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Abstract 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. Graphical Abstract 

Abstract The prebiotic formation of RNA building blocks is well‐supported experimentally, yet the emergence of sequence‐ and structure‐specific RNA oligomers is generally attributed to biological selection via Darwinian evolution rather than prebiotic chemical selectivity. In this study, we used deep sequencing to investigate the partitioning of randomized RNA overhangs into ligated products by either splinted ligation or loop‐closing ligation. Comprehensive sequence‐reactivity profiles revealed that loop‐closing ligation preferentially yields hairpin structures with loop sequences UNNG, CNNG, and GNNA (where N represents A, C, G, or U) under competing conditions. In contrast, splinted ligation products tended to be GC rich. Notably, the overhang sequences that preferentially partition to loop‐closing ligation significantly overlap with the most common biological tetraloops, whereas the overhangs favoring splinted ligation exhibit an inverse correlation with biological tetraloops. Applying these sequence rules enables the high‐efficiency assembly of functional ribozymes from short RNAs without template inhibition. Our findings suggest that the RNA tetraloop structures that are common in biology may have been predisposed and prevalent in the prebiotic pool of RNAs, prior to the advent of Darwinian evolution. We suggest that the one‐step prebiotic chemical process of loop‐closing ligation could have favored the emergence of the first RNA functions.