RESUMO
The nonspecific atmosphere around nucleic acids, often termed the ion atmosphere, encompasses a collection of weak ion-nucleic acid interactions. Although nonspecific, the ion atmosphere has been shown to influence nucleic acid folding and structural stability. Studies investigating the composition of the ion atmosphere have shown competitive occupancy of the atmosphere between metal ions in the same solution. Many studies have investigated single ion effects on nucleic acid secondary structure stability; however, no comprehensive studies have investigated how the competitive occupancy of mixed ions in the ion atmosphere influences nucleic acid secondary structure stability. Here, six oligonucleotides were optically melted in buffers containing molar quantities, or mixtures, of either XCl (X = Li, K, Rb, or Cs) or NaCl. A correction factor was developed to better predict RNA duplex stability in solutions containing mixed XCl/NaCl. For solutions containing a 1:1 mixture of XCl/NaCl, one alkali metal chloride contributed more to duplex stability than the other. Overall, there was a 54% improvement in predictive capabilities with the correction factor compared with the standard 1.0 M NaCl nearest-neighbor models. This correction factor can be used in models to better predict RNA secondary structure in solutions containing mixed XCl/NaCl.
RESUMO
Thermodenaturation (melting) curves of macromolecules are used to determine folding thermodynamic parameters. Notably, this insight into RNA and DNA stability underlies nearest neighbor theory and diverse structure prediction tools. Analysis of UV-detected absorbance melting curves is complex and multivariate, requiring many data preprocessing, regression, and error analysis steps. The absorbance melting curve-fitting software MeltWin, introduced in 1996, provided a consistent and facile melting curve analysis platform used in a generation of folding parameters. Unfortunately, MeltWin software is not maintained and relies on idiosyncratic choices of baselines by the user. Herein, we provide MeltR, an open-source, curve-fitting package for analysis of macromolecular thermodynamic data. The MeltR package provides the facile conversion of melting curve data to parameters provided by MeltWin while offering additional features including global fitting of data, auto-baseline generation, and two-state melting analysis. MeltR should be a useful tool for analyzing the next generation of DNA, RNA, and nonnucleic acid macromolecular melting data.
RESUMO
The prediction of RNA secondary structure and thermodynamics from sequence relies on free energy minimization and nearest neighbor parameters. Currently, algorithms used to make these predictions are based on parameters from optical melting studies performed in 1 M NaCl. However, many physiological and biochemical buffers containing RNA include much lower concentrations of monovalent cations and the presence of divalent cations. In order to improve these algorithms, thermodynamic data was previously collected for RNA duplexes in solutions containing 71, 121, 221, and 621 mM Na+. From this data, correction factors for free energy (ΔG°37) and melting temperature (Tm) were derived. Despite these newly derived correction factors for sodium, the stabilizing effects of magnesium have been ignored. Here, the same RNA duplexes were melted in solutions containing 0.5, 1.5, 3.0, and 10.0 mM Mg2+ in the absence of monovalent cations. Correction factors for Tm and ΔG°37 were derived to scale the current parameters to a range of magnesium concentrations. The Tm correction factor predicts the melting temperature within 1.2°C, and the ΔG°37 correction factor predicts the free energy within 0.30 kcalmol. These newly derived magnesium correction factors can be incorporated into algorithms that predict RNA secondary structure and stability from sequence.
