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Binding of Biotinylated DNA to Streptavidin-Coated Polystyrene Latex: Effects of Chain Length and Particle Size
ANALYTICAL BIOCHEMISTRY ARTICLE NO.
237, 115–122 (1996)
0208
Binding of Biotinylated DNA to Streptavidin-Coated Polystyrene Latex: Effects of Chain Length and Particle Size Shao-Chie Huang,* Mark D. Stump,† Robert Weiss,† and Karin D. Caldwell*,1 *Department of Chemical and Fuels Engineering, and †Department of Human Genetics, University of Utah, Salt Lake City, Utah 84112
Received December 8, 1995
The binding of 5*-end biotinylated DNA, ranging in size from 100 to 5000 base pairs, was studied using streptavidin-coated polystyrene latex particles with diameters between 0.944 and 0.090 mm. The experimental binding constants and forward rate constants of this solid-phase reaction were determined to be several orders of magnitude lower than values for the biotin–streptavidin interaction in solution as expected and were shown to depend on the size of both ligand and substrate. An observed inflection in the binding constant of the biotinylated DNA appeared around 1000 base pairs, possibly indicating different surface orientations of the macroligand above and below this critical size. This effect was more pronounced for the smaller latex particles used in this study and highlighted possible differences in the surface arrangement of streptavidin on the differently sized particles. Diffusion limitation to the binding reaction was found to be significant in all cases. In this present work, an exponential relationship was established between the experimental binding constant and the number of base pairs in the biotinylated DNA. This relationship possibly provides a means to predict capacity and binding speed in cases where adsorption, purification, and release of larger DNA chains are required. q 1996 Academic Press, Inc.
Streptavidin, a protein produced by Streptomyces avidinii, binds d-biotin with an extremely high affinity (the KA of biotin – streptavidin in solution is around 1015 M01); the formation of this complex can be regarded as practically irreversible, as the binding energy is comparable to that of a covalent bond. The 1 To whom correspondence should be addressed. Fax: (801) 5855151.
strong and specific binding feature of this biotin – streptavidin system not only offers many powerful bioanalytical applications but also generates considerable interest as a versatile model in studying macromolecule – ligand interactions (1 – 4). The binding kinetics of several biotin–streptavidin systems have been well studied in solution (1). The experimental results from such studies conform with classical chemical theories, i.e., the law of mass action with its rigorous thermodynamic underpinnings. However, a few reports exist in which the focus has been the interaction between either biotin or streptavidin in the form of a solid-phase reagent. These studies show that the heterogeneous phase kinetics differ substantially from the theoretical models that have been developed to explain the results of biotin–streptavidin reactions in solution (5–7). The initial forward reaction often becomes diffusion limited at the interface, possibly due to steric hindrance and the inflexibility of the immobilized ligand. On the other hand, the dissociation rate of the bound molecule is often faster than that characteristic of the complex in solution as a result of surface-induced conformational shifts in the binding sites presented by the immobilized molecules. Thus, both the forward and reverse reactions tend to reduce the magnitude of the binding constant (7). Uniform latex particles have been used in biomedical research for more than two decades. Since the surface area per mass of particles increases with smaller diameters, the amount of immobilized ligand relative to the total mass of the particles also increases. Furthermore, in binding large biotinylated macromolecules (e.g., biotinylated DNA) to streptavidin-coated surfaces, the size of the biotinylated ligand is likely to affect not only the equilibrium binding level, but also the rate at which this binding occurs. Since mass transfer limitations will govern this process at the fast binding rates inher115
0003-2697/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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ently provided by the biotin–streptavidin system, kinetic arguments postulate that the actual binding rate will decrease with increasing hydrodynamic size of the ligand as well as with increasing size of the solid phase. Therefore, a timely processing of ever larger ligands will require reducing the size of the particulate phase. In a previous report (5) from this laboratory, streptavidin was shown to adsorb readily and irreversibly to polystyrene (PS)2 colloidal substrates and thereby confer on their surfaces a specific affinity for biotin. In the present work we have selected PS latex particles with a wide variety of discrete sizes, ranging from 0.944 to 0.090 mm. For the same amount of bulk, these particles present several-fold higher surface areas than that associated with the commercially available larger (2.8 mm) M-280 beads from Dynal. Here, separation of particle-bound macromolecules from unbound debris and impurities is achieved by centrifugal forces, instead of the magnetic forces employed while working with the Dynal beads. In binding large biotinylated macromolecules to streptavidin-coated solid surfaces, the size of the ligand is thus expected to affect both the equilibrium binding and the binding rate. Due to (i) the mass transfer limitations discussed above and (ii) the kinetic arguments which dictate that the actual binding rate will decrease both with increasing hydrodynamic size of the ligand and with an increase in particle size, the timely processing of ever larger ligands will require decreasing the size of the solid substrate. The present study is undertaken to further investigate the equilibrium binding and binding kinetics for ligand-coupled particles on solid surfaces, as they vary with the size of both ligand and particle. MATERIALS
PS latex particles with diameters ranging from 0.944 to 0.090 mm were obtained as 10% (w/v) suspensions according to the manufacturer’s datasheet. Particles with diameters of 0.261 and 0.090 mm were purchased from Seradyn while PS-944, PS-214, and PS-165 were from Bangs Laboratories. These particles were routinely used in other similar studies within our laboratory. The adsorption results are quite consistent throughout the experiments. Streptavidin was purchased from Sigma (catalog no. S-4762), as was Streptavidin–fluorescein isothiocyanate (FITC) (catalog no. S-3762). Fluorescein biotin (F-B, 5-((N-(5-(N-(6-(biotinoyl)amino)hexanoyl)amino)pentyl)thioureidyl) fluorescein) was obtained from Molecular Probes (catalog no. B-1370). The phosphate-buffered saline (PBS, 101) stock solution contained 87.7 g sodium chloride, 15.5 g 2
Abbreviations used: PS, polystyrene; PBS, phosphate-buffered saline.
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monobasic sodium phosphate, and 40.9 g dibasic sodium phosphate, diluted to 1000 ml with deionized water, pH 7.2. Biotinylated fluorescent DNA was prepared according to the procedure described in next section. METHODS
Streptavidin solution (2 mg/ml) was added to the particle suspension. The adsorption reaction was allowed to proceed for 1 h at room temperature with vortexing, and the streptavidin-coated latex particles were then washed and resuspended in PBS buffer by repeated centrifugations (Eppendorf centrifuge 5415C from Brinkmann Instruments or Optima TL ultracentrifuge from Beckman). For binding capacity measurements, a series of biotinylated DNA solutions were reacted with one and the same amount of streptavidin-coated latex particles for 10 min at room temperature under continuous shaking. Biotinylated fluorescent DNA, not bound to the surfaces, was retrieved and quantitated by means of a Perkin–Elmer luminescence spectrometer (Model LS50) after sedimenting the suspension by centrifugation and its fluorescence intensity was determined at an excitation wavelength of 484 nm and an emission wavelength of 504 nm. For the kinetic study, an appropriate amount of fluorescent DNA solution was added to a suspension of streptavidin-coated particles in a 1.7-ml polypropylene microcentrifuge tube (National Scientific), and the reaction was carried out at 47C for 30 s to 15 min under continuous mixing. The reactions were stopped by centrifugation. The amount of DNA left in the supernatant was quantified as mentioned before (5), and the amount bound to the particles was determined by subtraction from the total amount originally added. Double-stranded DNA molecules, ranging from 100 to 5000 bp, are used for binding of large biotinylated ligands to streptavidin-coated beads. The reaction mixture of 100 ml contained 300 ng M13mp18 DNA template, 40 mM 5*-end biotinylated primer, 50 mM fluorescent universal primer, the sequences FL-1 (5*-CAGGAAACAGCTATGACC-3*), FL-2 (5*-ACAACACTCAACCCTATCT-3*), FL-3 (5*-TGCGTGATGGACAGACTCT-3*), FL-4 (5*-AGCCCTACTGTTGAGCGT-3*), and FL-5 (5*-AGCCTCTGTAGCCGTTGCT3*), 4 units Taq (Thermus aquaticus) DNA polymerase, 0.2 mM deoxynucleoside triphosphates (dNTP) in a buffer containing 1.5 mM MgCl2 , 50 mM Tris–HCl, pH 8.4, 25 mM KCl, 5 mM NH4Cl, and 150 mg/ml bovine serum albumin. The reaction mixture was covered with a layer of Nujol mineral oil (Perkin–Elmer). The in vitro amplification was carried out on the PTC-100 MJ Research thermal cycler. Thirty cycles were performed with denaturation at 957C for 60 s, annealing at 627C for 2 min, and extension at 727C for 20 s. Unreacted
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primers were removed by centrifugation using Centricon-30 microconcentrators (Amicon) with three washings in water.
TABLE 1
Measurements of the Specific Binding of Fluorescein Biotin to Streptavidin-Coated Latex for Each of the Five Types of Particles
RESULTS AND DISCUSSION Particle type
Equilibrium Binding If one assumes that nonspecific interactions are absent, the bimolecular binding reaction of a specific ligand containing biotin (B) to biotin binding sites (S) can be expressed as (8) kf
S / B S SB, kr
[1]
where kf is the forward and kr is the reverse rate constants. Thus, the binding rate equation is d[SB]/dt Å kf [S][B] 0 kr[SB],
[2]
where [S] is the concentration of biotin binding sites (streptavidin subunits), [B] the concentration of biotin, and [SB] the concentration of complex. At equilibrium
KA Å
[SB] kf , Å [S][B] kr
[3]
where KA is the binding constant. Since the total number of biotin binding sites is conserved, and the number of biotinylated ligands in the bulk solution is in large excess compared to the number of available ligands on the surfaces, Eq. [3] can be rearranged as 1 1 1 1 Å , / [SB] [S]0 [S]0 KA [B]
[4]
where [S]0 is the initial effective concentration of biotin binding sites. In this manner, the binding constant KA can be determined from a double-reciprocal plot of 1/[SB] versus 1/[B]. It is well documented that one streptavidin molecule in solution has four biotin binding sites (1–3). In the present study the streptavidin–biotin binding reaction occurs on a solid surface. It should also be noted that the biotinylated DNAs employed in this study are quite large compared to the biotin molecule itself. Therefore, a certain degree of decreased specific streptavidin–biotin binding should be anticipated for this solid-phase reaction. A summary of the binding of fluorescein-labeled biotin to each of the five streptavidin-coated PS
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Moles F-B per mole SA
PS-90 PS-165 PS-214 PS-261 PS-944
0.78 0.72 0.55 0.42 0.45
{ { { { {
0.04 0.02 0.01 0.01 0.02
latex substrates of interest here is shown in Table 1. Expressed on a biotin per streptavidin basis, higher specific binding is observed for particles with smaller diameters. This result is quite consistent with data presented in our previous report (5). The reduction in specific binding capability resulting from the immobilization procedure used here is expected to be due to either or both of the effects of decreased accessibility and steric hindrance on the solid surface. The calculated KA values for a variety of biotinylated fluorescent DNAs and their interactions with streptavidin-coated PS particles of different sizes are shown in Table 2. Figure 1 is a graphic representation of the binding constant as a function of the number of base pairs in the biotinylated fluorescent DNAs. As expected, the binding constants decrease while increasing the size of either ligand or substrate (carrier particle). The calculated binding constants were found to be in the range of 1.1 1 1010 to 0.6 1 108 M01, depending on ligand and substrate size. These data are substantially lower by about five to six orders of magnitude than the reported 1 1 1015 M01 for the streptavidin–biotin interaction in a homogeneous solution. In addition, the binding capacity of the streptavidin-coated particles for biotinylated DNA is considerably reduced as a result of the bead-immobilization of streptavidin. This, in turn, is likely to produce steric hindrance toward the binding of bulky ligands and possibly also conformational changes in the binding site which weakens the interaction. Alternatively, the KA of this bimolecular binding reaction can also be calculated using Scatchard analysis which is widely employed in computing the affinity of antibody–antigen systems (9–11). From the mass action equilibrium law, Eq. [3] can be expressed as r Å nKA 0 rKA , [B]
[5]
where r is the number of moles of biotinylated DNA bound per mole of immobilized streptavidin, and n is
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HUANG ET AL. TABLE 2
Binding Constants (KA , 1 108
M
01
) of Biotinylated DNA to Streptavidin-Coated PS Particles at Equilibrium PS particle size (nm)
DNA (base pairs) 100 500 1000 2500 5000
90 108.1 53.8 23.9 12.0 5.7
{ { { { {
165 1.3 1.5 0.7 0.4 0.1
57.2 26.3 13.3 7.6 3.1
{ { { { {
214 0.9 0.6 0.5 0.3 0.2
18.5 10.4 6.3 2.3 0.9
{ { { { {
261 0.9 0.7 0.4 0.1 0.1
11.1 4.8 3.0 1.8 0.6
{ { { { {
944 0.5 0.2 0.2 0.3 0.1
8.0 3.6 3.0 1.9 0.6
{ { { { {
0.4 0.3 0.2 0.2 0.1
Note. Reaction conditions are as follows: 10 min at room temperature in a 1.7-ml tube.
the total number of available binding sites. Hence, in this form of Scatchard plot, r/[B] versus r should yield a straight line with a slope of 0KA and an intercept on the abscissa corresponding to the concentration of streptavidin-binding sites (n). Based on the analysis mentioned above, a Scatchard plot for binding of biotinylated DNA to streptavidin-coated PS-261 is shown in Fig. 2. Both the binding constant and the streptavidinbinding site concentration are determined at least
FIG. 1. Particle size effects on binding constant KA as a function of the number of base pairs in the DNA ligand. Curve-fitting analyses result in coefficients of determination (R 2) of ca. 0.95 for the five types of PS particles. Symbols: PS-90 (s), PS-165 (l), PS-214 (h), PS-261 (n), and PS-944 (m).
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twice and are found to be quite consistent with the results calculated from Eq. [4]. Using linear fitting, the adsorbed streptavidin has been found to bind about 0.4 mol per mole of biotin ligand. This reduced binding capacity is one-tenth of that determined in solution. Nevertheless, the concave Scatchard plot indicates that a certain degree of heterogeneity of affinity is associated with this system. An interesting feature evident in Fig. 1 is the dramatic increase in KA when streptavidin is adsorbed to
FIG. 2. A Scatchard plot for binding of biotinylated DNA, 300 bp in length, to streptavidin-coated PS-261. This ‘‘concave-up’’ curve clearly demonstrates a certain degree of heterogeneity in the binding affinity of this system.
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EFFECT OF DNA AND PARTICLE SIZE ON BIOTIN–STREPTAVIDIN BINDING
119
the smaller particles (PS-90 and PS-165) compared to what is seen for the larger ones (PS-261 and PS-944). This is despite the fact that for each DNA chain length the ratio of surface concentrations for any pair of particles remains nearly constant. Moreover, this figure shows that under the conditions tested an exponential relationship exists between the binding constant KA and the number of base pairs in the biotinylated DNA for each type of streptavidin-coated particle studied here. Curve-fitting analyses yield coefficients of determination (R 2) around 0.95 for each of the five types of particles. Thus, the data are fitted to an expression of the form Y Å A 0 B log X,
[6]
where Y is the binding constant for each type of particle, X is the number of base pairs of biotinylated DNA, and A and B are parameters depending on ligand and substrate in the system. This finding has the practical consequence of potentially allowing an estimation of the capacity of any particle size to bind DNA molecules of a specified length. It is interesting to note, in the semilogarithmic plot shown in Fig. 3, that the relationships between binding constants of streptavidin-coated particles and the number of base pairs in the biotinylated DNA display inflections for PS-90 and PS-165, whereas for PS-214, PS-261, and PS-944 such inflections are less pronounced. This observed inflection appears at a ligand size corresponding to 1000 bp of biotinylated DNA. It may indirectly suggest that there is a tangible difference in the surface orientation of streptavidin for these two groups of particles which, in turn, translates into differences in DNA binding. In such a case there are at least two possible explanations for this significant phenomenon. First, the different protein-coated surfaces may have induced different conformations in the bulky DNA molecules, as they bind to the different surfaces. Second, from studies of the molecular weight (Mr) dependence of DNA viscosity, the polymer is known to experience rod-like behavior at Mr values below 2 million daltons or 3000 base pairs, while it behaves as a flexible random coil for Mr values above this level (12). These Mr-dependent differences in hydrodynamic shape may correlate with the Mr-dependent differences in surface packing, seen in this work. Kinetic Study In solution, the rate constants kf and kr for the biotin–steptavidin system are reported to be 107 M01 s01 and 1008 s01, respectively (13). As a consequence of the slow desorption rate, the value of the second term in Eq. [2], which accounts for the dissociation, can be considered negligible compared to the first term, at least
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FIG. 3. A semilogarithmic plot of the data from Fig. 1. Inflections, which are significant for both PS-90 and PS-165 and less pronounced for others, are observed in the vicinity of 1000 bp DNA length. Symbols are the same as in Fig. 1.
in the initial stages of the reaction (14). As a result, the kinetics of association of biotinylated DNA to streptavidin-coated particles can be modeled as a secondorder reaction of the form d[SB]/dt Å kf [S][B].
[7]
The differential Eq. [7] is readily solved to give ln
([S]0 0 [SB])[B]0 Å ([S]0 0 [B]0)kf t, [S]0([B]0 0 [SB])
[8]
where [B]0 is the initial concentration of biotin, and t is the reaction time. From this relationship, kf can be determined, given accurate values of the starting concentrations [S]0 and [B]0 , as well as of the amount of complex [SB] formed at time t. The calculated characteristic forward rate constants kf of biotinylated DNA to streptavidin-coated particles are listed in Table 3. The initial forward rate constants for the five different biotinylated DNA and streptavidin-coated particles studied here range from 0.8 1 107 to 0.6 1 105 M01 s01.
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HUANG ET AL. TABLE 3
Forward Rate Constants (kf , 1106
M
01
) of Biotinylated DNA to Streptavidin-Coated PS Particles PS particle size (nm)
DNA (base pairs) 100 500 1000 2500 5000
90 8.25 4.73 2.68 0.81 0.35
{ { { { {
165 0.17 0.08 0.04 0.03 0.02
4.58 2.44 0.89 0.32 0.14
{ { { { {
214
0.20 0.13 0.09 0.06 0.03
2.30 1.02 0.51 0.26 0.11
{ { { { {
0.11 0.06 0.07 0.05 0.01
261 1.24 0.69 0.38 0.19 0.06
{ { { { {
0.06 0.05 0.06 0.02 0.01
944 1.15 0.66 0.41 0.17 0.06
{ { { { {
0.04 0.03 0.05 0.03 0.01
Note. The value of each kf is determined after 30 s at 47C in a 1.7-ml tube.
This implies that the forward rate constants for the various forms of biotinylated DNA to streptavidincoated particles in this system are about one to two orders of magnitude smaller than those reported for the biotin–streptavidin (or biotin–avidin) system in solution, a result consistent with several studies observed by other research groups (6, 7). Considering the binding kinetics for a reaction involving a solid surface, a useful parameter describing the degree of diffusion limitation in the unstirred reaction is called the Sherwood number (Sh) which is defined by (15) Sh Å kf d/D,
[9]
where kf is the mass transfer coefficient, D is the diffusion constant which is inversely proportional to Mr (i.e., D Å k(Mr)00.65), and d is the diameter of the particle. The Sherwood number can be interpreted as the ratio between the association reaction rate at the interface and the rate of diffusional mass transfer across the depletion layer at the surface. A high Sh means that the surface reaction is fast compared to the diffusional mass transfer and that diffusion is rate limiting. In fact, the Sherwood number in a heterogeneous, unstirred, diffusionally controlled reaction has been determined experimentally to be a constant, with a value of about 2 (15). Given a typical forward rate constant kf of ca. 1 1 106 M01 s01, a DNA diffusion coefficient of 5 1 1008 cm2/s, and a surface concentration in the range of 1 pmol/cm2, the Sherwood number is found to be larger than one even for particles with a diameter of 0.05 mm. In the present work, utilizing PS particles with diameters ranging from 0.944 to 0.090 mm, the values for Sh indicate that diffusion limitation is significant in all cases. In fact, for the system containing a 5-kbp DNA chain even the small PS-90 particles show a Sherwood number of 1.73 calculated from Table 3. Thus, mass transfer aspects are important when optimizing the kinetics of this macromolecular solid-phase reaction. In the other words, the very short time con-
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stant in this reaction dictates that the initial binding of biotin to streptavidin at solid–liquid interfaces often becomes diffusion limited due to a depletion of reactants close to the surface, even though the intrinsic bimolecular reaction at the interface is reaction rate limited. A realistic, and by necessity quite complex model of the binding reaction studied here should indeed account for the fact that both diffusion and binding may be geometry dependent. Surface Packing Figure 4a schematically depicts the sequence of events upon which the specific adsorption of biotinylated DNA is based: First, streptavidin solution is added to a suspension of latex particles. After washing, fluorescent biotinylated DNA is bound to streptavidin-immobilized latex particles. From the fluorescence intensity in the supernatant and that stripped from the surface, the surface’s specific binding capability can be quantitated for this system. The experimental results for the binding capacity (pmol DNA bound/mg particle) of biotinylated DNA to streptavidin-coated latex are listed in Table 4. They demonstrate that the surface concentration of bound DNA decreases with increasing particle size and DNA chain length as expected. Clearly the reduction of DNA surface concentration with increasing chain length is the result of sterically reduced access to the binding sites at the surface. Furthermore, a reduction of the particle size clearly results in a higher surface concentration of bound DNA. This saturation level of binding is likely governed by either or both of the following effects: (i) saturation of the biotin-binding sites according to a simple Langmuir isotherm or (ii) steric hindrance of available sites by bound DNA molecules. Throughout this study we have assumed that all bound molecules occupy an equal area on the surface regardless of conformation. This is indeed an oversimplification. There is a significant functional relationship between the increased blocking of the surface due to increasing cross sectional areas of the longer DNA
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given particle size results in a twofold change between 100 and 5000 bp DNA while the calculated binding constant decreases by about one order of magnitude. Obviously the binding constant, as shown in Eq. [3], depends on the concentrations of both available biotin binding sites and formed complex. Since measurements of the specific binding of fluorescein biotin to streptavidin-coated latex for each of the five types of particles of interest here indicate that different concentrations of biotin binding sites are present on the different particle types (see Table 1), the relationship between the binding constant and the binding capacity is not immediately obvious. In the case of the more commonly used streptavidincoated magnetic beads (M-280), the binding capacity is frequently found to be reduced by a factor of about 10 when increasing the size of biotinylated DNA from 100 to 5000 bp (unpublished data). Unlike the case for PS latex particles, M-280 beads represent the only particle size (2.8 mm) in this category. It thus means that the variable of substrate size as a parameter to study is out of question. In addition, as pointed out in our previous work (5), the surface concentration of streptavidin on these M-280 beads is about four times the theoretical monolayer coverage, which suggests attachment via a loosely crosslinked gel matrix. This matrix may well present different degrees of steric hindrance to affinity ligands of different size. The order of magnitude difference observed here when increasing the ligand size from 100 to 5000 bp is therefore not unexpected. By contrast, the adsorption-coated latex particles of interest here with their monolayer, or bilayer in the case of PS-90 and PS-165 particles, surface coverage presents much less steric hindrance than the M-280 beads. In fact, the data in Table 4 show that in the binding capacity for each of the five different types of PS particles there is only a twofold reduction in capacity in going from the small to the large DNA chain. Our observation of higher binding capacity on the smaller particles may shed some light on the question of surface arrangement of the bound DNA chains. It has previously been reported that the radius of gyra-
FIG. 4. (a) Schematic description of this study. (b) Two types of surface arrangement: (i) mushroom mode (dash lines denote the outer perimeters of the chain model) and (ii) brush mode (adapted from Ref. 17).
chains and the steric hindrance resulting from increasing coverage on the surface. For the antibody–antigen system, Nygren and co-workers have reported that antibody binding at levels above 1 pmol/cm2 is influenced by steric interactions to a degree where this factor, rather than the intrinsic antibody binding, limits the binding rate (16). In view of the much larger DNA molecules under investigation here, steric hindrance will undoubtedly play a significant role. It is interesting to compare the results between Table 2 and Table 4. The observed binding capacity for a
TABLE 4
Summary of the Amount Bound (pmol DNA/mg Particle) for DNAs of Different Chain Length Quantified from Each Type of Five Particle Substrates PS particle size (nm) pmol DNA/mg particle 100 500 1000 2500 5000
90 1428 1352 1345 857 666
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165 26 32 22 10 8
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214 11 15 17 8 6
374 366 334 238 200
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{ { { { {
261 13 12 14 11 9
263 252 223 160 133
{ { { { {
944 9 11 6 9 7
74 70 59 43 37
{ { { { {
6 7 4 3 5
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HUANG ET AL.
tion of a 5-kbp native DNA in solution is about 107 nm (12). In the case of PS-90, the experimental binding results for biotinylated DNA clearly demonstrate that the surface concentration of bound biotinylated DNA is at least 10 times higher than that expected from a theoretical calculation based on the solution size of the molecule. For reasons as yet unknown to us, streptavidin forms a monolayer on PS particles with diameters above 200 nm, while on the smaller particle the protein adsorbs to form a more or less complete bilayer. This implies that the surface arrangement of bulky DNA ligands may differ between large and small particles. Considering the results presented in this study, regarding both binding constants and surface concentrations, the surface orientation of DNA fragments is thought to be better represented by a rigid ‘‘brush’’ arrangement for PS-90 than by the ‘‘mushroom-like’’ arrangement for PS-944 suggested in Fig. 4b. The two types of surface arrangement were originally proposed by de Gennes (17) to describe the differences in structure formed for polymer chains tethered to a surface. In the mushroom case, the chains are sufficiently far apart to assume their solution radius, while in the brush case they are more closely spaced and osmotic effects forced them to extend themselves in the direction bound to the surface. This apparently is the situation for DNA bound to PS-90 particles, where the close packing far exceeds that estimated from the solution size of the molecule (300 molecules of 5000-bp DNA, as displayed in Table 4, versus 20 molecules computed based on a solution radius of 107 nm). However, for PS-944, the DNA chains appear to repel each other and their orientations may therefore be arranged like adjacent mushrooms. CONCLUSIONS
Hybridization of DNA onto solid surfaces has been extensively used for the isolation, identification, and genetic analysis of specific DNA sequences. For these and other applications, particle-assisted processing is shown in this work to offer an efficient approach to DNA analysis. However, bulky DNA molecules present sizable orientational constraints and decreased mass transfer. To compensate for those intrinsic disadvantages that stem from the need to bind bulky ligands, it is suggested that nonporous latex substrates with small diameter show significant promise for increased speed and increased ability to process large amounts of DNA specimens at one time. The experimental data for the binding of biotinylated DNA on streptavidincoated solid surfaces shown here indicate that both the equilibrium binding constant and the initial forward
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rate constant have been reduced drastically compared to the homogeneous reaction. The binding kinetics in the present study show considerable mass transfer limitations. Judging from the present results, several different surface arrangements of bound bulky DNA may be displayed on the solid surface, depending on whether adsorption has occurred on a large or a small latex particle. These findings also clearly indicate that the binding reaction of biotinylated DNA to streptavidin-coated latex particles demonstrates an exponential relationship between the observed binding constants and the number of base pairs in the biotinylated DNA. This relationship can serve as a useful predictor of rates and capacities in the processing of ever larger DNA chains (e.g., cosmid and genomic DNA) using latex particles of a particular size. ACKNOWLEDGMENTS The financial support for this work from the Center for Biopolymers at Interfaces at the University of Utah and the Human Genome Center through the National Institute of Health (Grant P50HG00199) is gratefully acknowledged. The authors thank Mr. Robert P. Black for preparing the DNA samples.
REFERENCES 1. Wilchek, M., and Bayer, E. A. (Eds.) (1990) Methods in Enzymology, Vol. 184, Academic Press, San Diego. 2. Green, M. N., Konieczny, L., Toms, E. J., and Valentine, R. C. (1971) Biochem. J. 125, 781–791. 3. Bayer, E. A., and Wilchek, M. (1980) Methods Biochem. Anal. 26, 1–46. 4. Leckband, D. E., Schmitt, F. J., Israelachvili, J. N., and Knoll, W. (1994) Biochemistry 33, 4611–4624. 5. Huang, S. C., Swerdlow, H., and Caldwell, K. D. (1994) Anal. Biochem. 222, 441–449. 6. Ku, C. A., and Lentrichia, B. B. (1989) J. Colloid Interface Sci. 132, 578–584. 7. Fujita, K., and Silver, J. (1993) BioTechniques 14, 608–617. 8. Stenberg, M., and Nygren, H. (1988) J. Immunol. Methods 113, 3–15. 9. Berzofsky, J. A., and Berkower, I. J. (1984) in Fundamental Immunology (Paul, W. E., Ed.), pp. 595–644, Raven Press, New York. 10. Herron, J. N. (1984) in Fluorescein Hapten: An Immunological Probe (Voss, E. W., Ed.), pp. 49–73, CRC Press, Baton Rouge. 11. Nygren, G., Kaartinen, M., and Stenberg, M. (1986) J. Immunol. Methods 92, 219–225. 12. Eigner, J., and Doty, P. (1965) J. Mol. Biol. 12, 549–580. 13. Livnah, O., Bayer, E. A., Wilchek, M., and Sussman, J. L (1993) Proc. Natl. Acad. Sci. USA 90, 5076–5080. 14. Nygren, H., and Stenberg, M. (1989) Immunology 66, 1–6. 15. Smith, J. M. (1981) Chemical Engineering Kinetics, McGrawHill, New York. 16. Nygren, H., Werthen, M., and Stenberg, M. (1987) J. Immunol. Methods 101, 63–71. 17. de Gennes, P. G (1987) Adv. Colloid Interface Sci. 27, 189–209.
AP-Anal Bio
237, 115–122 (1996)
0208
Binding of Biotinylated DNA to Streptavidin-Coated Polystyrene Latex: Effects of Chain Length and Particle Size Shao-Chie Huang,* Mark D. Stump,† Robert Weiss,† and Karin D. Caldwell*,1 *Department of Chemical and Fuels Engineering, and †Department of Human Genetics, University of Utah, Salt Lake City, Utah 84112
Received December 8, 1995
The binding of 5*-end biotinylated DNA, ranging in size from 100 to 5000 base pairs, was studied using streptavidin-coated polystyrene latex particles with diameters between 0.944 and 0.090 mm. The experimental binding constants and forward rate constants of this solid-phase reaction were determined to be several orders of magnitude lower than values for the biotin–streptavidin interaction in solution as expected and were shown to depend on the size of both ligand and substrate. An observed inflection in the binding constant of the biotinylated DNA appeared around 1000 base pairs, possibly indicating different surface orientations of the macroligand above and below this critical size. This effect was more pronounced for the smaller latex particles used in this study and highlighted possible differences in the surface arrangement of streptavidin on the differently sized particles. Diffusion limitation to the binding reaction was found to be significant in all cases. In this present work, an exponential relationship was established between the experimental binding constant and the number of base pairs in the biotinylated DNA. This relationship possibly provides a means to predict capacity and binding speed in cases where adsorption, purification, and release of larger DNA chains are required. q 1996 Academic Press, Inc.
Streptavidin, a protein produced by Streptomyces avidinii, binds d-biotin with an extremely high affinity (the KA of biotin – streptavidin in solution is around 1015 M01); the formation of this complex can be regarded as practically irreversible, as the binding energy is comparable to that of a covalent bond. The 1 To whom correspondence should be addressed. Fax: (801) 5855151.
strong and specific binding feature of this biotin – streptavidin system not only offers many powerful bioanalytical applications but also generates considerable interest as a versatile model in studying macromolecule – ligand interactions (1 – 4). The binding kinetics of several biotin–streptavidin systems have been well studied in solution (1). The experimental results from such studies conform with classical chemical theories, i.e., the law of mass action with its rigorous thermodynamic underpinnings. However, a few reports exist in which the focus has been the interaction between either biotin or streptavidin in the form of a solid-phase reagent. These studies show that the heterogeneous phase kinetics differ substantially from the theoretical models that have been developed to explain the results of biotin–streptavidin reactions in solution (5–7). The initial forward reaction often becomes diffusion limited at the interface, possibly due to steric hindrance and the inflexibility of the immobilized ligand. On the other hand, the dissociation rate of the bound molecule is often faster than that characteristic of the complex in solution as a result of surface-induced conformational shifts in the binding sites presented by the immobilized molecules. Thus, both the forward and reverse reactions tend to reduce the magnitude of the binding constant (7). Uniform latex particles have been used in biomedical research for more than two decades. Since the surface area per mass of particles increases with smaller diameters, the amount of immobilized ligand relative to the total mass of the particles also increases. Furthermore, in binding large biotinylated macromolecules (e.g., biotinylated DNA) to streptavidin-coated surfaces, the size of the biotinylated ligand is likely to affect not only the equilibrium binding level, but also the rate at which this binding occurs. Since mass transfer limitations will govern this process at the fast binding rates inher115
0003-2697/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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ently provided by the biotin–streptavidin system, kinetic arguments postulate that the actual binding rate will decrease with increasing hydrodynamic size of the ligand as well as with increasing size of the solid phase. Therefore, a timely processing of ever larger ligands will require reducing the size of the particulate phase. In a previous report (5) from this laboratory, streptavidin was shown to adsorb readily and irreversibly to polystyrene (PS)2 colloidal substrates and thereby confer on their surfaces a specific affinity for biotin. In the present work we have selected PS latex particles with a wide variety of discrete sizes, ranging from 0.944 to 0.090 mm. For the same amount of bulk, these particles present several-fold higher surface areas than that associated with the commercially available larger (2.8 mm) M-280 beads from Dynal. Here, separation of particle-bound macromolecules from unbound debris and impurities is achieved by centrifugal forces, instead of the magnetic forces employed while working with the Dynal beads. In binding large biotinylated macromolecules to streptavidin-coated solid surfaces, the size of the ligand is thus expected to affect both the equilibrium binding and the binding rate. Due to (i) the mass transfer limitations discussed above and (ii) the kinetic arguments which dictate that the actual binding rate will decrease both with increasing hydrodynamic size of the ligand and with an increase in particle size, the timely processing of ever larger ligands will require decreasing the size of the solid substrate. The present study is undertaken to further investigate the equilibrium binding and binding kinetics for ligand-coupled particles on solid surfaces, as they vary with the size of both ligand and particle. MATERIALS
PS latex particles with diameters ranging from 0.944 to 0.090 mm were obtained as 10% (w/v) suspensions according to the manufacturer’s datasheet. Particles with diameters of 0.261 and 0.090 mm were purchased from Seradyn while PS-944, PS-214, and PS-165 were from Bangs Laboratories. These particles were routinely used in other similar studies within our laboratory. The adsorption results are quite consistent throughout the experiments. Streptavidin was purchased from Sigma (catalog no. S-4762), as was Streptavidin–fluorescein isothiocyanate (FITC) (catalog no. S-3762). Fluorescein biotin (F-B, 5-((N-(5-(N-(6-(biotinoyl)amino)hexanoyl)amino)pentyl)thioureidyl) fluorescein) was obtained from Molecular Probes (catalog no. B-1370). The phosphate-buffered saline (PBS, 101) stock solution contained 87.7 g sodium chloride, 15.5 g 2
Abbreviations used: PS, polystyrene; PBS, phosphate-buffered saline.
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monobasic sodium phosphate, and 40.9 g dibasic sodium phosphate, diluted to 1000 ml with deionized water, pH 7.2. Biotinylated fluorescent DNA was prepared according to the procedure described in next section. METHODS
Streptavidin solution (2 mg/ml) was added to the particle suspension. The adsorption reaction was allowed to proceed for 1 h at room temperature with vortexing, and the streptavidin-coated latex particles were then washed and resuspended in PBS buffer by repeated centrifugations (Eppendorf centrifuge 5415C from Brinkmann Instruments or Optima TL ultracentrifuge from Beckman). For binding capacity measurements, a series of biotinylated DNA solutions were reacted with one and the same amount of streptavidin-coated latex particles for 10 min at room temperature under continuous shaking. Biotinylated fluorescent DNA, not bound to the surfaces, was retrieved and quantitated by means of a Perkin–Elmer luminescence spectrometer (Model LS50) after sedimenting the suspension by centrifugation and its fluorescence intensity was determined at an excitation wavelength of 484 nm and an emission wavelength of 504 nm. For the kinetic study, an appropriate amount of fluorescent DNA solution was added to a suspension of streptavidin-coated particles in a 1.7-ml polypropylene microcentrifuge tube (National Scientific), and the reaction was carried out at 47C for 30 s to 15 min under continuous mixing. The reactions were stopped by centrifugation. The amount of DNA left in the supernatant was quantified as mentioned before (5), and the amount bound to the particles was determined by subtraction from the total amount originally added. Double-stranded DNA molecules, ranging from 100 to 5000 bp, are used for binding of large biotinylated ligands to streptavidin-coated beads. The reaction mixture of 100 ml contained 300 ng M13mp18 DNA template, 40 mM 5*-end biotinylated primer, 50 mM fluorescent universal primer, the sequences FL-1 (5*-CAGGAAACAGCTATGACC-3*), FL-2 (5*-ACAACACTCAACCCTATCT-3*), FL-3 (5*-TGCGTGATGGACAGACTCT-3*), FL-4 (5*-AGCCCTACTGTTGAGCGT-3*), and FL-5 (5*-AGCCTCTGTAGCCGTTGCT3*), 4 units Taq (Thermus aquaticus) DNA polymerase, 0.2 mM deoxynucleoside triphosphates (dNTP) in a buffer containing 1.5 mM MgCl2 , 50 mM Tris–HCl, pH 8.4, 25 mM KCl, 5 mM NH4Cl, and 150 mg/ml bovine serum albumin. The reaction mixture was covered with a layer of Nujol mineral oil (Perkin–Elmer). The in vitro amplification was carried out on the PTC-100 MJ Research thermal cycler. Thirty cycles were performed with denaturation at 957C for 60 s, annealing at 627C for 2 min, and extension at 727C for 20 s. Unreacted
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primers were removed by centrifugation using Centricon-30 microconcentrators (Amicon) with three washings in water.
TABLE 1
Measurements of the Specific Binding of Fluorescein Biotin to Streptavidin-Coated Latex for Each of the Five Types of Particles
RESULTS AND DISCUSSION Particle type
Equilibrium Binding If one assumes that nonspecific interactions are absent, the bimolecular binding reaction of a specific ligand containing biotin (B) to biotin binding sites (S) can be expressed as (8) kf
S / B S SB, kr
[1]
where kf is the forward and kr is the reverse rate constants. Thus, the binding rate equation is d[SB]/dt Å kf [S][B] 0 kr[SB],
[2]
where [S] is the concentration of biotin binding sites (streptavidin subunits), [B] the concentration of biotin, and [SB] the concentration of complex. At equilibrium
KA Å
[SB] kf , Å [S][B] kr
[3]
where KA is the binding constant. Since the total number of biotin binding sites is conserved, and the number of biotinylated ligands in the bulk solution is in large excess compared to the number of available ligands on the surfaces, Eq. [3] can be rearranged as 1 1 1 1 Å , / [SB] [S]0 [S]0 KA [B]
[4]
where [S]0 is the initial effective concentration of biotin binding sites. In this manner, the binding constant KA can be determined from a double-reciprocal plot of 1/[SB] versus 1/[B]. It is well documented that one streptavidin molecule in solution has four biotin binding sites (1–3). In the present study the streptavidin–biotin binding reaction occurs on a solid surface. It should also be noted that the biotinylated DNAs employed in this study are quite large compared to the biotin molecule itself. Therefore, a certain degree of decreased specific streptavidin–biotin binding should be anticipated for this solid-phase reaction. A summary of the binding of fluorescein-labeled biotin to each of the five streptavidin-coated PS
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Moles F-B per mole SA
PS-90 PS-165 PS-214 PS-261 PS-944
0.78 0.72 0.55 0.42 0.45
{ { { { {
0.04 0.02 0.01 0.01 0.02
latex substrates of interest here is shown in Table 1. Expressed on a biotin per streptavidin basis, higher specific binding is observed for particles with smaller diameters. This result is quite consistent with data presented in our previous report (5). The reduction in specific binding capability resulting from the immobilization procedure used here is expected to be due to either or both of the effects of decreased accessibility and steric hindrance on the solid surface. The calculated KA values for a variety of biotinylated fluorescent DNAs and their interactions with streptavidin-coated PS particles of different sizes are shown in Table 2. Figure 1 is a graphic representation of the binding constant as a function of the number of base pairs in the biotinylated fluorescent DNAs. As expected, the binding constants decrease while increasing the size of either ligand or substrate (carrier particle). The calculated binding constants were found to be in the range of 1.1 1 1010 to 0.6 1 108 M01, depending on ligand and substrate size. These data are substantially lower by about five to six orders of magnitude than the reported 1 1 1015 M01 for the streptavidin–biotin interaction in a homogeneous solution. In addition, the binding capacity of the streptavidin-coated particles for biotinylated DNA is considerably reduced as a result of the bead-immobilization of streptavidin. This, in turn, is likely to produce steric hindrance toward the binding of bulky ligands and possibly also conformational changes in the binding site which weakens the interaction. Alternatively, the KA of this bimolecular binding reaction can also be calculated using Scatchard analysis which is widely employed in computing the affinity of antibody–antigen systems (9–11). From the mass action equilibrium law, Eq. [3] can be expressed as r Å nKA 0 rKA , [B]
[5]
where r is the number of moles of biotinylated DNA bound per mole of immobilized streptavidin, and n is
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Binding Constants (KA , 1 108
M
01
) of Biotinylated DNA to Streptavidin-Coated PS Particles at Equilibrium PS particle size (nm)
DNA (base pairs) 100 500 1000 2500 5000
90 108.1 53.8 23.9 12.0 5.7
{ { { { {
165 1.3 1.5 0.7 0.4 0.1
57.2 26.3 13.3 7.6 3.1
{ { { { {
214 0.9 0.6 0.5 0.3 0.2
18.5 10.4 6.3 2.3 0.9
{ { { { {
261 0.9 0.7 0.4 0.1 0.1
11.1 4.8 3.0 1.8 0.6
{ { { { {
944 0.5 0.2 0.2 0.3 0.1
8.0 3.6 3.0 1.9 0.6
{ { { { {
0.4 0.3 0.2 0.2 0.1
Note. Reaction conditions are as follows: 10 min at room temperature in a 1.7-ml tube.
the total number of available binding sites. Hence, in this form of Scatchard plot, r/[B] versus r should yield a straight line with a slope of 0KA and an intercept on the abscissa corresponding to the concentration of streptavidin-binding sites (n). Based on the analysis mentioned above, a Scatchard plot for binding of biotinylated DNA to streptavidin-coated PS-261 is shown in Fig. 2. Both the binding constant and the streptavidinbinding site concentration are determined at least
FIG. 1. Particle size effects on binding constant KA as a function of the number of base pairs in the DNA ligand. Curve-fitting analyses result in coefficients of determination (R 2) of ca. 0.95 for the five types of PS particles. Symbols: PS-90 (s), PS-165 (l), PS-214 (h), PS-261 (n), and PS-944 (m).
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twice and are found to be quite consistent with the results calculated from Eq. [4]. Using linear fitting, the adsorbed streptavidin has been found to bind about 0.4 mol per mole of biotin ligand. This reduced binding capacity is one-tenth of that determined in solution. Nevertheless, the concave Scatchard plot indicates that a certain degree of heterogeneity of affinity is associated with this system. An interesting feature evident in Fig. 1 is the dramatic increase in KA when streptavidin is adsorbed to
FIG. 2. A Scatchard plot for binding of biotinylated DNA, 300 bp in length, to streptavidin-coated PS-261. This ‘‘concave-up’’ curve clearly demonstrates a certain degree of heterogeneity in the binding affinity of this system.
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EFFECT OF DNA AND PARTICLE SIZE ON BIOTIN–STREPTAVIDIN BINDING
119
the smaller particles (PS-90 and PS-165) compared to what is seen for the larger ones (PS-261 and PS-944). This is despite the fact that for each DNA chain length the ratio of surface concentrations for any pair of particles remains nearly constant. Moreover, this figure shows that under the conditions tested an exponential relationship exists between the binding constant KA and the number of base pairs in the biotinylated DNA for each type of streptavidin-coated particle studied here. Curve-fitting analyses yield coefficients of determination (R 2) around 0.95 for each of the five types of particles. Thus, the data are fitted to an expression of the form Y Å A 0 B log X,
[6]
where Y is the binding constant for each type of particle, X is the number of base pairs of biotinylated DNA, and A and B are parameters depending on ligand and substrate in the system. This finding has the practical consequence of potentially allowing an estimation of the capacity of any particle size to bind DNA molecules of a specified length. It is interesting to note, in the semilogarithmic plot shown in Fig. 3, that the relationships between binding constants of streptavidin-coated particles and the number of base pairs in the biotinylated DNA display inflections for PS-90 and PS-165, whereas for PS-214, PS-261, and PS-944 such inflections are less pronounced. This observed inflection appears at a ligand size corresponding to 1000 bp of biotinylated DNA. It may indirectly suggest that there is a tangible difference in the surface orientation of streptavidin for these two groups of particles which, in turn, translates into differences in DNA binding. In such a case there are at least two possible explanations for this significant phenomenon. First, the different protein-coated surfaces may have induced different conformations in the bulky DNA molecules, as they bind to the different surfaces. Second, from studies of the molecular weight (Mr) dependence of DNA viscosity, the polymer is known to experience rod-like behavior at Mr values below 2 million daltons or 3000 base pairs, while it behaves as a flexible random coil for Mr values above this level (12). These Mr-dependent differences in hydrodynamic shape may correlate with the Mr-dependent differences in surface packing, seen in this work. Kinetic Study In solution, the rate constants kf and kr for the biotin–steptavidin system are reported to be 107 M01 s01 and 1008 s01, respectively (13). As a consequence of the slow desorption rate, the value of the second term in Eq. [2], which accounts for the dissociation, can be considered negligible compared to the first term, at least
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FIG. 3. A semilogarithmic plot of the data from Fig. 1. Inflections, which are significant for both PS-90 and PS-165 and less pronounced for others, are observed in the vicinity of 1000 bp DNA length. Symbols are the same as in Fig. 1.
in the initial stages of the reaction (14). As a result, the kinetics of association of biotinylated DNA to streptavidin-coated particles can be modeled as a secondorder reaction of the form d[SB]/dt Å kf [S][B].
[7]
The differential Eq. [7] is readily solved to give ln
([S]0 0 [SB])[B]0 Å ([S]0 0 [B]0)kf t, [S]0([B]0 0 [SB])
[8]
where [B]0 is the initial concentration of biotin, and t is the reaction time. From this relationship, kf can be determined, given accurate values of the starting concentrations [S]0 and [B]0 , as well as of the amount of complex [SB] formed at time t. The calculated characteristic forward rate constants kf of biotinylated DNA to streptavidin-coated particles are listed in Table 3. The initial forward rate constants for the five different biotinylated DNA and streptavidin-coated particles studied here range from 0.8 1 107 to 0.6 1 105 M01 s01.
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Forward Rate Constants (kf , 1106
M
01
) of Biotinylated DNA to Streptavidin-Coated PS Particles PS particle size (nm)
DNA (base pairs) 100 500 1000 2500 5000
90 8.25 4.73 2.68 0.81 0.35
{ { { { {
165 0.17 0.08 0.04 0.03 0.02
4.58 2.44 0.89 0.32 0.14
{ { { { {
214
0.20 0.13 0.09 0.06 0.03
2.30 1.02 0.51 0.26 0.11
{ { { { {
0.11 0.06 0.07 0.05 0.01
261 1.24 0.69 0.38 0.19 0.06
{ { { { {
0.06 0.05 0.06 0.02 0.01
944 1.15 0.66 0.41 0.17 0.06
{ { { { {
0.04 0.03 0.05 0.03 0.01
Note. The value of each kf is determined after 30 s at 47C in a 1.7-ml tube.
This implies that the forward rate constants for the various forms of biotinylated DNA to streptavidincoated particles in this system are about one to two orders of magnitude smaller than those reported for the biotin–streptavidin (or biotin–avidin) system in solution, a result consistent with several studies observed by other research groups (6, 7). Considering the binding kinetics for a reaction involving a solid surface, a useful parameter describing the degree of diffusion limitation in the unstirred reaction is called the Sherwood number (Sh) which is defined by (15) Sh Å kf d/D,
[9]
where kf is the mass transfer coefficient, D is the diffusion constant which is inversely proportional to Mr (i.e., D Å k(Mr)00.65), and d is the diameter of the particle. The Sherwood number can be interpreted as the ratio between the association reaction rate at the interface and the rate of diffusional mass transfer across the depletion layer at the surface. A high Sh means that the surface reaction is fast compared to the diffusional mass transfer and that diffusion is rate limiting. In fact, the Sherwood number in a heterogeneous, unstirred, diffusionally controlled reaction has been determined experimentally to be a constant, with a value of about 2 (15). Given a typical forward rate constant kf of ca. 1 1 106 M01 s01, a DNA diffusion coefficient of 5 1 1008 cm2/s, and a surface concentration in the range of 1 pmol/cm2, the Sherwood number is found to be larger than one even for particles with a diameter of 0.05 mm. In the present work, utilizing PS particles with diameters ranging from 0.944 to 0.090 mm, the values for Sh indicate that diffusion limitation is significant in all cases. In fact, for the system containing a 5-kbp DNA chain even the small PS-90 particles show a Sherwood number of 1.73 calculated from Table 3. Thus, mass transfer aspects are important when optimizing the kinetics of this macromolecular solid-phase reaction. In the other words, the very short time con-
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stant in this reaction dictates that the initial binding of biotin to streptavidin at solid–liquid interfaces often becomes diffusion limited due to a depletion of reactants close to the surface, even though the intrinsic bimolecular reaction at the interface is reaction rate limited. A realistic, and by necessity quite complex model of the binding reaction studied here should indeed account for the fact that both diffusion and binding may be geometry dependent. Surface Packing Figure 4a schematically depicts the sequence of events upon which the specific adsorption of biotinylated DNA is based: First, streptavidin solution is added to a suspension of latex particles. After washing, fluorescent biotinylated DNA is bound to streptavidin-immobilized latex particles. From the fluorescence intensity in the supernatant and that stripped from the surface, the surface’s specific binding capability can be quantitated for this system. The experimental results for the binding capacity (pmol DNA bound/mg particle) of biotinylated DNA to streptavidin-coated latex are listed in Table 4. They demonstrate that the surface concentration of bound DNA decreases with increasing particle size and DNA chain length as expected. Clearly the reduction of DNA surface concentration with increasing chain length is the result of sterically reduced access to the binding sites at the surface. Furthermore, a reduction of the particle size clearly results in a higher surface concentration of bound DNA. This saturation level of binding is likely governed by either or both of the following effects: (i) saturation of the biotin-binding sites according to a simple Langmuir isotherm or (ii) steric hindrance of available sites by bound DNA molecules. Throughout this study we have assumed that all bound molecules occupy an equal area on the surface regardless of conformation. This is indeed an oversimplification. There is a significant functional relationship between the increased blocking of the surface due to increasing cross sectional areas of the longer DNA
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given particle size results in a twofold change between 100 and 5000 bp DNA while the calculated binding constant decreases by about one order of magnitude. Obviously the binding constant, as shown in Eq. [3], depends on the concentrations of both available biotin binding sites and formed complex. Since measurements of the specific binding of fluorescein biotin to streptavidin-coated latex for each of the five types of particles of interest here indicate that different concentrations of biotin binding sites are present on the different particle types (see Table 1), the relationship between the binding constant and the binding capacity is not immediately obvious. In the case of the more commonly used streptavidincoated magnetic beads (M-280), the binding capacity is frequently found to be reduced by a factor of about 10 when increasing the size of biotinylated DNA from 100 to 5000 bp (unpublished data). Unlike the case for PS latex particles, M-280 beads represent the only particle size (2.8 mm) in this category. It thus means that the variable of substrate size as a parameter to study is out of question. In addition, as pointed out in our previous work (5), the surface concentration of streptavidin on these M-280 beads is about four times the theoretical monolayer coverage, which suggests attachment via a loosely crosslinked gel matrix. This matrix may well present different degrees of steric hindrance to affinity ligands of different size. The order of magnitude difference observed here when increasing the ligand size from 100 to 5000 bp is therefore not unexpected. By contrast, the adsorption-coated latex particles of interest here with their monolayer, or bilayer in the case of PS-90 and PS-165 particles, surface coverage presents much less steric hindrance than the M-280 beads. In fact, the data in Table 4 show that in the binding capacity for each of the five different types of PS particles there is only a twofold reduction in capacity in going from the small to the large DNA chain. Our observation of higher binding capacity on the smaller particles may shed some light on the question of surface arrangement of the bound DNA chains. It has previously been reported that the radius of gyra-
FIG. 4. (a) Schematic description of this study. (b) Two types of surface arrangement: (i) mushroom mode (dash lines denote the outer perimeters of the chain model) and (ii) brush mode (adapted from Ref. 17).
chains and the steric hindrance resulting from increasing coverage on the surface. For the antibody–antigen system, Nygren and co-workers have reported that antibody binding at levels above 1 pmol/cm2 is influenced by steric interactions to a degree where this factor, rather than the intrinsic antibody binding, limits the binding rate (16). In view of the much larger DNA molecules under investigation here, steric hindrance will undoubtedly play a significant role. It is interesting to compare the results between Table 2 and Table 4. The observed binding capacity for a
TABLE 4
Summary of the Amount Bound (pmol DNA/mg Particle) for DNAs of Different Chain Length Quantified from Each Type of Five Particle Substrates PS particle size (nm) pmol DNA/mg particle 100 500 1000 2500 5000
90 1428 1352 1345 857 666
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165 26 32 22 10 8
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214 11 15 17 8 6
374 366 334 238 200
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{ { { { {
261 13 12 14 11 9
263 252 223 160 133
{ { { { {
944 9 11 6 9 7
74 70 59 43 37
{ { { { {
6 7 4 3 5
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HUANG ET AL.
tion of a 5-kbp native DNA in solution is about 107 nm (12). In the case of PS-90, the experimental binding results for biotinylated DNA clearly demonstrate that the surface concentration of bound biotinylated DNA is at least 10 times higher than that expected from a theoretical calculation based on the solution size of the molecule. For reasons as yet unknown to us, streptavidin forms a monolayer on PS particles with diameters above 200 nm, while on the smaller particle the protein adsorbs to form a more or less complete bilayer. This implies that the surface arrangement of bulky DNA ligands may differ between large and small particles. Considering the results presented in this study, regarding both binding constants and surface concentrations, the surface orientation of DNA fragments is thought to be better represented by a rigid ‘‘brush’’ arrangement for PS-90 than by the ‘‘mushroom-like’’ arrangement for PS-944 suggested in Fig. 4b. The two types of surface arrangement were originally proposed by de Gennes (17) to describe the differences in structure formed for polymer chains tethered to a surface. In the mushroom case, the chains are sufficiently far apart to assume their solution radius, while in the brush case they are more closely spaced and osmotic effects forced them to extend themselves in the direction bound to the surface. This apparently is the situation for DNA bound to PS-90 particles, where the close packing far exceeds that estimated from the solution size of the molecule (300 molecules of 5000-bp DNA, as displayed in Table 4, versus 20 molecules computed based on a solution radius of 107 nm). However, for PS-944, the DNA chains appear to repel each other and their orientations may therefore be arranged like adjacent mushrooms. CONCLUSIONS
Hybridization of DNA onto solid surfaces has been extensively used for the isolation, identification, and genetic analysis of specific DNA sequences. For these and other applications, particle-assisted processing is shown in this work to offer an efficient approach to DNA analysis. However, bulky DNA molecules present sizable orientational constraints and decreased mass transfer. To compensate for those intrinsic disadvantages that stem from the need to bind bulky ligands, it is suggested that nonporous latex substrates with small diameter show significant promise for increased speed and increased ability to process large amounts of DNA specimens at one time. The experimental data for the binding of biotinylated DNA on streptavidincoated solid surfaces shown here indicate that both the equilibrium binding constant and the initial forward
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rate constant have been reduced drastically compared to the homogeneous reaction. The binding kinetics in the present study show considerable mass transfer limitations. Judging from the present results, several different surface arrangements of bound bulky DNA may be displayed on the solid surface, depending on whether adsorption has occurred on a large or a small latex particle. These findings also clearly indicate that the binding reaction of biotinylated DNA to streptavidin-coated latex particles demonstrates an exponential relationship between the observed binding constants and the number of base pairs in the biotinylated DNA. This relationship can serve as a useful predictor of rates and capacities in the processing of ever larger DNA chains (e.g., cosmid and genomic DNA) using latex particles of a particular size. ACKNOWLEDGMENTS The financial support for this work from the Center for Biopolymers at Interfaces at the University of Utah and the Human Genome Center through the National Institute of Health (Grant P50HG00199) is gratefully acknowledged. The authors thank Mr. Robert P. Black for preparing the DNA samples.
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