Expanding the genetic code: DNA and RNA are naturally composed of four nucleotide bases that form hydrogen bonds in order to pair. Hoshika et al. added an additional four synthetic nucleotides to produce an eight-letter genetic code and generate so-called hachimoji DNA. Coupled with an engineered T7 RNA polymerase, this expanded DNA alphabet could be transcribed into RNA. Thus, new forms of DNA that add information density to genetic biopolymers can be generated that may be useful for future synthetic biological applications. Science , this issue p. 884

RNA enzymes or ribozymes catalyze some of the most important reactions in biology and are thought to have played a central role in the origin and evolution of life on earth. Catalytic function in RNA has evolved in crowded cellular environments that are different from dilute solutions in which most in vitro assays are performed. The presence of molecules such as amino acids, polypeptides, alcohols, and sugars in the cell introduces forces that modify the kinetics and thermodynamics of ribozyme-catalyzed reactions. Synthetic molecules are routinely used in in vitro studies to better approximate the properties of biomolecules under in vivo conditions. This review discusses the various forces that operate within simulated crowded solutions in the context of RNA structure, folding, and catalysis. It also explores ideas about how crowding could have been beneficial to the evolution of functional RNAs and the development of primitive cellular systems in a prebiotic milieu.

Catalytic RNAs or ribozymes are considered to be central to primordial biology. Most ribozymes require moderate to high concentrations of divalent cations such as Mg²⁺ to fold into their catalytically competent structures and perform catalysis. However, undesirable effects of Mg²⁺ such as hydrolysis of reactive RNA building blocks and degradation of RNA structures are likely to undermine its beneficial roles in ribozyme catalysis. Further, prebiotic cell-like compartments bounded by fatty acid membranes are destabilized in the presence of Mg²⁺, making ribozyme function inside prebiotically relevant protocells a significant challenge. Therefore, we sought to identify conditions that would enable ribozymes to retain activity at low concentrations of Mg²⁺. Inspired by the ability of ribozymes to function inside crowded cellular environments with <1 mM free Mg²⁺, we tested molecular crowding as a potential mechanism to lower the Mg²⁺ concentration required for ribozyme-catalyzed RNA assembly. Here, we show that the ribozyme-catalyzed ligation of phosphorimidazolide RNA substrates is significantly enhanced in the presence of the artificial crowding agent polyethylene glycol. We also found that molecular crowding preserves ligase activity under denaturing conditions such as alkaline pH and the presence of urea. Additionally, we show that crowding-induced stimulation of RNA-catalyzed RNA assembly is not limited to phosphorimidazolide ligation but extends to the RNA-catalyzed polymerization of nucleoside triphosphates. RNA-catalyzed RNA ligation is also stimulated by the presence of prebiotically relevant small molecules such as ethylene glycol, ribose, and amino acids, consistent with a role for molecular crowding in primordial ribozyme function and more generally in the emergence of RNA-based cellular life.