Predicting Stability of DNA Duplexes in Solutions Containing Magnesium and Monovalent Cations

Abstract

Accurate predictions of DNA stability in physiological and enzyme buffers are important for the design of many biological and biochemical assays. We therefore investigated the effects of magnesium, potassium, sodium, Tris ions, and deoxynucleoside triphosphates on melting profiles of duplex DNA oligomers and collected large melting data sets. An empirical correction function was developed that predicts melting temperatures, transition enthalpies, entropies, and free energies in buffers containing magnesium and monovalent cations. The new correction function significantly improves the accuracy of predictions and accounts for ion concentration, G-C base pair content, and length of the oligonucleotides. The competitive effects of potassium and magnesium ions were characterized. If the concentration ratio of [Mg 2+ ] 0.5 /[Mon + ] is less than 0.22 M -1/2 , monovalent ions (K + , Na + ) are dominant. Effects of magnesium ions dominate and determine duplex stability at higher ratios. Typical reaction conditions for PCR and DNA sequencing (1.5-5 mM magnesium and 20-100 mM monovalent cations) fall within this range. Conditions were identified where monovalent and divalent cations compete and their stability effects are more complex. When duplexes denature, some of the Mg 2+ ions associated with the DNA are released. The number of released magnesium ions per phosphate charge is sequence dependent and decreases surprisingly with increasing oligonucleotide length.Interactions of magnesium, sodium and potassium ions with DNA 1 and RNA molecules are important for essential functions of living cells and for molecular biology applications. Both divalent and monovalent cations bind to nucleic acid molecules and affect their physical properties (1). Magnesium cations stabilize nucleic acid duplexes and facilitate their folding into secondary and tertiary structures (2), which are biologically active. Mg 2+ ions are also necessary cofactors of many enzymatic reactions involving nucleic acids (3). The total magnesium concentrations in various cells range from 5 to 30 mM; however, free Mg 2+ concentrations are tightly controlled between 0.4 and 1.2 mM (4). Binding of monovalent cations also increases duplex stability. The major intracellular monovalent cation is K + , while the major monovalent cation in extracellular fluid is Na + .Several theoretical models have been proposed to describe the complex phenomena of associations between cations and nucleic acids. The most widely used approaches are the mean-field approximation of the Poisson-Boltzmann equation (5-8), the counterion condensation model (9, 10), and Monte Carlo simulations (11,12). There have been few experimental studies addressing effects of Mg 2+ ion on the stability of short DNA duplexes (13-16), and comprehensive experimental melting data are not available. The majority of melting studies in magnesium buffers investigated genomic DNAs (e.g., calf thymus) (5,(17)(18)(19), long polymeric repeats (20-22), or biologically active RNA molecules (2, 23). Pione...

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