Measuring thermodynamic preferences to form non-native conformations in nucleic acids using ultraviolet melting

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• Nucleic acid folding and function depend on thermodynamic preferences for non-native conformations.
• Experimentally measuring these minor (<10%) conformations is challenging.

Purpose of the Study:

• To develop a facile method for measuring thermodynamic preferences of non-native DNA and RNA conformations.
• To establish the delta-melt approach as a robust and cost-effective alternative to existing methods.

Main Methods:

• The delta-melt approach utilizes chemical modifications to shift the equilibrium towards minor non-native conformations.
• Melting experiments are employed to measure the thermodynamic preferences of these altered states.
• Results were validated against Nuclear Magnetic Resonance (NMR) spectroscopy under various conditions.

Main Results:

• Delta-melt accurately measures thermodynamic preferences for low-abundance non-native conformations.
• Significant conformational cooperativity was observed between adjacent Hoogsteen base pairs (1.0–2.5 kcal/mol).
• Sequence-specific variations in non-native conformations were quantified across 16 trinucleotide contexts.

Conclusions:

• The delta-melt method provides a rapid, cost-effective way to study nucleic acid thermodynamics beyond native states.
• This approach offers new insights into the sequence-specific landscape of DNA non-native conformations.
• Melting experiments can now probe previously inaccessible regions of biomolecular free energy landscapes.