Measuring Excess Heat Capacities of Deoxyribonucleic Acid (DNA) Folding at the Single-Molecule Level

Measurements of the thermodynamic properties of biomolecular folding (ΔG°, ΔH°, ΔS°, etc.) provide a wealth of information on the folding process and have long played a central role in biophysical investigation. In particular, the excess heat capacity of folding (ΔC_P) is crucial, as typically measured in bulk ensemble studies by differential scanning calorimetry (DSC) and isothermal titration calorimetry (ITC). Here, we report the first measurements of ΔC_P at the single-molecule level using the single-molecule fluorescence resonance energy transfer (smFRET) as well as the very first measurements of the heat capacity change associated with achieving the transition state (ΔC^‡_P) for nucleic acid folding. The deoxyribonucleic acid (DNA) hairpin used in these studies exhibits an excess heat capacity for hybridization (ΔC_P = -340 ± 60 J/mol/K per base pair) consistent with the range of literature expectations (ΔC_P = -100 to -420 J/mol/K per base pair). Furthermore, the measured activation heat capacities (ΔC^‡_P) for such hairpin unfolding are consistent with a folding transition state containing few fully formed base pairs, in agreement with prevailing models of DNA hybridization.