Direct Observation of Fluorescently Labeled Single-stranded lambda DNA Molecules in a Micro-Flow Channel

作者：Takahashi Shunsuke; Kawasaki Shohei; Yamaguchi Koji; Miyata Hidefumi; Kurita Hirofumi; Mizuno Takeshi; Matsuura Shun ichi; Mizuno Akira; Oshige Masahiko; Katsura Shinji^*

来源：Journal of Fluorescence, 2013, 23(4): 635-640.

DOI：10.1007/s10895-013-1210-1

• Single molecule
• Single-stranded DNA
• Single-stranded DNA recognition peptide
• Replication protein A
• Micro-flow channel

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 ss lambda DNA molecules were visualized over time in micro-flow channels. We report substantially different dynamics between these two labeling methods. When ss lambda DNA molecules were labeled with RPA-YFP, terminally bound fusion proteins were sheared from the free ends of the ss lambda DNA molecules unless 25-mer oligonucleotides were annealed to the free ends. RPA-YFP-ss lambda DNA 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-ss lambda DNA complexes, the ssBP-488-ss lambda DNA complexes behaved as rigid rods and were not dissociated even in 2 M NaCl.

• 出版日期2013-7