A small polymerase ribozyme that can synthesize itself and its complementary strand

Editor’s summary

For life to emerge, a primordial system must have been able to propagate genetic information without the complex machinery of modern biology. RNA has long been proposed to fulfill this role through self-replication; however, previously described catalytic RNAs (ribozymes) are too large to either copy themselves or arise readily from random RNA sequence pools. Gianni et al. report the discovery of a small ribozyme capable of assembling copies of both itself and its encoding template from short RNA building blocks. The demonstration of such complex activity in a compact RNA sequence supports the plausibility of spontaneously arising self-replicating RNAs at the origin of life. —Di Jiang

Structured Abstract

INTRODUCTION

The hypothesis that a single genetic polymer type can catalyze and template its own replication underpins present thinking about the origin of life. Requiring only one type of genetic polymer reduces the complexity of how life’s first genetic system began replicating from simple chemical building blocks. Such a capacity for self-replication, if found, also has fundamental implications for theories about self-organization and the design of lifelike nanomaterials. RNA has been proposed as the original genetic polymer to support primordial living systems because of its dual nature as both catalyst and template. Laboratory evolution experiments have successfully identified RNA polymerase ribozymes, which are RNA sequences that are capable of catalyzing the copying of RNA templates. However, these ribozymes are large [more than 150 nucleotides (nt) long] and structurally complex. Although this is seemingly necessary for their RNA copying activity, it presents challenges for both their self-replication and spontaneous emergence. The large size imposes a considerable synthetic burden and requires highly accurate replication to avoid mutational decay. Futhermore, complex, folded RNA structures can pose an obstacle for replication. This has thus far prevented experimental demonstration of self-replication in the laboratory. Finally, because the spontaneous chemical formation of RNA sequences favors shorter lengths, the emergence of large ribozymes from random sequence pools is less probable than that of smaller ones.

RATIONALE

The challenges of self-replication and emergence would be substantially reduced if RNA polymerase activity was found in much smaller and structurally less complex RNA motifs. We therefore performed a renewed search for this activity in random RNA sequence pools.

RESULTS

We carried out an in vitro selection for RNA polymerase activity in pools of short, random RNA sequences to discover small RNA motifs that could catalyze templated polymerization using activated RNA building blocks. We identified three ribozymes with RNA polymerase activity and carried out further directed evolution and engineering to improve their activity. This resulted in an unexpectedly small, 45-nt ribozyme (named QT45) with general RNA polymerase activity using activated RNA trinucleotide building blocks. We carried out a high-throughput mutation screen to map the fitness landscape of QT45, which revealed a densely functional, small catalytic core. Despite its small size, QT45 showed an ability to copy a variety of different RNA templates, including sequences with tightly folded secondary structure and those encoding a hammerhead endonuclease ribozyme. Most importantly, QT45 was able to synthesize a copy of both itself and its encoding template—the two key reactions necessary for self-replication.

CONCLUSION

The discovery of QT45 demonstrates that the complex and interlocking molecular functions needed for replication can be performed by an RNA motif of just 45 nt. The small size of QT45 enables it to synthesize both itself and its complementary strand—two previously intractable reactions. The discovery of general RNA polymerase activity in such a simple RNA motif increases the plausibility of RNA-based self-replication emerging spontaneously at the origins of life and provides a foundation for establishing such a system experimentally in the laboratory.

De novo evolution of a small RNA polymerase ribozyme that can copy itself and its template.
Starting from random RNA sequence pools, we discovered a polymerase ribozyme (named QT45, AlphaFold3 prediction shown in cyan), only 45 nt in length. This ribozyme displays a general RNA polymerase activity encoded in a small, functionally dense catalytic core. This advanced activity enables QT45 to synthesize an active copy of a hammerhead ribozyme, as well as a copy of itself and its encoding template strand.

Abstract

The emergence of a chemical system capable of self-replication and evolution is a critical event in the origin of life. RNA polymerase ribozymes can replicate RNA, but their large size and structural complexity impede self-replication and preclude their spontaneous emergence. Here, we describe QT45, a 45-nucleotide polymerase ribozyme, discovered from random sequence pools, that catalyzes general RNA-templated RNA synthesis using trinucleotide triphosphate (triplet) substrates in mildly alkaline eutectic ice. QT45 can synthesize both its complementary strand using a random triplet pool at 94.1% per-nucleotide fidelity and a copy of itself using defined substrates, both with yields of ~0.2% in 72 days. The discovery of polymerase activity in a small RNA motif suggests that polymerase ribozymes are more abundant in RNA sequence space than previously thought.

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