Protocell Effects on RNA Folding, Function, and Evolution

Abstract

Creating a living system from nonliving matter is a great challenge in chemistry and biophysics. The early history of life can provide inspiration from the idea of the prebiotic "RNA World" established by ribozymes, in which all genetic and catalytic activities were executed by RNA. Such a system could be much simpler than the interdependent central dogma characterizing life today. At the same time, cooperative systems require a mechanism such as cellular compartmentalization in order to survive and evolve. Minimal cells might therefore consist of simple vesicles enclosing a prebiotic RNA metabolism. The internal volume of a vesicle is a distinctive environment due to its closed boundary, which alters diffusion and available volume for macromolecules and changes effective molecular concentrations, among other considerations. These physical effects are mechanistically distinct from chemical interactions, such as electrostatic repulsion, that might also occur between the membrane boundary and encapsulated contents. Both indirect and direct interactions between the membrane and RNA can give rise to nonintuitive, "emergent" behaviors in the model protocell system. We have been examining how encapsulation inside membrane vesicles would affect the folding and activity of entrapped RNA. Using biophysical techniques such as FRET, we characterized ribozyme folding and activity inside vesicles. Encapsulation inside model protocells generally promoted RNA folding, consistent with an excluded volume effect, independently of chemical interactions. This energetic stabilization translated into increased ribozyme activity in two different systems that were studied (hairpin ribozyme and self-aminoacylating RNAs). A particularly intriguing finding was that encapsulation could rescue the activity of mutant ribozymes, suggesting that encapsulation could affect not only folding and activity but also evolution. To study this further, we developed a high-throughput sequencing assay to measure the aminoacylation kinetics of many thousands of ribozyme variants in parallel. The results revealed an unexpected tendency for encapsulation to improve the better ribozyme variants more than worse variants. During evolution, this effect would create a tilted playing field, so to speak, that would give additional fitness gains to already-high-activity variants. According to Fisher's Fundamental Theorem of Natural Selection, the increased variance in fitness should manifest as faster evolutionary adaptation. This prediction was borne out experimentally during in vitro evolution, where we observed that the initially diverse ribozyme population converged more quickly to the most active sequences when they were encapsulated inside vesicles. The studies in this Account have expanded our understanding of emergent protocell behavior, by showing how simply entrapping an RNA inside a vesicle, which could occur spontaneously during vesicle formation, might profoundly affect the evolutionary landscape of the RNA. Because of the exponential dynamics of replication and selection, even small changes to activity and function could lead to major evolutionary consequences. By closely studying the details of minimal yet surprisingly complex protocells, we might one day trace a pathway from encapsulated RNA to a living system.

Figure 1

Effects of protocell encapsulation on ribozymes. (a) Schematic drawing of a fatty acid vesicle (60 nm diameter) encapsulating a hairpin ribozyme (PDB: 2OUE, docked conformation at top right) and a 70S ribosome (PDB: 4V42, purple/green structure in center). The undocked conformation of the hairpin ribozyme is an artistic illustration and is not based on a solved structure. Encapsulation shifts the equilibrium toward the docked state of the hairpin ribozyme due to physical confinement effects. Such effects are expected to be greater for larger multisubunit structures such as a ribosome. The molecules are drawn approximately to scale; for comparison, the diameter of an A-form helix is 2.3 nm and the thickness of a membrane bilayer is approximately 3 nm, depending on the lipid. (b) Illustration of the excluded volume effect from confinement inside a boundary membrane. The native (N), folded conformation of a ribozyme (green) has a specific compact shape that can be configured at multiple positions. When a boundary is present, some configurations (gray) are disallowed due to steric clashes with the membrane. The membrane boundary creates a volume from which the center of the molecule is excluded (blue zone). For the unfolded conformations (U), many configurations are possible, differing in both position and conformation (red/orange). When a boundary is present, as with N, many of these configurations are disallowed due to steric clashes (gray). However, a greater fraction of U configurations is affected compared to N, due to the extended nature of the U conformations. For U, the precise exclusion zone (blue) depends on the conformation, but is generally larger for U compared to N. The relative decrease in the number of accessible configurations for U is the basis for reduced entropy, and thus higher free energy of U relative to N, when encapsulated. (c) Encapsulation increases the rate of evolutionary adaptation of ribozymes, compared to unencapsulated ribozymes. In this drawing, each dot corresponds to a mutant ribozyme, with different colors representing different sequences (magenta colors being the highest fitness sequences). Encapsulation is represented by a gray circle around the dot. Beginning with a diverse set, without encapsulation (top row), the population adapts slowly, requiring several rounds of in vitro selection to converge on the fittest sequences (magenta shades). In contrast, the population of encapsulated RNAs converges quickly onto the fittest sequences (bottom row).

Figure 2

Confinement and evolutionary adaptation. (a) Boltzmann sigmoidal curve for two-state equilibrium. The fraction in one state (e.g., folded conformation) is shown as a function of free energy difference ΔG (G_unfolded – G_folded). Confinement increases the energy of the unfolded state (G_unfolded) by + Δx, leading to an increase in the fraction of folded molecules (+Δf). If ribozymes are generally well-folded (high on the curve), confinement improves a better-folded variant (blue) less compared to a moderately folded variant (green) for the same increase Δx, i.e., Δf₄< Δf₃. On the other hand, if the ribozymes are generally poorly folded (low on the curve), confinement improves the better-folded variant (orange) more than it improves the poorly folded variant (red), i.e., Δf₂> Δf₁, consistent with our observations. (b) Illustrated comparison of a “peak” on a ribozyme fitness landscape with (purple) or without (blue) encapsulation. We suggest that confinement would sharpen the fitness landscape at low fitness, as observed for self-aminocylating RNAs, leading to greater slope in this region of sequence space. At the same time, confinement would rescue mutants close to high fitness, as observed for the hairpin ribozyme, leading to flattening of the peak apex.