DNA is a free-draining polymer. This subtle but "unfortunate" property of highly charged polyelectrolytes makes it impossible to separate nucleic acids by free-flow electrophoresis. This is why one must typically use a sieving matrix, such as a gel or an entangled polymer solution, in order to obtain some electrophoretic size separation. An alternative approach consists of breaking the charge to friction balance of free-draining DNA molecules. This can be achieved by labeling the DNA with a large, uncharged molecule (essentially a hydrodynamic parachute, which we also call a drag-tag) prior to electrophoresis; the resulting methodology is called end-labeled free-solution electrophoresis (ELFSE). In this article, we review the development of ELFSE over the last decade. In particular, we examine the theoretical concepts used to predict the ultimate performance of ELFSE for single-stranded (ssDNA) sequencing, the experimental results showing that ELFSE can indeed overcome the free-draining issue raised above, and the technological advances that are needed to speed the development of competitive ELFSE-based sequencing and separation technologies. Finally, we also review the reverse process, called free-solution conjugate electrophoresis (FSCE), wherein uncharged polymers of different sizes can be analyzed using a short DNA molecule as an electrophoretic engine.

We demonstrate the first use of a non-natural, genetically engineered protein polymer drag-tag to sequence DNA fragments by end-labeled free-solution electrophoresis (ELFSE). Fluorescently labeled DNA fragments resulting from the Sanger cycle sequencing reaction were separated by free-solution capillary electrophoresis, with much higher resolution and cleaner results than previously reported for this technique. With ELFSE, size-based separation of DNA in the absence of a sieving matrix is enabled by the end-on attachment of a polymeric "drag-tag" that modifies the charge-to-friction ratio of DNA in a size-dependent fashion. Progress in ELFSE separations has previously been limited by the lack of suitable large, monodisperse drag-tags. To address this problem, we designed, constructed, cloned, expressed, and purified a non-natural, genetically engineered 127mer protein polymer for use as an ELFSE drag-tag. The Sanger cycle sequencing reaction is performed with the drag-tag covalently attached to the sequencing primer, a major advance over previous strategies for ELFSE sequencing. The electrophoretic separation is diffusion-limited, without significant adsorption of the drag-tag to capillary walls. Although the read length (at about 180 bases) is still short, our results provide evidence that larger protein polymer drag-tags, currently under development, could extend the read length of ELFSE to more competitive levels. ELFSE offers the possibility of very rapid DNA sequencing separations without any of the difficulties associated with viscous polymeric sieving networks and hence will be amenable to implementation in microchannel and chip-based electrophoresis systems.

The development of innovative technologies designed to reduce the cost and increase the throughput of DNA separations continues to be important for large-scale sequencing and genotyping efforts. We report research aimed at the further development of a free-solution bioconjugate method of DNA size separation by capillary electrophoresis (CE), in particular, the determination of an optimal molecular architecture for polyamide-based "drag-tags". We synthesized several branched poly(N-methoxyethyl glycine)s (poly(NMEG)s, a class of polypeptoids) as novel friction-generating entities for end-on attachment to DNA molecules. A 30-mer poly(NMEG) "backbone," comprising five evenly spaced reactive epsilon-amino groups, was synthesized on solid phase, cleaved, and purified to monodispersity by RP-HPLC. Three different comblike derivatives of this backbone molecule were created by (1) acetylating the epsilon-amino groups or (2) appending small, monodisperse NMEG oligomers (a tetramer and an octamer). Grafting of the oligo(NMEG)s was done using solution-phase amide bond formation chemistry. Once purified to total monodispersity, the three different drag-tags were studied by free-solution electrophoresis to observe the effect of branching on their hydrodynamic drag or "alpha" and hence their ability to separate DNA. Drag was found to scale linearly with total molecular weight, regardless of branch length. The octamer-branched drag-tag-DNA conjugate was used to separate ssDNA products of 50, 75, 100, and 150 bases in length by free-solution CE in less than 10 min. Hence, the use of branched or comblike drag-tags is both a feasible and an effective way to achieve high frictional drag, allowing the high-resolution separation of relatively large DNA molecules by free-solution CE without the need to synthesize very long polymers.

We describe and illustrate a new cloning method for the production of long, size-controlled DNA concatemers, which can be expressed as protein polymers without an amino acid sequence requirement. Synthetic genes encoding different numbers of repeats of the amino acid sequence -(Gly-Lys-Gly-Ser-Ala-Gln-Ala) 3-were constructed using this cloning technique. The method enables the production of extremely long synthetic genes (e.g., up to 48 DNA repeats, 3024 bp), in a highly controlled fashion by providing higher-order multimers via the reconcatemerization of pre-multimerized genes and also eliminates the specific sequence requirement of the DNA monomer for restriction endonuclease cleavage. Moreover, the effect of intramolecular cyclization, which has a high probability of occurring during the self-ligation reaction and prevents the cloning of long DNA concatemers, is minimized by this approach. DNA multimers encoding a ladder of four differently sized protein polymers (6, 12, 24, and 48 repeats of a 63-bp segment) were expressed in E. coli upon induction with IPTG. Two of the resulting proteins, composed of 277 and 529 amino acid monomers, and each comprising a 10X histidine tag, were purified by immobilized metal affinity chromatography. During the course of the work, the sequence of the DNA concatemers was analyzed and confirmed to be correct by capillary electrophoresis, and the obtainment of the desired protein polymers was confirmed by MALDI-TOF mass spectrum analysis and amino acid compositional analysis. The resultant protein polymers are shown by CD to adopt a randomcoil conformation in aqueous solution and to be water-soluble between 20 and 95 °C. This polymer has lysine residues spaced evenly along the polymer backbone, which can be utilized either as derivatizable groups or as cross-linking sites for tissue engineering applications.

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.