Orientation of Cyanine Fluorophores Terminally Attached to DNA via Long, Flexible Tethers

Jonathan Ouellet, Stephanie Schorr, Asif Iqbal, Timothy J. Wilson, David M. J. Lilley

    Research output: Contribution to journalArticle

    34 Citations (Scopus)

    Abstract

    Cyanine fluorophores are commonly used in single-molecule FRET experiments with nucleic acids. We have previously shown that indocarbocyanine fluorophores attached to the 5'-termini of DNA and RNA via three-carbon atom linkers stack on the ends of the helix, orienting their transition moments. We now investigate the orientation of sulfoindocarbocyanine fluorophores tethered to the 5'-termini of DNA via 13-atom linkers. Fluorescence lifetime measurements of sulfoindocarbocyanine 3 attached to double-stranded DNA indicate that the fluorophore is extensively stacked onto the terminal basepair at 15 degrees C, with properties that depend on the terminal sequence. In single molecules of duplex DNA, FRET efficiency between sulfoindocarbocyanine 3 and 5 attached in this manner is modulated with helix length, indicative of fluorophore orientation and consistent with stacked fluorophores that can undergo lateral motion. We conclude that terminal stacking is an intrinsic property of the cyanine fluorophores irrespective of the length of the tether and the presence or absence of sulfonyl groups. However, compared to short-tether indocarbocyanine, the mean rotational relationship between the two fluorophores is changed by similar to 60 degrees for the long-tether sulfoindocarbocyanine fluorophores. This is consistent with the transition moments becoming approximately aligned with the long axis of the terminal basepair for the long-linker species.

    Original languageEnglish
    Pages (from-to)1148-1154
    Number of pages7
    JournalBiophysical Journal
    Volume101
    Issue number5
    DOIs
    Publication statusPublished - 7 Sep 2011

    Keywords

    • RESONANCE ENERGY-TRANSFER
    • DOUBLE-STRANDED DNA
    • FLUORESCENCE
    • FRET
    • CY3
    • PHOTOPHYSICS
    • ACIDS
    • TOOL

    Cite this

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    title = "Orientation of Cyanine Fluorophores Terminally Attached to DNA via Long, Flexible Tethers",
    abstract = "Cyanine fluorophores are commonly used in single-molecule FRET experiments with nucleic acids. We have previously shown that indocarbocyanine fluorophores attached to the 5'-termini of DNA and RNA via three-carbon atom linkers stack on the ends of the helix, orienting their transition moments. We now investigate the orientation of sulfoindocarbocyanine fluorophores tethered to the 5'-termini of DNA via 13-atom linkers. Fluorescence lifetime measurements of sulfoindocarbocyanine 3 attached to double-stranded DNA indicate that the fluorophore is extensively stacked onto the terminal basepair at 15 degrees C, with properties that depend on the terminal sequence. In single molecules of duplex DNA, FRET efficiency between sulfoindocarbocyanine 3 and 5 attached in this manner is modulated with helix length, indicative of fluorophore orientation and consistent with stacked fluorophores that can undergo lateral motion. We conclude that terminal stacking is an intrinsic property of the cyanine fluorophores irrespective of the length of the tether and the presence or absence of sulfonyl groups. However, compared to short-tether indocarbocyanine, the mean rotational relationship between the two fluorophores is changed by similar to 60 degrees for the long-tether sulfoindocarbocyanine fluorophores. This is consistent with the transition moments becoming approximately aligned with the long axis of the terminal basepair for the long-linker species.",
    keywords = "RESONANCE ENERGY-TRANSFER, DOUBLE-STRANDED DNA, FLUORESCENCE, FRET, CY3, PHOTOPHYSICS, ACIDS, TOOL",
    author = "Jonathan Ouellet and Stephanie Schorr and Asif Iqbal and Wilson, {Timothy J.} and Lilley, {David M. J.}",
    year = "2011",
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    doi = "10.1016/j.bpj.2011.07.007",
    language = "English",
    volume = "101",
    pages = "1148--1154",
    journal = "Biophysical Journal",
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    publisher = "Biophysical Society",
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    }

    Orientation of Cyanine Fluorophores Terminally Attached to DNA via Long, Flexible Tethers. / Ouellet, Jonathan; Schorr, Stephanie; Iqbal, Asif; Wilson, Timothy J.; Lilley, David M. J.

    In: Biophysical Journal, Vol. 101, No. 5, 07.09.2011, p. 1148-1154.

    Research output: Contribution to journalArticle

    TY - JOUR

    T1 - Orientation of Cyanine Fluorophores Terminally Attached to DNA via Long, Flexible Tethers

    AU - Ouellet, Jonathan

    AU - Schorr, Stephanie

    AU - Iqbal, Asif

    AU - Wilson, Timothy J.

    AU - Lilley, David M. J.

    PY - 2011/9/7

    Y1 - 2011/9/7

    N2 - Cyanine fluorophores are commonly used in single-molecule FRET experiments with nucleic acids. We have previously shown that indocarbocyanine fluorophores attached to the 5'-termini of DNA and RNA via three-carbon atom linkers stack on the ends of the helix, orienting their transition moments. We now investigate the orientation of sulfoindocarbocyanine fluorophores tethered to the 5'-termini of DNA via 13-atom linkers. Fluorescence lifetime measurements of sulfoindocarbocyanine 3 attached to double-stranded DNA indicate that the fluorophore is extensively stacked onto the terminal basepair at 15 degrees C, with properties that depend on the terminal sequence. In single molecules of duplex DNA, FRET efficiency between sulfoindocarbocyanine 3 and 5 attached in this manner is modulated with helix length, indicative of fluorophore orientation and consistent with stacked fluorophores that can undergo lateral motion. We conclude that terminal stacking is an intrinsic property of the cyanine fluorophores irrespective of the length of the tether and the presence or absence of sulfonyl groups. However, compared to short-tether indocarbocyanine, the mean rotational relationship between the two fluorophores is changed by similar to 60 degrees for the long-tether sulfoindocarbocyanine fluorophores. This is consistent with the transition moments becoming approximately aligned with the long axis of the terminal basepair for the long-linker species.

    AB - Cyanine fluorophores are commonly used in single-molecule FRET experiments with nucleic acids. We have previously shown that indocarbocyanine fluorophores attached to the 5'-termini of DNA and RNA via three-carbon atom linkers stack on the ends of the helix, orienting their transition moments. We now investigate the orientation of sulfoindocarbocyanine fluorophores tethered to the 5'-termini of DNA via 13-atom linkers. Fluorescence lifetime measurements of sulfoindocarbocyanine 3 attached to double-stranded DNA indicate that the fluorophore is extensively stacked onto the terminal basepair at 15 degrees C, with properties that depend on the terminal sequence. In single molecules of duplex DNA, FRET efficiency between sulfoindocarbocyanine 3 and 5 attached in this manner is modulated with helix length, indicative of fluorophore orientation and consistent with stacked fluorophores that can undergo lateral motion. We conclude that terminal stacking is an intrinsic property of the cyanine fluorophores irrespective of the length of the tether and the presence or absence of sulfonyl groups. However, compared to short-tether indocarbocyanine, the mean rotational relationship between the two fluorophores is changed by similar to 60 degrees for the long-tether sulfoindocarbocyanine fluorophores. This is consistent with the transition moments becoming approximately aligned with the long axis of the terminal basepair for the long-linker species.

    KW - RESONANCE ENERGY-TRANSFER

    KW - DOUBLE-STRANDED DNA

    KW - FLUORESCENCE

    KW - FRET

    KW - CY3

    KW - PHOTOPHYSICS

    KW - ACIDS

    KW - TOOL

    U2 - 10.1016/j.bpj.2011.07.007

    DO - 10.1016/j.bpj.2011.07.007

    M3 - Article

    VL - 101

    SP - 1148

    EP - 1154

    JO - Biophysical Journal

    JF - Biophysical Journal

    SN - 0006-3495

    IS - 5

    ER -