delta-Melt: Nucleic acid conformational penalties from melting experiments

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 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.