Spectroscopic properties of fluorescein and rhodamine dyes attached to DNA

Roberto F. Delgadillo, Lawrence J Parkhurst

Research output: Contribution to journalArticle

16 Citations (Scopus)

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 (τ0), 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 τ0 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 Å. We present evidence for the direct interaction of dyes attached to the termini of various single-stranded DNA sequences.

Original languageEnglish (US)
Pages (from-to)261-272
Number of pages12
JournalPhotochemistry and Photobiology
Volume86
Issue number2
DOIs
StatePublished - Mar 1 2010

Fingerprint

Rhodamines
rhodamine
Fluorescein
Coloring Agents
deoxyribonucleic acid
dyes
DNA
absorptivity
Avidin
Fluorescence
life (durability)
fluorescence
biotin
Single-Stranded DNA
DNA sequences
Quantum yield
Biotin
oligomers
Oligomers
strands

ASJC Scopus subject areas

  • Biochemistry
  • Physical and Theoretical Chemistry

Cite this

Spectroscopic properties of fluorescein and rhodamine dyes attached to DNA. / Delgadillo, Roberto F.; Parkhurst, Lawrence J.

In: Photochemistry and Photobiology, Vol. 86, No. 2, 01.03.2010, p. 261-272.

Research output: Contribution to journalArticle

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