Abstract
Catalytic RNAs are attractive objects for studying molecular evolution. To understand how RNA libraries can evolve from randomness toward highly active catalysts, we analyze the original samples that led to the discovery of Diels–Alderase ribozymes by next-generation sequencing. Known structure-activity relationships are used to correlate abundance with catalytic performance. We find that efficient catalysts arose not just from selection for reactivity among the members of the starting library, but from improvement of less potent precursors by mutations. We observe changes in the ribozyme population in response to increasing selection pressure. Surprisingly, even after many rounds of enrichment, the libraries are highly diverse, suggesting that potential catalysts are more abundant in random space than generally thought. To highlight the use of next-generation sequencing as a tool for in vitro selections, we also apply this technique to a recent, less characterized ribozyme selection. Making use of the correlation between sequence evolution and catalytic activity, we predict mutations that improve ribozyme activity and validate them biochemically. Our study reveals principles underlying ribozyme in vitro selections and provides guidelines to render future selections more efficient, as well as to predict the conservation of key structural elements, allowing the rational improvement of catalysts.
Highlights
RNA, a simple molecule, possesses a high catalytic potential
For each pool (DNA template of the respective selection round), sequences were sorted according to read numbers (Materials and Methods, Supplementary Dataset S1) and at first, sequence diversity was analyzed for the DAse selection (Figure 1C, Supplementary Figure S2), taking into account the relative selection pressure (Supplementary Text S1)
The most prominent reduction in complexity was observed in pool 6, and from pool 7 onward, the overall numeric pool diversity stagnated and remained constant the catalytic activity still increased markedly under increased selection pressure (Figure 1B and C, Supplementary Figure S2, Supplementary Tables S3 and S4)
Summary
Found to occur naturally [1,2], ribozymes catalyzing a wide range of chemical transformations [3,4,5,6,7] have been isolated using combinatorial in vitro selections [8,9] In these experiments, a population of different RNAs (typically $1014 sequences) is challenged for a specific task, and the selection process is designed such that few active sequences are retained and enzymatically amplified. To observe a significant enrichment of active sequences over background, 8–15 iterative rounds are usually conducted, and mutational errors in the amplification steps are assumed to make this a true evolutionary process in which species evolve that were not contained in the starting population [10]. There is no certainty about how RNA populations react to changes in selection pressure, and how precisely the composition and diversity vary over the selection cycles
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