RNA molecules have numerous functions including catalysis and small molecule recognition, which typically arise from a tertiary structure. There is increasing interest in mechanisms for the thermostability of functional RNA molecules. Sosnick, Pan, and co-workers introduced the notion of "functional stability" as the free energy of the tertiary (functional) state relative to the next most stable (nonfunctional) state. We investigated the extent to which secondary structure stability influences the functional stability of nucleic acids. Intramolecularly folding DNA triplexes containing alternating Tasterisk inside circle signAT and C+asterisk inside circle signGC base triples were used as a three-state model for the folding of nucleic acids with functional tertiary structures. A four-base-pair tunable region was included adjacent to the triplex-forming portion of the helix to allow secondary structure strength to be modulated. The degree of folding cooperativity was controlled by pH, with high cooperativity maintained by lower pH (5.5), and no cooperativity by higher pH (7.0). We find a linear relationship between functional free energy and the free energy of the secondary structure element adjacent to tertiary interactions, but only when folding is cooperative. We translate the definition of functional stability into equations and perform simulations of the thermodynamic data, which lend support to this model. The ability to increase the melting temperature of tertiary structure by strengthening base-pairing interactions separate from tertiary interactions provides a simple means for evolving thermostability in functional RNAs.
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