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Thermodynamic preferences to form non-native conformations are crucial for understanding how nucleic acids fold and function. However, they are difficult to measure experimentally because this requires accurately determining the population of minor low-abundance (<10%) conformations in a sea of other conformations. Here we show that melting experiments enable facile measurements of thermodynamic preferences to adopt non-native conformations in DNA and RNA. The key to this ‘delta-melt’ approach is to use chemical modifications to render specific minor non-native conformations the major state. The validity and robustness of delta-melt is established for four different non-native conformations under various physiological conditions and sequence contexts through independent measurements of thermodynamic preferences using NMR. delta-melt is fast, simple, cost-effective, and enables thermodynamic preferences to be measured for exceptionally low-populated conformations. Using delta-melt, we obtained rare insights into conformational cooperativity, obtaining evidence for significant cooperativity (1.0-2.5 kcal/mol) when simultaneously forming two adjacent Hoogsteen base pairs. We also measured the thermodynamic preferences to form G-C⁺ and A-T Hoogsteen and A-T base open states for nearly all sixteen trinucleotide sequence contexts and found distinct sequence-specific variations on the order of 2-3 kcal/mol. This rich landscape of sequence-specific non-native minor conformations in the DNA double helix may help shape the sequence-specificity of DNA biochemistry. Thus, melting experiments can now be used to …