sabato 4 maggio 2013

RNA: its constituents. a colossal BLUFF

Post n. 11 (English)

Imagine that you want to construct a house. You charge architects paying a conspicuous advance. After a few months you go and control the state of the work and you find only the roof held up by an enormous scaffolding. The architects explain to you that there is no problem, one can easily start with the roof. Foundations and basic walls are already in the project.
You look again, amazed, at the state of the construction and you observe that the scaffolding is beginning to vacillate.
What are your feelings?
Hence, for the peculiarity of their atomic structures, the biogenic elements are the only ones which through their compounds are adapted to carry out, in living organisms, the numerous biological functions. We know that between these compounds the most important are the nucleic acids, DNA and RNA, and the proteins. The nucleic acids are the carriers of information for the synthesis of the proteins. Between the two nucleic acids the RNA has given origin to the theory of the “RNA World”. This postulate that life had its origin through the appearance of self-reproducing  molecules of RNA.
The RNA is a large molecule whose   unities constitutive are four nucleotides.
These are the fundamental walls of RNA and they are constituted by a phosphate group, by D-Ribose (a pentose sugar) and one of the four nucleobases, Adenine, Guanine, Uracil, Cytosine.
The nucleotides, however, to be able to give origin to RNA must be activated, that is at only nucleotide it is necessary to link another two phosphate groups.
As we have already shown in the article concerning the “RNA World”, A. Graham Cairns-Smith has analysed the process through which, in laboratory,

 activated nucleotides were obtained. He has counted 140 events (pouring, agitating, decanting, distilling, etc.) which should have been realized, in the prebiotic era, not by chance but in the appropriate way; that is like throwing a dice and obtaining number 6 for 140 times straight. Seeing these immense difficulties of procedure, following the principle of Occam’s razor, the most simple and obvious supposition is that the nucleotides, in the prebiotic era, never existed. And in fact Cairns-Smith in “Sette indizi sull’origine della vita” 1986 concludes: «[…]. These have the complete aspect of molecules programed for particular designs».
However, as we have seen, the nucleotides are constituted of the synthesis of three different molecules, which are hence the fundaments of the RNA.
The phosphate group which, even if in small quantities, is diffused on the whole surface of the planet.
The D-Ribose (a pentose sugar) and one of the four nucleobases : Adenine, Guanine, belonging to the family of the purine; Uracil and Cytosine, belonging to the family of the pyrimidine. Now, over and above the procedural difficulties to obtain activated nucleotides, Ribose and nucleobases were present in prebiotic era?       

Those who thinks that these substances were present in the prebiotic era, are principally the supporters of the RNA World. So, through the publications of their most influential scientists, Ciryl Ponnamperuma, Manfred Eigen and Leslie Orgel, we go and discover their cards. (It is useful to point out that for ammonium cyanide one intends hydrocyanic acid, HCN, and ammonia, NH3; and over more, hydrocyanic acid is called also hydrogen cyanide).
Ciryl Ponnamperuma “Origine della vita” 1984: «The purines were synthesized  for the first time in conditions which simulated those of the primitive earth by Orò(v. Studies in …1963), who demonstrated that one could obtain the synthesis of Adenine by a concentrated solution of ammonium cyanide. In its broad lines the reaction can be represented as 5 molecules of hydrogen cyanide that in the presence of ammonia give place to Adenine. This synthesis has be confirmed by Lowe […]. Afterwards, Orò succeeded in synthesizing Guanine and Xanthine by bringing to a temperature of 100-140°C a solution with water of amminoimidazolcarbossiammide. The rendering was of 1,5% for both the purines. It is possible that this was one of the ways in which the synthesis of purines on the primitive Earth took place, but the concentrations used by Orò were very much too high to correspond to a prebiotic situation. If the experimental concentrations had been really like those prebiotic, if for example lower concentrations had been used, then these reactions would be a great help to the understanding of the origin of the purines in the condition present in the prebiotic phase of the Earth. In spite of the efforts made, it was not possible to identify with certainty purines and pyrimidines among the final products in experiments which used electric charge. As a great quantity of hydrocyanic acid is formed, it is difficult to understand how the purines could be absent. Very little has been done in the region of the synthesis of the pyrimidines. Fox and Harada (v., 1961) have demonstrated that Uracil can be obtained by the heating of malic acid and urea. Whereas  urea is easily formed in the experiments which simulate the primitive Earth, there does not exist any indication of the presence of malic acid. […] and others (v., Formation of…,1963) have obtained also the synthesis of adenine through electrons flux on methane, ammonia and water […]. The greatest part of the radiations produced by radioactive sources is absorbed by solids, and as the earth’s crust has a thickness of about 30 km, this type of radiation could have had no role in the synthesis of organic material in the primitive oceans». Hence Ponnamperuma affirms, in 1984 that these reactions are not of great help to the understanding of the synthesis of the nucleobases in the prebiotic phase. These however do not offer any indication of the presence of nucleobases in the prebiotic era. On the other hand reactions do not respect the first and the second procedural rule and they are hence laboratory experiments of no interest for prebiotic chemistry.
 With reference to Ribose, Ponnamperuma continues: «In contrast with the progress obtained in the field of primordial chemistry of the amino acids, the origin of another group of substances just as important biologically the monosaccharides (sugars), is even now fairly uncertain. Already in 1861 A. Butelow had shown that formaldehyde dissolved in water goes towards a process of condensation in an alkaline ambiance, giving place to a mixture of sugars. […]. There are for this reason various difficulties in accepting the hypothesis that formaldehyde could have been used as a precursor of the monosaccharides. Horowitz and Miller have made it clear that the high concentrations of formaldehyde used in some experiments do not reproduce in a realistic way the conditions of the primitive Earth. Objections have also been made to the use of very basic solutions. P. H. Abelson sustains that the concentration of ammonia free in the seas and in the atmosphere was not ever so elevated, and that a strong alkaline ocean never existed. Furthermore, formaldehyde polymerizes fairly rapidly giving paraformaldehyde, and so can go a certain distance from the ambiance […]. 
The fact of having reproduced in a satisfactory way the synthesis of the nucleobases and the sugars of the nucleic acids in conditions simulating those of the primitive Earth has inspired some researchers  to make searches on the abiotic formation of nucleosides». Sic!
It is not only one contradictions. Ponnamperuma falsifies the evidence of the facts by he himself listed. But why?
To understand such a conclusion, it is necessary to go back some years and refer to two affirmations by Manfred Eigen and others in “L’origine dell’informazione genetica” Le Scienze, 1981.  These quotations have already been amply introduced in the article concerning the “The RNA World”. With reference to the studies of Sol Spiegelman, Leslie Orgel and others Eigen affirms: «The fundamental conclusion of these studies is that the self-reproducing of the RNA in systems in vitro effectively takes place also without the intervention of sophisticated enzymes. It is possible to go ahead considering the evolutive consequence of self-reproducing of RNA without preoccupying oneself if this really happened in the prebiotic era: however it happened».
This affirmation was in reality the announcement of an announced disaster, because science does not proceed in this way.
And Manfred Eigen in the same article continues: «That which is important, here, is what these experiments reveal on Darwin’s processes. Natural selection and evolution, which are the consequences of self-reproducing, operates at the molecular level as at the level of the cells or the species».
But it was necessary to wait Ernst Mayr (L’unicità della biologia, 2005) to put into evidence, in a clear way, that the target of the selection is the phenotype and not the gene, and that this terminology is in flagrant conflict with Darwin’s basic thought?
At the end, in many researchers, sometime before, the idea had matured of extending Darwin’s theory also to molecules. In this way evolution through natural selection could be extended also to the origin of life. Everything should hence develop  according to this design. The idea slowly crystalized in their mind to the point of considering superfluous deepening the possibility of synthesis, in the prebiotic era, of nucleobases and ribose. And in fact Manfred Eigen in “Gradini verso la vita”.1992,with reference to the constitutive chemical unities necessary for life at the primordial era of our planet and in particular with reference to the nucleobases affirms: «To give a generic affirmative answer to this interrogation would mean, given the actual level of knowledge of organic chemistry, to deny the character of the basic question. […]. For the different constituents of the nucleic acids, methods of synthesis are known which in prebiotic conditions were fully realizable. The simplest to obtain is Alanine (the nucleobase A of the nucleic acids), for which different methods of synthesis exist, among which the most simple condensation of hydrocyanic acid […]. In ambiance without water that are, as Albert Eschenmoser has been able to demonstrate, other procedures of synthesis very elegant, efficient for all four nucleobases of the nucleic acids. With a degree of probability fairly close to certainty, the four nucleobases were, in the phase of their chemical synthesis, present in concentrations extremely different».
And so, after about 10 years, Eigen comes to the same false conclusion as Ponnamperuma. Moreover, as he could not give data to sustain the origin of nucleobases, he gives reactions in an ambiance without water that, in prebiotic chemistry, is not admitted.
In 1995 Christian De Duve, although a sustainer of the “RNA World” in “Polvere Vitale” affirms:« […] chemists have had a certain success in the production of the five organic components of the RNA, but with scarce rendering and in conditions at the same time very different from a prebiotic scenery and different for every substance. If one wants to combine the components in the right way, one comes across other problems, of such magnitude that no one has ever tried to do it in a prebiotic contest».
In 2004 Orgel, “Prebiotic Chemistry and the origin of the RNA World”, tries to unify the scene at least for the four nucleobases.
He shows how Adenine can be obtained from a eutectic solution, very concentrated, of NH3 and HCN at the temperature of -23,4 °C, that is the synthesis of Orò revisited. Orgel points out that among the products of this reaction, traces of Guanine were found.
Orgel observes how the Uracil can be obtained by the hydrolysis of Cytosine.
For the synthesis of Cytosine he takes first into consideration the reaction between cyan acetic aldehyde and urea concentrated, but he concludes that, in the prebiotic era, such a reaction is not plausible. However it seems to him more plausible the reaction of a eutectic solution of cyan acetylene with cyanide because it could proceed in parallel with the synthesis of adenine also in eutectic condition.
On paper under the word “eutectic” the synthesis of the four nucleobases were done with the same logic.  But temperature and concentrations indicated by Orgel are completely outside any prebiotic context. However, in which way all this could be translated in the prebiotic conditions of the primordial earth cannot be known. It is overmore necessary to keep in mind that these synthesis also cause the formation of many nucleobases without interest, even obstacles for the origin of life.
To be short, in 2004, that is after a half century of tentatives by the best chemists in the world, we can conclude that, in the prebiotic phase, no chemical process has produced the four nucleobases.
In 1994 in Le Scienze, “l’origine della vita sulla terra”, referring to Ribose L. Orgel writes: «Above all, in the lack of enzymes, it is a problem to synthesize Ribose in adequate quantities and with sufficient degree of purity».
In 2004, in “Prebiotic chemistry and the origin of the RNA World”, Orgel again takes up the now well-known reaction of Butlerow of 150 years ago. He writes that if this reaction could be addressed towards the synthesis of Ribose it would be ideal for the synthesis of sugar as a component of the nucleotides. Orgel quotes Zubey (1998, 2001) who repeated the reaction in the presence of lead. He also makes it clear how the calcium borate stabilises the pentoses (Ricardo and others, 2004). Orgel admits that all these reactions take place at pH to high, and with high concentrations of reagents. In the end he concludes that some progress has been mad, but there are still a series of obstacles to the synthesis which would produce significant and pure quantities of Ribose.  
Hence, after 150 years from Butlerow’s discovery, we can conclude that also Ribose in the prebiotic era never existed. And on nucleobases and Ribose, after 50 research, that which is left is only a handful of ashes.
Now it is clear how, to have tried to extend evolution by natural selection also to molecules, has extended the problem of the origin of life from mud to marsh. And yet, on nucleobases and sugars, that which was divulgated by the most respected scientific reviews, were the conclusion of these influential scientists. In truth there was some timid objection on the part of Freeman Dyson , Graham Cairns-Smith and some other  scientists.
 However the greater part of the scientists (and unfortunately we modest observers), for some ten years , remained convinced that, although with some difficulties, nucleobases and Ribose, in the prebiotic era were present on our planet.
And so, a poker of aces was divulgated, but it was only a colossal Bluff.
You meanwhile, incredulous, go away from the building site accompanied by a deafening sound which confirms you in your presentiment.
And in 2013? Now it is enough, let us go out of this marsh.
In the prebiotic era, on our planet no chemical-physical process has given origin to Ribose and nucleobases. The origin of these substances, in such an epoch, stays only in the head of those who sustain the “RNA World”. And in fact that which remains of their theory, after more than half a century of research, is only an amass of ruins.
Now, if we wish to imagine eventual scenes which go over our planet, it is useful to go through the principal passage necessary to arrive at the RNA. I apologize to those in possession of different knowledge; they will find the description too synthetic, and hence not easy to understand, but I hope it is possible to have the intuition of a global judgement as a matter of fact.
Let us admit that in another planet, in prebiotic conditions different from ours, the synthesis of nucleobases and sugars really happened.  
In the pentoses  three carbon atoms are asymmetric and thus we have three chiral centres. This implies that the number of possible molecules (stereoisomers) is equal to 23 that is 8, of which four D (right) and four L (left) and among these the Ribose. It is true that every couple D-L has chemical-physic characteristics slightly different with respect to others, but from an energy point of view in a prebiotic phase, they all have the same probability to being synthesized. Now only for the spontaneous assemblage a nucleotide illustrated below,

 the following passages are necessary:
1) The separation of the pentoses from all the other sugars.
2) The separation of the four pentoses D (right) from the L to avoid crossed reactions.
3) The separation of the D-Ribose  from the other three sugars D to avoid superposed reactions.
4) The separation of the four nucleobases necessary for the RNA from all the others.
5) Presence of a consistent source of phosphate (H2PO4-) in solution.
6) A particular orientation, to obtain the four nucleotides, of all the nucleobases on OH of C-1, and to avoid the reaction with OH from C-2, C-3, C-5, (as illustrated above).
7) A particular orientation of the phosphate group on OH of C-5 to the end of avoiding the reaction with the OH from C-2, C-3 (as illustrated above)
8) The orientation of all the nucleobases in the position β (that in upwards as in the figure).
9) The reaction of phosphate of the nucleotides with OH in the position C-3 of other nucleotides, with the exclusion of the position C-2, to give origin to the RNA.
In over 50 years of research, there have not been and there is not even now a chemist who has succeeded in corroding points 1, 2, 3, 4, 5. And there has not been and there is not even now a biologist who can give an explanation to points 6, 7, 8, 9 without the intervention of specific enzymes.
To conclude:
On our planet.
In the prebiotic era, on our planet, no chemical-physic process has given origin to Ribose and nucleobases; the laws of chemistry and physics stopped their formation. And if we rewind the film of the origin of life and we reproject it, in all probability we would see again the same story.
In the universe:
We cannot exclude that in different prebiotic conditions, on some other planet, sugars and nucleobases are formed. But life without water in a liquid state cannot exist. In such conditions, the laws of physics and chemistry being universal, in no planet, except for a miracle, can the 9 points, above mentioned, be realized spontaneously. Hence, in no part of the universe ever existed or will exist a “RNA World”.
And if there is still who wants to use the usual passe-partout phrase: one cannot exclude such a possibility, what to say: dreams cannot be prohibited.

                                                                                     Giovanni Occhipinti

Translated by Silvia Occhipinti  (04.10.2014)

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