This study presents the complete thermodynamic library of all 10 Watson–Crick DNA nearest-neighbor interactions. The researchers obtained thermodynamic data from calorimetric studies on 19 DNA oligomers and 9 DNA polymers. They demonstrate how these data can be used to calculate the stability and predict the temperature-dependent behavior of any DNA duplex structure based on its base sequence. The method is illustrated by predicting transition enthalpies and free energies for a series of DNA oligomers, which show excellent agreement with experimental values. This confirms that DNA duplex stability can be considered the sum of its nearest-neighbor interactions. The study highlights that DNA duplex stability depends primarily on the identity of the nearest-neighbor bases, not just the base composition. The researchers characterized the helix-to-coil transitions of 19 oligonucleotides and 9 polynucleotides using DSC and temperature-dependent UV absorption spectroscopy. They resolved thermodynamic profiles for all 10 DNA nearest-neighbor interactions and demonstrated that DNA duplex structures can be considered the sum of their nearest-neighbor interactions. The thermodynamic data allow the prediction of the stability and melting behavior of any DNA duplex from its primary sequence. This capability is valuable in various applications, such as predicting the stability of a probe–gene complex, selecting optimal conditions for hybridization experiments, determining the minimum length of a probe, and predicting the influence of specific mutations on DNA stability. The data also enable the prediction of the relative stabilities of local domains within a DNA duplex. The study emphasizes the importance of having a DNA thermodynamic database for evaluating sequence-dependent structural preferences in DNA molecules. The researchers developed a program to obtain the required DNA thermodynamic data using differential scanning calorimetry and UV spectroscopy. They demonstrated that DNA duplex stability can be predicted from its base sequence, which has important implications for understanding DNA structure and function. The thermodynamic data presented in this study provide a foundation for predicting the complete secondary structure of a DNA molecule based on its primary sequence.This study presents the complete thermodynamic library of all 10 Watson–Crick DNA nearest-neighbor interactions. The researchers obtained thermodynamic data from calorimetric studies on 19 DNA oligomers and 9 DNA polymers. They demonstrate how these data can be used to calculate the stability and predict the temperature-dependent behavior of any DNA duplex structure based on its base sequence. The method is illustrated by predicting transition enthalpies and free energies for a series of DNA oligomers, which show excellent agreement with experimental values. This confirms that DNA duplex stability can be considered the sum of its nearest-neighbor interactions. The study highlights that DNA duplex stability depends primarily on the identity of the nearest-neighbor bases, not just the base composition. The researchers characterized the helix-to-coil transitions of 19 oligonucleotides and 9 polynucleotides using DSC and temperature-dependent UV absorption spectroscopy. They resolved thermodynamic profiles for all 10 DNA nearest-neighbor interactions and demonstrated that DNA duplex structures can be considered the sum of their nearest-neighbor interactions. The thermodynamic data allow the prediction of the stability and melting behavior of any DNA duplex from its primary sequence. This capability is valuable in various applications, such as predicting the stability of a probe–gene complex, selecting optimal conditions for hybridization experiments, determining the minimum length of a probe, and predicting the influence of specific mutations on DNA stability. The data also enable the prediction of the relative stabilities of local domains within a DNA duplex. The study emphasizes the importance of having a DNA thermodynamic database for evaluating sequence-dependent structural preferences in DNA molecules. The researchers developed a program to obtain the required DNA thermodynamic data using differential scanning calorimetry and UV spectroscopy. They demonstrated that DNA duplex stability can be predicted from its base sequence, which has important implications for understanding DNA structure and function. The thermodynamic data presented in this study provide a foundation for predicting the complete secondary structure of a DNA molecule based on its primary sequence.