Langmuir 1996,11, 3772-3777
3772
Conformation of DNA Block Copolymer Molecules Adsorbed on Latex Particles As Revealed by Hydroxyl Radical Footprinting Harold W. Walker and Stanley B. Grant* Department of Civil and Environmental Engineering, University of California, Irvine, California 9271 7 Received April 24, 1995. I n Final Form: July 20, 1995@ DNA is an ideal polymer for adsorption studies due to the impressive array of biochemicaltools available for its synthesis, manipulation, and analysis. In this study, we use hydroxyl radical footprinting to probe the conformation of a single-stranded DNA analog adsorbed to the surface of latex microspheres in water. The DNA molecule has a diblock copolymer architecture with an uncharged block 20 nucleotides in length and an equally long negatively charged block. We find that this model block copolymer does not form a polymer brush layer when adsorbed to negatively charged latex at moderate salt concentrations (0.05 M NaC1). Furthermore, the exact nature of the DNA-surface interaction is found to depend on both the primary charge of the bare latex particles and on the surface density of adsorbed polymer.
Introduction The use of polymers to stabilize colloid-sized particles dates back to a t least 2500 BC when the ancient Egyptians attempted to stabilize carbon black fibers with casein and gum arabic for use as ink.l Today, over four millennia later, polymeric stabilization of colloids remains a topic of important practical and scientific interest. In recent years, block copolymers have emerged as the polymer of choice for a variety of colloid stabilization and membrane separation technologies. These molecules are characterized by an arrangement of polymer segments into regions (or blocks) with low solvent affinity (A) and blocks with high solvent affinity (B). Block copolymers are highly surface active, and upon adsorption they often adopt a three-dimensional conformation in which the A block anchors the molecule to the surface and the B block extends into the liquid, forming a “polymer brush”.2 When adsorbed to the surface of colloidal particles, these polymer brush layers create a repulsive barrier which prevents the particles from coagulating. While considerable theoretical work has been carried out to predict the conditions under which a polymer brush will form, experimental progress in this area has been slow due, in part, to limitations with existing experimental systems for investigating the conformation of polymers adsorbed at the solid-liquid interface. Here, we report on a model system for obtaining fundamental information about the conformation of adsorbed polymer chains. The system consists of singlestranded DNA as the model polymer and charge-modified polystyrene latex particles as the model adsorbent. There are several aspects of DNA synthesis and structure that make it well suited for fundamental studies of polymer adsorption. As shown in Figure 1, each DNA monomer (or “nucleotide”) consists of a five-carbon sugar, a nitrogenous base, and an internucleotide linkage. The chemical synthesis of short pieces of DNA (or “oligonucleotides”) has advanced to such a degree that molecules of specified base sequence, length, and linkage type can be synthesized in bulk quantities using automated phosphoramidite
* To whom correspondence should be addressed. @
Abstract published inAdvance ACSAbstracts, October 1,1995.
(1)Napper, D. H. Polymeric Stabilization of Colloidal Dispersions; Academic Press: London, 1983. (2) Halperin, A.; Tirrell, M.; Lodge, T. P.Adu. Polym. Sci. 1992,100, 31.
5’
X OC2H2
1a‘ b
0
internucleotide linkage
0-
Linkage Neutral ethylphosphonate (Class II) Negativelycharged phoophodiester (Class I)
deoxyribose 3’ sugar
Figure 1. Basic structure of DNA. Each DNA monomer, or nucleotide, contains a deoxyribose sugar, a nitrogenous base, and an internucleotide phosphate linkage. technology. Using this technology, model DNA compounds can be designed with specific chemical or electrical architectures. Furthermore, DNA fragments of a given size can be isolated using standard gel electrophoresis methods. When the isolated fragments are redissolved in water, the resulting DNA is not only monodisperse but also of a n exactly known length and chemical structure. These aspects of DNA synthesis and structure have already been exploited for investigating the physical and chemical properties of polyelectrolytes in s ~ l u t i o n . ~ In this report, we synthesized a DNA molecule (referred to as dT40B) that has a diblock copolymer architecture and investigated its adsorption to latex particles. To examine the conformation this molecule adopts upon adsorption we used a biochemical technique called hydroxyl radical footprinting (HRF). In the past, HRF has been used with great success in the field of biochemistry to investigate protein-DNA interaction^.^ The basic idea underlying this technique is that hydroxyl radical attack on the sugar moiety in the DNA backbone results in strand scission, essentially cutting the DNA molecule into two pieces at the site of attack. This process is diffusion-limited so that regions of a DNA molecule which are less accessible to hydroxyl radical are cut less frequently. In the case of protein-DNA studies, the region of DNA in direct contact with protein is protected from hydroxyl radical attack, and this “footprint” can be detected when the DNA fragments are separated by size using high-resolution polyacrylamide gel electrophoresis (PAGE). (3) Pecora, R. Science 1990,251, 893. (4) Tullius, T. D.; Dombrowski, B. A,; Churchill, M. E. A,; Kam, L. Methods Enzymol. 1987,155, 537.
0743-7463/95/2411-3772$09.00/0 0 1995 American Chemical Society
Conformation of DNA Block Copolymer Molecules
Langmuir, Vol. 11, No. 10, 1995 3773 ssDNA Molecule
OH)
\OH'
* 5'
* 5' * 5'
- -
3 3 3'
* 5' *S
3
-
3' 3' 3
-
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-
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ssDNA Fragmenls
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i
* 5'
3
-
-
*5'-
3'
.5'-
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I
I
ssDNA Fragmenls
Gel ElectrophoresisPattern
Figure 2. General strategy for determining the conformation of adsorbed DNA molecules using hydroxyl radical footprinting (HRF). Note that the radioactive isotope ofphosphorous (32P) is present on the 5'end of each ofthese molecules (shown schematically as a n asterisk).
In o u r application of HRF, the experiments are carried o u t in m u c h the same way, except that latex particles play the role of protein. As outlined schematically in Figure 2, DNA polymers are adsorbed to the surface of latex particles and then hydroxyl radicals a r e generated by the F e n t o n reaction (see Experimental Section). In principle, segments of the DNA a w a y from the surface-for instance, in a polymer brush-will be cut more frequently than segments directly adjacent to the surface. After the fragments a r e analyzed by PAGE, the cutting frequency of each DNA s e g m e n t should reveal t h e adsorbed molecule's conformation. In this paper, we employ this new method to investigate the surface conformation of t h e block copolymer molecule dT40B when adsorbed t o either negatively or positively charged latex microspheres i n an aqueous salt solution.
Molecule
Chemical Structure
I1 (0- p - dT) I 0-
I
0-
dT40C
impurities and stored a t 4 "C. All three DNA molecules are 40 nucleotides long and were synthesized with the base thymine at every nucleotide position. dT4OA was synthesized using only phosphodiester linkages, while the other two molecules (dT40B and dT40C) contain ethylphosphonate and phosphodiester internucleotide linkages. The 480 nm amidine and 470 nm sulfate polystyrene latex particles were purchased directly from Interfacial Dynamics Corp. (Portland, OR). These particles were synthesized in surfactantfree water by conventional free radical polymerization. Under the neutral pH conditions used here, the amidine and sulfate particles have a positive (13.1pC/cm2)and negative (4.6pC/cm2) surface charge, respectively. The density of surface functional groups on the amidine (NzHd+)and sulfate latex (SO1-) particles is approximately 0.8 and 0.3 sites/nm2, respectively. These surface charge densities were reported by the manufacturer; the average particle diameters were reported by the manufacturer and confirmed in our laboratory using a photon correlation spectrometer (Model N4 submicronparticle size analyzer, Coulter Scientific Instruments, Amherst, MA). All solutions were made with analytical grade reagents and deionized water (Mill-&water, 18 MQ cm, Millipore, Bedford, MA).
0
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Experimental Section Materials. The three model DNA molecules used in this study (see Figure 3) were purchased directly from Oligo Etc. (Portand, OR) and were received in lypholyzed form. Prior to use the molecules were purified by column chromatography to remove
40
0 dT40B
5'
3'
5 ' 0 dT40A
ElectrostaticArchitecture
Negatively charged
OH*
Figure 3. Chemical and electrical architecture of the three model DNA molecules used in this study. Hydroxyl radicals cleave DNA by attacking the 4'carbon of the deoxyribose moiety.
OctanoWater Partitioning. Oligonucleotides were radiolabeled on the 5' end in vitro using the enzyme polynucleotide kinase which catalyzes the transfer of a radioactive phosphate from y32P-2'-deoxyadenosine5'4riphosphate to the 5' hydroxyl on the nucleotides.5 After a 30 minincubation period, the labeling reaction was terminated by heating at 65 "C and the unincorporated nucleotides were removed by passage through a column containing Sephadex G50 (Sigma, St. Louis, MO). Two hundred p L of 1-octanol (Fisher, Pittsburgh, PA) was added to a 200 p L mixture ofDNA, and the solution was mixed on a rotating shaker. After at least 2 h, the total amount of radioactivity in both the aqueous phase and the 1-octanol phase was measured using a benchtop radioisotope counter (QCZOOO, Bioscan, Washington, DC). The octanol-water partition coefficient (&.,I was deter( 5 ) Sambrook, J.; Fritch, E. F.; Maniatis, T. Molecular Cloning A Laboratory Manual, 2nd ed.; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY,1989.
Walker and Grant
3774 Langmuir, Vol. 11, No. 10, 1995 mined as
(1) where DNAoctis the radioactivity in the 1-octanol phase and DNAH~o is the amount of radioactivity in the aqueous phase. Adsorption Isotherms. DNA molecules were heated at 65 “C to disrupt any aggregates present in the stock solution and diluted into an aqueous salt solution (pH = 6.6). Negatively charged latex particles were added, and the mixture was incubated for 1 h, after which time the latex particles were separated from solution by filtration through sterile 0.2pm pore size Millex GV disposable filters (Millipore, Bedford, MA). Control tubes containing DNA and no latex were treated in an identical manner. The fluid concentration of DNA, before and after adsorption, was determined by measuring the absorbance of the samples at 2 = 260 nm (Spectronic 601, Milton Roy, Rochester, NY). Extinction coefficients were provided by the DNA supplier (Oligo Etc., Portland, OR). The amount of DNA adsorbed (r)to the particles was determined as
r=
(DNA, - DNAf)V A
(2)
where DNAi and DNAf are the solution concentrations of DNA before and after exposure to latex, respectively, Vis the solution volume, and A is the total particle surface area. Desorption Experiments. DNA molecules were radiolabeled on the Fend by the enzymatic reaction described previously. Molecules exactly 40 nucleotides in length were isolated by electrophoresis on a denaturing polyacrylamide gel (20%acrylamide, 50%urea), excised from the gel, eluted by the “crush and s o a k m e t h ~ dand , ~ ethanol precipitated to remove any residual elution buffer. DNAmolecules were resuspended in 0.05 M NaCl solution, negatively charged latex particles were added, and the suspension was allowed to incubate for 1h. After 1h, the particles were pelleted by centrifugation at 7000 rpm for approximately 15 min, and the supernatant was removed. The amount of DNA in the supernatant was determined by measuring the radioactivity using a radioisotope counter (QC2000, Bioscan, Washington, DC). The particles were then resuspended in 0.05 M NaCl solution by gentle aggitation and centrifuged again. This process was repeated to determine the extent ofDNA desorption. Hydroxyl Radical Footprinting (HRF). The dT40B molecules were radiolabeled on the 5’ end, isolated by PAGE, and ethanol precipitated, as described above. Purified and radiolabeled dT40B was adsorbed to either positively or negatively charged latex particles in 10 p L of “cutting buffer” (HzO, 50 mM NaC1, pH = 6.6). The DNA was allowed to adsorb for 1 h, after which time the particles were pelleted by centrifugation at 7000 rpm and resuspended in 10 p L of DNA-free cutting buffer by gentle agitation. Pelleting was repeated until the supernatant recovered contained less than 2%of the radioactivity present on the particles. All of our HRF experiments were carried out a t polymer surface densities well below the theoretical surface density corresponding to monolayer coverage (approximately 150 ng/cm2). To initiate the generation of hydroxyl radicals, 5 p L each ofFe[EDTA12-(0.5 mM), HzOz (0.18%), and sodium ascorbate (50 mM) were added t o the side of an eppendorf tube and mixed. ThreepL ofthis solution was added to the DNA samples for final solution concentrations of Fe[EDTA12-, HzOz, and sodium ascorbate of 0.05 mM, 0.018%, and 5 mM, respectively. The cutting reaction was carried out for 5-7 min and then quenched by adding 4 pL of thiourea. Two hundred p L of elution buffer (0.1% SDS, 0.5 M ammonium acetate, 10mM magnesium acetate) was added to desorb the DNA from the particles. In preliminary experiments we found that the elution buffer recovers greater than 99% ofthe adsorbed fragments, and therefore the cutting patterns observed in the gels do not reflect fractionation ofpolymer fragments during the elution step. After centrifugation, the DNA was vacuum concentrated, resuspended in formamide loading buffer (80% formamide, 0.025%xylene cyanol, 0.025%bromophenol blue), and analyzed by PAGE. The same amount of radioactive DNA, as determined by a direct p radiation counter (QCZOOO, Bioscan, Washington, DC), was loaded into each lane of the gel. Elec-
trophoresis was carried out for 1.5 h using a sequencing gel apparatus (Gibco BRL, Gaithersburg, MD) run a t 50 W. The resulting gels were transferred onto Whatman filter paper, dried, and exposed to preflashed X-ray film with an intensifying screen at -70 “C, typically for 1-3 days. The length of exposure was controlled to produce a linear response in optical density. In the HRF experiments, hydroxyl radicals were generated by the Fenton reaction. The Fenton reaction involves a one-electron reduction of hydrogen peroxide by iron(I1) with the concomitant formation of hydroxyl radicals:
+
Fe2+ H,O,
-
+
Fe3+ OH-
+ OH’
The iron (11) in these experiments is added as an Fe[EDTA12complex, consistent with other applications of HRF.6 Analysis of HRF Cutting Patterns. The band densities in the autoradiograms were quantified by two-dimensional densitometry using a Biorad Model GS-670 imaging densitometer (Biorad, Hercules, CAI. Methods for analyzingfootprintingdata are well developed in the case of DNA-protein interaction^.^ Proteins bind to specific locations along the DNA backbone, and therefore, the extent of binding is measured by comparing the intensity of bands a t binding sites relative to the intensity of bands within the same lane where no binding occurs. The fractional protection is then linearly related to the fractional saturation of DNA binding sites. In the experiments described later, however, the entire dT40B molecule is at least partially protected from strand scission. Therefore, the analysis used for DNA-protein binding could not be directly applied to our system. Instead, we determined the relative degree of protection by comparing the cutting frequency of DNA adsorbed to latex particles (protection experiment)to the cutting frequency of DNA in solution (control experiment). Apart from the presence or absence oflatex particles in these two parallel experiments, they were conducted in an identical manner. The relative cutting frequency (RCF) for a particular block of nucleotides is then defined by RCF = alb, where a and b represent the band intensities corresponding to the protection and control experiments, respectively, integrated over the block in question. Defining the RCF in this manner requires that the same amount of total DNA be present in each set of control and protection lanes. In our experiments, we were careful to load the same amount of radioactive DNA (within f10%) into each lane of the gel. Assuming a f 1 0 8 loading error, the maximum error in the computed RCF would be &22%. By comparison, the differences in the RCF values between dT40B adsorbed to positively and negatively charged latex are between 250-5008 (see the Results).
Results Model DNAMolecules. When a single-stranded DNA molecule is suspended in w a t e r , ionization of sites on the linkages and bases results in a charge that is pH dependent. In this study, phosphodiester and ethylphosp h o n a t e linkages (class I and I1 in Figure 1,respectively) are utilized. Phosphodiester linkages ionize at pH > 1, resulting in one negative charge per nucleotide. Ethylphosphonates, o n the o t h e r hand, are uncharged over all pH values of experimental interest. Ionization equilibria for the four bases (adenine, cytosine, guanine, and thymine) h a v e been determined experimentally.* F r o m these acidity constants it c a n b e deduced that for pH values ranging between 3 a n d 10 the electrostatic characteristics of a DNAmolecule are derived from two sources: ionization of the phosphodiester linkage (if the DNA is class I) resulting in 1negative charge per nucleotide and ionization of the b a s e resulting in a charge that is “base dependent” (i.e., whether the b a s e is A, C, G , o r T). At neutral pH, all four bases are uncharged, implying that the charge (6) Tullius, T. D. Nucl. Acids Mol. Biol. 1989,3, 1. (7) Brenowitz, M.; Senear, D.; Jamison, E.; Dalma-Weishausz,D. In Footprinting of Nucleic Acid-Protein Complexes; Revzin, A,, Ed.; Academic Press: San Diego, 1993. (8) Lide, D. R. CRC Handbook of Chemistry and Physics, 73rd ed.; CRC Press: London, 1993.
Langmuir, .Vol. 11, No. 10, 1995 3775
Conformation of DNA Block Copolymer Molecules 1
11-11
I 1 1 1 1 1 1 1 1
I
IIIIIIII
1 1 1 1 1 1 1 1 1
I
A
n
OH. (+)Latex (-) Latex
4
- ++++ - - +-+ - ----
-
++++
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6
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301 20
1o
-~
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IO”
loo
[NaCl], M Figure 4. Maximum amount of dT40A (A), dT40B (01,and dT40C (0) adsorbed to negatively charged latex particles at different NaCl concentrations.
characteristics of single-stranded DNA under these conditions is dictated solely by the type of linkage employed. In this study we utilize three model DNA molecules with the chemical and electrical architectures shown in Figure 3. They are 40 nucleotides long and were synthesized with the base thymine a t every nucleotide position. The molecules differ in the nature of their internucleotide linkages. One molecule (dT40A) was synthesized using only phosphodiester linkages, the negatively charged linkages present in natural DNA. The other two molecules have ethylphosphonate linkages in which the negative charge on the phosphorus has been “turned off and replaced with a hydrophobic ethyl group. These linkages are arranged in dT40B so that the molecule has a diblock copolymer architecture with the phosphodiester and ethylphosphonate linkages forming blocks on the 5’ and 3’ ends of the molecule, respectively. Ethylphosphonates are the primary linkage type present in dT40C, and therefore, this molecule should behave like a mostly uncharged polymer. DNA Hydrophobicity and Adsorption Characteristics. The octanol-water partition coefficients (&,) of dT40A, dT40B, and dT40C are 1.1x 2.3 x and 6.9 x lop4,respectively. Molecules with a larger fraction of ethylphosphonatelinkages are clearly more hydrophobic (higher KW). Figure 4 showsthe maximum amount of dT40A, dT40B, and dT40C adsorbed to negatively charged latex particles at different NaCl concentrations. dT40C, which contains only two negatively charged phosphodiester linkages, adsorbs strongly a t all NaCl concentrations tested. The amount of adsorbed dT40A and dT40B increases sharply with increasing NaCl concentration. For salt concentrations between 0.05 and 0.1 M NaC1, approximately two to six times more dT40B adsorbs to negatively charged latex than dT40A. On the basis of these adsorption data, we conclude that regions of the DNA containing negatively charged phosphodiester linkages have less affinity for the negatively charged latex than regions containing the uncharged and relatively more hydrophobic ethylphosphonate linkages. Hence, one can conjecture that dT40B might form a polymer brush when adsorbed to negatively charged latex in a 0.05 M NaCl solution, with the ethylphosphonate linkages (or A block) anchoring the molecule to the surface and the phosphodiester linkages (or B block) forming a negatively charged brush layer. Hydroxyl Radical Footprinting. To investigate this hypothesis we used hydroxyl radical footprinting to probe the conformation of dT40B adsorbed to both positively
6
2 3 4 5
5‘
1
0.6
0.2 n Low
High
High
Low ~~
(+) Latex
(-) Latex
Figure 5. (A) Autoradiograms for two HRF experiments involving dT40B adsorbed either to positively charged latex particles (lanes 1-5) or to negatively charged latex particles (lanes 6- 10). Experiments were conducted a t both “low”(lanes 3 and 8) and “high” polymer surface density (lanes 5 and 101, corresponding to fractional saturations of approximately 0.1 and 1.0, respectively. Lanes 2, 4, 7, and 9 are control experiments where hydroxyl radicals were reacted with surfacefree suspensions of dT4OB. (B) The relative cutting frequency (RCF) of the A and B blocks of dT4OB adsorbed to positively (+) and negatively (-) charged latex.
and negatively charged latex particles. The two gels in Figure 5A show the results of these experiments. Lanes 1and 6 of the gels show dT40B after purification by PAGE. In these two lanes all of the DNA is contained in one distinct band a t the top of the gel, indicating that dT40B was 40 nucleotides long and highly monodisperseprior to exposure to hydroxyl radicals. Lanes 2 , 4 , 7 , and 9 show unadsorbed dT40B after exposure to hydroxyl radicals. Exposing dT40B to hydroxyl radicals generates DNA fragments ranging in size from 1-40 nucleotides, although most of the DNA (>70%) is still uncut. Poisson “single hit” statistics can be used to demonstrate that virtually no DNA molecules are cut more than once, provided that most of the DNA (>70%) is not cut a t all.9 Because the process of cutting an adsorbed DNA molecule might relax its conformation on the surface, it is critical that HRF experiments are carried out in the “single hit” regime, as was done here. Lanes 3 and 5 in Figure 5A represent a set of HRF experiments in which dT40B was adsorbed to positively charged latex particles a t 3 ng/cm2(lane 3) and 30 ng/cm2 (lane 5 ) polymer surface density. At low coverage (3 ng/ cm2), the amount of dT40B adsorbed is well below saturation (roughly 1order of magnitude), while a t high coverage (30 ng/cm2)the surface is saturated with adsorbed polymer. The corresponding control experimentsin which (9) Brenowitz, M.; Senear, D. F.; Shea, M. A.; Ackers, G. K. Methods Enzymol. 1986, 130,132.
3776 Langmuir, Vol. 11, No. 10, 1995 hydroxyl radicals were reacted with a surface-free suspension of dT40B molecules are shown in lanes 2 and 4, respectively. When dT40B is adsorbed to positively charged latex, the molecule is almost completely protected from hydroxyl radical attack a t both polymer surface coverages investigated. Lanes 8 and 10 represent a comparable set of experiments in which dT40B was adsorbed to negatively charged latex a t 1ng/cmz (lane 8 ) and 11ng/cmz (lane 10)polymer surface density. Again, a t low coverage (1ng/cm2)the amount of dT40B adsorbed is well below saturation while a t high coverage (11ng/ cm2)the surface is saturated with adsorbed polymer. Lanes 7 and 9 are the corresponding control experiments. When dT40B is adsorbed to negatively charged latex the amount of hydroxyl radical cleavage increases, especially a t the higher polymer surface density. To obtain more detailed information about the susceptibility of dT40B to hydroxyl radical attack, the bands in Figure 5A were quantified using two-dimensional densitometry. In each lane of the gel, the optical density of a band represents the cutting frequency a t that position of the molecule.6 For the two gels shown in Figure 5A, there is relatively little difference in cutting from band to band within a given lane. Therefore, instead of examining the cutting frequencies of individual bands, we computed the average cutting frequency for entire blocks of dT40B. For a particular surface condition the relative cutting frequency (RCF) for a block is defined as the sum of the optical density of all the bands in the block divided by the sum of the optical density of an equivalent block in solution. Figure 5B shows calculated RCF values for the A and B blocks of dT40B when adsorbed to either positively or negatively charged latex. When the bare particles are positively charged the RCF is less than 0.1 for both blocks a t both polymer surface densities tested. Put simply, all segments of the adsorbed DNA molecule are cut less than 1/10 as frequently as equivalent segments in solution under these conditions. When adsorbed to negatively charged sulfate latex, the RCF of both blocks is approximately 0.25 a t low coverage and approaches 0.5 a t high
