ROBERT SHAPIRO clarifies some important points. He was interviewed by J.Craig Venter in 2008:
I then spent decades running a laboratory in DNA chemistry, and so many people were working on DNA synthesis — which has been put to good use as you can see — that I decided to do the opposite, and studied the chemistry of how DNA could be kicked to Hell by environmental agents. Among the most lethal environmental agents I discovered for DNA — pardon me, I'm about to imbibe it — was water. Because water does nasty things to DNA. For example, there's a process called DNA animation, where it kicks off part of the coding part of DNA from the units — that was discovered in my laboratory. Another thing water does is help the information units fall off of DNA, which is called depurination and ought to apply only one of the subunits — but works under physiological conditions for the pyrimidines as well, and I helped elaborate the mechanism by which water helped destroy that part of DNA structure.
Since then, so-called prebiotic chemistry, which is of course falsely named, because we have no reason to believe that what they're doing would ever lead to life — I just call it 'investigator influenced abiotic organic chemistry' — has fallen into the same trap. In the proceedings of the National Academy of Sciences about two months ago there was a paper — I think it was theoretical — they showed that in certain hydro-thermal events, convection forces and other attractive forces, about which I am unable to comment, would serve to concentrate organic molecules so that organic molecules would get much more concentrated in the bottom of this than they would in the ordinary ocean. Very nice, perhaps it's a good place for the origin of life, and interesting finding, but then there was another commentary paper in the Proceedings by another invited commentator, who said,
Great advance for RNA world because if you put nucleotides in, they'll be concentrated enough to form RNA; and if you put RNA in, the RNA will come together and form aggregates, giving you much more chance of forming a ribosome or whatever. I looked at the paper and thought, How did nucleotides come in? How did RNA come in? How did anything come in? The point is, you would take whatever mess prebiotic chemistry gives you and you would concentrate that mess so it's relevant to RNA or the origin of life — it's all in the eye of the beholder. And almost all of prebiotic chemistry is like this; they take chemicals of their own selection.
People were talking about Steve Benner and his borate paper where he selected, of his own free will, the chemical formaldehyde, the chemical acid-aldehyde, and the mineral borate, and he decided to mix them together and got a product that he himself said was significant in leading to the origin of RNA world, and I, looking at the same thing, see only the hands of Steve Benner reaching to the shelf of organic chemicals, picking formaldehyde, and from another shelf, picking acidaldehyde, etc. Excluding them carefully. Picking a mineral that occurs only in selective places on the Earth and putting it in heavy doses. And at the end getting a complex of ribose and borate, which by itself would be of no use for making RNA, because the borate loves to hold onto the ribose, and as long as it holds onto the ribose it can't be used to make RNA. If it lets go of the ribose, then the ribose becomes vulnerable to destruction by all the other environmental agents. The half-life of pure ribose in solution, a different experiment and a very good one, by Stanley Miller is of the order of one or two hours, and all of the other sugars prominent in Earth biology have similar instability.
I was publishing papers like this and I got the reputation, or the nickname in the laboratory of the prebiotic chemist, of 'Dr. No'. If someone wanted a paper murdered, send it to me as a referee. At some point, someone said, Shapiro, you've got to be positive somewhere. So how did life start? And do we have any examples of authentic abiotic chemistry, not subject to investigator interference? The only true samples we have are those meteorites, which are scooped up quickly and often fallen in an unspoiled place — there was a famous meteorite that fell in France in a sheep field in the 1840s and led to dreadful chemistry of people seeing all sorts of biomolecules in it, not surprisingly. But if you took pristine meteorites and look inside, what you see are a predominance of simple organic compounds. The smaller the organic compound, the more likely it is to be present. The larger it is, the less likely it is to be present. Amino acids, yes, but the simplest ones. Over a hundred of them. All the simplest ones, some of which, coincidentally, overlap the unique set of 20 that coincide with Earth life, but not containing the larger amino acids that overlap with Earth life.
And no sample of a nucleotide, the building block of RNA or DNA, has ever been discovered in a natural source apart from Earth life. Or even take off the phosphate, one of the three parts, and no nucleoside has ever been put together. Nature has no inclination whatsoever to build nucleosides or nucleotides that we can detect, and the pharmaceutical industry has discovered this. Life had to start with the mess — a miscellaneous mixture of organic chemistry to begin with. How do you organize this? You have to have a preponderance of some chemicals or lacking others would be against the second law of thermo-dynamics — it violates a concept that as a non-physicist that I barely grasp called 'entropy'.
In the simplest case, and there may be many more elaborate cases, they found that the energy wouldn't be released unless some chemical transformations took place. If the chemical transformations took place then the energy was released, a lot of it is heat. If this just went on continuously, all you do is use up the energy. Release all of it and you've converted one chemical to another. Big deal. To get things interesting, you have to close the cycle where the chemicals can be recycled by processes of their own, and then go through it again, releasing more energy. And once you have that, you can then develop nodes — because organic chemistry is very robust, there are reaction pathways leading everywhere, which is why it's such a mess.
One doesn't need a freak set of perhaps a hundred consecutive reactions that will be needed to make an RNA, and life becomes a probable thing that can be generated through the action of the laws of chemistry and physics, provided certain conditions are met. You must have the energy. It's good to have some container or compartment because if your products just diffuse away from each other and get lost and cease to react with one another you'll eventually extinguish the cycle. You need a compartment, you need a source of energy, you need to couple the energy to the chemistry involved, and you need sufficiently rich chemistry to allow for this network of pathways to establish itself. Having been given this, you can then start to get evolution.
Shapiro wrote in: A Skeptic's Guide to the Creation of Life on Earth 1986, p.186:
In other words,' I said, `if you want to create life, on top of the challenge of somehow generating the cellular components out of non-living chemicals, you would have an even bigger problem in trying to it the ingredients together in the right way.' `Exactly! ... So even if you could accomplish the thousands of steps between the amino acids in the Miller tar-which probably didn't exist in the real world anyway-and the components you need for a living cell-all the enzymes, the DNA, and so forth-you's still immeasurably far from life. ... the problem of assembling the right parts in the right way at the right time and at the right place, while keeping out the wrong material, is simply insurmountable. 5
A. Graham Cairns-Smith also lists several hurdles that would have to be overcome in his book: Genetic takeover, page 64:
What is missing from this story of the evolution of life on earth is the original means of producing such sophisticated materials as RNA. The main problem is that the replication of RNA depends on a clean supply of rather complicated monomers—activated nucleotides. What was required to set the scene for an RNA world was a highly competent, long-term means of production of at least two nucleotides. In practice the discrimination required to make nucleotide parts cleanly, or to assemble them correctly, still seems insufficient.
The implausibility of prevital nucleic acid If it is hard to imagine polypeptides or polysaccharides in primordial waters it is harder still to imagine polynucleotides. But so powerful has been the effect of Miller’s experiment on the scientific imagination that to read some of the literature on the origin of life (including many elementary texts) you might think that it had been well demonstrated that nucleotides were probable constituents of a primordial soup and hence that prevital nucleic acid replication was a plausible speculation based on the results of experiments. There have indeed been many interesting and detailed experiments in this area. But the importance of this work lies, to my mind, not in demonstrating how nucleotides could have formed on the primitive Earth, but in precisely the opposite: these experiments allow us to see, in much greater detail than would otherwise have been possible, just why prevital nucleic acids are highly implausible. Let us consider some of the difficulties to make RNA & DNA
1. as we have seen, it is not even clear that the primitive Earth would have generated and maintained organic molecules. All that we can say is that there might have been prevital organic chemistry going on, at least in special locations.
2. high-energy precursors of purines and pyrimidines had to be produced in a sufficiently concentrated form (for example at least 0.01 M HCN).
3. the conditions must now have been right for reactions to give perceptible yields of at least two bases that could pair with each other.
4. these bases must then have been separated from the confusing jumble of similar molecules that would also have been made, and the solutions must have been sufficiently concentrated.
5. in some other locations a formaldehyde concentration of above 0.01 M must have built up.
6. this accumulated formaldehyde had to oligomerize to sugars.
7. somehow the sugars must have been separated and resolved, so as to give a moderately good concentration of, for example, D-ribose.
8. bases and sugars must now have come together.
9. they must have been induced to react to make nucleosides. (There are no known ways of bringing about this thermo dynamically uphill reaction in an aqueous solution: purine nucleosides have been made by dry phase synthesis, but not even this method has been successful for condensing pyrimidine bases and ribose to give nucleosides
10. Whatever the mode of joining base and sugar it had to be between the correct nitrogen atom of the base and the correct carbon atom of the sugar. This junction will fix the pentose sugar as either the a- or fl-anomer of either the furanose or pyranose forms. For nucleic acids, it has to be the fl-furanose. (In the dry-phase purine nucleoside syntheses referred to above, all four of these isomers were present with never more than 8 ‘Z, of the correct structure.)
11. phosphate must have been, or must now come to have been, present at reasonable concentrations. (The concentrations in the oceans would have been very low, so we must think about special situations—evaporating lagoons etc.
12. the phosphate must be activated in some way — for example as a linear or cyclic polyphosphate — so that (energetically uphill) phosphorylation of the nucleoside is possible.
13. to make standard nucleotides only the 5’- hydroxyl of the ribose should be phosphorylated. (In solid-state reactions with urea and inorganic phosphates as a phosphorylating agent, this was the dominant species to begin with. Longer heating gave the nucleoside cyclic 2’,3’-phosphate as the major product although various dinucleotide derivatives and nucleoside polyphosphates are also formed
14. if not already activated — for example as the cyclic 2’,3’-phosphate — the nucleotides must now be activated (for example with polyphosphate) and a reasonably pure solution of these species created of reasonable concentration. Alternatively, a suitable coupling agent must now have been fed into the system.
15. the activated nucleotides (or the nucleotides with coupling agent) must now have polymerized. Initially this must have happened without a pre-existing polynucleotide template (this has proved very difficult to simulate ; but more important, it must have come to take place on pre-existing polynucleotides if the key function of transmitting information to daughter molecules was to be achieved by abiotic means. This has proved difficult too. Orgel & Lohrmann give three main classes of problem.
(i) While it has been shown that adenosine derivatives form stable helical structures with poly(U) — they are in fact triple helixes — and while this enhances the condensation of adenylic acid with either adenosine or another adenylic acid — mainly to di(A) - stable helical structures were not formed when either poly(A) or poly(G) Were used as templates.
(ii) It was difficult to find a suitable means of making the internucleotide bonds. Specially designed water-soluble carbodiimides were used in the experiments described above, but the obvious pre-activated nucleotides — ATP or cyclic 2’,3’-phosphates — were unsatisfactory. Nucleoside 5'-phosphorimidazolides, for example N/\ n K/N/P-r’o%OHN/\N were more successful, but these now involve further steps and a supply of imidazole, for their synthesis.
(iii) Internucleotide bonds formed on a template are usually a mixture of 2’—5’ and the normal 3’—5’ types. Often the 2’—5’ bonds predominate although it has been found that Zn“, as well as acting as an eflicient catalyst for the template-directed oligomerization of guanosine 5’-phosphorimidazolide also leads to a preference for the 3’—5’ bonds.
16. the physical and chemical environment must at all times have been suitable — for example the pH, the temperature, the M2+ concentrations.
17. all reactions must have taken place well out of the ultraviolet sunlight; that is, not only away from its direct, highly destructive effects on nucleic acid-like molecules, but away too from the radicals produced by the sunlight, and from the various longer lived reactive species produced by these radicals.
18. unlike polypeptides, where you can easily imagine functions for imprecisely made products (for capsules, ionexchange materials, etc), a genetic material must work rather well to be any use at all — otherwise it will quickly let slip any information that it has managed to accumulate.
19. what is required here is not some wild one-off freak of an event: it is not true to say ‘it only had to happen once’. A whole set-up had to be maintained for perhaps millions of years: a reliable means of production of activated nucleotides at the least.
As the difficulties accumulate the stakes get higher: success would be all the more resounding, but it becomes less likely. Sooner or later it becomes wiser to put your money elsewhere. 2
M. Gargaud and colleagues detail the size of the problem:
One of the principal problems concerning the hypothesis of the RNA world is that it appears quite unlikely that a prebiotic environment could have existed containing the mixture of activated nucleotides favoring the formation and replication of ribozymes, as well as their evolution through natural selection. Even if there were several candidate reactions for the efficient prebiotic synthesis of nucleic bases, access to monomeric nucleotides by chemical pathways in fact comes up against several obstacles. If one goes no further than mimicking the biochemical pathway, the first difficulty that occurs is that of synthesizing ribose, which is formed in just negligible quantities within the complex mixture obtained by polymerization of formaldehyde, and, what is more, has a limited lifetime. The bond between a nucleic base and ribose that produces a nucleoside is then a very difficult reaction. There still remains the matter of obtaining a nucleotide by phosphorylation, which leads to mixtures because three positions remain available on the ribose, and then there is its activation. So there are two possibilities, either to envisage an easier pathway for the prebiotic synthesis of nucleotides or to squarely reject RNA as the initial bearer of information, in favor of an alternative bearer that has not left any evolutionary traces. 4
Unsolved issues regarding nucleic acid synthesis
How would the early Earth have generated and maintained organic molecules? All that can be said is that there might have been prebiotic organic chemistry going on, at least in special locations.
How would prebiotic processes have purified the starting molecules to make RNA and DNA which were grossly impure? They would have been present in complex mixtures that contained a great variety of reactive molecules.
How did the synthesis of the nitrogenic nucleobases in prebiotic environments occur?
How did fortuitous accidents select the five just-right nucleobases to make DNA and RNA, Two purines, and three pyrimidines?
How did unguided non-designed events select purines with two rings, with nine atoms, forming the two rings: 5 carbon atoms and 4 nitrogen atoms, amongst almost unlimited possible configurations?
How did lucky coincidence pick pyrimidines with one ring, with six atoms, forming its ring: 4 carbon atoms and 2 nitrogen atoms, amongst an unfathomable number of possible configurations?
How did random trial and error foresee that this specific atomic arrangement of the nucleobases is required to get the right strength of the hydrogen bond to join the two DNA strands and form Watson–Crick base-pairing?
How did mechanisms without external direction foresee that this specific atomic arrangement would convey one of, if not the best possible genetic system to store information?
How would these functional bases have been separated from the confusing jumble of similar molecules that would also have been made?
How were high-energy precursors to produce purines and pyrimidines produced in a sufficiently concentrated form and joined to the assembly site?
How could the adenine-uracil interaction function in any specific recognition scheme under the chaotic conditions of a "prebiotic soup" considering that its interaction is weak and nonspecific?
How could the ribose 5 carbon sugar rings which form the RNA and DNA backbone have been selected, if 6 or 4 carbon rings, or even more or less, are equally possible but non-functional?
How would the functional ribose molecules have been separated from the non-functional sugars?
How were the correct nitrogen atom of the base and the correct carbon atom of the sugar selected to be joined together?
How could right-handed configurations of RNA and DNA have been selected in a racemic pool of right and left-handed molecules? Ribose must have been in its D form to adopt functional structures ( The homochirality problem )
How could random events have brought all the 3 parts together and bonded them in the right position ( probably over one million nucleotides would have been required ?)
How could prebiotic reactions have produced functional nucleosides? (There are no known ways of bringing about this thermodynamically uphill reaction in aqueous solution)
How could prebiotic glycosidic bond formation between nucleosides and the base have occurred if they are thermodynamically unstable in water, and overall intrinsically unstable?
How could RNA nucleotides have accumulated, if they degrade at warm temperatures in time periods ranging from nineteen days to twelve years? These are extremely short survival rates for the four RNA nucleotide building blocks.
How was phosphate, the third element, concentrated at reasonable concentrations?. (The concentrations in the oceans or lakes would have been very low)
How would prebiotic mechanisms phosphorylate the nucleosides at the correct site (the 5' position) if, in laboratory experiments, the 2' and 3' positions were also phosphorylated?
How could phosphate have been activated somehow? In order to promote the energy dispendious nucleotide polymerization reaction, and (energetically uphill) phosphorylation of the nucleoside had to be possible.
How was the energy supply accomplished to make RNA? In modern cells, energy is consumed to make RNA.
How could a transition from prebiotic to biochemical synthesis have occurred? There are a huge gap and enormous transition that would be still ahead to arrive at a fully functional interlocked and interdependent metabolic network.
How could RNA have formed, if it requires water to make them, but RNA cannot emerge in water and cannot replicate with sufficient fidelity in water without sophisticated repair mechanisms in place?
How would the prebiotic synthesis transition of RNA to the highly regulated cellular metabolic synthesis have occurred? The pyrimidine synthesis pathway requires six regulated steps, seven enzymes, and energy in the form of ATP.
The starting material for purine biosynthesis is Ribose 5-phosphate, a product of the highly complex pentose phosphate pathway, which uses 12 enzymes. De novo purine synthesis pathway requires ten regulated steps, eleven enzymes, and energy in the form of ATP.
How did formaldehyde concentration of above 0.01 M build up?
How did accumulated formaldehyde oligomerise to sugars?
How were they induced to react to make nucleosides? (There are no known ways of bringing about this thermo dynamically uphill reaction in aqueous solution: purine nucleosides have been made by dry phase synthesis, but not even this method has been successful for condensing pyrimidine bases and ribose to give nucleosides
2. A. Graham Cairns-Smith: Genetic Takeover: And the Mineral Origins of Life 1988
5. Robert Shapiro: Origins : A Skeptic's Guide to the Creation of Life on Earth January 1, 1986
I then spent decades running a laboratory in DNA chemistry, and so many people were working on DNA synthesis — which has been put to good use as you can see — that I decided to do the opposite, and studied the chemistry of how DNA could be kicked to Hell by environmental agents. Among the most lethal environmental agents I discovered for DNA — pardon me, I'm about to imbibe it — was water. Because water does nasty things to DNA. For example, there's a process called DNA animation, where it kicks off part of the coding part of DNA from the units — that was discovered in my laboratory. Another thing water does is help the information units fall off of DNA, which is called depurination and ought to apply only one of the subunits — but works under physiological conditions for the pyrimidines as well, and I helped elaborate the mechanism by which water helped destroy that part of DNA structure.
Since then, so-called prebiotic chemistry, which is of course falsely named, because we have no reason to believe that what they're doing would ever lead to life — I just call it 'investigator influenced abiotic organic chemistry' — has fallen into the same trap. In the proceedings of the National Academy of Sciences about two months ago there was a paper — I think it was theoretical — they showed that in certain hydro-thermal events, convection forces and other attractive forces, about which I am unable to comment, would serve to concentrate organic molecules so that organic molecules would get much more concentrated in the bottom of this than they would in the ordinary ocean. Very nice, perhaps it's a good place for the origin of life, and interesting finding, but then there was another commentary paper in the Proceedings by another invited commentator, who said,
Great advance for RNA world because if you put nucleotides in, they'll be concentrated enough to form RNA; and if you put RNA in, the RNA will come together and form aggregates, giving you much more chance of forming a ribosome or whatever. I looked at the paper and thought, How did nucleotides come in? How did RNA come in? How did anything come in? The point is, you would take whatever mess prebiotic chemistry gives you and you would concentrate that mess so it's relevant to RNA or the origin of life — it's all in the eye of the beholder. And almost all of prebiotic chemistry is like this; they take chemicals of their own selection.
People were talking about Steve Benner and his borate paper where he selected, of his own free will, the chemical formaldehyde, the chemical acid-aldehyde, and the mineral borate, and he decided to mix them together and got a product that he himself said was significant in leading to the origin of RNA world, and I, looking at the same thing, see only the hands of Steve Benner reaching to the shelf of organic chemicals, picking formaldehyde, and from another shelf, picking acidaldehyde, etc. Excluding them carefully. Picking a mineral that occurs only in selective places on the Earth and putting it in heavy doses. And at the end getting a complex of ribose and borate, which by itself would be of no use for making RNA, because the borate loves to hold onto the ribose, and as long as it holds onto the ribose it can't be used to make RNA. If it lets go of the ribose, then the ribose becomes vulnerable to destruction by all the other environmental agents. The half-life of pure ribose in solution, a different experiment and a very good one, by Stanley Miller is of the order of one or two hours, and all of the other sugars prominent in Earth biology have similar instability.
I was publishing papers like this and I got the reputation, or the nickname in the laboratory of the prebiotic chemist, of 'Dr. No'. If someone wanted a paper murdered, send it to me as a referee. At some point, someone said, Shapiro, you've got to be positive somewhere. So how did life start? And do we have any examples of authentic abiotic chemistry, not subject to investigator interference? The only true samples we have are those meteorites, which are scooped up quickly and often fallen in an unspoiled place — there was a famous meteorite that fell in France in a sheep field in the 1840s and led to dreadful chemistry of people seeing all sorts of biomolecules in it, not surprisingly. But if you took pristine meteorites and look inside, what you see are a predominance of simple organic compounds. The smaller the organic compound, the more likely it is to be present. The larger it is, the less likely it is to be present. Amino acids, yes, but the simplest ones. Over a hundred of them. All the simplest ones, some of which, coincidentally, overlap the unique set of 20 that coincide with Earth life, but not containing the larger amino acids that overlap with Earth life.
And no sample of a nucleotide, the building block of RNA or DNA, has ever been discovered in a natural source apart from Earth life. Or even take off the phosphate, one of the three parts, and no nucleoside has ever been put together. Nature has no inclination whatsoever to build nucleosides or nucleotides that we can detect, and the pharmaceutical industry has discovered this. Life had to start with the mess — a miscellaneous mixture of organic chemistry to begin with. How do you organize this? You have to have a preponderance of some chemicals or lacking others would be against the second law of thermo-dynamics — it violates a concept that as a non-physicist that I barely grasp called 'entropy'.
In the simplest case, and there may be many more elaborate cases, they found that the energy wouldn't be released unless some chemical transformations took place. If the chemical transformations took place then the energy was released, a lot of it is heat. If this just went on continuously, all you do is use up the energy. Release all of it and you've converted one chemical to another. Big deal. To get things interesting, you have to close the cycle where the chemicals can be recycled by processes of their own, and then go through it again, releasing more energy. And once you have that, you can then develop nodes — because organic chemistry is very robust, there are reaction pathways leading everywhere, which is why it's such a mess.
One doesn't need a freak set of perhaps a hundred consecutive reactions that will be needed to make an RNA, and life becomes a probable thing that can be generated through the action of the laws of chemistry and physics, provided certain conditions are met. You must have the energy. It's good to have some container or compartment because if your products just diffuse away from each other and get lost and cease to react with one another you'll eventually extinguish the cycle. You need a compartment, you need a source of energy, you need to couple the energy to the chemistry involved, and you need sufficiently rich chemistry to allow for this network of pathways to establish itself. Having been given this, you can then start to get evolution.
Shapiro wrote in: A Skeptic's Guide to the Creation of Life on Earth 1986, p.186:
In other words,' I said, `if you want to create life, on top of the challenge of somehow generating the cellular components out of non-living chemicals, you would have an even bigger problem in trying to it the ingredients together in the right way.' `Exactly! ... So even if you could accomplish the thousands of steps between the amino acids in the Miller tar-which probably didn't exist in the real world anyway-and the components you need for a living cell-all the enzymes, the DNA, and so forth-you's still immeasurably far from life. ... the problem of assembling the right parts in the right way at the right time and at the right place, while keeping out the wrong material, is simply insurmountable. 5
A. Graham Cairns-Smith also lists several hurdles that would have to be overcome in his book: Genetic takeover, page 64:
What is missing from this story of the evolution of life on earth is the original means of producing such sophisticated materials as RNA. The main problem is that the replication of RNA depends on a clean supply of rather complicated monomers—activated nucleotides. What was required to set the scene for an RNA world was a highly competent, long-term means of production of at least two nucleotides. In practice the discrimination required to make nucleotide parts cleanly, or to assemble them correctly, still seems insufficient.
The implausibility of prevital nucleic acid If it is hard to imagine polypeptides or polysaccharides in primordial waters it is harder still to imagine polynucleotides. But so powerful has been the effect of Miller’s experiment on the scientific imagination that to read some of the literature on the origin of life (including many elementary texts) you might think that it had been well demonstrated that nucleotides were probable constituents of a primordial soup and hence that prevital nucleic acid replication was a plausible speculation based on the results of experiments. There have indeed been many interesting and detailed experiments in this area. But the importance of this work lies, to my mind, not in demonstrating how nucleotides could have formed on the primitive Earth, but in precisely the opposite: these experiments allow us to see, in much greater detail than would otherwise have been possible, just why prevital nucleic acids are highly implausible. Let us consider some of the difficulties to make RNA & DNA
1. as we have seen, it is not even clear that the primitive Earth would have generated and maintained organic molecules. All that we can say is that there might have been prevital organic chemistry going on, at least in special locations.
2. high-energy precursors of purines and pyrimidines had to be produced in a sufficiently concentrated form (for example at least 0.01 M HCN).
3. the conditions must now have been right for reactions to give perceptible yields of at least two bases that could pair with each other.
4. these bases must then have been separated from the confusing jumble of similar molecules that would also have been made, and the solutions must have been sufficiently concentrated.
5. in some other locations a formaldehyde concentration of above 0.01 M must have built up.
6. this accumulated formaldehyde had to oligomerize to sugars.
7. somehow the sugars must have been separated and resolved, so as to give a moderately good concentration of, for example, D-ribose.
8. bases and sugars must now have come together.
9. they must have been induced to react to make nucleosides. (There are no known ways of bringing about this thermo dynamically uphill reaction in an aqueous solution: purine nucleosides have been made by dry phase synthesis, but not even this method has been successful for condensing pyrimidine bases and ribose to give nucleosides
10. Whatever the mode of joining base and sugar it had to be between the correct nitrogen atom of the base and the correct carbon atom of the sugar. This junction will fix the pentose sugar as either the a- or fl-anomer of either the furanose or pyranose forms. For nucleic acids, it has to be the fl-furanose. (In the dry-phase purine nucleoside syntheses referred to above, all four of these isomers were present with never more than 8 ‘Z, of the correct structure.)
11. phosphate must have been, or must now come to have been, present at reasonable concentrations. (The concentrations in the oceans would have been very low, so we must think about special situations—evaporating lagoons etc.
12. the phosphate must be activated in some way — for example as a linear or cyclic polyphosphate — so that (energetically uphill) phosphorylation of the nucleoside is possible.
13. to make standard nucleotides only the 5’- hydroxyl of the ribose should be phosphorylated. (In solid-state reactions with urea and inorganic phosphates as a phosphorylating agent, this was the dominant species to begin with. Longer heating gave the nucleoside cyclic 2’,3’-phosphate as the major product although various dinucleotide derivatives and nucleoside polyphosphates are also formed
14. if not already activated — for example as the cyclic 2’,3’-phosphate — the nucleotides must now be activated (for example with polyphosphate) and a reasonably pure solution of these species created of reasonable concentration. Alternatively, a suitable coupling agent must now have been fed into the system.
15. the activated nucleotides (or the nucleotides with coupling agent) must now have polymerized. Initially this must have happened without a pre-existing polynucleotide template (this has proved very difficult to simulate ; but more important, it must have come to take place on pre-existing polynucleotides if the key function of transmitting information to daughter molecules was to be achieved by abiotic means. This has proved difficult too. Orgel & Lohrmann give three main classes of problem.
(i) While it has been shown that adenosine derivatives form stable helical structures with poly(U) — they are in fact triple helixes — and while this enhances the condensation of adenylic acid with either adenosine or another adenylic acid — mainly to di(A) - stable helical structures were not formed when either poly(A) or poly(G) Were used as templates.
(ii) It was difficult to find a suitable means of making the internucleotide bonds. Specially designed water-soluble carbodiimides were used in the experiments described above, but the obvious pre-activated nucleotides — ATP or cyclic 2’,3’-phosphates — were unsatisfactory. Nucleoside 5'-phosphorimidazolides, for example N/\ n K/N/P-r’o%OHN/\N were more successful, but these now involve further steps and a supply of imidazole, for their synthesis.
(iii) Internucleotide bonds formed on a template are usually a mixture of 2’—5’ and the normal 3’—5’ types. Often the 2’—5’ bonds predominate although it has been found that Zn“, as well as acting as an eflicient catalyst for the template-directed oligomerization of guanosine 5’-phosphorimidazolide also leads to a preference for the 3’—5’ bonds.
16. the physical and chemical environment must at all times have been suitable — for example the pH, the temperature, the M2+ concentrations.
17. all reactions must have taken place well out of the ultraviolet sunlight; that is, not only away from its direct, highly destructive effects on nucleic acid-like molecules, but away too from the radicals produced by the sunlight, and from the various longer lived reactive species produced by these radicals.
18. unlike polypeptides, where you can easily imagine functions for imprecisely made products (for capsules, ionexchange materials, etc), a genetic material must work rather well to be any use at all — otherwise it will quickly let slip any information that it has managed to accumulate.
19. what is required here is not some wild one-off freak of an event: it is not true to say ‘it only had to happen once’. A whole set-up had to be maintained for perhaps millions of years: a reliable means of production of activated nucleotides at the least.
As the difficulties accumulate the stakes get higher: success would be all the more resounding, but it becomes less likely. Sooner or later it becomes wiser to put your money elsewhere. 2
M. Gargaud and colleagues detail the size of the problem:
One of the principal problems concerning the hypothesis of the RNA world is that it appears quite unlikely that a prebiotic environment could have existed containing the mixture of activated nucleotides favoring the formation and replication of ribozymes, as well as their evolution through natural selection. Even if there were several candidate reactions for the efficient prebiotic synthesis of nucleic bases, access to monomeric nucleotides by chemical pathways in fact comes up against several obstacles. If one goes no further than mimicking the biochemical pathway, the first difficulty that occurs is that of synthesizing ribose, which is formed in just negligible quantities within the complex mixture obtained by polymerization of formaldehyde, and, what is more, has a limited lifetime. The bond between a nucleic base and ribose that produces a nucleoside is then a very difficult reaction. There still remains the matter of obtaining a nucleotide by phosphorylation, which leads to mixtures because three positions remain available on the ribose, and then there is its activation. So there are two possibilities, either to envisage an easier pathway for the prebiotic synthesis of nucleotides or to squarely reject RNA as the initial bearer of information, in favor of an alternative bearer that has not left any evolutionary traces. 4
Unsolved issues regarding nucleic acid synthesis
How would the early Earth have generated and maintained organic molecules? All that can be said is that there might have been prebiotic organic chemistry going on, at least in special locations.
How would prebiotic processes have purified the starting molecules to make RNA and DNA which were grossly impure? They would have been present in complex mixtures that contained a great variety of reactive molecules.
How did the synthesis of the nitrogenic nucleobases in prebiotic environments occur?
How did fortuitous accidents select the five just-right nucleobases to make DNA and RNA, Two purines, and three pyrimidines?
How did unguided non-designed events select purines with two rings, with nine atoms, forming the two rings: 5 carbon atoms and 4 nitrogen atoms, amongst almost unlimited possible configurations?
How did lucky coincidence pick pyrimidines with one ring, with six atoms, forming its ring: 4 carbon atoms and 2 nitrogen atoms, amongst an unfathomable number of possible configurations?
How did random trial and error foresee that this specific atomic arrangement of the nucleobases is required to get the right strength of the hydrogen bond to join the two DNA strands and form Watson–Crick base-pairing?
How did mechanisms without external direction foresee that this specific atomic arrangement would convey one of, if not the best possible genetic system to store information?
How would these functional bases have been separated from the confusing jumble of similar molecules that would also have been made?
How were high-energy precursors to produce purines and pyrimidines produced in a sufficiently concentrated form and joined to the assembly site?
How could the adenine-uracil interaction function in any specific recognition scheme under the chaotic conditions of a "prebiotic soup" considering that its interaction is weak and nonspecific?
How could the ribose 5 carbon sugar rings which form the RNA and DNA backbone have been selected, if 6 or 4 carbon rings, or even more or less, are equally possible but non-functional?
How would the functional ribose molecules have been separated from the non-functional sugars?
How were the correct nitrogen atom of the base and the correct carbon atom of the sugar selected to be joined together?
How could right-handed configurations of RNA and DNA have been selected in a racemic pool of right and left-handed molecules? Ribose must have been in its D form to adopt functional structures ( The homochirality problem )
How could random events have brought all the 3 parts together and bonded them in the right position ( probably over one million nucleotides would have been required ?)
How could prebiotic reactions have produced functional nucleosides? (There are no known ways of bringing about this thermodynamically uphill reaction in aqueous solution)
How could prebiotic glycosidic bond formation between nucleosides and the base have occurred if they are thermodynamically unstable in water, and overall intrinsically unstable?
How could RNA nucleotides have accumulated, if they degrade at warm temperatures in time periods ranging from nineteen days to twelve years? These are extremely short survival rates for the four RNA nucleotide building blocks.
How was phosphate, the third element, concentrated at reasonable concentrations?. (The concentrations in the oceans or lakes would have been very low)
How would prebiotic mechanisms phosphorylate the nucleosides at the correct site (the 5' position) if, in laboratory experiments, the 2' and 3' positions were also phosphorylated?
How could phosphate have been activated somehow? In order to promote the energy dispendious nucleotide polymerization reaction, and (energetically uphill) phosphorylation of the nucleoside had to be possible.
How was the energy supply accomplished to make RNA? In modern cells, energy is consumed to make RNA.
How could a transition from prebiotic to biochemical synthesis have occurred? There are a huge gap and enormous transition that would be still ahead to arrive at a fully functional interlocked and interdependent metabolic network.
How could RNA have formed, if it requires water to make them, but RNA cannot emerge in water and cannot replicate with sufficient fidelity in water without sophisticated repair mechanisms in place?
How would the prebiotic synthesis transition of RNA to the highly regulated cellular metabolic synthesis have occurred? The pyrimidine synthesis pathway requires six regulated steps, seven enzymes, and energy in the form of ATP.
The starting material for purine biosynthesis is Ribose 5-phosphate, a product of the highly complex pentose phosphate pathway, which uses 12 enzymes. De novo purine synthesis pathway requires ten regulated steps, eleven enzymes, and energy in the form of ATP.
How did formaldehyde concentration of above 0.01 M build up?
How did accumulated formaldehyde oligomerise to sugars?
How were they induced to react to make nucleosides? (There are no known ways of bringing about this thermo dynamically uphill reaction in aqueous solution: purine nucleosides have been made by dry phase synthesis, but not even this method has been successful for condensing pyrimidine bases and ribose to give nucleosides
2. A. Graham Cairns-Smith: Genetic Takeover: And the Mineral Origins of Life 1988
5. Robert Shapiro: Origins : A Skeptic's Guide to the Creation of Life on Earth January 1, 1986