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

• Published in: Biophysical journal Vol. 89; no. 5; pp. L37 - L39
• Main Authors: Zhu, Peizhi, Clamme, Jean-Pierre, Deniz, Ashok A.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.11.2005; Biophysical Society
• Subjects: Base Sequence; Biophysical Letters; Biophysics; Biophysics - methods; Deoxyribonucleic acid; DNA; DNA - chemistry; DNA - drug effects; Fluorescence; Fluorescence Polarization - methods; Fluorescent Dyes - pharmacology; Free Radicals; Iodides - chemistry; Kinetics; Models, Molecular; Molecular Sequence Data; Molecules; Nitric Oxide - chemistry; Proteins; Rhodamines - chemistry; Rhodamines - pharmacology; RNA - chemistry; Spectrometry, Fluorescence - methods
• ISSN: 0006-3495; 1542-0086
• DOI: 10.1529/biophysj.105.071027

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 10–30Å range. The results demonstrate that TEMPO-5-TAMRA fluorescence quenching is a promising method to measure short distance changes within single biomolecules.