Real-time single-molecule observations of T7 Exonuclease activity in a microflow channel

Shunsuke Takahashi; Tomohiro Usui; Shohei Kawasaki; Hidefumi Miyata; Hirofumi Kurita; Shun-ichi Matsuura; Akira Mizuno; Masahiko Oshige; Shinji Katsura

doi:10.1016/j.ab.2014.04.012

Real-time single-molecule observations of T7 Exonuclease activity in a microflow channel

Anal Biochem. 2014 Jul 15:457:24-30. doi: 10.1016/j.ab.2014.04.012. Epub 2014 Apr 19.

Authors

Shunsuke Takahashi¹, Tomohiro Usui¹, Shohei Kawasaki¹, Hidefumi Miyata¹, Hirofumi Kurita², Shun-ichi Matsuura³, Akira Mizuno², Masahiko Oshige¹, Shinji Katsura⁴

Affiliations

¹ Department of Chemical and Environmental Engineering, Graduate School of Engineering, Gunma University, 1-5-1 Tenjin-cho, Kiryu, Gunma 376-8515, Japan.
² Department of Environmental and Life Sciences, Graduate School of Engineering, Toyohashi University of Technology, Aichi 441-8580, Japan.
³ Research Center for Compact Chemical System, National Institute of Advanced Industrial Science and Technology (AIST), Miyagi 983-8551, Japan.
⁴ Department of Chemical and Environmental Engineering, Graduate School of Engineering, Gunma University, 1-5-1 Tenjin-cho, Kiryu, Gunma 376-8515, Japan. Electronic address: katsura@cee.gunma-u.ac.jp.

PMID: 24751469
DOI: 10.1016/j.ab.2014.04.012

Abstract

T7 Exonuclease (T7 Exo) DNA digestion reactions were studied using direct single-molecule observations in microflow channels. DNA digestion reactions were directly observed by staining template DNA double-stranded regions with SYTOX Orange and staining single-stranded (digested) regions with a fluorescently labeled ssDNA-recognizing peptide (ssBP-488). Sequentially acquired photographs demonstrated that a double-stranded region monotonously shortened as a single-stranded region monotonously increased from the free end during a DNA digestion reaction. Furthermore, DNA digestion reactions were directly observed both under pulse-chase conditions and under continuous buffer flow conditions with T7 Exo. Under pulse-chase conditions, the double-stranded regions of λDNA monotonously shortened by a DNA digestion reaction with a single T7 Exo molecule, with an estimated average DNA digestion rate of 5.7 bases/s and a processivity of 6692 bases. Under continuous buffer flow conditions with T7 Exo, some pauses were observed during a DNA digestion reaction and double-stranded regions shortened linearly except during these pauses. The average DNA digestion rate was estimated to be 5.3 bases/s with a processivity of 5072 bases. Thus, the use of our direct single-molecule observations using a fluorescently labeled ssDNA-recognizing peptide (ssBP-488) was an effective analytic method for investigating DNA metabolic processes.

Keywords: Double-stranded DNA; Single-molecule observation; Single-stranded DNA; T7 Exonuclease.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Exodeoxyribonucleases / metabolism*
Fluorescent Dyes
Microfluidic Analytical Techniques / methods*
Nucleic Acid Denaturation
Organic Chemicals
Time Factors

Substances

Fluorescent Dyes
Organic Chemicals
SYTOX Orange dye
Exodeoxyribonucleases
gene b exonuclease