Separation efficiency of free‐solution conjugated electrophoresis with drag‐tags incorporating a synthetic amino acid

DNA sequencing or separation by conventional capillary electrophoresis with a polymer matrix has some inherent drawbacks, such as the expense of polymer matrix and limitations in sequencing read length. As DNA fragments have a linear charge‐to‐friction ratio in free solution, DNA fragments cannot be separated by size. However, size‐based separation of DNA is possible in free‐solution conjugate electrophoresis (FSCE) if a “drag‐tag” is attached to DNA fragments because the tag breaks the linear charge‐to‐friction scaling. Although several previous studies have demonstrated the feasibility of DNA separation by free‐solution conjugated electrophoresis, generation of a monodisperse drag‐tag and identification of a strong, site‐specific conjugation method between a DNA fragment and a drag‐tag are challenges that still remain. In this study, we demonstrate an efficient FSCE method by conjugating a biologically synthesized elastin‐like polypeptide (ELP) and green fluorescent protein (GFP) to DNA fragments. In addition, to produce strong and site‐specific conjugation, a methionine residue in drag‐tags is replaced with homopropargylglycine (Hpg), which can be conjugated specifically to a DNA fragment with an azide site.