Prebiotic Oligomer Assembly: What Was the Energy Source?

Abstract

Polymerization of nucleotides and amino acids to form large, complex, and potentially functional products was an early and essential event on the paths leading to life's origin. The standard Gibbs energies of the condensation reactions are uphill, however, and at equilibrium will yield only declining sequences of small, nonfunctional oligomers. Geochemically produced condensing agents such as carbonyl sulfide, cyanamide, and polyphosphates have been proposed to invert the unfavorable condensation Gibbs energies and thereby activate exergonic condensation. We argue, however, that although activators may provide modest yields of oligomers, the inherently episodic nature of their sources throttles their effectiveness, and the fundamental hydrolytic instabilities of oligonucleotides and peptides ultimately prevail to yield decreasing product sequences. Notably, the Gibbs energy governing oligomer formation is antientropic. Accordingly, we propose that declining progression can be surmounted in evaporating pools in which a favorable entropy change is produced when high surface/volume ratios concentrate reactants at the air/water interface in continuous cycles of wetting and drying. The severely reduced configurational freedom of the solutes then inverts the antientropic nature of the condensation reactions, pivoting them to exergonic states and thus to the production of ascending sequences of complex polymeric products.

When thermodynamics says “no,” it means exactly that. When it says “yes,” it means “maybe.” Stephen Lower ^*

1. Introduction

Over the past century, studies of life's origins have considered broad spans of venues, including deep-sea hydrothermal vents (Martin and Russell, 2007), fluctuating hydrothermal pools (DeGuzman et al., 2014; Damer and Deamer, 2015), and organic aerosols (Donaldson et al., 2004). The corresponding range of chemical motifs runs from the replication-first primordial soup (Bada and Lazcano, 2002) and the RNA worlds (Robertson and Joyce, 2012) to the notion of metabolism-first and the iron/sulfur world (Huber and Wächtershäuser, 2006). Whatever the chemical route, oligomerization was a requisite path to life's first functional polymers. The RNA world scenario, for example, proposes that the first primitive form of cellular life used RNA both as a catalyst and to store and process genetic information (Gilbert, 1986; see Robertson and Joyce, 2012, for review). This notion grew out of the discovery of ribozymes in 1983, and small biological ribozymes such as the hammerheads have nucleotide sequences ranging from 70 to 90 bases, although RNA strings as short as five nucleotides such as the GUGGC-3′ sequence reported by Illangasekare and Yarus (1999) have displayed catalytic ability.

The oligomerizations must therefore have delivered ascending product sequences, that is, sequences in which R_n _+ 1 > R_n _, and thus must have been exergonic. It is commonly recognized, however, that amino acid and nucleotide oligomerizations are endergonic (Bada and Lazcano, 2002), and on that basis will yield declining product sequences (R ₁ > R ₂ > R ₃…), with the progressions terminating at relatively small oligomers, dimers, and monomers. Thus, as recently described by Guseva et al. (2017), a question central to life's origins arises: How did prebiotic processes avoid what they termed the Flory Length Problem, that is, the tendency of endergonic polymerization to favor shorter rather than longer chains? That check on growth stands as a particularly conspicuous restraint on the advance of self-replication since nucleic acid polymers from size-limited templates will necessarily include no more than small segments of bases in the face of the need for broadly diverse and ultimately complex base sequences.

It is apparent that the energetic landscape directing prebiotic oligomerization must have been upended in some manner, such that oligomerization became exergonic and was thereby positioned to deliver the required ascending and prolonged sequences. However, the means by which the transposition occurred remains an open question, and over the past decades, researchers have settled on activated polymerization. The activation agents used include cyanamide (Steinman et al., 1964), polyphosphates (Rabinowitz et al., 1969), and carbonyl sulfide (Leman et al., 2004), posited in most cases to have been produced by volcanic geochemistry. The activators are consumed in the reactions and accordingly are not catalysts; rather they are stoichiometric components and thus in principle can act as agents of an energy inversion.

Prebiotic activation is most often a presumed participant in origins' accounts and is largely accepted without judgment. There are some perceived shortcomings to the notion, however, as was noted in a review of the RNA world by Robertson and Joyce (2012). The authors observed that although the most chemically reasonable candidates for activation are polyphosphates, their prebiotic source remains unidentified. They pointed out moreover that hydrolysis of the product oligonucleotides should in the end kinetically swamp polymerization. Indeed, although there is a large literature describing activated chain elongation (Tam et al., 2018, and references therein), as we illustrate, the ultimate application of activation to the prebiotic formation of the large, complex polymers required for life cannot be realized owing to an inherent and significant kinetic obstacle. Here we address that shortcoming and expand on a recent modeling exercise supporting the notion that dry/wet cycling can yield the required energy reversal (Ross and Deamer, 2016).

2. Discussion

2.1. Activation is problematic

The reaction recognized as pivotal to life's origins, the aqueous elongation of peptides and nucleotides, is simply represented by the equilibrium sequence \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document} \begin{align*}{R_m} + {R_n} \; \rightleftarrows \;{R_m}_{ + n} + {\rm H}_2 {\rm O} \tag{1} \end{align*} \end{document}

The primary difficulty, its endergonic character, is reflected in the standard thermodynamic values for aqueous peptide and ribose/phosphate ester formation listed in Table 1. The introduction of an activating partner (X) provides a reactive monomer and enhances the process as shown in (2) and (3) \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document} \begin{align*} X + {R_1} \to X - {R_1} + {\rm H}_2 {\rm O} \tag{2} \end{align*} \end{document} \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document} \begin{align*} X - {R_1} + {R_n} \to {R_n}_{ + 1} + {\rm HO} - X \tag{3} \end{align*} \end{document}

Table 1.

Standard Thermodynamic Parameters for Peptide, Phosphate/Sugar Ester Formation at 25°C and pH 7

Equilibrium	ΔG^o (kcal/mol)	ΔH^o (kcal/mol)	ΔS^o (cal/K)	References
2 Glycine ⇄ diglycine + H₂O	3.5	1.7	−4.8	Johnson et al. (1992)
Adenosine + HPO₄ ²⁻ ⇄ AMP²⁻ + H₂O	2.7	−0.2	−9.6	LaRowe and Helgeson (2006)

transporting the system to an exergonic state and thereby triggering synthesis of the appropriate oligomeric products.

In the absence of a continuous supply of activator, the sequence of reactions (2) and (3) is no more than a transient kinetic overlay, however, and in the end the response to the endergonic equilibrium (1) cannot be avoided. The eventual breakdown arises from two factors involving reaction rates (r). First, r ₂ is a function of the concentration of the activator X, and since volcanism is by its very nature episodic (Moore and Web, 2013), at some point activator concentrations and thus r ₂ must decline. Then because r _{1 rev} increases directly with oligomer growth, ultimately r _{1 rev} > r ₂, and at that point equilibrium (1) overwhelms the activation sequence (2) and (3). The fundamental endergonic nature of the equilibrium then emerges as the controlling feature of oligomerization, and monomers and short oligomers dominate the system.

We conclude that the chemical activation approach to prebiotic oligomer formation is unlikely to succeed, and its deficiencies are illustrated in a simulation of the sequence (1)–(3) in Fig. 1A. The figure, generated to 10mers with the kinetic simulation application Kintecus (Ianni, 2003), typifies a wide range of starting conditions and rate constants and reflects the general case. It confirms that although activation does indeed increase oligomeric products at first, the concentration of oligomers decreases as the activator is exhausted, and hydrolysis dominates the reaction.

FIG. 1.

Simulations of reactions (1)–(3) with k ₋₁ ₌ 10^–6.1 s⁻¹ at pH 7 and 90°C, developed from the data of Oivanen et al. (1998). (A) With activation; a starting solution 5 and 10 mM, respectively, in activator X and R ₁; assuming reactions (2) and (3) to be instantaneous. The monomer prevails as the activator is consumed. (B) Without activation; a starting solution 10 mM in R ₁; ignoring reactions (2) and (3); ΔG ₁ = −6 kcal/mol. An inversion of the product profile ultimately arises.

An outcome such as that in Fig. 1A is exhibited in the findings of Kawamura and Ferris (1994) in their account of oligonucleotide synthesis in the imidazole-activated elongation of adenosine monophosphate on Na⁺-montmorillonite. Product sequences up to 10mer were initiated with the 5′-phosphorimidazolide of adenosine, in which imidazole served as a leaving group so that adenosine monophosphate then engaged in reaction (3). The result, however, was a substantial yield decrease from monomer to nmer in the final mixture by a factor of about 1/2ⁿ, or a reduction by ∼1000 at the 10mer, and provided a clear reflection of the evolving influence of r _1 rev over the reaction course.

The consequence of the problematic trend of equilibrium (1) is even more marked for circumstances in which the conversion to an activated monomer is relatively slow. An example of that case is provided in an account by Leman et al. (2004), in which carbonyl sulfide was used as an activator in phenylalanine elongation. Reactions conducted with a range of additives yielded dipeptide and tripeptide in yields of up to 60% and 12%, respectively, but no longer peptides were detected.

2.2. Molecular crowding and interfacial regions can surmount the Flory length problem

Activation thus cannot provide indefinitely long oligomers. Some insight into a resolution of the issue can be gained by considering the values for the thermodynamic parameters of peptide bond synthesis and nucleoside phosphorylation in Table 1. It is apparent that the endergonic Gibbs energies of both equilibria have significant entropic components as would be expected from their 2 to 1 stoichiometry. The task of creating an energy inversion for equilibrium (1) is thus reduced largely to entropy management that can lead in turn to the conversion of the equilibrium in a bond-forming direction to an entropy-positive process.

That approach has been addressed in a number of ways that have included adjusting local stoichiometry (Greenwald et al., 2016), composite prebiotic networks (Nghe et al., 2015; Semenov et al., 2016), and thermophoresis (Kreysing et al., 2015). [The authors are grateful to a reviewer for directing us to an account by Sievers et al. (2004), highlighting the significant benefits, in this case kinetic, to ribosome-catalyzed peptide formations that are delivered entirely through entropy augmentation.] We focus here on evaporative condensation and dry/wet cycling of solutions of mononucleotides and amino acids that appear to achieve that objective, yielding, respectively, RNA-like products (Verlander et al., 1973; Sleeper and Orgel, 1979; Rajamani et al., 2008; DeGuzman et al., 2014; Morasch et al., 2014; Da Silva et al., 2015) and polypeptides (Fox et al., 1959; Rodriguez-Garcia et al., 2015; Forsythe et al., 2017). We have recently described those results as emerging directly from the developing premium on free volume and resulting molecular crowding and compartmentalization in the drying steps (Ross and Deamer, 2016), and on the basis of recent observations of spontaneous ribose phosphate formation in microdroplets (Nam et al., 2018), we expand on that notion here to include the significance of the air/water interface.

The expression for the Gibbs energy for equilibrium (1) is presented in Equation (4), in which ΔG ^o is the standard value for either peptide or ester formation in Table 1 and a refers to activity. \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document} \begin{align*}{ \rm { \Delta } } { G_ { \rm { 1 } } } { \rm { = \Delta } } { G^ { \rm { o } } } { \rm { + RTln } } { \frac { { a_ { { R_ { m { \rm { + } } n } } \, } } { a_w } } { { a_ { { R_m } \, } } { a_ { { R_n } \, } } } } \tag { 4 } \end{align*} \end{document}

As discussed earlier, the molecular crowding that develops with evaporation and the growth of solute concentrations elicit both a Raoult's law decline in the activity of water and a growth of solute activities to values that can exceed their molar concentrations by orders of magnitude (Ross and Deamer, 2016). The latter can be critical in evaporative settings where activity coefficients, reflections of deviations from Henry's law, can grow increasingly sensitive to solvation differences among solutes (Moore and Pearson, 1981). The activity fraction thus falls strongly with evaporation and at some point oligomer growth becomes exergonic—indeed increasingly so with oligomer growth—and large, complex oligomeric products are the result.

Accounts describing the significance of crowding and confined spaces in an origins' context include those of Zhou and Dill (2001), Donaldson et al. (2004), Hansma (2014), Munoz-Santiburcio and Marx (2017), and Guseva et al. (2017). In these settings, the surface excess effect (Mitropoulos, 2008) becomes a pronounced feature of the dynamics, and the hyperconcentration of organic solutes at the interface can generate both marked rate enhancements (Li et al., 2016) and shifts to a condensation-favoring equilibrium position (Fallah-Araghi et al., 2014).^† Indeed, in an application of interfacial effects tied directly to life's origins, Nam et al. (2018) recently reported studies in which aqueous microdroplets of reactants were injected at atmospheric pressure into a mass spectrometer. They described the rapid and spontaneous phosphorylation of ribose in meaningful yields at ambient temperatures, representing a sizeable reduction in the Gibbs energy from 5.4 down to −1.1 kcal/mol.

These results make it clear that the entropic obstacle facing condensation reactions in bulk solution can be largely overcome in settings with high surface area/volume ratios, venues that necessarily extend well beyond microdroplets to include large evaporating systems. That condition is proposed here to be an essential component of prebiotic elongation, and while the mechanism is yet to be developed (Lee et al., 2015), the overall effects can be rationalized in terms of a simple pairing of thermodynamic and kinetic gradients along paths from the bulk medium to the interface. With the union of the surface excess effect and Equation (4), ΔG ₁ will fall with approach to the interface and equilibrium (1) is then shifted to the right. At the same time, from the model for “on-water” interfacial catalysis by Jung and Marcus (2007), it can be reasoned that the activation barrier separating the right and left sides of equilibrium (1) will fall by perhaps 7 kcal/mol at the interface, eliciting reaction rate increases by factors of up to 10⁵. Thus, in those regions, condensation is both realized and rapid. In turn, the hydrolysis of the condensation products, favored once again in subsequent transport from the interface, will be suppressed by the resumed kinetic barrier, and in the end, the oligomers are trapped kinetically in the bulk medium.

As we have described, this route to elongation must ultimately yield a mounting oligomer sequence (Ross and Deamer, 2016), and the effect is illustrated in Fig. 1B for equilibrium (1) to the 10mer with no added activator, and thus with reactions (2) and (3) bypassed. The shift to an exergonic condensation will necessarily establish an inversion in the product sequence, as shown in the figure (with ΔG ₁ set to −6 kcal/mol for purposes of illustration). Also, while these results are functions of the specific choice of conditions, the contrasts in Fig. 1A and 1B reflect the general case. They underscore both the shortcomings of the activation model and the essential requirement of an entropy-promoting large surface/volume ratio.^‡

3. Summary and Conclusions

By increasingly downshifting configurational freedom to a more confining volume that progressively tends to a two-dimensional space, evaporation and a mounting air/water interface overturn otherwise unfavorable antientropic factors and invert Gibbs energies to favor condensation. It is worth noting further that folding, an inherently antientropic process, is similarly advanced by molecular crowding (Dupuis et al., 2014). This rationale can be enhanced to the limiting case at the interface itself where the loss of a full degree of freedom can then promote assembly and molecular organization through the formation of Langmuir monolayers (Oliveira et al., 2004; Ariga and Hill, 2011). Significantly and in contrast to the commonly presumed random oligomer assembly (Eigen, 1971; Robertson and Joyce, 2012), this scenario leads to a thermodynamics-governed prebiotic biomolecule synthesis, the potential advantages of which have been discussed (Ross and Deamer, 2016).

Finally, the points raised here go beyond issues of terrestrial origins and can be considered in discussions of extraterrestrial life (Deamer and Damer, 2017). A recent NASA landing site workshop for its forthcoming Mars 2020 sample-return mission, for example, settled on three sites (http://mars.nasa.gov/news/2017/scientists-shortlist-three-landing-sites-for-mars-2020). One of the choices, Columbia Hills, is thought to have been associated with hot springs and geyser activity, qualities that on the basis of the origin factors described here should weigh heavily in its favor. As for the presumed internal oceans of Europa and Enceladus, their celebrated geyser activity suggests the presence of interfacial regions and thus sites for polymer synthesis, although possibly limited kinetically by the minimal thermal energy available at low temperatures.

Footnotes

Author Disclosure Statement

No competing financial interests exist.

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