Constraint on an RNA world on montmorillonite clay

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The RNA world hypothesis proposes that RNA played central roles before the emergence of life (1). While acting as a storage of genetic information, RNA can also catalyze chemical reactions, as evidenced in the ribosome, RNAse P, and splicesome. A growing body of research has discovered functional RNAs, some of which promote fundamental biological processes such as their own replication and metabolism, at least in part (2). To assess the validity of the RNA world scenario, understanding where and how RNA could have developed and functioned on the early Earth is crucial. Mineral surfaces, geochemical environments ubiquitous on Earth, may have served as a site for RNA world establishment, given the enormous catalytic capabilities of minerals in the complexification of organic molecules, including RNA (3). In this regard, prior studies have explored the potential influence of minerals on RNA synthesis, function, and evolution, revealing the apparent compatibility between minerals and RNA development. Writing in Biophysical Journal, Saha and colleagues present a different perspective; the authors revealed a new aspect of montmorillonite clay, an extensively studied and widely distributed mineral, that may actually constrain an RNA world (4).

Diverse interactions between minerals and RNA are recognized, providing support for the RNA world hypothesis. For example, certain minerals facilitated the synthesis of RNA components and oligonucleotides (5,6), and various minerals concentrated RNA on the surfaces with selectivity for longer sequences (7). Minerals may also play an evolutionary role by restricting RNA diffusion on surfaces, thereby protecting functional RNAs from unfavorable interactions with parasitic molecules (8). Among the diverse minerals available, montmorillonite clay has been most extensively studied for its interactions with RNA, as well summarized in the supporting material of the associated article by Saha et al. (4).

A hammerhead ribozyme, a self-cleaving RNA, has been the primary focus of investigating RNA catalysis on mineral surfaces. For example, it was shown that a hammerhead ribozyme maintained its activity, albeit at a reduced level, when adsorbed on montmorillonite, suggesting that the RNA could fold into an active structure on the surface (9). The Azoarcus recombinase ribozyme also functioned in the presence of minerals, although its catalysis when adsorbed on the surfaces remained uncertain (7). Whereas these observations indicated some compatibility of minerals and catalytic RNAs, in the latest study, Saha and colleagues questioned the generality of the tolerance of functional RNAs to interactions with mineral surfaces (4). Using montmorillonite K10 clay and the malachite green (MG) RNA aptamer as models, the authors revealed a detrimental effect of montmorillonite on the structure and function of the RNA, underscoring a potential constraint on the development of an RNA world on mineral surfaces.

The MG aptamer folds into a structure with two stems, with a binding pocket for MG in between (Fig. 1). Upon binding, the aptamer substantially enhances the fluorescence of MG. Saha and colleagues first observed that not only did the aptamer adsorb onto montmorillonite, as expected based on previous studies, but the fluorescence in the presence of MG decreased with increasing concentrations of montmorillonite. The hindered interaction of the aptamer with MG resulted from the competitive binding of montmorillonite to MG. Interestingly, montmorillonite displayed significantly stronger binding to MG compared to the aptamer’s binding to MG. Perhaps surprisingly, the concentration of montmorillonite required to sequester MG was too low to cause substantial adsorption of the aptamer onto the surface. How could montmorillonite prevent the aptamer-MG association without apparent interactions with the RNA, such as a tight binding for disrupting RNA folding? Substances potentially leached from montmorillonite were confirmed not to be the cause.

Figure 1.

Montmorillonite clay weakly interacted with the 38-nucleotide MG aptamer and disrupted its folding, including the binding pocket for MG, and hence the function of the RNA. To see this figure in color, go online.

Saha and colleagues hypothesized that montmorillonite denatures RNA folding but without strong RNA-montmorillonite adsorption. To test the hypothesis, the authors first determined the melting curve of the aptamer-MG complex with a low concentration of montmorillonite. This concentration was well below the apparent dissociation constant between the aptamer and montmorillonite and low enough to maintain a fraction of the intact aptamer-MG complex. While the aptamer barely bound the surface, the temperature of melting the aptamer-MG complex decreased in the presence of montmorillonite, confirming that the mineral perturbed the interactions between the aptamer and MG. The folding of the aptamer with MG in the presence or absence of montmorillonite was also determined by NMR. The aptamer formed a known stable structure without montmorillonite. In contrast, the addition of montmorillonite denatured the terminal stem, affecting the adjacent binding pocket of MG, while the other structures remained intact (Fig. 1). In sum, montmorillonite did not merely compete with the aptamer to bind MG but also destabilized the RNA structure and facilitated the release of MG. Overall, the study revealed the inhibitory effect of montmorillonite on RNA function without strong adsorption, providing previously unknown evidence that diminishes the plausibility of RNA world development on mineral surfaces.

While most previous studies focused on self-cleaving ribozymes to investigate interactions between functional RNAs and mineral surfaces, the impact of minerals on such intramolecular functions may differ from that on intermolecular functions such as ligand binding of an RNA aptamer adopted by the authors. Many RNAs, as exemplified by the aptamer, interact with organic molecules or substrate RNAs that may also interact with mineral surfaces. Such competitive interactions complicate the understanding of the effect of minerals on RNA functions, and this is perhaps a reason why self-cleaving ribozymes have historically been chosen for detailed analysis. Saha and colleagues overcame the challenge by employing multiple biophysical approaches, demonstrating an unexpected inhibitory effect of well-studied mineral montmorillonite on the intermolecular RNA function.

Does this discovery make us pessimistic about the contribution of minerals to putative RNA world formation on the early Earth? Even if incompatible with certain RNA functions, minerals could still have played crucial roles, such as supplying, concentrating, and protecting RNA, as briefly mentioned above. In addition, the generality of the authors’ discovery, the types of RNA structures and functions that minerals constrain, remains to be studied. Given the diversity of functional RNAs and minerals, some RNAs may be active on certain mineral surfaces. This has been demonstrated in at least the case of a hammerhead ribozyme, yet, in partial agreement with the findings of Saha and colleagues, the activity is reduced (9). In addition to the MG aptamer, the authors examined the structural denaturation of a simple RNA duplex by montmorillonite, but they observed no significant structural changes. Based on these findings, it is conceivable that RNA could maintain its function if a catalytic or binding site is supported by stable terminal structures, such as relatively long stems, that mitigate the denaturation effect by minerals. Such RNA may be found through in vitro selection with minerals (10). Future exploration into how minerals influence diverse functional RNAs would provide valuable insights into the potential environments where an RNA world may have developed before the emergence of living systems.

Author contributions

R.M. wrote the paper.

Editor: Jason Kahn.