Measuring thermodynamic preferences to form non-native conformations in nucleic acids using melting experiments reveals a rich sequence-specific DNA conformational landscape

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 (+ 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 access thermodynamic information regarding regions of the free energy landscape of biomolecules beyond the native folded and unfolded conformations.Significance StatementThermodynamic preferences of nucleic acids to adopt non-native conformations are crucial for understanding how they function but prove difficult to measure experimentally. As a result, little is known about how these thermodynamic preferences vary with sequence and structural contexts, physiological conditions, and chemical modifications. Here, we show that modifications stabilizing non-native conformations and rendering them the major state, in conjunction with melting experiments, enable facile measurements of thermodynamic preferences to form various non-native conformations in DNA and RNA. delta-melt provided rare insights into the cooperativity of forming tandem Hoogsteen base pairs and revealed large and distinct sequence-specific preferences to form G-C+ and A-T Hoogsteen and A-T base open conformations in DNA, which may contribute to sequence-specific DNA biochemistry.