Free Solution Mobility Research Articles

Flanking AT base pairs affect the localization of monovalent cations in DNA A-tracts.

Capillary electrophoresis has been used to measure the free solution mobilities of a series of 26-base pair (bp) DNA oligomers containing two phased A4T1in-tracts embedded in flanking sequences containing 0 to 11 additional AT bps. A random-sequence 26-bp oligomer with 12 isolated AT bps was used as the reference. Mobility ratios (A-tract/reference) were measured in background electrolytes (BGEs) containing mixtures of small monovalent cations and tetrabutylammonium (TBA+ ) or tetrapropylammonium (TPA+ ) ions. The mobility ratios observed in 0.3M TBA+ were >1.00, suggesting that the TBA+ ions had formed electrostatic contact pairs with the AT bp in the reference and in the A-tract flanking sequences, decreasing the mobilities of both oligomers. The TBA-AT pairing interactions could be eliminated by increasing the concentration of small monovalent cations in the BGE. In 0.3M TPA+ , electrostatic contact pairs were formed with the AT bps in the flanking sequences and in the A-tracts. Interestingly, the shapes of the mobility ratio profiles observed for the A4T1in-tract oligomers depended on the total number of A+T residues in the oligomer.

Monovalent cation localization in DNA A-tracts with different sequences.

The free solution mobilities of 26-base pair (bp) DNA oligomers containing A-tracts with and without internal ApT steps have been measured by capillary electrophoresis, using the mobility of a 26-bp random-sequence oligomer as a reference. The background electrolytes (BGEs) contained mixtures of Li+ and tetrapropylammonium (TPA+ ) ions, keeping the total cation concentration constant at 0.3M. The mobility ratios equaled 1.00 in 0.3M TPA+ , indicating that the A-tract and reference oligomers had the same B-form conformation in this BGE. With increasing [Li+ ], the mobility ratio decreased as Li+ ions became localized in the A-tract minor groove, suggesting that the A-tract was now in the B* conformation. If the A-tract contained an internal ApT step and the oligomer contained less than ∼50% A+T, the mobility ratio reached a reduced plateau value that remained constant as the [Li+ ] increased to 0.3M. However, for A-tracts without an internal ApT step and for A-tracts embedded in oligomers containing more than 50% A+T, the mobility ratios increased again at high [Li+ ], eventually reaching a plateau value of 1.00. Hence, DNA A-tracts in solution appear to exist as mixtures of the B and B* conformations, with the fractional concentration of each conformer depending on the [Li+ ], the A-tract sequence, and the total A+T content of the oligomer.

Examining the Effects of Netropsin on the Mobility of DNA using Capillary Electrophoresis

Netropsin binds the minor groove of DNA, particularly at AT-rich sequences. The presence of DNA A-tracts, contiguous repeated adenine base sequences, causes a natural curve of the helix axis. Orienting multiple A-tracts on a 98-base pair DNA fragment either in-phase or out-of-phase creates different shapes in the DNA. These molecular shape differences alter DNA mobility. Capillary electrophoresis (CE) has been widely used to examine the free solution mobility of negatively charged analytes. Here we attempt to better understand how netropsin binding affects the mobility in free solution of double-stranded DNA fragments with different A-tract arrangements. Results to date are presented.

Electrophoretic Mobility of DNA in Solutions of High Ionic Strength

The free-solution mobilities of small single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) have been measured by capillary electrophoresis in solutions containing 0.01–1.0 M sodium acetate. The mobility of dsDNA is greater than that of ssDNA at all ionic strengths because of the greater charge density of dsDNA. The mobilities of both ssDNA and dsDNA decrease with increasing ionic strength until approaching plateau values at ionic strengths greater than ∼0.6 M. Hence, ssDNA and dsDNA appear to interact in a similar manner with the ions in the background electrolyte. For dsDNA, the mobilities predicted by the Manning electrophoresis equation are reasonably close to the observed mobilities, using no adjustable parameters, if the average distance between phosphate residues (the b parameter) is taken to be 1.7 Å. For ssDNA, the predicted mobilities are close to the observed mobilities at ionic strengths ≤0.01 M if the b-value is taken to be 4.1 Å. The predicted and observed mobilities diverge strongly at higher ionic strengths unless the b-value is reduced significantly. The results suggest that ssDNA strands exist as an ensemble of relatively compact conformations at high ionic strengths, with b-values corresponding to the relatively short phosphate-phosphate distances through space.

DNA Electrophoretic Mobilities in High Ionic Strength Solutions

The free solution mobilities of small dsDNA and ssDNA oligomers have been measured as a function of ionic strength by capillary electrophoresis. The mobilities of both types of DNA decrease with increasing ionic strength, approaching limiting plateau values at high ionic strengths. The observed mobilities of the dsDNAs are similar to the mobilities calculated from the Manning electrophoresis equation, although the shapes of the mobility curves are different. The observed ssDNA mobilities cannot be described by the Manning electrophoresis equation using a single value of the axial charge spacing.

Electrophoretic ratcheting of spherical particles in well/channel microfluidic devices: Making particles move against the net field.

We examine the electrophoresis of spherical particles in microfluidic devices made of alternating wells and narrow channels, including a system previously used to separate DNA molecules. Our computer simulations predict that such systems can be used to separate spherical particles of different sizes that share the same free-solution mobility. Interestingly, the electrophoretic velocity shows an inversion as the field intensity is increased: while small particles have higher velocities at low field, the situation is reversed at high fields with the larger particles then moving faster. The resulting nonlinearity suggests that asymmetric pulsed electric fields could be used to build separation ratchets: particles then have a net size-dependent velocity in the presence of a zero-mean external field. Exploiting the inversion mentioned above, we show how to design pulsed field sequences that make particles move against the mean field (an example of negative mobility). Finally, we demonstrate that it is possible to use pulsed fields to make particles of different sizes move in opposite directions, even though their charge have the samesign.

Database of free solution mobilities for 276 metabolites

Although databases are available that provide mass spectra and chromatographic retention information for small-molecule metabolites, no publicly available database provides electrophoretic mobility for common metabolites. As a result, most compounds found in electrophoretic-based metabolic studies are unidentified and simply annotated as “features”. To begin to address this issue, we analyzed 460 metabolites from a commercial library using capillary zone electrophoresis coupled with electrospray mass spectrometry. To speed analysis, a sequential injection method was used wherein six compounds were analyzed per run. An uncoated fused silica capillary was used for the analysis at 20 °C with a 0.5% (v/v) formic acid and 5% (v/v) methanol background electrolyte. A Prince autosampler was used for sample injection and the capillary was coupled to an ion trap mass spectrometer using an electrokinetically-pumped nanospray interface. We generated mobility values for 276 metabolites from the library (60% success rate) with an average standard deviation of 0.01 × 10−8 m2V−1s−1. As expected, cationic and anionic compounds were well resolved from neutral compounds. Neutral compounds co-migrated with electro-osmotic flow. Most of the compounds that were not detected were neutral and presumably suffered from adsorption to the capillary wall or poor ionization efficiency.

Determination of electroosmotic and electrophoretic mobility of DNA and dyes in low ionic strength solutions.

Nanocoding, a genome analysis platform, relies on very low ionic strength conditions to elongate DNA molecules up to 1.06 (fully stretched DNA=1). Understanding how electroosmotic and electrophoretic forces vary, as ionic strength decreases, will enable better Nanocoding devices, or other genome analysis platforms, to be developed. Using gel electrophoresis to determine overall mobility (includes contributions from electrophoretic and electroosmotic forces) in different ionic strength conditions, linear DNA molecules (pUC19 (2.7 kb), pBR322 (4.4 kb), ΦX174 (5.4 kb), and PSNAPf-H2B (6.2 kb)) were analyzed in varying gel concentrations (1.50, 1.25, 1.00, 0.75, and 0.50%). Additionally, buffer concentration (Tris-EDTA, TE) was varied to determine free solution mobility at different ionic strength solutions. As ionic strength decreased from 13.8 to 7.3mM, overall mobility increased. As TE buffer decreased (< 7.3mM), overall mobility drastically decreased as ionic strength decreased. Rhodamine B dye was utilized to determine the electroosmotic mobility. As the ionic strength decreased, electroosmotic mobility increased. The experimental electrophoretic mobility was compared to theoretical considerations for electrophoretic mobility (Pitts and Debye-Hückel-Onsager). Electroosmotic forces decreased the overall mobility of DNA molecules and bromophenol blue migration in a gel matrix as ionic strength decreased.

The Charge Variants of DNA and other Polyelectrolytes: Comparison of Experimental Mobilities with Electrophoresis Theories

The free solution mobilities of the charge variants of single- and double-stranded DNA oligomers, small aromatic molecules, peptides, proteins and synthetic copolymers depend on the logarithm of the number of charged residues, not the first power of the charge as predicted by classic electrophoresis theories. The mobility ratios observed for the charge variants of all polyelectrolytes except the synthetic copolymers exhibit a common dependence on the logarithm of fractional charge. The different results observed for the synthetic copolymers can probably be attributed to the flexibility and hydrophobicity of the copolymer backbone. By contrast, the other polyelectrolytes have hydrophilic and relatively rigid backbones. The mobilities observed for the charge variants of the DNA oligomers are well predicted by the Manning electrophoresis equation, while the mobilities predicted by zeta potential theories are too high. However, the mobility ratios observed for the protein charge variants are better described by zeta potential theories.

Counterion Condensation vs. Zeta Potential: Can Either Theory Describe the Electrophoresis of DNA and other Polyions?

Many textbooks state that the electrophoretic mobility of a polyion is directly proportional to the number of charged residues and inversely proportional to its frictional coefficient. If a series of polyions differ only in the number of charged residues, the frictional coefficients will be approximately constant. Hence, the free solution mobilities are expected to be proportional to the effective charge. However, the mobilities of the charge variants of a given polyion are proportional to the logarithm of the linear charge density, not the first power of the charge. The semilogarithmic relationship between the mobility and the fractional charge of the polyion is observed for single- and double-stranded DNA oligomers, small organic molecules, protein “ladders” and protein sequence mutants.Manning has used counterion condensation theory to derive an equation describing DNA electrophoretic mobility. This equation predicts that the mobilities should depend on the logarithm of the linear charge density, as observed. Surprisingly, the same equation can be used to predict the fractional mobilities of the charge variants of other types of polyions, if the square root of the surface charge density is used as the variable instead of the linear charge density. Mobilities calculated from theories of the zeta potential are less accurate than those calculated from the Manning equation. Hence, the Manning electrophoresis equation appears to have a wider validity than commonly recognized.

Determination of Electrophoretic Forces in a Gel Matrix in Different Ionic Strength Conditions

Nanocoding, a genome analysis platform, relies on very low ionic strength conditions to elongate DNA molecules up to 1.06 (fully stretched DNA molecule = 1)1, which is the largest stretch reported in the literature. Understanding how electroosmotic and electrophoretic forces vary, as ionic strength decreases, will enable better Nanocoding devices to be developed. Using two different gel assays to determine electroosmotic and overall mobility (includes contributions from electrophoretic forces and electroosmotic forces), we were able to determine electrophoretic mobility in different ionic strength solutions. Our first gel assay relied on electrophoresising linear DNA molecules [pUC19 (2.7 kb), pBR322 (4.4 kb), φX174 (5.4 kb), and PSNAPf-H2B (6.2 kb)] in varying gel concentrations (1.5%, 1.25%, 1%, 0.75%, 0.5%) to determine the free solution mobility (overall mobility) of these molecules in dilutions of TE buffer. As the buffer concentration decreases from 2X (Ionic strength = 13.8 mM) to 1X (Ionic strength = 7.3 mM), the overall mobility increased. As we further diluted TE (< 1X TE), the overall mobility drastically decreased as the ionic strength decreased.1. Kounovsky-Shafer, K.L., Hernandez-Ortiz, J.P., Jo, K., Odijk, T., de Pablo, J.J., and David C. Schwartz, D.C. Presentation of large DNA molecules for analysis as nanoconfined dumbbells, Macromolecules, 2013, 46 (20), 8356-8368.

Use of Capillary Electrophoresis for Polysaccharide Studies and Applications.

CE applications to charged polysaccharides are briefly reported. A simple procedure is presented to determine the esterification degree of a hyaluronan derivative. In this case the degree of substitution was as low as 14 %.The molecular weight distribution of mannuronic oligosaccharides mixture produced by hydrolysis of native polymannuronic is readily calculated from peak area of the species resolved by CE on the basis of a specific degree of polymerization.The influence of the applied electric field strength on the free solution mobility of hyaluronan samples is briefly addressed for molar masses of the order of 10(5) and 10(6) g/mol. The data are compared with the results obtained for a 50 % galactose substituted HA.Mobility data obtained as a function of buffer pH for a native HA sample as well as for two galactose-amide HA derivatives, having slightly different degrees of substitution, are presented and discussed in terms of the polymer charge density parameters ξ.In most cases, more questions than answers arise from the application of CE to charged polysaccharides. However, perspectives are disclosed for a further understanding of the reliability of CE applied for the structural elucidation of such macromolecules.

Flanking A·T Basepairs Destabilize the B∗ Conformation of DNA A-Tracts

Capillary electrophoresis has been used to characterize the interaction of monovalent cations with 26-basepair DNA oligomers containing A-tracts embedded in flanking sequences with different basepair compositions. A 26-basepair random-sequence oligomer was used as the reference; lithium and tetrabutylammonium (TBA+) ions were used as the probe ions. The free solution mobilities of the A-tract and random-sequence oligomers were identical in solutions containing <∼100 mM cation. At higher cation concentrations, the A-tract oligomers migrated faster than the reference oligomer in TBA+ and slower than the reference in Li+. Hence, cations of different sizes can interact very differently with DNA A-tracts. The increased mobilities observed in TBA+ suggest that the large hydrophobic TBA+ ions are preferentially excluded from the vicinity of the A-tract minor groove, increasing the effective net charge of the A-tract oligomers and increasing the mobility. By contrast, Li+ ions decrease the mobility of A-tract oligomers because of the preferential localization of Li+ ions in the narrow A-tract minor groove. Embedding the A-tracts in AT-rich flanking sequences markedly alters preferential interactions of monovalent cations with the B∗ conformation. Hence, A-tracts embedded in genomic DNA may or may not interact preferentially with monovalent cations, depending on the relative number of A·T basepairs in the flanking sequences.

The Relationship between Electrophoretic Mobility and Polyelectrolyte Charge

Many textbooks state that the electrophoretic mobility of a polyelectrolyte is proportional to its effective charge divided by its frictional coefficient. We have tested this relationship by analyzing the mobilities of single- and double-stranded DNA molecules containing the same number of bases or base pairs and different numbers of negatively charged phosphate linkers. Small organic molecules containing different numbers of charged residues were also analyzed,using data taken from the literature. In each case, the free solution mobilities of the charge variants of a given molecule were divided by the mobility of the charge variant with the highest number of charged residues, measured under the same conditions. The mobility ratios were then plotted as a function of the fractional charge of each molecule. The results indicate that the fractional mobilities of polyelectrolytes of the same size are proportional to the logarithm of the fractional charge, not the first power of the charge as commonly assumed. The Manning theory of DNA electrophoresis and electrophoretic theories based on the zeta potential both predict a logarithmic dependence of the mobility on charge density. The experimental mobility ratios will be compared with the predictions of these two theories.

The free solution mobility of DNA and other analytes varies as the logarithm of the fractional negative charge

The free solution mobilities of ssDNA and dsDNA molecules with variable charge densities have been measured by CE. DNA charge density was modified either by appending positively or negatively charged groups to the thymine residues in a 98 bp DNA molecule, or by replacing some of the negatively charged phosphate internucleoside linkers in small ssDNA or dsDNA oligomers with positively charged phosphoramidate linkers. Mobility ratios were calculated for each dataset by dividing the mobility of a charge variant by the mobility of its unmodified parent DNA. Mobility ratios essentially eliminate the effect of the BGE on the observed mobility, making it possible to compare analytes measured under different experimental conditions. Neutral moieties attached to the thymine residues in the 98-bp DNA molecule had little or no effect on the mobility ratios, indicating that bulky substituents in the DNA major groove do not affect the mobility significantly. The mobility ratios observed for the thymine-modified and linker-modified DNA charge variants increased approximately linearly with the logarithm of the fractional negative charge of the DNA. Mobility ratios calculated from previous studies of linker-modified DNA charge variants and small multicharged organic molecules also increased approximately linearly with the logarithm of the fractional negative charge of the analyte. The results do not agree with the Debye-Hückel-Onsager theory of electrophoresis, which predicts that the mobility of an analyte should depend linearly on analyte charge, not the logarithm of the charge, when the frictional coefficient is held constant.

Nanoparticle gel electrophoresis: Soft spheres in polyelectrolyte hydrogels under the Debye–Hückel approximation

A mathematical model for electrophoresis of polyelectrolyte coated nanoparticles (soft spheres) in polyelectrolyte hydrogels is proposed, and evaluated by comparison to literature models for bare-sphere gel electrophoresis and free-solution electrophoresis. The utilities of approximations based on the bare-particle electrophoretic mobility, free-solution mobility, and electroosmotic flow in hydrogels are explored. Noteworthy are the influences of the particle–core dielectric constant and overlap of the polyelectrolyte shell. The present theory, which neglects ion-concentration and charge-density perturbations, indicates that the gel electrophoretic mobilities of metallic-core nanoparticles in polyelectrolyte gels can be qualitatively different than for their non-metallic counterparts. These insights will be beneficial for interpreting nanoparticle gel-electrophoresis data, optimizing electrophoretic separations, and engineering nanoparticles for technological applications.

Electrophoretic Mobility of DNA is Controlled by the Surface Charge Density, not the Linear Charge Density as Expected from Counterion Condensation Theory

The free solution mobilities of small single- and double-stranded DNA molecules with variable charge densities have been measured by capillary electrophoresis. The charge density was modified either by appending charged groups to the thymine residues or by replacing some of the negatively charged phosphate internucleoside linkers with neutral or positively charged phosphoramidate linkers. Mobility ratios were calculated for each data set by dividing the mobility of the charge variant by the mobility of the unmodified parent DNA. Mobility ratios eliminate the effect of the background electrolyte on the observed mobility, making it possible to compare data obtained in different buffers. The mobility ratios observed for ss- and dsDNA charge variants increase logarithmically with increasing fractional charge density, as expected from Manning's theory of DNA electrophoresis. Surprisingly, however, the mobility ratios are directly related to the surface charge densities of the ss- and ds-DNA molecules, not the linear charge density as expected from counterion condensation theory. The results therefore suggest that surface charge density may play a role in counterion condensation, as well as in electrostatic interactions between DNA and other charged molecules in the cell.

DNA A-tracts are not curved in solutions containing high concentrations of monovalent cations.

The intrinsic curvature of seven 98 bp DNA molecules containing up to four centrally located A6-tracts has been measured by gel and capillary electrophoresis as a function of the number and arrangement of the A-tracts. At low cation concentrations, the electrophoretic mobility observed in polyacrylamide gels and in free solution decreases progressively with the increasing number of phased A-tracts, as expected for DNA molecules with increasingly curved backbone structures. Anomalously slow electrophoretic mobilities are also observed for DNA molecules containing two pairs of phased A-tracts that are out of phase with each other, suggesting that out-of-phase distortions of the helix backbone do not cancel each other out. The mobility decreases observed for the A-tract samples are due to curvature, not cation binding in the A-tract minor groove, because identical free solution mobilities are observed for a molecule with four out-of-phase A-tracts and one with no A-tracts. Surprisingly, the curvature of DNA A-tracts is gradually lost when the monovalent cation concentration is increased to ∼200 mM, regardless of whether the cation is a hydrophilic ion like Na+, NH4+, or Tris+ or a hydrophobic ion like tetrabutylammonium. The decrease in A-tract curvature with increasing ionic strength, along with the known decrease in A-tract curvature with increasing temperature, suggests that DNA A-tracts are not significantly curved under physiological conditions.

Rapid separation of synthetic oligonucleotides on polymer modified capillary surfaces using short-end injection capillary electrophoresis in free solution

Here we use short-end electrokinetic injection capillary electrophoresis (CE) to investigate the free solution mobility of short strands of double-stranded oligonucleotides (dsODNs) on polymer modified capillaries. Single base pair (bp) resolution (Rs) of dsODNs ranging from 16-20 bp was achieved in free solution on an 8 cm capillary dynamically coated with poly(ethylpyrrolidine methacrylate-co-methyl methacrylate) (PEPyM-co-PMMA) random copolymer. Interestingly, separation of a dsODN mixture containing two 16 bp strands of different sequences resulted in partial resolution (0.52) implying that the free solution mobility of dsODNs was sequence dependent. The single bp resolution achieved for the complementary sequence strands (the sequence of all strands in the mixtures contained the same 16 bp sequence) was improved by up to 37% for separation of dsODNs containing non-complementary sequences. The 16 bp peak was not additive within each mixture, indicating the presence of ODN-ODN interactions. Investigation of these interactions (and ODN-buffer interactions) showed that they can be influenced by the ionic strength and conductivity of the background electrolyte (BGE). Increasing the ionic strength reduced the ODN-ODN interactions and improved the resolution, whereas, increasing the conductivity reduced ODN-buffer interactions, increasing the mobility, at the consequence of promoting ODN-ODN interactions, and hence decreasing the resolution.

Tandem GA Residues on Opposite Sides of the Loop in Molecular Beacon-Like DNA Hairpins Compact the Loop and Increase Hairpin Stability

The free solution electrophoretic mobilities and thermal stabilities of hairpins formed by two complementary 26-nucleotide oligomers have been measured by capillary electrophoresis. The oligomers are predicted to form molecular beacon-like hairpins with 5 base paired stems and 16 nucleotides in the loop. One hairpin, called hairpin2 (hp2), migrates with a relatively fast free solution mobility and exhibits melting temperatures that are reasonably well predicted by the popular structure-prediction program Mfold. Its complement, called hairpin1 (hp1), migrates with a slower free solution mobility and forms a stable hairpin only in solutions containing ≥200 mM Na+. The melting temperatures observed for hp1 are ∼18°C lower than observed for hp2 and ∼20°C lower than predicted by Mfold. The greater thermal stability of hp2 is due to the presence of tandem GA residues on opposite sides of the loop. If the corresponding TC residues in the hp1 loop are replaced by tandem GA residues, the melting temperatures of the modified hairpin are close to the values observed for hp2. Eliminating the tandem GA residues in the hp2 loop significantly decreases the thermal stability of hp2. If the loops are replaced by a loop of 16 thymine residues, the free solution mobilities and thermal stabilities of the T-loop hairpin are equal to those observed for hp1. Hence, the hp1 loop appears to be relatively unstructured, with few base-base stacking interactions. Interactions between tandem GA residues on opposite sides of the hp2 loop compact the loop and increase hairpin stability.