The RNA Dream World

The RNA Dream World (cont.)

A published procedure for attaching imidazoles to nucleotides (Page 281 of Joyce G, Inoue T, Orgel L. Journal of Molecular Biology, 1984, 176, 279-306) involves dissolving them in dimethylformamide and dimethylsulfoxide, and using 2,2’-dipyridyldisulfide and triphenylphosphine as condensing agents. The prebiotic implausibility of this procedure is directly stated by the authors themselves (Page 304 of Joyce G, Inoue T, Orgel L. Journal of Molecular Biology, 1984, 176, 279-306).

This report from 1984 is still cited today as the standard scenario for attaching imidazoles to nucleotides (see, for example, page 312 of Ferris J. Origins of Life and Evolution of the Biosphere, 2002, 32, 311-332). Professors Orgel and Schwartz wrote: “In previous experiments on template-directed synthesis, specific activated nucleoside 5’-phosphates were usually used as substrates, and 3’,5’-linked oligonucleotides were sought as products. However, it is unlikely that chemically pure substrates of this kind, for example 2-MeImpG, could have accumulated on the primitive earth” (Schwartz A, Orgel, L. Science, 1985, 228, 585-587). 2-MeImpG is a nucleotide with 2-methylimidazole attached to its 5’ phosphate.

Moreover, Professor Ferris stated that significant amounts of imidazoles were probably not available on the prebiotic earth (Page 4332 of Prabahar K, Ferris J. Journal of the American Chemical Society, 1997, 119, 4330-4337).

“I do not consider the 5’-phosphorimidazolides to be plausible in prebiotic times. I view them as providing a model system that is amenable to experimentation. Perhaps 5’-polyphosphates were a more plausible form of activation in prebiotic times, but unfortunately they are so unreactive that they are difficult to study in the laboratory” (Professor Gerald Joyce, January 2003, Personal Communication).

Most of the experiments published in the literature for oligomerization of nucleotides involve imidazoles attached to the nucleotides (see, for example, Ferris J. Origins of Life and Evolution of the Biosphere, 2002, 32, 311-332; Kanavarioti A, Monnard P, Deamer D. Astrobiology, 2001, 1, 271-281; Ferris J. et al. Nature, 1996, 381, 59-61).

A published procedure for attaching the compound 2-methyladenine to a nucleotide involves the organic solvents dimethylformamide and dimethylsulfoxide and the condensing agents 2,2’-dipyridyldisulfide and triphenylphosphine: “A mixture of 5’-NMP-H2O (free acid, 0.5 mmol) and heterocyclic base 3 (2-methyladenine, 0.5 mmol) was dissolved in DMF (dimethylformamide, 10 mL) and DMSO (dimethylsulfoxide, 5 mL) in a 50 mL flask, and the solvents were evaporated to 2 mL at a reduced pressure to remove H2O. The evaporation was repeated twice with DMF (2 x 10 mL). The residue was dissolved in DMF (10 mL) and cooled to -15 C in an ice-salt mixture. Triethylamine (2 mL) was added to the reaction mixture with stirring followed by a solution of 2,2’- dipyridyldisulfide (0.333 g, 1.5 mmol) and triphenylphosphine (0.393 g, 1.5 mmol) in DMF (5 mL). The stirring was continued for 4 h…” (Page 4331 of Prabahar K, Ferris J. Journal of the American Chemical Society, 1997, 119, 4330-4337). As discussed above, this procedure involving dimethylformamide and dimethylsulfoxide and the condensing agents 2,2’-dipyridyldisulfide and triphenylphosphine was not plausible in prebiotic times.

Professors Kanavarioti, Monnard and Deamer used eutectic (freezing) concentration to bind nucleotides together to form RNA, but they also used imidazoles attached to the nucleotides (Kanavarioti A, Monnard P, Deamer D. Astrobiology, 2001, 1, 271-281). In response to my question, Professor Kanavarioti wrote in April 2003 that without imidazoles, no binding together of nucleotides is expected.

Minerals have been shown to enhance nucleotide oligomerization, but in all cases amines were attached to the nucleotides (see, for example, Ferris J. Origins of Life and Evolution of the Biosphere, 2002, 32, 311-332; Prabahar K, Ferris J. Journal of the American Chemical Society, 1997, 119, 4330-4337; Ferris J. et al. Nature, 1996, 381, 59-61).

Professors Orgel and Joyce concluded that the series of reactions four billion years ago needed to form even short nucleic acids (which are called oligonucleotides) “would have been a near miracle” (Page 68 of Joyce G, Orgel L. in The RNA World, Second Edition, 1999, editor R. Gesteland, New York: Cold Spring Harbor Lab). Professor Orgel confirmed this in a later report (Orgel L. Science, 2000, 290, 1306-1307).

Professor Gustaf Arrhenius of the University of California at San Diego and colleagues wrote: “It is at the present time unknown how such a complex molecule as even a single nucleotide could arise in a lifeless world, and without an obvious autocatalytic and selective driving force” (Arrhenius G, Sales B, Mojzsis S, Lee T. Journal of Theoretical Biology 1997, 187, 503-522). In a letter to me in April 2003, Professor Arrhenius confirmed this. In keeping with this, nucleosides or nucleotides have never been detected in spark-discharge experiments, meteorites, comets, or interstellar space (Sephton M. Nature Product Report, 2002, 19, 292-311; Cronin J, Chang S. in The Chemistry of Life’s Origins, editor J. Greenberg, Kluwer Publishers, 1993, pages 209-258; Shapiro R. Origins: A Skeptic’s Guide to the Creation of Life on Earth, 1986, New York: Summit Books).

Professor Joyce wrote in a recent publication: “If the building blocks of RNA were available in the prebiotic environment, if these combined to form polynucleotides, and if some of the polynucleotides began to self-replicate, then the RNA world may have emerged as the first form of life on earth. But based on current knowledge of prebiotic chemistry, this is unlikely to have been the case” (Page 215 of Joyce G. Nature, 2002, 418, 214-221).

An important point is that the conditions needed to produce nucleotides must also have produced a “much larger amount of various nucleotide analogs.” Oligomerization of the nucleotides and their analogs would have resulted in “a combinatorial mixture of 2',5'-, 3',5'- and 5',5'-phosphodiester linkages, a variable number of phosphates between the sugars, D- and L-stereoisomers of the sugars, alpha- and beta-anomers at the glycosidic bond, and assorted modifications of the sugars, phosphates and bases. It is difficult to visualize a mechanism for self-replication that either would be impartial to these compositional differences or would treat them as sequence information in a broader sense and maintain them as heritable features” (Page 215 of Joyce G. Nature, 2002, 418, 214-221).

This combination of various sugars can lead to irregular nucleic-acid backbones, and some sugars can even terminate chain growth: “Our results suggest that the activated arabinosyl nucleotides, because their cyclization is substantially slower than their hydrolysis, could have reacted with growing oligoribonucleotide chains and acted as chain terminators. Proponents of RNA as the first informational macromolecule must explain why arabinosyl nucleosides were much less abundant than the ribonucleotides in the prebiotic soup, or how they were excluded from the ends of growing oligonucleotide chains” (Page 359 of Harada and Orgel, Journal of Molecular Evolution, 1991, 32, 358-359).

One of the most effective chain terminators is L-ribose. It has been repeatedly observed that, during the process of oligomerization of nucleotides to produce nucleic acids, when the 5’ end of a nucleotide containing L-ribose binds to the 3’ end of a chain of nucleotides, further addition of nucleotides to the 3’ end of the chain can not occur. In other words, L-ribose terminates chain growth. For example, an aqueous solution containing the D-enantiomer of guanosine-5’-phosphoro-2-methylimidazole forms oligonucleotides up to 30 units long in the presence of a poly(C) template, but a racemic (meaning equal amounts of L and D enantiomers) mixture of these mononucleotides forms only small amounts of dimers and trimers (Joyce et al Proceedings of the National Academy of Sciences USA, 1987, 84, 4398-4402; Joyce et al Nature, 1984, 310, 602-604). Although these two reports are nearly two decades old, they are still cited repeatedly today as exhaustive and conclusive studies on this subject. Consider the following report, which was written in 1999: “The effect of L-guanosine-5’-phosphoro-2-methylimidazole (L-2-MeImpG) on the nonenzymatic oligomerization of the D-enantiomer on a poly(D-C) template has been studied in some detail because of its relevance to prebiotic chemistry (Joyce et al Nature, 1984, 310, 602-604). It was shown that the L-enantiomer is a potent inhibitor of the oligomerization and is incorporated as a chain terminator in short oligo(D-G) products. A similar result was obtained when the poly(D-C) template was replaced by an achiral peptide nucleic acid (PNA) C10 template (Schmidt et al Journal of the American Chemical Society, 1997, 119, 1494-1495). …Enantiomeric cross-inhibition is due to the ability of L-2-MeImpG to compete with the D-enantiomer for the binding site on the C residue adjacent to the 3’-terminus of the growing oligo(G) chain. If L-2-MeImpG, after binding, is unable to form a covalent bond to the growing oligo(G) chain, it will behave as a competitive inhibitor; however, if it does form a covalent bond, it will terminate the chain irreversibly” (Kozlov et al Journal of the American Chemical Society, 1999, 121, 1108-1109).

The polymerization of formaldehyde in the Formose Reaction produces a complex mixture of sugars, of which ribose is only a small part. Professor Joyce and Professor Orgel wrote: “This reaction does not provide a reasonable route to the ribonucleotides” (Page 67 of Joyce G, Orgel L. in The RNA World, Second Edition, 1999, editor R. Gesteland, New York: Cold Spring Harbor Lab). Professor Stanley Miller and coworkers wrote that the Formose Reaction “produces too many interfering sugars” (Page 555 of Kolb V, Dworkin J, and Miller S. Journal of Molecular Evolution, 1994, 38, 549-557). Professors Alan Schwartz and R. de Graaf of the University of Nijmegen in the Netherlands wrote: “We agree with Reid and Orgel (1967) and Shapiro (1988) that this process is unlikely to have contributed to chemical evolution on the primitive earth” (Page 105 of Schwartz A and de Graaf R. Journal of Molecular Evolution, 1993, 36, 101-106). “The specificity of the Formose Reaction is not increased by catalysis on hydroxylapatite or other minerals” (Orgel L. Proceedings of the National Academy of Sciences USA, 2000, 97, 12503-12507). The Formose Reaction produces racemic ribose.

What about the role of minerals in the synthesis of ribose? The positively-charged interlayer of certain double-layer metal-hydroxide (DLH) minerals attracts negatively-charged ions such as glycolaldehyde-phosphate and glyceraldehyde-2- phosphate. By this process, the concentration of glycolaldehyde-phosphate has been found to increase from 2 x 10-5 moles/liter in the external solution to ~10 moles/liter in the interlayer (Page 506 of Arrhenius G, Sales B, Mojzsis S, Lee T. Journal of Theoretical Biology, 1997, 187, 503-522). In the interlayer, glycolaldehyde-phosphate and glyceraldehyde-2-phosphate combine to form racemic pentose-2,4-bisphosphates. Conditions were established (including neutral pH) in which the following percentages were obtained: 48% ribose-2,4-bisphosphate, 16% arabinose-2,4-bisphosphate, 25% lyxose-2,4-bisphosphate, and 11% xylose-2,4-bisphosphate. Under these conditions, practically no tetrose-phosphates or hexose-phosphates are said to be produced (Krishnamurthy R, Pitsch S, Arrhenius G. Origins of Life and Evolution of the Biosphere, 1999, 29, 139-152). Still, racemic ribose is produced.

What about an extraterrestrial source of D-ribose? Ribose has never been detected in any extraterrestrial source (Professor Andre Brack, University of Orleans, France, Personal Communication, May 2003; Sephton M. Nature Product Report, 2002, 19, 292-311; Cooper G, et al. Nature, 2001, 414, 879-882; Cronin J, Chang S. “Organic matter in meteorites: molecular and isotopic analysis of the Murchison meteorite.” In The Chemistry of Life’s Origins, ed. J. Greenberg, Kluwer Publishers, 1993, pages 209-258; Cronin J., et al. in Meteorites and the early solar system, ed. Kerridge, J., University of Arizona Press, 1988, pages 819-857).

Another problem with the mineral-induced production of ribose is that glycolaldehyde-phosphate and glyceraldehyde-2-phosphate can be displaced from the interlayer. For example, glycolaldehyde-phosphate molecules in the mineral interlayer are replaced in a few hours at pH 8 and 25° C by CO32- absorbed from a 0.1 moles/liter external solution; even after condensing to racemic hexose-phosphates they are still replaced in several weeks by CO32- (Page 319 of Pitsch S, Eschenmoser A, Gedulin B, Hui S, Arrhenius G. Origins of Life and Evolution of the Biosphere, 1995, 25, 297-334). This was confirmed by Professor Krishnamurthy in a letter to a friend of mine at the University of Tartu in Estonia dated 8 April 2003. Note that glyceraldehyde-2-phosphate and glycolaldehyde-phosphate have identical charge of minus 2 and differ in mass by only 20%, yet this small difference alone makes the interlayer affinity for glycolaldehyde- phosphate three times greater than for glyceraldehyde-2-phosphate (Page 151 of Krishnamurthy R, Pitsch S, Arrhenius G. Origins of Life and Evolution of the Biosphere, 1999, 29, 139-152). In a letter to me in April 2003, Professor Arrhenius wrote: “Naturally occurring DLH minerals are interlayer substituted with a variety of anions, chloride, sulfate and carbonate, the latter most common since the flat sp2 ion forms strong hydrogen bonds.”

There are a number of anions (including S2-, CO32-, SiO44-, SO42-, PO43-) with charge greater than or equal to that of glyceraldehyde-2-phosphate and glycolaldehyde- phosphate and much less mass. Their combined concentration may have exceeded that of glyceraldehyde-2-phosphate or glycolaldehyde-phosphate in practically all of the hydrosphere in prebiotic times, in which case they would have occupied most of the positively-charged mineral interlayers instead of these aldehyde-phosphates. In this case, very little ribose-2,4-bisphosphate would have been produced on earth in prebiotic times. Professor Arrhenius and colleagues remind us that: “It would also be necessary, before characterizing these model experiments as ´prebiotic reactions´, to measure the distribution in the aqueous mineral interlayer of other competitive anions in natural solutions, primarily bisulfite, sulfide species, carbonate, nitrate, nitrite, hexacyano- ferroate” (Page 500 of Kolb V, Zhang S, Xu Y, Arrhenius G. Origins of Life and Evolution of the Biosphere, 1997, 27, 485-503).

90% of the pentose-2,4-bisphosphate molecules in the mineral interlayer remained unaltered after three months, and the other 10% decomposed (Page 149-150 of Krishnamurthy R, Pitsch S, Arrhenius G. Origins of Life and Evolution of the Biosphere, 1999, 29, 139-152). There is absolutely no evidence of oligomerization of these molecules to form the backbone of RNA. This was confirmed by Professor Krishnamurthy in a letter to a friend of mine at the University of Tartu in Estonia dated 31 March 2003.

Professor Krishnamurthy wrote in this letter that the nucleobases have never been observed to be attracted into the interlayer of the mineral that forms ribose and hence could not have combined with ribose there. He also stated that if, prior to entering the mineral interlayer, glycolaldehyde-phosphate or glyceraldehyde-2-phosphate had a nucleobase attached at the 1’-carbon-position (where it must be in the final nucleotide), then these aldehyde-phosphates would have been unable to combine to form tetrose-phosphates or pentose-phosphates. He wrote: “Work in this area has been difficult and there are no recent papers with tangible success in introducing the nucleobases” (Letter from Professor Krishnamurthy dated 31 March 2003).

Thus, the evidence indicates that the presence of even short nucleic acids four billion years ago “would have been a near miracle” (Page 68 of Joyce G, Orgel L. in The RNA World, Second Edition, 1999, editor R. Gesteland, New York: Cold Spring Harbor Lab). But there is something even worse than that, which will be described in the next section.

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