A short peptide capable of self-replicating while absorbed on mineral surfaces

UNIVERSITY OF SEVILLE FACULTY OF PHARMACY UNDERGRADUATE DISSERTATION A SHORT PEPTIDE CAPABLE OF SELF-REPLICATING WHILE ADSORBED ON MINERAL SURFACES AUTHOR: JOSÉ CARLOS CACHINERO PADILLA SUPERVISOR: RAFAEL PRADO GOTOR University of Seville Faculty of Pharmacy Physical Chemistry Department Degree in Pharmacy A SHORT PEPTIDE CAPABLE OF SELF-REPLICATING WHILE ADSORBED ON MINERAL SURFACES UNDERGRADUATE DISSERTATION Literature review. Author: José Carlos Cachinero Padilla. Supervisor: Rafael Prado Gotor. Seville, June 2018. ABSTRACT A single small peptide (from 5 to 9 residues long) is one of the finest candidates for being the first replicator that appeared on the primitive Earth and ultimately led to the emergence of life. However, it is not recognized as so, and replication-first theories tend to automatically disregard very small peptides as possible replicators. Their small size makes them seem unable to efficiently store and replicate information, but this might just be a false impression. In this dissertation, a hypothetical self-replication mechanism that can be carried out by a small peptide is presented and it shows that only a slight selectivity and catalytic activity are needed for replication to take place, even if many errors are made along the self-replication process. The fundamental role of minerals is also underlined as they could catalyze certain chemical reactions and provide the appropriate conditions that allow this small peptide to self-replicate. Keywords Origin of life, Peptide, Self-replication, Replication-first, Abiogenesis. 1 Contents Page 1. Introduction. 3 2. Objectives. 5 3. Methodology. 5 4. Results and discussion. 6 4.1 The RNA world and the role of peptides. 6 4.2 A hypothetical mechanism for peptide self-replication. 10 4.3 Why small peptides and minerals? 18 4.4 The two greatest adversaries of small self-replicating peptides: thermodynamics of peptide bond formation and diketopiperazine formation. 22 5. Conclusion. 27 6. References. 28 2 3 1. Introduction On the search for the origin of life one of the most important challenges yet to overcome is finding the first replicator, a molecule with the ability to self-replicate and evolve in a hypothetical prebiotic scenario that can ultimately lead to the emergence of complex life forms. At the same time, this first replicator has to be simple enough to have been randomly synthesized by the previously existing chemicals. However, even if the majority view among scientists is that the spontaneous appearance of a replicator led to the emergence of life (Dyson, 1999)⁠, it is a hypothesis that poses some serious difficulties (Shapiro, 2000; Shapiro, 2007; Kun et al., 2015)⁠ and is viewed with skepticism by a significant number of scientists. They advocate for a metabolismfirst approach where “replication (and therefore a genetic apparatus) could not have arisen until more or less complicated molecules needed for the replication process had accumulated, with a metabolism based either on simple prebiotic organic molecules or on inorganic carbon sources such as CO2” (Anet, 2004)⁠. These metabolism-first approaches are not endorsed in this dissertation because very consistent arguments against them exist (Anet, 2004; Orgel, 2008; Vasas et al., 2010)⁠. On the other hand, there are many experimental efforts that demonstrate the plausibility of a replication-first scenario: several self-replicating nucleic acids (Robertson and Joyce, 2014)⁠, nucleic acid analogues (Plöger and von Kiedrowski, 2014)⁠, peptides (Lee et al., 1996)⁠ and other kinds of molecules (Clixby and Twyman, 2016)⁠ have been observed and synthesized in the recent years. Unfortunately, none of them has been considered by the scientific community to be a firm candidate that can explain the rise of life on Earth. This lack of consensus exists due to the fact that open-ended evolution has never been accomplished by these self-replicating systems (Duim and Otto, 2017)⁠. The concept of open-ended evolution is explained in Duim and Otto’s paper and it refers to a chemical system that “can diversify and increase in complexity and invent new functions indefinitely”. So, what should be done to accelerate the process of finding this first replicator? Extensive empirical work, although expensive, is indispensable in a field where possibilities are infinite and experimental efforts scarce. Worse still, the few experimental efforts that 10 The only major objection against 5-9 residues long peptides being the first replicator is that there are no hints about how they could self-replicate by using simple building blocks; there are no known natural or artificial systems which behave in a similar way. However, that is not a good enough reason for casting them aside because they are very attractive for fulfilling the role of a first replicator and there might exist a plausible mechanism that allows them to self-replicate. 4.2 A hypothetical mechanism for peptide self-replication To find such a mechanism we have to go back to 1969, when Melvin Calvin imagined in his book Chemical Evolution a peptide (P1) that is able to catalyze the synthesis of a specific polypeptide (P2), without a template. This is possible by following two “simple” steps: (1) P1 consecutively catalyzes the condensation of free amino acids (or dipeptides, tripeptides, etc., it is not specified) in one terminus or residue. (2) P1 cleaves from itself (with the help of a metal complex that allows for a stereospecific and amino acid specific peptide bond hydrolysis) the new polypeptide that is formed, and is available to start again with the first step (Calvin, 1969). It must be pointed out that P1 is able to specify the correct amino acid that needs to be condensed so, instead of a random peptide, P2 is formed. This is what Calvin calls “growing-end control”, and he cites two examples where it has been documented, one “artificial” and the other “natural”. The artificial one can be found in a paper written by Steinman and Cole, where they explain that “with all other factors being equal, the probability of union of individual amino acids with one another is determined by the size of the side chains” (Steinman and Cole 1967)⁠. That means that the reaction of different amino acids in aqueous solution is not random, it has a certain degree of selectivity (for example, even at the same relative abundance, it is much more probable for glycine to react with alanine than with phenylalanine). The natural example is the bacterial synthesis of the pentapeptide (UDP-GNAc-Lactic- L-Ala-D-Glu-L-Lys-D-Ala-D-Ala) (Ito and Strominger, 1960)⁠, that does not need a templating mechanism. It is an example of “growing-end control” because the amino acid 11 that needs to be added to the growing peptide is chosen depending on the residue in the end. The enzyme that catalyzes this reaction will only attach D-glutamic acid to the L- alanine residue, then it will only attach L-lysine to the D-glutamic acid residue, etc. Nowadays hundreds of enzymes like this have been identified: they belong to the large family of non-ribosomal peptide synthetases (Süssmuth and Mainz, 2017)⁠. A more recent discussion about their implications in the role of the origin of life can be found in this review (Poole, 2011).⁠ However, this bacterial synthesis only serves as the proof that growing-end control can be extremely precise. We cannot forget that the hypothetical method for producing a specific peptide that we are looking for must occur in a prebiotic scenario, so there is no place for something as complex as an enzyme. Calvin himself acknowledges that a nonenzymatic growing-end control mechanism based only in the partial selectivity intrinsic to the condensation reaction of the different amino acids would not be very accurate. To that end he proposed other theoretical methods to overcome this problem (Bjornson, 1970).⁠ These theoretical methods are not useful for us because what we want to do is to adapt the mechanism imagined by Calvin to a small self-replicating peptide. To do this, it could be theorized that: (1) the sequence of P1 can be functional while being 5-9 amino acid long, and (2) the sequence of P2 is the same as the sequence of P1 (Figure 3). In this scenario accuracy is not a big problem, because as long as P1 produces two copies of itself during its whole existence its net-replication rate will be positive and exponential growth will occur. To illustrate how a small self-replicating peptide like the one in Figure 3 could function, even if a great number of errors are made along the replicating process, we can perform a small thought experiment. Imagine a prebiotic 7-residue long peptide which is able to catalyze, as explained above, the condensation of free amino acids into one of its terminus and then cleave the newly formed 7-residue long peptide. Then imagine that the only free amino acids available are those that could have existed in the prebiotic Earth (there is no consensus about which were these amino acids (Zaia et al., 2008)⁠, but Longo and Blaber have proposed a prebiotic set of 10 α-amino acids (Longo and Blaber, 2012), a number that we can arbitrarily raise to 12 for our calculations, owing to the lack of consensus). 12 Figure 3. A hypothetical mechanism by which small peptides might be able to self-replicate. Each colored ball represents an amino acid. 13 The number of possible combinations for this 7 amino acid long peptide that can be made of 12 different amino acids is 12⁷ = 3.58 · 10⁷. If the half-life of the peptide bond ranges between 350 and 600 years at 25 ºC and neutral pH (Radzicka and Wolfenden, 1996)⁠, then the half-life of a peptide made of 7 amino acids (and thus 6 peptide bonds) should be: 𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃 ℎ𝑎𝑎𝑎𝑎𝑎𝑎 − 𝑎𝑎𝑃𝑃𝑎𝑎𝑃𝑃: 350 𝑦𝑦𝑃𝑃𝑎𝑎𝑦𝑦𝑦𝑦 ⋅ 1 𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃 𝑏𝑏𝑏𝑏𝑏𝑏𝑃𝑃 6 𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃 𝑏𝑏𝑏𝑏𝑏𝑏𝑃𝑃𝑦𝑦 =58 𝑦𝑦𝑃𝑃𝑎𝑎𝑦𝑦𝑦𝑦 However, it turns out that for small peptides with a free N-terminus the main cause of their degradation is not peptide bond hydrolysis, but a much faster reaction: the successive formation of diketopiperazines in their N-terminus (Figure 4) (Steinberg and Bada, 1983)⁠. The half-life of this reaction can be calculated from its estimated rate constant at 25 ºC (8 · 10⁻⁸ s⁻¹) (Radzicka and Wolfenden, 1996)⁠: 𝐻𝐻𝑎𝑎𝑎𝑎𝑎𝑎 − 𝑎𝑎𝑃𝑃𝑎𝑎𝑃𝑃: ln (2) 8⋅10−8 𝑦𝑦−1 = 8.66 ⋅106 𝑦𝑦𝑃𝑃𝑠𝑠𝑏𝑏𝑏𝑏𝑃𝑃𝑦𝑦; ; 8.66 ⋅106 𝑦𝑦𝑃𝑃𝑠𝑠𝑏𝑏𝑏𝑏𝑃𝑃𝑦𝑦 ⋅ 1 𝑚𝑚𝑃𝑃𝑏𝑏𝑚𝑚𝑃𝑃𝑃𝑃 60 𝑦𝑦𝑃𝑃𝑠𝑠𝑏𝑏𝑏𝑏𝑃𝑃𝑦𝑦 ⋅1 ℎ𝑏𝑏𝑚𝑚𝑦𝑦 60 𝑚𝑚𝑃𝑃𝑏𝑏𝑚𝑚𝑃𝑃𝑃𝑃𝑦𝑦 ⋅1 𝑃𝑃𝑎𝑎𝑦𝑦 24 ℎ𝑏𝑏𝑚𝑚𝑦𝑦𝑦𝑦 =100 𝑃𝑃𝑎𝑎𝑦𝑦𝑦𝑦 When a diketopiperazine is formed two residues are cleaved from the N-terminus, so according to these calculations a 7 amino acid long peptide would be degraded into a 5 amino acid long peptide in “only” 100 days. Fortunately, thanks to the self-replication process, our peptide is constantly growing on its N-terminus so its half-life would be much longer. In addition, a very simple and plausible mechanism that makes it very resistant (more than 10 years) to decomposition by formation of diketopiperazines might exist. This protective mechanism will be explained in the section “Possible solutions to two challenging fields: thermodynamics and diketopiperazine formation”.⁠ 14 Figure 4. Hydrolysis due to diketopiperazine formation of the hexapeptide Phe-Gly-Leu-Gly- Val-Gly at pH 7.26 and 100ªC. Reprinted by permission from (Steinberg and Bada, 1983), Copyright © 1983, American Chemical Society.⁠ 15 From now on it will be assumed that the half-life of the self-replicating peptide is 10 years. Remember that it is a 7 amino acid long peptide that can be made of 12 different amino acids (12⁷ = 3.58 · 10⁷), thus, if the addition of each amino acid is random and the concentration of each amino acid is the same, it would need to produce 3.58 · 10⁷ peptides in 10 years to generate its own sequence (replicate) only once. These conditions have not been observed in experiments that simulate the primitive Earth environment, since Gly and Ala seem to give a much higher yield (even from different simulated sources of amino acids, such as hydrothermal vents and UV radiation) than the rest of amino acids (Zaia et al., 2008)⁠. It could be easily imagined then, that 3 out of the 7 residues of our selfreplicating peptide are Gly residues. Also, because of the higher concentration of Gly in the solution, it would have a higher chance of being condensed into the growing peptide than the other free amino acids. For simplicity, it could be stated that Gly has a 50% chance (0.5) of being added to the new peptide, and the 50% left belongs to the other 11 amino acids (0.5/11) (the number 22 is used in the denominator so the numerator will be a whole number): 𝑂𝑂𝑃𝑃𝑃𝑃𝑦𝑦 𝑎𝑎𝑏𝑏𝑦𝑦 𝐺𝐺𝑎𝑎𝑦𝑦 𝑏𝑏𝑃𝑃𝑃𝑃𝑏𝑏𝑏𝑏 𝑎𝑎𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃 𝑃𝑃𝑏𝑏 𝑃𝑃ℎ𝑃𝑃 𝑏𝑏𝑃𝑃𝑛𝑛 𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃 = 0.5 = 11 22 𝑂𝑂𝑃𝑃𝑃𝑃𝑦𝑦 𝑎𝑎𝑏𝑏𝑦𝑦 𝑃𝑃ℎ𝑃𝑃 𝑏𝑏𝑃𝑃ℎ𝑃𝑃𝑦𝑦 11 𝑎𝑎𝑚𝑚𝑃𝑃𝑏𝑏𝑏𝑏 𝑎𝑎𝑠𝑠𝑃𝑃𝑃𝑃𝑦𝑦 𝑏𝑏𝑎𝑎 𝑏𝑏𝑃𝑃𝑃𝑃𝑏𝑏𝑏𝑏 𝑎𝑎𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃 𝑃𝑃𝑏𝑏 𝑃𝑃ℎ𝑃𝑃 𝑏𝑏𝑃𝑃𝑛𝑛 𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃 =0.5 11 =1 22 𝑂𝑂𝑃𝑃𝑃𝑃𝑦𝑦 𝑏𝑏𝑎𝑎 𝑃𝑃ℎ𝑃𝑃 𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃 𝑏𝑏𝑃𝑃𝑃𝑃𝑏𝑏𝑏𝑏 𝑦𝑦𝑃𝑃𝑃𝑃𝑎𝑎𝑃𝑃𝑠𝑠𝑎𝑎𝑃𝑃𝑃𝑃𝑃𝑃 =11 22 ⋅11 22 ⋅11 22 ⋅1 22 ⋅1 22 ⋅1 22 ⋅1 22 =1 1.87 ⋅10⁶ That means that out of every 1.87·10⁶ peptides synthesized our peptide will create a copy of itself. Until now that is the result that would be obtained if the effect of concentration was taken into account and the free amino acids randomly reacted with our peptide. It can, however, get even lower if we theorize that our peptide has a low selectivity for adding the correct amino acid (besides the intrinsic partial selectivity of the condensation reaction between amino acids (Steinman and Cole, 1967; Weber and Orgel, 1979)⁠ which, for simplicity, will not be considered). To represent this low selectivity we have arbitrarily raised the chance of adding the correct amino acid from 1/22 to 6/22, while leaving intact the 11/22 chance of adding a correct Gly residue: 16 𝑂𝑂𝑃𝑃𝑃𝑃𝑦𝑦 𝑏𝑏𝑎𝑎 𝑃𝑃ℎ𝑃𝑃 𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃 𝑏𝑏𝑃𝑃𝑃𝑃𝑏𝑏𝑏𝑏 𝑦𝑦𝑃𝑃𝑃𝑃𝑎𝑎𝑃𝑃𝑠𝑠𝑎𝑎𝑃𝑃𝑃𝑃𝑃𝑃 =11 22 ⋅11 22 ⋅11 22 ⋅6 22 ⋅6 22 ⋅6 22 ⋅6 22 =1 1446 Therefore, by accounting for concentration and selectivity effects the number of one replication every 3.58 · 10⁷ peptides produced has been lowered to one replication every 1446 peptides produced (more favorable and yet realistic scenarios can be imagined where this number is even lower). This means if we hypothesize that our peptide, which has a half-life of 10 years (3650 days), could produce one peptide per day, it would replicate once every 1446 days; producing more than two copies of itself before its degradation and thus beginning a process of exponential self-replication. But is the production of one peptide per day a reasonable rate? Well, this implies the formation of 7 peptide bonds and the hydrolysis of 1 to separate the newly formed peptide from its maker. It could therefore be said that peptide bond formation, knowing the estimate of its uncatalyzed rate constant at 25 ºC (~ 3 · 10⁻⁴ M⁻¹ s⁻¹) (Weber and Orgel, 1979)⁠, would be fast enough, even with a low amino acid concentration, to allow the formation of 7 peptide bonds per day. On the other hand, the hydrolysis of 1 peptide bond would be too slow, as can be deduced from its low uncatalyzed rate constant at 25 ºC (~ 6.3 · 10⁻¹¹ s⁻¹) (half life = ~ 350 years) (Radzicka and Wolfenden, 1996). Fortunately, many well-documented metal catalysts which can raise the rate constant up to 6.4 · 10⁻⁵ s⁻¹ (half-life = 3 hours) at 50 ºC exist for this reaction (Wezynfeld et al., 2016)⁠. From this it could be deduced that one peptide per day is a reasonable rate. However, it would be pointless spending time considering the rate of peptide production per day because at this point a lot has already been speculated. The range of possible variables in the prebiotic Earth are enormous, and the results can differ depending on which variables are taken into account. These calculations were only made to show that, for small oligomers, very little selectivity is required for producing a specific sequence, even if a huge amount of errors are made along the way. This is not an easy-to-realize idea because the number of errors seems very high for self-replication (and information transfer, and evolution) to occur, but this is not true. 17 It also needs to be noted that currently, there are no known small peptides which are able to catalyze the peptide bond formation/hydrolysis reaction. The peptide Ser-His was believed for a few years to be capable of carrying out this task (Li et al., 2000)⁠, but a recent article has reevaluated its presumed catalytic activity and concluded that “previously reported results cannot be interpreted as evidence that Ser-His or any other short Hiscontaining peptide is competent to catalyze amide hydrolysis” (Macdonald et al., 2016)⁠. We acknowledge that the catalysis of this reaction by 5 to 9 amino acid long peptides in solution is not a very likely event, and that is the exact reason that we emphasize the importance of the role of minerals so much. It is very difficult for a small peptide alone to catalyze any kind of reaction because they lack the preorganized environment found in enzymes which is responsible for the major part of their catalytic power: “In water the free energy of moving from the ground state to the transition state is larger than in the enzyme since the water dipoles have to reorganize and pay for the reorganization energy, whereas in the enzyme the polar groups or the catalytic groups are preorganized in the correct direction and thus do not have to pay large preorganization energy. The difference between the large reorganization energy in water and the small reorganization of the protein groups is the reason for catalysis” (Jindal and Warshel, 2017)⁠. Organization of water molecules is also commonly seen on mineral surfaces (Perry IV et al., 2007; Khatib et al., 2016)⁠, where they have a reduced orientational and translational entropy (Wang et al., 2006)⁠. This could mimic, in a basic way, the preorganized environment found in enzymes and allow a small peptide to catalyze (at least at a low rate) the peptide bond formation reaction. It does not stop here; there are many different ways in which minerals could help small peptides to self-replicate, but they will be discussed later. 18 4.3 Why small peptides and minerals? The mechanism of self-replication described above (Figure 3) could also be, theoretically, carried out by an oligoribonucleotide (also known as a small RNA strand) instead of an oligopeptide. The preference for small peptides rather than for oligoribonucleotides lies mostly in structural reasons. Because of their simplicity, amino acids are very easy to synthesize from inorganic compounds, so there are a great deal of very plausible ways in which they could have been formed on the prebiotic Earth: by electric discharges, as shown by the Miller experiment (Miller, 1953)⁠ and more modern experiments that were carried out after our understanding of the composition of the early atmosphere changed (Plankensteiner et al., 2006; Cleaves et al., 2008)⁠, by proton irradiation (Kobayashi et al., 1990)⁠, in submarine hydrothermal vents (Marshall, 1994)⁠, by the Strecker reaction (Strecker, 1850)⁠ and many more reviewed in (Brack, 2007; Ruiz-Mirazo et al., 2014)⁠. However there is also more direct evidence that amino acids were present in the prebiotic Earth. They have been found in various meteorites (Cobb and Pudritz, 2014), specifically carbonaceous chondrites, a type of stony meteorites formed in the early Solar System which have a well-preserved composition. The Murchinson meteorite is the most studied of this group and it has been shown to contain a great variety of amino acids (Figure 5). Therefore it is accepted that, in one way or another, amino acids were widely available in the prebiotic Earth. On top of that, since they are the building blocks that peptides are made of, the synthesis of peptides was also possible. There are numerous processes by which this synthesis could have been carried out: “One may cite salt-induced peptide formation (SIPF) (Rode et al. 1991; Eder and Rode 1994;Rode 1999), hydrothermal synthesis (Imai et al. 1999), impact polymerization (Lyons and Vasavada 1999; Blank et al. 2001), polymerization by coupling to metaphosphate hydrolysis (Yamagata and Inomata 1998), and polymerization in the adsorbed phase (...)—several solutions may be combined in one scenario, e.g. surface polymerization+SIPF (Le Son et al. 1998)”. (Lambert, 2008). 19 Figure 5. “Venn diagram showing the number of amino acids represented in different categories of chemical space. Abiotic refers to the 66 amino acids reported in the Murchison meteorite (including 8 members of the standard alphabet). Non-a refers to the 16 amino acids reported from the Murchison meteorite that have longer carbon ‘‘backbones’’ than those used in genetic coding. Coded refers to the 20 amino acids used within the standard genetic code. Biosynthetic refers to the additional 12 members of the standard alphabet and a further 14 amino acids that are produced as intermediates in their production”. (Philip and Freeland 2011)⁠. Color images available online at www.liebertonline.com/ast. Even though the real importance that each process had is difficult to infer (mainly because of the many uncertainties about the prebiotic Earth’s nature), it is reasonable to believe that small peptides were produced on a regular basis by at least one of them. The rate of formation of each peptide and the maximum length that could be obtained with each process is also a difficult guess, but linear sequences made of up to 8 amino acids could certainly be formed (Gulik et al., 2009)⁠. On the other hand, because of their more complex structure, the prebiotic synthesis of oligoribonucleotides is much less plausible than the prebiotic synthesis of peptides. Its low feasibility has even earned it the name of "The Prebiotic Chemist's Nightmare”, and it is believed by many scientists that “the de novo appearance of oligonucleotides on the primitive earth would have been a near miracle” (Joyce and Orgel, 1993).⁠ In addition, nucleosides are less versatile than amino acids as basic building blocks because of their bigger size and less variable structure (Figure 6). We should not forget that the molecule 26 Figure 8. A self-replicating peptide might be able to protect itself from degradation by diketopiperazine formation by synthesizing a 2 amino acid long cap before starting to produce new peptides. 27 5. Conclusion In the first instance it is not very intuitive to think about a short peptide (from 5 to 9 residues long) as a potential self-replicating entity that was responsible for the origin of life. It has never been demonstrated that a peptide so small can catalyze peptide bond formation, a necessary requirement for the synthesis of new peptides. Moreover, even if a peptide was capable of catalyzing this reaction two things would still be required for it to be considered a self-replicating molecule: the ability to connect the free amino acids in the right order so a copy of itself is formed, and the ability to hydrolyze one specific peptide bond so the newly formed peptide is set free. Of course, all of this has to happen while the right thermodynamic conditions that favor peptide bond formation are met and before the fast degradation of the peptide due to diketopiperazine formation in its N- terminus occurs. However, the plausibility of small peptides as possible self-replicating molecules can be appreciated when one realizes that these problems can be solved with relative ease: a selfreplicating peptide which is constantly growing in its N-terminus is resistant to degradation by diketopiperazine formation; there are many ways for making the thermodynamic conditions favorable to peptide bond formation; metals can catalyze peptide bond hydrolysis; if given enough time, only a little selectivity is needed for forming a few copies of a small peptide; and that while it is true that there are no known peptides which can catalyze the peptide bond formation, the catalytic properties of small peptides in the presence of minerals are poorly studied (minerals might change the catalytic properties of peptides by allowing them to adopt new conformations or by providing them with a specific microenvironment). On top of that, other hypothetical first replicators such as bigger peptides/proteins, RNA, or an RNA-protein combined hybrid come with more problems than small peptides. Bigger peptides/proteins face the same problems as smaller peptides, and because of their bigger size they need a much more robust replication system. The formation of RNA is also thermodynamically unfavorable, and the hydrolysis of each phosphodiester bond occurs with a half-life of only 4 years (Thompson et al., 1995).⁠ Furthermore, the use of activating agents in phosphodiester bond formation “leads to backbones with mixed 3′,5′ and 2′,5′ linkages, misincorporated (i.e., non-Watson-Crick pairing) bases, and truncation 28 products resulting from strand cyclization” (Bean et al., 2009).⁠ Therefore, in view of the plausibility of a small self-replicating peptide as a hypothetical first replicator, more experimental work is needed to either prove or disprove this hypothesis. For example, screening large arrays of small peptides in the presence of different minerals and free amino acids at different temperatures and pHs while looking for the formation of new peptide bonds would be a nice starting point. 6. References Anet F Al. The place of metabolism in the origin of life. Curr Opin Chem Biol. 2004;8(6):654–9. Basiuk VA, Sainz-Rojas J. Catalysis of Peptide Formation by Inorganic Oxides: High Efficiency of Alumina Under Mild Conditions on the Earth-like Planets. Advances in Space Research. 2001;27(2):225–30. Bean HD, Lynn DG, Hud NV. Self-assembly and the origin of the first RNA-like polymers. In: Zaikowski L, Friedrich JM, Seidel SR (eds.). Chemical Evolution II: From the Origins of Life to Modern Society. 1st ed. Washington DC: American Chemical Society; 2009. Belmonte L, Mansy SS. Metal catalysts and the origin of life. Elements. 2016;12(6):413– 8. Bernal JD. The Physical Basis of Life. Proc. Phys. Soc. 1949;62(A):537–58. Bernhardt HS. The RNA world hypothesis: the worst theory of the early evolution of life (except for all the others). Biol Direct. 2012;7(23):1–10. Bertrand M, Brack A. Conformational Transition of Acidic Peptides Exposed to Minerals in Suspension. Chem Eur J. 2000;6:3452–5. Biebricher CK, Eigen M. The error threshold. Virus Res. 2005;107(2):117–27. Bjornson. Chemical Evolution: Condensation reactions of nucleotides and amino acids in aqueous solution. [PhD thesis]. Berkeley: California Univ., Berkeley. Lawrence Radiation Lab; 1970. Brack A. From Interstellar Amino Acids to Prebiotic Catalytic Peptides: A Review. Chem Biodivers. 2007;4:665–79. 29 Bruce Martin R. Free energies and equilibria of peptide bond hydrolysis and formation. Biopolymers. 1998;45(5):351–3. Cairns-Smith AG. Genetic takeover and the mineral origins of life. 1st ed. England: Cambridge University Press; 1982. Calvin M. Chemical Evolution. 1st ed. Oxford and New York: Oxford University Press; 1969. Cleaves HJ, Chalmers JH, Lazcano A, Miller SL, Bada JL. Prebiotic organic synthesis in neutral planetary atmospheres. ACS Symp Ser. 2008;981:282–92. Cleaves HJ, Scott AM, Hill FC, Leszczynski J, Sahaide N, Hazen R. Mineral–organic interfacial processes: potential roles in the origins of life. Chem Soc Rev. 2012;41(16):5365–568. Clixby G, Twyman L. Self-replicating systems. Org Biomol Chem. 2016;14(18):4170– 84. Cobb AK, Pudritz RE. Nature’s starships. I. observed abundances and relative frequencies of amino acids in meteorites. Astrophys J. 2014;783(2):1–12. Danger G, Plasson R, Pascal R. Pathways for the formation and evolution of peptides in prebiotic environments. Chem Soc Rev. 2012;41:5416–29 Dawson P, Muir T, Clark-Lewis I, Kent S. Synthesis of proteins by native chemical ligation. Science. 1994;266(5186):776–9. Duim H, Otto S. Towards open-ended evolution in self-replicating molecular systems. Beilstein J Org Chem. 2017;13:1189–203. Dunetz JR, Magano J, Weisenburger GA. Large-Scale Applications of Amide Coupling Reagents for the Synthesis of Pharmaceuticals. Org Process Res Dev. 2016;20(2):140–77. Dyson F. Origins of Life. 2nd ed. Cambridge: Cambridge University Press; 1999. Eigen M. Self-organization of matter and the evolution of biological macromolecules. Naturwissenschaften. 1971;58(10):465–523. El-Faham A, Albericio F. Peptide Coupling Reagents, More than a Letter Soup. Chem Rev. 2011;111(11):6557–602. Erastova V, Degiacomi MT, G. Fraser D, Greenwell HC. Mineral surface chemistry control for origin of prebiotic peptides. Nat Commun. 2017;8(1):2033. 30 Falagas ME, Pitsouni EI, Malietzis GA, Pappas G. Comparison of PubMed, Scopus, Web of Science, and Google Scholar: strengths and weaknesses. FASEB J. 2007;22(2):338–42. Fox SW, Harada K. Thermal copolymerization of amino acids to a product resembling protein. Science. 1958;128(3333):1214. Gilbert W. The RNA world. Nature. 1986;319:618. Gulik P Van Der, Massar S, Gilis D, Buhrman H, Rooman M. The first peptides: The evolutionary transition between prebiotic amino acids and early proteins. J Theor Biol. 2009;261(4):531–9. Gupta SS, Bhattacharyya KG. Kinetics of adsorption of metal ions on inorganic materials: A review. Adv Colloid Interface Sci. 2011;162(1–2):39–58. Haldane JBS. Origin of life. Ration. Annu. 1929;148:3–10. Hazen RM. Genesis: Rocks, Minerals, and the Geochemical Origin of Life. Elements. 2005;1(3):135–7. Hazen RM, Filley TR, Goodfriend GA. Selective adsorption of L- and D-amino acids on calcite: Implications for biochemical homochirality. Proc Natl Acad Sci. 2001;98(10):5487–90. Hazen RM, Sverjensky DA. Mineral surfaces, geochemical complexities, and the origins of life. Cold Spring Harb Perspect Biol. 2010;2(5):1–22. Iqubal MA, Sharma R, Jheeta S, Kamaluddin. Thermal Condensation of Glycine and Alanine on Metal Ferrite Surface: Primitive Peptide Bond Formation Scenario. Life. 2017;7(2):15. Ito E, Strominger JL. Enzymatic Synthesis of the Peptide in a Uridine Nucleotide from Staphylococcus aureus. J Biol Chem. 1960;235(2):5–7. Jindal G, Warshel A. Misunderstanding the Preorganization Concept can lead to Confusions about the Origin of Enzyme Catalysis. Proteins. 2017;85:2157–61. Joyce GF. The antiquity of RNA-based evolution. Nature. 2002;418(6894):214–21. Joyce GF, Orgel LE. Prospects for understanding the origin of the RNA world. In: Gesteland R.F, Atkins J.F. (eds.). The RNA world. 1st ed. New York: Cold Spring Harbor Laboratory Press; 1993. Kauffman SA. Autocatalytic sets of proteins. J Theor Biol. 1986;119(1):1–24. 31 Khatib R, Backus EHG, Bonn M, Perez-Haro MJ, Gaigeot MP, Sulpizi M. Water orientation and hydrogen-bond structure at the fluorite/water interface. Sci Rep. 2016;6:1–10. Kobayashi K, Tsuchiya M, Oshima T, Yanagawah. Abiotic synthesis of amino acids and imidazole by proton irradiation of simulated primitive Earth atmospheres. Orig Life Evol Biosph. 1990;20(2):99–109. Kruger K, Grabowski PJ, Zaug AJ, Sands J, Gottschling DE, Cech TR. Self-splicing RNA: Autoexcision and autocyclization of the ribosomal RNA intervening sequence of tetrahymena. Cell. 1982;31(1):147–57. Kun Á, Szilágyi A, Könnyu B, Boza G, Zachar I, Szathmáry E. The dynamics of the RNA world: Insights and challenges. Ann N Y Acad Sci. 2015;1341(1):75–95. Lambert JF. Adsorption and polymerization of amino acids on mineral surfaces: A review. Orig Life Evol Biosph. 2008;38(3):211–42. Lee DH, Granja JR, Martinez JA, Severin K, Ghadiri MR. A self-replicating peptide. Nature. 1996;382,:525–8. Li Y, Zhao Y, Hatfield S, Wan R, Zhu Q, Li X, et al. Dipeptide seryl-histidine and related oligopeptides cleave DNA, protein, and a carboxyl ester. Bioorganic Med Chem. 2000;8(12):2675–80. Longo LM, Blaber M. Protein design at the interface of the pre-biotic and biotic worlds. Arch Biochem Biophys. 2012;526(1):16–21. Macdonald MJ, Lavis LD, Hilvert D, Gellman SH. Evaluation of the Ser-His Dipeptide, a Putative Catalyst of Amide and Ester Hydrolysis. Org Lett. 2016;18(15):3518– 21. Marshall W. Hydrothermal synthesis of amino acids. Geochim Cosmochim Acta. 1994;58(9):2099–106. Miller SL. A Production of Amino Acids Under Possible Primitive Earth Conditions. 1953;117(3046):528–9. Oparin AI. The Origin of Life. 1st ed. Moscow: Moscow Worker publisher; 1924 [in Russian]. Orgel LE. The implausibility of metabolic cycles on the prebiotic earth. PLoS Biol. 2008;6(1):5-13. 32 Perry IV TD, Cygan RT, Mitchell R. Molecular models of a hydrated calcite mineral surface. Geochim Cosmochim Acta. 2007;71(24):5876–87. Philip GK, Freeland SJ. Did Evolution Select a Nonrandom “Alphabet” of Amino Acids? Astrobiology. 2011;11(3):235–40. Plankensteiner K, Reiner H, Rode BM. Amino acids on the rampant primordial Earth: Electric discharges and the hot salty ocean. Mol Divers. 2006;10(1):3–7. Plöger TA, von Kiedrowski G. A self-replicating peptide nucleic acid. Org Biomol Chem. 2014;12(35):6908–14. Poole AM. Origins of Life: The Primal Self-Organization. In: Egel R, Lankenau DH, Mulkidjanian A (eds.). Origins of Life: The Primal Self-Organization. 1st ed. Berlin: Heidelberg Springer; 2011. Quirk S. Triggered release of small molecules from proteinoid microspheres. J Biomed Mater Res. 2009;91A:391–9. Radzicka A, Wolfenden R. Rates of uncatalyzed peptide bond hydrolysis in neutral solution and the transition state affinities of proteases. J Am Chem Soc. 1996;118(26):6105–9. Ricardo A, Carrigan MA, Olcott AN, Benner SA. Borate Minerals Stabilize Ribose. Science. 2004;303(5655):196. Robertson MP, Joyce GF. The Origins of the RNA World. 2012;1–22. Robertson MP, Joyce GF. Highly efficient self-replicating RNA enzymes. Chem Biol. 2014;21(2):238–45. Rubinov B, Wagner N, Rapaport H, Ashkenasy G. Self-Replicating Amphiphilic β-Sheet Peptides. Angew Chemie. 2009;121(36):6811–4. Ruiz-Mirazo K, Briones C, De La Escosura A. Prebiotic systems chemistry: New perspectives for the origins of life. Chem Rev. 2014;114(1):285–366. Ryakhovsky V V., Ivanov AS. Study of intramolecular aminolysis in peptides containing N-alkylamino acids at position 2. Tetrahedron. 2012;68(35):7070–6. Segre D, Ben-Eli D, Lancet D. Compositional genomes: Prebiotic information transfer in mutually catalytic noncovalent assemblies. Proc Natl Acad Sci. 2000;97(8):4112– 7. Shapiro R. A replicator was not involved in the origin of life. IUBMB Life. 2000;49(3):173–6. 33 Shapiro R. A Simpler Origin for Life. Scientific American; 2007;296:46–53. Shock EL. Stability of peptides in high-temperature aqueous solutions. Geochim Cosmochim Acta. 1992;56(9):3481–91. Sievers A, Beringer M, Rodnina M V, Wolfenden R. The ribosome as an entropy trap. 2004;101(21):12397–8. Sivasankar N, Vasudevan S. Stabilization of a distorted conformation of cyclohexane adsorbed in the voids of silicalite-1. Catal Letters. 2004;97(1–2):53–8. Sowerby SJ, Cohn CA, Heckl WM, Holm NG. Differential adsorption of nucleic acid bases: Relevance to the origin of life. Proc Natl Acad Sci. 2001;98(3):820–2. Steinberg SM, Bada JL. Peptide Decomposition in the Neutral pH Region via the Formation of Diketopiperazines. J Org Chem. 1983;48,:2295–8. Steinman G, Cole MN. Synthesis of biologically pertinent peptides under possible primordial conditions. Proc Natl Acad Sci USA. 1967;58(2):735–42. Stojanoski K, Zdravkovski Z. On the Formation of Peptide Bonds. J Chem Educ. 1993;70(2):134. Strecker A. Ueber die künstliche Bildung der Milchsäure und einen neuen, dem Glycocoll homologen Körper. Justus Liebigs Ann Chem. 1850;75(1):27–45. Süssmuth RD, Mainz A. Nonribosomal Peptide Synthesis—Principles and Prospects. Angew Chemie. 2017;56(14):3770–821. Takahashi Y, Mihara H. Construction of a chemically and conformationally selfreplicating system of amyloid-like fibrils. Bioorganic Med Chem. 2004;12(4):693– 9. Thompson JE, Kutateladze TG, Schuster MC, Venegas FD, Messmore JM, Raines RT. Limits to Catalysis by Ribonuclease A. Bioorg Chem. 1995;23(4):471–81. Toniolo C, Temussi P. Conformational flexibility of aspartame. Biopolymers. 2016;106(3):376–84. Valeur E, Bradley M. Amide bond formation: beyond the myth of coupling reagents. Chem Soc Rev. 2009;38(2):606–31. Vasas V, Szathmary E, Santos M. Lack of evolvability in self-sustaining autocatalytic networks constraints metabolism-first scenarios for the origin of life. Proc Natl Acad Sci. 2010;107(4):1470–5. 34 Wang J, Kalinichev AG, Kirkpatrick RJ. Effects of substrate structure and composition on the structure, dynamics, and energetics of water at mineral surfaces: A molecular dynamics modeling study. Geochim Cosmochim Acta. 2006;70(3):562–82. Weber AL, Orgel LE. The formation of dipeptides from amino acids and the 2’(3’)-glycyl ester of an adenylate. J Mol Evol. 1979;13:185–92. Westall F, Hickman-Lewis K, Hinman N, Gautret P, Campbell KA, Bréhéret JG, et al. A Hydrothermal-Sedimentary Context for the Origin of Life. Astrobiology. 2018;18(3):259–93. Wezynfeld NE, Frączyk T, Bal W. Metal assisted peptide bond hydrolysis: Chemistry, biotechnology and toxicological implications. Coord Chem Rev. 2016;327– 328:166–87. Zaia DAM, Zaia CTB V, De Santana H. Which amino acids should be used in prebiotic chemistry studies? Orig Life Evol Biosph. 2008;38(6):469–88.