THE "FATAL FLAWS" WITH RICHARD CARRIER'S PNA SELF-REPLICATOR EXAMPLES
The "Fatal Flaw" with Richard Carrier's
PNA Self-Replicator Examples
Richard Carrier’s claim:
"In actual fact it’s more likely RNA is an evolved structure, and that life originated with a simpler molecule like PNA, which is more robust and far easier to assemble in nature (Singhal et al. 2014). Simple self-replicating PNA strands are an established fact (the Lee peptide being just one of them; we now have the Plöger-Kiedrowski peptide, the Singhal-Nielsen peptide, and so on). Indeed PNA automatically assembles in the expected conditions (Leman et al., “Dynamic Chemical Assembly of Peptide Nucleic Acids” in XVIIIth International Conference on Origin of Life 2017; and Rodriguez et al., “Nitrogen Heterocycles in Miller-Urey Spark-Discharge Mixtures: Using Chemical Trends to Elucidate Plausible pre-RNAs on the Early Earth”; and now Liu et al. 2020, Frenkel-Pinter et al. 2020 and Scognamiglio et al. 2021). So the probability of natural biogenesis is very much higher than even Totani calculates. All based on evidence."
Q's Verdict:
Peptide nucleic acids are artificial, synthetic molecules that do not exist in nature. Richard cites a mix of articles ranging from irrelevant ones that have nothing to do with PNA self-replicators to proof of concept/principle lab-designed PNA self-replicators that are not prebiotically plausible.
Lee et al. (1996):
Source citation: Lee, David H., et al. (1996). A self-replicating peptide. Nature 382.6591: 525-528.
Summary: Richard writes, "Simple self-replicating PNA strands are an established fact (the Lee peptide being just one of them)." But Richard is wrong. The "Lee peptide" is a polypeptide, not a PNA. (For more information, see, "Richard's First Mistake: a 'Lee peptide' is not a PNA"). We have also already seen, "The 'Fatal Flaw' with Richard Carrier's '1 in 10^41' Argument for Abiogenesis".
Plöger & Kiedrowski (2014):
Source citation: Plöger, T. A., & von Kiedrowski, G. (2014). A self-replicating peptide nucleic acid. Organic & biomolecular chemistry, 12(35), 6908-6914.
Summary: Richard writes, "we now have the Plöger-Kiedrowski peptide," Here, Richard makes the opposite mistake: this is a PNA, not a "peptide." Richard links to the "Supplementary Material" of this study by Plöger & Kiedrowski (2014). This noteworthy proof of concept/principle study is very first lab-designed self-replicating PNA, demonstrating in 2014 that self-replicating PNAs can exist in principle (This should have been Richard's first clue that the 1996 "Lee peptide" is not a self-replicating PNA!). This self-replicator is not a candidate for the origin of life, however. For one, it does not self-replicate by "chaining" single building block monomers together, but two PNA half-strands of itself that are already pre-assembled in the lab. PNAs are also synthetic molecules that are not known to exist in nature.
The "Ploger-Kiedrowski peptide" is, of course, not a 'thing' anymore than "Lee peptide" or "Singhal-Nielsen peptide". These are not scientific names, but merely Richard's designations. Nor are these magical "life-sparking" molecules. "Ploger-Kiedrowski peptide" is also a misnomer, because it is not a peptide (which is a short chain of amino acids), but a more complex Peptide Nucleic Acid (PNA).
The "Ploger-Kiedrowski PNA" and "Lee peptide" do have at least one thing in common, though. They are both "minimal self-replicating systems" and represent, "The simplest form of a self-replicating system....in which the replicator acts as catalyst for its own formation from a set of basic building blocks." (Duim, H., & Otto, S. (2017). Towards open-ended evolution in self-replicating molecular systems. Beilstein journal of organic chemistry, 13(1), 1189-1203.).
The significance of this 2014 study is that it is the first "case of self-replication of PNA" in which there is "template directed synthesis of a self-complementary hexa-PNA from two trimeric building blocks." That should have been Richard's first clue that the 1996 "Lee peptide" is not a "self-replicating PNA". The study further reported that "Kinetic modeling…revealed parabolic growth characteristics. The observed template effect, as well as the rate of ligation, was significantly influenced by nucleophilic catalysts, pH value, and uncharged co-solvents...Our findings contribute to the hypothesis that PNA may have served as a primordial genetic molecule and was involved in a potential precursor of a RNA world.” The subtle distinction should not be missed. The findings "contribute" to the PNA-first hypothesis, but do not provide direct empirical evidence of it. "Needs/wish fulfillment" is not the same as evidence that it happened. Just because RNA is hard to make and it would be helpful to start with something easier and PNA fulfills that need, doesn't mean "therefore" this "proves" it "must" have happened that way. It is a proof of concept/principle study.
Once again, we're not dealing with a single PNA molecule, but three: a hexamer PNA strand that serves as a template to assist the joining of two trimer PNA strands to form another hexamer PNA. And the case is actually worse with this one, because even if all three spontaneously formed in the same location around the same time, the hexamer PNA template strand still would not connect the two trimer PNAs together. This is because the template only assists by physically orienting the two trimer PNA strands in a favorable position to promote bonding. The hexamer PNA template "self-replicator" does not actually catalyze the reaction that connects the two trimer PNA strands. In point of fact, it is a "condensing agent" called EDC (Ethylene Dichloride) that catalyzes the chemical bonding of the two trimer PNAs. This is why the investigators found “the yield of the ligation product is limited by the hydrolysis of the condensing agent: Increasing the EDC concentration from 0.2 to 0.4 M doubles the yield of hexa-PNA T." EDC is, in fact, commonly used as a "condensing agent" in lab experiments to connect nucleotides. But the problem with that is that EDC is not a prebiotically plausible reagent; which is why, once again, it is a proof of concept/principle study:
“Coupling reactions were carried out using EDC, well known to be efficient for the phosphoramidate ligation of nucleotides. Though it does not represent a plausible reagent in a prebiotic environment.” (Liu, Z., Ajram, G., Rossi, J. C., & Pascal, R. (2019). The chemical likelihood of ribonucleotide-α-amino acid copolymers as players for early stages of evolution. Journal of molecular evolution, 87(2), 83-92.)
The investigators further noted that, "The observed template effect, as well as the rate of ligation, was significantly influenced by nucleophilic catalysts, pH value, and uncharged co-solvents," which is hardly surprising. The controlled laboratory conditions and tolerances under which biologically relevant organic molecules form and exist are usually quite narrow, and unlikely to be sustained in natural environments. One cannot just assume that lab results can transfer to natural settings without additional evidence for this.
Finally, the investigators also reported, “The kinetics of the reaction...reveal[ed] parabolic growth according to the square-root law"—which we also observed with the "Lee peptide" and which indicates reaction inhibition. This is a common problem with many "self-replicating systems" which often exhibit "parabolic" instead of runaway, exponential growth. The inhibition is usually caused by failure of the product (in this case, the new hexamer that forms from joining the two trimers) to dissociate from the template strand:
"If this complex does not dissociate, the newly formed template molecule cannot lead to further enhancement of the reaction rate, effectively arresting the autocatalytic cycle. Such product inhibition is an important limiting factor in many synthetic replicator systems and prevents them from attaining exponential growth." (Duim, H., & Otto, S. (2017). Towards open-ended evolution in self-replicating molecular systems. Beilstein journal of organic chemistry, 13(1), 1189-1203.
In other words, this system cannot sustain an exponential growth self-replication reaction.
Singhal et al. (2014):
Summary: Richard writes, "In actual fact it’s more likely RNA is an evolved structure, and that life originated with a simpler molecule like PNA, which is more robust and far easier to assemble in nature (Singhal et al. 2014)." But this study is not evidence of assembling PNA in nature or even a prebiotic simulation of assembling PNA in the lab from scratch, but starts with lab-designed PNA strands that were already pre-assembled. It is another proof of concept/principle study.
Richard cites Singhal et al. (2014), but this study doesn’t actually support what he claims. Singhal et al. (2014) designed a 20mer PNA oligomer to serve as a template in template-directed “self-replication” to help join two 10mer PNA oligomers to make another 20mer PNA that is complementary to the template (See, Figure 5). In simple terms, the 20mer PNA template helps connect two 10mer halves to make another 20mer PNA. In reality, the 20mer PNA template does not catalyze the joining of the 10mer halves, per se, but only physically orients them in a favorable position for them to connect on their own (hence, “template assisted”). Importantly, one end of each 10mer half was specially designed to bond with the other via “aminoacyl transfer of phenylalanine” using “simple ester aminolysis chemistry primitively analogous to the ribosomal peptidyl transferase reaction in the absence of anchimeric assistance from ribose and ribose catalysis”. In other words, the substrates weren’t free-floating amino acids or even free-floating peptide-nucleobase monomers, but were specially designed 10mer oligomer strands where the end of one 10mer PNA was already amino-acylated to provide the nucleophilic NH2 group to “attack the ester linkage of the phenylalanine moiety to form a peptide bond”.
 |
| Singhal et al. (2014) |
So once again, we’re not dealing with a single “self-replicating” molecule, but three separate PNA strands—one template, and two substrates, complementary to each other—that would have to spontaneously form in the same location around the same time (with specially formed ends conducive to aminoacyl transfer of phenylalanine) just to complete a single round of “self-replication”. And once again, even if this occurred, so what? Without a continuous supply of more 10mer PNA substrates to continue template-assisted synthesis the reaction stops. However, the problems run deeper than this, and call into question the prebiotic plausibility of such a replicating system.
Specifically, “the efficiency of the PNA directed acyl transfer in terms of yield is quite low [10%] and...this to a large extent is limited by the high rate of hydrolysis (low stability) of the ester relative to amide formation. Therefore, shielding of the ester from water would increase ester half-life and consequently ‘translation’ efficiency (yield) as observed by including organic solvent (DMSO, NMAA) in the medium….Indeed, a parallel may be drawn to contemporary ribosomes in which a pocket of reduced water activity at the peptidyl transferase center is formed.”
Thus, “In order to optimize the conditions for intramolecular Phetransfer, a series of experiments were performed exploring the effect of organic solvent (DMSO 10−90%), pH (8−11) and temperature (50−90 °C). The reaction was strongly and nonlinearly dependent on the DMSO concentration, and no product could be detected below 25% DMSO (Figure 2B,i). Likewise, increased pH (Figure 2B,ii) and temperature (Figure 2B,iii) strongly favored Phe-transfer. However, increasing temperature as well as pH, and the presence of organic solvents including DMSO, weaken the stability of PNA duplexes, and therefore, conditions that favor chemical acyl transfer disfavor hybridization including hairpin formation.”
In English, the reaction is inhibited by water, and performs optimally in pure organic solvents such as DMSO (used in place of water)—which has not been demonstrated to be prebiotically plausible. But also paradoxically, the use of organic solvents, while favoring “chemical acyl transfer” to connect the 10mer PNA halves, “disfavors hybridization” because “increasing temperature as well as pH, and the presence of organic solvents including DMSO, weaken the stability of PNA duplexes” that form in the process.
Singhal & Nielsen (2014):
Summary: Richard writes, "the Singhal-Nielsen peptide," and so on)." This is also a PNA, not a "peptide." It is similarly not a viable candidate for the origin of life, because among other things, it is a cross-catalytic replicating system of six different pre-assembled PNA strands that would all have to spontaneously originate at the same time and place—four of them continuously.
Like "Ploger-Kiedrowski peptide," Richard's "Singhal-Nielsen peptide" is also a misnomer, because it is not a peptide (which is a short chain of amino acids), but a more complex Peptide Nucleic Acid (PNA). Like Lincoln & Joyce (2009), this study similarly employs a "cross catalytic self-replicating" strategy, whereby "one template strand catalyzes the formation of the other template strand and vice versa." (See, Figure 1(A)). And like Lincoln & Joyce (2009), your "Singhal-Nielsen peptide" is not a single "self-replicating" molecule, but a self-replicating system that requires six different molecules: "two [decameric] template PNAs [ab & cd] and four pentameric precursor PNAs [a & b, which are complementary to c & d]," such that template ab assists the joining of c & d to make template cd, and template cd assists the joining of a & b to make template ab, via "carbodiimide assisted amide ligation."
Thus, once again a single "self-replicating" molecule is not sufficient. We need all six of these complementary molecules to spontaneously form in the same location around the same time for just a single round of "self-replication," and even if this occurs, so what? The reaction promptly stops without additional pentamer substrates that are complementary to the templates. But in addition, and as with most reactions in organic chemistry, the mere presence of molecules in the same location is not sufficient to cause chemical bonding. Naturally occurring organic compounds exist because they are thermodynamically stable, and if they're already thermodynamically they're not going to participate in further reactions unless they are energetically activated. The same applies here. The two decamer PNA "self-replicators" only serve as templates that assist by physically orienting the pentamer PNA substrates in a favorable position via complementary base-pairing, but they don't actually catalyze formation of the chemical bond that connects the pentamers to make decamers. For this to happen, we need the substrates to be energetically activated in a specific way that promotes the chemical bond we are after. Hence, the caveat that this is "carbodiimide assisted amide ligation." Specifically, the investigators report that "the reactive ends of the respective PNA precursors are set up for ligation by amide bond formation via EDC activation." (emphasis added). But as we've already seen in Ploger & Kierdowski (2014), EDC (Ethylene Dichloride), while effective as a nucleotide "condensing agent" in labs, is not a prebiotically plausible reagent:
“Coupling reactions were carried out using EDC, well known to be efficient for the phosphoramidate ligation of nucleotides. Though it does not represent a plausible reagent in a prebiotic environment.” (Liu, Z., Ajram, G., Rossi, J. C., & Pascal, R. (2019). The chemical likelihood of ribonucleotide-α-amino acid copolymers as players for early stages of evolution. Journal of molecular evolution, 87(2), 83-92.)
But there are more problems still. Organic chemistry is complicated and messy, and invariably characterized by a myriad of possible cross reactions and side reactions proportional to the number of different chemical bonds that can potentially form in addition to the single one we're after. Prebiotic syntheses typically ignore and/or mitigate against these annoying "interfering cross-reactions," which constitutes a "cheat; which again is why it is a proof of concept/principle study, not a proof of prebiotic plausibility.
As Bains (2020) notes, "the issue in OOL research is not just 'can we make it' but also 'can we stop making everything else'". (See, Supplementary Information, in Bains, W. (2020). Getting beyond the Toy Domain. Meditations on David Deamer’s “Assembling Life”.).
Here we see an example of this. Specifically, the investigators report that "In order to favor cross-catalysis and avoid side products, the amino-end of two carboxyl PNA precursors b and c were N-terminally acetylated, while the carboxy-end of the two amine PNA precursors a and d was C-terminally amidated." This, of course, calls into question the prebiotic plausibility of the "self-replicating" system. But in fairness to the investigators, they never made such a claim (only Richard seems to!). They were simply trying to "demonstrate that simpler nucleobase replication systems than natural oligonucleotides are feasible." Their goal wasn't to demonstrate prebiotic plausibility, nor your outrageous claim that this "single self-replicator" (which it is not) in the "right environment" is sufficient to guarantee biogenesis. This study is nowhere near close to demonstrating that. It's not even in the same ball-park. As the investigators noted: "Although our findings demonstrate the feasibility of simple chemical PNA replication systems of comparable properties to oligonucleotide replicators, and thereby can support models of PNA as a prebiotic genetic material [i.e., it is a proof of concept/principle study], much further work is required for validation of such a hypothetical scenario." But even more, not only do such N-terminal and C-terminal modifications limit side products (and effectively provide only a single way for the pentamers to link in this system), but they also effectively prevent additive reactions that could potentially lengthen the PNA strand beyond 10mer (i.e., the system is designed to make 10mer copies of the templates and nothing more).
But there are more problems still. The reaction was, of course, conducted in a closed reaction system under controlled laboratory conditions that included buffered pH, carefully controlled temperature, and the like, but the yields were still low. "Cross-catalytic product formation followed product inhibited kinetics." "This closed system composed of four pentameric precursors and two decameric templates (and thus also products) yields approximately two replication rounds (starting with 10 fold excess of the precursors) despite showing product inhibited kinetics." That is, even though "cross-catalytic product formation followed product inhibited kinetics," the investigators were still pleased that not one, but two rounds of "self-replication" were achieved. But of course this required "starting with 10 fold excess of the precursors,'' for chemistry in the real-world typically requires high reactant concentrations to drive reactions (See, "The 'Concentration Threshold' Problem"). A mere six molecules in "toy model land"—even if they did spontaneously form in the same location around the same time—is woefully insufficient. And even with a 10-fold excess, only two replication rounds were achieved.
So we must ask, how do we get life out of that?
Leman et al. (2017):
Summary: Richard writes, "Indeed PNA automatically assembles in the expected conditions (Leman et al., “Dynamic Chemical Assembly of Peptide Nucleic Acids” in XVIIIth International Conference on Origin of Life 2017;" This is the most promising article so far, and is a proof of concept/principle study that seems to show that some of the steps of nonenzymatic PNA assembly can happen in principle, but it is difficult to assess because Richard only links to the article abstract. The study certainly doesn't demonstrate that "PNA automatically assembles in the expected conditions," because among other things, the molecules don't automatically assemble but must be energetically activated in a specific way. Also, the peptide 'backbone' is a homopolymer comprised of a single type of amino acid—cysteine—which we've seen is considered prebiotically rare or restricted in occurrence (plus, it's unrealistic to expect such a purified concentration of a single type of amino acid; much less a synthetically modified form of cysteine)
Rodriguez et al. (2019):
Summary: Richard writes, "and Rodriguez et al., “Nitrogen Heterocycles in Miller-Urey Spark-Discharge Mixtures: Using Chemical Trends to Elucidate Plausible pre-RNAs on the Early Earth”;" This article has nothing to do with "automatic assembl[y]" of PNAs, but is about the hypothetical role of nitrogen heterocycles (N-heterocycles)—which are not unique to PNA and which "probably existed in complex mixtures like those generated by spark discharge experiments" (which is a bad thing; see, "Asphalt Paradox")—as possible pre-precursors of PNA (i.e., hypothetical precursors of the precursors of PNA).
This study has nothing to do with “PNA automatically assembl[ing] in the expected conditions,” but is about possible PNA 'pre-precursors' (i.e., it's not even about nucleoamino acid precursors). It simply documents the occurrence of different reactive “nitrogen heterocycles (N-heterocycles)”—i.e., non-canonical and canonical nucleobases (including purines and pyrimidines)—in Miller-Urey type spark discharge experiments under reducing and neutral redox conditions:
“We show here that multiple prebiotically plausible pathways exist for the robust formation of carbonylated N-heterocycles in Miller-Urey mixtures and discuss how these structures could serve as precursors for the formation of PNAs on the early Earth.” (emphasis added)
But nitrogen heterocycles (N-heterocycles) are not unique to PNAs. They are “components of [genetic] polymers” like “the extant genetic macromolecules RNA and DNA”. Nucleobases are components of RNA, DNA, and PNA. There is no indication in this study that the results are exclusive to PNA (Nor any demonstration or discussion of how one goes from complex Miller-Urey type mixtures that include N-heterocycles to peptide-nucleobase assembly to form nucleoamino acids, and then polymerization to form PNA oligomers). The investigators just exclusively focus on PNA.
They also, “did not determine yields,” which is important information to know. For just because an organic molecule can form under reducing and neutral conditions doesn’t automatically make it prebiotically relevant, especially if the yields are low; for then the "The 'Concentration Threshold' Problem" rears its ugly head again.
Fig. 2 Rodriguez et al. (2019) Fig. 3 Rodriguez et al. (2019) Fig. 5 Rodriguez et al. (2019)
The study further reports that their “observations imply that the canonical nucleobases and other N-heterocycles—whether they formed in situ or were delivered to primordial Earth—probably existed in complex mixtures like those generated by spark discharge experiments.” But not only is this hardly surprising, and the understatement of the year, their existence in “complex mixtures like those generated by spark discharge experiment” is not necessarily a positive, but is more likely a negative. See, e.g., Benner, S. A. (2014). Paradoxes in the origin of life. Origins of Life and Evolution of Biospheres, 44(4), 339-343.:
"The Asphalt Paradox (Neveu et al. 2013). An enormous amount of empirical data have established, as a rule, that organic systems, given energy and left to themselves, devolve to give uselessly complex mixtures, 'asphalts'. Theory that enumerates small molecule space, as well as Structure Theory in chemistry, can be construed to regard this devolution a necessary consequence of theory. Conversely, the literature reports (to our knowledge) exactly zero confirmed observations where RIRI ["replication involving replicable imperfections"] evolution emerged spontaneously from a devolving chemical system. Furthermore, chemical theories, including the second law of thermodynamics, bonding theory that describes the 'space' accessible to sets of atoms, and structure theory requiring that replication systems occupy only tiny fractions of that space, suggest that it is impossible for any non-living chemical system to escape devolution to enter into the Darwinian world of the 'living'. Such statements of impossibility apply even to macromolecules not assumed to be necessary for RIRI evolution. Again richly supported by empirical observation, material escapes from known metabolic cycles that might be viewed as models for a 'metabolism first' origin of life, making such cycles short-lived. Lipids that provide tidy compartments under the close supervision of a graduate student (supporting a protocell first model for origins) are quite non-robust with respect to small environmental perturbations, such as a change in the salt concentration, the introduction of organic solvents, or a change in temperature."
Most prebiotic syntheses are subject to the "Asphalt Paradox," and especially "the complex mixtures like those generated by spark discharge experiments." Only a few purport to solve the paradox (See, e.g., Benner et al. (2012). Asphalt, water, and the prebiotic synthesis of ribose, ribonucleosides, and RNA. Accounts of chemical research, 45(12), 2025-2034.).
Liu et al. (2020):
Summary: Richard writes, "and now Liu et al. 2020," This is not even a PNA, and is the same article that Richard linked to for his "P(any other PNA origin)," and that I also already reviewed. See, my review in "The 'Fatal Flaws' with Richard Carrier's Probability Calculus."
 |
| "Abstract." Liu et al. (2020). |
Frenkel-Pinter et al. (2020):
Source citation: Frenkel-Pinter, M., Samanta, M., Ashkenasy, G., & Leman, L. J. (2020). Prebiotic peptides: Molecular hubs in the origin of life. Chemical reviews, 120(11), 4707-4765.
Summary: Richard writes, "Frenkel-Pinter et al. 2020." Richard only links to the article abstract, so this is similarly difficult to assess. Additionally, the abstract says nothing about PNA. Richard may have confused "peptides" in the title ("Prebiotic Peptides: Molecular Hubs for the Origin of Life") with PNA.
Richard only posted a link to the abstract, so like Leman et al. (2017) it is difficult to assess. According to the abstract, the investigators discuss "non-covalent interactions of peptides with other peptides as well as with nucleic acids, lipids, carbohydrates, metal ions, and aromatic molecules...in relation to the possible roles of such interactions in chemical evolution of structure and function," and "describe research involving structural alternatives to peptides and covalent adducts between amino acids/peptides and other classes of molecules," but beyond this doesn't specify with regard to PNA (which is not a peptide).
Scognamiglio et al. (2021):
Summary: Richard writes, "and Scognamiglio et al. 2021)." This article simply summarizes various applications of nucleopeptides, and includes a short section on "Self-Assembling Nucleopeptides as Potential Prebiotic Genetic Materials," but with no indication of whether or not this includes nucleopeptide monomer (i.e., nucleoamino acid) synthesis as part of the process.
Fig. 2 Scognamiglio et al. (2021)
Comments
Post a Comment