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Driving Forces for DNA Adsorption to Silica in Perchlorate Solutions
JOURNAL OF COLLOID AND INTERFACE SCIENCE ARTICLE NO.
181, 635–644 (1996)
0421
Driving Forces for DNA Adsorption to Silica in Perchlorate Solutions KATHRYN A. MELZAK, CHRIS S. SHERWOOD, ROBIN F. B. TURNER,*
AND
CHARLES A. HAYNES† ,1
Biotechnology Laboratory, *Department of Electrical Engineering, and †Department of Chemical Engineering, 237 Wesbrook Building, 6174 University Boulevard, The University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada Received January 22, 1996; accepted February 26, 1996
The adsorption of both plasmid and chromosomal duplex DNA to silica is investigated with the aim of determining the dominant forces involved in the binding reaction. Changes in the initial slopes and plateau values of adsorption isotherms for DNA on microcrystalline silica particles are used to establish the sensitivity of the binding reaction to ionic strength, temperature and pH, and DNA size and conformation. Binding is driven by an increase in entropy, with little or no enthalpic contribution. Adsorption isotherm results indicate that three effects, namely: ( i ) shielded intermolecular electrostatic forces, ( ii ) dehydration of the DNA and silica surfaces, and ( iii ) intermolecular hydrogen bond formation in the DNA – silica contact layer, make the dominant contributions to the overall driving force for adsorption. q 1996 Academic Press, Inc. Key Words: DNA-silica interactions; DNA adsorption; adsorption isotherms; DNA conformation; DNA purification; DNA binding.
INTRODUCTION
Understanding the nature of DNA interactions with silica and SiOx surfaces is becoming increasingly important as industrial efforts to automate and miniaturize DNA manipulation and purification technologies intensify (1). Chaotropic-salt-induced adsorption of DNA to silica is one of the most common methods for purifying both chromosomal and plasmid DNA from cell homogenates (2). The binding reaction is usually carried out in 4 M sodium iodide solution, buffered to pH 7.5 or 8, using a silica dispersion or, in some cases, pressed glass fibers which provide a high specific surface area for DNA binding. Since the adsorption process is reproducible for sufficiently large segments of DNA, several commercial kits, including the Geneclean and S&S EluQuick technologies (3, 4), are readily available. However, there is little scientific information to explain why the silica/ aqueous–solvent interface under these high-ionic-strength conditions selectively binds duplex DNA when exposed to aqueous mixtures containing protein, lipid, carbohydrate, and RNA contaminants. 1
To whom correspondence should be addressed.
Previous studies of DNA adsorption to silica or to any other solid are extremely limited. The most extensive studies are those of Lorenz and Wackernagel (5, 6) and others (7), who investigated the adsorption of plasmid DNA to mineral surfaces with the general aim of determining the increase in the stability of genetic information against nucleolytic inactivation due to adsorption. These studies built on a limited set of initial studies by Miller and colleagues (8, 9). Our poor understanding of duplex DNA–SiOx interactions is, in part, responsible for the slow progress made to date in the development of automated micromachined systems for DNA amplification, sequencing, etc., despite considerable industrial effort. The United States Department of Commerce identified the development of new tools for DNA diagnostics as a critical area of research for international competitiveness (10). Over the period from 1994 to 1999, the agency will commit up to $145 million toward the development of new technologies for DNA diagnostics. Many of these technologies involve the microfabrication of existing, larger-volume DNA manipulation or diagnostics systems in silicon wafers. The preferred approach would be to micromachine a monolithic device which provides for chemical or physical separation of the target DNA from cellular contaminants, followed by micromachined elements designed to sort out the sample’s chemical constituents, sequence DNA, search for mutations in a gene, etc., aligned in the electrophoretic carrier stream either in series or in parallel (11, 12). Although significant progress has been made in the development of the diagnostics and (bio)chemical processing (e.g., polymerase chain reaction) elements of these devices (13– 16), relatively little attention has been given to the crucial upstream procedures required to purify the target DNA. Silicon processing technology has advanced to the point where the SiOx channels formed in micromachined silicon devices can have a wide range of surface properties. Methodologies are available for varying surface potential and charge sign, hydrophobicity, and hydrogen-bonding capability (17). Thus, in principal, it is possible to tailor-make SiOx channels in a micromachined device with surface properties similar to the silica dispersions used for batch DNA purification. This paper addresses both plasmid and chromosomal
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0021-9797/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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duplex DNA adsorption to silica from a colloid–chemical perspective with the aim of determining the dominant forces involved in the binding reaction and the properties of the DNA and silica surfaces which control adsorption. Changes in the initial slopes and plateau values of adsorption isotherms for DNA on microcrystalline silica particles are used to establish the sensitivity of the binding reaction to ionic strength, temperature, and pH. The molecular weight and tertiary structure of the DNA substrate are also varied to determine their influence on the binding affinity under otherwise constant conditions. Adsorption of duplex DNA to silica will occur when DNA, silica, water, and a sufficient concentration of chaotropic salt are present in solution or suspension, usually with the addition of buffer to control the pH. Before discussing results, we present a brief description of each of these system components, including duplex DNA structures and surface properties, to establish potential contributions to the overall driving force for adsorption. CHARACTERISTICS OF SUSPENSION COMPONENTS
The phosphate diesters on the backbone of DNA are strong acids, making duplex DNA a strong polyelectrolyte carrying two univalent negative charges per base pair at most solution pH (18). In multivalent electrolyte solutions, this large negative surface-charge density is compensated by the site-specific binding of polyvalent cations, particularly magnesium and various polyamines. These cations, which are usually associated with water molecules, tend to bridge between adjacent phosphate anions. Monovalent cations may also associate with the backbone; in general, they do not bind to specific sites, but instead neutralize the negatively charged DNA surface through counterion condensation. The dynamic nature of counterion condensation results in partial occupancy of specific sites at regions of high electrostatic potential, with the level of occupancy a function of ionic strength (19). This interplay between free and fixed charges is one of the factors contributing to the dependence of DNA conformation on ionic strength (see below). The surface of silica, although more difficult to characterize, is also negatively charged at basic and near neutral pH values due to the weakly acidic silanol groups; surface silanol groups typically titrate over a rather broad pH range from ca. 4 to 8 with an average pKa ranging from 5 to 7, suggesting that the silica surface is heterogeneous (20, 21). Intermolecular electrostatic forces may therefore make an important contribution to the adsorption mechanism, particularly near and above neutral pH, where the net electrostatic repulsion between fixed charges on the DNA and silica surfaces will strongly disfavor adsorption at low ionic strength. Base pairs in the interior of the DNA double helix are uncharged at neutral pH. Complete titration of the nitrogen atoms on the purine and pyrimidine rings at low pH results
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in denaturation of the double helix. Unfolding is also initiated when the inside of the helix acquires an excess negative charge due to dissociation of protons from the |N{C(OH){ groups of guanine and thymine. Thus, the structural stability of duplex DNA is pH dependent, with a maximum stability of ca. DGd Å 3–8 kJ/mol base pair near pH 7 (22). Although electrostatic effects are partially responsible for the low structural stability of duplex DNA, the restricted conformational degrees of freedom of the furanose–phosphate backbone and the ribonucleotide side chains also destabilize the double helix relative to the single-strand randomcoil configuration. Linear duplex DNA, for instance, behaves as a worm-like chain. It is relatively inflexible due to steric restrictions in the backbone and to intramolecular repulsion between the charges on the phosphate residues. Helix stiffness is dependent on ionic strength; below 10 02 M ionic strength, DNA becomes less flexible and the persistence length increases sharply (23, 24). An increase in conformational entropy could therefore contribute to the driving force for duplex DNA adsorption provided the adsorption process results in (partial) denaturation, and a concomitant increase in the average rotational mobility of each strand. Circularizing DNA reduces the number of possible helix configurations, resulting in an entropy decrease proportional to the length of the DNA in the circle (25). Supercoiling increases the free energy of DNA still further, as can be seen by the fact that the introduction of a nick leads to spontaneous removal of superhelices. This free energy increase is due to the distortion required to bend the DNA helix into supercoils and to the entropy loss from placing the DNA in a more ordered structure. Ligands generally bind more strongly to supercoiled DNA because they can disrupt the supercoiling and thereby cause a greater decrease in free energy (26). A similar result may be expected for adsorption of supercoiled DNA to silica. The exterior of B-form duplex DNA binds about 0.6 g of water per gram of DNA, which corresponds to a ratio of approximately 20 water molecules per nucleotide (27). Each phosphate residue is capable of forming multiple hydrogen bonds with water or potentially with other groups. Decreasing the water activity in solution through addition of either a chaotropic salt or an alcohol changes the helical structure of B-DNA either continuously to C-DNA or through a sharp cooperative transition to A-DNA (28). Either transition is accompanied by a decrease in solvent-accessible surface area. For example, Alden and Kim (29) determined that the number of bound water molecules which can be accommodated within the minor and major grooves around one nucleotide drops from 19.3 to 10.5 during the B-DNA to A-DNA transition. Both of these transitions are thought to be at least partially driven by the increase in entropy which accompanies the release of bound water from the DNA surface. The silanol groups on the silica surface are also hydrogen
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bonded to water through one or more hydration layers (30). Thus, sorbent and DNA dehydration may also contribute to the driving force for adsorption provided the process results in a significant release of bound water from one or both surfaces. Perchlorate is present in solution as a tetrahedral symmetric anion with the negative charge distributed evenly over all four oxygens. Dissolution of sodium perchlorate in water is a spontaneous, endothermic process, indicating that the entropy change for the process is positive and, thus, that the perchlorate anion is chaotropic. Four to five water molecules are bound per perchlorate anion; infrared spectroscopy measurements show that the stretching band due to water not hydrogen bonded to other water molecules increases with increasing perchlorate concentration, while the stretching band for water hydrogen bonded to itself decreases (31, 32). An increase in the concentration of perchlorate in solution therefore results in a decrease in both the length scale for electrostatic interactions and the concentration of free (bulk) water. The former effect reduces the electrostatic penalty for close interaction of the two negatively charged macrosurfaces, while the latter should increase the energetic reward of sorbent and DNA dehydration during binding. EXPERIMENTAL
Materials a. Silica source and preparation. Microcrystalline silica particles (BDH Chemicals) were cleaned at 807C for 1 h in a chromic acid bath containing 5 g K2Cr2O7 . The particles were then cooled and centrifuged to remove excess acid, followed by rinses with two volumes of water, one volume of concentrated hydrochloric acid, and again with five volumes of distilled deionized water, before being dried at room temperature on a vacuum line. Cleaned particles were stored in a desiccator at room temperature before use. The specific surface areas of three cleaned silica preparations were measured by nitrogen adsorption using a Quantasorb BET adsorption apparatus. A value of 5.6 ( {0.2) m2 / g was obtained for each sample and was taken as constant for all remaining samples. Photographs taken with a Phillips transmission electron microscope show that the silica particles are flat chips with nominal diameters ranging from ca. 0.1 to 10 mm. b. Salmon and plasmid DNA preparation. Salmon sperm DNA was purchased from Sigma Chemicals Inc. (Cat. No. 01626; St. Louis, MO). One hundred-milligram aliquots of salmon DNA were dissolved in 5 ml of sterile water or buffer and sonicated for 20 min (at 20% full power) in a SON-IM Model XL2020 sonicator to give a polydisperse, relatively high-molecular-weight DNA mixture as shown in lane 3 (from l.h.s.) of the 0.8% agarose gel shown in Fig.
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FIG. 1. Agarose gel (0.8%) of purified linearized pUC18 DNA (lane 2), purified and sonicated salmon sperm DNA (lane 3), and purified closedcircular pUC18 DNA (lane 6) for which a very large fraction is supercoiled. Lane 1 shows 1-kb DNA ladder (Life Technologies, Inc.). Lane 4 shows a lesser loading (1/10 of that in Lane 6) of the purified closed-circular pUC18 DNA.
1. Figure 1 also shows pure linearized pUC18 plasmid DNA in lane 2, and in lane 6 pure closed-circular pUC18 DNA which is predominantly supercoiled in structure. Contaminating RNA was removed by treatment with DNase-free RNase (Boehringer Mannheim Biochemica) according to the method of Maniatis et al. (33) followed by extraction with a mixture of phenol, chloroform, and isoamyl alcohol to remove protein. The A260 :A280 ratio of the final preparation was 1.8, indicating that the DNA stock was substantially free of protein contaminants. Closed-circular pUC18 DNA was prepared from lysates of Escherichia coli DH10B harboring plasmids that carry ColE1 replicons according to the procedure of Maniatis et al. (33). Linearized pUC18 DNA was prepared by digestion with the restriction enzyme HindIII (Cat. No. 15615-024; Life Technologies, Burlington, Ontario, Canada), followed by treatment with RNase and a phenol/chloroform extraction. No RNA contaminant was detected on 0.8% agarose gels of the pUC18 stock solutions. c. Perchlorate solutions. Sodium perchlorate (BDH Chemicals) solutions were made up with water that was distilled and then deionized using a Corning Mega-pure system. Solutions were filtered through a 0.22-mm filter and then autoclaved to denature any DNases before use. Buffered perchlorate solutions were prepared by mixing 50 mM Tris:HCl and Tris base in appropriate ratios to achieve the desired pH at the desired temperature in the absence of perchlorate. High concentrations of perchlorate alter the pH by affecting the dissociation of the buffer and, thus, the proton activity coefficient. Perchlorate will also affect the measured pH by changing the double layer at the pH meter electrode. Solution pH was therefore taken as that measured by the
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FIG. 2. Ascending (increasing sorbate concentration in the bulk) adsorption isotherms for both linearized pUC18 DNA (filled circles), with one distinct size (2686 bp), and sonicated salmon DNA (open squares), containing fragments ranging from ca. 500 bp to 12 kb, on silica in 6 M perchlorate solution at pH 8.0 and 377C.
electrode. Buffer solutions were titrated to the appropriate measured pH with 0.1 M HCl or NaOH. Methods DNA adsorption measurements. The amount of DNA adsorbed to silica under various conditions was determined by solution depletion as measured by absorbance at 260 nm using a Hewlett–Packard 8452A spectrophotometer. Silica particles were weighed into 1.5-ml poly(ethylene) centrifuge tubes with sample weights ranging from 2.8 to 3.1 mg of clean dry silica. Silica solutions containing 50 mM Tris buffer in varying amounts of perchlorate were sonicated with a Heat Systems sonicator microtip for ca. 5 min at a 2.5 power setting to fully suspend the silica. DNA was then added as a stock solution, containing 50 mM Tris buffer and an identical concentration of perchlorate, and the resulting mixture was then equilibrated by rotating end-over-end for 4 h at the specified binding temperature (37 or 227C). The equilibrated suspension was then centrifuged at 12,000 rpm for 1 min to remove the silica partices, and an aliquot of supernatant was collected. Initial DNA concentrations were calculated knowing the concentration of the DNA stock solution and the volumes of DNA solution and silica suspension that were mixed together. The concentration of the equilibrated supernatant was determined by absorbance at 260 nm. Supernatant samples did not contain silica particles, as determined by the shape of the absorbance spectra (from 200 to 800 nm). RESULTS
Adsorption Isotherms and the Mode(s) of DNA Binding Figure 2 shows ascending (increasing sorbate concentration in the bulk) adsorption isotherms for both linearized
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pUC18 DNA, with one distinct size (2686 bp), and sonicated salmon DNA, containing fragments ranging from ca. 500 bp to 12 kb, on silica in 6 M perchlorate solution at pH 8.0. The surface concentration of DNA at saturation ( G sat ) is similar for both DNA samples, indicating that the mode of DNA binding is insensitive to average length and polydispersity. This suggests that the DNA adsorbs in a side-on orientation, such that changing the length of the DNA makes a corresponding proportional change in the area occupied per macromolecule. Side-on orientation is consistent with the magnitude of G sat (ca. 500 mg/m 2 ). If duplex DNA is considered a cylin˚ , as determined from the crystal der with a diameter of 20 A structure for B-DNA, then the projected area of adsorbed DNA at G sat corresponds to ca. 25% of the available surface area, indicating that the adsorbed DNA remains loosely packed on the sorbent up to saturation. Fractional surface coverages at saturation tend to be substantially higher than 25% for both protein and (random-coil) polymer adsorption (34, 35). For homopolymer adsorption on well-defined sorbents, surface coverages are often in excess of 70%; both experiment (36) and theory (37) have been used to connect these results with a general binding configuration where only a fraction of monomer units is in direct contact with the sorbent, such that the remaining unbound stretches extend into the bulk solution in a series of loops and tails. Apparent contact density is then increased by extension of the adsorbed chain in the direction normal to the surface and by the conformational freedom of the (unbound) polymer backbone which allows proximal chains to overlap and entangle on the surface. The low fractional surface coverages observed for linear DNA on silica may result from the rigidity of the double helix, as compared to random-coil homopolymers, which limits its ability to form extended loops and tails and, to a lesser extent, molecular overlap. Fluorescence polarization decay curves for duplex DNA (38) reveal a stiff structure ˚ toward helix bending with a persistence length of ca. 600 A at low ionic strength. Steric interactions, primarily caused by base pairing in duplex DNA, limit the range of possible rotational angles along the sugar–phosphate backbone (39). Computer simulations by Vigil and Ziff (40) of a twodimensional random sequential adsorption (an irreversible process in which sorbate molecules are sequentially placed on a surface in random orientation under the condition that they do not overlap) show jamming coverages (i.e., maximum surface coverages) between 25 and 35% for rod-shaped molecules with a high aspect ratio. Although initial adsorption orientations are random, the simulation predicts significant ordering of molecules such as DNA at saturation since a high-aspect-ratio rod must have almost identical angular orientations with its nearest neighbors in order to successfully adsorb at high surface concentrations. As shown in Fig. 2, saturation levels for both sonicated salmon and linear
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FIG. 3. Ascending adsorption isotherms and desorption by dilution data for (a) linear and (b) closed-circular supercoiled pUC18 DNA on silica particles in 6 M perchlorate at pH 8 and 377C.
pUC18 DNA at pH 8 are consistent with the random sequential adsorption model. However, as shown below, saturation levels increase for both types of DNA with decreasing pH, suggesting that limited amounts of inter- and intramolecular overlap occur in the adsorbed layer. Provided the duplex structure remains intact during adsorption, the bound structure consistent with these results is a worm-like chain adsorbed side-on such that there are regions of near continuous contact with the sorbent surface. Linearized pUC18 DNA has a contour length of ca. 0.7 mm, which is long with respect to some of the silica particles. The longest fragments of the salmon DNA have a contour length of about ca. 3 mm. Since the silica particles have nominal diameters in the range 0.1–10 mm, bound DNA in continuous contact with a (single) silica particle must exhibit curvature along the center-line axis of the helix and, in some cases, chain direction reversal on the surface. End-region detachment of the chain, breakdown of the double-helix structure, or bridging between silica particles may also occur. Binding Reversibility with Respect to Dilution Thermodynamic reversibility requires the absence of hysteresis between the ascending and descending (decreasing bulk sorbate concentration at otherwise constant conditions) branches of the adsorption isotherm (41). In 6 M perchlorate solution at 377C, adsorption of either linearized pUC18 DNA or sonicated salmon sperm DNA is irreversible with respect to dilution. As shown in Fig. 3a, washing of silica particles with 6 M perchlorate after adsorption results in no appreciable desorption of linear pUC18 DNA at any surface coverage. Irreversible adsorption is very often observed in both polymer and protein adsorption to solids, and is thought to be the result of the large number of contacts made between polymer or protein segments and the sorbent surface such that simultaneous disruption of all contacts is energetically unfavorable (36). In a side-on orientation, linear duplex
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DNA can form a near continuous series of favorable contacts with the sorbent. In contrast, Fig. 3b shows that adsorption of closed-circular supercoiled pUC18 DNA is reversible under the same conditions. Washing with 6 M perchlorate results in near complete removal of supercoiled DNA from the silica surface. Thus, DNA superstructure can have a dramatic influence on adsorption characteristics. Supercoiled DNA, for instance, may be limited in its ability to form long stretches of favorable contacts with the flat silica because of the topology of the supercoil. Initial Slopes of Adsorption Isotherms a. Supercoiling effects on binding affinity. Irreversible adsorption precludes the determination of binding constants using conventional adsorption models such as Langmuir theory. However, initial slopes of adsorption isotherms provide an unambiguous indication of relative binding affinities under conditions where the surface coverage is low enough to ignore the influence of lateral sorbate interactions on the strength of the sorbate–sorbent interaction. Steeper initial slopes indicate higher affinity. Figure 4 compares initial slopes of binding isotherms for linearized and supercoiled pUC18 DNA on silica in 6 M perchlorate (pH 8.0) at 377C. The initial slope for linearized pUC18 binding is nearly twice that for supercoiled pUC18, indicating that linearization increases the binding affinity, which agrees with our argument that linearization increases the number of favorable energetic contacts between the furanose–phosphate exterior of the double helix and the silica surface. Analogous to protein adsorption systems, denaturation and a concomitant increase in the rotational mobilities of the two furanose–phosphate backbones and their nucleotide side chains can provide a strong entropic driving force for duplex DNA adsorption. Cyclization of linear DNA causes
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of pUC18 DNA to silica in 6 M perchlorate solution indicates that the irreversible process is largely driven by an increase in entropy. Potential interfacial events which would result in an increase in entropy include the release of bound ordered water on the sorbate and sorbent surfaces, or an increase in the overall rotational or translational mobility of the DNA through, for instance, disruption of the double-helix structure. The latter event is inconsistent with our adsorption isotherm data, leaving dehydration effects as the most probable driving force for adsorption. Dependence on pH
FIG. 4. Comparison of initial slopes of binding isotherms for linearized and supercoiled pUC18 DNA on silica in 6 M perchlorate (pH 8.0) at 377C.
a decrease in the number of accessible configurations of the DNA and a decrease in conformational entropy which is proportional to the logarithm of the 03/2 power of the length of the DNA in the loop. Thus, supercoiled DNA occupies a higher Gibbs energy state (and lower entropic state) than linear DNA, and denaturation and relaxation of chain stresses should provide a stronger driving force for adsorption. However, as shown in Fig. 4, supercoiling results in a decrease in binding affinity. This lower binding affinity of the supercoiled pUC18 DNA suggests that denaturation of the duplex DNA structure, if it occurs, does not make a strong contribution to the driving force for binding. b. Effect of changing the temperature. Adsorption isotherms at several fixed pH values between 4 and 8 were measured at 23 and at 377C for both sonicated salmon and pUC18 DNA. For both DNA samples, the initial slope of the isotherm is near infinite, making it difficult to identify a dependence on the binding temperature. A temperature dependence can be seen for the somewhat weaker binding of supercoiled pUC18 DNA to silica in 6 M perchlorate. As shown in Fig. 5, the initial slope of the adsorption isotherm increases slightly with increasing temperature for binding at pH 8, indicating that the binding reaction is slightly endothermic under these conditions. At pH 7, binding affinity increases at both temperatures such that the initial slope is the same at 23 and 377C, indicating that the binding reaction is athermal at neutral pH. Comparison of adsorption isotherms below pH 7 suggests that the binding reaction is slightly exothermic, although the steepness of the initial slopes makes it more difficult to draw a firm conclusion. At all adsorption pH, analysis of initial slopes suggests that the enthalpy of binding is small. For adsorption to be thermodynamically favored and spontaneous, the Gibbs energy change for the adsorption process DadsG must be negative, where DadsG Å Dads H 0 T Dads S. The relatively small enthalpy change for adsorption
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a. Plateau values. If DNA and silica dehydration effects drive the binding reaction, they must be of sufficient strength to overcome the net repulsive force caused by overlap of the negative electrostatic potentials on the DNA and sorbent surfaces. The silanol groups on the surface of silica are weakly acidic. Reported pKa values for surface silanol groups are in the range of 5 to 7 (20, 21). Thus, silica possesses a negative surface-charge density at neutral to alkaline pH which is reduced as the pH becomes more acidic. Electrophoretic mobility data for our silica particles in 0.025 M NaCl solution titrated with 0.1 M HCl show a steady reduction in negative surface charge as the pH is decreased from 9.0 to 5.0. Figure 6 shows G sat values for closed-circular (supercoiled) pUC18 as a function of pH in 6 M perchlorate solution and a fixed DNA concentration above that required to saturate the silica surface at pH 8.0. The surface concentration increases three- to fourfold when the pH is decreased from 8 to ca. 5.5. The surface concentration then remains constant at ca. 800 mg/m 2 from pH 5 to pH 3. Results very similar to those shown in Fig. 6 were observed for the pH dependence of salmon DNA adsorption. Since the phosphate anion of each diester in the backbone exterior of soluble duplex DNA remains deprotonated between pH 9 and pH
FIG. 5. Comparison of initial slopes of adsorption isotherms (pH 8) for supercoiled pUC18 DNA at 23 and at 377C.
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FIG. 6. G sat values for closed-circular (supercoiled) pUC18 DNA as a function of pH in 6 M perchlorate solution at 377C.
3, most of the pH dependence observed in Fig. 6 is likely due to changes in the sorbent surface protonation state. The observed increase in bound DNA between pH 8 and 5 may be related in the following ways to protonation of negative charge on the silica surface: (1) a decrease in the negative potential at the sorbent surface diminishes the net electrostatic repulsion force with the DNA, (2) an increase in the density of surface hydroxyl residues increases the ability of the sorbent to form favorable intermolecular hydrogen bonds with bound DNA, and (3) a decrease in free hydroxyl residues diminishes solution competition with DNA for hydrogen-bonding sites on the silica surface. Accumulation of excess negative charge in the solventpoor contact layer between the silica and bound DNA surfaces is energetically unfavorable due to the relatively low dielectric permittivity of the regions where DNA is in close contact with the surface. Excess negative charge on the contacting surfaces must therefore be neutralized by coadsorption of either protons or low-molecular-weight cations of background electrolyte. Assuming that the number of protons required to neutralize the contact area of each bound DNA molecule remains constant, the increase in binding capacity observed between pH 8 and 5 can be related to the change in free protons required to neutralize the sorbent. Thus, the variable region between pH 8 and 5 in Fig. 6 represents a crude titration curve of the silica surface, from which an effective pKa of ca. 7.5 can be established for the surface silanol groups in contact with adsorbed DNA. This is somewhat higher than the measured pKa values for bare silica reported in the literature and reflects the influence of the negative surface potential of the DNA adjacent to the silica surface, and possibly the low dielectric permittivity of the contact layer. Adsorption pH values above 8.0 are not included in Fig. 6 because of competing effects from the primary amines on the bases of duplex DNA, which have an average pKa of ca. 9.5. For plasmid DNA adsorption to chemically pure sand,
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which has surface properties and titratable groups similar to the silica used here, Romanowski et al. (6) also observed a large reduction in G sat with increasing pH from 5 to 9. In further agreement with our results, Romanowski et al. (6) found G sat values for supercoiled plasmid DNA to be slightly lower than those for linearized plasmid DNA under otherwise constant conditions. b. Initial slopes. Adsorption isotherms were measured at 377C for sonicated salmon DNA both at pH 5 and at pH 8. The initial slope is near infinite for both solution pH, indicating a high affinity for the sorbent surface at both adsorption pH, but not allowing for discrimination of a pH dependence. In contrast, a substantial increase in initial slope, and thus binding affinity, with decreasing solution pH is observed for the adsorption of supercoiled pUC18 DNA which, relative to linear DNA, has an overall lower affinity for silica (Fig. 7). The increase in binding affinity for supercoiled pUC18 DNA with decreasing solution pH is consistent with the concomitant loss of negative charge and increase in hydroxyl groups on the silica surface and suggests that, although they do not dominate the adsorption process, intermolecular electrostatic forces contribute to the overall driving force for adsorption. Perchlorate Concentration Effects and the Role of Electrostatics and pH The influence of ionic strength on surface saturation levels was determined at 377C by measuring G sat for both pUC18 DNA and salmon DNA as a function of perchlorate concentrations. The resulting titration curves are essentially the same for the two systems. As shown in Fig. 8, adsorption of salmon DNA to silica at pH 8 is not observed at perchlorate concentrations below 1 M; the adsorbed amount then gradually increases with perchlorate concentrations between 1 and 5 M to a saturation level of 460 ( {45) mg/m 2 . Decreasing
FIG. 7. Comparison of initial slopes of adsorption isotherms measured at pH 5 and at pH 8 for supercoiled pUC18 DNA at 377C.
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binds ca. 4 water molecules (30). In addition to its shielding effect, the perchlorate therefore reduces the amount of free water available to solvate the DNA, which sequesters ca. 20 water molecules per nucleotide in the B-form duplex conformation. An appropriate description of the adsorption reaction occurring at the silica surface is therefore given by DNA (hydrated) / silica (hydrated) / counterions S neutral DNA–silica complex / water.
FIG. 8. The influence of ionic strength on surface saturation G sat levels for salmon DNA determined as a function of perchlorate concentration at 377C and pH 4.8 (filled circles) and pH 8 (open circles).
the solution pH increases G sat and decreases the perchlorate concentration required to initiate adsorption. At pH 4.8 (Fig. 8), G sat increases rapidly from 0 to 700 mg/m 2 over the NaClO4 concentration range of 0 to 2 M. The gradual increase in G in the pH 8 system is similar to a previous report of DNA adsorption to filter papers, which showed a somewhat sharper increase in adsorption at an NaClO4 concentration centered about 3 M (42). Figure 8 also shows that, in 6 M perchlorate solution, G sat for DNA on silica is limited by the solution pH and not by the perchlorate concentration. At higher solution pH, the surface concentration of DNA on the silica cannot be increased to that found at lower pH by increasing the perchlorate concentration above 6 M. This indicates that G sat is not determined by the sorbent surface potential. Other effects, such as steric effects, sorbent and DNA dehydration, and intermolecular hydrogen bonds to protonated surface silanols, must determine the maximum loading of DNA on the silica at a given solution pH.
Decreasing the concentration of free water in solution through addition of perchlorate drives the reaction to the right. Dehydration effects have been identified as a primary contributor to the driving forces for a number of macromolecular adsorption processes in water, particularly protein adsorption processes (43). In most of these systems, the macromolecule surface, the sorbent surface, or both are significantly more hydrophobic than those of the duplex DNA and silica dispersion used in this study. As a result, adsorption is generally observed in the absence of high (water-sorbing) salt concentrations, even under conditions where the macromolecule and sorbent surface are of the same charge sign. As shown in Fig. 8, adsorption of highly charged duplex DNA to hydrophilic negatively charged silica is strongly dependent on salt concentration and, thus, the fraction of unbound water available for solvating the DNA and silica surfaces. For aqueous sodium perchlorate solutions, Fig. 9 shows calculated shielding lengths rDH and unbound water fractions (i.e., water not associated with perchlorate) based on Debye–Hu¨ckel theory and infrared spectroscopy results at 257C (32). Perchlorate titration curves such as those shown in Fig. 8 indicate, over the adsorption pH range 6 õ pH õ 8, that silica is not a sorbent for DNA at perchlorate
DISCUSSION
The isotherm data presented here indicate that duplex DNA adsorption to silica is controlled by three competing effects: (i) weak electrostatic repulsion forces, (ii) dehydration, and (iii) hydrogen bond formation. The Debye–Hu¨ckel ˚ in 6 M perchlorate solution at shielding length is ca. 1.2 A 257C; this high-ionic-strength condition reduces the negative potential at the silica surface, thereby greatly reducing the electrostatic penalty for placing a negatively charged DNA molecule adjacent to the sorbent. As a result, sorbent and DNA dehydration effects and intermolecular hydrogen bond formation overcompensate this net electrostatic repulsion and drive DNA adsorption to the silica surface. Each perchlorate anion in aqueous 6 M NaClO4 solution
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FIG. 9. Calculated shielding lengths rDH and unbound water fractions (i.e., water not associated with perchlorate) as a function of ionic strength for aqueous sodium perchlorate solutions at 257C; calculations based on Debye–Hu¨ckel theory and infrared spectroscopy results (32).
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is observed at all solution pH, hydrogen-bonding effects do not dominate the adsorption process. Since the DNA adsorption process is partially driven by an increase in entropy of water molecules released from the DNA and silica surfaces, binding affinity should be proportional to the fraction of each duplex DNA molecule which is in direct contact with the silica surface. Open duplexes (i.e., linear DNA) can establish a continuous line of contact with the silica surface, and thus, bind more strongly than supercoiled DNA under otherwise identical conditions (see Fig. 3). ACKNOWLEDGMENTS FIG. 10. Ionic strength I* and mole fraction of free water (xfree )* values determined from the midpoint of perchlorate titration curves (where hydrated and bound DNA are in equilibrium such that half of the binding sites on the silica surface are occupied) for salmon DNA as a function of adsorption pH.
This work was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC). The authors are grateful to Dr. Donald E. Brooks for helpful discussions and for permitting access to some of the equipment used in these experiments.
REFERENCES
concentrations less than 0.5 M, the region over which large changes in rDH are observed. Beyond an ionic strength of 1 M, where adsorbed DNA concentration increases with increasing perchlorate concentration, the electrostatic ˚ and little change in its value shielding length is below 3 A is observed with increasing perchlorate concentration. Instead, as shown in Fig. 9, the dominant effect of adding perchlorate above a concentration of 1 M is to decrease the fraction of unbound water in the system. This suggests that, although a critical salt concentration is required to shield the net electrostatic repulsion between the DNA and silica surfaces, the dependence of adsorbed DNA on perchlorate concentration (see Fig. 8) is primarily due to perchlorate anion hydration effects and a loss of free water. Figure 9 shows how little free water is available in a 6 M sodium perchlorate solution; only ca. 42% of the water in the system is available for hydrating the DNA and bare silica surfaces. The perchlorate titration curves in Fig. 8 are reversible. The midpoint of the titration curve, where G Å 0.5 G sat , therefore defines the ionic strength I* and mole fraction of free water (xfree )*, where hydrated and bound DNA are in equilibrium such that half of the binding sites on the silica surface are occupied. Figure 10 shows I* and (xfree )* values determined from perchlorate titration curves for salmon DNA as a function of adsorption pH. From pH 8 to pH 5, where the weakly acidic silanol groups titrate and thereby increase the density of hydrogen bond accepting hydroxyl residues on the silica surface, (xfree )* and I* steadily increases and decreases, respectively. Below pH 5, where all surface silanol groups are protonated, constant values are observed for both (xfree )* and I*. This suggests that intermolecular hydrogen bonding contributes to the driving force for adsorption. However, since adsorption in 6 M perchlorate
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1. Fodor, S. P. A., Rava, R. P., Huang, X. C., Pease, A. C., Holmes, C. P., and Adams, C. P., Nature 364, 555 (1993). 2. Buffone, G. J., Demmler, G. J., Schimbor, C. M., and Greer, J., Clin. Chem. 37, 1945 (1991). 3. Marko, M. A., Chipperfield, R., and Birnboim, H. C., Anal. Biochem. 121, 382 (1982). 4. Willis, E. K., Mardis, E. R., James, W. L., and Little, M. C., Biotechniques 9, 92 (1990). 5. Lorenz, M. G., and Wackernagel, W., Appl. Environ. Microbiol. 53, 2948 (1987). 6. Romanowski, G., Lorenz, M. G., and Wackernagel, W., Appl. Environ. Microbiol. 57, 1057 (1991). 7. Ogram, A. V., Mathot, M. L., Harsh, J. B., Boyle, J., and Pettigrew, C. A., Appl. Environ. Microbiol. 60, 393 (1994). 8. Miller, I. R., Biochem. Biophys. Acta 103, 219 (1965). 9. Frommer, M. A., and Miller, I. R., J. Phys. Chem. 72, 2862 (1968). 10. Gershon, D., Nature Med. 1(2), 102 (1995). 11. Connolly, P., TIBTECH 12, 123 (1994). 12. Service, R. F., Science 268, 26 (1995). 13. Jacobson, S. C., Hergenro¨der, R., Moore, A. W., and Ramsey, J. M., ‘‘Solid State Sensor and Actuator Workshop, Hilton Head, South Carolina, June 13–16, 1994,’’ p. 65. 14. Volkmuth, W. D., and Austin, R. H., Nature 358, 600 (1992). 15. Northrup, M. A., Ching, M. T., White, R. M., and Watson, R. T., ‘‘Transducers ’93: 7th International Conference on Solid-State Sensors and Actuators, Tokyo, 1993,’’ p. 924. 16. Jacobson, S. C., Hergenro¨der, R., Moore, A. W., and Ramsey, J. M., Anal. Chem. 66, 4127 (1994). 17. Woolley, A. T., and Mathies, R. A., Proc. Natl. Acad. Sci. USA 91, 11348 (1994). 18. Cantor, C. R., and Schimmel, P. R., ‘‘Biophysical Chemistry Part I: The Conformation of Biological Macromolecules.’’ W. H. Freeman, San Francisco, 1980. 19. Matthew, J. B., and Richards, F. M., Biopolymers 23, 2743 (1984). 20. van Wagenen, R. A., Andrade, J. D., and Hibbs, J. B., J. Electrochem. Soc. 123, 1438 (1976). 21. Hair, M. L., and Hertl, W., J. Phys. Chem. 74, 91 (1970). 22. Klump, H., and Ackermann, T., Biopolymers 10, 513 (1971). 23. Odijk, T., Tijelschr. Natuurk A 50, 53 (1984). 24. Hearst, J. E., and Stockmayer, W. H., J. Chem. Phys. 37, 1425 (1962).
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25. Cantor, C. R., and Schimmel, P. R., ‘‘Biophysical Chemistry Part III: The Behavior of Biological Macromolecules,’’ p. 1266–1290. W. H. Freeman, San Francisco, 1980. 26. Chou, W. Y., Marky, L. A., Zaunczkowski, D., and Breslauer, K. J., J. Biomol. Struct. Dyn. 5, 345 (1987). 27. Dickerson, R. E., J. Mol. Biol. 166, 419 (1983). 28. Harvey, S. C., Nucleic Acids Res. 11, 4867 (1983). 29. Alden, C. J., and Kim, S.-H., J. Mol. Biol. 132, 411 (1979). 30. Pashley, R. M., and Kitchener, J. A., Interfacial Sci. 713, 491 (1979). 31. Symons, M. C. R., and Waddington, D., J. Chem. Soc. Faraday Trans. 71, 22 (1975). 32. James, D. W., and Armishaw, R. F., Inorg. Nucl. Chem. Lett. 12, 425 (1976). 33. Maniatis, T., Sambrook, J., and Fritsch, E. F., ‘‘Molecular Cloning: A Laboratory Manual,’’ p. 89. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1982.
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34. Haynes, C. A., and Norde, W., Colloids Surf. B: Biointerfaces 2, 517 (1994). 35. Grant, W. H., Smith, L. E., and Stromberg, R. R., Faraday Disc. Chem. Soc. 59, 209 (1975). 36. Fleer, G. J., Cohen Stuart, M. A., Scheutjens, J. M., Cosgrove, T., and Vincent, B., ‘‘Polymers at Interfaces,’’ p. 31. Chapman & Hall, London, 1993. 37. Scheutjens, J. M., and Fleer, G. J., J. Phys. Chem. 83, 1619 (1979). 38. Barkley, M. D., and Zimm, B. H., J. Chem. Phys. 70, 2991 (1979). 39. Olson, W. K., and Flory, P. J., Biopolymers 11, 1 (1972). 40. Vigil, R. D., and Ziff, R. M., J. Chem. Phys. 91(4), 2599 (1989). 41. Haynes, C. A., and Norde, W., Proteins Interfaces II, ACS Symp. Ser. 602, 26 (1995). 42. Chen, C. W., and Thomas, C. A., Anal. Biochem. 101, 339 (1980). 43. Haynes, C. A., Sliwinsky, E., and Norde, W., J. Colloid Interface Sci. 164, 394 (1994).
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0421
Driving Forces for DNA Adsorption to Silica in Perchlorate Solutions KATHRYN A. MELZAK, CHRIS S. SHERWOOD, ROBIN F. B. TURNER,*
AND
CHARLES A. HAYNES† ,1
Biotechnology Laboratory, *Department of Electrical Engineering, and †Department of Chemical Engineering, 237 Wesbrook Building, 6174 University Boulevard, The University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada Received January 22, 1996; accepted February 26, 1996
The adsorption of both plasmid and chromosomal duplex DNA to silica is investigated with the aim of determining the dominant forces involved in the binding reaction. Changes in the initial slopes and plateau values of adsorption isotherms for DNA on microcrystalline silica particles are used to establish the sensitivity of the binding reaction to ionic strength, temperature and pH, and DNA size and conformation. Binding is driven by an increase in entropy, with little or no enthalpic contribution. Adsorption isotherm results indicate that three effects, namely: ( i ) shielded intermolecular electrostatic forces, ( ii ) dehydration of the DNA and silica surfaces, and ( iii ) intermolecular hydrogen bond formation in the DNA – silica contact layer, make the dominant contributions to the overall driving force for adsorption. q 1996 Academic Press, Inc. Key Words: DNA-silica interactions; DNA adsorption; adsorption isotherms; DNA conformation; DNA purification; DNA binding.
INTRODUCTION
Understanding the nature of DNA interactions with silica and SiOx surfaces is becoming increasingly important as industrial efforts to automate and miniaturize DNA manipulation and purification technologies intensify (1). Chaotropic-salt-induced adsorption of DNA to silica is one of the most common methods for purifying both chromosomal and plasmid DNA from cell homogenates (2). The binding reaction is usually carried out in 4 M sodium iodide solution, buffered to pH 7.5 or 8, using a silica dispersion or, in some cases, pressed glass fibers which provide a high specific surface area for DNA binding. Since the adsorption process is reproducible for sufficiently large segments of DNA, several commercial kits, including the Geneclean and S&S EluQuick technologies (3, 4), are readily available. However, there is little scientific information to explain why the silica/ aqueous–solvent interface under these high-ionic-strength conditions selectively binds duplex DNA when exposed to aqueous mixtures containing protein, lipid, carbohydrate, and RNA contaminants. 1
To whom correspondence should be addressed.
Previous studies of DNA adsorption to silica or to any other solid are extremely limited. The most extensive studies are those of Lorenz and Wackernagel (5, 6) and others (7), who investigated the adsorption of plasmid DNA to mineral surfaces with the general aim of determining the increase in the stability of genetic information against nucleolytic inactivation due to adsorption. These studies built on a limited set of initial studies by Miller and colleagues (8, 9). Our poor understanding of duplex DNA–SiOx interactions is, in part, responsible for the slow progress made to date in the development of automated micromachined systems for DNA amplification, sequencing, etc., despite considerable industrial effort. The United States Department of Commerce identified the development of new tools for DNA diagnostics as a critical area of research for international competitiveness (10). Over the period from 1994 to 1999, the agency will commit up to $145 million toward the development of new technologies for DNA diagnostics. Many of these technologies involve the microfabrication of existing, larger-volume DNA manipulation or diagnostics systems in silicon wafers. The preferred approach would be to micromachine a monolithic device which provides for chemical or physical separation of the target DNA from cellular contaminants, followed by micromachined elements designed to sort out the sample’s chemical constituents, sequence DNA, search for mutations in a gene, etc., aligned in the electrophoretic carrier stream either in series or in parallel (11, 12). Although significant progress has been made in the development of the diagnostics and (bio)chemical processing (e.g., polymerase chain reaction) elements of these devices (13– 16), relatively little attention has been given to the crucial upstream procedures required to purify the target DNA. Silicon processing technology has advanced to the point where the SiOx channels formed in micromachined silicon devices can have a wide range of surface properties. Methodologies are available for varying surface potential and charge sign, hydrophobicity, and hydrogen-bonding capability (17). Thus, in principal, it is possible to tailor-make SiOx channels in a micromachined device with surface properties similar to the silica dispersions used for batch DNA purification. This paper addresses both plasmid and chromosomal
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duplex DNA adsorption to silica from a colloid–chemical perspective with the aim of determining the dominant forces involved in the binding reaction and the properties of the DNA and silica surfaces which control adsorption. Changes in the initial slopes and plateau values of adsorption isotherms for DNA on microcrystalline silica particles are used to establish the sensitivity of the binding reaction to ionic strength, temperature, and pH. The molecular weight and tertiary structure of the DNA substrate are also varied to determine their influence on the binding affinity under otherwise constant conditions. Adsorption of duplex DNA to silica will occur when DNA, silica, water, and a sufficient concentration of chaotropic salt are present in solution or suspension, usually with the addition of buffer to control the pH. Before discussing results, we present a brief description of each of these system components, including duplex DNA structures and surface properties, to establish potential contributions to the overall driving force for adsorption. CHARACTERISTICS OF SUSPENSION COMPONENTS
The phosphate diesters on the backbone of DNA are strong acids, making duplex DNA a strong polyelectrolyte carrying two univalent negative charges per base pair at most solution pH (18). In multivalent electrolyte solutions, this large negative surface-charge density is compensated by the site-specific binding of polyvalent cations, particularly magnesium and various polyamines. These cations, which are usually associated with water molecules, tend to bridge between adjacent phosphate anions. Monovalent cations may also associate with the backbone; in general, they do not bind to specific sites, but instead neutralize the negatively charged DNA surface through counterion condensation. The dynamic nature of counterion condensation results in partial occupancy of specific sites at regions of high electrostatic potential, with the level of occupancy a function of ionic strength (19). This interplay between free and fixed charges is one of the factors contributing to the dependence of DNA conformation on ionic strength (see below). The surface of silica, although more difficult to characterize, is also negatively charged at basic and near neutral pH values due to the weakly acidic silanol groups; surface silanol groups typically titrate over a rather broad pH range from ca. 4 to 8 with an average pKa ranging from 5 to 7, suggesting that the silica surface is heterogeneous (20, 21). Intermolecular electrostatic forces may therefore make an important contribution to the adsorption mechanism, particularly near and above neutral pH, where the net electrostatic repulsion between fixed charges on the DNA and silica surfaces will strongly disfavor adsorption at low ionic strength. Base pairs in the interior of the DNA double helix are uncharged at neutral pH. Complete titration of the nitrogen atoms on the purine and pyrimidine rings at low pH results
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in denaturation of the double helix. Unfolding is also initiated when the inside of the helix acquires an excess negative charge due to dissociation of protons from the |N{C(OH){ groups of guanine and thymine. Thus, the structural stability of duplex DNA is pH dependent, with a maximum stability of ca. DGd Å 3–8 kJ/mol base pair near pH 7 (22). Although electrostatic effects are partially responsible for the low structural stability of duplex DNA, the restricted conformational degrees of freedom of the furanose–phosphate backbone and the ribonucleotide side chains also destabilize the double helix relative to the single-strand randomcoil configuration. Linear duplex DNA, for instance, behaves as a worm-like chain. It is relatively inflexible due to steric restrictions in the backbone and to intramolecular repulsion between the charges on the phosphate residues. Helix stiffness is dependent on ionic strength; below 10 02 M ionic strength, DNA becomes less flexible and the persistence length increases sharply (23, 24). An increase in conformational entropy could therefore contribute to the driving force for duplex DNA adsorption provided the adsorption process results in (partial) denaturation, and a concomitant increase in the average rotational mobility of each strand. Circularizing DNA reduces the number of possible helix configurations, resulting in an entropy decrease proportional to the length of the DNA in the circle (25). Supercoiling increases the free energy of DNA still further, as can be seen by the fact that the introduction of a nick leads to spontaneous removal of superhelices. This free energy increase is due to the distortion required to bend the DNA helix into supercoils and to the entropy loss from placing the DNA in a more ordered structure. Ligands generally bind more strongly to supercoiled DNA because they can disrupt the supercoiling and thereby cause a greater decrease in free energy (26). A similar result may be expected for adsorption of supercoiled DNA to silica. The exterior of B-form duplex DNA binds about 0.6 g of water per gram of DNA, which corresponds to a ratio of approximately 20 water molecules per nucleotide (27). Each phosphate residue is capable of forming multiple hydrogen bonds with water or potentially with other groups. Decreasing the water activity in solution through addition of either a chaotropic salt or an alcohol changes the helical structure of B-DNA either continuously to C-DNA or through a sharp cooperative transition to A-DNA (28). Either transition is accompanied by a decrease in solvent-accessible surface area. For example, Alden and Kim (29) determined that the number of bound water molecules which can be accommodated within the minor and major grooves around one nucleotide drops from 19.3 to 10.5 during the B-DNA to A-DNA transition. Both of these transitions are thought to be at least partially driven by the increase in entropy which accompanies the release of bound water from the DNA surface. The silanol groups on the silica surface are also hydrogen
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bonded to water through one or more hydration layers (30). Thus, sorbent and DNA dehydration may also contribute to the driving force for adsorption provided the process results in a significant release of bound water from one or both surfaces. Perchlorate is present in solution as a tetrahedral symmetric anion with the negative charge distributed evenly over all four oxygens. Dissolution of sodium perchlorate in water is a spontaneous, endothermic process, indicating that the entropy change for the process is positive and, thus, that the perchlorate anion is chaotropic. Four to five water molecules are bound per perchlorate anion; infrared spectroscopy measurements show that the stretching band due to water not hydrogen bonded to other water molecules increases with increasing perchlorate concentration, while the stretching band for water hydrogen bonded to itself decreases (31, 32). An increase in the concentration of perchlorate in solution therefore results in a decrease in both the length scale for electrostatic interactions and the concentration of free (bulk) water. The former effect reduces the electrostatic penalty for close interaction of the two negatively charged macrosurfaces, while the latter should increase the energetic reward of sorbent and DNA dehydration during binding. EXPERIMENTAL
Materials a. Silica source and preparation. Microcrystalline silica particles (BDH Chemicals) were cleaned at 807C for 1 h in a chromic acid bath containing 5 g K2Cr2O7 . The particles were then cooled and centrifuged to remove excess acid, followed by rinses with two volumes of water, one volume of concentrated hydrochloric acid, and again with five volumes of distilled deionized water, before being dried at room temperature on a vacuum line. Cleaned particles were stored in a desiccator at room temperature before use. The specific surface areas of three cleaned silica preparations were measured by nitrogen adsorption using a Quantasorb BET adsorption apparatus. A value of 5.6 ( {0.2) m2 / g was obtained for each sample and was taken as constant for all remaining samples. Photographs taken with a Phillips transmission electron microscope show that the silica particles are flat chips with nominal diameters ranging from ca. 0.1 to 10 mm. b. Salmon and plasmid DNA preparation. Salmon sperm DNA was purchased from Sigma Chemicals Inc. (Cat. No. 01626; St. Louis, MO). One hundred-milligram aliquots of salmon DNA were dissolved in 5 ml of sterile water or buffer and sonicated for 20 min (at 20% full power) in a SON-IM Model XL2020 sonicator to give a polydisperse, relatively high-molecular-weight DNA mixture as shown in lane 3 (from l.h.s.) of the 0.8% agarose gel shown in Fig.
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FIG. 1. Agarose gel (0.8%) of purified linearized pUC18 DNA (lane 2), purified and sonicated salmon sperm DNA (lane 3), and purified closedcircular pUC18 DNA (lane 6) for which a very large fraction is supercoiled. Lane 1 shows 1-kb DNA ladder (Life Technologies, Inc.). Lane 4 shows a lesser loading (1/10 of that in Lane 6) of the purified closed-circular pUC18 DNA.
1. Figure 1 also shows pure linearized pUC18 plasmid DNA in lane 2, and in lane 6 pure closed-circular pUC18 DNA which is predominantly supercoiled in structure. Contaminating RNA was removed by treatment with DNase-free RNase (Boehringer Mannheim Biochemica) according to the method of Maniatis et al. (33) followed by extraction with a mixture of phenol, chloroform, and isoamyl alcohol to remove protein. The A260 :A280 ratio of the final preparation was 1.8, indicating that the DNA stock was substantially free of protein contaminants. Closed-circular pUC18 DNA was prepared from lysates of Escherichia coli DH10B harboring plasmids that carry ColE1 replicons according to the procedure of Maniatis et al. (33). Linearized pUC18 DNA was prepared by digestion with the restriction enzyme HindIII (Cat. No. 15615-024; Life Technologies, Burlington, Ontario, Canada), followed by treatment with RNase and a phenol/chloroform extraction. No RNA contaminant was detected on 0.8% agarose gels of the pUC18 stock solutions. c. Perchlorate solutions. Sodium perchlorate (BDH Chemicals) solutions were made up with water that was distilled and then deionized using a Corning Mega-pure system. Solutions were filtered through a 0.22-mm filter and then autoclaved to denature any DNases before use. Buffered perchlorate solutions were prepared by mixing 50 mM Tris:HCl and Tris base in appropriate ratios to achieve the desired pH at the desired temperature in the absence of perchlorate. High concentrations of perchlorate alter the pH by affecting the dissociation of the buffer and, thus, the proton activity coefficient. Perchlorate will also affect the measured pH by changing the double layer at the pH meter electrode. Solution pH was therefore taken as that measured by the
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FIG. 2. Ascending (increasing sorbate concentration in the bulk) adsorption isotherms for both linearized pUC18 DNA (filled circles), with one distinct size (2686 bp), and sonicated salmon DNA (open squares), containing fragments ranging from ca. 500 bp to 12 kb, on silica in 6 M perchlorate solution at pH 8.0 and 377C.
electrode. Buffer solutions were titrated to the appropriate measured pH with 0.1 M HCl or NaOH. Methods DNA adsorption measurements. The amount of DNA adsorbed to silica under various conditions was determined by solution depletion as measured by absorbance at 260 nm using a Hewlett–Packard 8452A spectrophotometer. Silica particles were weighed into 1.5-ml poly(ethylene) centrifuge tubes with sample weights ranging from 2.8 to 3.1 mg of clean dry silica. Silica solutions containing 50 mM Tris buffer in varying amounts of perchlorate were sonicated with a Heat Systems sonicator microtip for ca. 5 min at a 2.5 power setting to fully suspend the silica. DNA was then added as a stock solution, containing 50 mM Tris buffer and an identical concentration of perchlorate, and the resulting mixture was then equilibrated by rotating end-over-end for 4 h at the specified binding temperature (37 or 227C). The equilibrated suspension was then centrifuged at 12,000 rpm for 1 min to remove the silica partices, and an aliquot of supernatant was collected. Initial DNA concentrations were calculated knowing the concentration of the DNA stock solution and the volumes of DNA solution and silica suspension that were mixed together. The concentration of the equilibrated supernatant was determined by absorbance at 260 nm. Supernatant samples did not contain silica particles, as determined by the shape of the absorbance spectra (from 200 to 800 nm). RESULTS
Adsorption Isotherms and the Mode(s) of DNA Binding Figure 2 shows ascending (increasing sorbate concentration in the bulk) adsorption isotherms for both linearized
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pUC18 DNA, with one distinct size (2686 bp), and sonicated salmon DNA, containing fragments ranging from ca. 500 bp to 12 kb, on silica in 6 M perchlorate solution at pH 8.0. The surface concentration of DNA at saturation ( G sat ) is similar for both DNA samples, indicating that the mode of DNA binding is insensitive to average length and polydispersity. This suggests that the DNA adsorbs in a side-on orientation, such that changing the length of the DNA makes a corresponding proportional change in the area occupied per macromolecule. Side-on orientation is consistent with the magnitude of G sat (ca. 500 mg/m 2 ). If duplex DNA is considered a cylin˚ , as determined from the crystal der with a diameter of 20 A structure for B-DNA, then the projected area of adsorbed DNA at G sat corresponds to ca. 25% of the available surface area, indicating that the adsorbed DNA remains loosely packed on the sorbent up to saturation. Fractional surface coverages at saturation tend to be substantially higher than 25% for both protein and (random-coil) polymer adsorption (34, 35). For homopolymer adsorption on well-defined sorbents, surface coverages are often in excess of 70%; both experiment (36) and theory (37) have been used to connect these results with a general binding configuration where only a fraction of monomer units is in direct contact with the sorbent, such that the remaining unbound stretches extend into the bulk solution in a series of loops and tails. Apparent contact density is then increased by extension of the adsorbed chain in the direction normal to the surface and by the conformational freedom of the (unbound) polymer backbone which allows proximal chains to overlap and entangle on the surface. The low fractional surface coverages observed for linear DNA on silica may result from the rigidity of the double helix, as compared to random-coil homopolymers, which limits its ability to form extended loops and tails and, to a lesser extent, molecular overlap. Fluorescence polarization decay curves for duplex DNA (38) reveal a stiff structure ˚ toward helix bending with a persistence length of ca. 600 A at low ionic strength. Steric interactions, primarily caused by base pairing in duplex DNA, limit the range of possible rotational angles along the sugar–phosphate backbone (39). Computer simulations by Vigil and Ziff (40) of a twodimensional random sequential adsorption (an irreversible process in which sorbate molecules are sequentially placed on a surface in random orientation under the condition that they do not overlap) show jamming coverages (i.e., maximum surface coverages) between 25 and 35% for rod-shaped molecules with a high aspect ratio. Although initial adsorption orientations are random, the simulation predicts significant ordering of molecules such as DNA at saturation since a high-aspect-ratio rod must have almost identical angular orientations with its nearest neighbors in order to successfully adsorb at high surface concentrations. As shown in Fig. 2, saturation levels for both sonicated salmon and linear
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FIG. 3. Ascending adsorption isotherms and desorption by dilution data for (a) linear and (b) closed-circular supercoiled pUC18 DNA on silica particles in 6 M perchlorate at pH 8 and 377C.
pUC18 DNA at pH 8 are consistent with the random sequential adsorption model. However, as shown below, saturation levels increase for both types of DNA with decreasing pH, suggesting that limited amounts of inter- and intramolecular overlap occur in the adsorbed layer. Provided the duplex structure remains intact during adsorption, the bound structure consistent with these results is a worm-like chain adsorbed side-on such that there are regions of near continuous contact with the sorbent surface. Linearized pUC18 DNA has a contour length of ca. 0.7 mm, which is long with respect to some of the silica particles. The longest fragments of the salmon DNA have a contour length of about ca. 3 mm. Since the silica particles have nominal diameters in the range 0.1–10 mm, bound DNA in continuous contact with a (single) silica particle must exhibit curvature along the center-line axis of the helix and, in some cases, chain direction reversal on the surface. End-region detachment of the chain, breakdown of the double-helix structure, or bridging between silica particles may also occur. Binding Reversibility with Respect to Dilution Thermodynamic reversibility requires the absence of hysteresis between the ascending and descending (decreasing bulk sorbate concentration at otherwise constant conditions) branches of the adsorption isotherm (41). In 6 M perchlorate solution at 377C, adsorption of either linearized pUC18 DNA or sonicated salmon sperm DNA is irreversible with respect to dilution. As shown in Fig. 3a, washing of silica particles with 6 M perchlorate after adsorption results in no appreciable desorption of linear pUC18 DNA at any surface coverage. Irreversible adsorption is very often observed in both polymer and protein adsorption to solids, and is thought to be the result of the large number of contacts made between polymer or protein segments and the sorbent surface such that simultaneous disruption of all contacts is energetically unfavorable (36). In a side-on orientation, linear duplex
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DNA can form a near continuous series of favorable contacts with the sorbent. In contrast, Fig. 3b shows that adsorption of closed-circular supercoiled pUC18 DNA is reversible under the same conditions. Washing with 6 M perchlorate results in near complete removal of supercoiled DNA from the silica surface. Thus, DNA superstructure can have a dramatic influence on adsorption characteristics. Supercoiled DNA, for instance, may be limited in its ability to form long stretches of favorable contacts with the flat silica because of the topology of the supercoil. Initial Slopes of Adsorption Isotherms a. Supercoiling effects on binding affinity. Irreversible adsorption precludes the determination of binding constants using conventional adsorption models such as Langmuir theory. However, initial slopes of adsorption isotherms provide an unambiguous indication of relative binding affinities under conditions where the surface coverage is low enough to ignore the influence of lateral sorbate interactions on the strength of the sorbate–sorbent interaction. Steeper initial slopes indicate higher affinity. Figure 4 compares initial slopes of binding isotherms for linearized and supercoiled pUC18 DNA on silica in 6 M perchlorate (pH 8.0) at 377C. The initial slope for linearized pUC18 binding is nearly twice that for supercoiled pUC18, indicating that linearization increases the binding affinity, which agrees with our argument that linearization increases the number of favorable energetic contacts between the furanose–phosphate exterior of the double helix and the silica surface. Analogous to protein adsorption systems, denaturation and a concomitant increase in the rotational mobilities of the two furanose–phosphate backbones and their nucleotide side chains can provide a strong entropic driving force for duplex DNA adsorption. Cyclization of linear DNA causes
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of pUC18 DNA to silica in 6 M perchlorate solution indicates that the irreversible process is largely driven by an increase in entropy. Potential interfacial events which would result in an increase in entropy include the release of bound ordered water on the sorbate and sorbent surfaces, or an increase in the overall rotational or translational mobility of the DNA through, for instance, disruption of the double-helix structure. The latter event is inconsistent with our adsorption isotherm data, leaving dehydration effects as the most probable driving force for adsorption. Dependence on pH
FIG. 4. Comparison of initial slopes of binding isotherms for linearized and supercoiled pUC18 DNA on silica in 6 M perchlorate (pH 8.0) at 377C.
a decrease in the number of accessible configurations of the DNA and a decrease in conformational entropy which is proportional to the logarithm of the 03/2 power of the length of the DNA in the loop. Thus, supercoiled DNA occupies a higher Gibbs energy state (and lower entropic state) than linear DNA, and denaturation and relaxation of chain stresses should provide a stronger driving force for adsorption. However, as shown in Fig. 4, supercoiling results in a decrease in binding affinity. This lower binding affinity of the supercoiled pUC18 DNA suggests that denaturation of the duplex DNA structure, if it occurs, does not make a strong contribution to the driving force for binding. b. Effect of changing the temperature. Adsorption isotherms at several fixed pH values between 4 and 8 were measured at 23 and at 377C for both sonicated salmon and pUC18 DNA. For both DNA samples, the initial slope of the isotherm is near infinite, making it difficult to identify a dependence on the binding temperature. A temperature dependence can be seen for the somewhat weaker binding of supercoiled pUC18 DNA to silica in 6 M perchlorate. As shown in Fig. 5, the initial slope of the adsorption isotherm increases slightly with increasing temperature for binding at pH 8, indicating that the binding reaction is slightly endothermic under these conditions. At pH 7, binding affinity increases at both temperatures such that the initial slope is the same at 23 and 377C, indicating that the binding reaction is athermal at neutral pH. Comparison of adsorption isotherms below pH 7 suggests that the binding reaction is slightly exothermic, although the steepness of the initial slopes makes it more difficult to draw a firm conclusion. At all adsorption pH, analysis of initial slopes suggests that the enthalpy of binding is small. For adsorption to be thermodynamically favored and spontaneous, the Gibbs energy change for the adsorption process DadsG must be negative, where DadsG Å Dads H 0 T Dads S. The relatively small enthalpy change for adsorption
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a. Plateau values. If DNA and silica dehydration effects drive the binding reaction, they must be of sufficient strength to overcome the net repulsive force caused by overlap of the negative electrostatic potentials on the DNA and sorbent surfaces. The silanol groups on the surface of silica are weakly acidic. Reported pKa values for surface silanol groups are in the range of 5 to 7 (20, 21). Thus, silica possesses a negative surface-charge density at neutral to alkaline pH which is reduced as the pH becomes more acidic. Electrophoretic mobility data for our silica particles in 0.025 M NaCl solution titrated with 0.1 M HCl show a steady reduction in negative surface charge as the pH is decreased from 9.0 to 5.0. Figure 6 shows G sat values for closed-circular (supercoiled) pUC18 as a function of pH in 6 M perchlorate solution and a fixed DNA concentration above that required to saturate the silica surface at pH 8.0. The surface concentration increases three- to fourfold when the pH is decreased from 8 to ca. 5.5. The surface concentration then remains constant at ca. 800 mg/m 2 from pH 5 to pH 3. Results very similar to those shown in Fig. 6 were observed for the pH dependence of salmon DNA adsorption. Since the phosphate anion of each diester in the backbone exterior of soluble duplex DNA remains deprotonated between pH 9 and pH
FIG. 5. Comparison of initial slopes of adsorption isotherms (pH 8) for supercoiled pUC18 DNA at 23 and at 377C.
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FIG. 6. G sat values for closed-circular (supercoiled) pUC18 DNA as a function of pH in 6 M perchlorate solution at 377C.
3, most of the pH dependence observed in Fig. 6 is likely due to changes in the sorbent surface protonation state. The observed increase in bound DNA between pH 8 and 5 may be related in the following ways to protonation of negative charge on the silica surface: (1) a decrease in the negative potential at the sorbent surface diminishes the net electrostatic repulsion force with the DNA, (2) an increase in the density of surface hydroxyl residues increases the ability of the sorbent to form favorable intermolecular hydrogen bonds with bound DNA, and (3) a decrease in free hydroxyl residues diminishes solution competition with DNA for hydrogen-bonding sites on the silica surface. Accumulation of excess negative charge in the solventpoor contact layer between the silica and bound DNA surfaces is energetically unfavorable due to the relatively low dielectric permittivity of the regions where DNA is in close contact with the surface. Excess negative charge on the contacting surfaces must therefore be neutralized by coadsorption of either protons or low-molecular-weight cations of background electrolyte. Assuming that the number of protons required to neutralize the contact area of each bound DNA molecule remains constant, the increase in binding capacity observed between pH 8 and 5 can be related to the change in free protons required to neutralize the sorbent. Thus, the variable region between pH 8 and 5 in Fig. 6 represents a crude titration curve of the silica surface, from which an effective pKa of ca. 7.5 can be established for the surface silanol groups in contact with adsorbed DNA. This is somewhat higher than the measured pKa values for bare silica reported in the literature and reflects the influence of the negative surface potential of the DNA adjacent to the silica surface, and possibly the low dielectric permittivity of the contact layer. Adsorption pH values above 8.0 are not included in Fig. 6 because of competing effects from the primary amines on the bases of duplex DNA, which have an average pKa of ca. 9.5. For plasmid DNA adsorption to chemically pure sand,
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which has surface properties and titratable groups similar to the silica used here, Romanowski et al. (6) also observed a large reduction in G sat with increasing pH from 5 to 9. In further agreement with our results, Romanowski et al. (6) found G sat values for supercoiled plasmid DNA to be slightly lower than those for linearized plasmid DNA under otherwise constant conditions. b. Initial slopes. Adsorption isotherms were measured at 377C for sonicated salmon DNA both at pH 5 and at pH 8. The initial slope is near infinite for both solution pH, indicating a high affinity for the sorbent surface at both adsorption pH, but not allowing for discrimination of a pH dependence. In contrast, a substantial increase in initial slope, and thus binding affinity, with decreasing solution pH is observed for the adsorption of supercoiled pUC18 DNA which, relative to linear DNA, has an overall lower affinity for silica (Fig. 7). The increase in binding affinity for supercoiled pUC18 DNA with decreasing solution pH is consistent with the concomitant loss of negative charge and increase in hydroxyl groups on the silica surface and suggests that, although they do not dominate the adsorption process, intermolecular electrostatic forces contribute to the overall driving force for adsorption. Perchlorate Concentration Effects and the Role of Electrostatics and pH The influence of ionic strength on surface saturation levels was determined at 377C by measuring G sat for both pUC18 DNA and salmon DNA as a function of perchlorate concentrations. The resulting titration curves are essentially the same for the two systems. As shown in Fig. 8, adsorption of salmon DNA to silica at pH 8 is not observed at perchlorate concentrations below 1 M; the adsorbed amount then gradually increases with perchlorate concentrations between 1 and 5 M to a saturation level of 460 ( {45) mg/m 2 . Decreasing
FIG. 7. Comparison of initial slopes of adsorption isotherms measured at pH 5 and at pH 8 for supercoiled pUC18 DNA at 377C.
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binds ca. 4 water molecules (30). In addition to its shielding effect, the perchlorate therefore reduces the amount of free water available to solvate the DNA, which sequesters ca. 20 water molecules per nucleotide in the B-form duplex conformation. An appropriate description of the adsorption reaction occurring at the silica surface is therefore given by DNA (hydrated) / silica (hydrated) / counterions S neutral DNA–silica complex / water.
FIG. 8. The influence of ionic strength on surface saturation G sat levels for salmon DNA determined as a function of perchlorate concentration at 377C and pH 4.8 (filled circles) and pH 8 (open circles).
the solution pH increases G sat and decreases the perchlorate concentration required to initiate adsorption. At pH 4.8 (Fig. 8), G sat increases rapidly from 0 to 700 mg/m 2 over the NaClO4 concentration range of 0 to 2 M. The gradual increase in G in the pH 8 system is similar to a previous report of DNA adsorption to filter papers, which showed a somewhat sharper increase in adsorption at an NaClO4 concentration centered about 3 M (42). Figure 8 also shows that, in 6 M perchlorate solution, G sat for DNA on silica is limited by the solution pH and not by the perchlorate concentration. At higher solution pH, the surface concentration of DNA on the silica cannot be increased to that found at lower pH by increasing the perchlorate concentration above 6 M. This indicates that G sat is not determined by the sorbent surface potential. Other effects, such as steric effects, sorbent and DNA dehydration, and intermolecular hydrogen bonds to protonated surface silanols, must determine the maximum loading of DNA on the silica at a given solution pH.
Decreasing the concentration of free water in solution through addition of perchlorate drives the reaction to the right. Dehydration effects have been identified as a primary contributor to the driving forces for a number of macromolecular adsorption processes in water, particularly protein adsorption processes (43). In most of these systems, the macromolecule surface, the sorbent surface, or both are significantly more hydrophobic than those of the duplex DNA and silica dispersion used in this study. As a result, adsorption is generally observed in the absence of high (water-sorbing) salt concentrations, even under conditions where the macromolecule and sorbent surface are of the same charge sign. As shown in Fig. 8, adsorption of highly charged duplex DNA to hydrophilic negatively charged silica is strongly dependent on salt concentration and, thus, the fraction of unbound water available for solvating the DNA and silica surfaces. For aqueous sodium perchlorate solutions, Fig. 9 shows calculated shielding lengths rDH and unbound water fractions (i.e., water not associated with perchlorate) based on Debye–Hu¨ckel theory and infrared spectroscopy results at 257C (32). Perchlorate titration curves such as those shown in Fig. 8 indicate, over the adsorption pH range 6 õ pH õ 8, that silica is not a sorbent for DNA at perchlorate
DISCUSSION
The isotherm data presented here indicate that duplex DNA adsorption to silica is controlled by three competing effects: (i) weak electrostatic repulsion forces, (ii) dehydration, and (iii) hydrogen bond formation. The Debye–Hu¨ckel ˚ in 6 M perchlorate solution at shielding length is ca. 1.2 A 257C; this high-ionic-strength condition reduces the negative potential at the silica surface, thereby greatly reducing the electrostatic penalty for placing a negatively charged DNA molecule adjacent to the sorbent. As a result, sorbent and DNA dehydration effects and intermolecular hydrogen bond formation overcompensate this net electrostatic repulsion and drive DNA adsorption to the silica surface. Each perchlorate anion in aqueous 6 M NaClO4 solution
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FIG. 9. Calculated shielding lengths rDH and unbound water fractions (i.e., water not associated with perchlorate) as a function of ionic strength for aqueous sodium perchlorate solutions at 257C; calculations based on Debye–Hu¨ckel theory and infrared spectroscopy results (32).
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is observed at all solution pH, hydrogen-bonding effects do not dominate the adsorption process. Since the DNA adsorption process is partially driven by an increase in entropy of water molecules released from the DNA and silica surfaces, binding affinity should be proportional to the fraction of each duplex DNA molecule which is in direct contact with the silica surface. Open duplexes (i.e., linear DNA) can establish a continuous line of contact with the silica surface, and thus, bind more strongly than supercoiled DNA under otherwise identical conditions (see Fig. 3). ACKNOWLEDGMENTS FIG. 10. Ionic strength I* and mole fraction of free water (xfree )* values determined from the midpoint of perchlorate titration curves (where hydrated and bound DNA are in equilibrium such that half of the binding sites on the silica surface are occupied) for salmon DNA as a function of adsorption pH.
This work was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC). The authors are grateful to Dr. Donald E. Brooks for helpful discussions and for permitting access to some of the equipment used in these experiments.
REFERENCES
concentrations less than 0.5 M, the region over which large changes in rDH are observed. Beyond an ionic strength of 1 M, where adsorbed DNA concentration increases with increasing perchlorate concentration, the electrostatic ˚ and little change in its value shielding length is below 3 A is observed with increasing perchlorate concentration. Instead, as shown in Fig. 9, the dominant effect of adding perchlorate above a concentration of 1 M is to decrease the fraction of unbound water in the system. This suggests that, although a critical salt concentration is required to shield the net electrostatic repulsion between the DNA and silica surfaces, the dependence of adsorbed DNA on perchlorate concentration (see Fig. 8) is primarily due to perchlorate anion hydration effects and a loss of free water. Figure 9 shows how little free water is available in a 6 M sodium perchlorate solution; only ca. 42% of the water in the system is available for hydrating the DNA and bare silica surfaces. The perchlorate titration curves in Fig. 8 are reversible. The midpoint of the titration curve, where G Å 0.5 G sat , therefore defines the ionic strength I* and mole fraction of free water (xfree )*, where hydrated and bound DNA are in equilibrium such that half of the binding sites on the silica surface are occupied. Figure 10 shows I* and (xfree )* values determined from perchlorate titration curves for salmon DNA as a function of adsorption pH. From pH 8 to pH 5, where the weakly acidic silanol groups titrate and thereby increase the density of hydrogen bond accepting hydroxyl residues on the silica surface, (xfree )* and I* steadily increases and decreases, respectively. Below pH 5, where all surface silanol groups are protonated, constant values are observed for both (xfree )* and I*. This suggests that intermolecular hydrogen bonding contributes to the driving force for adsorption. However, since adsorption in 6 M perchlorate
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