In vitro selections are the only known methods to generate catalytic RNAs (ribozymes) that do not exist in nature. Such new ribozymes are used as biochemical tools, or to address questions on early stages of life. In both cases, it is helpful to identify the shortest possible ribozymes since they are easier to deploy as a tool, and because they are more likely to have emerged in a prebiotic environment. One of our previous selection experiments led to a library containing hundreds of different ribozyme clusters that catalyze the triphosphorylation of their 5'-terminus. This selection showed that RNA systems can use the prebiotically plausible molecule cyclic trimetaphosphate as an energy source. From this selected ribozyme library, the shortest ribozyme that was previously identified had a length of 67 nucleotides. Here we describe a combinatorial method to identify short ribozymes from libraries containing many ribozymes. Using this protocol on the library of triphosphorylation ribozymes, we identified a 17-nucleotide sequence motif embedded in a 44-nucleotide pseudoknot structure. The described combinatorial approach can be used to analyze libraries obtained by different in vitro selection experiments.
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