Nucleic acids are incapable of self-replication

Nucleic acids are incapable of self-replication

Consider an RNA template, which consists of a sequence of nucleotides, each of which contains one of the four nucleobases, adenine (A), guanine (G), cytosine (C) or uracil (U). Nucleotides floating free in solution tend to bind to complementary nucleotides on the template according to the following scheme: A binds to U, and C binds to G. If, for example, the template sequence is AUUGCGGCCCCGACCUG, then, in principal, the sequence of nucleotides bound to it is UAACGCCGGGGUGGAC. This sequence is a complementary copy of the template. Thus, RNA replication requires first a complementary copy of the template, and then a complementary copy of this complementary copy to make an exact duplicate of the original template. The first complementary copy can be made in special cases, but not the second complementary copy, which means replication fails: “The sequence CCGCC, for example, has been shown to catalyze the synthesis of GGCGG from a mixture of activated G and C nucleotides. But the reciprocal reaction that is needed to complete a replication cycle has not been demonstrated” (Page 203 of Orgel, L. Nature, 1992, 358, 203-209). “We are unlikely to find a pair of complementary sequences each of which facilitates the synthesis of the other using nucleoside-5´-phosphoro-2-methylimidazolides as substrates.” [These are the best substrates (nucleotides) found after years of sustained searching]. “The chief obstacles are the formation of intermolecular complexes involving a tetrahelix formed by sequences of consecutive G residues, and the formation of intramolecular complexes in which the molecules fold back on themselves and form Watson-Crick double-helical segments. In summary, [complementary] copying with information transfer can be achieved in this relatively simple system, but there are severe obstacles to replication. Some of the obstacles may be attributable to the choice of reagents and reaction conditions, but others seem to be intrinsic to oligonucleotide replication” (Page 206 of Orgel, L. Nature, 1992, 358, 203-209). (Please note that these reagents and reaction conditions are the culmination of decades of experiments designed to find the optimum conditions). Other severe obstacles include enantiomeric inhibition, cyclization of the monomers, strand separation after copying, and fidelity of replication (Page 207 of Orgel, L. Nature, 1992, 358, 203-209).

This was confirmed seven years later by Professors Orgel and Joyce, who are the world’s foremost experts on prebiotic oligonucleotide self-replication: “The first major conclusion is that most activated nucleotides do not undergo efficient, regiospecific template-directed reactions. In general, only a small proportion of template molecules succeed in directing the synthesis of a complement… After a considerable search, a set of activated nucleotides was found [nucleoside-5´-phosphoro-2-methylimidazolides] that undergo efficient…template-directed reactions…. Self-replication, however, is unlikely, mainly because poly(C,G) molecules that do not contain an excess of C residues tend to form stable self-structures that prevent them from acting as templates. The self-structures are of two types: the standard Watson-Crick variety based on C-G pairs, and a quadrahelix structure that results from the association of four G-rich sequences. As a consequence, any C-rich oligonucleotide that can serve as a good template will give rise to G-rich complementary products that tend to be locked in self-structure and so can not act as templates. Overcoming the self-structure problem is very difficult because it requires the discovery of conditions that favor the binding of mononucleotides in order to allow template-directed synthesis to occur, but suppress the formation of long duplex regions that would exclude activated monomers from the template. …In light of the available evidence, it seems unlikely that a pair of complementary sequences can be found, each of which facilitates the synthesis of the other using nucleoside-5´-phosphoro-2- methyl- imidazolides as substrates. Some of the obstacles to self-replication may be attributable to the choice of reagents and reaction conditions, but others seem to be intrinsic to the template-directed condensation of activated mononucleotides” (Pages 53 and 54 of Joyce G, Orgel L. in The RNA World, Second Edition, 1999, editor R. Gesteland, New York: Cold Spring Harbor Lab).

A later report confirmed this: “Nonenzymatic template-directed copying of RNA sequences rich in cytidylic acid using nucleoside 5’-(2-methylimidazol-1-yl phosphates) as substrates is substantially more efficient than the copying of corresponding DNA sequences. However, many sequences can not be copied, and the prospect of replication in this system is remote, even for RNA” (Page 1678 of Zielinski, Kozlov and Orgel, Helvetica Chimica Acta, 2000, 83, 1678-1684).

It has been suggested that this problem might be overcome by using short oligonucleotides instead of nucleotides as substrates. But the problem is that the template forms self-structures that block access by the substrates. There is no reason to believe that short oligonucleotides have greater access to the template than nucleotides; indeed, the opposite is likely to be true.

I wrote to Professor Joyce in June of 2003 about this, and he wrote to me that no one has found a way to make nucleic acids capable of self-replication. At this time, I also wrote a separate letter to Professor Orgel, who wrote to me that self-replication of nucleic acids has never been demonstrated.

Thus, the most devastating conclusion from fifty years of research in prebiotic chemistry at the world’s best universities is that, even under the most favorable conditions, which were practically impossible four billion years ago (high concentrations of nucleotides, all of which contain the same sugar and the needed enantiomer of this sugar, as well as imidazole activation), nucleic acids are still incapable of self-replication!

The above discussion revealed the importance of cytosine (C) in self-replication, but “owing to the chemical instability of cytosine, which readily deaminates to uracil, a primitive genetic system composed of the bases A, U, G and C may have been difficult to establish” (Reader J, Joyce G. Nature, 2002, 420, 841-844). Note that cytosine was not detected in the Murchison meteorite (although the other nucleobases were), and there appears to be no plausible scenario for its prebiotic production (Orgel L. Origins of Life and Evolution of the Biosphere, 2002, 32, 279-281; Shapiro R. Origins of Life and Evolution of the Biosphere, 2002, 32, 275-278; Shapiro R. Proceedings of the National Academy of Sciences USA, 1999, 96, 4396-4401).

Professor David Bartel and coworkers at the Massachusetts Institute of Technology have spent years trying to create an RNA molecule that can replicate itself and other RNA molecules. Such a molecule is called a replicase. An RNA molecule with a specific catalytic ability is called a ribozyme. Thus, a replicase is a special kind of ribozyme. Their latest attempt at a replicase uses nucleoside triphosphates (abbreviated as NTPs) to extend a short RNA molecule called a primer. The 3’ terminus of the primer must pair (align complementary nucleotides) with the template, which is the RNA molecule to be replicated. This “replicase” can not add more than 14 nucleotides to the primer. “Polymerization is too slow for more extension to be observed within 24 hours, and longer incubations yield limiting returns, because buffer and ionic conditions optimal for polymerization also promote ribozyme and template degradation” (Page 1323 of Johnston W, Unrau P, Lawrence M, Glasner M, Bartel D. Science, 2001, 292, 1319–1325). Thus, after this “replicase” adds its maximum of 14 nucleotides, it decomposes along with the RNA molecule it is supposed to copy (the template), which means that it is not capable of self-replication or template replication. Bartel and colleagues wrote: “General template- directed RNA polymerization requires recognition of the generic features of a primer- template complex in addition to ever-changing NTP specificity, as dictated by the next template residue. It is a complex reaction—one of the more sophisticated reactions catalyzed by single polypeptides. …. Our shortest construct retaining activity was 165 nucleotides, with about 90 nucleotides involved in important Watson-Crick pairing and at least another 30 critical nucleotides. Ribozymes with the efficiency, accuracy, and other attributes of a [true] RNA replicase might have to be even larger than this” (Page 1324 of Johnston W, Unrau P, Lawrence M, Glasner M, Bartel D. Science, 2001, 292, 1319–1325). Bartel and coworkers admitted that this “replicase” lacks the very important capability of separating the copy from the template after polymerization, and to achieve this function would probably necessitate a longer replicase.