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Fluorescence Quenching by TEMPO: A Sub-30 Å Single-Molecule Ruler

Department of Molecular Biology, The Scripps Research Institute, La Jolla, California 92037

Correspondence: Address reprints and inquiries to Ashok A. Deniz, Tel.: 858-784-9192; E-mail: deniz{at}scripps.edu.

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 measurements both show an exponential decrease in the quenching rate constant with the dye-quencher distance in the 10–30 Å range. The results demonstrate that TEMPO-5-TAMRA fluorescence quenching is a promising method to measure short distance changes within single biomolecules.