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Langmuir 2002, 18, 8616-8619
On the Mechanism of Electrophoretic Migration of DNA in Pluronic Gels Roine Svingen,† Paschalis Alexandridis,‡ and Bjo¨rn Åkerman*,† Department of Chemistry and Bioscience, Chalmers University of Technology, Gothenburg, Sweden, Department of Chemical Engineering, University of Buffalo, The State University of New York, Buffalo, New York 14260, and Physical Chemistry 1, Lund University, Lund S22100, Sweden Received April 15, 2002. In Final Form: July 30, 2002
Capillary electrophoresis of nucleic acids has recently employed gels of self-assembled uncharged triblock copolymers as sieving media. Pluronic F127 contains poly(ethylene oxide) (EO) and poly(propylene oxide) (PO) with the block structure (EO)106(PO)70(EO)106. Aqueous solutions of 30% w/w of this polymer are liquids at low temperatures, but above 11 °C the polymers assemble to micelles that pack into a locally cubic lattice forming a gel-like lyotropic liquid crystal phase. Here we use linear dichroism spectroscopy to study the orientation dynamics of double-stranded DNA molecules during the electrophoresis. In 30% Pluronic F127, a 5400 bp DNA migrates with substantial perpendicular orientation of the helix axis, which is in contrast to electrophoresis in agarose gels where the helix axis of DNA is aligned parallel to the field direction. Comparison between linear and circular DNA indicates that neither DNA form enters the cubic microcrystals at low fields, and when combined with velocity measurements the kinetics of alignment buildup and relaxation suggests that migration instead occurs in grain boundaries between domains of microcrystals.
1. Introduction Gel electrophoresis is an important method to separate biomolecules, such as DNA, according to charge, size, and conformation and has played an important role as an analytical tool in for instance genetic research for a long time. More recently, development has focused on increasing separation efficiency by use of capillary electrophoresis (CE) and on preparative applications by exploring novel gels, which allow simple sample recovery.1 Capillary electrophoresis has improved separation efficiency remarkably with regard to running time by use of high field strength. However, for effective separation of different sizes of DNA a sieving medium is needed. Polymer solutions often act as sieving media, and a drawback is laborious procedures for replacing the viscous polymer solution in the capillary. Pluronic F127 is an amphiphilic triblock copolymer2 with a center hydrophobic poly(propylene oxide) block flanked by hydrophilic poly(ethylene oxide) blocks and the molecular formula (EO)106(PO)70(EO)106. Below 11 °C, a 30% F127 aqueous solution is a liquid,3 but when heated the polymer becomes more hydrophobic due to conformation changes and micelles pack into a face-centered cubic (fcc) lattice4 creating a gel-like state. The capillary can be easily filled and emptied since gel formation is reversible.2 This property also makes pluronic gels promising candidates for preparative applications since the separated components can be recovered in solution by just decreasing the temperature. * Corresponding author. † Chalmers University of Technology. ‡ The State University of New York and Lund University. (1) Cole, K. D. Biotechniques 1999, 26 (4), 748-756. (2) Alexandridis, P.; Hatton, T. A. Colloids Surf., A 1995, 96, 1-46. Alexandridis, P. Curr. Opin. Colloid Interface Sci. 1997, 2, 478-479. Ivanova, R.; Lindman, B.; Alexandridis, P. J. Colloid Interface Sci. 2002, 252, 226-235. (3) Wu, C.; Liu, T.; Chu, B. Electrophoresis 1998, 19, 231-241. (4) Wu, C.; Liu, T.; Chu B. J. Non-Cryst. Solids 1998, 253-237, 605611.
Pluronic gels have a local pore structure that is welldefined compared to that of conventional gels. The micelle has a hydrophobic core of polypropylene oxide chains approximately 9 nm in diameter, and the polyethylene oxide chains extend in a corona to give a total micelle diameter of 18 nm.5 When the micelles pack in a cubic lattice, the interstitial spaces form aqueous pores about 0.5-2 nm in diameter6 in a 20% solution of Pluronic F127. On geometrical grounds, it is thus possible for doublestranded (ds) DNA to migrate end-on (reptate) through these pores, since the helix diameter is 2 nm. Pluronic F127 gels indeed function as an effective separation medium for linear ds DNA shorter than 800 bp,5 but the reptation model fails to describe the molecular-weight dependence of the velocity, which suggests that the mechanism of ds DNA migration in Pluronic F127 is different from that of migration in conventional gels such as agarose. Here we use a spectroscopic approach to monitor perturbation of DNA coils during migration, which has been important for understanding the mode of DNA migration in conventional gels. 2. Method 2.1. Materials. Double-stranded relaxed circular DNA φX174, 5386 bp, was linearized with restriction enzyme Eco1471 (both from MBI Fermentas). Pluronic F127 was obtained as a dry powder from BASF. The electrophoresis buffer was TBE (50 mM in boric acid and 50 mM in Tris, 1,25 mM in EDTA in deionized water, pH ) 8.2). This buffer has negligible effects on Pluronic F127 gel properties4 compared to those in water. The Pluronic F127 powder was mixed with TBE buffer by stirring at 4 °C, and the solutions were stored at 5 °C for at least 4 days before use. Agarose D-1 Low EEO was from Pronadisa. 2.2. Electrophoresis. The electrophoresis experiments in F127 were performed on the relaxed circular and linear DNA forms according to procedures described for agarose in detail in (5) Rill, R. L.; Locke, B. R.; Liu, Y.; Van Winkle, D. H. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 1534-1539. (6) Rill, R. L.; Liu, Y.; Ramey, B. A.; Van Winkle, D. H.; Locke, B. R. Chromatographia Supplement I 1999, 49, 65-71.
10.1021/la025835x CCC: $22.00 © 2002 American Chemical Society Published on Web 10/02/2002
Electrophoretic Migration of DNA in Pluronic Gels ref 7. A birefringence-free silica cuvette with internal dimensions of 3 × 30 × 100 mm3 was mounted vertically between the electrophoresis buffer chambers in contact at the top and the bottom of the cuvette, in which an electric field was generated using platinum electrodes. To prevent leaking of the Pluronic F127 solution before gelation, a 30 mm high 1% agarose plug was formed at the bottom of the cuvette. A 5 °C solution of 30% Pluronic F127 in TBE buffer was then poured into the cell, which was subsequently kept on ice for 2 h. The device was then put in a 45 °C water bath for 15 min, hence creating a gel. A sample well was created by forming a cavity in the top of the gel. The loaded sample volume was 20 µL containing 1 µg of DNA and 3% Ficoll in TBE buffer. After loading, electrophoresis was performed at 7.5 V/cm for 4 h. Reference experiments were performed in 1% agarose, in the same electrophoresis buffer. 2.3. Spectroscopy. The vertical electrophoresis cell was used for both DNA orientation and velocity measurements, using an elevator device to position the gel section containing the DNA zone in the horizontal beam. Orientation was monitored using a Jasco J-500 spectropolarimeter, with an achromatic quarterwave device that converts the incoming light beam from circular polarization into linear.7 Linear dichroism (LD) is an efficient technique7 to study coil deformation of DNA undergoing electrophoresis. LD is the difference in absorption of light polarized parallel (A|) and perpendicular (A⊥) to the direction of the electric field:
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Figure 1. Orientation response of linear φX174 DNA to an applied electric field in Pluronic F127 and agarose gels, given as LDr (see text). Two top curves, 30% Pluronic F127 gel: thin black line, 3.75 V/cm; thick gray line, 7.5 V/cm. Bottom curve, 1% agarose: thick black line, 7.5 V/cm. DNA concentration: 50 µM phosphate. Arrow down, field applied; double arrow, field reversed 180°; arrow up, field off.
LD ) A| - A⊥ To quantify the orientation of a macroscopically aligned sample, the reduced linear dichroism (LDr) is calculated:
LDr ) LD/Aiso where Aiso is absorbance of the isotropic sample. LDr is related to the angle R between the transition moment of and the orientation axis of the molecule and to an orientation factor S that describes the average orientation of this axis relative to the electrophoresis direction as
LDr ) 3S(3 〈cos2 R〉 - 1)/2 S ) (3 〈cos2 θ〉 - 1)/2 where θ is the angle between the helix axis and the electrophoresis direction. For B-form DNA, an average value of 86° was used for R.8,9 The orientation factor varies between 0 for an isotropic coil to +1 (LDr ) -3/2) for perfect parallel alignment of the helix axis along the field and -1/2 (LDr ) +3/4) for perfect perpendicular helix axis alignment. DNA velocity (v) at different field strengths was obtained by a new approach, which allows in situ measurements in the same cuvette as for the LD experiments, using a Cary 219 UV-vis spectrophotometer. The absorption gradient (dA/dx) on one edge of the DNA band was obtained at 260 nm by scanning the cuvette through the light beam at a known velocity. Then, after the beam had been positioned at a fixed position on the gradient the DNA band was moved by electrophoresis through the beam to produce an absorption time-profile slope (dA/dt). The DNA migration velocity could then be calculated as
v ) (dA/dt)/(dA/dx) ) dx/dt using a portion of the zone where both slopes were constant.
3. Results 3.1. Linear Dichroism Measurements. Figure 1 compares the LDr time profile (260 nm) of linear φX174 DNA in 30% Pluronic F127 and in 1% agarose at two (7) Jonsson, M.; Åkerman, B.; Norde´n, B. Biopolymers 1988, 27, 381414. (8) Norde´n, B. Appl. Spectrosc. Rev. 1978, 14 (2), 157-248. (9) Åkerman, B.; Jonsson, M. J. Phys. Chem. 1990, 94 (9), 38283838.
Figure 2. Orientation response (LDr) of circular φX174 DNA to an applied electric field in 30% Pluronic F127 gel. Thin black line, 3.75 V/cm; thick gray line, 7.5 V/cm. DNA concentration: 50 µM. Arrows are as in Figure 1.
different field strengths applied at t ) 0. To avoid net migration of the zone out of the beam, the LD measurements were carried out by first applying the field for 60 s and then reversing it for another 60 s. In F127, the LDr response of linear φX174 is seen to be positive. This in contrast to the negative LDr response in 1% agarose, which reflects a parallel field alignment of the helix axis that is known to occur during migration in this gel.7 In addition to the sign, the kinetics of the LD provides important information on the mode of migration. In F127, a steady-state LD alignment is reached within a few tens of seconds after field application. As the field is turned off, the LD relaxation is much slower, and the initial LD value is not reached until after more than 25 min (data not shown). Both observations are in contrast to data for agarose where both buildup and relaxation occur on the subsecond time scale.10 The LD response from circular φX174 DNA (Figure 2) displays a more complex behavior. There is a positive LD as for linear DNA, but at the higher field there is also a negative LD contribution. The two components can be resolved due to their different rates of relaxation. As the field is turned off, there is a fast relaxation of the negative LD followed by a slow relaxation of the positive contribution, as for the linear form. We characterize the buildup kinetics of the positive LD by the time constant, τss, to reach a steady-state LD (Table 1). (10) Magnu´sdo´ttir, S.; Åkerman, B.; Jonsson, M. J. Phys. Chem. 1994, 98 (10), 2624-2633.
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Table 1. Steady-State Orientation Parameters for OX174 DNA in 30% Pluronic F127 Gel DNA form
field (V/cm)
τss (s)a
Lτ (µm)b
linear linear circular circular
3.75 7.5 3.75 7.5
38.0 29.7 59.8 35.3
3.0 7.4 2.7 1.9
a Time to reach steady perpendicular alignment. b Migrated distance obtained as Lτ ) τssv, where velocity, v, is taken from Figure 3.
Figure 3. Electrophoretic velocity vs field strength of linear (triangles) and circular (circles) φX174 DNA in 30% Pluronic F127 gel and of linear φX174 DNA in 1% agarose (squares). Notice the scale factor of 1/5 for velocity in agarose.
3.2. Velocity Measurements. In agreement with earlier observations,5 the DNA velocity is greatly reduced in 30% Pluronic F127 compared to 1% agarose (Figure 3). In 30% Pluronic F127, both linear and circular φX174 exhibit two regimes of linear dependence on field strength (E), with a break point at approximately 11 ( 1 V/cm. This in contrast to the gradual increase in velocity in 1% agarose (Figure 3), where below 10 V/cm the increase is approximately linear in the field strength, corresponding to an almost constant electrophoretic mobility, µ ) v/E, in agreement with earlier observations for DNA of similar size.11 At higher fields, the distinct upward curvature shows that the mobility increases with increasing field. In the lower field regime studied by Hervet et al.,11 a similar mobility increase was observed for longer DNA. This behavior in agarose is mostly due to a reduced friction because of the parallel coil alignment in this gel, which is stronger the higher the field and the longer the DNA.7 By use of the velocity data, the τss values can be converted into the corresponding migrated distance, which is required to reach steady-state LD (Table 1). 4. Discussion 4.1. B-Form DNA. The positive LD response observed in the Pluronic gel for linear φX174 DNA is surprising since a negative LD is expected if DNA is aligned along the field, as is indeed observed in both agarose (Figure 1) and polyacrylamide gels.7 The positive LD therefore suggests a mode of migration with the DNA helix axis preferentially oriented perpendicular to the field direction, (11) Hervet et al. Biopolymers 1989, 26, 727-742.
but before discussing the implications some alternative explanations must be ruled out. The negative LD sign in agarose is because the DNA bases are perpendicular to the helix axis8 in the B-form, which DNA is known to attain in this gel.9 One possible explanation for the positive LD thus is that the DNA secondary structure in the Pluronic gel is different such that the bases are strongly tilted, resulting in a positive LD if the helix axis is fieldaligned also in this gel (such DNA forms occur in some nonaqueous solutions12). However, circular dichroism spectra strongly indicate that the DNA is in the B-form also in the Pluronic F127 gel (not shown). Second, a positive LD could arise from the gel itself since gel contributions to the LD at 260 nm with opposite sign to that of DNA do occur in agarose gels7 as the gel becomes deformed during the electrophoresis. This possibility could be ruled out by LD measured at 320 nm (not shown) where DNA does not absorb but the gel still does. There was no detectable LD at this wavelength under the same conditions that produce positive LD at 260 nm. We conclude that the LD signal of Figure 1 reflects the helix alignment of DNA in its B-form and thus that the positive LD reveals a preferential perpendicular helix alignment. The degree of alignment is weak, but the unexpected preferred direction of alignment points to an unusual mode of migration. An important observation in the velocity measurements (Figure 3) is the break point at 11 ( 1 V/cm, where the higher slope (electrophoretic mobility) at high fields indicates that the friction is reduced compared to migration in weak fields. The two-regime behavior indicates a distinct change in the nature of the friction at the break point, possibly due to a migration-induced perturbation of the gel structure. By contrast, the gradual nonlinear increase in agarose is consistent with a gradual coil deformation in an essentially rigid network. Here we concentrate on the behavior at fields below 11 ( 1 V/cm and base the LD analysis on the assumption that the cubic structure is unperturbed under these conditions. 4.2. Confinement. The perpendicular helix orientation in 30% F127 shows that the mode of DNA migration is different compared to that of agarose. The parallel field alignment of the helix observed during DNA migration in agarose is due to a field-induced perturbation of the DNA coils. Such deformations occur if the DNA is confined in a gel with a pore size that is comparable to or smaller than the coil size.9 The average pore radius in 1% agarose of about 250 nm13,14 is in fact somewhat larger than the radius of gyration, Rg, of linear φX174 (170 nm). This explains why coil deformation (LDr) is weak (Figure 1) but the confinement still is significant, evidenced by the fact that field-free LD relaxation15 and the electrophoretic migration16 are slow compared to those in unconfined (free) solution. The even slower migration (Figure 3) and orientation buildup and relaxation in Pluronic F127 (Figure 1) show that the DNA is migrating in an even more confining structure than that of agarose. This is consistent with the present picture of the cubic phase that F127 forms under our conditions,4 where the aqueous channels between packed micelles are only a few nanometers in diameter and thus much smaller than the radius of gyration (Rg) of the DNA. Interestingly, migration of DNA end-on through the channels is still possible since (12) Norde´n, B.; Seth, S.; Tjerneld, F. Biopolymers 1978, 17, 523525. (13) Pernodet, N.; Maaloum, M.; Tinland, B. Electrophoresis 1997, 18, 55-58. (14) Maaloum, M.; Pernodet, N.; Tinland, B. Electrophoresis 1998, 19, 1606-1610. (15) Stellwagen, N. C. J. Biomol. Struct. Dyn. 1985, 3 (2), 299-314. (16) Åkerman, B. Electrophoresis 1996, 17, 1027-1036.
Electrophoretic Migration of DNA in Pluronic Gels
the helix diameter is only 2 nm, but clearly the positive LD in Figure 1 contradicts that migration occurs end-on, because a parallel helix alignment would be expected for such strongly confined DNA.7 The hypothesis that DNA enters the cubic phase was therefore tested by using circular φX174 DNA with the Rg of 119 nm, comparable to the linear form and again orders of magnitudes larger than the pore dimensions in the cubic phase. The circular DNA lacks free ends and thus always has to form a loop between obstacles in order to migrate through a gel. The energy penalty ∆G for a semicircular DNA loop of radius Rp is given by ∆G ) (kTP)/R,17 where P is the persistence length. The circular form is therefore not expected to enter the cubic phase because the persistence length is much longer (P ) 50 nm) than the channel width (Rp ) 2 nm), giving a barrier for penetration of about 25 kT. The circular DNA exhibits a more complex LD behavior than the linear form (Figure 2), but this is not unique to F127 gels. Also in polyacrylamide gels do circles exhibit both positive and negative LD contributions due to the presence of two coil deformation mechanisms.18 Impalement of circles on open-ended gel fibers results in negative LD (parallel alignment). The positive LD component (perpendicular alignment) has been ascribed to circles being pushed against dense regions of the gel which are known to have pore sizes of a few nanometers and are thus also too small to allow circular DNA to enter. We are presently investigating the mechanisms behind the fast negative component and here focus on the positive contribution, which dominates the LD behavior below the break point in velocity. The facts that the positive contribution exhibits the same slow buildup and relaxation that characterize the orientation response of the linear form and that both DNA forms exhibit a biphasic field dependency (Figure 3) in velocity strongly indicate that the linear and circular molecules migrate by the same mechanism at low fields. Given that circles cannot enter the pores of the cubic phase, this shared LD and velocity behavior indicates that the linear form does not enter the cubic phase either and thus that the perpendicular alignment arises in another type of confined geometry in the Pluronic gel. This hypothesis is supported by calculations of the average distance, Lτ ) τssv, which is obtained by combining data of buildup time (Table 1) and velocity (Figure 3) in F127. The parameter Lτ is the average distance the molecules have covered when they reach steady deformation, and it has been useful in understanding DNA migration in other gel systems. For linear DNA in agarose, Lτ is approximately 25% of the contour length (Lc) because this is the distance the center of mass has moved when the DNA chain has formed a maximally field-aligned U-conformation hooked on a gel fiber.16 For circles in (17) Cohen, G.; Eisenberg, H. Biopolymers 1966, 4, 429-440. (18) Åkerman, B. J. Phys. Chem. 1998, 102, 8909-8922.
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agarose, on the other hand, Lτ is not related to DNA size but is much longer, about 100 µm.19 In this case, alignment is due to the trapping of DNA in dangling gel fibers, and Lτ reflects the distance between such obstacles, a gel property. The calculated Lτ values in Pluronic F127 (Table 1) are a few micrometers for both linear and circular DNA. For φX174, Lc/4 is 0.4 µm so the Lc values are not compatible with a U-formation mechanism, which is also contradicted by the positive LD. In fact, Lτ is larger than any DNA length scale, such as Rg ) 120 nm (circular φX174), so it is more likely that Lτ is determined by a gel property, as is the case for circles in agarose. Because Lτ is much longer than the nanometer length scale of the micelles and channels between them in the cubic phase, the responsible gel obstacles involved must correspond to a much longer length scale. Pluronic gels are known to be polycrystalline when prepared by simple heating of a fluid solution,20 as used here. The sizes of the crystalline domains are only known to be much larger than the micellar size, so we cannot judge if the micrometer length scale we observe is related to them, but one possibility is that the DNA chains migrate in the “grain boundaries” between such domains. Interestingly, those defects are involved in shear deformation of the cubic phase at low shear rate, where the fcc structure is locally preserved in the domains,21 as assumed here at low fields. A possible mechanism behind the perpendicular helix orientation is then that DNA coils are flattened out against cubic-phase domains, which blocks a downfield path, and that τss (and Lτ) is determined by the average domain size which has to be covered until the next blocking domain is encountered. The low velocity would then be due to these strong obstructions, and the strongly hindered rotational motion we observe is still expected if the space available to the DNA in the grain boundaries is small compared to the Rg of the DNA. Our results on DNA conformation dynamics show that the mode of DNA migration in Pluronic gels is different in nature from that in conventional gels, in agreement with proposals based on separation data.5 The similarity in the behavior for linear and circular DNA indicates that DNA molecules of the plasmid sizes and field strengths (below 11 ( 1 V/cm) studied here do not migrate in the cubic monocrystal domains, but possibly in grain boundaries. Acknowledgment. Financial support from the Swedish Natural and Technical Research Councils is thankfully acknowledged. LA025835X (19) Cole, K. D.; Åkerman, B. Biomacromolecules 2000, 1, 771-781. (20) Wanka, G.; Hoffmann, H.; Ulbricht, W. Colloid Polym. Sci. 1990, 268, 101-117. (21) Molino, F. R.; Berret, J.-F.; Porte, G.; Diat, O.; Lindner, P. Eur. Phys. J. B 1998, 3, 59-72.
