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Measurement of the specific and non-specific binding energies of Mg2+ to RNA

  • A. Martinez-Monge
    Affiliations
    Small Biosystems Lab, Departament de Física de la Matèria Condensada, Facultat de Física, Universitat de Barcelona, Carrer de Martí i Franquès, 1, 08028 Barcelona, Spain
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  • Isabel Pastor
    Affiliations
    Small Biosystems Lab, Departament de Física de la Matèria Condensada, Facultat de Física, Universitat de Barcelona, Carrer de Martí i Franquès, 1, 08028 Barcelona, Spain

    Institut de Nanociència i Nanotecnologia (IN2UB), Universitat de Barcelona, 08028 Barcelona, Spain
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  • Carlos Bustamante
    Affiliations
    Departments of Chemistry, Physics and Molecular and Cell Biology, University of California Berkeley, Berkeley, California

    Howard Hughes Medical Institute University of California Berkeley, Berkeley, California

    Kavli Energy Nanosciences Institute, University of California Berkeley, Berkeley, California
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  • Maria Manosas
    Correspondence
    Corresponding author
    Affiliations
    Small Biosystems Lab, Departament de Física de la Matèria Condensada, Facultat de Física, Universitat de Barcelona, Carrer de Martí i Franquès, 1, 08028 Barcelona, Spain

    Institut de Nanociència i Nanotecnologia (IN2UB), Universitat de Barcelona, 08028 Barcelona, Spain
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  • Felix Ritort
    Correspondence
    Corresponding author
    Affiliations
    Small Biosystems Lab, Departament de Física de la Matèria Condensada, Facultat de Física, Universitat de Barcelona, Carrer de Martí i Franquès, 1, 08028 Barcelona, Spain

    Institut de Nanociència i Nanotecnologia (IN2UB), Universitat de Barcelona, 08028 Barcelona, Spain
    Search for articles by this author

      Abstract

      Determining the non-specific and specific electrostatic contributions of magnesium binding to RNA is a challenging problem. We introduce a single-molecule method based on measuring the folding energy of a native RNA in magnesium and at its equivalent sodium concentration. The latter is defined so that the folding energy in sodium equals the non-specific electrostatic contribution in magnesium. The sodium equivalent can be estimated according to the empirical 100/1 rule (1 M NaCl is equivalent to 10 mM MgCl2), which is a good approximation for most RNAs. The method is applied to an RNA three-way junction (3WJ) that contains specific Mg2+ binding sites and misfolds into a double hairpin structure without binding sites. We mechanically pull the RNA with optical tweezers and use fluctuation theorems to determine the folding energies of the native and misfolded structures in magnesium (10 mM MgCl2) and at the equivalent sodium condition (1 M NaCl). While the free energies of the misfolded structure are equal in magnesium and sodium, they are not for the native structure, the difference being due to the specific binding energy of magnesium to the 3WJ, which equals Δ G 10 kcal/mol. Besides stabilizing the 3WJ, Mg2+ also kinetically rescues it from the misfolded structure over timescales of tens of seconds in a force-dependent manner. The method should generally be applicable to determine the specific binding energies of divalent cations to other tertiary RNAs.
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