By revealing that RNA base pairs and tertiary contacts open at low free energies (0.5–3 kcal/mol), PRIME resolves a decades-long debate, overturns assumptions of structural rigidity, and makes nucleotide-resolution thermodynamics scalable to any RNA.
Chemical probing kinetics reveals that RNA nucleotides ubiquitously undergo low-energy structural fluctuations (0.5-3 kcal/mol) at physiological conditions, with nucleotide-resolution energetic measurements demonstrating long-range coupling through secondary structure dynamics and energetic compensation between secondary and tertiary interactions during RNA folding.
RNA function is governed by RNA folding, but strategies for measuring RNA folding thermodynamics are limited, and fundamental questions such as the energy of base pair opening remain debated. Here we introduce Probing-Resolved Inference of Molecular Energetics (PRIME) to extract nucleotide-resolution RNA structural energetics from scalable chemical probing experiments. Applying PRIME to diverse RNAs, we find that RNA base pairs and tertiary interactions dynamically open with free energies of 0.5-3 kcal/mol, revealing that RNA nucleotides ubiquitously sample open conformations at biologically accessible energies. PRIME further resolves energetic coupling across RNAs, providing an energetic understanding of RNA structural dynamics and long-range coordination in RNA folding. PRIME represents a widely accessible strategy for interrogating RNA thermodynamics, enabling mechanistic understanding and engineering of RNA biology.