title: Direct observation of fluorescently labeled single-stranded λDNA molecules in a micro-flow channel.
abstract: We developed two labeling methods for the direct observation of single-stranded DNA (ssDNA), using a ssDNA binding protein and a ssDNA recognition peptide. The first approach involved protein fusion between the 70-kDa ssDNA-binding domain of replication protein A and enhanced yellow fluorescent protein (RPA-YFP). The second method used the ssDNA binding peptide of Escherichia coli RecA labeled with Atto488 (ssBP-488; Atto488-IRMKIGVMFGNPETTTGGNALKFY). The labeled ssλDNA molecules were visualized over time in micro-flow channels. We report substantially different dynamics between these two labeling methods. When ssλDNA molecules were labeled with RPA-YFP, terminally bound fusion proteins were sheared from the free ends of the ssλDNA molecules unless 25-mer oligonucleotides were annealed to the free ends. RPA-YFP-ssλDNA complexes were dissociated by the addition of 0.2 M NaCl, although complex reassembly was possible with injection of additional RPA-YFP. In contrast to the flexible dynamics of RPA-YFP-ssλDNA complexes, the ssBP-488-ssλDNA complexes behaved as rigid rods and were not dissociated even in 2 M NaCl.
authors.affiliation: Department of Chemical and Environmental Engineering, Graduate School of Engineering, Gunma University, 1-5-1 Tenjin-cho, Kiryu, Gunma, 376-8515, Japan.
authors.forename: Shunsuke
authors.initials: S
authors.lastname: Takahashi
authors.forename: Shohei
authors.initials: S
authors.lastname: Kawasaki
authors.forename: Koji
authors.initials: K
authors.lastname: Yamaguchi
authors.forename: Hidefumi
authors.initials: H
authors.lastname: Miyata
authors.forename: Hirofumi
authors.initials: H
authors.lastname: Kurita
authors.forename: Takeshi
authors.initials: T
authors.lastname: Mizuno
authors.forename: Shun-ichi
authors.initials: S
authors.lastname: Matsuura
authors.forename: Akira
authors.initials: A
authors.lastname: Mizuno
authors.forename: Masahiko
authors.initials: M
authors.lastname: Oshige
authors.forename: Shinji
authors.initials: S
authors.lastname: Katsura
mesh_terms: D000595:Amino Acid Sequence; D010582:Bacteriophage lambda; D001483:Base Sequence; D004277:DNA, Single-Stranded / Q000235:genetics / Q000378:metabolism*; D004279:DNA, Viral / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism*; D046210:Microfluidic Analytical Techniques; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011938:Rec A Recombinases / Q000737:chemistry; D013194:Staining and Labeling / Q000379:methods*
chemical_list: D004277:DNA, Single-Stranded; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D005456:Fluorescent Dyes; D010446:Peptide Fragments; D011938:Rec A Recombinases