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Experiments on “Self-Replicating” RNA Indicate the Need for Intelligent Agency in Life’s Origin

Brian Miller
Photo credit: Dariusz Bartosik, CC BY-SA 3.0 , via Wikimedia Commons.

In an article yesterday, I critiqued the RNA World hypothesis by examining the required investigator interventions in an experiment on “self-replicating” nucleotide sequences:

  • Careful control of temperature variations and other experimental conditions 
  • Supply of information in nucleotide sequences 
  • Supply of large numbers of templates and individual nucleotide strands 

Now I will describe how all other experiments on self-replicating RNAs require a comparable level of investigator interference. 

Studies typically approach the problem of self-replication in one of two ways. Either they start with RNA enzymes (aka ribozymes) that are engineered to join (“self-assemble”) two or more fragments of ribozymes together (herehereherehere). Or they start with ribozymes termed polymerases that can bind to a template RNA and use it to link individual nucleotides, or smaller strands of nucleotides, together to replicate the template nucleotide sequence (hereherehere, here). 

Standard Interventions 

All such studies require similar interventions to those listed above. Experiments invariably require highly orchestrated physical and chemical conditions to produce the desired results. They also consistently start with ribozymes from cells and carefully modify them to perform such targeted actions as combining RNA fragments or replicating an RNA template. And, they regularly use concentrations of ribozymes and templates that correspond to at least trillions of RNAs in 1 ml of solution. The replication rates would drop below the degradation rates if concentrations dropped below a million RNAs per ml. Replication would then continuously decrease until it ceased. 

Even with all of this assistance, the error rate for polymerases is typically 3-10  percent per nucleotide. The Attwater et al. 2018 study reported the lowest error rate at 2.6 percent per nucleotide. Yet, this claim must be greatly qualified. The experiment did not use individual nucleotides as the building blocks for new RNA. It instead used nucleotide triplets (three joined nucleotides) at concentrations corresponding to quadrillions of triplets per ml. In addition, the investigators had to manually link 25 percent of the triplets together, so they could bind to a template as a larger chain. In other words, the polymerase failed to replicate a template without the researchers linking sets of nucleotides together beforehand. 

Of key importance, the high error rates in all experiments guarantee that the information in any sequence longer than 30 nucleotides would be completely lost after several rounds of replication. Polymerases have sequence lengths that are typically close to 200 nucleotides, so replicated copies — assuming that polymerases could copy themselves — would become entirely nonfunctional after only a few generations. This challenge was well articulated in the Adamski et al. 2020 review article:

To date, the fidelities (replication accuracies) of synthetic self-replicating systems rely solely on molecular recognition and lack the sophisticated error-correction machinery that promotes fidelity in DNA replication. Thus, error-prone replication appears unavoidable and must be accommodated in any scenario that involves self-replicators becoming more complex, which requires a greater amount of information to be copied during replication. Increasing the amount of information in self-replicators leads to Eigen’s paradox — self-replicators must contain a lot of information to replicate accurately, yet obtaining self-replicators containing a lot of information already requires accurate replication. 

The Polymerase Paradox

Even more problematic, no polymerase has ever been constructed, or likely will be constructed, that could replicate more than a small fraction of itself. The challenge is that polymerases cannot efficiently replicate long RNAs with the complex folded structures that are essential for ribozyme functions. Attwater et al. clearly describe the problem: 

However, even the most highly-evolved RPRs [RNA polymerase ribozymes] are substantially impeded by template secondary structures. Such structures are ubiquitous in larger, functional RNAs (including the RPRs themselves) and generally indispensable for function. The strong inhibitory role of this central feature of RNA leads to an antagonism between the degree to which an RNA sequence is able to fold into a defined three-dimensional structure to encode function (such as catalysis) and the ease with which it can be replicated.

In addition, vast numbers of very long nucleotide sequences must be synthesized for at least one to have any chance of acting as a fully functional polymerase. Even to create the highly limited polymerases used in experiments today, researchers must start with ribozymes in cells and then modify them for improved performance (herehere). Specifically, they create hundreds of trillions of altered versions of the initial RNA sequence. They then select those that most efficiently and accurately replicate different templates. After multiple rounds of selection, the best performing RNAs are reproduced in mass quantities for their experiment. 

The challenge of simply improving an existing polymerase demonstrates that finding one in a pool of random sequences would require an enormous number of trials. The paradox is that a fully functional polymerase is required to generate large numbers of long RNA sequences. Yet, large numbers of RNAs must be generated before a polymerase would have any chance of emerging. 

Even if polymerases and nucleotides were seeded on the ancient earth, the polymerases would most efficiently replicate RNAs that lacked the complex folds required for ribozyme function. Any evolving system of RNAs would quickly include almost exclusively RNAs that performed no biologically useful actions. The polymerases and other RNAs would quickly degrade, followed by the nucleotides, and the entire process would have to start again from scratch. This challenge and those mentioned above reinforce the conclusion that the RNA World hypothesis is entirely nonviable. The origin of highly accurate replication requires intelligent direction.