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Investigation of dye characteristics in various environments for fluorescence resonance energy transfer (FRET) on a DNA molecule
Author(s) -
Tavisala Srilatha,
Bruist Michael F.
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.5.a946-a
Subject(s) - förster resonance energy transfer , quantum yield , acceptor , chemistry , fluorescence , molecule , dna , molar absorptivity , radius , resonance (particle physics) , quantum , oligonucleotide , yield (engineering) , chemical physics , atomic physics , analytical chemistry (journal) , physics , optics , quantum mechanics , thermodynamics , biochemistry , computer security , organic chemistry , chromatography , computer science
Fluorescence resonance energy transfer can be used for measuring the distance between two points on a molecule, which are 10–100 Å apart. The points of interest are labeled with two different fluorophores. In our study tetramethylrhodamine (TMR) is the donor (D) and indocarbocyanine5 (Cy5) is the acceptor (A). The D and A are tethered at the 3′ and 5′ ends respectively of a 20‐mer oligonucleotide. Our objective is to measure the energy transfer (ET) for our single‐stranded DNA by itself and for double‐stranded DNA hybridized to complementary strands of varying lengths, with and without magnesium in the buffer. ET lowers the quantum yield of the donor (Q D ). But quantum yield also varies considerably depending on the environment surrounding the probe. In order to accurately determine the distance between the D and A on a molecule from ET measurements, the quantum yield of the donor and the acceptor extinction coefficient must be known for that specific environment. The Föster radius (R o ) is the distance at which the ET is 50% efficient, which is dependent on these parameters. The value of R o is specific to a given set of D and A molecules in a particular environment. We observed a 60% decrease in the Q D for tetramethylrhodamine in the absence of Cy5 on going from single‐stranded to double‐stranded environment. The extinction coefficient also dropped by 9%. These values were not affected by the presence of 10mM Mg 2+ . Similar measurements for Cy5 are under way. The information available from the sensitivity of dyes in various environments is helpful in characterizing the conformational changes of DNA in solution.