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Spectroscopic Properties of Fluorescein and Rhodamine Dyes Attached to DNA
Author(s) -
Delgadillo Roberto F.,
Parkhurst Lawrence J.
Publication year - 2010
Publication title -
photochemistry and photobiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.818
H-Index - 131
eISSN - 1751-1097
pISSN - 0031-8655
DOI - 10.1111/j.1751-1097.2009.00663.x
Subject(s) - rhodamine , molar absorptivity , fluorescence , absorbance , photochemistry , chemistry , rhodamine b , fluorescein , dna , quenching (fluorescence) , quantum yield , analytical chemistry (journal) , chromatography , organic chemistry , biochemistry , optics , photocatalysis , physics , catalysis
Abstract We report the spectroscopic properties of fluorescein, x‐rhodamine, tetramethyl‐rhodamine, attached to single strand, duplex DNA, and to the digestion products by DNAse I. The properties reported include: molar absorptivity, quantum yield, absorbance and fluorescence spectra, fluorescence lifetime, intrinsic lifetime (τ 0 ), static quenching (S) and the Förster critical distances ( R 0 ) between fluorescein and x‐rhodamine or tetramethyl‐rhodamine (acceptors). These spectroscopic properties depend strongly on the local dye environment. Fluorescein was studied: (1) attached to biotin (BF), (2) BF bound to avidin; and attached to two positions in DNA. X‐rhodamine and tetramethyl‐rhodamine were studied as free dyes and attached at the 5′‐end of DNA. We propose a general method to determine the molar absorptivity and τ 0 of a dye attached to DNA based on the reaction of a biotinylated and dye‐labeled oligomer with standardized avidin. The molar absorptivity of a second dye attached to a DNA duplex can be obtained by comparing spectra of doubly and singly labeled sequences. S, arising from dye–DNA interactions can then be determined. R 0 for free and attached dyes showed differences from 1.1 to 4.2 Å. We present evidence for the direct interaction of dyes attached to the termini of various single‐stranded DNA sequences.