Spectroscopic Properties of Fluorescein and Rhodamine Dyes Attached to DNA

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 (tau0), static quenching (S) and the Förster critical distances (R0) 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 tau0 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. R0 for free and attached dyes showed differences from 1.1 to 4.2 A. We present evidence for the direct interaction of dyes attached to the termini of various single-stranded DNA sequences.

Probing the Interaction Between Fluorophores and DNA Nucleotides by Fluorescence Correlation Spectroscopy and Fluorescence Quenching†

“…In addition to the aforementioned spectral shifts, interactions between the dye and the DNA bases have been reported to result in changes in the extinction coefficient of the dye (7,21,46). Figure S2 shows the change in absorbance observed for Cy3B upon increasing additions of dNMPs.…”

“…Analogous experiments with TAMRA and Alexa 546 were not performed because the absorption spectrum of rhodamines has a more complex solvent-dependence due to specific solvent-dye interactions that involve H-bonding and solvation of the amino and carboxylic groups (50,51). In addition to the aforementioned spectral shifts, interactions between the dye and the DNA bases have been reported to result in changes in the extinction coefficient of the dye (7,21,46). Figure S2 shows the change in absorbance observed for Cy3B upon increasing additions of dNMPs.…”

“…Despite this ubiquity, the quantitative interpretation of the measured data is often hampered by lack of knowledge of how the photophysical and spectroscopic properties of the dyes depend on their microenvironment within the biomolecule. Dyebiomolecule interactions can affect the absorption and emission spectra of the dye (4,5), as well as its extinction coefficient (6,7), fluorescence lifetime (8,9) and fluorescence quantum yield (8)(9)(10). In addition, interactions influence the rotational freedom of the dye within the macromolecule, with important consequences in many quantitative applications of fluorescence in biochemistry and biophysics (8,11,12).…”

Transition metal ion FRET to measure short-range distances at the intracellular surface of the plasma membrane

“…FRET is steeply distance dependent, and each donor–acceptor pair has a characteristic distance at which the FRET efficiency is 50% ( R 0 ), where the amount of FRET is optimally sensitive to changes in distance. The R 0 value for fluorescein and rhodamine, for example, is ∼60 Å ( Delgadillo and Parkhurst, 2010 ), making them an ideal FRET pair for quantifying membrane fusion ( Keller et al, 1977 ).…”

Spectroscopic and Photophysical Properties of dUTP and Internally DNA Bound Fluorophores for Optimized Signal Detection in Biological Formats

“…At present, however, ssystematic studies focusing on a methodical dye comparison are relatively rare and are usually conducted on chemically synthesized 5¢ bound fluorophores (14)(15)(16), while enzymatically based labeling stragies like polymerase chain reaction (PCR), result in internal labeling (17). Yet, the position of the dyes in the DNA strand can alter their spectroscopic properties and lead to a flawed interpretation of the results, that is, for FRET calculations (18 Photochemistry and Photobiology, 2012, 88: 867-875 study could provide a road map towards the design and understanding of experiments using such methodically assessed fluorescent reporters within DNA of known sequence. Also, the fluorescence properties of many labels can depend dramatically on their molecular environment within the biomolecule.…”