Nonenzymatic assembly of active chimeric ribozymes from aminoacylated RNA oligonucleotides

Abstract

Aminoacylated transfer RNAs, which harbor a covalent linkage between amino acids and RNA, are a universally conserved feature of life. Because they are essential substrates for ribosomal translation, aminoacylated oligonucleotides must have been present in the RNA world prior to the evolution of the ribosome. One possibility we are exploring is that the aminoacyl ester linkage served another function before being recruited for ribosomal protein synthesis. The nonenzymatic assembly of ribozymes from short RNA oligomers under realistic conditions remains a key challenge in demonstrating a plausible pathway from prebiotic chemistry to the RNA world. Here, we show that aminoacylated RNAs can undergo template-directed assembly into chimeric amino acid–RNA polymers that are active ribozymes. We demonstrate that such chimeric polymers can retain the enzymatic function of their all-RNA counterparts by generating chimeric hammerhead, RNA ligase, and aminoacyl transferase ribozymes. Amino acids with diverse side chains form linkages that are well tolerated within the RNA backbone and, in the case of an aminoacyl transferase, even in its catalytic center, potentially bringing novel functionalities to ribozyme catalysis. Our work suggests that aminoacylation chemistry may have played a role in primordial ribozyme assembly. Increasing the efficiency of this process provides an evolutionary rationale for the emergence of sequence and amino acid–specific aminoacyl-RNA synthetase ribozymes, which could then have generated the substrates for ribosomal protein synthesis.

Template-Free Assembly of Functional RNAs by Loop-Closing Ligation

“…A compatible in situ activation chemistry that could maintain sets of oligonucleotides in an activated state − might drive these ligation reactions closer to completion and thus favor ribozyme assembly by iterated loop-closing ligations. The efficiency of loop-closing ligation could also be increased by using 3′-amino terminated oligonucleotides, as suggested by our previous work with the splinted ligation of 3′-amino or 2′/3′-aminoacylated nucleotides. , …”

“…The efficiency of loop-closing ligation could also be increased by using 3′-amino terminated oligonucleotides, as suggested by our previous work with the splinted ligation of 3′-amino or 2′/ 3′-aminoacylated nucleotides. 10,17 The noncovalent assembly of fragments into partly functional ensembles can be seen as an evolutionary precursor to the assembly of full-length ribozymes by loop-closing ligation. [21][22][23][24]45 Notably, it has been demonstrated experimentally that compared to fully random pools of RNA, compact structured pools are superior sources of functional RNAs in in vitro selection experiments.…”

“…However, ribozymes assembled in this manner are inevitably subject to inhibition by a full-length template or even by shorter splint templates. − , Either product purification to remove the template or the design of splints that bind strongly enough to allow for ligation but weakly enough to avoid inhibition are needed to enable RNA function. Previous efforts to assemble ribozymes by splinted ligation also made use of 3′-amino terminated or 2′,3′-aminoacylated oligonucleotides to increase the efficiency of ligation. A further problem with the generation of complex-structured RNAs is that the final structure is only realized during the folding stage (unfolded ligation product in Figure , top pathway).…”

The Photophysics of Nucleic Acids: Consequences for the Emergence of Life

“…During these early “nonenzymatic” times, the chemical template‐directed RNA copying is thought to have played a key role in the origin of translation [80] where the aminoacylation of short RNA oligonucleotides helped in their assembly into functional ribozymes [81] . Prior to the evolution of ribosomal translation, such ribozymes, with their distinct sequences, would have been subject to the UV‐induced sequence selection as described in this review.…”

Selective Synthesis of Lysine Peptides and the Prebiotically Plausible Synthesis of Catalytically Active Diaminopropionic Acid Peptide Nitriles in Water

“…Specific interactions between nucleic acids (e.g., DNA and RNA) and peptides may have been essential at the origin of life. 17,18,[31][32][33][34][35]54,55 It is even possible that evidence for this primordial interaction is preserved today in the core of the ribosome, 56 making the interrogation of the available prebiotic composition of (cationic) peptides an especially important undertaking. By investigating the viability of Lys homologues in aqueous peptide nitrile ligations, we have observed a pronounced differentiation of Lys from both Orn and Dab.…”