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Gene editing technologies sparked a renewed interest in the hybridization of DNA/RNA duplexes, yet little improvement on nearest-neighbour parameters was made over the past two decades. For low sodium concentration no parameter set was yet calculated. Here, we revised the existing experimental datasets and used an expanded set of sequences from which we recalculated the nearest-neighbour parameters, reducing the average temperature prediction uncertainty to 1.6 °C. Two experimental sets using temperatures extracted via different methods were used with similar results, with the curve-fitting method achieving a slight advantage in prediction quality over other methods. Additionally, we obtained new parameters for low salt with an average uncertainty of 0.98 °C. We also tested several types of salt correction factors and concluded that it is advisable to use those originally developed for RNA/RNA rather than for DNA/DNA.

Melting temperature, T(m), is an important property of nucleic acid duplexes. It is typically determined from spectroscopic or calorimetric melting experiments. More than one analytical method has been used to extract T(m) values from experimental melting data. Unfortunately, different methods do not give the same results; the same melting data can be assigned different T(m) values depending upon which method is used to process that data. Inconsistencies or systematic errors between T(m)s reported in published data sets can be significant and add confusion to the field. Errors introduced from analysis can be greater than experimental errors, ranging from a fraction of degree to several degrees. Of the various methods, the most consistent and meaningful approach defines melting temperature as the temperature at the transition midpoint where half of the base pairs are melted and standard free energy is zero. Assuming a two-state melting behavior, we present here a set of general equations that can be used to reconcile these analytical T(m) differences and convert results to the correct melting temperatures at the transition midpoint. Melting temperatures collected from published sources, which were analyzed using different methods, can now be corrected for these discrepancies and compared on equal footing. The similar corrections apply to T(m) differences between calorimetric and spectroscopic melting curves. New algorithm for selection of linear sloping baselines, 2nd derivative method, is suggested, which can be used to automate melting curve analysis.

Cyanine dyes are important chemical modifications of oligonucleotides exhibiting intensive and stable fluorescence at visible light wavelengths. When Cy3 or Cy5 dye is attached to 5′ end of a DNA duplex, the dye stacks on the terminal base pair and stabilizes the duplex. Using optical melting experiments, we have determined thermodynamic parameters that can predict the effects of the dyes on duplex stability quantitatively (ΔG°, Tm). Both Cy dyes enhance duplex formation by 1.2kcal/mol on average, however, this Gibbs energy contribution is sequence-dependent. If the Cy5 is attached to a pyrimidine nucleotide of pyrimidine–purine base pair, the stabilization is larger compared to the attachment to a purine nucleotide. This is likely due to increased stacking interactions of the dye to the purine of the complementary strand. Dangling (unpaired) nucleotides at duplex terminus are also known to enhance duplex stability. Stabilization originated from the Cy dyes is significantly larger than the stabilization due to the presence of dangling nucleotides. If both the dangling base and Cy3 are present, their thermodynamic contributions are approximately additive. New thermodynamic parameters improve predictions of duplex folding, which will help design oligonucleotide sequences for biophysical, biological, engineering, and nanotechnology applications.

β 2 -microglobulin (β 2 m) is the smallest building block of molecules belonging to the immunoglobulin superfamily. By comparing thermodynamic and structural characteristics of chicken β 2 m with those of other species, we seek to elucidate whether it is possible to pinpoint features that set the avian protein apart from other β 2 m. The thermodynamic assays revealed that chicken β 2 m exhibits a lower melting temperature than human β 2 m, and the H/D exchange behavior observed by infrared spectroscopy indicates a more flexible structure of the former protein. To understand these differences at a molecular level, we determined the structure of free chicken β 2 m by X-ray crystallography to a resolution of 2.0 A. Our comparisons indicate that certain biophysical characteristics of the chicken protein, particularly its conformational flexibility, diverge considerably from those of the other β 2 m analyzed, although basic structural features have been retained through evolution.

The effect of high supercoil densities on the melting characteristics of a supercoiled DNA has been studied. It is found that although the melting temperature increases abruptly on converting a linear DNA merely into the relaxed circular form, it falls back substantially at high supercoil densities. It is further predicted, in such cases, that the number of melted base pairs should be significantly enhanced even at the physiological temperature, which may facilitate the binding of other molecules to the highly supercoiled DNA.

The order-disorder transitions of the double helices formed by the ribo-oligoadenylic acids rA 8 and rA 10 at pH 4.2 have been investigated in a series of organic/aqueous mixed solvents. Melting temperature data, T m derived from the uv melting curves were used to define the stability of the double helices in the different mixed solvent systems. It was found that the extent of helix destabilization depended in a predictable fashion on both the quantity and the nature of the added organic solvents. For the C 1 through C 4 aliphatic alcohols, the longer, less branched alcohols proved to be more effective destabilizers of the helical structure. Significantly, the amides proved to be more powerful destabilizers than the alcohols. Analysis of the melting curves provided the Van't Hoff enthalpy change for each transition. The data are interpreted in terms of the role of solvent in the stabilization of ribonucleic acid structure.