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.