Nanomechanical Effect of Enzymatic Manipulation of DNA on

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Nanomechanical Effect of Enzymatic Manipulation of DNA on Microcantilever Surfaces Karen A. Stevenson,* Adosh Mehta, Pavlo Sachenko, Karolyn M. Hansen, and Thomas Thundat Life Sciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831 Received July 18, 2002. In Final Form: September 6, 2002 Microcantilevers functionalized with DNA incorporating a Hind III restriction endonuclease site were digested with Hind III to produce DNA with a single-stranded end on the cantilever surface. Ligase was then used to link a second DNA molecule with a compatible end to the DNA on the cantilever. Nanomechanical motion of the cantilever was monitored throughout the digestion and ligation. Fluorescently labeled DNA was used to confirm that ligation and digestion occurred. The DNA was attached to the silicon side because Hind III and DNA ligase both require dithiothreitol to retain their activity. We therefore avoided the possibility that thiolated DNA on the gold side of the cantilever would be displaced by thiol-containing compounds in solution. Our results show that any natural DNA containing a restriction endonuclease site could be digested and attached to a cantilever functionalized with a compatible DNA. Our results also show that the ligated DNA can be removed, regenerating the cantilever for future use.

1. Introduction Nanomechanical deflection due to biomolecular interactions is the characteristic signal in cantilever biosensors. The origin of nanomechanical cantilever motion has been attributed to the change in surface stress induced by molecular interaction events that are restricted to one surface of the cantilevers.1 Capitalizing on this motion, microcantilevers have been used as sensors in a wide variety of physical, chemical, and biological applications, for example, humidity,2 pH,3 chemical sensing,4-9 and biomolecular affinity10-13 reactions. Manipulation of biomolecules immobilized on cantilever surfaces is reflected in nanomechanical cantilever motion when the biochemical reaction is well defined and reproducible. At present, nanomechanical detection of biomolecular interactions on cantilevers has been limited to affinity interactions, specifically DNA hybridization10,11 and antibody/antigen interactions.12,13 Here we report the first study describing the enzymatic manipulation of immobilized DNA on gold/silicon micro(1) Wu, G.; Ji, H.; Hansen, K.; Thundat, T.; Datar, R.; Cote, R.; Hagan, M. F.; Chakraborty, A. K.; Majumdar, A. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 1560-1564. (2) Thundat, T.; Warmack, R. J.; Chen, G. Y.; Allison, D. P. Appl. Phys. Lett. 1994, 64, 2894-2896. (3) Ji, H.-F.; Hansen, K. M.; Hu, Z.; Thundat, T. Sens. Actuators, B 2001, 72, 233-238. (4) Cherian, S.; Mehta, A.; Thundat, T. Langmuir 2002, 18, 69356939. (5) Berger, R.; Gerber, Ch.; Gimzewski, J. K.; Meyer, E.; Gu¨ntherodt, H. J. Appl. Phys. Lett. 1996, 69, 40-42. (6) Gimzewski, J. K.; Gerber, Ch.; Meyer, E.; Schlittler, R. R. Chem. Phys. Lett. 1994, 217, 589-593. (7) Thundat, T.; Wachter, E. A.; Sharp, S. L.; Warmack, R. J. Appl. Phys. Lett. 1995, 66, 1695-1697. (8) Lang, H. P.; Berger, R.; Battiston, F.; Ramseyer, J.-P.; Meyer, E.; Andreoli, C.; Brugger, J.; Vettiger, P.; Despont, M.; Mezzacasa, T.; Scandella, L.; Gu¨ntherodt, H. J.; Gerber, Ch.; Gimzewski, J. K. Appl. Phys. A 1998, 66, S61-S64. (9) Jensenius, H.; Thaysen, J.; Rasmussen, A. A.; Veje, L. H.; Hansen, O.; Boisen, A. Appl. Phys. Lett. 2000, 76, 2615-2617. (10) Fritz, J.; Baller, M. K.; Lang, H. P.; Rothuizen, H.; Vettiger, P.; Meyer, E.; Gu¨ntherodt, H.-J.; Gerber, Ch.; Gimzewski, J. K. Science 2000, 288, 316-318. (11) Hansen, K. M.; Ji, H.-F.; Wu, G.; Datar, R.; Cote, R.; Majumdar, A.; Thundat, T. Anal. Chem. 2001, 73, 1567-1571. (12) Wu, G.; Datar, R. H.; Hansen, K. M.; Thundat, T.; Cote, R. J.; Majumdar, A. Nat. Biotechnol. 2001, 19, 856-860. (13) Raiteri, R.; Nelles, G.; Butt, H.-J.; Knoll, W.; Ska´dal, P. Sens. Actuators, B 1999, 61, 213-217.

cantilevers and the resultant mechanical motion. Two enzymatic reactions were conducted: a restriction digestion of immobilized DNA and subsequent ligation of a compatible DNA sequence to the remaining immobilized DNA. Figure 1 shows a schematic of the digestion and ligation reactions. Double-stranded (ds) DNA incorporating a Hind III restriction endonuclease recognition site was attached to the silicon side of a gold-coated cantilever. DNA-coated cantilevers were then exposed to Hind III restriction enzyme, and deflection was monitored before, during, and after exposure to Hind III enzyme. Hind III cuts the DNA on the cantilever at the specific recognition site, leaving a 5-base single-stranded “sticky end” that can be used to attach a piece of DNA with a complementary end. To verify that the restriction digestion did occur, a fluorescently labeled dsDNA was ligated onto the remaining immobilized DNA while monitoring cantilever deflection. Ligase catalyzes the joining of the strand of DNA on the cantilever to the compatible DNA in solution. For both restriction and ligation reactions, a permanent deflection of the cantilever was observed even after the enzymes were washed away. Fluorescence microscopy showed that the ligation had occurred; hence, the restriction digestion was also successful. The fluorescent DNA was then redigested from the cantilever with Hind III restriction enzyme; fluorescence imaging verified that the digestion had occurred, regenerating the DNA on the cantilever for another round of ligation. This repeatable digestion/ligation procedure opens up a range of possibilities for control and manipulation of DNA-coated microcantilevers for use not only in molecular interaction sensing devices but also as tunable actuators and nanomechanical motors. 2. Experimental Section DNA incorporating a Hind III restriction endonuclease recognition site was purchased from Oligos Etc. (Wilsonville, OR). This oligonucleotide (DNA 1) contained a 5′ amino group to facilitate attachment to the microcantilever and is shown in Table 1 with the Hind III site in bold. Plasmid pSP72 (Promega) was digested with restriction enzymes Hind III and Bgl II to produce a 69 bp fragment (DNA 2; Table 1) compatible with the Hind III digested DNA fragment on the cantilever. This 69 base pair fragment was ligated to the Hind III site exposed by digestion. DNA 3 (Table 1) was labeled with a 5′ fluorescein and was used

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Figure 1. Schematic of the digestion and ligation experiments. DNA on the cantilever surface incorporating a Hind III site was first cut with Hind III endonuclease, leaving a single-stranded end. Then DNA with a compatible end was ligated to the DNA on the cantilever, producing a longer DNA on the cantilever surface. X’s and Y’s represent base pairs not involved in the digestion/ ligation site. Subscripts denote that a variable number of uninvolved base pairs could be used. Table 1. DNA Sequences Used

to confirm that ligation had occurred. Prior to use, oligonucleotides were annealed by mixing equimolar amounts of single strands, heating to 90° for 1 min, and allowing the mixture to cool to room temperature. All enzymes were purchased from Promega. Reagent-grade chemicals were purchased from Sigma-Aldrich. Commercially available rectangular silicon cantilevers (NTMDT, Portland, OR) coated with aluminum on one side were used in this study. The cantilevers were 350 or 300 µm long, 35 µm wide, and 1 µm thick with a spring constant of 0.03-0.05 N/m. The aluminum was removed to prevent reaction with silane by washing sequentially with 1 N NaOH, 3 N HCl, and then 1 N NaOH. After they were washed with water, the cantilevers were rinsed with ethanol and dried at 80 °C. Chromium (2.5 nm) and then gold (25 nm) were deposited on the upper surface of the cantilevers using vacuum e-beam evaporation (Thermionics, Port Townsend, WA). The cantilevers were stored in a vacuum desiccator. Immediately prior to use, the cantilevers were washed for approximately 10 min each with acetone and ethanol and then immersed in piranha solution (3 parts hyrogen peroxide, 7 parts sulfuric acid) for 10 s. They were then rinsed with water and ethanol, and the silicon side was functionalized. Cantilevers were immersed in 2% of (3-aminopropyl)triethoxysilane (APTS) in ethanol overnight and then washed with ethanol and dried in a vacuum desiccator. Cantilevers were then incubated in 1% aqueous glutaraldehyde for 1 h, rinsed in phosphate buffered saline (PBS, pH 7.4), and immersed for at least 24 h in 1 mg/mL DNA 1 containing 10 mM potassium hydroxide. Cantilever optical deflection measurements were conducted using a four-quadrant AFM head with integrated laser and position-sensitive detector (Digital instruments, Santa Barbara, CA) as previously described.11 Flow was controlled using a syringe pump (IITC, Inc., Woodland Hills, CA) equipped with a lowpressure liquid chromatography injector valve and a 2 mL injection loop (Upchurch Scientific, Oak Harbor, WA). Cantilever deflection was monitored throughout each experiment using HP Data Logger Model 34970A. Throughout this Letter, a positive deflection refers to bending toward the gold side, while a negative deflection means the cantilever bent toward the silicon/DNA side. For the restriction endonuclease experiments, cantilevers were removed from the DNA solution after 24-72 h, washed in Hind III buffer, and then soaked overnight in Hind III buffer containing 6 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1 mM DTT, and 6 mM MgCl2. The cantilever was placed in the flow cell in the same buffer, and a syringe pump was set up to deliver the buffer at 2 mL/h. Hind III has optimum activity at 37 °C, so temperature was maintained at 37 ( 2 °C using a Thermolyne Dri-Bath (Dubuque, IA) and monitored throughout the experiment. After

a stable baseline was obtained, typically 1.5-2.5 h, 2 mL of Hind III buffer containing 100 units of Hind III enzyme was injected, at a flow rate of 2 mL/h. After 1 h, the syringe pump resumed delivering buffer as a wash solution. After the cantilever was washed in the flow cell for at least 1 h, the cantilever was removed, rinsed with ligase buffer (30 mM Tris-HCl, pH 7.8, 10 mM DTT, 10 mM MgCl2, and 1 mM ATP), and then stored in ligase buffer at least overnight prior to use in a ligation experiment. The cantilever was then placed in the flow cell with ligase buffer, and the syringe pump was set up to deliver the buffer at 2 mL/h. After a stable baseline was obtained, 2 mL of ligase buffer containing 75 units of Ligafast enzyme and 2 µg of DNA with a compatible Hind III end was injected. After 1 h the syringe pump resumed delivering ligation buffer as a wash solution. Because ligase is active at room temperature, the temperature was not controlled for ligation experiments but was monitored continuously. Fluorescence measurements were made on a Nikon Eclipse TE300 inverted microscope in epi-illumination configuration with a 1.4 N.A. 100X oil immersion collection objective. The cantilevers were placed on a fused quartz cover-glass and continuously illuminated with the 488.5 nm line from an argon ion laser and imaged using a long-pass filter with a band edge (50% transmission) at 570 nm. The image was captured using a 512 EFTB frame transfer back-illuminated CCD (Roper Scientific, Princeton, NJ).

3. Results and Discussion The silicon sides of the cantilevers were functionalized by attaching sequentially APTS, glutaraldehyde, and then an amino-modified 40 base pair oligonucleotide containing a Hind III restriction enzyme recognition site. The silicon side of the cantilever was used because most enzymatic manipulations of DNA require solutions containing dithiothreitol (DTT) or mercaptoethanol to maintain the enzyme in an active state. These thiol containing compounds may produce complications if the DNA is immobilized on the gold side of the cantilever using a thiol group. Many studies have addressed the possibility of thiol exchange occurring on a surface when additional thiol groups are available in solution.14-18 We have shown that thiolated DNA attached to the gold side of a cantilever desorbs after exposure to DTT.19 By covalently attaching the DNA to the silicon side through an amide bond, we avoided the possibility of DNA being displaced from the cantilever by a thiolated compound in solution. Before any functionalized cantilever was used, it was soaked overnight in the (14) Collard, D. M.; Fox, M. A. Langmuir 1991, 7, 1192-1197. (15) Chidsey, C. E. D.; Bertozzi, C. R.; Putvinski, T. M.; Mujsce, A. M. J. Am. Chem. Soc. 1990, 112, 4301-4306. (16) Hostetler, M. J.; Templeton, A. C.; Murray, R. W. Langmuir 1999, 15, 3782-3789. (17) Schlenoff, J. B.; Li, M.; Ly, H. J. Am. Chem. Soc. 1995, 117, 12528-12536. (18) Fleming, M. S.; Walt, D. R. Langmuir 2001, 17, 4836-4843. (19) Stevenson, K. A.; Mehta, A.; Hansen, K. M.; Thundat, T. G. In Proceedings on MEMS-V, a Symposium of the 2002 Spring Meeting of the Electrochemical Society, Pennington, NJ, pp 218-225.

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Figure 2. Temperature-corrected plot of cantilever deflection vs time for Hind III digest of DNA on the cantilever surface. A 40 base pair DNA on the cantilever surface incorporating a Hind III site was cut with Hind III endonuclease, leaving DNA with a single-stranded end on the cantilever. Arrows show the point of injection of Hind III and the point where wash begins. Sinusoidal cantilever motion (amplitude 5 nm) due to temperature control (37 ( 2 °C) was filtered out in this curve.

appropriate buffer containing DTT. In effect, this passivated the gold side of the cantilever by coating it with DTT. Figure 2 shows the temperature-corrected deflection versus time response of a DNA-functionalized cantilever that was exposed to Hind III endonuclease at 37 ( 2°. Digestion with Hind III should release a 22 base fragment from the DNA on the cantilever, leaving an 18 base pair fragment with a single-stranded end. In Figure 2, the interval before the first arrow corresponds to flow of Hind III buffer and establishment of a stable baseline. The first arrow shows the point at which Hind III restriction endonuclease was injected. Enzyme was flowing through the cell until the point designated by the second arrow. At that point, the flow of buffer only resumed, which corresponded to the wash period. Upon injection of the enzyme, compressive stress on the DNA/silicon side of the cantilever is seen, causing a 20 nm deflection toward the silicon/DNA side, corresponding to binding of the enzyme to its recognition sequence on the DNA. The cantilever remains deflected toward the silicon side until the beginning of the wash period, when it partially recovers, and then stabilizes so that the net deflection of the cantilever is approximately 12 nm toward the silicon/ DNA side. The net negative deflection indicates that there is tensile stress on the gold side of the cantilever and/or compressive stress on the silicon side, where the DNA is attached. The partial recovery at the beginning of the wash, after a 1 h exposure to the enzyme, could correspond to the release of the DNA that is digested by the enzyme or to release of the enzyme itself or a combination of both. A control experiment, data not shown, used a cantilever that was first functionalized with APTS, as was the cantilever in Figure 2, but instead of attaching amino-

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Figure 3. Ligation experiments on the cantilever surface. (A) Plot of cantilever deflection vs time for ligation of DNA on the cantilever surface to DNA in solution. DNA on the cantilever surface has a single-stranded end produced by Hind III digestion. A 69 base DNA with a compatible end was injected along with ligase to link the two DNA molecules. Arrows show the point of injection of compatible DNA and ligase and the point where the wash begins. (B) Deflection vs time for control ligation experiments. Curve A shows the plot for the injection of compatible DNA with no ligase. Curve B shows the injection of ligase and DNA with an incompatible end. Arrows show the point of injection and the point where wash begins.

modified DNA to the silane, an amino-modified alkane, methylamine, was bound to the cantilever. Injection of Hind III enzyme produced a slow negative bending of the cantilever that remained unchanged even after more than 1 h of washing. This deflection may be due to nonspecific binding of the Hind III enzyme to the cantilever. The cantilevers used in the digestion experiments were routinely removed and soaked overnight in ligase buffer. Figure 3A shows the deflection versus time response of a ligation reaction. After a baseline was established, ligase and a 69 base pair DNA fragment containing a compatible Hind III end were injected into the flow cell (first arrow). Upon injection, there was a compressive stress on the silicon/DNA side of the cantilever reflected in a sharp 20 nm deflection of the cantilever toward the silicon/DNA side. During the time that the reactants were present in the flow cell, the deflection was fairly stable. When the wash began (second arrow), a tensile stress on the silicon/ DNA side of the cantilever was observed with a sharp deflection toward the gold side of approximately 30 nm. The net deflection, after 1 h of washing the cantilever, was positive about 10 nm. This indicates a compressive stress on the gold side of the cantilever or a tensile stress on the silicon side where the DNA was bound. The net positive deflection not only indicates that a ligation occurred but also confirms that the digestion took place,

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since no compatible end for ligation would exist if the digestion had not occurred. Various controls were done, and two are shown in Figure 3B. Using DNA-functionalized cantilevers after digestion with Hind III, compatible DNA was injected without ligase (curve A). Upon injection, a compressive stress on the silicon/DNA side of the cantilever is observed, reflected in a slow negative deflection of the cantilever that is unchanged even after 1 h of washing with buffer. This is in sharp contrast to Figure 3A, which confirms that, in the presence of ligase, the enzymatic reaction is taking place on the cantilever surface. Control reactions were also carried out by injecting ligase without compatible DNA and by injecting ligase and DNA using a cantilever functionalized with methylamine instead of DNA. All these reactions produced a slow drift of the cantilever toward the silicon/DNA side that continued even after the wash. These results suggest that there is nonspecific adsorption of DNA and/or ligase to the gold side of the cantilever. Injection of ligase and DNA cut with the incompatible enzyme Bgl II produced a deflection upon injection, a recovery to the preinjection baseline when the wash began, and then a slow drift toward the silicon/DNA side as the wash continued (curve B). There was no net deflection toward the gold side as seen when DNA with a compatible end was used. To confirm that the changes in cantilever deflection were indicative of digestion and ligation, a fluorescently labeled DNA was ligated to the DNA on the cantilever. First, a DNA-functionalized cantilever containing a Hind III site was digested and washed in the flow cell. After the cantilever was soaked in ligase buffer for 48 h, the cantilever was replaced in the flow cell. Injection of a fluorescein-labeled DNA and ligase over a 1-h period was followed by 1 h of washing in the flow cell. The cantilever was removed from the flow cell and stored in ligase buffer. It was then dried and imaged with a fluorescence microscope, as was a control cantilever that had been digested with Hind III, and then stored in ligase buffer. Figure 4A shows a fluorescence image of the cantilever that was used in the ligation, while Figure 4B shows a fluorescence image of the control cantilever. All fluorescence images are shown using the same gray scale for comparison purposes. The cantilever treated with fluorescently labeled DNA shows a marked increase in fluorescence as compared to that of the control cantilever. The fluorescence was intense over the entire illumination area (approximately 10 µm), whereas the control sample showed isolated low-intensity spots that are probably due to Hind III autofluorescence. The fluorescence imaging implies a continuous coverage of the initial doublestranded DNA substrate. Further studies are underway to characterize the surface coverage of DNA immobilized using APTS and glutaraldehyde. We next tested whether a restriction digest could be used to remove ligated DNA in order to regenerate the cantilever surface. The cantilever imaged in Figure 4C is the same as the cantilever in Figure 4A. It was rehydrated in Hind III buffer, placed in the flow cell, and digested with Hind III as described for Figure 2. The cantilever was removed from the flow cell after washing, and then it was dried and imaged with the fluorescence microscope. The fluorescence image in Figure 4C looks very similar to that of the control cantilever seen in Figure 4B. This suggests that the fluorescently labeled DNA that had been ligated to the DNA on the cantilever was effectively removed by the Hind III digest, thus regenerating the cantilever for future use. We attribute the nanomechanical motion generated by digestion and ligation reactions to the change in surface

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Figure 4. Fluorescence images of cantilevers. The fluorescence images are shown using the same gray scale for comparison purposes. The size of the images is 1.7 µm × 1.7 µm. (A) Fluorescence image of a DNA-functionalized cantilever after digestion with Hind III and ligation to fluorescein-labeled DNA 3. (B) Fluorescence image of a control cantilever after digestion with Hind III. (C) Fluorescence image of the cantilever in Figure 4A after digestion with Hind III.

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stress and surface charge on the silicon/DNA side of the cantilever versus the gold side. The net deflection toward the silicon/DNA side after digestion of DNA on the cantilever was reproducible over many experiments, as was the net deflection toward the gold side after ligation of the DNA. Undigested (longer) DNA has a greater overall charge density due to the number of phosphate groups in the DNA backbone. Ligation of a 69 base fragment of DNA could lead to less dense packing of the DNA on the cantilever surface, causing the deflection away from the DNA side of the cantilever. Digestion of the DNA on the cantilever reduces the length of the DNA, which could lead to tighter packing on the cantilever surface and cause deflection toward the DNA side. However, digestion also leaves a single-stranded end on the DNA, and one might expect that would prevent tighter packing. The salt concentration in the restriction endonuclease buffer may provide a counterion shield that prevents repulsion of the single-stranded ends. There is a compressive stress on the DNA/silicon side of the cantilever upon binding of either Hind III or ligase. Wu et al.1 have suggested that entropy changes are responsible for cantilever bending in DNA hybridization assays. It is known that large numbers of water molecules are released upon nonspecific binding of a restriction enzyme to DNA.20 It is also known that a conformation change takes place in the enzyme upon transition from the nonspecific initial complex with the DNA to the specific DNA binding complex with its recognition sequence.20 It is unclear whether the net entropy change due to the DNA/enzyme interactions and the release of water molecules is positive or negative. Using Wu’s argument, if the unfavorable entropy of binding outweighs the entropy gain from release of the water molecules, the cantilever should bend away from the DNA/ silicon side. If the favorable entropy from the water release predominates, the cantilever should bend toward the DNA/ gold side. It is possible that the cantilever bending is at least partially a response to these entropy changes. Another possibility is that the enzyme shields adjacent DNA molecules from the negatively charged DNA backbone, decreasing the repulsion between neighboring molecules. The fact that it is relatively simple to ligate a compatible strand of DNA to DNA on the cantilever surface means that any piece of DNA containing a restriction enzyme site can be bound to DNA on the cantilever surface with no modifications necessary. Until now, DNA has been (20) Pingoud, A.; Jeltsch, A. Nucleic Acids Res. 2001, 29, 3705-3727.

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attached to the gold side of cantilevers through thiol groups, precluding the attachment of natural, unmodified DNA. Using this method, any DNA sample can be digested with a restriction enzyme and ligated to a cantilever that has been functionalized with an oligonucleotide containing a compatible single-stranded end. After an assay is carried out on the cantilever, the DNA can be digested and removed from the cantilever, regenerating it for further use. Developing the ability to control and manipulate cantilever deflection using biomolecular interactions offers the prospect of tunable nanomechanical systems in a variety of settings, both in vivo and ex situ. Modulation of cantilever motion by biochemical reactions involving immobilized biomolecules such as DNA opens up an array of possibilities for control in nanomechanical systems. It could lead to a tunable actuator directed by biomolecular interactions that would have potential applications in micro- and nanofluidic delivery systems. 4. Conclusions We have demonstrated that it is possible to detect both the digestion and the ligation of DNA on the silicon side of a microcantilever surface. The biomolecular reactions of a restriction endonuclease and a DNA ligase are manifested in the nanomechanical motion of the cantilever. Our ligation results show that it should be possible to attach any DNA containing a restriction enzyme site to a cantilever containing the appropriate compatible DNA end. Our digestion results suggest that the DNA could also be removed, regenerating a restriction site on the cantilever, allowing it to be reused. We have, in effect, developed a universal DNA sensor surface. Our results also suggest the possibility that other enzymatic reactions could be detected by monitoring the nanomechanical motion of cantilevers. Acknowledgment. The authors wish to thank Drs. Suman Cherian and Jianhong Pei for helpful discussions and Dr. Roberto Raiteri for technical assistance. K.A.S., A.M., P.S., and K.M.H. acknowledge support from the Oak Ridge Associated Universities. This research was sponsored by the Office of Biological and Environmental Research (OBER), U.S. DOE. Oak Ridge National Laboratory (ORNL) is managed by UT-Battelle, LLC, for the U.S. Department of Energy under Contract No. DE-AC05-00OR22725. LA0262654