Fluorescence quenching by TEMPO: a sub-30 A single-molecule ruler.
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 A range. The results demonstrate that TEMPO-5-TAMRA fluorescence quenching is a promising method to measure short distance changes within single biomolecules.
Zhu
Peizhi
P
Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.
Clamme
Jean-Pierre
JP
Deniz
Ashok A
AA
D001483:Base Sequence; D001703:Biophysics / Q000379:methods; D004247:DNA / Q000737:chemistry* / Q000187:drug effects; D005454:Fluorescence Polarization / Q000379:methods*; D005456:Fluorescent Dyes / Q000494:pharmacology; D005609:Free Radicals; D007454:Iodides / Q000737:chemistry; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009569:Nitric Oxide / Q000737:chemistry; D012313:RNA / Q000737:chemistry; D012235:Rhodamines / Q000737:chemistry / Q000494:pharmacology*; D013050:Spectrometry, Fluorescence / Q000379:methods*
C437523:5-carboxytetramethylrhodamine; D005456:Fluorescent Dyes; D005609:Free Radicals; D007454:Iodides; D012235:Rhodamines; D009569:Nitric Oxide; D012313:RNA; D004247:DNA