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Abstract The intricate landscape of tRNA modification presents persistent analytical challenges, which have impeded efforts to simultaneously resolve sequence, modification, and aminoacylation state at the level of individual tRNAs. To address these challenges, we introduce “aa-tRNA-seq”, an integrated method that uses chemical ligation to sandwich the amino acid of a charged tRNA in between the body of the tRNA and an adaptor oligonucleotide, followed by high throughput nanopore sequencing. Our approach reveals the identity of the amino acids attached to all tRNAs in a cellular sample, at the single molecule level. We describe machine learning models that enable the accurate identification of amino acid identities based on the unique signal distortions generated by the interactions between the amino acid in the RNA backbone and the nanopore motor protein and reader head. We apply aa-tRNA-seq to characterize the impact of the loss of specific tRNA modification enzymes, confirming the hypomodification-associated instability of specific tRNAs, and identifying additional candidate targets of modification. Our studies lay the groundwork for understanding the efficiency and fidelity of tRNA aminoacylation as a function of tRNA sequence, modification, and environmental conditions. 

Abstract The prebiotic formation of RNA building blocks is well‐supported experimentally, yet the emergence of sequence‐ and structure‐specific RNA oligomers is generally attributed to biological selection via Darwinian evolution rather than prebiotic chemical selectivity. In this study, we used deep sequencing to investigate the partitioning of randomized RNA overhangs into ligated products by either splinted ligation or loop‐closing ligation. Comprehensive sequence‐reactivity profiles revealed that loop‐closing ligation preferentially yields hairpin structures with loop sequences UNNG, CNNG, and GNNA (where N represents A, C, G, or U) under competing conditions. In contrast, splinted ligation products tended to be GC rich. Notably, the overhang sequences that preferentially partition to loop‐closing ligation significantly overlap with the most common biological tetraloops, whereas the overhangs favoring splinted ligation exhibit an inverse correlation with biological tetraloops. Applying these sequence rules enables the high‐efficiency assembly of functional ribozymes from short RNAs without template inhibition. Our findings suggest that the RNA tetraloop structures that are common in biology may have been predisposed and prevalent in the prebiotic pool of RNAs, prior to the advent of Darwinian evolution. We suggest that the one‐step prebiotic chemical process of loop‐closing ligation could have favored the emergence of the first RNA functions.