Analysis of natural structures and chemical mapping data reveals local stability compensation in RNA.

RNA molecules adopt complex structures that perform essential biological functions across all forms of life, making them promising candidates for therapeutic applications. However, our ability to design new RNA structures remains limited by an incomplete understanding of their folding principles. While global metrics such as the minimum free energy are widely used, they are at odds with naturally occurring structures and incompatible with established design rules. Here, we introduce local stability compensation (LSC), a principle that RNA folding is governed by the local balance between destabilizing loops and their stabilizing adjacent stems, challenging the focus on global energetic optimization. Analysis of over 100 000 RNA structures revealed that LSC signatures are particularly pronounced in bulges and their adjacent stems, with distinct patterns across different RNA families that align with their biological functions. To validate LSC experimentally, we systematically analyzed thousands of RNA variants using DMS chemical mapping. Our results demonstrate that stem folding, as measured by reactivity, correlates with LSC (R² = 0.458 for hairpin loops) and that instabilities show no significant effect on folding for distal stems. These findings demonstrate that LSC can be a guiding principle for understanding RNA function and for the rational design of custom RNAs.