Limits of Neutral Drift: Lessons From the In Vitro Evolution of Two Ribozymes

Abstract

The relative contributions of adaptive selection and neutral drift to observed genetic change are unknown, but likely depend on the inherent abundance of functional genotypes in sequence space and how accessible those genotypes are to one another. To better understand the relative roles of selection and drift in evolution, local fitness landscapes for two different RNA ligase ribozymes were examinedusing a continuous in vitro evolution system under conditions that foster the capacity for neutral drift to mediate genetic change.The exploration of sequence spacewas acceleratedby increasing the mutation rate using mutagenic nucleotide analogs. Drift wasencouraged by carrying out evolution within millions of separate compartments to exploit the founder effect. Deep sequencing of individuals from the evolved populations revealed that the distribution of genotypes did not escape the starting local fitness peak, remaining clustered around the sequenceused to initiate evolution.This is consistent with a fitness landscape where high-fitness genotypes are sparse and well isolated, and suggests, at least in this context, that neutral drift alone is not a primary driver of genetic change. Neutral driftdoes, however, provide a repository of genetic variation upon which adaptive selection can act.

Origin of life in a digital microcosm

“…Empirical studies of RNA-based fitness landscapes, biochemical model systems for the origin of life, also show that these landscapes consist of isolated fitness peaks with many non-viable genotypes [19,20]. The fact that both RNAbased landscapes [19,20] and these digital landscapes have similar structures suggests that the evolutionary patterns we see in these Avida experiments may be similar to those one would have seen in the origin of life on Earth. The presence of isolated genotype clusters in both digital and RNA fitness landscapes further suggests that the identity of the first self-replicator may determine life's future evolution, as other evolutionary trajectories are not accessible.…”

“…While the fitness landscapes of some RNA-based genotypes have been mapped [19,20] (and other RNA replicators have been evolved experimentally [21]), in all such cases evolution already had the chance to shape the landscape for these organisms and "dictate", as it were, the sequences most conducive for evolution.…”

Elucidating the molecular architecture of adaptation via evolve and resequence experiments

“…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 .…”

“…It is worth noting that these epistasic interactions are easily detected in this system because the distances between interacting sites in the linear nucleotide chain are typically shorter than sequencing read lengths, hence interaction sites can be collectively analyzed in single sequencing reads. It is important to appreciate that all evolutionary change occurring in the stem part of a ribozyme or an aptamer is epistatic in nature, thus epistasis is pervasive in this system 77,78,116 .…”

The RNA World as a Model System to Study the Origin of Life

“…Nevertheless, the success of these selections suggests that there must be numerous functional families in the totality of sequence space [138,139]. More recently, extensive studies of the fitness landscapes of various functional RNAs have shown mostly isolated, sharp peaks with distinct structural motifs linked by generally unfavorable paths of mutation [140][141][142]. One recently proposed hypothesis is that molecular crowding and confinement may have played a role in increasing pathways of evolution [143], since crowding effects contribute to the folding stability of ribozymes [144][145][146][147][148] and reduce the effect of otherwise deleterious mutations [149].…”