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Thermodynamic stability and mutational robustness of secondary structure are critical to the function and evolutionary longevity of RNA molecules. We hypothesize that natural and artificial selection for functional molecules favors the formation of structures that are stable to both thermal and mutational perturbation. There is little direct evidence, however, that functional RNA molecules have been selected for their stability. Here we use thermodynamic secondary structure prediction algorithms to compare the thermal and mutational robustness of over 1000 naturally and artificially evolved molecules. Although we find evidence for the evolution of both types of stability in both sets of molecules, the naturally evolved functional RNA molecules were significantly more stable than those selected in vitro, and artificially evolved catalysts (ribozymes) were more stable than artificially evolved binding species (aptamers). The thermostability of RNA molecules bred in the laboratory is probably not constrained by a lack of suitable variation in the sequence pool but, rather, by intrinsic biases in the selection process.

T heoretical studies focusing on the nature of landscapes that correlate molecular sequences to molecular function have mainly been carried out in silico due to the vast amounts of data that are needed. Automated in vitro selection is capable of producing significant amounts of data in a short time, making theoretical modeling with real experimental data attainable. A Biomek 2000 Laboratory Automation Workstation has been outfitted to carry out multiple in vitro nucleic acid selections in parallel, yielding substantial amounts of data for theoretical studies. A random sequence population of nucleic acids is initially generated by a combination of chemical synthesis and enzymatic amplification. On the workstation, this population is parsed for its ability to bind a protein, lysozyme. After each round of selection, the selected nucleic acid binding species (also known as aptamers) are amplified by a combination of reverse transcription polymerase chain reaction (PCR) and in vitro transcription. All eight pools that have undergone selection have yielded different sequences.