In an exciting advancement at the frontier of chemical biology, researchers have unveiled a groundbreaking method for synthesizing peptidyl-RNA in aqueous environments, employing thioester-mediated aminoacylation. This novel approach hinges on finely tuned chemical reactivities between nucleoside diols and aminoacyl-thiols, enabling selective attachment of amino acids to RNA molecules in water without the need for enzymatic machinery or protecting groups. Such a discovery significantly deepens our understanding of the chemical underpinnings of peptide-RNA formation, a vital process that echoes the origins of biological translation.
Central to this breakthrough is the observation that aminoacyl-thiols — thioester-activated amino acids — can chemoselectively couple to the 2′,3′-diol groups of nucleosides, forming aminoacyl-RNA conjugates with remarkable specificity at neutral pH. Unlike traditional peptide bond formation, which requires activation of carboxylic acids and proceeds via nucleophilic attack of amines, the thioester substrates employed do not promote peptide formation directly under these mild conditions. This orthogonality between thioester activation and peptide synthesis is crucial, as it provides a unique chemical switch: aminoacylation proceeds without formation of peptidyl-RNA intermediates, underscoring a new mechanistic control for stepwise biomimetic synthesis.
To dissect the subtleties of this control, the researchers probed the direct synthesis of peptidyl-RNA via reactions between pre-formed thioester-activated peptides and RNA diol substrates. Strikingly, acylation of the nucleoside diol was prevented when the thioester’s amine was blocked by N-acylation, eliminating peptidyl-RNA formation. Moreover, combining aminoacyl-thiols and peptide thioesters in the same reaction mixture led exclusively to aminoacyl-RNA, with no detectable peptidyl-RNA. These experiments elegantly demonstrated that the free amine functionality of aminoacyl-thiols is essential to selectively modify the RNA diol, effectively precluding premature peptide bond formation.
The implications of this selective aminoacylation go beyond mere chemical curiosity. Aminoacylated nucleotides have been shown previously to catalyze their own conversion into peptides through iterative non-enzymatic condensation cycles, powered by carbodiimide coupling reagents. However, such systems rely on highly reactive carboxyl activators that incite non-specific reactions and side products. By contrast, the present thioester chemistry offers a non-enzymatic, protecting-group-free platform for peptide synthesis tightly regulated by intrinsic chemical reactivity differences. Intriguingly, despite the presence of excess thioester, aminoacyl-RNAs produced via this method do not self-condense into peptidyl-RNA spontaneously, hinting at a profound mechanistic barrier linked to the nature of the activating group.
Exploring this barrier further, the scientists hypothesized that the mode of acyl activation is the critical factor dictating whether peptide bond formation ensues. While carbodiimide-activated carboxylic acids are highly electrophilic and readily engage amines to form peptides in aqueous media, aminoacyl-thiols exhibit comparatively muted reactivity, preventing premature peptide synthesis. Testing this concept, the study turned to peptide thioacids — structurally related yet distinct acyl donors that can be generated from thioesters by treatment with hydrogen sulfide under neutral conditions.
Peptide thioacids emerged as the key to unlocking controlled peptidyl-RNA synthesis. Upon selective activation by mild oxidants such as ferricyanide in aqueous buffers near neutral pH, thioacids promoted efficient, chemoselective ligation to aminoacyl-RNA substrates. This orthogonal activation circumvented the issues of non-specific acylation associated with traditional carbodiimide chemistry and was compatible with all twenty canonical amino acids. The elegant orchestration of orthogonal reactivities enabled the selective formation of peptidyl-RNA conjugates in near-quantitative yields.
What makes this advance particularly notable is the preservation of stereochemical integrity throughout the multistep process. The mild reaction conditions prevented racemization of chiral aminoacyl-thiols, aminoacyl-RNAs, and peptide thioacids, resulting in peptidyl-RNA products formed as single homochiral isomers. This fidelity mirrors the exquisite stereochemical control characteristic of enzymatic protein synthesis, highlighting the potential relevance of this chemistry to prebiotic peptide assembly and the early evolution of the translation apparatus.
A highlight of the study is the demonstration of one-pot synthesis of peptidyl-RNA in water. By incubating nucleosides with aminoacyl-thiols and peptide thioacids alongside a mild oxidant, the sequential formation of aminoacyl-RNA followed by conversion into peptidyl-RNA was achieved seamlessly. Importantly, no hydrolysis of thioacids or undesired peptide bond formation occurred prior to oxidation, underscoring the tight temporal and chemical control afforded by this method. The orthogonal reactivity profile of thioester and thioacid intermediates allows distinct stages of RNA aminoacylation and peptide bond formation to be orchestrated precisely under identical conditions.
The reported findings decisively reveal that the selective aminoacylation of RNA diols mediated by thioesters cannot be simply ascribed to protonation states of amines at physiological pH. Instead, the interplay of acyl donor reactivity and nucleophile availability governs the selectivity and outcome of these reactions. Such mechanistic insights open new avenues to chemically model the ribosomal peptide synthesis process in a prebiotic context. By harnessing chemical differentiation rather than solely relying on nucleic acid templating, this approach offers a powerful toolset for biomimetic synthesis and synthetic biology applications.
Beyond its fundamental scientific significance, the method holds promise for advancing synthetic approaches toward RNA-peptide conjugates, which are essential intermediates in understanding protocellular evolution and the origins of life’s translational machinery. The researchers’ strategy capitalizes on simple yet robust chemistries compatible with water as a solvent and neutral pH, conditions closely resembling plausible early Earth environments. This democratizes access to complex biopolymer assemblies without requiring sophisticated enzymatic catalysts or protective group maneuvers.
In sum, this landmark study introduces a chemically elegant and operationally straightforward method for directing RNA aminoacylation and peptide bond formation within a single aqueous system. The clever exploitation of thioester and thioacid orthogonality provides a critical chemical switch that precisely controls stepwise peptidyl-RNA synthesis. Such discoveries pave the way for reconstituting life’s core translational processes outside the bounds of cellular machinery, shedding light on key questions about the origin and evolution of protein synthesis.
Looking ahead, the versatile chemistry demonstrated here could also underpin novel synthetic biology platforms for constructing tailor-made peptidyl-RNA constructs with applications in therapeutics and nanotechnology. More broadly, the findings encourage revisiting classical biochemical paradigms through the lens of subtle chemical control, inspiring fresh interpretations of how life’s molecular complexity emerged from the primordial organic milieu.
The implications extend far beyond academic curiosity: this innovative chemistry may also catalyze advances in designing RNA-based catalysts and materials, expanding the toolkit for bottom-up assembly of functional biopolymers. Ultimately, by delineating a clean, efficient pathway to assemble peptidyl-RNA conjugates non-enzymatically, the work fundamentally redefines our understanding of early peptide synthesis chemistry and its integration with RNA function.
Subject of Research: Mechanistic and synthetic investigation of thioester- and thioacid-mediated aminoacylation and peptidyl-RNA synthesis in aqueous media.
Article Title: Thioester-mediated RNA aminoacylation and peptidyl-RNA synthesis in water.
Article References:
Singh, J., Thoma, B., Whitaker, D. et al. Thioester-mediated RNA aminoacylation and peptidyl-RNA synthesis in water. Nature 644, 933–944 (2025). https://doi.org/10.1038/s41586-025-09388-y
Image Credits: AI Generated
DOI: https://doi.org/10.1038/s41586-025-09388-y
Tags: aminoacyl-thiols in nucleoside chemistryaqueous environment peptide synthesischemical biology advancementschemical reactivities in RNA synthesisenzymatic-free peptide synthesismechanistic control in biomimetic synthesisnucleoside diols and aminoacyl-thiolsorigins of biological translationpeptidyl-RNA synthesisselective amino acid attachment to RNAthioester activation in peptide bond formationthioester-mediated aminoacylation
