Next-generation sequencing reveals how RNA catalysts evolve from random space

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.

Elucidating the molecular architecture of adaptation via evolve and resequence experiments

“…NGS allows testing the selected population in earlier rounds and provides a much more complete picture of the final array of winning genotypes 44 . In fact, direct experimental measurement of the fitness landscapes of RNA ligase 15 , kinase 45 , Diels-Alderase 46 , and self-splicing ribozymes 47 are obtained at much higher resolution than ever before. Combined with microfluidic analytical platforms, NGS can be directly coupled to activity measurement, revealing the fitness landscape of a functional RNA 48,49 .…”

“…In one example, selection for GTP aptamers directly yielded an optimal family of aptamer sequences, suggesting that these sequences were present in the initial pool; by contrast, other aptamer families improved their binding affinity by several orders of magnitude upon mutagenesis and re-selection, suggesting that the initial sequences of these aptamers were relatively far from optimal and required subsequent newly arising mutations for optimal activity 35,77 . To date, the evidence from NGS of in vitro selected pools suggest that individual families of functional RNAs exist in the starting pools, but greatly benefit from mutagenesis 15,46,77 or synthetic shuffling 45 to uncover the most active variants, which then do not tend to drift towards other peaks in the fitness landscape 78 .…”

Viroid quasispecies revealed by deep sequencing

“…Such mutations may allow fast adaptation to new Figure 4. Dependence of the calculated error thresholds ( t max ; see equation (3)) on selective advantage ( s ; see equation (2)) of the master copy for individual PSTVd variants based on the P error values calculated from analysis of the respective vsRNA populations (see Table 1 environments, as shown for example for ribozyme selection, 44 and may also increase the spectrum of host targets susceptible to post-transcriptional gene silencing. Thus, a PLMVd variant that contains a specific hairpin insertion sequence and induces a loss of pigmentation known as 'peach calico', expresses a vsRNA that targets the mRNA of chloroplastic heat-shock protein 90 leading to chloroplast malformation.…”

Synthetic, Switchable Enzymes

“…Candidate catalytic antibodies also include those that catalyze the Diels-Alder reaction [Hilvert et al, 1989; Marti et al, 2008; Romesberg et al, 1998]. Candidate ribozymes also exist and include a set of artificial ribozymes that catalyze the Diels-Alder reaction and that have already been characterised [Ameta et al, 2014]. …”