In vitroevolution of distinct self-cleaving ribozymes in diverse environments

Abstract

In vitro evolution experiments have long been used to evaluate the roles of RNA in both modern and ancient biology, and as a tool for biotechnology applications. The conditions under which these experiments have been conducted, however, do not reflect the range of cellular environments in modern biology or our understanding of chemical environments on the early earth, when the atmosphere and oceans were largely anoxic and soluble Fe2+ was abundant. To test the impact of environmental factors relevant to RNA's potential role in the earliest forms of life, we evolved populations of self-cleaving ribozymes in an anoxic atmosphere with varying pH in the presence of either Fe2+ or Mg2+. Populations evolved under these different conditions are dominated by different sequences and secondary structures, demonstrating global differences in the underlying fitness landscapes. Comparisons between evolutionary outcomes and catalytic activities also indicate that Mg2+ can readily take the place of Fe2+ in supporting the catalysis of RNA cleavage at neutral pH, but not at lower pH. These results highlight the importance of considering the specific environments in which functional biopolymers evolve when evaluating their potential roles in the origin of life, extant biology, or biotechnology.

Evolution of ribozymes in the presence of a mineral surface

“…5A) are comparable to the differences between a previously generated pair of replicate evolutionary trajectories (Supplemental Fig. S2; Popovićet al 2015). The differences between populations (Fig.…”

“…4, 5). Critically, this similarity is not an inevitable consequence of using this starting population or technique, as evidenced by the large differences that were previously observed (Popovićet al 2015) between populations evolved from the same starting population used here and the same partitioning method. The differences between the [+]clay and [−]clay populations are relatively small compared to the differences between our previously evolved self-cleaving ribozyme populations in which pH and ion identity were varied ( (Fig.…”

“…2015). Additionally, a conserved, three-way junction motif, DCGUY-3WJ (Popovićet al 2015), is also common within both the [−]clay and [+]clay populations (Fig. 5).…”

“…The RNA populations were evolved to catalyze RNA cleavage, a function catalyzed by several different RNA structures present in biology (Hammann et al 2012) and in in vitro evolved populations (Jayasena and Gold 1997;Tang and Breaker 2000;Salehi-Ashtiani and Szostak 2001;Popovićet al 2015). We recently used this in vitro evolution approach to investigate the impact of pH and ion identity on RNA function (Popovićet al 2015), and showed that some structures that are highly favored in one environment are disfavored in others. In contrast, in the study described here we find that the outcomes of parallel in vitro evolution experiments conducted either in the presence or absence of montmorillonite are strikingly similar.…”

Crucial steps to life: From chemical reactions to code using agents

“…This is not the primary goal of interacting RNAs, however. The primary goal is the interaction to become part of cooperative networks that may also be competing and restricting (Popovic et al, 2015), as documented in the predominant ligase function in contrast to the nuclease function (Hayden and Lehman, 2006;Díaz Arenas and Lehman 2010). This means the turn to life is also a behavioural pattern between becoming part of an identity of RNA groups that reject those that do not fit into this identity.…”