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Biophys. J. BioFAST: First Published September 30, 2005. doi:10.1529/biophysj.105.071027
© 2005 by the Biophysical Society.

A more recent version of this article appeared on November 1, 2005.
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BIOPHYSICAL LETTERS

Fluorescence quenching by TEMPO: a sub-30 Å single molecule ruler

Peizhi Zhu 1, Jean-Pierre Clamme 1 and Ashok Deniz 1*

1 The Scripps Research Institute

* To whom correspondence should be addressed. E-mail: deniz{at}scripps.edu.

Submitted on July 18, 2005
Revised on September 5, 2005
Accepted on 13 September 2005


  Abstract
A series of DNA molecules labeled with 5-carboxytetramethylrhodamine (5-TAMRA) and the small nitroxide radical TEMPO were synthesized and tested to investigate whether the intramolecular quenching efficiency can be used to measure short intramolecular distances in small ensemble and single molecule experiments. In combination with distance calculations using molecular mechanics modeling, the experimental results from steady-state ensemble fluorescence and fluorescence correlation spectroscopy (FCS) measurements both show an exponential decrease in the quenching efficiency with the dye-quencher distance in the 10 to 30 Å range. The results demonstrate that TEMPO-5-TAMRA fluorescence quenching is a promising method to measure short distance changes within single biomolecules.

Key Words: Single molecule, fluorescence correlation spectroscopy, fluorescence quenching, molecular ruler, protein/nucleic acid structure




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Copyright © 2005 by the Biophysical Society.