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New Article Purports to Help Explain the Origin of the Genetic Code

Image credit: Vossman/Wikimedia Commons, via Flickr (cropped).

The RNA World hypothesis is the most popular model for the origin of life. Its primary selling point is that RNA can both store information in its four-letter nucleotide sequence and fold into complex three-dimensional structures. A foundational assumption is that large numbers of RNAs with long sequences materialized on the ancient earth. Some eventually appeared that were able to self-replicate or replicate other strands. RNA evolution then commenced where mutations occurred in the nucleotide sequences, and natural selection preserved and multiplied those that folded into RNA enzymes (aka ribozymes) that could drive biologically relevant reactions. RNA then played the role of both DNA and protein enzymes. 

James Tour in his video series on the origin of life (hereherehere) detailed the enormous obstacles inhibiting RNA formation on the early earth. In this article I will describe how research into DNA “self-replication” further discredits the RNA World hypothesis and demonstrates the need for intelligent agency in life’s origin.  

Self-Replicating Templates

recent paper by origin-of-life researchers Alexandra Kühnlein, Simon Lanzmich, and Dieter Braun purports to give credence to the claim that a system of evolving RNAs could transform into primitive protein translation machinery. The experiment actually used DNA as a surrogate for RNA since the two molecules interact very similarly. 

The researchers generated DNA strands with two ends that could bind to the complementary ends of other strands. Residing between the end regions was a center region that acted as an “information domain.” Different versions of the DNA were synthesized with different information domain nucleotide sequences. Each sequence was assigned either a number with a subscript (0A, 0B, 0C, 0D, 1A, 1B, 1C, 1D) or the complement of a number (0A-com, 0B-com, …). Each number and its complement corresponded to complementary sequences that would spontaneous bind together. The ends of multiple strands were linked together to form a chain that represented a “binary code” based on the order of the information domains such as 0010. The chain served the role of a template.  

During the experiment, the information domains of the DNA strands in a template first bound with those of complementary strands. The ends of the complementary DNA then linked together, and a new template emerged. The binary code in the original template was replicated in the new one through the information domains’ specific ordering in the chain. For instance, a template with the domain order (binary code) 0010 would create a new template with the complementary code (0-com 0-com 1-com 0-com). The new template could then direct the formation of the original template (see Figure 1).

Describing this process as replication is highly questionable since the RNA strands were all provided, and they simply linked together. More specifically, nucleotide sequences were not being replicated, but only the few bits of information associated with the binary code were passed on to the next generation. Nevertheless, the researchers speculate that such a binary code could eventually transition into a genetic code for proteins. In reality, the experiment strongly suggests that self-replication could never have commenced without intelligent intervention.

Sequence Information

The DNA strands could only perform the required actions because they were engineered to do so. The researchers started with tRNA sequences from cells and then modified them, so their ends would link together:

We designed a set of cooperatively replicating DNA strands using the program package NUPACK (Zadeh et al., 2011). The sequences are designed to have self-complementary double hairpins and are pairwise complementary within the molecule pool, such that the 3’ hairpin of one strand is complementary to the 5’ hairpin of the next. Their structure resembles the secondary structure of proto-tRNAs proposed by stereochemical theories (Figure 1a), comprising two hairpin loops that surround the anticodon with a few neighboring bases (Krammer et al., 2012).

In addition, the information domains were chosen, so each domain matched a complementary sequence guaranteeing they would bind together. Random domains would have only bound together on very rare occasions. Further, the sequences were specifically “selected for optimal homogeneity of binding energies and melting temperatures.” The success of the experiment depended on the investigators supplying the required information. 

Enormous Numbers of DNA

The investigators also supplied the experiment with templates and individual DNA strands at concentrations that correspond to trillions of copies in a volume the size of a single drop of water. At these concentrations the experiment increased the number of templates by 40 percent within 10 minutes. Such large concentrations were required since much lower concentrations could not have driven template replication at a sustainable rate. 

The minimal required concentration to sustain replication for actual RNA can be estimated by comparing the rates at which replication occurs in this and other studies with the rate at which RNA spontaneously degrades. The researchers determined that the rate at which new templates are generated drops proportionally with the decrease in template concentration. The rate also drops with the concentration of individual strands. 

In comparison, the bond that links nucleotides in RNA spontaneously breaks after a few years on average under conditions comparable to those used in the experiment. Even DNA under ideal conditions will degrade after a few years. Since RNA is far less stable than DNA, it could only last for months at most before degrading. For the experiment, the concentration of templates and of free strands could not likely be reduced below millions of copies per milliliter without sacrificing sustainable replication.    

The implausibility of so many similar versions of the same RNA existing at the same time in the same locale cannot be overstated. Even a single RNA of only a few dozen nucleotides existing in the entire history of the earth would be close to miraculous. For two polymers the length of tRNA — let alone millions with similar sequences — to exist in close proximity is beyond the realm of the imagination. The authors simply note, optimistically, that based on our current understanding of “prebiotic chemistry regarding polymerization and ligation, the creation of >80nt RNA is not yet understood.”

Additional Assistance

Even with all of the aforementioned investigator intervention, the replication process was still not sustainable without additional assistance. The experiment required highly orchestrated changes in temperature:

Template sequences were prepared using a two-step protocol. Annealing from 95 ̊C to 70 ̊C within 1 hr, followed by incubation at 70  ̊C for 30 min. Afterwards, samples were cooled to 2  ̊C and stored on ice. When assembling complexes containing paired information domains (Figure 2), samples were slowly cooled down from 70 to 25  ̊C within 90 min before being transferred onto ice. DNA double hairpins were quenched into monomolecular state by heating to 95  ̊C and subsequent fast transfer into ice water. 

The temperature changes were essential for the complementary strands to properly bind to a template, link together, and then break free as a new template. The experiment also required other carefully controlled environmental and chemical conditions that could not possibly have occurred with such exquisite exactness on the ancient earth. 

In addition, the accuracy of replication corresponded to 85-90 percent per nucleotide, so the error rate was 10-15 percent per nucleotide. The error rate beyond which information is guaranteed to be lost after several rounds of replication is called the error threshold. The threshold is reached for a 10 percent error rate by chains with a length of only 10 nucleotides, but the error threshold for the DNA used in the study or the information in the templates is less than 2 percent. As a result, any information contained in the template would quickly degrade, even under the favorable conditions of the experiment. To maintain the integrity of the information being replicated, the investigators had to constantly resupply it by synthesizing new templates. Without all of the described investigator interventions, a system of replicating RNAs could never emerge or even sustain itself.