Population dynamics of ribozymes during in vitro selection

Abstract

Ribozymes are central to models of RNA-based primordial life. Understanding how ribozymes emerge from RNA populations under changing selection pressures can help delineate the biochemical constraints and capabilities of early RNA-based biology. In vitro selection has been instrumental in isolating ribozymes with diverse functions from combinatorial populations; however, their population dynamics during selection remain poorly characterized. By analyzing high-throughput sequencing data produced from all rounds of an in vitro selection experiment, consisting of over 5 million unique sequences, we tracked how sequences rose and fell in abundance during selection. We found that the most abundant sequences emerged early and maintained their dominance, suggesting that only a few cycles of selection-amplification may be sufficient to isolate well-adapted ribozymes. Characterization of the catalytic activities of the dominant sequences in each selection round indicated that abundance did not always predict activity. This observation was reinforced through nucleotide conservation analysis and activity assays of close sequence variants of the most dominant ribozyme. Our analyses show that sequence complementarity with the substrate emerged as a common feature as early as the first round and persisted throughout, highlighting early convergence of diverse sequence populations on a beneficial catalytic strategy. By combining bioinformatics and experimental approaches, our work presents a detailed description of ribozyme population dynamics during an in vitro selection experiment and provides a high-resolution view of how functional RNA sequences compete for survival and propagation under selection pressure.