Analysis of branched nucleic acid structure using comparative gel electrophoresis

    Research output: Contribution to journalReview article

    20 Citations (Scopus)

    Abstract

    Electrophoresis in polyacrylamide gels provides a simple yet powerful means of analyzing the relative disposition of helical arms in branched nucleic acids. The electrophoretic mobility of DNA or RNA with a central discontinuity is determined by the angle subtended between the arms radiating from the branchpoint. In a multi-helical branchpoint, comparative gel electrophoresis can provide a relative measure of all the inter-helical angles and thus the shape and symmetry of the molecule. Using the long-short arm approach, the electrophoretic mobility of all the species with two helical arms that are longer than all others is compared. This can be done as a function of conditions, allowing the analysis of ion-dependent folding of branched DNA and RNA species. Notable successes for the technique include the four-way (Holliday) junction in DNA and helical junctions in functionally significant RNA species such as ribozymes. Many of these structures have subsequently been proved correct by crystallography or other methods, up to 10 years later in the case of the Holliday junction, just as important, the technique has riot failed to date. Comparative gel electrophoresis can provide a window on both fast and slow conformational equilibria such as conformer exchange in four-way DNA junctions. But perhaps the biggest test of the approach has been to deduce the structures of complexes of four-way DNA junctions with proteins. Two recent crystallographic structures show that the global structures were correctly deduced by electrophoresis, proving the worth of the method even in these rather complex systems. Comparative gel electrophoresis is a robust method for the analysis of branched nucleic acids and their complexes.

    Original languageEnglish
    Pages (from-to)139
    Number of pages39
    JournalQuarterly Reviews of Biophysics
    Volume41
    Issue number1
    DOIs
    Publication statusPublished - Feb 2008

    Keywords

    • 4-WAY DNA JUNCTION
    • RESONANCE ENERGY-TRANSFER
    • HUMAN-IMMUNODEFICIENCY-VIRUS
    • NATURAL HAMMERHEAD RIBOZYME
    • T7 ENDONUCLEASE-I
    • KINK-TURN RNA
    • HOLLIDAY JUNCTION
    • CRYSTAL-STRUCTURE
    • HAIRPIN RIBOZYME
    • RESOLVING ENZYME

    Cite this

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    title = "Analysis of branched nucleic acid structure using comparative gel electrophoresis",
    abstract = "Electrophoresis in polyacrylamide gels provides a simple yet powerful means of analyzing the relative disposition of helical arms in branched nucleic acids. The electrophoretic mobility of DNA or RNA with a central discontinuity is determined by the angle subtended between the arms radiating from the branchpoint. In a multi-helical branchpoint, comparative gel electrophoresis can provide a relative measure of all the inter-helical angles and thus the shape and symmetry of the molecule. Using the long-short arm approach, the electrophoretic mobility of all the species with two helical arms that are longer than all others is compared. This can be done as a function of conditions, allowing the analysis of ion-dependent folding of branched DNA and RNA species. Notable successes for the technique include the four-way (Holliday) junction in DNA and helical junctions in functionally significant RNA species such as ribozymes. Many of these structures have subsequently been proved correct by crystallography or other methods, up to 10 years later in the case of the Holliday junction, just as important, the technique has riot failed to date. Comparative gel electrophoresis can provide a window on both fast and slow conformational equilibria such as conformer exchange in four-way DNA junctions. But perhaps the biggest test of the approach has been to deduce the structures of complexes of four-way DNA junctions with proteins. Two recent crystallographic structures show that the global structures were correctly deduced by electrophoresis, proving the worth of the method even in these rather complex systems. Comparative gel electrophoresis is a robust method for the analysis of branched nucleic acids and their complexes.",
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    author = "Lilley, {David M. J.}",
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    language = "English",
    volume = "41",
    pages = "139",
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    Analysis of branched nucleic acid structure using comparative gel electrophoresis. / Lilley, David M. J.

    In: Quarterly Reviews of Biophysics, Vol. 41, No. 1, 02.2008, p. 139.

    Research output: Contribution to journalReview article

    TY - JOUR

    T1 - Analysis of branched nucleic acid structure using comparative gel electrophoresis

    AU - Lilley, David M. J.

    PY - 2008/2

    Y1 - 2008/2

    N2 - Electrophoresis in polyacrylamide gels provides a simple yet powerful means of analyzing the relative disposition of helical arms in branched nucleic acids. The electrophoretic mobility of DNA or RNA with a central discontinuity is determined by the angle subtended between the arms radiating from the branchpoint. In a multi-helical branchpoint, comparative gel electrophoresis can provide a relative measure of all the inter-helical angles and thus the shape and symmetry of the molecule. Using the long-short arm approach, the electrophoretic mobility of all the species with two helical arms that are longer than all others is compared. This can be done as a function of conditions, allowing the analysis of ion-dependent folding of branched DNA and RNA species. Notable successes for the technique include the four-way (Holliday) junction in DNA and helical junctions in functionally significant RNA species such as ribozymes. Many of these structures have subsequently been proved correct by crystallography or other methods, up to 10 years later in the case of the Holliday junction, just as important, the technique has riot failed to date. Comparative gel electrophoresis can provide a window on both fast and slow conformational equilibria such as conformer exchange in four-way DNA junctions. But perhaps the biggest test of the approach has been to deduce the structures of complexes of four-way DNA junctions with proteins. Two recent crystallographic structures show that the global structures were correctly deduced by electrophoresis, proving the worth of the method even in these rather complex systems. Comparative gel electrophoresis is a robust method for the analysis of branched nucleic acids and their complexes.

    AB - Electrophoresis in polyacrylamide gels provides a simple yet powerful means of analyzing the relative disposition of helical arms in branched nucleic acids. The electrophoretic mobility of DNA or RNA with a central discontinuity is determined by the angle subtended between the arms radiating from the branchpoint. In a multi-helical branchpoint, comparative gel electrophoresis can provide a relative measure of all the inter-helical angles and thus the shape and symmetry of the molecule. Using the long-short arm approach, the electrophoretic mobility of all the species with two helical arms that are longer than all others is compared. This can be done as a function of conditions, allowing the analysis of ion-dependent folding of branched DNA and RNA species. Notable successes for the technique include the four-way (Holliday) junction in DNA and helical junctions in functionally significant RNA species such as ribozymes. Many of these structures have subsequently been proved correct by crystallography or other methods, up to 10 years later in the case of the Holliday junction, just as important, the technique has riot failed to date. Comparative gel electrophoresis can provide a window on both fast and slow conformational equilibria such as conformer exchange in four-way DNA junctions. But perhaps the biggest test of the approach has been to deduce the structures of complexes of four-way DNA junctions with proteins. Two recent crystallographic structures show that the global structures were correctly deduced by electrophoresis, proving the worth of the method even in these rather complex systems. Comparative gel electrophoresis is a robust method for the analysis of branched nucleic acids and their complexes.

    KW - 4-WAY DNA JUNCTION

    KW - RESONANCE ENERGY-TRANSFER

    KW - HUMAN-IMMUNODEFICIENCY-VIRUS

    KW - NATURAL HAMMERHEAD RIBOZYME

    KW - T7 ENDONUCLEASE-I

    KW - KINK-TURN RNA

    KW - HOLLIDAY JUNCTION

    KW - CRYSTAL-STRUCTURE

    KW - HAIRPIN RIBOZYME

    KW - RESOLVING ENZYME

    U2 - 10.1017/S0033583508004678

    DO - 10.1017/S0033583508004678

    M3 - Review article

    VL - 41

    SP - 139

    JO - Quarterly Reviews of Biophysics

    JF - Quarterly Reviews of Biophysics

    SN - 0033-5835

    IS - 1

    ER -