Electrokinetic stretching of tethered DNA

During electrophoretic separations of DNA in a sieving medium, DNA molecules stretch from a compact coil into elongated conformations when encountering an obstacle and relax back to a coil upon release from the obstacle. These stretching dynamics are thought to play an important role in the separation mechanism. In this article we describe a silicon microfabricated device to measure the stretching of tethered DNA in electric fields. Upon application of an electric field, electro-osmosis generates bulk fluid flow in the device, and a protocol for eliminating this flow by attaching a polymer brush to all silicon oxide surfaces is shown to be effective. Data on the steady stretching of DNA in constant electric fields is presented. The data corroborate the approximate theory of hydrodynamic equivalence, indicating that DNA is not free-draining in the presence of both electric and nonelectric forces. Finally, these data provide the first quantitative test of a Stigter and Bustamante's detailed theory of electrophoretic stretching of DNA without adjustable parameters. The agreement between theory and experiment is good.     

Free solution mobility of oligomeric DNA

The free solution electrophoretic mobility of a charged oligomer in an ionic solvent that approximately takes into account relaxation field effects, screening of the velocity field, and the hydrodynamic interactions resulting from motions of the charges due to an electric field is described. For double‐stranded DNA, the free solution electrophoretic mobility under ionic strengths determined by the buffer and pH conditions relevant to capillary electrophoresis increases with increasing molecular weight up to few hundred base pairs. © 1999 John Wiley & Sons, Inc. Biopoly 49: 209–214, 1999     

Orientation of DNA molecules in agarose gels by pulsed electric fields

The electric birefringence of DNA restriction fragments of three different sizes, 622, 1426, and 2936 base pairs, imbedded in agarose gels of different concentrations, was measured. The birefringence relaxation times observed in the gels are equal to the values observed in free solution, if the median pore diameter of the gel is larger than the effective hydrodynamic length of the DNA molecule in solution. However, if the median pore diameter is smaller than the apparent hydrodynamic length, the birefringence relaxation times increase markedly, becoming equal to the values expected for the birefringence relaxation of fully stretched DNA molecules. This apparent elongation indicates that end-on migration, or reptation is a likely mechanism for the electrophoresis of large DNA molecules in agarose gels. The relaxation times of the stretched DNA molecules scale with molecular weight (or contour length) as N2.8, in reasonable agreement with reptation theories.     

Theory for the hydrodynamic and electrophoretic stretch of tethered B-DNA.

We have developed a theory for the extension and force of B-DNA tethered at a fixed point in a uniform hydrodynamic flow or in a uniform applied electric field. The chain tethered in an electric field is considered to be subject to free electrophoresis compensated by free sedimentation in the opposite direction. This allows the use of results of free electrophoresis for including the effects of small ions. The force on the chain is derived for a sequence of ellipsoidal segments, each twice the persistence length of the wormlike chain. Hydrodynamic interaction between these segments is based on the long-range limit of flow around the prolate ellipsoids, as derived from equivalent Stokes spheres. The chain extension is derived by applying the entropic elasticity relation of Marko and Siggia (1995 Macromolecules. 28:8759-8770) to each segment for polymer chains under constant tension. We justify this procedure by comparing with extension results based on the Boltzmann averaged orientation of straight, freely jointed segments. Predicted results agree well with recent extension-flow experiments by Perkins et al., 1995. Science. 258:83-87, and with electrophoretic stretch experiments by Smith and Bendich (1990 Biopolymers. 29:1167-1173) on fluorescently stained B-DNA. We find that the equivalence of hydrodynamic and electrophoretic stretch, proposed by Long et al. (1996 Phys. Rev. Lett. 76:3858-3861; 1996 Biopolymers 39:755-759), is valid only for very small chain deformations, but not in general.     

Orientation of DNA Molecules in Agarose Gels By Pulsed Electric Fields

Abstract

The electric birefringence of DNA restriction fragments of three different sizes, 622,1426, and 2936 base pairs, imbedded in agarose gels of different concentrations, was measured. The birefringence relaxation times observed in the gels are equal to the values observed in free solution, if the median pore diameter of the gel is larger than the effective hydrodynamic length of the DNA molecule in solution. However, if the median pore diameter is smaller than the apparent hydrodynamic length, the birefringence relaxation times increase markedly, becoming equal to the values expected for the birefringence relaxation of fully stretched DNA molecules. This apparent elongation indicates that end-on migration, or reptation is a likely mechanism for the electrophoresis of large DNA molecules in agarose gels. The relaxation times of the stretched DNA molecules scale with molecular weight (or contour length) as N^2.8, in reasonable agreement with reptation theories.     

Relaxation effects in the gel electrophoresis of DNA in intermittent fields

The electrophoretic mobility of restriction fragments of lambda DNA in agarose gels declines if the field is intermittent rather than continuous, with a greater effect on the longer fragments. The changes are compatible with the assumption of two exponential relaxation processes for field-dependent configurational changes, one when the field is turned on and another when it terminates. The length dependence at the extrapolated limit of mobility for short pulses with long intervals corresponds closely to the simple inverse proportionality to length expected from theoretical considerations when the molecular configuration is not affected by the electric field. Simple intermittent fields would allow separation of longer molecules than can ordinarily be resolved. The relaxation times for both the change in conformation imposed by the field and the return to field-free conformation vary as approximately the second power of the length of the molecule, independent of the salt concentration or field strength and varying only slightly with gel density. These relations are not in good agreement with properties expected from reputation theory, and they suggest that a different mechanism must be invoked for the electrophoretic migration of long DNA molecules at ordinary values of field strength.     

High-resolution field-cycling NMR studies of a DNA octamer as a probe of phosphodiester dynamics and comparison with computer simulation

Phosphorus-spin longitudinal relaxation rates of the DNA duplex octamer [d(GGAATTCC)](2) have been measured from 0.1 to 17.6 T by means of conventional and new field-cycling NMR methods. The high-resolution field-cycling method is identical to a conventional relaxation experiment, except that after preparation the sample is moved pneumatically from its usual position at the center of the high-resolution magnet upward to a lower field above its normal position and then returned to the center for readout after it has relaxed for the programmed relaxation delay at the low field. This is the first measurement of all longitudinal relaxation rates R(1) of a nuclear species in a macromolecule over virtually the entire accessible magnetic field range. For detailed analysis, three magnetic field regions can be delineated: (i) dipolar relaxation dominates at fields below 2 T, (ii) chemical shift anisotropy (CSA) relaxation is roughly constant from 2 to 6 T, and (iii) a square-law increasing dependence is seen at fields higher than approximately 6 T due to internal motion CSA relaxation. The analysis provides a rotational correlation time (tau(r) = 4.1 +/- 0.3 ns) for the duplex at both 1.5 and 0.25 mM concentrations (of duplex) at 22 degrees C. For comparison, extraction of tau(r) in the conventional way from the ratio of T(1)/T(2) at 14 T yields 3.2 ns. The tau(r) discrepancy disappears when we exclude the contribution of internal motion from the R(1) in the ratio. The low-field dipolar relaxation provides a weighted inverse sixth power sum of the distances from the phosphorus to the protons responsible for relaxation. This average is similar for all phosphates in the octamer and similar to that in previous B-DNA structures (its inverse sixth root is about 2.40 A for two different concentrations of octamer). The CSA relaxation at intermediate field provides an estimate of the order parameter squared, S(c)(2), for each phosphorus. S(c)(2) is about 0.7-1, clearly different for different phosphate linkages in the octamer duplex. The increasing R(1) at high fields reflects CSA relaxation due to internal motions, for which a correlation time, tau(hf), can be approximately extracted with the aid of additional measurements at 14.0 and 17.6 T. We conclude that tau(hf) values are relatively large, in the range of about 150 ps. Insight into the motions leading to this correlation time was gained by a 28 ns molecular dynamics simulation of the molecule. S(2) and tau(s) (corresponding to tau(hf)) predicted by this simulation were in good agreement with the experimental values from the field-cycling data. Both the effect of Mg(2+) on the dynamic parameters extracted from (31)P relaxation rates and the field dependence of relaxation rates for several protons of the octamer were measured. High-resolution field cycling opens up the possibility of monitoring residue-specific dipolar interactions and dynamics for the phosphorus nuclei of diverse oligonucleotides.     

Nuclear magnetic resonance studies of amino acids and proteins. Rotational correlation times of proteins by deuterium nuclear magnetic resonance spectroscopy

We show that measurement of the spin-lattice (T1) and spin-spin (T2) relaxation times (or line widths) of irrotationally bound 2H nuclei in macromolecules undergoing isotropic rotational motion outside of the extreme narrowing limit (i.e., for the case omega 02 tau R2 much greater than 1) permits determination of both the rotational correlation time (tau R) of the macromolecule and the electric quadrupole coupling constant (e2qQ/h) of the 2H label. The technique has the advantage over 13C nuclear magnetic resonance (NMR) that no assumptions about bond lengths (which appear to the sixth power in 13C relaxation studies) or relaxation mechanisms need to be made, since relaxation will always be quadrupolar, even for aromatic residues at high field. Asymmetry parameter (eta) uncertainties are shown to cause negligible effects on tau R determinations, and in any case it is shown that both e2qQ/h and eta may readily be determined in separate solid-state experiments. By way of example, we report 2H NMR results on aqueous lysozyme (EC 3.2.1.17) at 5.2 and 8.5 T (corresponding to 2H-resonance frequencies of 34 and 55 MHz). Interpretation of the results in terms of the isotropic rigid-rotor model yields e2qQ/h values of approximately equal to 170 or approximately equal to 190 kHz, respectively, for the imidazolium and free-base forms of [epsilon 1-2H] His-15 lysozyme in solution, in excellent agreement with e2qQ/h values of approximately 167 and approximately 190 kHz obtained for the free amino acids in the solid state. In principle, the method may in suitable cases permit comparison between the dynamic structures of proteins in solution and in the crystalline solid state.     

Orientational dynamics of T2 DNA during agarose gel electrophoresis: influence of gel concentration and electric field strength

The understanding, on a molecular level, of the mechanisms responsible for the improved separation in DNA gel electrophoresis when using modulated electric fields requires detailed information about conformational distribution and dynamics in the DNA/gel system. The orientational order due to electrophoretic migration ("electrophoretic orientation") is an interesting piece of information in this context that can be obtained through linear dichroism spectroscopy [M. Jonsson, B. Akerman, and B. Nordén, (1988) Biopolymers 27, 381-414]. The technique permits measurement of the orientation factor S of DNA (S = 1 corresponds to perfect orientation) within an electrophoretic zone in the gel during the electrophoresis. It is reported that the degree of orientation of T2 DNA [170 kilo base pairs (kpb)] is considerable (S = 0.17 in 1% agarose at 10 V/cm) compared to relatively modest orientations of short fragments found earlier (for 23-kbp DNA, S = 0.03 in 1% agarose at 10 V/cm), showing that large DNA coils are substantially deformed during the migration. Growth and relaxation dynamics of the orientational order of the T2 DNA are also reported, as functions of gel concentration (0.3-2%), electric field strength (0-40 V/cm), and pulse characteristics. The rise profile of the DNA orientation, when applying a constant field, is a nonmonotonic function that displays a pronounced overshoot, followed by a minor undershoot, before it reaches steady-state orientation (after 12 s in 1% agarose, 9 V/cm). The orientational relaxation in absence of field shows a multiexponential decay in a time region of some 10 s, when most of the DNA anisotropy has disappeared. A surprising phenomenon is a memory over minutes of the DNA/gel system to previous pulses: with two consecutive rectangular pulses (of the same polarity), the orientational overshoot and undershoot as a response to the second pulse are significantly reduced compared to the first pulse. The time required to recover 90% of their amplitudes is typically 1200 s (1% agarose, 9 V/cm), which may be compared to the time required to relax 90% of the DNA orientation, which is only 6 s. The major part of the over- and undershoot recovery is thus a reorganization of a system in which DNA is already randomly oriented. The different response amplitudes and relaxation times, including the amplitude and recovery time of the overshoot, of the orientational order of DNA in the electrophoretic gel have been studied as functions of gel concentration and field strength. The results are discussed against relevant theories of polymer dynamics.     

Extensional properties of hydroxypropyl ether guar gum solutions

The extensional properties of 2-hydroxypropyl ether guar gum solutions were investigated using a capillary breakup extensional rheometer (CaBER). Optimization of the geometric parameters of this device allowed for the measurement of the characteristic relaxation times and the apparent extensional viscosities of a series of dilute to semidilute guar gum solutions. The measured relaxation times were compared with predicted Zimm relaxation times, assuming that the hydrophobically modified guar was in a good solvent. Good agreement was found at low concentrations (0.01 wt % approximately 0.17 c*, where c* is the polymer overlap concentration), and this technique allowed for relaxation times on the order of 1 ms to be measured for solutions with shear viscosities of 2 mPa.s. Both the shear and (apparent) steady-state extensional viscosities of this set of industrially relevant fluids exhibited two regions of dependency on polymer concentration: linear up to concentrations of 0.2 wt % ( c/ c* approximately 3) and power law thereafter, where interchain interactions became significant. The extracted relaxation times followed the same trend (i.e., having a near linear dependency on concentration up to 0.2 wt % and a power-law dependency on concentration up to 9 c*). The results indicate that the transition from dilute to semidilute behavior occurs at a nominal concentration of approximately 3 c* instead of c*. The results presented suggest that interchain interactions for this modified guar are weak overall, and the solutions investigated are absent of entanglements over the whole range of frequencies and concentrations explored ((0.17-9) c*).     

Conformational analysis of single DNA molecules undergoing entropically induced motion in nanochannels

We have used the interface between a nanochannel and a microchannel as a tool for applying controlled forces on a DNA molecule. A molecule, with a radius of gyration larger than the nanochannel width, that straddles such an interface is subject to an essentially constant entropic force, which can be balanced against other forces such as the electrophoretic force from an applied electric field. By controlling the applied field we can position the molecule as desired and observe the conformation of the molecule as it stretches, relaxes, and recoils from the nanochannel. We quantify and present models for the molecular motion in response to the entropic, electrophoretic, and frictional forces acting on it. By determining the magnitude of the drag coefficients for DNA molecules in the nanostructure, we are able to estimate the confinement-induced recoil force. Finally, we demonstrate that we can use a controlled applied field and the entropic interfacial forces to unfold molecules, which can then be manipulated and positioned in their simple extended morphology.     

Effect of pH on protein distribution in electrospun PVA/BSA composite nanofibers

We examine the protein distribution within an electrospun polymer nanofiber using polyvinyl alcohol and bovine serum albumin as a model system. We hypothesize that the location of the protein within the nanofiber can be controlled by carefully selecting the pH and the applied polarity of the electric field as the pH affects the net charge on the proteins. Using fluorescently labeled BSA and surface analysis, we observe that the distribution of BSA is affected by the pH of the electrospinning solution. Therefore, we further probe the relevant forces on the protein in solution during electrospinning. The role of hydrodynamic friction was assessed using glutaraldehyde and HCl as a tool to modify the viscosity of the solution during electrospinning. By varying the pH and the polarity of the applied electric field, we evaluated the effects of electrostatic forces and dielectrophoresis on the protein during fiber formation. We surmise that electrostatic forces and hydrodynamic friction are insignificant relative to dielectrophoretic forces; therefore, it is possible to separate species in a blend using polarizability contrast. A coaxial distribution of protein in the core can be obtained by electrospinning at the isoelectric point of the protein, whereas surface enrichment can be obtained when the protein carries a net charge.     

Intrinsic curvature in the VP1 gene of SV40: comparison of solution and gel results

DNA restriction fragments that are stably curved are usually identified by polyacrylamide gel electrophoresis because curved fragments migrate more slowly than normal fragments containing the same number of basepairs. In free solution, curved DNA molecules can be identified by transient electric birefringence (TEB) because they exhibit rotational relaxation times that are faster than those of normal fragments of the same size. In this article, the results observed in free solution and in polyacrylamide gels are compared for a highly curved 199-basepair (bp) restriction fragment taken from the VP1 gene in Simian Virus 40 (SV40) and various sequence mutants and insertion derivatives. The TEB method of overlapping fragments was used to show that the 199-bp fragment has an apparent bend angle of 46 +/- 2 degrees centered at sequence position 1922 +/- 2 bp. Four unphased A- and T-tracts and a mixed A3T4-tract occur within a span of approximately 60 bp surrounding the apparent bend center; for brevity, this 60-bp sequence element is called a curvature module. Modifying any of the A- or T-tracts in the curvature module by site-directed mutagenesis decreases the curvature of the fragment; replacing all five A- and T-tracts by random-sequence DNA causes the 199-bp mutant to adopt a normal conformation, with normal electrophoretic mobilities and birefringence relaxation times. Hence, stable curvature in this region of the VP1 gene is due to the five unphased A- and T- tracts surrounding the apparent bend center. Discordant solution and gel results are observed when long inverted repeats are inserted within the curvature module. These insertion derivatives migrate anomalously slowly in polyacrylamide gels but have normal, highly flexible conformations in free solution. Discordant solution and gel results are not observed if the insert does not contain a long inverted repeat or if the long inverted repeat is added to the 199-bp fragment outside the curvature module. The results suggest that long inverted repeats can form hairpins or cruciforms when they are located within a region of the helix backbone that is intrinsically curved, leading to large mobility anomalies in polyacrylamide gels. Hairpin/cruciform formation is not observed in free solution, presumably because of rapid conformational exchange. Hence, DNA restriction fragments that migrate anomalously slowly in polyacrylamide gels are not necessarily stably curved in free solution.     

Fluorescence correlation spectroscopy for ultrasensitive DNA analysis in continuous flow capillary electrophoresis

Continuous flow capillary electrophoresis (CFCE) is non-separations based analytical technique based on the free solution electrophoretic mobility of biological molecules such as DNA, RNA, peptides, and proteins. The electrophoretic mobilities and translational diffusion constants of the analyte molecules are determined using single molecule detection methods, including fluorescence correlation spectroscopy (FCS). CFCE is used to resolve multiple components in a mixture of analytes, measure electrophoretic mobility shifts due to binding interactions, and study the hydrodynamic and electrostatic properties of biological molecules in solution. Often this information is obtained with greater speed and sensitivity than conventational separations-based capillary-zone electrophoresis. This paper will focus on the application of two-beam fluorescence cross-correlation spectroscopy as a versatile detection method for CFCE and explore several applications to the study of the solution properties of single-stranded DNA.     

The flexibility of alternating dA-dT sequences

The flexibility of alternating poly (dA-dT) has been investigated by the technique of transient electric dichroism. Rotational relaxation times, which are very sensitive to changes in the end-to-end length of flexible polymers, are determined from the field free dichroism decay curves of four, well defined fragments of poly (dA-dT) ranging in size from 136 to 270 base pairs. Persistence lengths, calculated from the results of Hagerman and Zimm (Biopolymers (1981) 29, 1481-1502), are in the range 200-250 A. This makes alternating dA-dT sequences about twice as flexible as naturally occurring, "random" sequence DNA. Considering a bend around a nucleosome, for example, this difference in persistence length translates to an energy difference between poly (dA-dT) and random sequence DNA of 0.17 kT/base pair or 1 kcal per 10 base pair stretch. This energy difference is sufficiently large to suggest that dA-dT sequences could serve as markers in DNA packaging, for example, at sites where DNA must tightly bend to accommodate structures.     

Probing surface structures of Shewanella spp. by microelectrophoresis

Long-range electrostatic forces substantially influence bacterial interactions and bacterial adhesion during the preliminary steps of biofilm formation. The strength of these forces depends strongly on the structure of the bacterium surfaces investigated. The latter may be addressed from appropriate analysis of electrophoretic mobility measurements. Due to the permeable character of the bacterium wall and/or surrounding polymer layer, bacteria may be regarded as paradigms of soft bioparticles. The electrophoretic motion of such particles in a direct-current electric field differs considerably from that of their rigid counterparts in the sense that electroosmotic flow takes place around and within the soft surface layer. Recent developments of electrokinetic theories for soft particles now render possible the evaluation of the softness degree (or equivalently the hydrodynamic permeability) from the raw electrokinetic data. In this article, the electrophoretic mobilities of three Shewanella strains (MR-4, CN32, and BrY) presenting various and well-characterized phenotypes of polymer fringe are reported over a wide range of pH and ionic strength conditions. The data are quantitatively analyzed on the basis of a rigorous numerical evaluation of the governing electrostatic and hydrodynamic equations for soft particles. It is clearly shown how the peculiar surface structures of the bacteria investigated are reflected in their electrohydrodynamic properties.     

The mobility minima in pulsed-field capillary electrophoresis of large DNA.

Pulsed-field capillary electrophoresis represents a new tool for rapid and highly efficient separations of large biopolymers. The method has been utilized here to study dependencies of the electrophoretic mobility upon the frequency and pulse shape of applied voltage for large, double-stranded DNA molecules (5-100 kb) migrating in neutral polymer solutions. Two different shapes of alternating electric field (sine- and square-wave impulses) were examined with the frequency values ranging from 1 to 30 Hz. The linear dependence between duration of the forward pulse (at which the DNA molecule experiences a minimum mobility) and the product N.In(N) (where N is the number of base pairs) was experienced in field-inversion gel electrophoresis, while exponential dependence was found with the sinusoidal electric field. The mobility minima were lower in field-inversion electrophoresis than with the biased sinusoidal-field technique. The DNA (5 kb concatamers) was adequately separated using a ramp of frequency in the square-wave electric field, in approximately 1 h. The migration order of DNA fragments was referenced through adding a monodisperse DNA (48.5 kb) into the sample. The band inversion phenomena were not observed under any experimental conditions used in this work.     

Numerical simulation of the electrophoretic transport of a biopolymer through a synthetic nano-pore

We employed a hybrid approach to study numerically the translocation of a biopolymer through an artificial nano-pore driven by an external electric field in the presence of an explicit solvent. The motion of the polymer is simulated by the 3D Langevin dynamics technique. The hydrodynamic interactions (HI) between the polymer and the fluid are taken into account by the lattice Boltzmann equation. Our polymer chain model representing the double-stranded DNA was first validated by comparing the diffusion coefficient obtained from the numerical results with the experimental and theoretical results. Then, we conducted numerical simulations of the biopolymer's translocation process by applying a theoretical formula for the net electrophoretic force acting on the part of the polymer residing in the pore. We compared quantitatively the translocation times and the velocities of different DNA lengths with the corresponding experimental results. Our simulation results are in good agreement with the experimental ones when the HI are considered explicitly.     

Light scattering of aqueous solutions of DNA, poly(acrylic acid), and tobacco mosaic virus under alternating electric field

Two new techniques, amplitude modulation (AM) and frequency modulation (FM) of an electric field, are developed for the light-scattering study of polymer solutions under ac electric fields. The AM technique makes it possible to observe accurately the frequency dependence of the intensity changes of scattered light due to the electric field. The FM one allows us to obtain directly the frequency derivative of the intensity change. The techniques are applied to DNA, poly(acrylic acid), and tobacco mosaic virus in the frequency range from 10 Hz to 100 kHz. A low-frequency relaxation is found for both DNA and poly(acrylic acid). The obsersved relaxation time of DNA agrees with that in the dielectric relaxation of DNA, which has been attributed to the rotation of the molecule with a quasipermanent dipole. In the case of poly(acrylic acid), the relaxation strength increases with increasing degree of neutralization. TMV at a concentration of 0.1% exhibits a negative relaxation at low frequencies, which indicates the rotation of TMV aggregate with a permanent dipole along its minor axis.