THE "FATAL FLAWS" WITH RICHARD CARRIER'S RNA SELF-REPLICATOR EXAMPLES

   

The "Fatal Flaw" with Richard Carrier's 

RNA Self-Replicator Examples

(See, "Q's Response to Richard Carrier: About the Debate," for context)

(See also, "Q's Open Challenge to Richard Carrier")

Richard Carrier’s claim:  

"From this he estimates, counting all comparable planets and moons with suitable environs abundant evidence entails for the known universe, the probability of a self-replicating chain of RNA (which we know exist:  Tews & Meyers 2017, Robertson & Joyce 2014, Lincoln et al. 2009, etc.) arising in those environs somewhere in the universe."

Q's Verdict:

The first study has absolutely nothing whatsoever to do with the origin of life, and the remaining two are proof of concept/principle studies for lab-engineered RNA self-replicators that are too complex to spontaneously emerge by chance, but that demonstrate such could exist in principle. While RNA replicases are known to exist in nature (in living organisms), self-replicating RNA replicases are not known to exist in nature.

Tews & Meyers (2017):

Source citation: Tews, B. A., & Meyers, G. (2017). Self-replicating RNA. RNA Vaccines, 15-35.

Summary: This study has absolutely nothing whatsoever to do with the origin of life, but concerns viral RNA in vaccine production that requires a living cell to replicate, which, of course, would not exist prior to the origin of life. 

Richard uncritically cites this study because it has "Self-replicating RNA" in the title, when in fact this study has absolutely nothing whatsoever to do with the origin of life, but concerns the use of "self-replicating" viral RNA in vaccine production. This "self-replicating [viral] RNA" cannot actually replicate itself, but requires a living cell to replicate; which, of course, we would not have prior to the origin of life. The viral RNA can only replicate with the help of proteins and cellular machinery, so it must be transferred into a living cell in a process known as transfection in order to replicate. It is, thus, completely irrelevant to the origin of life and doesn't help Richard's arguments in the least.

"Replication" can mean anything from "production of a new organism from a genetic blueprint (as in "viruses replicate") or kinetically-determined self-catalysis (as in "crystal defects replicate")." (Bains, W. (2020). Getting beyond the Toy Domain. Meditations on David Deamer's "Assembling Life"). Here, the first usage is in mind.

The "self-replicating RNA" in this study has nothing to do with the origin of life, but concerns viral RNA replication in vaccine design and production. The "self-replicating RNA derived from the genomes of positive strand RNA viruses" in this study is "self-replicating" in that “upon introduction into cells is able to promote a full replication cycle including release of infectious virus particles.” (Tews & Meyers 2017). It essentially contains all the information needed to direct its own synthesis, but like a blueprint without the corresponding factory assembly line, it cannot physically replicate itself, and, thus, needs viral and host cell "machinery" such as replicase and polymerase enzymes to "read" and "translate" the genetic information. 

Also, "[t]ranslation of this RNA leads to a polyprotein that is co-translationally and posttranslationally processed by viral and host cellular proteases." In other words, as stated above, it essentially contains all the information needed to replicate itself, but not quite. The protein products must be further processed by "viral and host cellular proteases" to become functional. The details are in the Tews & Meyers (2017) article Richard cites.

(Fig. 2, Tews & Meyers 2017)

"Figure 2: Lower part: From full length plasmids containing a eukaryotic promoter vRNA will be transcribed by the cellular machinery upon transfection of the cDNA construct. After export of the RNA into the cytoplasm its translation will provide the viral proteins necessary for replication....The resulting RNA is transfected into cells where it is translated. In all cases translation of the RNA within transfected cells generates the viral replicase proteins that are necessary and sufficient to initiate virus replication and production of viral particles." (Tews & Meyers 2017)

Lincoln & Joyce (2009):

Source citation: Lincoln, T. A., & Joyce, G. F. (2009). Self-sustained replication of an RNA enzyme. Science, 323(5918), 1229-1232.

Summary: Self-replicating RNAs are not known to exist in nature. However, this historic, breakthrough "proof of concept/principle" study demonstrated for the first time that self-replicating RNA enzymes can exist in principle. However, the self-replicator is not prebiotically plausible and is considered too complex to spontaneously emerge by chance.

This breakthrough study demonstrated for the first time that RNA enzymes that can self-replicate "indefinitely" (with an unending supply of substrates) and without the need for proteins or other cellular components can exist in principle (i.e., self-replicating RNAs are not known to exist in nature). But this "proof of principle"—as important and historic as it is—is not proof of prebiotic plausibility. In fact, no one believes the cross-catalytic self-replicating RNA system Lincoln & Joyce designed is prebiotically plausible, nor did Lincoln & Joyce ever make such a claim, because that was not the point of their research. Their replicating system is not a viable candidate for the origin of life, because for one it is considered too complex to form by spontaneous random assembly. A single round of self-replication requires the spontaneous origin of six molecules—two enzymes and four substrates—totaling 284 nucleotides. But even if such a system did miraculously form, nothing further would happen without a continuous supply of substrates to sustain self-replication "indefinitely." This is the same "fatal flaw" that the "Lee peptide" encounters. In fact, the problem is far worse, because while "Lee peptide" self-replication requires the spontaneous origin of a continuous supply of two, specifically sequenced substrates totaling 32 amino acids, Lincoln-Joyce replication requires the spontaneous origin of a continuous supply of four, specifically sequenced substrates totaling 132 nucleotides—an impossible feat by any standard. Of course, Lincoln & Joyce (2009) never make such an outrageous claim. It is Richard Carrier who is misrepresenting the scope and significance of their research.

As a 2009 press release explained:

“The scientists [Lincoln & Joyce] have synthesized for the first time RNA enzymes that can replicate themselves without the help of any proteins or other cellular components, and the process proceeds indefinitely....The replicating system actually involves two enzymes, each composed of two subunits and each functioning as a catalyst that assembles the other. The replication process is cyclic, in that the first enzyme binds the two subunits that comprise the second enzyme and joins them to make a new copy of the second enzyme; while the second enzyme similarly binds and joins the two subunits that comprise the first enzyme. In this way the two enzymes assemble each other — what is termed cross-replication. To make the process proceed indefinitely requires only a small starting amount of the two enzymes and a steady supply of the subunits...[Joyce] is quick to point out that, while the self-replicating RNA enzyme systems share certain characteristics of life, they are not themselves a form of life…The subunits in the enzymes the team constructed each contain many nucleotides, so they are relatively complex and not something that would have been found floating in the primordial ooze.” (“How Did Life Begin? RNA That Replicates Itself Indefinitely Developed For First Time”) (emphasis added)

Self-replicating RNAs are not known to exist in nature (See, e.g., Piette & Heddle 2020: "The simplest RNA World theory requires only a self-replicating ribozyme. This could be an RNA strand with ligase activity; that is, self-templating using pre-existing large fragments of complimentary sequences. Ligase (but not self-replicating ligase) ribozymes do exist in nature" (emphasis added)). Lincoln & Joyce (2009) is a "proof of principle" study that demonstrates they could exist....in principle. But as explained below, the replicating system they designed is too complex to be a viable candidate for the origin of life. It is not prebiotically plausible.

Self-replicators are also not immortal 'magical molecules' that can sustain their replication "indefinitely." This is a common misconception that Richard seems to share. The oft missed caveat (noted in the press release above) is that self-replication can be sustained "indefinitely" only if "a steady supply of" substrates is maintained (See, also, Lincoln & Joyce, 2009): “Exponential growth can be continued indefinitely so long as a supply of the four substrates is maintained.” (emphasis added)). But that's just it: a steady supply cannot be maintained indefinitely, so exponential growth cannot be sustained indefinitely—even in theory. For even if we imagine some hypothetical endless supply of substrates, the mass of product produced in the reaction would increase exponentially to absurd proportions. As Lincoln & Joyce (2009) note, "In this system, exponential growth continues indefinitely at constant temperature, with a doubling time of about 1 h." At that rate the mass of product produced would be greater than the mass of the earth itself after just one week! (For more information, see, "The 'Concentration Threshold' Problem"). But as explained below, the spontaneous origin of molecules needed for just a single round of replication is extremely unlikely.

Lincoln & Joyce employed a strategy called cross-catalytic replication, whereby, “two [RNA] enzymes catalyze each other's synthesis from a total of four oligonucleotide substrates." (Lincoln & Joyce, 2009). The four oligonucleotide substrates are 52 (A), 52 (A’), 14 (B), and 14 (B’) nucleotides long, and are complementary to each other (See, Figure 1 & Supplementary Information). The two RNA enzymes (E & E’) are each 76 nucleotides long, and are essentially the A & B (or A’ & B’) pieces combined with an extra 10 nucleotides tacked on. Enzyme E’ catalyzes a single chemical bond reaction to connect A & B together to make Enzyme E, and Enzyme E catalyzes a single chemical bond reaction to join A’ & B’ together to make Enzyme E’. Thus, a single round of “self-replication” requires six molecules (not one molecule): four oligonucleotides totaling 132 nucleotides (i.e., 52nt + 52nt + 14nt + 14nt = 132nt); and two enzymes totaling 152 nucleotides (i.e., 76nt + 76nt = 152nt) for a grand total of 284 nucleotides.

"Fig. 1. Scheme for cross-catalytic replication of RNA enzymes. (A) The enzyme E′ (gray) catalyzes ligation of substrates A and B (black) to form the enzyme E, while E catalyzes ligation of A′ and B′ to form E′. The two enzymes dissociate to provide copies of both E and E′ that each can catalyze another reaction. (B) Sequence and secondary structure of the complex formed between the cross-replicating RNA enzyme and its two substrates (E′, A, and B are shown; E, A′, and B′ are the reciprocal). Curved arrow indicates the site of ligation. Boxed residues indicate the sites of critical wobble pairs that provide enhanced catalytic activity compared to the parental R3C ligase." (Lincoln & Joyce, 2009)

These six molecules can’t have just any nucleotide sequence, but must have specific nucleotide sequences that are complementary to each other, and they must further spontaneously form in the same location at the same time. Now what is the probability of that happening in order to have just one single round of self-replication? A crude "toy model" estimate based on the correct sequencing of the four RNA nucleotides A, U, G & C in each of 284 positions (i.e., 4^284) gives a probability of 1 chance in 10^170. The probability of the four substrates spontaneously forming again in order to have a second round of self-replication (and each subsequent round of self-replication) is [an additional] 1 in 4^132 = 10^79; which all combined gives a probability of (10^170) x (10^79) = 10^25. 

This, of course, ignores numerous complicating factors. Our above scenario for just two rounds of “self-replication” is just “toy model” chemistry “on paper”. That is not how chemistry actually works, which is driven by thermodynamics and kinetics, and usually highly dependent on sufficient reactant concentration to drive forward reactions (Again, see, "The 'Concentration Threshold' Problem"). A mere six molecules is not enough to drive a reaction in mass action chemistry. Indeed, the study required hundreds of trillions of reactant molecules (See, Supplementary Information in Lincoln & Joyce 2009). And even having hundreds of trillions of substrate molecules is not sufficient. The substrates must also be phosphorylated—that is, they must be energetically activated or they won’t participate in chemical reactions. Lincoln & Joyce designed complex substrates that were already phosphorylated (energetically activated).

Lincoln & Joyce also designed 12 pairs of cross-replicating enzymes (i.e., different variations of Enzyme E & E’ with slightly different nucleotide sequences) to model “evolution”. “Populations of various cross-replicating enzymes were constructed and allowed to compete for a common pool of substrates, during which recombinant replicators arose and grew to dominate the population.” But this wasn’t true open-ended evolution via random mutation, because Lincoln & Joyce pre-designed the mutant variants (i.e., the mutations didn’t arise during the reaction), and the mutations weren’t truly random, but restricted to specific regions of the enzymes by design, and in a way that ultimately limited further evolution. As noted by Duim, H., & Otto, S. (2017). Towards open-ended evolution in self-replicating molecular systems. Beilstein journal of organic chemistry, 13(1), 1189-1203.):

“This study by Joyce et al. demonstrates how selection pressure can lead to certain replicators dominating a population in a cross-catalytic replication process. However, the environmental conditions in this experiment are static and the system lacks open-endedness because the number of building blocks that is provided to the system restricts the total diversity of the newly formed species, in this case 12 × 12 different possible replicators. This will cause the system to reach a steady state in which no novel forms of the replicator can be explored anymore.”

If that weren’t enough, the products (E & E’) are also restricted in size, and cannot grow larger than their initial 76 nucleotide length. This is because the molecules Lincoln & Joyce designed were “capped” on the ends to maintain the structural integrity of the molecules—like aglets on the end of shoelaces to prevent fraying and unraveling.

As the follow-up study (below) by Robertson & Joyce 2014 noted about the Lincoln & Joyce 2009 study: 

"Those variants that have faster exponential growth rates enjoy a selective advantage, resulting in the self-sustained Darwinian evolution of the fittest replicators (Lincoln and Joyce, 2009). The self- and cross-replicating RNA enzymes are the only known informational macromolecules that bring about their own exponential amplification." 

However:

"The cross-replicating RNA enzymes have not yet demonstrated the capacity for inventive Darwinian evolution. Existing function can be optimized within the system, but the invention of novel function requires more genetic information than currently can be supported (Sczepanski and Joyce, 2012). The chief reason for this limitation is that the Km of the enzymes for their oligonucleotide substrates is in the range of 1–8 μM, but supplying complex mixtures of substrates at this concentration becomes problematic when there are thousands of variants. In addition, the substrates tend to form nonproductive complexes that sequester these materials and reduce their effective concentration (Ferretti and Joyce, 2013). As a result, the enzymes do not operate close to saturation, which causes their observed rate of reaction to be substantially slower than their inherent catalytic rate."

Robertson & Joyce (2014):

Source citation: Robertson, M. P., & Joyce, G. F. (2014). Highly efficient self-replicating RNA enzymes. Chemistry & biology, 21(2), 238-245.

Summary: This is another "proof of concept/principle" and follow-up study that optimized the catalytic efficiency of the cross-catalytic self-replicating system developed by Lincoln & Joyce 2009. However, like  Lincoln & Joyce 2009 it is not prebiotically plausible and is also considered too complex to spontaneously emerge by chance.   "proof of concept/principle" 

In this later study Joyce et al. note the limitations in the aforementioned 2009 study and problems with RNA “self-replication” in general:

“Thus far, however, these enzymes are not sufficiently robust to enable the replication of RNA, let alone replication of the RNA enzyme that catalyzes the polymerization reaction…The self- and cross-replicating RNA enzymes are the only known informational macromolecules that bring about their own exponential amplification. They can do so indefinitely, so long as an ongoing supply of substrates is made available...The cross-replicating RNA enzymes have not yet demonstrated the capacity for inventive Darwinian evolution. Existing function can be optimized within the system, but the invention of novel function requires more genetic information than currently can be supported…The chief reason for this limitation is…[the substrate concentration]…[also] supplying complex mixtures of substrates at this concentration becomes problematic when there are thousands of variants. In addition, the substrates tend to form non-productive complexes that sequester these materials and reduce their effective concentration…As a result, the enzymes do not operate close to saturation, which causes their observed rate of reaction to be substantially slower than their inherent catalytic rate… The replicating RNA enzyme is the only known molecule that can undergo self-sustained Darwinian evolution, but it has limited genetic complexity, and therefore limited capacity for the invention of novel function. Recent kinetic studies have pointed out the key shortcomings of the original form of this enzyme.” (emphasis added).

These shortcomings “motivated the present study to develop an improved version [of the enzyme] that could replicate faster and/or in the presence of lower concentrations of substrates…Such an improvement would be reflected exponentially, and therefore would have a dramatic effect on the tempo of self-sustained evolution.” And Joyce et al. succeeded in designing an optimized form of the aforementioned Lincoln & Joyce (2009) RNA enzyme. They further used the optimized enzyme to achieve an “[a]mplification of 10^100-fold…over a period of 37.5 hours.” Although, in terms of prebiotic plausibility they employed a “cheat” to achieve this 10^100-fold increase via “serial transfer” by “periodically transferring a portion of a completed reaction mixture to a new reaction mixture that contains a fresh supply of substrates” to “provide what is effectively an infinite supply of substrates.” In other words, the reaction was discontinuous, involving human interference. The serial transfer is similar in effect to how networked enzyme-catalyzed biochemical reactions in living cells continually remove products to sustain continuous reactions. By contrast, chemical reactions in non-biological systems run to completion when reactants are used up, and/or the reaction system attains equilibrium between reactants and products, and can even reverse direction.

But all that aside, the replication system is effectively the same as the cross-catalytic replication strategy employed in Lincoln & Joyce (2009), and still requires six different molecules (not one) to achieve a single round of “self-replication”; including, two enzymes that catalyze each other’s synthesis from the same four oligonucleotide substrates of the 2009 study (A, A’, B & B’). The primary difference is the slightly smaller enzymes—66 nucleotides long instead of 76—that have been optimized to speed the rate of reaction, but again only “so long as an ongoing supply of substrates is made available,” which requires spontaneous formation of all six molecules in the same location around the same time just to achieve a single round of “self-replication.”

Figure 3: Sequence and secondary structure of the evolved enzyme, bound to the substrates used in self-replication. Mutations in F1 relative to the E1 enzyme are highlighted by black circles. Nucleotide positions 10, 20, 30, 40, and 50 are numbered. Boxed regions indicate nucleotides that differ in F1′, the partner for F1 in cross-replication. (Robertson & Joyce 2014)

Figure 4: Self-replication with exponential growth, comparing the starting and evolved enzymes. Reactions were performed using either E1 at 44 °C (gray) or F1 at 48 °C (black), in the presence of either (A) 10 μM or (B) 2 μM substrates. Inset in (A) shows the behavior of F1 over the first 10 min of the reaction. The data were fit to the logistic growth equation, which gave an exponential growth rate of 0.035 and 0.14 min−1 for E1 and F1, respectively, in the presence of 10 μM substrates, and 0.019 and 0.0070 min−1 for E1 and F1, respectively, in the presence of 2 μM substrates. Reaction conditions: 25 mM MgCl2, pH 8.5. (Robertson & Joyce 2014)

Figure 5: Exponential growth deduced from the initial rate of reaction as a function of the starting concentration of enzyme. Reactions were carried out using either 5 (gray) or 10 (black) μM substrates. Initial velocity was measured over the first 10% of the reaction, and the data were fit to an equation with reaction order 1.0. Reaction conditions: 25 mM MgCl2, pH 8.5, 48 °C. (Robertson & Joyce 2014)

Figure 6: Cross-replication and sustained exponential growth. (A) One round of cross-replication, beginning with 0.02 μM each of F1 (black) and F1′ (gray). The data were fit to the logistic growth equation, which gave an exponential growth rate of 0.11 min−1 for both enzymes. Dashed vertical line indicates the yield at 45 min, which was the time of serial transfer. (B) Fifty successive rounds of cross- replication, with transfer of 1% of reacted materials (100-fold dilution) after each round. The yield of newly-synthesized F1 and F1′ was measured after each round and the compounded yield was plotted as a function of time, giving a growth rate of 2.67 logs/h−1. Reaction conditions: 5 μM each substrate, 25 mM MgCl2, pH 8.5, 47 °C. (Robertson & Joyce 2014)

"Cross-replication employing 5 μM substrates reaches a maximum extent of about 4 μM (75%). Similar behavior is seen with the E1 and E1′ enzymes, which has been attributed to sequence heterogeneity at the 5′ end of the 5′-triphosphorylated substrates (Olea et al., 2012). When the substrates were prepared synthetically, a maximum extent of >90% was achieved. In addition, there is some degradation of the RNA during the course of the reaction, occurring at a constant rate of ~0.1% min−1 for both the enzymes and substrates."

Supplementary Information: 

https://ars.els-cdn.com/content/image/1-s2.0-S1074552113004262-mmc1.pdf 

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3943892/bin/NIHMS550544-supplement-01.pdf