Real-time direct observation of single-molecule DNA hydrolysis by exonucleaseIII

Hirofumi Kurita, Ken Ichi Inaishi, Ken Torii, Madoka Urisu, Michihiko Nakano, Shinji Katsura, Akira Mizuno

Research output: Chapter in Book/Report/Conference proceedingConference contribution

1 Citation (Scopus)

Abstract

Real time direct observation of single molecule using fluorescent technique has elucidated various biological phenomena, because not only average but also distributions and fluctuations of molecules can be characterized directly by this method, on the other hand, conventional methods can observe only average behaviors of molecules. In recent years, single-molecule analyses of various DNA-protein interactions by using florescent techniques were performed. In this study, single-molecule DNA hydrolysis by exonucleaseIII was observed, which has 3′ → 5′ exonuclease activity. This single-molecule observation method was based on optical trap and two-layer laminar flow, in which mixing between the two layers was negligible. First of all, one DNA-bead complex was optically trapped in a layer without exonucleaseIII. This trapping permitted stretching of DNA by flow. ExonucleaseIII reaction was initiated by moving the trapped DNA-bead complex to another flow layer containing exonucleaseIII, then shortening of fluorescently stained DNA was observed in real time. The sequentially captured photographs demonstrate that the digested DNA molecule linearly shortened with reaction time. The digestion rate from single-molecule experiment was determined and compared to in vitro experiment. As a result, digestion rate of exonucleaseIII from single-molecule experiment was ∼20 times higher than in vitro digestion rate.

Original languageEnglish
Title of host publication2006 IEEE International Symposium on Micro-Nano Mechanical and Human Science, MHS
DOIs
Publication statusPublished - 2006
Externally publishedYes
Event2006 IEEE International Symposium on Micro-Nano Mechanical and Human Science, MHS - Nagoya, Japan
Duration: Nov 5 2006Nov 8 2006

Other

Other2006 IEEE International Symposium on Micro-Nano Mechanical and Human Science, MHS
CountryJapan
CityNagoya
Period11/5/0611/8/06

All Science Journal Classification (ASJC) codes

  • Computational Mechanics
  • Control and Systems Engineering
  • Human Factors and Ergonomics

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    Kurita, H., Inaishi, K. I., Torii, K., Urisu, M., Nakano, M., Katsura, S., & Mizuno, A. (2006). Real-time direct observation of single-molecule DNA hydrolysis by exonucleaseIII. In 2006 IEEE International Symposium on Micro-Nano Mechanical and Human Science, MHS [4110383] https://doi.org/10.1109/MHS.2006.320277