Fluorescence properties and photophysics of the sulfoindocyanine Cy3 linked covalently to DNA

Matthew E. Sanborn, Brian K. Connolly, Kaushik Gurunathan, Marcia Levitus

Research output: Contribution to journalArticle

160 Citations (Scopus)

Abstract

The sulfoindocyanine Cy3 is one of the most commonly used fluorescent dyes in the investigation of the structure and dynamics of nucleic acids by means of fluorescence methods. In this work, we report the fluorescence and photophysical properties of Cy3 attached covalently to single-stranded and duplex DNA. Steady-state and time-resolved fluorescence techniques were used to determine fluorescence quantum yields, emission lifetimes, and fluorescence anisotropy decays. The existence of a transient photoisomer was investigated by means of transient absorption techniques. The fluorescence quantum yield of Cy3 is highest when attached to the 5' terminus of single-stranded DNA (Cy3-5′ ssDNA), and decreases by a factor of 2.4 when the complementary strand is annealed to form duplex DNA (Cy3-5′ dsDNA). Substantial differences were also observed between the 5′-modified strands and strands modified through an internal amino-modified deoxy uridine. The fluorescence decay of Cy3 became multiexponential upon conjugation to DNA. The longest lifetime was observed for Cy3-5′ ssDNA, where about 50% of the decay is dominated by a 2.0-ns lifetime. This value is more than 10 times larger than the fluorescence lifetime of the free dye in solution. These observations are interpreted in terms of a model where the molecule undergoes a trans-cis isomerization reaction from the first excited state. We observed that the activation energy for photoisomerization depends strongly on the microenvironment in which the dye is located. The unusually high activation energy measured for Cy3-5′ ssDNA is an indication of dye-ssDNA interactions. In fact, the time-resolved fluorescence anisotropy decay of this sample is dominated by a 2.5-ns rotational correlation time, which evidences the lack of rotational freedom of the dye around the linker that separates it from the terminal 5′ phosphate. The remarkable variations in the photophysical properties of Cy3-DNA constructs demonstrate that caution should be used when Cy3 is used in studies employing DNA conjugates.

Original languageEnglish (US)
Pages (from-to)11064-11074
Number of pages11
JournalJournal of Physical Chemistry B
Volume111
Issue number37
DOIs
StatePublished - Sep 20 2007

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Fluorescence
fluorescence
Dyes
dyes
Coloring Agents
strands
life (durability)
decay
Quantum yield
Anisotropy
Activation energy
activation energy
Photoisomerization
anisotropy
Uridine
Nucleic acids
nucleic acids
Isomerization
conjugation
Fluorescent Dyes

ASJC Scopus subject areas

  • Physical and Theoretical Chemistry

Cite this

Fluorescence properties and photophysics of the sulfoindocyanine Cy3 linked covalently to DNA. / Sanborn, Matthew E.; Connolly, Brian K.; Gurunathan, Kaushik; Levitus, Marcia.

In: Journal of Physical Chemistry B, Vol. 111, No. 37, 20.09.2007, p. 11064-11074.

Research output: Contribution to journalArticle

Sanborn, Matthew E. ; Connolly, Brian K. ; Gurunathan, Kaushik ; Levitus, Marcia. / Fluorescence properties and photophysics of the sulfoindocyanine Cy3 linked covalently to DNA. In: Journal of Physical Chemistry B. 2007 ; Vol. 111, No. 37. pp. 11064-11074.
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abstract = "The sulfoindocyanine Cy3 is one of the most commonly used fluorescent dyes in the investigation of the structure and dynamics of nucleic acids by means of fluorescence methods. In this work, we report the fluorescence and photophysical properties of Cy3 attached covalently to single-stranded and duplex DNA. Steady-state and time-resolved fluorescence techniques were used to determine fluorescence quantum yields, emission lifetimes, and fluorescence anisotropy decays. The existence of a transient photoisomer was investigated by means of transient absorption techniques. The fluorescence quantum yield of Cy3 is highest when attached to the 5' terminus of single-stranded DNA (Cy3-5′ ssDNA), and decreases by a factor of 2.4 when the complementary strand is annealed to form duplex DNA (Cy3-5′ dsDNA). Substantial differences were also observed between the 5′-modified strands and strands modified through an internal amino-modified deoxy uridine. The fluorescence decay of Cy3 became multiexponential upon conjugation to DNA. The longest lifetime was observed for Cy3-5′ ssDNA, where about 50{\%} of the decay is dominated by a 2.0-ns lifetime. This value is more than 10 times larger than the fluorescence lifetime of the free dye in solution. These observations are interpreted in terms of a model where the molecule undergoes a trans-cis isomerization reaction from the first excited state. We observed that the activation energy for photoisomerization depends strongly on the microenvironment in which the dye is located. The unusually high activation energy measured for Cy3-5′ ssDNA is an indication of dye-ssDNA interactions. In fact, the time-resolved fluorescence anisotropy decay of this sample is dominated by a 2.5-ns rotational correlation time, which evidences the lack of rotational freedom of the dye around the linker that separates it from the terminal 5′ phosphate. The remarkable variations in the photophysical properties of Cy3-DNA constructs demonstrate that caution should be used when Cy3 is used in studies employing DNA conjugates.",
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