Critical Reviews in Biochemistry and Molecular Biology, 39:99–123, 2004
Copyright
c
Taylor & Francis Inc.
ISSN: 1040-9238 print / 1549-7798 online
DOI: 10.1080/10409230490460765
Prebiotic Chemistry and the Origin of the RNA World
Leslie E. Orgel
The Salk Institute, La Jolla, California, USA
The demonstration that ribosomal peptide synthesis is a
ribozyme-catalyzed reaction makes it almost certain that there was
once an RNA World. The central problem for origin-of-life studies,
therefore, is to understand how a protein-free RNA World became
established on the primitive Earth. We first review the literature on
the prebiotic synthesis of the nucleotides, the nonenzymatic synthe-
sis and copying of polynucleotides, and the selection of ribozyme
catalysts of a kind that might have facilitated polynucleotide repli-
cation. This leads to a brief outline of the Molecular Biologists’
Dream, an optimistic scenario for the origin of the RNA World. In
the second part of the review we point out the many unresolved
problems presented by the Molecular Biologists’ Dream. This in
turn leads to a discussion of genetic systems simpler than RNA that
might have “invented” RNA. Finally, we review studies of prebiotic
membrane formation.
Keywords nucleotide synthesis, polynucleotide formation, RNA
replication, prebiotic membranes
INTRODUCTION
The ideas behind the hypothesis of an RNA World origi-
nated in the late 1960s in response to a profound puzzle.
The basic principles of molecular biology were well un-
derstood, and it was clear that the replication of nucleic
acids was dependent on protein enzymes and the synthe-
sis of protein enzymes was dependent on nucleic acids.
Even if one allowed for every possible simplification of
the system, for example, by postulating a nucleic acid with
only two bases and proteins assembled from a very limited
suite of amino acids, what remained was too complicated
to have arisen de novo from an assembly of abiotic organic
molecules. The only way out was to regard the dilemma as
Editor: Michael M. Cox.
Address correspondence to Leslie E. Orgel, The Salk Institute,
10010 N. Torrey Pines Road, La Jolla, CA 92037, USA. E-mail: orgel@
salk.edu
a “chicken and egg problem” and to ask which came first,
proteins or nucleic acids? At the time, it was well recog-
nized that natural selection through replication and muta-
tion was the only mechanism for evolving complex bio-
chemical systems from simpler ones. Trying to solve the
“chicken and egg” problem, therefore, was equivalent to
asking whether proteins or nucleic acids were more plausi-
ble as the components of a self-contained replicating sys-
tem. The answer seemed obvious: nucleic acids. Watson-
Crick base-pairing provided a very plausible mechanism
by which a polynucleotide could direct the synthesis of
its complement from mononucleotides or short oligonu-
cleotides, while no equivalent mechanism was known for
the replication of a polypeptide. These arguments were de-
veloped in some detail in three overlapping papers (Woese,
1967; Crick, 1968; Orgel, 1968), and a program to explore
nonenzymatic copying of nucleic acid sequences was ini-
tiated (Sulston et al., 1968a, 1968b).
The authors of the three early papers clearly recognized
that an autonomous RNA “organism” would be possi-
ble only if RNA could take on several of the functions
presently performed by proteins, for example, the func-
tions of RNA polymerases and nucleases. They speculated
that coenzymes incorporating nucleotides in their structure
were fossils from a time when RNA functioned without
the help of proteins (Woese, 1967; Orgel, 1968; Orgel &
Sulston, 1971), an idea that was subsequently developed
in some detail (White, 1976). In one instance it was spec-
ulated that the original ribosome was composed entirely
of RNA (Crick, 1968). However, in none of the papers
was it suggested that RNA catalysis was still important in
contemporary biology. It was taken for granted that pro-
tein enzymes could always outperform RNA catalysts and
had, therefore, completely replaced them in contemporary
organisms.
The unanticipated discovery of catalytic RNA mole-
cules, ribozymes, that perform enzyme-like reactions
(Kruger et al., 1982; Guerrier-Takada et al., 1983) marked
the beginning of the present interest in a proteinless biolog-
ical world. In the few years following Cech and Altman’s
discoveries, ribozymes were shown to be able to catalyze
99
100 L. E. ORGEL
a significant number of diverse chemical reactions. This
led to an increased interest in the hypothesis that an RNA
World, a term introduced by Gilbert (Gilbert, 1986), pre-
ceded the DNA/RNA/Protein world (Gesteland et al.,
1999). The determination of the structure of the ribosome,
showing that it is a ribozyme (Steitz & Moore, 2003),
seems to clinch the case for an RNA World, although it
leaves open almost all questions about the origin and “bio-
chemistry” of the RNA World. The RNA World hypothesis
does not deny that peptides may have been involved in the
origin of life. It does, however, exclude the possibility that
any peptides that were involved were formed by ribosomal
protein synthesis or a closely related mechanism.
The knowledge that an RNA World preceded our famil-
iar biochemical world has profound implications for those
interested in the origin of life. It may be claimed, without
too much exaggeration, that the problem of the origin of
life is the problem of the origin of the RNA World, and
that everything that followed is in the domain of natural
selection. If this is accepted, studies of the chemistry of the
origin of life are, in principle, greatly simplified because
they need only be concerned with the origin of RNA and
do not need to deal with the origins of most other features
of biochemistry. Of course, the origin of protein synthe-
sis and of DNA are also of the greatest interest, but their
appearance can be regarded as the consequences of selec-
tion acting on populations of autonomous RNA organisms.
The focus of this review, therefore, will be the origin of
the RNA World and its evolution prior to the development
of protein synthesis. We will have little to say about the
prebiotic synthesis of amino acids, peptides, cofactors, etc.
While acceptance of an RNA World greatly simplifies
the problem of the origin of life, it also has a negative aspect
(Orgel, 2003). If the origin of the RNA World preceded the
origin of protein synthesis, little can be learned about the
chemistry of the origin of life from the study of protein en-
zyme mechanisms. The justification of prebiotic syntheses
by appealing to their similarity to enzymatic mechanisms
has been routine in the literature of prebiotic chemistry.
Acceptance of the RNA World hypothesis invalidates this
type of argument. If the RNA World originated de novo
on the primitive Earth, it erects an almost opaque barrier
between biochemistry and prebiotic chemistry.
It is possible that the RNA World was the first organized
biochemical world on the primitive Earth. If we suppose
that this is the case, the problem of the origin of life can
conveniently be divided into a number of subproblems:
1. The nonenzymatic synthesis of nucleotides.
2. The nonenzymatic polymerization of nucleotides to
give random-sequence RNA.
3. The nonenzymatic copying or replication or both, of
RNA.
4. The emergence through natural selection of a set of
functional RNA catalysts that together could sustain
exponential growth in the prebiotic environment.
The first three topics are part of the traditional field of
prebiotic chemistry, while the fourth is the subject matter
of the newer field of RNA evolution. We begin this review
by covering the first three topics in some detail. Since the
fourth topic falls outside the scope of traditional prebiotic
chemistry, only a very brief overview will be given. From
our discussion of prebiotic chemistry we will conclude
that the abiotic synthesis of RNA is so difficult that it is
unclear that the RNA World could have evolved de novo
on the primitive Earth, a conclusion that was first em-
phasized by Cairns-Smith (Cairns-Smith & Davies, 1977;
Cairns-Smith, 1982). Consequently, we will have to con-
sider different routes to the RNA World. We will explore
the possibility that a simpler replicating molecule could
have formed on the primitive Earth and that organisms
with a genetic system based on that simpler polymer could
have “invented” RNA.
PREBIOTIC SYNTHESIS OF NUCLEOTIDES
Prebiotic Synthesis
Prebiotic chemistry is concerned with molecules that are
interesting to students of the origin of life which, they
believe, could have been formed on the primitive Earth.
Since we know very little about the availability of start-
ing materials on the primitive Earth or about the physical
conditions at the site where life began, it is often difficult
to decide whether or not a synthesis is plausibly prebi-
otic. Not surprisingly, claims of the type, “My synthesis
is more prebiotic than yours” are common. Nonetheless,
there is fairly general agreement about the following re-
strictions on organic synthesis imposed by the requirement
for prebioticity:
It must be plausible, at least to the proposers of a prebiotic
synthesis, that the starting materials for a synthesis
could have been present in adequate amounts at the
site of synthesis.
Reactions must occur in water or in the absence of a
solvent.
The yield of the product must be “significant,” at least in
the view of the proposers of the synthesis.
Clearly “prebiotic” is a very elastic term, and it would not
be wise to try to define it too closely.
Just as many people have been speaking prose all their
lives without realizing it, many organic chemists of the
19th and the first half of the 20th century were prebi-
otic chemists without realizing it. If it were discovered
for the first time today, Wohler’s synthesis of urea from
ammonium cyanate (Wohler, 1828) would certainly merit
PREBIOTIC CHEMISTRY AND ORIGIN OF RNA WORLD 101
publication in Science or Nature as an important contribu-
tion to prebiotic chemistry. Butlerow’s synthesis of sugars
from formaldehyde (Butlerow, 1861) is still one of the cor-
nerstones of the subject. However, these and other early
experiments on the synthesis of biochemicals from sim-
ple starting materials were never motivated by an interest
in the origin of life. Stanley Miller’s classic experiment
demonstrating the synthesis of amino acids in an electric
discharge (Miller, 1953) marks the beginning of prebiotic
chemistry as an enterprise directed to understanding the
chemistry of the origin of life.
Miller and Urey believed that the atmosphere of the
primitive Earth was strongly reducing, containing large
amounts of methane and ammonia. Miller showed that
formaldehyde and hydrogen cyanide (HCN) were key in-
termediates in the synthesis of glycine from such a mixture
(Miller, 1957). Although not directly relevant to the origin
of the RNA World, these observations led Juan Oro and his
coworkers to study the products formed when ammonium
cyanide is refluxed in aqueous solution. His remarkable
discovery that adenine is a product of cyanide polymer-
ization (Oro & Kimball, 1960), together with the earlier
results reported by Butlerow and Miller, determined the di-
rection of research on prebiotic chemistry for many years.
The relevance of all of this early work to the origin of life
has been questioned because it now seems very unlikely
that the Earth’s atmosphere was ever as strongly reducing
as Miller and Urey assumed. However, it still seems possi-
ble that that the Earth’s atmosphere was once sufficiently
reducing to have supported Miller/Urey chemistry to some
extent (Kasting & Brown, 1998).
The Butlerow (Formose) Synthesis of Sugars
from Formaldehyde
The polymerization of formaldehyde in the presence of
simple mineral catalysts to form a mixture of sugars—
the formose reaction—originally discovered by Butlerow
in the 19th century (Butlerow, 1861), has been investi-
gated in considerable detail (Mizuno & Weiss, 1974). The
reaction is of great interest as a unique, cyclic autocat-
alytic process that takes place in aqueous solution and
converts a very simple substrate, formaldehyde, to a mix-
ture of complex molecules, many of which are important
biochemicals.
It is fortunate that Butlerow did not completely purify
the formaldehyde he used before initiating the reaction,
because the formation of sugars from formaldehyde is de-
pendent on the presence of trace amounts of one of a num-
ber of common impurities (Socha et al., 1980; Kieboom &
VanBekkum, 1984). Glycolaldehyde, the first product of
the polymerization reaction, is an efficient initiator and
is often used in this role. In the absence of an initiator,
formaldehyde in alkaline solution undergoes the
Cannizaro reaction, yielding methanol and formic acid.
The Butlerow synthesis of sugars is usually carried out in
alkaline solution in the presence of a catalyst. Most studies
have employed heterogeneous catalysts, particularly sus-
pensions of calcium hydroxide, but some homogeneous
catalysts are known, for example, Pb
++
and Tl
+
ions.
A few investigations of the reaction under near-neutral
conditions in the presence of minerals have been reported
(Gabel & Ponnamperuma, 1967; Reid & Orgel, 1967).
The most intriguing feature of the formose reaction is
the long induction period that precedes the formation of
detectable products. Under many conditions the polymer-
ization, once started, is completed in a time shorter than the
induction period, but the induction period can be reduced
progressively by adding increasing amounts of an initiator.
The first product of the polymerization is glycolaldehyde,
which is later converted to glyceraldehyde and a variety
of tetrose, pentose, and hexose sugars. Under the condi-
tions usually used to bring about the reaction, the sugars
decompose to hydroxy-acids and related compounds on a
timescale similar to that of their appearance.
Cycles of the type shown in Figure 1 best explain most
of the experimental findings (Breslow, 1959). Two types
of reaction are involved, forward and reverse aldol reac-
tions and tautomerizations that interconvert aldehydes and
ketones. The scheme in the figure is no doubt a gross over-
simplification. Many related cycles involving reverse al-
dol reactions of different representatives of the tetrose,
pentose, and hexose sugars must contribute to the total re-
action. Furthermore, the addition of formaldehyde to glyc-
eraldehyde and similar molecules leads to the formation of
branched chain sugars, and the Cannizaro reduction of sug-
ars to polyols is also an important side reaction (Mizuno &
Weiss, 1974). Despite these complications, the major con-
clusion to be drawn from the scheme in Figure 1 is correct;
FIG. 1. The simplest hypothetical autocatalytic formose reac-
tion cycle. In each turn of the cycle, a glycolaldehyde molecule
facilitates the synthesis of a second glycolaldehyde molecule
from two formaldehyde molecules. The stereochemistry at the
asymmetric carbon atoms (marked with asterisks in the diagram)
is not specified.
102 L. E. ORGEL
as the consequence of traversing a cycle of the type shown,
a single input glycolaldehyde molecule leads, in principle,
to the production of two output glycolaldehyde molecules.
This ensures an exponentially growing rate of product for-
mation until the concentration of formaldehyde begins to
decline.
The Butlerow reaction, if it could be directed to the syn-
thesis of ribose, would provide an ideal route to the sugar
component of the nucleotides. However, until recently it
had not been possible to channel the Butlerow reaction
to the synthesis of any particular sugar, and ribose usually
has been a notoriously minor product (Decker et al., 1982).
More recently, Zubay has studied in detail the progress of
the Pb
++
catalyzed formose reaction (Zubay, 1998; Zubay
& Mui, 2001). He has shown that more than 30% of the
input formaldehyde can be converted to a mixture of the
aldopentoses and provides evidence suggesting that ribose
is the first pentose sugar formed, and that the other pen-
toses are formed from it by Pb
++
-catalyzed isomerization.
These studies suggest that a satisfactory prebiotic synthe-
sis of ribose may be possible. In a very recent report it has
been claimed that the four pentose sugars are stabilized by
the presence of calcium borate minerals (Ricardo et al.,
2004).
The production of ribose in the formose reaction de-
pends, at least in part, on the aldol reaction of glycolalde-
hyde with glyceraldehyde. Eschenmoser and his cowork-
ers showed that the pattern of products could be greatly
simplified if glycolaldehyde and glyceraldehyde were re-
placed by their monophosphates (Mueller et al., 1990).
Under alkaline conditions glycolaldehyde phosphate
alone yields a relatively simple mixture of tetrose-2-4-
diphosphates and hexose-2-4-6-triphosphates. Most
interestingly, ribose–2-4-diphosphate was the major sugar
product from the reaction of glycolaldehyde phos-
phate with glyceraldehyde-2-phosphate. Ribose-2-4-
diphosphate was also a major product of an equivalent
reaction involving formaldehyde and two molecules of
glycolaldehyde-phosphate. In these reactions the phos-
phate groups prevent the rearrangements that are charac-
teristic of triose, tetrose, and pentose sugars under alkaline
conditions and that lead directly or indirectly to much of
the complexity of the formose product mixture. Eschen-
moser’s synthesis would provide a first step in a plausible
route to the nucleotides if ribose-2-4-diphosphate could be
converted to a 5-phosphate or a 1-5-diphosphate.
The reactions described above occur in solution only
at high pHs and with high concentrations of the reactants.
However, certain layer hydroxides such as magnesium alu-
minium hydroxide are powerful catalysts for the reaction.
Negatively charged organic phosphates are absorbed so
strongly between the positively charged metal-hydroxide
layers that they can be concentrated from very dilute so-
lution. Furthermore, once in the environment between the
metal-hydroxide layers, they react rapidly to form sugar
phosphates even if the pH of the external solution is close to
7 (Pitsch et al., 1995). Since layer hydroxides are abundant
minerals, this version of Eschenmoser’s synthesis may be
considered as a promising prebiotic reaction. However, it
is less specific than the solution reaction for the production
of ribose-2-4-diphosphate.
We conclude that some progress has been made in the
search for an efficient and specific prebiotic synthesis of ri-
bose and its phosphates. However, in every scenario, there
are still a number of obstacles to the completion of a syn-
thesis that yields significant amounts of sufficiently pure
ribose in a form that could readily be incorporated into
nucleotides.
Purine Synthesis
In a series of seminal papers published in the 1950s, Juan
Oro and his coworkers showed that adenine is produced
in appreciable yield by refluxing a solution of ammonium
cyanide, and that 4-amino-5-cyanoimidazole (II) is an in-
termediate in the synthesis (Oro & Kimball, 1960, 1961,
1962; Oro, 1961a). This and closely related reactions have
been investigated repeatedly under different reaction con-
ditions, and the products have been analyzed using im-
proved analytical techniques. In addition to adenine, small
amounts of guanine have been detected among the prod-
ucts of HCN polymerization (Miyakawa et al., 2002a,
2002b). In a particularly striking experiment, adenine has
been obtained in 20% yield by heating HCN with liquid
ammonia in a sealed tube (Wakamatsu et al., 1966). Here
we can only review the literature on HCN polymerization
that is most relevant to prebiotic chemistry.
The first reasonably stable product of the polymeriza-
tion of HCN in aqueous solution is the HCN tetramer,
diaminomaleodinitrile (I). Subsequent steps in the poly-
merization are complex and are not well understood. The
tetramer, once formed, initiates a further polymerization
reaction that leads to the precipitation of a dark intractable
solid from which adenine, guanine, and numerous other
mostly uncharacterized compounds can be released by hy-
drolysis with acids or bases. In some experiments a small
quantity of adenine is also present in the solution phase
(Miyakawa et al., 2002a, 2002b). Very little is known about
the structure of the insoluble polymer or about the way in
which adenine is incorporated into it. While some ade-
nine may be released directly from the solid on hydroly-
sis by acid, much of it is released initially as adenine-8-
carboxamide and related compounds (Voet & Schwartz,
1983).
Several reactions that might contribute to the synthesis
of adenine from HCN via the HCN tetramer
(Figure 2) have been studied (Ferris & Orgel, 1965, 1966a;
Sanchez et al., 1967, 1968). It has been shown that
PREBIOTIC CHEMISTRY AND ORIGIN OF RNA WORLD 103
FIG. 2. Steps in possible prebiotic syntheses of adenine from HCN. (a) The formation of the HCN tetramer. (b) The conversion of
HCN tetramer to AICN. (c) The formation of purines from AICN or from its hydrolysis product 4-amino-imidazole-5-carboxamide
(III).
formamidine can be formed by the addition of ammonia to
HCN, and that formamidine reacts with the HCN tetramer
to give 4-amino-5-cyano-imidazole (AICN) (Figure 2b),
which, in turn, reacts with a second molecule of formami-
dine to yield adenine. In other experiments it has been
shown that HCN adds to AICN in aqueous solution to
give adenine directly (Figure 2c). Heating HCN tetramer
or AICN with ammonium formate—the hydrolysis prod-
uct of HCN—in the solid state is another way of obtain-
ing adenine (Zubay & Mui, 2001; Hill & Orgel, 2002).
Adenine has also been obtained directly by heating
formamide, a synthesis that may involve HCN as an in-
termediate (Saladino et al., 2001). However, none of the
reactions has been proven to contribute directly to adenine
synthesis under the conditions employed by Oro and his
coworkers.
AICN and its hydrolysis product 4-amino-imidazole-5-
carboxamide (III) are readily converted to hypoxanthine
and a variety of 2, 6-disubstituted purines in aqueous so-
lution by reaction with simple one-carbon molecules (Fig-
ure 2c) (Sanchez et al., 1968). Thus AICN and the related
carboxamide, if they could be obtained under prebiotic
conditions, would offer very plausible routes to the purines
that are important in biochemistry. We must therefore re-
view attempts to obtain HCN tetramer and to convert it to
AICN under plausibly prebiotic conditions.
Detailed kinetic studies show that hydrolysis of HCN to
formamide and ultimately ammonium formate competes
very effectively with tetramer synthesis if the HCN con-
centration falls below 10
−1
to 10
−2
M (Sanchez et al.,
1967). It would have been impossible to reach such a
high concentration of HCN in the bulk oceans, while
