between DNA:Au conjugates. Nanoparticle:DNA hybridization experiments are typically carried out in high ionic strength buffer solutions (e.g., 0.3 M NaCl) to overcome electrostatic repulsion between the charged phosphates on the backbone of the closely spaced DNA molecules. Decreasing the ionic strength, which has been used to increase stringency for mismatch discrimination in nanoparticle-based biosensing (48) In studying this system, it was determined that Tm in polymerfree hybridization buffer for a given batch of DNA:Au conjugates can vary by up to (1 °C, possibly due to slight variations in DNA surface coverage from batch to batch. The effect of this variability in the baseline Tm for the system is apparent in the Y intercepts for the plots for the PEG data (Figure 2). To minimize the effect of this variation on the determination of the crowding effect of the polymers, all Tm measurements for a given molecular weight polymer were determined using the same batch of DNA:Au conjugate. (49) Woolley, P.; Wills, P. R. Biophys. Chem. 1985, 22, 89-94.

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Figure 4. Effect of increasing salt concentration on the percent colloid nonspecifically aggregated in 15% PEG 12 kDa.

Figure 5. Deriviative plot of absorbance versus temperature for DNA:Au conjugates in 15% PEG 12 kDa at various NaCL concentrations: (a) no salt, (b) 0.030 M, (c) 0.075 M, (d) 0.150 M, (e) 0.225 M, and (f) 0.30 M. (Inset: plot of Tm as a function of salt concentration.)

protocols,3 also restores specificity in the presence of polymeric crowding agents. Figure 4 illustrates the effect of decreasing the NaCl concentration on nonspecific aggregation in 15% PEG 12 kDa solutions. At 0.3 M NaCl, the concentration typically used for hybridization, roughly 80% of the bioconjugates have nonspecifically aggregated. This effect can be eliminated by lowering the concentration of NaCl in the polymer solution to 0.07 M. Lowering the salt concentration to prevent nonspecific interactions will also effect the Tm of the DNA:Au aggregates in the presence of a complementary linker. This can be seen in a plot of the first derivative of the melting transition of the bioconjugates (Figure 5). Reducing the salt concentration of the polymer solution results in a decrease in the Tm of the conjugates. Thus, increased stability of DNA:Au conjugate aggregates in polymeric solutions can be offset by a decreased NaCl concentration. It will be important to balance these solution conditions in future assembly applications. While macromolecular crowding often appears to result largely from physical volume exclusion, chemical effects are also important. For example, different macromolecular solutes may interact differently with solvent molecules or with the biomolecule of interest.17 Minsky and co-workers have demonstrated that PEGs effect not only the entropy (as is expected from volume exclusion) but also the enthalpy of DNA association.18 They ascribe the enthalpic terms to changes in DNA hydration. Spink and Chaires have discussed the effect of hydration, ion release, and volume exclusion on DNA dissociation in the presence of PEG.19 Because both the conformation and hydration of polymeric crowding agents depend on their chemical structure, some differences in crowding effectiveness are expected for different polymers.

Figure 6. Effect of dextran 144 kDa, dextran 10 kDa, and dextran 1 kDa on the Tm of DNA:Au conjugates. Lines represent the linear least-squares fit for the data.

To investigate the importance of polymer chemistry in our experiments, we performed thermal denaturation experiments in the presence of dextrans. The average Tm values for the DNA:Au aggregates as a function of weight percent for 144 kDa, 10 kDa, and 1 kDa dextrans are presented in Figure 6. The magnitude of the change in Tm with increasing polymer weight percent is much less for dextran (Figure 6) as compared with PEG (Figure 2). The effect of increasing the dextran concentration from 0 to 10 wt % in solution is less than 1 degree for both dextran 10 kDa and dextran 144 kDa. In comparison, the change in Tm resulting from an increase in PEG 12 kDa from 0 to 10 wt % is approximately 7 °C. The significant difference in slope for PEG and dextran polymers indicates that PEG and dextran have very different crowding efficiencies. This difference in crowding ability has been shown previously with RNA double helixes,49 as well as for DNA duplexes and triplexes.18 Minsky et al. report that dextrans led to approximately one-third the increase in Tm as compared with PEGs.18 Interestingly, the Tm data for 1 kDa dextran (Figure 6) has a negative slope. This indicates that the low molecular weight dextran is actually destabilizing the DNA duplex. This is most likely due to the fact that the molecule is too small to effectively crowd the DNA and is simply reducing the activity coefficient of water in the system, thus destabilizing the duplexes. This result correlates well with previously reported data using small PEGs and dextrans as crowding agents.18,21,50 To understand the effects of conjugation to Au nanoparticles on (50) Laurent, T. C.; Preston, B. N.; Carlsson, B. Eur. J. Biochem. 1974, 43, 231-235.

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containing various weight percents of PEG and dextran polymers. The results are presented in Figure 7. For the PEG system, the Tm of the duplex increases with increasing PEG concentration, while for the dextran there appears to be a significant increase in the observed Tm that was nearly independent of the polymer concentration. The difference in the Tm due to increasing the molecular weight of the PEG is again minimal. Thus, the effect of macromolecular crowding in the DNA:Au bioconjugate system is qualitatively similar to that for DNA duplexes, with PEG being a more effective crowding agent than dextran. Conclusions

Figure 7. Effect of PEG 12 kDa (b), PEG 35 kDa (9), dextran 480 kDa (9), and dextran 10 kDa (b) on the 24-mer DNA:DNA duplex melting in the absence of Au nanoparticles. Lines represent the linear least squares regression for each data set.

sensitivity to macromolecular crowding, we have also performed melting experiments on free oligonucleotides in buffer. A 24-mer DNA duplex was melted in buffer

The effect of macromolecular crowding by aqueous polymer solutions on DNA-directed nanoparticle assembly has been investigated. We have demonstrated that macromolecular crowding by polymer solutions can impact the thermodynamics of DNA-directed nanoparticle assembly, under some conditions even driving association in the absence of a complementary linking oligonucleotide. Selectivity could be retained in these experiments by adjusting the solution ionic strength to offset the effect of crowding reagents. Acknowledgment. This work was supported by the National Science Foundation (MCB 0074845), DARPA/ ONR (N00014-01-1-0659), the National Institutes of Health (EB 00268), and Penn State University. LA048434L