Nucleic Acid Conformational Penalties from Optical Melting Experiments

Abstract

Measuring the thermodynamic propensities of biomolecules to undergo conformational changes is important for understanding how they fold and function. However, this often requires accurately measuring the relative populations of low-abundance short-lived conformational states in the apo ensemble that may fall outside the detection limits of conventional biophysical methods. NMR methods for characterizing such states are costly, laborious, technically demanding, and have detection limits. Here, we developed an approach for measuring the thermodynamic propensities of nucleic acids to adopt alternative conformations using optical melting experiments and chemical modifications that are an order of magnitude faster and lower in cost compared to NMR, that in theory have no limits on population and timescale, and molecular size. A broad range of applications validated by NMR data establish the generality of the approach, which enabled thermodynamic mapping of the sequence-dependence of the energetics of DNA conformational substates and the characterization of ultra-low abundance conformational states invisible to NMR.