The molecular end effect and its critical impact on the behavior of charged-uncharged polymer conjugates during free-solution electrophoresis

Recently two novel techniques using free-solution electrophoresis to separate charged-uncharged polymer conjugates have proven successful: end-labeled free-solution electrophoresis (ELFSE) for DNA sequencing, and free-solution conjugate electrophoresis (FSCE) for molar mass profiling of uncharged polymers. The approach taken to analyze the experimental data was an extension of the theory of Long and co-workers (Long, D., Dobrynin, A. V., Rubinstein, M., Ajdari, A., J. Chem. Phys. 1998, 108, 1234-1244) for the electrophoresis of molecules with varying charge distributions. This theory also predicts that the ends of the polymers play a large role in determining the polymer's overall mobility; however, this aspect of the theory was neglected in previous work. Until now this "end effect" has, to the knowledge of the authors, not been recognized in experimental data. Through a careful investigation of the predicted end effect and a reanalysis of the experimental data, we demonstrate that indeed this effect critically impacts on the behavior of charged-uncharged polymer conjugates during electrophoresis. This work indicates that not only does the end effect need to be taken into account to avoid significant errors in data analysis, but also it provides novel system optimization approaches.     

Molecular deformation and free-solution electrophoresis of DNA-uncharged polymer conjugates at high field strengths: theoretical predictions. Part 1: hydrodynamic segregation

Recent advancements in DNA sequencing by end-labeled free-solution electrophoresis (ELFSE) show the promise of this novel technique which overcomes the need for a gel by using a label (or drag-tag) to render the free solution mobility of the DNA size-dependent. It is the attachment of an uncharged drag-tag molecule of a set size to various lengths of DNA in the sample that selectively slows down smaller DNA chains which have less force to pull the drag-tag than larger DNA. So far, only globally random coil conformations have been associated with ELFSE, i.e., the DNA and the label together form a single, undeformed hydrodynamic unit. This paper investigates the conditions under which the DNA and label will segregate into two hydrodynamically distinct units, based on a theoretical approach developed for the electrophoresis of polyampholytes. Optimal experimental conditions tailored to the available label sizes and voltages are predicted along with insight into ideal label architecture.     

A theoretical study of the possible use of electroosmotic flow to extend the read length of DNA sequencing by end-labeled free solution electrophoresis

End-labeled free solution electrophoresis (ELFSE) provides a means of separating DNA with free-solution CE, eliminating the need for gels and polymer solutions which increase the run time and can be difficult to load into a capillary. In free-solution electrophoresis, DNA is normally free-draining and all fragments reach the detector at the same time, whereas ELFSE uses an uncharged label molecule attached to each DNA fragment in order to render the electrophoretic mobility size-dependent. With ELFSE, however, the larger molecules are not separated enough (limiting the read length in the case of ssDNA sequencing) while the smaller ones are overseparated; the larger ones are too fast while the shorter ones are too slow, which is the opposite of traditional gel-based methods. In this article, we show how an EOF could be used to overcome these problems and extend the DNA sequencing read length of ELFSE. This counterflow would allow the larger, previously unresolved molecules more time to separate and thereby increase the read length. Through our theoretical investigation, we predict that an EOF mobility of approximately the same magnitude as that of unlabeled DNA would provide the best results for the regime where all molecules move in the same direction. Even better resolution would be possible for smaller values of EOF which allow different directions of migration; however, the migration times then would become too large. The flow would need to be well controlled since the gain in read length decreases as the magnitude of the counterflow increases; an EOF mobility double that of unlabeled DNA would no longer increase the read length, although ELFSE would still benefit from a reduction in migration time.     

Branched polymeric labels used as drag-tags in free-solution electrophoresis of ssDNA

In the framework of the classical blob theory of end-labeled free-solution electrophoresis of ssDNA, and based on recent experimental data with linear and branched polymeric labels (or drag-tags), the present study puts forward design principles for the optimal type of branching that would give, for a given total number of monomers, the highest effective frictional drag for ssDNA sequencing purposes. The hydrodynamic radii of the linear and branched labels are calculated using standard models like the freely jointed chain model and the Kratky-Porod worm-like chain model. Based on comparisons of the theory with the experimental data, we propose that the design of new branched labels should use either side chains whose length is comparable to the distance between the branching points or two long branches located near the ends of the molecule's backbone.     

Protein polymer drag-tags for DNA separations by end-labeled free-solution electrophoresis

We demonstrate the feasibility of end-labeled free-solution electrophoresis (ELFSE) separation of DNA using genetically engineered protein polymers as drag-tags. Protein polymers are promising candidates for ELFSE drag-tags because their sequences and lengths are controllable not only to generate monodisperse polymers with high frictional drag, but also to meet other drag-tag requirements for high-resolution separations by microchannel electrophoresis. A series of repetitive polypeptides was designed, expressed in Escherichia coli, and purified. By performing an end-on conjugation of the protein polymers to a fluorescently labeled DNA oligomer (22 bases) and analyzing the electrophoretic mobilities of the conjugate molecules by free-solution capillary electrophoresis (CE), effects of the size and charge of the protein polymer drag-tags were investigated. In addition, the electrophoretic behavior of bioconjugates comprising relatively long DNA fragments (108 and 208 bases) and attached to uncharged drag-tags was observed, by conjugating fluorescently labeled polymerase chain reaction (PCR) products to charge-neutral protein polymers, and analyzing via CE. We calculated the amount of friction generated by the various drag-tags, and estimated the potential read-lengths that could be obtained if these drag-tags were used for DNA sequencing in our current system. The results of these studies indicate that larger and uncharged drag-tags will have the best DNA-resolving capability for ELFSE separations, and that theoretically, up to 233 DNA bases could be sequenced using one of the protein polymer drag-tags we produced, which is electrostatically neutral with a chain length of 337 amino acids. We also show that denatured (unfolded) polypeptide chains impose much greater frictional drag per unit molecular weight than folded proteins, such as streptavidin, which has been used as a drag-tag before.     

Free-solution electrophoresis of DNA modified with drag-tags at both ends

In end-labeled free-solution electrophoresis (ELFSE), DNA molecules are labeled with a frictional modifier or "drag-tag", allowing their size-based electrophoretic separation in free solution. Among the interesting observations from early work with dsDNA using streptavidin as a drag-tag was that the drag induced by including a streptavidin label at both ends was significantly more than double that from a single streptavidin (Heller, C. et al.., J. Chromatogr. A 1998, 806, 113-121). This finding was assumed to be in error, and subsequent work focused on experiments in which only a single drag-tag is appended to one end of the DNA molecule. Recent theoretical work (McCormick, L. C., Slater, G. W., Electrophoresis 2005, 26, 1659-1667) has examined the contribution of end-effects to the free-solution electrophoretic mobility of charged-uncharged polymer conjugates, reopening the question of enhanced drag from placing a drag-tag at both ends. In this study, this effect is investigated experimentally, using custom-synthesized ssDNA oligonucleotides allowing the attachment of drag-tags to one or both ends, as well as dsDNA PCR products generated with primers appropriate for the attachment of drag-tags at one or both ends. A range of sizes of drag-tags are used, including synthetic polypeptoid drag-tags as well as genetically engineered protein polymer drag-tags. The enhanced drag arising from labeling both ends has been confirmed, with 6-9% additional drag for the ssDNA and 10-23% additional drag for the dsDNA arising from labeling both ends than would be expected from simply doubling the size of the drag-tag at one end. The experimental results for ssDNA labeled at both ends are compared to the predictions of the recent theory of end-effects, with reasonably good quantitative agreement. These experimental findings demonstrate the feasibility of enhancing ELFSE separations by labeling both ends of the DNA molecule, leading to greater resolving power and a wider range of applications for this technique.     

Length-dependent DNA separations using multiple end-attached peptide nucleic acid amphiphiles in micellar electrokinetic chromatography

End-labeled free-solution electrophoresis (ELFSE) is an alternative approach to gel-based methods for size-based electrophoretic separation of DNA. In ELFSE, an electrically neutral "drag-tag" is appended to DNA to add significant hydrodynamic drag, thereby breaking its constant charge-to-friction ratio. Current drag-tag architecture relies on covalent attachment of polymers to each DNA molecule. We have recently proposed the use of micellar drag-tags in conjunction with sequence-specific hybridization of peptide nucleic acid amphiphiles (PNAAs). This work investigates the effect of multiple PNAA attachment on DNA resolution using MEKC. Simultaneous PNAA hybridization allows for the separation of long DNA targets, up to 1012 bases, using micellar drag-tags. Each PNAA handle independently interacts with the micellar phase, reducing the overall mobility of this complex relative to individual PNAA binding. The sequence- and size-based dependence of this separation technique is maintained with multiple PNAA binding over a range of DNA sizes. Results are accurately described by ELFSE theory, yielding alpha=54 for single-micelle tagging and alpha=142 for dual-micelle tagging. This method is the first example of a non-covalent drag-tag used to separate DNA of 1000 bases based on both size and sequence.     

Chiral separation in capillary electrophoresis using dual neutral cyclodextrins: theoretical models of electrophoretic mobility difference and separation selectivity

Simple equations and theoretical models, related to enantioselectivity (kappa) and C, have been developed for prediction of electrophoretic mobility difference (Deltamu) and separation selectivity (alpha) for enantiomers in CE using dual CDs, where alpha and kappa are defined as the ratio of mu and the ratio of binding constant (K) for enantiomers to each CD, respectively, C the CD concentration, and the average K for enantiomers and each CD. Experiments were carried out using dual CDs as beta-CD and dimethyl-beta-cyclodextrin (DM-beta-CD) and test analytes as five pairs of amphetamine drug enantiomers. A change in observed Deltamu and alpha of enantiomers in dual CDs was found to be in excellent agreement with the theoretical models. For example, in comparison with single CD1, dual CDs can enhance Deltamu and alpha up to the maximum value when enantiomers migrate with the same order in CD1 and CD2, and have the value of rho > 1.0, where rho is the enantioselectivity ratio for CD2 to CD1, while worse Deltamu and alpha are obtained for enantiomers with rho < 1.0.     

High-throughput separation of DNA and proteins by three-dimensional geometry gel electrophoresis: feasibility studies

We report a novel separation method that is applicable to both DNA and protein samples, based on electrophoresis in a three-dimensional (3-D) geometry. In contrast to conventional electrophoresis, samples are applied in a two-dimensional, planar array to one of the surfaces of a 3-D geometry separation medium. Loading onto a plane results in a very high sample capacity. Sample migration and separation occur along the third spatial dimension, which is perpendicular to the loading plane. The key problem of electrophoresis in a 3-D geometry separation setup is that temperature gradients are caused by Joule's heat, affecting the electrical conductivity and viscosity of the separation medium. A means of achieving straight sample migration under these circumstances is to force heat flow through the separation medium parallel to the axis of sample migration. This can be done by dissipating the heat via the electrode sides of the gel and blocking any other heat transfer. The separation of DNA and proteins by this method has been tested using agarose gel electrophoresis, polyacrylamide gel electrophoresis, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Data were acquired off-line by conventional staining methods as well as on-line by detection of laser-induced fluorescence. We describe how to excise samples from the separation medium for preparative purposes. Possible unique applications of this 3-D geometry electrophoresis separation method are also discussed.