Abstract: Using a single-stranded region tracing system, single-molecule DNA synthesis reactions were directly observed in microflow channels. The direct single-molecule observations of DNA synthesis were labeled with a fusion protein consisting of the ssDNA-binding domain of a 70-kDa subunit of replication protein A and enhanced yellow fluorescent protein (RPA-YFP). Our method was suitable for measurement of DNA synthesis reaction rates with control of the ssλDNA form as stretched ssλDNA (+flow) and random coiled ssλDNA (−flow) via buffer flow. Sequentially captured photographs demonstrated that the synthesized region of an ssλDNA molecule monotonously increased with the reaction time. The DNA synthesis reaction rate of random coiled ssλDNA (−flow) was nearly the same as that measured in a previous ensemble molecule experiment (52 vs. 50 bases/s). This suggested that the random coiled form of DNA (−flow) reflected the DNA form in the bulk experiment in the case of DNA synthesis reactions. In addition, the DNA synthesis reaction rate of stretched ssλDNA (+flow) was approximately 75% higher than that of random coiled ssλDNA (−flow) (91 vs. 52 bases/s). The DNA synthesis reaction rate of the Klenow fragment (3’-5’exo–) was promoted by DNA stretching with buffer flow.
Keywords: single-molecule observation; single-stranded DNA; replication protein A (RPA); DNA polymerase; DNA synthesis
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Takahashi, S.; Kawasaki, S.; Miyata, H.; Kurita, H.; Mizuno, T.; Matsuura, S.-I.; Mizuno, A.; Oshige, M.; Katsura, S. A New Direct Single-Molecule Observation Method for DNA Synthesis Reaction Using Fluorescent Replication Protein A. Sensors 2014, 14, 5174-5182.
Takahashi S, Kawasaki S, Miyata H, Kurita H, Mizuno T, Matsuura S-I, Mizuno A, Oshige M, Katsura S. A New Direct Single-Molecule Observation Method for DNA Synthesis Reaction Using Fluorescent Replication Protein A. Sensors. 2014; 14(3):5174-5182.
Takahashi, Shunsuke; Kawasaki, Shohei; Miyata, Hidefumi; Kurita, Hirofumi; Mizuno, Takeshi; Matsuura, Shun-ichi; Mizuno, Akira; Oshige, Masahiko; Katsura, Shinji. 2014. "A New Direct Single-Molecule Observation Method for DNA Synthesis Reaction Using Fluorescent Replication Protein A." Sensors 14, no. 3: 5174-5182.