Full text and figures at: http://www.chemsoc.org/chembytes/ezine/2000/evans_may00.htm

It's alive – isn't it? Speculations on the origin of life have, until recently, been purely theoretical. The latest experimental findings, however, are raising more questions than they're answering. Jon Evans reports Astronomers believe that the Earth was formed, along with the rest of the solar system, around 4600m years ago. For the next 500m years the Earth suffered under an intense bombardment of comets and meteorites, the leftover debris from the formation of the solar system, which kept the Earth's surface in a constant state of molten upheaval. However, the fossil record shows that a mere 600m years after the bombardment stopped (ie around 3500m years ago), organisms very similar to present-day cyanobacteria (blue-green algae) abounded in the Earth's seas. Indeed, some forms of life may have existed even earlier: rocky sediment from Greenland dating back 3800m years is enriched with the isotope 12C, a sign of the biological processing of carbon (inorganic carbon chemistry does not distinguish 12C from 13C, whereas biological processes preferentially use 12C). So life may have been present only 200m to 300m years after the Earth could first possibly have supported it. And, perhaps even more astonishingly, there is fossil evidence that life as we would recognise it today, probably using DNA and proteins as its basis for replication and metabolism, was thriving only 300m years later (about 3500m years ago). This all points to the possibility that the formation and development of simple life was not an especially difficult process; at least not when compared to the evolution of land plants, which only appeared some 3000m years after the ancestral cyanobacteria. It seems as though simple chemical reactions among the compounds that scientists envisage were available on the early Earth were sufficient to drive the process that led inexorably to life (see Box 1). There is, however, one problem with this assumption: scientists still have no firm idea of what the mechanics of this kind of 'prebiotic' chemistry were or how the whole ascent to life actually happened. Recently, new experimental methods have shed some light on the possible processes, but have also served to add to the general uncertainty. One thing that scientists working in this field do know, however, is that whatever form early life took it didn't possess DNA or proteins. It's a small world Every living cell on the Earth today uses DNA to store its genetic information and uses proteins to catalyse its metabolic reactions. DNA contains all the information needed to manufacture proteins, and some of these proteins (enzymes) catalyse the reactions that the cell needs to live and replicate. The one cannot operate without the other; DNA cannot naturally catalyse reactions and proteins cannot manufacture themselves. And therein lies the chicken-and-egg problem: if DNA and proteins cannot operate without each other, how did the system first evolve? One obvious answer is that early life used a single molecule for both information storage and catalysis, a form of self-replicating enzyme. Exactly what form that self-replicating enzyme might have taken was first suggested 30 years ago, when RNA was put forward as the precursor to DNA and proteins in early lifeforms. In cells today, RNA is the go-between for DNA and proteins: a protein is manufactured from an RNA template, which has itself been created from a DNA template. The idea remained purely speculative until the early 1980s when Thomas Cech at the University of Colorado and Sydney Altman at Yale University independently discovered RNA molecules with catalytic ability, now known as ribozymes. This discovery immediately put on a much firmer footing the idea that RNA could have been used for both storing information and catalysing reactions in early forms of life, and in 1986 the term 'RNA world' was coined. Nevertheless, despite the fact that most scientists working in this field accept the validity of the idea, the RNA world hypothesis is still far from being proved. For one thing, in almost 20 years only seven types of natural ribozymes have been discovered: two remove introns (parts of RNA that don't code for proteins) from themselves; four cut themselves in two; and one trims off the end of an RNA precursor. So, as David Bartel and Peter Unrau, researchers in the Whitehead Institute at Massachusetts Institute of Technology (MIT), Cambridge, US, say in a recent review (Trends Cell Biol., 1999, 9(12), M9): 'Although the reactions of natural ribozymes are fascinating and impressive, they do not approach the sophistication of the key reactions assumed by the RNA world hypothesis'. This is not exactly surprising; enzymes, being superior catalysts, would have usurped RNA's catalytic role as life evolved, and thus few examples of natural ribozymes remain. So Bartel, Unrau and other researchers are having to turn to a new experimental technique to determine what types of ribozymes might have been active in the RNA world. Test tube evolution By putting a large pool of random RNA sequences through a process of directed mutation, choosing and enriching those sequences that best perform some pre-defined function, researchers are now creating artificial ribozymes. They start with an initial pool of around 1015 different RNA fragments, each able to occupy hundreds of random positions on a strand. Then the researchers link these random-sequence pool molecules to a specific substrate and select those that convert the substrate to a desired product. The selected molecules are then amplified using protein replicases, and this selection-amplification procedure is repeated until sequences with the desired activity dominate the pool. By using this method, researchers have created a whole host of new ribozymes that catalyse a variety of reactions, such as RNA ligation (joining RNA units together with a phosphodiester bond), RNA phosphorylation, RNA branch formation, and amide and peptide bond formation - all important biochemical reactions that are catalysed by proteins in today's cells. However, the development of this method has been a decidedly mixed blessing for the RNA world hypothesis. As Bartel and Unrau explain: 'Before the technology of in vitro selection existed, it was easy to proclaim boldly that RNA could catalyse the reactions required in the RNA world - no one expected experimental verification. However, now the onus is not merely to propose a key reaction of the RNA world, but also to propose an RNA molecule that can perform such a reaction'. The kind of proof that the RNA world hypothesis really needs is the creation of an RNA molecule that can replicate either itself or another RNA molecule, the kind of self-replicating ribozyme that really could have performed the roles of both DNA and enzymes in early life. Unfortunately, this has not yet happened. Ribozymes have been created that perform different aspects of that function, but not one that performs all of them. 'Three key features of an RNA replicase currently reside in three different ribozymes and reactions', say Bartel and Unrau. 'One efficiently catalyses the proper chemistry, another uses nucleoside triphosphates [the individual units of RNA] in a templated fashion, and the third recognises an RNA duplex without regard for sequence. To prove the replicase assumption of the RNA world hypothesis, these features must be united into a single ribozyme'. Bartel and Unrau managed to produce a ribozyme that goes some way towards this goal. In a paper in Nature in 1998 (395, 260), they reported that they had created an RNA molecule with the ability to catalyse the formation of a glycosidic bond joining a ribose sugar to a base (uracil) to make a nucleotide, the main building block of DNA and RNA. Nevertheless, the construction of a self-replicating RNA molecule, even if possible, is still a long way off. More disconcerting still for proponents of the RNA world hypothesis is the fact that even if one of these is eventually created it will not necessarily mean that an RNA strand with that particular sequence performed the same function on the early Earth. Indeed, it wouldn't even prove the validity of the RNA world hypothesis. 'Even if ribozymes for all the essential activities of an RNA world were generated and assembled into RNA-based life, this would only show that the fundamental properties of RNA are compatible with the "RNA world" scenario', say Bartel and Unrau. As well as being hard to prove, the RNA world hypothesis will also be hard to disprove. Only a minute fraction of RNA sequences can be sampled in an experiment, so just because a sequence that performs a certain catalytic function can't be found, doesn't mean that there isn't one available. Other evidence can come from 'molecular fossils', putative remnants of the RNA world that are still active in modern-day cells. The seven ribozymes that have been discovered so far are examples of these 'fossils'; another has turned up from recent studies of the structure of the ribosome, the cellular component where proteins are manufactured. These studies have shown that a large proportion of the ribosome is constructed from RNA, meaning that the component's structure may have remained largely unchanged from the RNA world. Even so, hard proof of the RNA world hypothesis is probably not going to be immediately forthcoming. What may turn out to be easier to prove or disprove are the underlying assumptions on which the hypothesis is based. Links in a chain If early life was based on RNA, then the first ribozymes must initially have formed abiotically on the early Earth before going on to help form the first lifeforms. Researchers propose that these first RNA sequences must have been produced by the gradual stringing together of individual nucleotide building blocks of RNA that arose naturally in the environment. When this has been attempted in the laboratory, only a few nucleotides have joined together before the RNA chain snaps - far short of the 50 nucleotides that would be needed before a sequence could show any kind of catalytic activity. One alternative method for how an RNA sequence of this length could have been produced on the early Earth has been suggested by James Ferris at Rensselaer Polytechnic Institute in Troy, New York, US. Ferris has performed experiments showing that RNA sequences of up to 50 nucleotides in length can be formed using a type of clay known as montmorillonite (a hydrated aluminosilicate) as both a template and catalyst for linking the nucleotides together. This is not the first time that clay has played a role in origin of life theories (see Box 2). Ferris has shown that when a solution of activated nucleotides (where the energy needed for them to link together is already provided in the form of a phosphate bond) is washed over montmorillonite clay, the nucleotides bind to the clay particles, eventually forming chains of RNA up to 14 nucleotides in length. As more nucleotides are washed over the clay surface, these chains can extend to up to 50 nucleotides in length. Furthermore, the clay doesn't just catalyse the formation of RNA strands, it also acts as a template for them, dictating the sequence of the nucleotide units. Ferris says he still isn't sure how the clay does this, although he and his team are doing experiments at the moment to try to find the answer, but he argues that the fact that it does is of great importance. The reason for this is that there wouldn't have been enough organic material on the early Earth to create all possible RNA sequences, therefore by directing the nature of the products formed the clay ensured that only a specific set of RNA sequences, some with catalytic ability, would have built up in the seas of the early Earth. At some point, Ferris believes, a sequence that could catalyse its own replication was created, either directly from the clay or after interacting with other ribozymes. Thus the RNA world would have been born. Once again, however, despite its intellectual appeal, this scenario still has a number of problems. One is that although Ferris gives his nucleotides all the energy they need to link together, in the form of phosphate bonds, there is no evidence that such bonds can form abiotically. Another more fundamental and intractable problem that strikes at the very heart of the RNA world hypothesis is, as Ferris himself admits, the prebiotic formation of the nucleotide units. At first glance, this doesn't really seem to be too much of a problem. An RNA nucleotide is made up of a phosphorylated ribose sugar linked to one of the four RNA bases, and a variety of plausible prebiotic synthetic routes for creating all of these have been suggested. For instance, one of the simplest prebiotic methods for creating ribose is the polymerisation of formaldehyde. Adenine can be formed from ammonia and hydrogen cyanide, as can guanine. Cytosine can be formed by reacting cyanoacetylene with cyanate, cyanogen or urea, and uracil can be produced by the hydrolysis of cytosine. Looking for the right path But would these kinds of reactions have been plausible on the early Earth? One scientist who thinks not is Robert Shapiro at New York University, US. He argues that many of the prebiotic routes suggested for the synthesis of nucleotide bases are so artificial that it is unlikely that they ever took place on the early Earth, and that, even if they did, any yields from the reactions would have been so small and the products would have decayed so rapidly that there would have been little chance of them getting together to form nucleotides. For instance, the adenine nucleotide, adenosine, can theoretically be produced entirely abiotically. However, according to Shapiro, ribose can only be derived from formaldehyde in relatively small yields, and a whole bunch of closely related products are produced as well; this is also the case for the synthesis of adenine from ammonia and hydrogen cyanide. To produce adenosine abiotically in the laboratory, however, prebiotic chemists extract ribose and adenine from the other compounds, and react them together under optimum conditions. As Shapiro says in his recent book Planetary dreams: 'It would be much more realistic to heat together the entire formaldehyde and cyanide products, which would furnish the mother of all messes. Better yet, the chemist should simply mix the cyanide and formaldehyde starting materials. But we know what happens in that case; the two substances have a great affinity for each other and their reaction takes off in a direction that bears no relation to life as we know it'. At least a theoretical prebiotic synthesis path has been developed for adenosine: no such path has yet been defined for the pyrimidine nucleotides. Even proponents of the RNA world hypothesis admit that there are major problems with the prebiotic synthesis of RNA nucleotides. Writing in The RNA world, Gerald Joyce, a professor in the departments of chemistry and molecular biology at the Scripps Research Institute, California, US, and Leslie Orgel of the Salk Institute for Biological Studies, San Diego, US, state: 'Scientists interested in the origins of life seem to divide neatly into two classes. The first, usually but not always molecular biologists, believe that RNA must have been the first replicating molecule and that chemists are exaggerating the difficulties of nucleotide synthesis. The second group of scientists are much more pessimistic. They believe that the de novo appearance of oligonucleotides on the primitive Earth would have been a near miracle. Time will tell which is correct'. The world that time forgot The description of the appearance of life on the Earth as 'a near miracle' does not square, however, with the relatively short length of time that it took to appear (at most 600m years). There are therefore two ways around this problem. Either more plausible abiotic synthesis paths need to be discovered for the creation of the nucleotides, such as those being developed by Geoffrey Zubay at Columbia University, New York, US, or other possibilities for the development of life need to be considered. One proposal that is now gaining ground is that RNA was not the first replicating molecule; that there was a pre-RNA world from which the RNA world developed. Suggestions for what form this pre-RNA world may have taken are many and varied. But the general idea behind almost all of them is that the first replicating molecule was not RNA, at least not as we would recognise it today, but that whatever form this replicator took it eventually spawned self-replicating RNA, which then went on to usurp its role as the prime replicator on the early Earth. This hypothesis thus neatly bypasses the problem inherent in the prebiotic synthesis of RNA, because the first RNA would have been produced by the original replicators. Some of the proposed inhabitants of the pre-RNA world are merely simplified versions of RNA, which researchers envisage might have been easier to produce abiotically. For instance, it may be that a form of RNA that could self-replicate without needing all four nucleotide bases would have arisen initially. Evidence that this idea might at least be feasible came at the end of last year, when Joyce, together with another Scripps researcher, Jeff Rogers, used RNA in vitro selection methods to develop a ribozyme that joined together strands of RNA even though it lacked the cytosine nucleotide, cytidine (Nature, 1999, 402, 323). Joyce and Rogers chose to do away with cytidine for two reasons: it is the least stable of the four RNA nucleotides because of its tendency to undergo spontaneous deamination to uridine; and the uracil nucleotide, uridine, is able to bond with both adenosine and guanosine, which should be sufficient to form the complex RNA structures required for catalytic ability. In the same vein, researchers have suggested forms of RNA where the ribose sugar backbone of RNA is replaced by another sugar, or in which the furanose form of ribose is replaced by the pyranose form. Indeed, RNA strands that possess a pyranosyl analogue of ribose, known as p-RNA, seem to be better replicators than normal RNA: p-RNA is more stable and less likely to form multiple strand structures that inhibit replication. However, if one accepts that this molecule inhabited the pre-RNA world, it then becomes difficult to see how RNA would ever have gained the upper hand. Another example is peptide nucleic acid (PNA), where the ribose-phosphate backbone is replaced by amide bonds. PNA can form a stable double helix with complementary RNA, and information can be passed from RNA to PNA and vice versa, showing that the two could have co-existed until RNA gained the upper hand. Merger or takeover If the replicators in a pre-RNA world were simplified forms of RNA, then the changeover to an RNA world would probably have been gradual, with RNA initially acting solely as the genetic material before also developing catalytic abilities and then taking over. A more aggressive changeover would have happened if the original replicators were based on a completely different system to RNA. In this instance, a pre-existing self-replicating system would sow the seeds of its own destruction by evolving, initially for its own selective advantage, the mechanism for synthesising and polymerising the components of a completely different genetic system, eventually being taken over by it. A whole host of organic compounds not found in RNA or DNA have been proposed as the basis for possible early replicators, including hydroxy acids, amino acids, phosphomonoesters of polyhydric alcohols, aminoaldehydes, thioesters and molecules containing two sulfhydryl groups. Replication amongst inorganic compounds has also been suggested (see Box 2). It has even been proposed that systems of high complexity can develop without any need for a distinct store of genetic information. In this scenario, a set of simple small molecules, such as enzymes and amino acid residues, carrying out cycles of mutually reinforcing reactions gradually evolve into greater complexity. This is the model for the origin of life to which Shapiro adheres. However, the existence and make-up of the pre-RNA world will be even harder to prove and define than that of the RNA world. As Joyce says: 'There is no direct evidence for a pre-RNA world. This is a conjecture based on what seems to be overwhelming difficulties with the prebiotic synthesis and replication of polynucleotides. Unlike the case for an RNA world, there are no 'molecular fossils' within biology to support the existence of a pre-RNA world'. Nevertheless, despite all the difficulties with the prebiotic synthesis of RNA and the practical impossibility of determining what a pre-RNA world might have been like, proponents of the RNA world hypothesis argue that it is still the most credible theory for the origin of life. Ferris claims that experimental proof of the hypothesis will eventually be forthcoming: 'We just haven't done enough experiments yet to show how to get plausible [chemical] pathways', he says. Joyce agrees; however, he believes that proof will come from the opposite direction, from the 'molecular fossils' in modern-day cells. 'The RNA world hypothesis is on the verge of receiving a huge boost as the crystal structure of the ribosome emerges', he says. 'Thomas Steitz and colleagues at Yale have a 2.7Šresolution structure of the large subunit, which includes the "peptidyl transfer site", where peptide bond formation occurs. That site is composed entirely of RNA. Thus, even today, the translation apparatus is an RNA machine.' He does admit, however, that 'the problem remains as to how the RNA world arose'. So, despite the obvious advantages of the RNA world hypothesis, the chemistry of how it all began remains its Achilles' heel. Geologists believe that life only took 600m years to arise on the Earth; let's hope that an explanation for how that happened arises even faster. Further reading A. C. Cairns-Smith, Genetic takeover and the mineral origins of life. Cambridge: CUP, 1982. Raymond F. Gesteland, Thomas R. Cech and John F. Atkins (eds), The RNA world, 2nd Edn. New York: Cold Spring Harbour Laboratory Press, 1999. Robert Shapiro, Planetary dreams: the quest to discover life beyond Earth. New York: Wiley, 1999.