Chemical Fueling Enables Molecular Complexification of Self-Replicators*

Abstract

Unravelling how the complexity of living systems can (have) emerge(d) from simple chemical reactions is one of the grand challenges in contemporary science. Evolving systems of self-replicating molecules may hold the key to this question. Here we show that, when a system of replicators is subjected to a regime where replication competes with replicator destruction, simple and fast replicators can give way to more complex and slower ones. The structurally more complex replicator was found to be functionally more proficient in the catalysis of a model reaction. These results show that chemical fueling can maintain systems of replicators out of equilibrium, populating more complex replicators that are otherwise not readily accessible. Such complexification represents an important requirement for achieving open-ended evolution as it should allow improved and ultimately also new functions to emerge.

Keywords: dissipative systems; dynamic combinatorial libraries; self-assembly; self-replication; systems chemistry.

Figure 1

Schematic representation of assembly‐driven self‐replication in a replication–destruction regime. A) Mechanism of self‐replication. Dithiol building block 1 is oxidized to give rise to a mixture of interconverting disulfides of different ring size. Slow nucleation of a stack of one particular ring size is followed by elongation of the stack. When the stack is sufficiently long to be susceptible to mechanical energy the system enters a breakage–elongation cycle leading to exponential growth of the fibers and the macrocycles from which they are constituted. B) Simplified representation of the replication–destruction regime achieved upon constant simultaneous addition of oxidant and reductant. NaBO₃ oxidizes the dithiol building block into a mixture of different disulfide macrocycles, from which two competing replicators can grow. TCEP reduces the disulfides in the non‐assembled macrocycles as well as in the assembled replicating macrocycles back to the thiol building block. The thickness of the arrows indicates the magnitude of the fluxes (in units of 1) through the various pathways in a kinetic model of the reaction network (SI Section S5). The flux through the short‐circuiting reaction of perborate with TCEP (not shown) accounts for less than 0.1 % of the total flux.

Figure 2

Self‐assembly‐driven self‐replication of 1 ₃ and 1 ₆. A) Change in product distribution with time of a mixture made from dithiol building block 1 (0.19 mm) in borate buffer (pH 8.2) in the presence of 2.5 m guanidinium chloride. B) TEM analysis of the mixture dominated by trimers after shaking at 1200 rpm at room temperature for two weeks (scale bar=200 nm); Change in product distribution with time of a pre‐oxidized sample made from 1 (0.19 mm) in borate buffer pH 8.2 in the presence of 1.5 m guanidinium chloride in the absence and presence of various initial amounts of seeds of C) 1 ₃ replicator and D) 1 ₆ replicator. Seeding % are expressed in units of 1 relative to the total number of units of 1. Note that the data in (C) and (D) cannot be compared directly as the experiments are started at different oxidation levels (see SI Figure S4 for details). Lines are drawn to guide the eye.

Figure 3

Comparison of the growth and/or decline of replicators 1 ₃ and 1 ₆ under different conditions. A) In a mixture of replicators 1 ₃ and 1 ₆ and non‐assembled 1 ₃ and 1 ₄ macrocycles (in a 15:30:55 ratio in units of building block) 1 ₃ replicates faster than 1 ₆. The 0.50 mL sample was shaken at 1200 rpm in the presence of oxygen from the air. B) Change in product distribution with time of a mixture made from replicators 1 ₃ and 1 ₆ (approximately equimolar in units of 1) in 1.5 m guanidinium chloride in the presence of 10 mol % dithiol 1. Total [1]=0.19 mm. C) Decrease in UPLC peak area of replicators 1 ₃ (blue triangles) and 1 ₆ (red circles) and corresponding increase in peak area of monomer 1 (black squares) upon reduction of a mixture of these replicators (0.095 mm each in units of building block 1) to different extents by adding 8, 20, and 40 mol % TCEP (with respect to units of 1). Error bars show the standard deviations of three independent repeats. For a statistical analysis, see SI Section S4.5. Note that hexamer‐to‐trimer conversion is insignificant on the timescale of the reduction experiments. All samples were prepared in borate buffer (50 mm, pH 8.2) containing 1.5 m guanidinium chloride. Lines in (A) and (B) are drawn to guide the eye.

Figure 4

Population of a disfavored and slow replicator is possible in a chemically fueled replication–destruction regime. A) Potential energy landscape in which replicators 1 ₃ and 1 ₆ compete for building block 1 qualitatively showing the energy barriers for the replication (black line) and destruction (blue line) pathways. The formation of each replicator from building block 1 is coupled to the conversion of oxidant (ox) into waste (w), while the disassembly of replicators back into building block is coupled to the conversion of reducing agent (rd) into waste. It proved hard to unambiguously determine the relative thermodynamic stability of replicators 1 ₃ and 1 ₆; see SI Figure S7 and the discussion below this Figure. B) Evolution of the product distribution with time upon continuous and simultaneous addition of TCEP and NaBO₃ solutions to a mixture initially containing replicators 1 ₃ and 1 ₆ and non‐assembled 1, 1 ₃ and 1 ₄ (overall 0.19 mm in 1) in 50 mm borate buffer (pH 8.2) containing 1.5 m guanidinium chloride. The black arrow indicates the moment that the addition of NaBO₃ was stopped. Lines are drawn to guide the eye. Five repeats of this experiment show that the behavior is qualitatively reproducible (see SI Figure S2).

Figure 5

The more complex replicator is a more proficient catalyst. A) Retro‐aldol reaction used as a model reaction to assess the catalytic proficiencies of the competing replicators. B) Kinetic data, averaged over three repeats, comparing the production of retro‐aldol product 3 catalyzed by replicator 1 ₆ (red circles) with the effects of replicator 1 ₃ (blue triangles), a mixture of non‐assembled 1 ₃ and 1 ₄ (green triangles), and monomer 1 (black squares). The background reaction in the absence of 1 or any of its oligomers is shown in blue circles and coincides with the data for the reaction in the presence of 1. The concentrations of the various species were 25 μm (in units of 1) in borate buffer (50 mm, pH 8.12) containing 1.5 m guanidinium chloride and 0.20 mm substrate 2. Shaded areas show the standard deviation and lines are drawn to guide the eye. For a detailed mechanistic analysis of the retro‐aldol reaction catalyzed by 1 ₆, see ref. [34]. For a repeat of the experiment at higher concentrations and temperature to give a higher conversion, see SI Figure S25.