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Cellular Redox Profiling Using High-content Microscopy
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DNA Microenvironment Monitored by Controlling Redox Blinking.

Kiyohiko Kawai1,2, Kenji Higashiguchi3,4, Atsushi Maruyama5

  • 1The Institute of Scientific and Industrial Research, Osaka University, Mihogaoka 8-1, Ibaraki, Osaka, 567-0047, Japan. kiyohiko@sanken.osaka-u.ac.jp.

Chemphyschem : a European Journal of Chemical Physics and Physical Chemistry
|October 1, 2015
PubMed
Summary

Researchers enhanced monitoring of DNA conformational changes using redox blinking. By employing ascorbic acid 2-phosphate (VcP) and a bulky oxidant (FeDTPA), they achieved sensitive detection of subtle DNA structure variations.

Keywords:
DNAelectron transferfluorescence correlation spectroscopyradical ionsredox chemistry

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Spectroscopy

Background:

  • Bimolecular reaction rates are influenced by molecular accessibility.
  • Previously, R6G blinking monitored DNA duplex-hairpin transitions, but O2's small size limited sensitivity.
  • Sensitive monitoring of microenvironment changes is crucial for understanding molecular interactions.

Purpose of the Study:

  • To develop a more sensitive method for monitoring DNA conformational changes.
  • To investigate the use of redox blinking with controlled reductants and oxidants for enhanced microenvironment sensing.
  • To detect subtle DNA conformational changes, including those caused by single nucleotide variations.

Main Methods:

  • Utilized redox blinking of a fluorophore (R6G) controlled by ascorbic acid 2-phosphate (VcP) as a reductant.
  • Employed a bulky oxidant, FeDTPA, to modulate the electron-transfer rate.
  • Correlated changes in electron-transfer rates with DNA conformational states.

Main Results:

  • The electron-transfer rate between the R6G radical anion (R6G(.-)) and FeDTPA was highly sensitive to microenvironment changes.
  • This method provided enhanced sensitivity compared to using molecular oxygen (O2).
  • Subtle DNA conformational changes, even those induced by a single nucleotide difference, were successfully monitored.

Conclusions:

  • Controlling redox blinking with VcP and FeDTPA offers a sensitive approach to probe microenvironment dynamics.
  • This technique significantly improves the ability to detect small-scale DNA conformational alterations.
  • The enhanced sensitivity opens new avenues for studying DNA structure-function relationships at a single-nucleotide level.