Critical Reviews in Biochemistry and Molecular Biology, 39:99–123, 2004
Copyright c ? Taylor & Francis Inc.
ISSN: 1040-9238 print / 1549-7798 online
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
once an RNA World. The central problem for origin-of-life studies,
therefore, is to understand how a protein-free RNA World became
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
nucleotide synthesis, polynucleotide formation, RNA
replication, prebiotic membranes
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
suite of amino acids, what remained was too complicated
Editor: Michael M. Cox.
Address correspondence to Leslie E. Orgel, The Salk Institute,
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
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
1967;Crick, 1968; Orgel, 1968),andaprogramtoexplore
nonenzymatic copying of nucleic acid sequences was ini-
tiated (Sulston et al., 1968a, 1968b).
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-
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
The unanticipated discovery of catalytic RNA mole-
cules, ribozymes, that perform enzyme-like reactions
ical world. In the few years following Cech and Altman’s
discoveries, ribozymes were shown to be able to catalyze
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
does not deny that peptides may have been involved in the
origin of life. It does, however, exclude the possibility that
protein synthesis or a closely related mechanism.
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
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-
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
While acceptance of an RNA World greatly simplifies
origin of protein synthesis, little can be learned about the
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.
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.
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
have “invented” RNA.
PREBIOTIC SYNTHESIS OF NUCLEOTIDES
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-
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
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 WORLD101
publication in Science or Nature as an important contribu-
tion to prebiotic chemistry. Butlerow’s synthesis of sugars
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-
(Miller, 1957). Although not directly relevant to the origin
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
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
ble that that the Earth’s atmosphere was once sufficiently
extent (Kasting & Brown, 1998).
The Butlerow (Formose) Synthesis of Sugars
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
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-
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,
Cannizaro reaction, yielding methanol and formic acid.
The Butlerow synthesis of sugars is usually carried out in
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-
induction period, but the induction period can be reduced
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-
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-
Weiss, 1974). Despite these complications, the major con-
inalkaline solution undergoesthe
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
is not specified.
The simplest hypothetical autocatalytic formose reac-
102 L. E. ORGEL
mation until the concentration of formaldehyde begins to
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
More recently, Zubay has studied in detail the progress of
& Mui, 2001). He has shown that more than 30% of the
input formaldehyde can be converted to a mixture of the
is the first pentose sugar formed, and that the other pen-
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.,
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-
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-
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
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.
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
minerals, this version of Eschenmoser’s synthesis may be
considered as a promising prebiotic reaction. However, it
We conclude that some progress has been made in the
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
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,
been investigated repeatedly under different reaction con-
ditions, and the products have been analyzed using im-
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
drolysis with acids or bases. In some experiments a small
quantity of adenine is also present in the solution phase
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,
Several reactions that might contribute to the synthesis
Sanchez et al., 1967, 1968). It has been shown that
PREBIOTIC CHEMISTRY AND ORIGIN OF RNA WORLD103
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
synthesis under the conditions employed by Oro and his
carboxamide (III) are readily converted to hypoxanthine
and a variety of 2, 6-disubstituted purines in aqueous so-
ure 2c) (Sanchez et al., 1968). Thus AICN and the related
carboxamide, if they could be obtained under prebiotic
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.
formamide and ultimately ammonium formate competes
very effectively with tetramer synthesis if the HCN con-
centration falls below 10−1to 10−2M (Sanchez et al.,
1967). It would have been impossible to reach such a
high concentration of HCN in the bulk oceans, while
104 L. E. ORGEL
evaporation of lakes or tide pools could not adequately
sible prebiotic method for concentrating HCN is by “eu-
tectic is obtained at −23.4◦C that contains 74.5 (moles)%
of HCN. This very concentrated solution slowly deposits
a typical dark HCN polymer. Schwartz and his coworkers
hydrolysis of this polymer or in 0.02% yield if glycoloni-
trile was added to the reaction mixture before freezing
(Schwartz et al., 1982). Miller and his coworkers have
obtained very similar results (Miyakawa et al., 2002a,
2002b). At present, the most plausible routes to adenine
from HCN involve an initial synthesis of HCN tetramer in
solutions concentrated by freezing.
The route from HCN tetramer to AICN by reaction
with formamidine (Figure 2b) is somewhat problemati-
cal, since the formation of formamidine would require a
high concentration of ammonia, and it is questionable that
ammonia was ever present on the primitive Earth in sig-
nificant amounts. The photochemical isomerization of the
HCN tetramer (Figure 2b) provides an alternative route
to AICN (Ferris & Orgel, 1966b). The reaction occurs
readily in sunlight and gives almost quantitative yields of
to AICN (Figure 2b), and reaction of AICN with HCN to
synthesis of adenine that is independent of ammonia.
Completely different schemes for the accumulation of
adenine and other purines on the primitive Earth have
been discussed. Miyakama and his coworkers suggested
that purines were formed in the atmosphere but by mech-
anisms that are independent of HCN (Miyakawa et al.,
2000). Substantial amounts of adenine have been found
in carbonaceous chondrites, so it has been suggested that
purines were formed elsewhere in the solar system, per-
haps by Oro-type chemistry, and brought to the Earth in
meteorites (Oro, 1961b; Chyba & Sagan, 1992).
Most of the published work on prebiotic pyrimidine syn-
thesis is concerned with a series of closely related reac-
tions between cyanoacetylene (IV) or its hydrolysis prod-
uct, cyanoacetaldehyde (V), and cyanate ions, cyanogen
or urea (Figure 3) (Ferris et al., 1968, 1974; Robertson &
Miller, 1995a, 1995b; Nelson et al., 2001). The product,
cytosine, is obtained in good yield in several of these re-
actions. Since cyanoacetylene is a major product formed
when an electric discharge is passed through a mixture
of nitrogen and methane (Sanchez et al., 1966b) and hy-
these two molecules have been claimed to be potentially
prebiotic (Orgel, 2002). Uracil is formed from cytosine
by hydrolysis, and this has been proposed as a prebiotic
The highest yields of cytosine, up to 50%, are obtained
when cyanoacetaldehyde is incubated with a saturated so-
lution of urea (Robertson & Miller, 1995a, 1995b).
However, the so-called drying lagoon model, which pos-
tulates that lagoons of saturated urea existed on the primi-
tive Earth does not seem plausible (Shapiro, 1999, 2002).
More modest yields of cytosine (about 5%) are obtained
when cyanoacetylene reacts with 1.0 M cyanate or when
either cyanoacetylene or cyanoacetaldehyde reacts with
hyde with urea or of cyanoacetylene with cyanate in eu-
tectic solution seem the most plausible prebiotic routes to
cytosine. Syntheses of this kind are particularly attractive
because they could proceed in parallel with the synthesis
of adenine from HCN (Orgel, 2004).
The synthesis of nucleosides from ribose and the nucle-
oside bases is the weakest link in the chain of prebiotic
(a) The hydrolysis of cyanoacetylene to cyanoacetaldehyde.
(b) The reaction between cyanoacetylene and two molecules
of cyanic acid. (c) The condensation of cyanoacetaldehyde with
Steps in proposed prebiotic syntheses of cytosine.
PREBIOTIC CHEMISTRY AND ORIGIN OF RNA WORLD105
of hydrolysis of cyclocytidine caused by the presence of a neighboring phosphate group.
(a) The synthesis of cytosine arabinoside from arabinose, cyanamide, and cyanoacetylene. (b) Modification of the mode
directly with hypoxanthine in the presence either of mag-
in seawater, up to 8% of authentic β-D-inosine is formed,
along with a somewhat smaller amount of the α-isomer
(Fuller et al., 1972). The product mixture formed from
adenine under the same conditions is more complex, since
the major reaction occurs at the amino-group of the base,
and nucleoside formation is a relatively minor side reac-
tion. However, hydrolysis of the reaction products under
relatively mild conditions leaves behind a small yield of a
mixture of adenosine isomers including up to 3% of β-D-
No direct synthesis of pyrimidine nucleosides from ri-
bose and uracil or cytosine has been reported. An indirect
cyanamide, and cyanoacetylene in aqueous solution has
ing reaction of arabinose yields β-cytosine arabinoside
ribose-5-phosphate in this reaction sequence α-cytidine-
photo-anomerize α-cytidine to β-cytidine but only in 5%
yield (Sanchez & Orgel, 1970). A similar reaction occurs
with a α-cytidine-5?-phosphate.
Nagivary prepared the 3?-phosphate of cyclo-cytidine
(VII) by standard laboratory synthesis and showed that it
hydrolyzes in water, via a cyclic phosphate, to give a good
yield of cytidine 2?(3?)-phosphate (Figure 4b) along with a
smaller amount of cytosine arabinoside-3?-phosphate. He
speculated that these two reactions together might pro-
vide a prebiotic route to cytidine-3?-phosphate (Tapiero
& Nagyvary, 1971). More recently, Sutherland and his
coworkers have succeeded in combining the two reac-
tion sequences, thus obtaining cytidine-3?-phosphate di-
rectly, in aqueous solution, from arabinose-3-phosphate,
106 L. E. ORGEL
cyanamide and cyanoacetylene (Ingar et al., 2003).
is unclear, but this work nonetheless suggests that the
prebiotic synthesis of pyrimidine nucleotides may be
involves the displacement of phosphate from α-ribose-
1-phosphate or of pyrophosphate from α-ribose-5-
phosphate-1-pyrophosphate by a nucleoside base. This
seems to provide a promising approach to the correspond-
ing prebiotic syntheses. A few preliminary and unpub-
lished experiments attempted in our laboratory using
periments reported by Zubay and his coworkers suggest
may be possible (Zubay & Mui, 2001).
Phosphorylation of Nucleosides
reduced forms of phosphorus have occasionally been con-
sidered in this context (Schwartz, 1997; Peyser & Ferris,
2001). Only orthophosphates are abundant in rocks and
minerals, principally insoluble calcium phosphates, but
there is evidence that condensed phosphates are products
of volcanism (Yamagata et al., 1991). A number of ap-
phates or polyphosphates have been explored.
Many of the earliest attempts to phosphorylate nu-
cleosides utilized organic condensing agents such as
cyanamide, cyanamide dimer, or cyanate. These reactions
are prebiotic equivalents of the much-used phosphory-
lation protocols that employ carbodiimides as activating
agents in organic solvents. Unfortunately, such reactions
are usually inefficient in aqueous solution because of the
competition of water for the activated phosphate inter-
mediate (Lohrmann & Orgel, 1968). Appreciable yields
of cyclic phosphates can occasionally be obtained, for
example, from monophosphates of cis glycols, because
the cis hydroxyl group can compete efficiently with wa-
ter for the activated phosphate moiety. Nucleoside-2?- or
3?-phosphates sometimes give nucleoside-2?- or 3?-cyclic
phosphates in good yield in this way. More recently it has
been shown that AMP can be converted to ADP and ATP
by heating in the solid state with acidic phosphates such
as NaH2PO4. These reactions require fairly high temper-
atures and are not very efficient (Beck et al., 1967). How-
is used as the inorganic component. Presumably, ammo-
nium phosphate is particularly effective in this reaction
because it loses ammonia on heating and thus generates a
ponent, heating ammonium phosphate with urea yields a
mixture of high molecular-weight linear polyphosphates
Nucleosides can be converted to a complex mixture of
products containing one or more phosphate groups in ex-
monium phosphate and urea (Lohrmann & Orgel, 1971).
When uridine, for example, is heated with excess urea and
ammonium phosphate at 100◦C, about 70% of the input is
converted to a complex mixture of phosphorylated prod-
ucts (Figure 5). Attempts to direct this reaction to the syn-
thesis of a particular phosphate or polyphosphate, for ex-
ample, a nucleoside-5?-phosphate or 5?-triphosphate, have
et al., 1973; Reimann & Zubay, 1999). These solid-state
reactions have been studied in considerable detail, but the
reaction mechanism is not known. It is unlikely to involve
carbamoyl phosphate or a phosphoramidate intermediate,
so acid-base catalysis seems most probable (Osterberg &
as insoluble calcium phosphates, and this is likely to have
slowly when ammonium phosphate is replaced by cal-
as 20% were obtained when hydroxylapatite was heated
phosphate, ammonium chloride, ammonium bicarbonate, and
urea at 100◦C. U, —; Up!, — – – – —; pU, — – – —;Up, —
— — —; pUp!, — – —; pUp – – – – – –; total incorporation of
inorganic phosphate, —. Redrawn from Figure 6 in Lohrmann
and Orgel (1971).
PREBIOTIC CHEMISTRY AND ORIGIN OF RNA WORLD107
with urea, ammonium chloride, and a nucleoside at 100◦C
(Lohrmann & Orgel, 1971).
with a catalyst such as urea leads to the formation of a
mixture of linear polyphosphates. These compounds can
also be formed by heating other acidic phosphates such as
NaH2PO4strongly and, perhaps for this reason, are pro-
duced in volcanoes (Yamagata et al., 1991). While linear
polyphosphates have never been shown to be good phos-
readily to cyclic triphosphates, the trimetaphosphates, un-
der alkaline conditions in the presence of divalent metal
tially prebiotic phosphorylating agents.
Cis glycols react with trimetaphosphate in strongly
alkaline solution to yield cyclic phosphates that subse-
a mixture of 2?- and 3?-phosphates, often in excellent yield
(Schwartz, 1969; Saffhill, 1970; Tsuhako et al., 1984).
The same reaction occurs less efficiently under less al-
kaline conditions in the presence of Mg++(Yamagata
et al., 1995) or in a cycle of wetting and drying reactions
highly alkaline conditions to yield triphosphates, for ex-
The phosphorylation of α-hydroxyaldehydes by the amidotriphosphate anion.
triphosphate from thymidine (Etaix & Orgel, 1978).
N-triphosphates. An ingenious modification of this re-
action provides a simple procedure for phosphorylating
action of ammonia with the trimetaphosphate anion yields
carbonyl group of the hydroxyaldehyde. Next the
2-hydroxyl group attacks the α-phosphate, expelling py-
rophosphate and forming a cyclic phosphoramidate. Fi-
nally the cyclic phosphoramidate undergoes hydrolysis to
yield the 2-phosphate of the aldehyde (Figure 6). This
reaction sequence is of considerable interest for prebi-
While most effort has been devoted to the phospho-
rylation of preformed nucleosides, the direct phospho-
rylation of ribose is also interesting because the reac-
tion of α-ribofuranose-1-phosphate with nucleoside bases
is a plausible route to the nucleosides. Ribose can be
108 L. E. ORGEL
atively pure β-ribofuranose-1 phosphate, not the desired
thesis is that there is at present no convincing, prebiotic
total synthesis of any of the nucleotides. Many individual
steps that might have contributed to the formation of nu-
cleotides on the primitive Earth have been demonstrated,
but few of the reactions give high yields of products, and
those that do tend to produce complex mixtures of prod-
ucts. It should also be realized that any prebiotic synthe-
sis of a nucleotide would yield a racemic product, not
the biologically important D-nucleotide. Recent publica-
above), suggest that the search for a convincing prebiotic
synthesis of the nucleotides is not hopeless. However, the
appearance of RNA on the primitive Earth deserve serious
consideration. The succeeding sections of this review, in
thetical source of prebiotic nucleotides, will also consider
other ways in which the RNA World could have appeared.
INPUT IN METEORITES OR COMETS
A substantial proportion of the meteorites that presently
fall on the Earth belong to a class known as carbonaceous
chondrites. These interesting stones may contain more
than 3% of carbon, much of it organic. Numerous detailed
chemical analyses of carbonaceous chondrites have led
to the identification of a very large number of hydrocar-
bons, carboxylic acids, amino acids, hydroxy acids, sul-
fonic acids, phosphonic acids, poly-hydroxy compounds
(Cooper et al., 2001), etc. Very little is known in detail
about the mechanisms that produced the various classes
of molecules. It is thought that primary processes in the
interstellar or stellar medium produced organic precursors
that were incorporated into the parent bodies of the mete-
orites. Then further processing on the parent bodies modi-
fied and extended the inventory of organics. The details of
this complex subject are beyond the scope of the present
plicated chemistry are available (Anders, 1989; Chyba &
The presence of adenine and the other nucleoside bases
in the carbonaceous chondrites at a level of about one
part per million, however they may have formed, is ob-
viously relevant to discussions of the origin of the RNA
There can be no doubt that material analogous to that
making up the carbonaceous chondrites contribute
substantially to the formation of the Earth, and that the
influx of meteoritic material was much greater during the
early history of the Earth than it is today. The extent to
which the organic material present in meteorites could
have survived passage through the atmosphere and impact
on the surface of the Earth is unclear. It is also difficult
to estimate the importance of micrometeorites and comets
(Goo, 1961), which are thought by some to have brought
in the bulk of the organic material that was present on
the early Earth. Despite the uncertainties, many scientists
believe that meteorites, comets, and interplanetary dust
particles were the major source of organic material for the
origin of life. If so, life must have originated in a mix-
Miller/Urey chemistry but at least as complicated.
SYNTHESIS IN THE DEEP SEA VENTS
Wachtershauser initiated a novel approach to the problem
of the origin of life (Wachtershauser, 1988). He proposed
that the original source of organic material for the origin
of life was provided by the reduction of carbon dioxide
using hydrogen sulfide (H2S) over ferrous sulfide (FeS)
as the reducing agent. He further proposed that the prod-
ucts of this reaction never entered free aqueous solution
but set up a complex “metabolism” while confined to the
surface on which they were synthesized. In more detail,
he noted that the conversion of FeS to FeS2, pyrite, by
H2S could provide the reducing power needed to produce
organic material from CO2. The self-organization of the
reductive citric acid cycle on an iron sulfide surface with-
out the help of enzymes or other informational molecules
was also a central part of his scheme. Chemistry of this
kind could most plausibly occur in the deep-sea vents,
where superheated water containing dissolved H2S and
transition-metal sulfides is mixed suddenly with a large
excess of cold seawater, causing sulfides, including large
amounts of FeS, to precipitate. Scenarios for the origin
of life that involve this or related hydrothermal chemistry
are now popular. As far as I am aware, these scenarios
have not been shown to be directly relevant to the origin
of the RNA World, but they may be relevant to the origin
of membrane-forming organic material.
Wachtershauser’s imaginative suggestion that the for-
mation of FeS2from H2S and FeS could drive reduction
and his coworkers. They have, for example, demonstrated
the reduction of acetylene and mercaptans by H2S over
FeS (Blochl et al., 1992). Most impressively, it has re-
cently been reported that freshly precipitated ferrous sul-
fide in the presence of H2S is able to reduce molecular
nitrogen to ammonia (Dorr et al., 2003). Thus, the reac-
tion of FeS with H2S to form pyrite provides the driving
PREBIOTIC CHEMISTRY AND ORIGIN OF RNA WORLD109
force for many reductions, including the very difficult re-
duction of N2to ammonia in just the way predicted by
The reduction of CO2has not been achieved, but an
interesting alternative synthesis, the reaction of carbon
sulfide to give an activated derivative of acetic acid, prob-
ably the methyl thioester, has been described (Huber &
Wachtershauser, 1997). In another paper, Huber and
acids by activation with carbon monoxide in the pres-
ence of Fe/Ni S and H2S or methyl mercaptan (Huber &
may be an intermediate in this reaction, its hydrolysis to
carbon dioxide and hydrogen sulfide in the presence of
transition-metal sulfides providing the free energy needed
to drive peptide synthesis. The N-carboxyanhydrides of
the amino acids are probably intermediates in the reaction
Cody and his coworkers have reported another poten-
tially important synthesis. They were able to obtain pyru-
vic acid from formic acid, a source of carbon monoxide,
in the presence of nonylmercaptan and FeS (Cody et al.,
2000). The reaction was carried out at 250◦C and a high
pressure, without the addition of water in excess of that
generated by the decomposition of the formic acid. The
synthesis of a significant yield of pyruvic acid, a rela-
tively unstable molecule at such a high temperature, is
unexpected. It is not clear whether nonylmercaptan has an
essential role in the reaction or if it could be omitted or
replaced by a lower molecular weight thiol such as methyl
mercaptan, nor is it known whether the synthesis occurs
In addition to these recent studies, earlier work on the
Fischer-Tropsch reaction, which leads to the production
of long straight-chain hydrocarbons and their oxygenated
derivatives from carbon monoxide and hydrogen in the
presence of a suitable catalyst, may well be relevant to
the chemistry of the deep-sea vents. The conditions and
catalysts typically employed in the chemical industry are
unlikely to have been present on the primitive Earth, but
there is some evidence that similar results can be obtained
with plausible prebiotic catalysts (Zolotov et al., 2001).
The production of straight-chain fatty acids might pro-
vide a source of membrane-forming organic material (see
of some or all of the organic material needed for the origin
of life. However, it seems unlikely that nucleotides could
have formed directly in the vents, so Wachtershauser’s
theory is best suited to a scenario in which some other
genetic polymer preceded RNA (see below).
In the above discussion I have treated reactions that oc-
cur on metal sulfides as prebiotic syntheses that support a
novel scenario for the formation of the organic substrates
that contributed to the origin of life. Wachtershauser in-
sists that they are more than that and that they support his
theory of a complex surface metabolism in which the re-
on the surface of a metal sulfide. I am unaware of any
experimental evidence for such self-organization, and I
have argued elsewhere that on theoretical grounds it is ex-
ceedingly unlikely that such self-organization could occur
(Orgel, 2000). If life originated in the deep-sea vents, I
think it more likely that the vents provided the compo-
nents of a relatively simple informational polymer. Unfor-
under anaerobic conditions requires the use of specialized
hydrothermal synthesis on the surfaces of transition metal
sulfides is adequately explored.
POLYMERIZATION OF ACTIVATED NUCLEOTIDES
The polymerization of nucleotides in aqueous solution is
an uphill reaction and does not occur spontaneously to a
significant extent. Evaporation of acidic solutions of nu-
cleotides and subsequent heating leads to the formation of
2?-5?-, or 3?-5?-phosphodiester linkages occur more or less
at random (Moravek, 1967). Consequently, attempts to
polymerize nucleotides from aqueous solution must nec-
essarily make use of external activating agents. Attempts
along these lines using cyanamide and similar activating
agents or water-soluble carbodiimides have been disap-
pointing, at best leading to poor yields of dinucleotides
and very short oligonucleotides.
greater success. Unfortunately, nucleoside-5?-polyphos-
phates react so slowly in aqueous solution at moderate
temperatures and pHs that their polymerization cannot
easily be studied in the laboratory. Instead, nucleotides
activated as phosphoramidates, usually phosphorimida-
zolides, have been used as substrates in most experiments
(Figure 7a). They can be obtained in fairly good yield
from nucleoside-5?-polyphosphates and amines or imida-
zoles and have, therefore, been claimed to be prebiotic
(Lohrmann, 1977). However, it is unclear that phospho-
primitive Earth, so these experiments form only a rough
guide to the classes of reaction that might have been rele-
vant to chemical evolution. The source of the free energy
needed to drive the uphill polymerization of nucleotides
is unclear. One plausible suggestion involves the initial
formation of nucleoside polyphosphates from nucleosides
110 L. E. ORGEL
diagram showing activated mononucleotides aligned on a d(CCCGCCCGCCCGCC) template. (c) Electrophoresis of the products
formed from activated monomers aligned on the d(CCCGCCCGCCCGCC) template shown in (b) above. The composition of the
oligomers corresponding to major products is indicated. Degradation with specific RNAases showed that each major product is
complementary to a subsequence of the template.
(a) The structures of some activated nucleotides used in the prebiotic synthesis of oligonucleotides. (b) Oversimplified
they could have been converted to phosphoramidates that
polymerized more rapidly.
Nucleoside 5?-phosphorimidazolides oligomerize in
aqueous solution, but in the absence of a catalyst yield
only a complex mixture of short linear and cyclic prod-
ucts. A number of metal ions are effective catalysts for
this class of polymerization (Sawai & Orgel, 1975; Sawai,
1976), in particular Pb++(Sawai, 1976; Sleeper & Orgel,
1979). Long homo- and hetero-oligomers are obtained us-
ing this metal ion when the reaction is carried out in eu-
tectic solution (Kanavarioti et al., 2001; Monnard et al.,
2003). The uranyl ion is another very efficient catalyst in
aqueous solution, leading to the synthesis of oligomers
up to at least 16mers. These oligonucleotide products are
predominantly 2?-5?-linked (Sawai et al., 1989, 1992).
siderable detail a remarkable series of reactions in which
an abundant clay mineral, montmorillonite, catalyzes the
synthesis of long oligonucleotides from relatively dilute
solutions of nucleoside phosphoramidates including the
5?phosphorimidazolides (Ferris et al., 2003). In some of
their more recent experiments using a phosphoramidate
based on 1-methyladenine (X; Figure 7a), they were able
to identify oligomers up to 40 residues long (Huang &
PREBIOTIC CHEMISTRY AND ORIGIN OF RNA WORLD111
pressive examples of the catalysis of a prebiotic reaction
by a mineral that has been reported.
structure that readily expands to permit large molecules to
enter the interlayers, and it is probable that new phospho-
in the interlayers (Ertem & Ferris, 1998). An extensive
body of experimental work suggests that there are spe-
cific catalytic sites somewhere on or in the mineral that
can be blocked by adsorbed but unreactive analogs of the
substrates, for example dimethyladenine (Wang & Ferris,
The regiospecificity of the polymerization is very de-
pendent on the nature of the nucleoside base and the ac-
tivating group. In some cases it can favor the formation
of 3?-5?- over 2?-5?-phosphodiester linkages by as much
as a factor of four (Prabahar & Ferris, 1997), but in other
cases the regiospecificity is modest or favors the forma-
action is limited (Joshi et al., 2000). Clearly, this im-
pressive example of mineral-based catalysis is efficient
but not particularly specific with respect to the distribu-
tion of isomeric products. Nonetheless, products of the
(Ertem & Ferris, 1997).
In summary, two procedures for the efficient oligomer-
phosphoramidates have been reported, montmorillonite
catalysis and metal ion catalysis by Pb++or uranyl ions.
not likely to be prebiotic molecules.
The replication of RNA without the help of protein en-
without the help of any informational catalyst, played a
review experiments on nonenzymatic template-directed
synthesis here. Later we will discuss briefly ribozyme-
catalyzed template-directed synthesis, which is likely to
have evolved in the RNA World before the “invention” of
The basic principle of template-directed synthesis is a
simple one (Figure 7b). If a polynucleotide is incubated
with an appropriate mixture of complementary mononu-
cleotides or short oligonucleotides at a sufficiently low
plex is formed (Howard et al., 1966). These complexes
are structurally similar to double- or triple-stranded nu-
cleic acids, but one chain is interrupted. Thus the tem-
plate brings complementary activated monomers or short
a complementary complex is formed, the template influ-
ences the ligation reaction, but the devil is in the details.
lead to a greater or lesser excess of 3?-5?-phosphodiester
linkages over 2?-5?-linkages. The objective of most re-
search programs in this field has been to find conditions
that lead to the efficient synthesis of predominantly 3?-5?-
linked oligonucleotides for as broad a range of template
sequences as possible.
The first experiments on template-directed synthesis of
showed that a dA12template catalyzed the synthesis of
T12 from two T6 molecules when a water-soluble car-
bodiimide was used as the condensing agent (Naylor &
Gilham, 1966). Zoe Shabarova and her coworkers have
reported a large number of related experiments using dif-
ferent deoxy-templates, deoxy-substrates, and activating
agents (Shabarova, 1988). We will not discuss this inter-
esting and extensive literature because it is unlikely to be
relevant to the origin of the RNA World. Experiments on
RNA synthesis necessarily used activated ribonucleotide
monomers or short oligomers as substrates, but often used
DNA templates when RNA heteropolymers were not eas-
ily available. Water-soluble carbodiimides were initially
used as activating agents (Sulston et al., 1968a, 1968b,
short oligonucleotide products were obtained.
More recent work in the area of template-directed syn-
thesis from mononucleotides has employed preactivated
substrates, phosphorimidazolides, or closely related phos-
ature on this topic has been reviewed (Joyce, 1987; Orgel,
1992; Kozlov & Orgel, 2000), so only the main conclu-
sions will be presented here. The cited reviews should
be consulted for experimental procedures and quantitative
results. Nucleoside-5?-triphosphates, which might seem
an obvious choice of substrates in experiments with nu-
cleotide monomers, cannot easily be used in laboratory
experiments because they react too slowly at temperatures
form. Studies of the spontaneous template-directed liga-
tion of oligonucleotides terminated by a 5?-triphosphate
group, however, have led to interesting conclusions, as we
The first efficient and regiospecific polymerization re-
actions to be reported were the syntheses of long oli-
goguanylic acids (oligoGs) on poly(C) templates using
guanosine-5?-phosphorimidazolide (VIII; ImpG) as the
112 L. E. ORGEL
activated monomer. The reaction showed a remarkably
specific dependence on divalent metal ions. In the pres-
ence of Mg++and Pb++, the products were almost exclu-
sively 2?-5?-linked (Lohrmann & Orgel, 1980), while in
the presence of Mg++and Zn++ions virtually pure 3?-5?-
linked oligomers were formed (Bridson & Orgel, 1980).
If neither Pb++nor Zn++was present, no long oligomers
were obtained. This reaction was restricted to the synthe-
sis of oligo(G)s on a poly(C) template and could not be
extended to incorporate bases other than G on homo- or
It was found, surprisingly, that when 2-methyl imida-
zole replaced imidazole in the activated nucleotide (IX;
to give 2-MeImpG) the synthesis of long oligo(G)s on
a poly(C) template no longer required the presence of
any metal ion other than Mg++. In this context, 2-Me-
imidazole seems unique since neither imidazole nor its
2-ethyl derivative can substitute for it. Furthermore, the
clusively 3?-5?-linked (Inoue & Orgel, 1981). One further
advantage of using the 2-methyl imidazolides is that it al-
lows the copying of heteropolymers containing all four
bases, but only if the template contains at least 60% of
C residues (Joyce, 1987). This latter restriction rules out
the possibility of repeated rounds of replication, since the
product of a successful template-directed oligomerization
contains at most 40% of C residues and cannot, therefore,
act efficiently as a template.
There is now substantial evidence that the template-
directed oligomerization of 2-Me-imidazole derivatives
proceeds best in double helices that adopt the A-form nu-
cleic acid structure (Kurz et al., 1997, 1998; Kozlov et al.,
1999b, 2000b). RNA sequences are superior to DNA se-
quences as templates, but the general features of the reac-
tions are similar for RNA and DNA templates (Zielinski
et al., 2000). Nucleic acid analogs that tend to preorga-
nize in the A-form DNA structure are usually excellent
templates (Kozlov et al., 1999b, 2000b).
The sequence dependence of the reactions has been
studied extensively using hairpin oligodeoxynucleotides
as substrates (Wu & Orgel, 1992a, 1992b, 1992c; Hill
et al., 1993). Incorporation of G opposite C in the tem-
plate is most efficient, while incorporation of U opposite
A is least efficient. Incorporation of A opposite U or of C
A residues in the template is an almost complete barrier to
further synthesis. The fidelity of these reactions is usually
very good, but with one notable exception: wobble pair-
ing of G opposite U leads to extensive misincorporation
of G, particularly on some RNA templates. The results
of a long series of detailed studies show, therefore, that
a wide variety of DNA or RNA sequences can be copied,
The results of copying an especially favorable sequence,
deoxy(CCGCCCGCCCGCCC), are shown in Figure 7c
(Acevedo & Orgel, 1987).
Any prebiotic synthesis that yields ribonucleotides
would produce racemates. Unfortunately, the L-enant-
iomers of activated nucleotides are efficient inhibitors of
template-directed synthesis using the naturally occuring
D-enantiomers (Joyce et al., 1984). This difficulty, of-
ten described as enantiomeric cross-inhibition, is not eas-
ily overcome without making substantial changes to the
nature of the backbone of the template (Kozlov et al.,
cleotide replication from plausibly prebiotic, monomeric
phorimidazolides has been studied less extensively. When
very short oligomers activated as phosphorimidazolides
are used as substrates, the efficiency and regiospecificity
of the reaction depends very strongly and unpredictably
on the sequences of the substrates (Ninio & Orgel, 1978).
However, with somewhat longer 3?,-5?-linked sequences
ligation is efficient and yields predominantly 3?-5?-linked
ity of using phosphorus-labeled substrates of high specific
activity, has made possible a detailed study of the liga-
tion of substrates activated as 5?-triphosphates (Rohatgi
et al., 1996a, 1996b). Encouragingly, the ligation of 5?-
triphosphates, a close analog of enzymatic ligation, yields
almost exclusively 3?-5?-linked products. In the context of
prebiotic chemistry, 3?-5?-linked oligonucleotides are su-
perior to mononucleotides as substrates with respect to
regiospecificity, and they permit ligation over wider tem-
perature ranges. However, it is not obvious that homochi-
ral, exclusively 3?-5?-linked oligomers are plausible prebi-
otic molecules (see previous section), and the fidelity of
template-directed ligation of oligomers is lower than the
fidelity of oligomerization of monomers.
If one believes that the RNA World was the first organized
biological world, one must postulate that a library of RNA
strands with different sequences formed spontaneously on
the primitive Earth and that this family of sequences in-
cluded catalysts able to support self-replication of RNA.
The idea of an RNA that performed some of the functions
of an RNA polymerase is, therefore, an essential feature
catalysts for some metabolic reactions were required for
the RNA World to become self-sustaining, but it is hard
to guess which metabolic reactions. Instead of discussing
ribozymes in general, we will review very briefly what is
PREBIOTIC CHEMISTRY AND ORIGIN OF RNA WORLD113
known about the ribozyme-catalyzed reactions that seems
most relevant to the origin of the RNA World. The whole
tal protocols used in directed RNA-evolution experiments
fall outside the scope of this article. Excellent accounts of
these topics are available in two general reviews (Wilson
& Szostak, 1999; Joyce, 2004).
portant, are rather limited in the range of reactions that
they catalyze. The initial discoveries of Cech and Altman
identified ribozymes that cut and ligate naturally occur-
ring RNA (Kruger et al., 1982; Guerrier-Takada et al.,
1983). With one important exception, the ribozymes that
have been discovered subsequently in nature perform the
same kind of functions. There is no evidence that tran-
scription or RNA replication involve ribozyme catalysis.
The most important ribozyme in nature is the ribosome. It
is now clear that the peptide-bond–forming step of protein
This is the smoking gun that has led to the more general
acceptance of the RNA World hypothesis. Unfortunately,
our rapidly improving understanding of protein synthesis
is unlikely to throw much light on the origin of the RNA
World, since the invention of protein synthesis marked the
beginning of its decline.
In vitro methods have been used to isolate RNA mole-
cules that bind with high specificity to selected small or-
ganic molecules or to peptides and proteins (Puglisi &
Williamson, 1999). In principle, it should be possible to
extend this type of procedure to obtain RNAs that bind
to almost any water-soluble polymer or to any structured
rocks. Selection of RNAs that perform catalytic functions
required the invention of more complicated experimen-
tal protocols but is now a routine laboratory procedure
(Wilson & Szostak, 1999; Joyce, 2004). It is clear that a
great variety of reactions can be catalyzed by RNA. On
the one hand, an RNA that catalyzes the racemization of a
the other, the RNA-catalyzed synthesis of 4-thiouridine-
5?-phosphate from 4-thiouracil and 5-phosphoribosyl-1-
tion that, according to the RNA-first scenario, might have
evolved when the availability of prebiotic nucleotides de-
ning of the evolution of the RNA World only one function
was essential, namely catalysis of RNA replication. Re-
markable progress has been made in this area.
Bartel and his coworkers have been able to evolve a
catalytic RNA that has many of the essential properties of
an RNA polymerase (Johnston et al., 2001). This rather
polymerase. Redrawn with modification from Robertson et al.
complicated molecule (Figure 8), when presented with an
arbitrary, single-stranded RNA template, an RNA primer,
and a mixture of the four nucleoside triphosphates, will
synthesize the complement of the template. At present,
templates that contain more than 14 residues cannot be
copied effectively, but it seems likely that further in vitro
evolution will lead to the isolation of a ribozyme capable
of copying much longer RNAs. The isolation of such a
ribozyme would constitute a major advance in attempts
to understand the origin of life. However, the formidable
problem of separating the double-stranded product of the
would remain to be solved.
THE MOLECULAR BIOLOGISTS?DREAM
The RNA-first scenario for the origin of the RNA World
that we have described as the ‘Molecular Biologists’
Dream (Joyce & Orgel, 1999) can be strung together from
optimistic extrapolations of the various achievements of
above. First we suppose that nucleoside bases and sug-
ars were formed by prebiotic reactions on the primitive
Earth and/or brought to the Earth in meteorites, comets,
etc. Next, nucleotides were formed from prebiotic bases,
sugars, and inorganic phosphates or polyphosphates, and
they accumulated in an adequately pure state in some
special little “pool.” A mineral catalyst at the bottom of
the pool—for example, montmorillonite—then catalyzed
the formation of long single-stranded polynucleotides,
some of which were then converted to complementary
114L. E. ORGEL
double strands by template-directed synthesis. In this way
a library of double-stranded RNAs accumulated on the
We suppose that among the double-stranded RNAs
there was at least one that on melting yielded a (single-
stranded) ribozyme capable of copying itself and its com-
plement. Copying the complement would then have pro-
duced a second ribozyme molecule, and then repeated
copying of the ribozyme and its complement would have
lead to an exponentially growing population. In this sce-
gested that all life is descended from one or a few simple
organisms that evolved on the Earth long ago. According
to the more radical scenario of the Molecular Biologists’
Dream, the whole biosphere descends from one or a few
replicating polynucleotides that formed on the primitive
Earth about four billion years ago. Of course, there are
still a few problems in prebiotic chemistry that must be
solved before the Dream can be turned into a convincing
theory! In addition, a plausible prebiotic mechanism for
keeping together ribozymes and the products of their ac-
tivity, for example, enclosure within a membrane, must be
demonstrated (see below).
Although tentative solutions to most of the problems that
have been offered, nearly every one of them, as we have
seen, is problematic. The synthesis of ribose leads to a
complex mixture of sugars, with ribose as only a mi-
nor constituent under most conditions. The synthesis of
purines nucleosides directly from ribose and a base is
inefficient, while the only available prebiotic synthesis
of the pyrimidine nucleosides starts from arabinose-3-
phosphate, a marginally prebiotic molecule. Phosphory-
lation of nucleosides leads to a complex mixture of iso-
meric mono- and polyphosphates, while polymerization
even of pure nucleoside-5?-phosphates leads to a product
with mixed phosphodiester linkages. The phosphorimida-
zolides used in most studies of both template-directed and
of template-independent synthesis are unlikely to be pre-
biotic molecules. It is possible that all of these, and many
other difficulties will one day be overcome and that a con-
However, many researchers in the field, myself included,
think that this is unlikely and that there must be a different
kind of solution to the problem of the origin of the RNA
Graham Cairns-Smith was the first person to empha-
size the complexity of RNA and how improbable it is
that RNA could have formed de novo on the primitive
Earth. He suggested that the first system on the primitive
Earth that was capable of evolving by natural selection
was a self-reproducing clay (Cairns-Smith, 1982), but he
also mentioned the possibility of a genetic system based
on a linear polymer simpler than RNA (Cairns-Smith &
Davies, 1977). He thought that the original mineral (or
simple organic) genetic system “invented” RNA and was
subsequently displaced by it, and he introduced the term
netic material by another. There is as yet no experimental
mineral. The possibility of genetic takeover from a sim-
issue in discussions of the origin of the RNA World.
The idea that some simpler genetic system preceded
RNA opens Pandora’s box. There is very little to con-
strain the type of molecule involved or the environment in
which it first functioned. Perhaps RNA is the “invention”
of a completely different earlier world that operated under
Perhaps the original genetic system was inorganic or in-
cluded an essential inorganic component. Perhaps repli-
cation could initially take place only on the surface of
some particular mineral or only at a particular kind of
complex defect site on a particular mineral. Perhaps—
experimental work has not begun to explore these pos-
sibilities. Instead, a few polymers fairly closely related to
that could have acted as precursors of RNA. Here, we can
only review briefly some of the more important results
insofar as they are related to prebiotic chemistry. Many
nucleic-acid analogs have been investigated as potential
antisense inhibitors of protein synthesis. Since they form
stable heteroduplexes with RNA it seems likely that two
complementary strands of almost any one of them would
form a more or less stable double helix, but this has rarely
been studied experimentally.
The most extensive body of experimental work is that
of polymers in which the 3?-5?-linked ribose–phosphate
backbone of RNA (Figure 9a) was replaced by some other
sugar–phosphate backbone. Here, we can only review
briefly some of their more striking results. In early experi-
stable double helices held together by standard Watson-
Crick base pairing (Groebke et al., 1998). These dou-
ble helices have a very different structure from those of
double-stranded RNA, since they turn about the helix axis
chimeric double helices between a strand of homo-DNA
and a strand of RNA. The discovery of a structurally regu-
lar isomer of RNA, pyranosyl RNA, or pRNA (Figure 9c)
was very surprising. If the furanose isomers of the nu-
cleotides are replaced by their pyranose equivalents, it is
PREBIOTIC CHEMISTRY AND ORIGIN OF RNA WORLD 115
The structures of single strands of (a) RNA, (b) homo-DNA, (c) p-RNA, (d) TNA, and (e) PNA.
still possible to form a double-stranded, base-paired dou-
ble helix (Pitsch et al., 2003). Again, the structure of the
helix is incompatible with that of RNA.
The analogs discussed above are different from but
not simpler than RNA. More recently it has been shown
that threose-based nucleotides are also capable of forming
base-paired double helices (Schoning et al., 2000). Thre-
ose nucleic acids (TNAs), unlike the nucleic acid analogs
discussed above, form stable heteroduplexes with RNA.
This is surprising, because the number of atoms in the
backbone-repeat of TNA is five (Figure 9d) rather than
the six (or seven) that occur in RNA and in all previously
imally extending the threose–phosphate moiety it can be
stretched out to roughly match the repeat length in RNA.
TNA is the first base-pairing analog of RNA that is sim-
pler than RNA in the sense that it is based on monomers,
which can be synthesized more easily than the standard
nucleotides. While it is not suggested that TNA was a pre-
cursor of RNA on the primitive Earth, the results obtained
with TNA are encouraging because they suggest that even
that some of them may be able to adopt structures very
similar to that of RNA.
Peptide nucleic acids (PNAs) are another extensively
studied group of antisense nucleic acid analogs (Figure
9e) that have been discussed in the context of prebiotic
chemistry (Egholm et al., 1992). The monomers contain
the usual nucleic acid bases but are achiral and free of
phosphate. Pairs of complementary PNA oligomers form
Watson-Crick base-paired double helices in aqueous so-
lution. PNAs, besides forming these double helices, also
form very stable chimeric double helices with nucleic
acids (Egholm et al., 1993). Solutions of double-helical
homoduplexes formed by underivatized PNA must con-
tain equal proportions of left- and right-handed helices
since PNA is achiral. However, the covalent attachment of
to the termini of one of the strands of a PNA double helix
has been shown to bias the ratio of the concentrations of
the two mirror-image helices.
PNA has been investigated as a model of a potential
genetic material that is free of phosphate. PNA templates
catalyze the oligomerization of activated nucleotides and
the ligation of complementary oligonucleotides (Schmidt
an intrinsically achiral PNA double-helix (Kozlov et al.,
2000a). These experiments suggest one way in which ge-
netic takeover might occur. It has been claimed that PNAs
are plausible prebiotic molecules and that PNA may have
been a precursor of RNA (Miller, 1997), but this seems
doubtful because no straightforward prebiotic synthesis
of the PNA monomers has been reported, and the activa-
tion of the carboxyl group of a PNA monomer would be
expected to lead to rapid cyclization.
Diederichsen and his coworkers have described a novel
pairing structure based on an alternating sequence of D-
and L-amino acids (Figure 10). Sequences based on
116 L. E. ORGEL
B designates a standard nucleotide base. (b) The proposed base-
with slight modifications from, Diederichsen, U. (1997). Alanyl
PNA: evidence for linear band structures based on guanine-
cytosine base pairs, Angew Chem Intl Ed Engl 36(17):1886.)
α-amino acids of a single chirality cannot form pairing
structures because of geometric constraints, but peptides
in which the two enantiomers alternate are ideally suited
for interchain interactions. Taking advantage of this ob-
servation, Diederichsen and his coworkers have made a
detailed study of ANAs, alternating peptides based on rel-
atively simple α-amino acids that incorporate one of the
standard nucleic-acid bases (Figure 10a). They find that
complementary sequences form very stable antiparallel
double helices (Figure 10b; Diederichsen, 1996).
Diederichsen does not seem to have discussed ANAs in
to see how a single enantiomer of any chiral organic com-
pound could have formed on the primitive Earth, except
perhaps in some chiral microenvironment. The copying of
an alternating polymer is, in principle, no more difficult
than the copying of a homochiral polymer if the substrates
are racemic. Both are subject to inhibition through the in-
sertion of the “incorrect” enantiomer in much the same
way. If a substantial proportion of the substrate monomers
plate with a racemic substrate is advantageous. The incor-
a homochiral template biases the composition of the sys-
tem against further synthesis, but no such difficulty arises
in the case of an alternating template.
All of the experimental studies described above envis-
age genetic materials that incorporate the standard nu-
mer to a later one is usually assumed to have taken place
with conservation of sequence information, presumably
via chimeric intermediates. There is a completely differ-
structure completely unrelated to RNA “invented” RNA.
In this scenario it must be assumed that nucleotides or
closely related molecules were synthesized and polymer-
ized by the earlier system for some nongenetic function,
based on very simple monomers.
One of the outstanding challenges in the field is, there-
fore, to design pairing structures based on monomers that
are much more easily synthesized than the standard nu-
pends on nonspecific stacking interactions between bases
as well as on specific hydrogen-bonding interactions. It
is not difficult to imagine simple informational polymers
The specificity of interaction might depend on charge—
for example, with aspartic acid pairing with arginine—or
on size—for example, with asparagine pairing with glu-
ordination to shared metal ions are also attractive mech-
anisms for associating two chains in a sequence-specific
manner. However, it is not easy to suggest simple, pre-
biotically plausible monomers that provide for stacking
as well as for these potential interchain interactions. The
possibility that simple, stable pairing structures can exist
without the need for stacking, perhaps on the surface of a
mineral, needs to be explored.
In summary, a number of polymers that form double-
been reported, but none of them is very much simpler in
structure than RNA. The idea that RNA was “invented”
by a simpler genetic system is now a popular one, but no
convincing precursor system has been described.
PREBIOTIC CHEMISTRY AND ORIGIN OF RNA WORLD 117
some kind of single-celled organism, a packet of proteins
and nucleic acids, cofactors and so on, enclosed in a rela-
of the RNA World enclosed in a similar membrane from
the very beginning, or was the cell membrane a late “in-
vention” of the RNA World or an early “invention” of the
up the arguments in favor of compartmentalization. There
are two important and distinct aspects of this generaliza-
the other concerning macromolecules. Evolution is at a
severe disadvantage in enhancing the performance of an
by the producing macromolecules (or their near relatives).
There is no place in evolution for charity, and to synthe-
size a useful molecule and hand it over to an unrelated
competitor would constitute molecular charity. However,
it must be recognized that molecular isolationism is likely
to be a double-edged weapon as far as small molecules
are concerned, for it inhibits the acceptance of any useful
small molecules that are freely available in the environ-
ment. When we consider genetic and functional macro-
molecules with defined sequences, whether ribozymes or
protein enzymes, rather than small-molecule metabolites,
the situation is simpler. Since these macromolecules can
never be supplied as free goods from outside, it almost
always pays to keep them together, whether or not they
have free access to the external environment.
The arguments presented so far suggest that it is always
advantageous to keep together macromolecules that are
involved in each others’ synthesis, whether they are ge-
netic or metabolic, but that compartmentalization within
an impermeable membrane will only be useful when an
organism has reached the stage of synthesizing for itself
some or most of its metabolites. Intuitively, one might ex-
pect, therefore, that compartmentalization within a some-
a more impermeable membrane would develop gradually
as “enzymatic” synthesis took over from the use of pre-
biotically available small organic molecules. This seems
to be a very reasonable scenario, but it clearly isn’t the
only scenario and it does assume the availability of suit-
evolution, it was only necessary to keep macromolecules
together and it was not necessary to contain small organic
molecules, there are alternatives to encapsulation.
As we have seen, the easiest modification of an imper-
meable lipid membrane that would meet the requirements
membrane with holes or pores large enough to pass small
molecules such as nucleotides but too small to pass RNA
polymers. The existence of such vesicles in a prebiotic en-
vironment may be possible (see below). A less-organized
structure, a coacervate or organic colloid to which RNA
easily. In the latter case a strong interaction would be
needed to keep the RNA and the organic colloid together.
Rocks and minerals, which must have been ubiquitous on
There are strong theoretical arguments (Orgel, 1998) and
some experimental evidence (Hill et al., 1998) showing
that sufficiently long negatively charged oligomers adsorb
almost irreversibly on anion-exchanging minerals. Since
many mineral surfaces (Schwartz & Orgel, 1984), mineral
particles might support replication until they became sat-
urated with the descendents of a single ancestral RNA
molecule. Further expansion would depend on the occa-
sional colonization of a vacant mineral particle.
I do not believe that there is at present enough evidence
to justify a choice among these possibilities. A scenario
replicators were “naked genes” adsorbed on the surface of
mineral particles, and in which impermeable membrane
caps were “invented” by the genetic system as it became
enabled by the development of a closed spherical mem-
brane would occur at a relatively late stage in evolution.
However, most published experimental studies concerned
with encapsulation assume that encapsulation in a more
or less impermeable membrane was important from the
David Deamer and his coworkers (Deamer et al., 2002)
have explored the possibility of forming membrane struc-
paper they described experiments in which they extracted
separated the mixture of extracted organic components by
paper chromatography, and then examined the structures
formed when the separated components were mixed with
water. Many different structures were observed, most in-
terestingly the membranous vesicles obtained with some
but not all of the separated components. In a few cases,
vesicles were observed that seemed to consist of an in-
terior compartment surrounded by a double membrane;
fluorescent molecules could be trapped in the interior of
the structure (Deamer & Pashley, 1989). More recently,
very similar results have been obtained with amphiphylic
material obtained in simulations of organic synthesis in
118 L. E. ORGEL
2001). These studies suggest that the prebiotic formation
of vesicles may be possible.
cerns chemistry that occurs within vesicles formed from
simple, but not prebiotically synthesized, organic mol-
ecules. Not surprisingly, protein enzymes function more
or less normally within large-enough bilayer vesicles, and
the enzymatic synthesis of nucleic acids within vesicles
has been described (Chakrabarti et al., 1994; Oberholzer
of the clay mineral montmorillonite catalyze the forma-
tion of closed vesicles from micelles composed of simple
aliphatic carboxylic acids and that particles of the clay
become encapsulated within the vesicles (Hanczyc et al.,
2003). Since montmorillonite is an excellent catalyst for
the oligomerization of a number of activated nucleotides
this might point to a route to a nucleic-acid–synthesizing
system enclosed within a vesicle. It remains to be shown
that montmorillonite catalysis of polynucleotide synthesis
can occur within the vesicles and lead to the formation of
The generation of an autonomous self-replicating sys-
tem of RNA within a lipid vesicle requires the vesicle, as
well as its contents, to be capable of exponential growth.
In one series of experiments it was shown that vesicles
composed of caprylic acid were effective catalysts for the
hydrolysis of ethyl caprylate. The newly formed caprylic
acid never appeared in solution but was incorporated di-
rectly into the vesicle walls, causing the vesicles to grow
and ultimately to divide (Bachmann et al., 1992). Similar
behavior was observed with suspensions of the insoluble
In summary, it seems almost certain that RNA organ-
isms as complicated as those that “invented” protein syn-
thesis must have been enclosed in relatively impermeable
membranes. However, it is not clear whether the very first
attached to organic colloids, or adsorbed on mineral sur-
faces. Perhaps they were adsorbed to mineral particles
within lipid membranes (Hanczyc et al., 2003).
This review has focussed on experimental work designed
to explain how RNA, directly or indirectly, could have
appeared on the primitive Earth. It has catalogued the
achievements in each pertinent area of research and also
emphasized the gaps in our understanding. A more global
summary may be useful.
The most impressive advances in the past decade or
so have come in the field of RNA selection. Enough is
already known to suggest that each of the steps needed
to evolve from a library of randomly sequenced double-
stranded RNAs to a self-sustaining RNA organism can
be demonstrated in laboratory experiments. An advanced
RNA organism would presumably need to be enclosed in
a membrane. Attempts to develop prebiotically plausible
promising. There are already hints that a membrane capa-
along with them could be put together from molecules as
simple as monocarboxylic acid.
Considerable progress has also been made in under-
standing how a library of random sequence double-
stranded RNA molecules might have arisen from a pool
of activated nucleotides. The catalysis of the formation of
long RNA strands by the clay mineral montmorillonite is
remarkable. It suggests that mineral catalysis may provide
the solution to many problems. Nonenzymatic, template-
directed copying of single-stranded RNA to generate dou-
ble strands has been explored in detail. While exponential
replication cannot be achieved using presently available
methods, a wide range of single-stranded RNAs can be
converted to double strands.
The prebiotic synthesis of nucleotides in a sufficiently
pure state to support RNA synthesis cannot be achieved
chemistry, but the reactions were inefficient, nonspecific,
specific prebiotic syntheses, but formidable difficulties re-
main. This has led some researchers to explore a major
new approach to the problem of molecular evolution—
the search for polymers that could function as alternative
It is now clear that there are numerous double-stranded
tion of these structures is a novel and fruitful branch of or-
ganic chemistry (and Astrobiology) regardless of whether
also seems possible that there are pairing structures much
simpler than RNA in the sense that their monomeric com-
ponents can be synthesized much more easily than nu-
as yet unknown.
Prebiotic chemistry remains so diverse a field that it is by
no means clear where the next important advances will
occur. It seems likely that adsorption on and catalysis by
increasing efforts to study heterogeneous reactions are to
PREBIOTIC CHEMISTRY AND ORIGIN OF RNA WORLD119
and structure, combinatorial methods will be required. It
will be necessary to study each potentially important re-
action in parallel on tens or hundreds of different mineral
samples. Whether or not this approach will lead to the dis-
covery of a plausible prebiotic route to the nucleotides,
as the believers in the Molecular Biologists’ Dream hope,
remains to be seen, but it is likely that many novel mineral
catalysts will be discovered in this way.
The search for pairing structures based on monomeric
nucleotides and, hopefully, that polymerize more readily
has just begun. No doubt it will remain an active and ex-
panding field. Whether or not it leads to a plausible sce-
nario for a simple pre-RNA World, as advocates of “RNA
the problem of the origin of the RNA World is far from
I thank Gerald Joyce for many useful comments on an
earlier version of this review and Kathleen Murray for
Acevedo, O.L. and Orgel, L.E. 1987. Non-enzymatic transcription of
an oligodeoxynucleotide 14 residues long. J Mol Biol 197:187–193.
Anders, E. 1989. Pre-biotic organic matter from comets and asteroids.
Bachmann, P.A., Luisi, P.L., and Lang, J. 1992. Autocatalytic self-
replicating micelles as models for prebiotic structures. Nature
Beck, A., Lohrmann, R., and Orgel, L.E. 1967. Phosphorylation with
inorganic phosphates at moderate temperatures. Science 157:952.
Blochl, E., Keller, M., Wachtershauser, G., and Stetter, K.O. 1992.
Reactions depending on iron sulfide and linking geochemistry with
biochemistry. Proc Natl Acad Sci USA 89:8117–8120.
Botta, O. and Bada, J.L. 2002. Extraterrestrial organic compounds in
meteorites. Surveys Geophys 23:411–467.
Breslow, R. 1959. On the mechanism of the formose reaction. Tetrahe-
dron Lett 21:22–26.
Bridson, P.K. and Orgel, L.E. 1980. Catalysis of accurate poly(C)-
directed synthesis of 3?-5?-linked oligoguanylates by Zn2+. J Mol
Butlerow, A. 1861. Formation synthetique d’une substance sucree.
Compt Rend Acad Sci 53:145–147.
Cairns-Smith, A.G. 1982. Genetic Takeover and the Mineral Origin of
Life. Cambridge: Cambridge University Press.
Cairns-Smith, A.G. and Davies, C.J. 1977. The design of novel repli-
cating polymers. In Encyclopaedia of Ignorance. Duncan, R. and
Weston-Smith, M., Eds., New York: Pergamon Press.
Chakrabarti, A.C., Breaker, R.R., Joyce, G.F., and Deamer, D.W. 1994.
Production of RNA by a polymerase protein encapsulated within
phospholipid vesicles. J Mol Evol 39:555–559.
Cheng, C., Fan, C., Wan, R., Tong, C., Miao, Z., Chen, J., and Zhao,
Y. 2002. Phosphorylation of adenosine with trimetaphosphate under
simulated prebiotic conditions. Orig Life Evol Biosph 32:219–224.
Chyba, C. and Sagan, C. 1992. Endogenous production, exogenous de-
for the origins of life. Nature 355:125–132.
Cody, G.D., Boctor, N.Z., Filley, T.R., Hazen, R.M., Scott, J.H.,
Sharma, A., and Yoder, H.S., Jr. 2000. Primordial carbonylated iron-
sulfur compounds and the synthesis of pyruvate. Science 289:1337–
Cooper, G., Kimmich, N., Belisle, W., Sarinana, J., Brabham, J., and
organic compounds for the early Earth. Nature 414:879–883.
Crick, F.H.C. 1968. The origin of the genetic code. J Mol Biol 38:367–
Cronin, J.R. and Chang, S. 1993. Organic matter in meteorites: molec-
ular and isotopic analysis of the Murchison meteorite. In The Chem-
istry of Life’s Origin, pp. 209–258. Greenberg, J.M., Mendoza-
Gomez, C.X., and Pirronello, V., Eds., The Netherlands: Kluwer
Deamer, D., Dworkin, J.P., Sandford, S.A., Bernstein, M.P., and
Allamandola, L.J. 2002. The first cell membranes. Astrobiology
Deamer, D.W. and Pashley, R.M. 1989. Amphiphilic components of
brane formation. Orig Life Evol Biosph 19:21–38.
Decker, P., Schweer, H., and Pohlmann, R. 1982. Identification of
formose sugars, presumable prebiotic metabolites, using capillary
gas chromatography/gas chromatography-mass spectrometry of n-
butoxime trifluoroacetates on OV-225. J Chromatog 244:281–291.
Diederichsen, U. 1996. Pairing properties of alanyl peptide nucleic
acids containing an amino acid backbone with alternating configu-
ration. Angew Chem Int Ed Engl 35:445–448.
R.A., Geilmann, H., Appel, C., Robl, C., and Weigand, W. 2003. A
possible prebiotic formation of ammonia from dinitrogen on iron
sulfide surfaces. Angew Chem Int Ed Engl 42:1540–1543.
assembling amphiphilic molecules: synthesis in simulated interstel-
lar/precometary ices. Proc Natl Acad Sci USA 98:815–819.
Egholm, M., Buchardt, O., Christensen, L., Behrens, C., Freier, S.M.,
Driver, D.A., Berg, R.H., Kim, S.K., Norden, B., and Nielsen, P.E.
1993. PNA hybridizes to complementary oligonucleotides obeying
the Watson-Crick hydrogen-bonding rules. Nature 365:566–568.
Ertem, G. and Ferris, J.P. 1997. Template-directed synthesis using the
heterogeneous templates produced by montmorillonite catalysis. A
possible bridge between the prebiotic and RNA worlds. J Am Chem
Ertem, G. and Ferris, J.P. 1998. Formation of RNA oligomers on mont-
morillonite: site of catalysis. Orig Life Evol Biosph 28:485–499.
Eschenmoser, A. 1999. Chemical etiology of nucleic acid structure.
120 L. E. ORGEL
Etaix, E. and Orgel, L.E. 1978. Phosphorylation of nucleosides in
aqueous solution using trimetaphosphate: formation of nucleoside
triphosphates. J Carbohydrates-Nucleosides-Nucleotides 5:91–110.
oligomers. Adv Space Res in press.
Ferris, J.P. and Orgel, L.E. 1965. Aminomalononitrile and 4-amino-
5-cyanoimidazole in hydrogen cyanide polymerization and adenine
synthesis. J Am Chem Soc 87:4976–4977.
Ferris, J.P. and Orgel, L.E. 1966a. Studies on prebiotic synthesis. I.
Aminomalononitrile and 4-amino-5-cyanoimidazole. J Am Chem
Ferris, J.P. and Orgel, L.E. 1966b. An unusual photochemical re-
arrangement in the synthesis of adenine from hydrogen cyanide.
J Amer Chem Soc 88:1074.
Ferris, J.P., Sanchez, R.A., and Orgel, L.E. 1968. Studies in prebi-
otic synthesis. 3. Synthesis of pyrimidines from cyanoacetylene and
cyanate. J Mol Biol 33:693–704.
ical evolution. 18. Synthesis of pyrimidines from guanidine and
cyanoacetaldehyde. J Mol Evol 3:301–309.
Fuller, W.D., Sanchez, R.A., and Orgel, L.E. 1972. Studies in prebiotic
synthesis: VII. Solid-state synthesis of purine nucleosides. J Mol
Gabel, N.W. and Ponnamperuma, C. 1967. Model for origin of
monosaccharides. Nature 216:453–455.
ed. Cold Springs Harbor, NY: Cold Springs Harbor Press.
Gilbert, W. 1986. The RNA World. Nature 319:618.
Goo, J. 1961. Comets and the formation of biochemical compounds on
the primitive Earth. Nature 190:389–390.
Groebke, K., Hunziker, J., Fraser, W., Peng, L., Diederichsen, U.,
Zimmermann, K., Holzner, A., Leumann, C., and Eschenmoser,
A. 1998. Why pentose- and not hexose-nucleic acids? Part V.
Purine-purine pairing in homo-DNA: Guanine, isoguanine, 2,6-
diaminopurine, and xanthine. Helv Chim Acta 81:375–474.
Guerrier-Takada, C., Gardiner, K., Marsh, T., Pace, N., and Altman, S.
1983. The RNA moiety of ribonuclease P is the catalytic subunit of
the enzyme. Cell 35:849–857.
Halmann, M., Sanchez, R.A., and Orgel, L.E. 1969. Phosphorylation
of D-ribose in aqueous solution. J Org Chem 34:3702–3703.
models of primitive cellular compartments: encapsulation, growth,
and division. Science 302:618–622.
Handschuh, G.J., Lohrmann, R., and Orgel, L.E. 1973. The effect of
Mg2+and Ca2+on urea-catalyzed phosphorylation reactions. J Mol
and ammonium formate. Orig Life Evol Biosph 32:99–102.
Hill, A.R., Jr., Bohler, C., and Orgel, L.E. 1998. Polymerization on the
rocks: negatively-charged alpha-amino acids. Orig Life Evol Biosph
Hill, A.R., Jr., Orgel, L.E., and Wu, T. 1993. The limits
of template-directed synthesis with nucleoside-5?-phosphoro(2-
methyl)imidazolides. Orig Life Evol Biosph 23:285–290.
and nucleotides. II. J Mol Biol 16:415–439.
Huang, W. and Ferris, J.P. 2003. Synthesis of 35-40 mers of RNA
world. Chem Commun (Camb) 12:1458–1459.
fixation on (Fe,Ni)S under primordial conditions. Science 276:245–
acids with CO on (Ni,Fe)S surfaces: implications for the origin of
life. Science 281:670–672.
Ingar, A.A., Luke, R.W., Hayter, B.R., and Sutherland, J.D. 2003. Syn-
thesis of cytidine ribonucleotides by stepwise assembly of the hete-
rocycle on a sugar phosphate. Chembiochem 4:504–507.
Inoue, T. and Orgel, L.E. 1981. Substituent control of the poly(C)-
directed oligomerization of guanosine 5-phosphoroimidazolide.
J Amer Chem Soc 103:7666–7667.
Johnston, W.K., Unrau, P.J., Lawrence, M.S., Glasner, M.E., and
general RNA-templated primer extension. Science 292:1319–1325.
Joshi, P.C., Pitsch, S., and Ferris, J.P. 2000. Homochiral selection in
the montmorillonite-catalyzed and uncatalyzed prebiotic synthesis
of RNA. Chem Com 24:2497–2498.
Joyce, G.F. 1987. Nonenzymatic template-directed synthesis of in-
formational macromolecules. Cold Spring Harb Symp Quant Biol
Joyce, G.F. 2004. Directed evolution of nucleic acid enzymes. Ann Rev
of Biochem 73:791–836.
R.F., Cech, T.R., and Atkins, J.F., Eds., Cold Spring Harbor: Cold
Spring Harbor Press.
Joyce, G.F., Visser, G.M., van Boeckel, C.A., van Boom, J.H., Orgel,
synthesis of oligo(G). Nature 310:602–604.
Kanavarioti, A., Monnard, P.A., and Deamer, D.W. 2001. Eutectic
phases in ice facilitate nonenzymatic nucleic acid synthesis. Astro-
Kasting, J.F. and Brown, L.L. 1998. The early atmosphere as a source
Brack, A., Ed., New York: Cambridge University Press.
Kieboom, A. and VanBekkum, H. 1984. Aspects of the chemical con-
version of glucose. Rec Tr Chim Pays-Bas 103:1–12.
Koppitz, M., Nielsen, P.E., and Orgel, L.E. 1998. Formation of
oligonucleotide-PNA-chimeras by template-directed ligation. J Am
Chem Soc 120:4563–4569.
Kozlov, I.A. and Orgel, L.E. 2000. Nonenzymatic template-directed
synthesis of RNA from monomers (translated from Molekulamaya
Biologlya). Molecular Biology 34:781–789.
Kozlov, I.A., Orgel, L.E., and Nielsen, P.E. 2000a. Remote enantio
selection transmitted by an achiral PNA backbone. Angew Chem Int
Kozlov, I.A., Politis, P.K., Pitsch, S., Herdewijn, P., and Orgel, L.E.
1999a. A highly enantio-selective hexitol nucleic acid template for
nonenzymatic oligoguanylate synthesis. J Am Chem Soc 121:1108–
Kozlov, I.A., Politis, P.K., Van Aerschot, A., Busson, R., Herdewijn, P.,
and Orgel, L.E. 1999b. Nonenzymatic synthesis of RNA and DNA
oligomers on hexitol nucleic acid templates: the importance of the
A structure. J Am Chem Soc 121:2653–2656.
PREBIOTIC CHEMISTRY AND ORIGIN OF RNA WORLD121
Kozlov, I.A., Zielinski, M., Allart, B., Kerremans, L., Van Aerschot,
A., Busson, R., Herdewijn, P., and Orgel, L.E. 2000b. Nonenzymatic
template-directed reactions on altritol oligomers, preorganized ana-
logues of oligonucleotides. Chemistry 6:151–155.
tive α-phosphorylation of aldoses in aqueous solution. Angew Chem
Int Ed Engl 39:2281–2285.
Cech, T.R. 1982. Self-splicing RNA: autoexcision and autocycliza-
tion of the ribosomal RNA intervening sequence of Tetrahymena.
Kurz, M., Gobel, K., Hartel, C., and Gobel, M. 1997. Nonenzymatic
pression of the self-pairing of guanosine. Angew Chem Int Ed Engl
Kurz, M., Gobel, K., Hartel, C., and Gobel, M. 1998. Acridine-labeled
Helv Chim Acta 81:1156–1180.
Lohrmann, R. 1977. Formation of nucleoside 5?-phosphoramidates un-
der potentially prebiological conditions. J Mol Evol 10:137–154.
Lohrmann, R. and Orgel, L.E. 1968. Prebiotic synthesis: phosphoryla-
tion in aqueous solution. Science 161:64–66.
as prebiotic phosphorylating agents. Science 171:490–494.
Lundstrom, F.O. and Whittaker, C.W. 1937. Chemical reactions in fer-
tilizer mixtures. Effect of ammoniation on urea component of super-
phosphate mixtures. Ind Eng Chem 29:61–68.
earth conditions. Science 117:528–529.
Miller, S.L. 1957. The mechanism of synthesis of amino acids by elec-
tric discharges. Biochim Biophys Acta 23:480–489.
Miller, S.L. 1997. Peptide nucleic acids and prebiotic chemistry. Nat
Struct Biol 4:167–169.
Miyakawa, S., Cleaves, H.J., and Miller, S.L. 2002a. The cold origin of
life: A. Implications based on the hydrolytic stabilities of hydrogen
cyanide and formamide. Orig Life Evol Biosph 32:195–208.
Miyakawa, S., Cleaves, H.J., and Miller, S.L. 2002b. The cold origin
of life: B. Implications based on pyrimidines and purines produced
from frozen ammonium cyanide solutions. Orig Life Evol Biosph
Abiotic synthesis of guanine with high-temperature plasma. Orig
Life Evol Biosph 30:557–566.
Mizuno, T. and Weiss, A.H. 1974. Synthesis and utilization of formose
sugars. In: Advances in Carbohydrate Chemistry and Biochemistry,
v. 29, pp. 173–227. Tipson, R.W., and Horton, D., Eds., New York
London: Academic Press.
polymerization of activated ribonucleotide mixtures yields quasi-
equimolar incorporation of purine and pyrimidine nucleobases.
J Am Chem Soc 125:13734–13740.
Moravek, J. 1967. Formation of oligonucleotides during heating of a
mixture of uridine 2?(3?)-phosphate and uridine. Tetrahedron Lett
Mueller, D., Pitsch, S., Kittaka, A., Wagner, E., Wintner, C.E.,
and Eschenmoser, A. 1990. Chemistry of alpha aminonitriles. Al-
domerization of glycolaldehyde phosphate to racemic hexose 2,4,6-
triphosphates and (in presence of formaldehyde) racemic pentose
2,4-diphosphate are the main reaction products. Helvetica Chimica
Naylor, R. and Gilham, P.T. 1966. Studies on some interactions and
reactions of oligonucleotides in aqueous solution. Biochemistry
Nelson, K.E., Robertson, M.P., Levy, M., and Miller, S.L. 2001. Con-
centration by evaporation and the prebiotic synthesis of cytosine.
Orig Life Evol Biosph 31:221–229.
Ninio, J. and Orgel, L.E. 1978. Heteropolynucleotides as templates for
nonenzymatic polymerizations. J Mol Evol 12:91–99.
Oberholzer, T., Albrizio, M., and Luisi, P.L. 1995. Polymerase chain
reaction in liposomes. Chem Biol 2:677–682.
of life. In Prebiotic and Biochemical Evolution, pp. 89–94. Kimball,
A., and Oro, J., Eds., Amsterdam: North-Holland Publishing
Orgel, L.E. 1968. Evolution of the genetic apparatus. J Mol Biol
Orgel, L.E. 1992. Molecular replication. Nature 358:203–209.
Orig Life Evol Biosph 28:227–234.
Orgel, L.E. 2000. Self-organizing biochemical cycles. Proc Natl Acad
Sci USA 97:12503–12507.
Orgel, L.E. 2002. Is cyanoacetylene prebiotic? Orig Life Evol Biosph
Orgel, L.E. 2003. Some consequences of the RNA world hypothesis.
Orig Life Evol Biosph 33:211–218.
istry. Orig Life Evol Biosph in press.
Oro, J. 1961a. Mechanism of synthesis of adenine from hydrogen
Oro, J. 1961b. Comets and the formation of biochemical compounds
on the primitive Earth. Nature 190:389–390.
Oro, J. and Kimball, A. 1960. Synthesis of adenine from ammonium
cyanide. Biochem Biophys Res Commun 2:407–412.
Oro, J. and Kimball, A.P. 1961. Synthesis of purines under possible
primitive earth conditions. I. Adenine from hydrogen cyanide. Arch
Biochem Biophys 94:217–227.
Oro, J. and Kimball, A.P. 1962. Synthesis of purines under possible
primitive earth conditions. II. Purine intermediates from hydrogen
cyanide. Arch Biochem Biophys 96:293–313.
Osterberg, R. and Orgel, L.E. 1972. Polyphosphate and trimetaphos-
phate formation under potentially prebiotic conditions. J Mol Evol
Osterberg, R., Orgel, L.E., and Lohrmann, R. 1973. Further studies of
urea-catalyzed phosphorylation reactions. J Mol Evol 2:231–234.
Peyser, J.R. and Ferris, J.P. 2001. The rates of hydrolysis of thymidyl-
3?,5?-thymidine-H-phosphonate: the possible role of nucleic acids
linked by diesters of phosphorous acid in the origins of life. Orig
Life Evol Biosph 31:363–380.
Pitsch, S., Eschenmoser, A., Gedulin, B., Hui, S., and Arrhenius, G.
Bolli, M., et al. 2003. Pentopyranosyl oligonucleotide systems, 9th
122L. E. ORGEL
communication. The beta-D-ribopyranosyl-(4?-2?)-oligonucleotide
system (“Pyranosyl-RNA”): synthesis and resume of base-pairing
properties. Helv Chim Acta 86:4270–4363.
Prabahar, K.J. and Ferris, J.P. 1997. Adenine derivatives as phosphate-
activating groups for the regioselective formation of 3?,5?-linked
oligoadenylates on montmorillonite: possible phosphate-activating
Prudent, J.R., Uno, T., and Schultz, P.G. 1994. Expanding the scope of
RNA catalysis. Science 264:1924–1927.
Puglisi, J.D. and Williamson, J.R. 1999. RNA interaction with small
ligands and peptides. In The RNA World, 2nd ed., pp. 403-425.
Gesteland, R.F., Cech, T.R., and Atkins, J.F., Eds., Cold Spring Har-
bor: Cold Spring Harbor Laboratory Press.
Reid, C. and Orgel, L.E. 1967. Synthesis of sugars in potentially pre-
biotic conditions. Nature 216:455.
Reimann, R. and Zubay, G. 1999. Nucleoside phosphorylation: a fea-
sible step in the prebiotic pathway to RNA. Orig Life Evol Biosph
Ricardo, A., Carrigan, M.A., Olcott, A.N., and Benner, S.A. 2004.
Borate minerals stabilize ribose. Science 303:196.
of cytosine and uracil. Nature 375:772–774.
Robertson, M.P. and Miller, S.L. 1995b. Correction. an Efficient Prebi-
otic Synthesis of Cytosine and Uracil. Nature 377:257.
Rohatgi, R., Bartel, D.P., and Szostak, J.W. 1996a. Kinetic and mech-
anistic analysis of nonenzymatic, template-directed oligoribonu-
cleotide ligation. J Am Chem Soc 118:3332–3339.
Rohatgi, R., Bartel, D.P., and Szostak, J.W. 1996b. Nonenzymatic,
template-directed ligation of oligoribonucleotides is highly regiose-
lective for the formation of 3?-5?phosphodiester bonds. J Am Chem
Saffhill, R. 1970. Selective phosphorylation of the cis-2?,3?-diol of un-
protected ribonucleosides with trimetaphosphate in aqueous solu-
tion. J Org Chem 35:2881–2883.
Saladino, R., Crestini, C., Costanzo, G., Negri, R., and Di Mauro, E.
4(3H)-pyrimidinone from formamide: implications for the origin of
life. Bioorg Med Chem 9:1249–1253.
Sanchez, R., Ferris, J.P., and Orgel, L.E. 1966a. Conditions for purine
Sanchez, R.A., Ferris, J.P., and Orgel, L.E. 1966b. Cyanoacetylene in
prebiotic synthesis. Science 154:784–785.
Sanchez, R.A., Ferris, J.P., and Orgel, L.E. 1967. Studies in prebiotic
synthesis. II. Synthesis of purine precursors and amino acids from
aqueous hydrogen cyanide. J Mol Biol 30:223–253.
Sanchez, R.A., Ferris, J.P., and Orgel, L.E. 1968. Studies in prebi-
otic synthesis. IV. Conversion of 4-aminoimidazole-5-carbonitrile
derivatives to purines. J Mol Biol 38:121–128.
Sanchez, R.A. and Orgel, L.E. 1970. Studies in prebiotic synthesis. V.
Synthesis and photoanomerization of pyrimidine nucleosides. J Mol
metal ions. J Am Chem Soc 98:7037–7039.
Sawai, H., Higa, K., and Kuroda, K. 1992. Synthesis of cyclic and
acyclic oligocytidylates by uranyl catalysis in aqueous solution.
Chem Soc Perkin Trans 1:505–508.
Sawai, H., Karoda, K., and Hojo, T. 1989. Uranyl ion as a highly effec-
tive catalyst for internucleotide bond formation. Bull Chem Soc Jpn
Sawai, H. and Orgel, L.E. 1975. Oligonucleotide synthesis catalyzed
by the Zn2+ion. J Am Chem Soc 97:3532-3533.
Schmidt, J.G., Christensen, L., Nielsen, P.E., and Orgel, L.E. 1997.
directed syntheses. Nucleic Acids Res 25:4792–4796.
Schoning, K., Scholz, P., Guntha, S., Wu, X., Krishnamurthy, R., and
Eschenmoser, A. 2000. Chemical etiology of nucleic acid structure:
the alpha-threofuranosyl-(3?→ 2?) oligonucleotide system. Science
Schwartz, A.W. 1969. Specific phosphorylation of the 2?- and 3?posi-
tions in ribonucleotides. J Am Chem Soc 23:1393.
Schwartz, A.W. 1997. Prebiotic phosphorus chemistry reconsidered.
Orig Life Evol Biosph 27:505–512.
Schwartz, A.W., Joosten, H., and Voet, A.B. 1982. Prebiotic adenine
synthesis via HCN oligomerization in ice. Biosystems 15:191–193.
Schwartz, A.W. and Orgel, L.E. 1984. Template-directed polynu-
cleotide synthesis on mineral surfaces. J Mol Evol 21:299–
cleotide probes for binding to DNA and RNA. Biochimie 70:1323–
Shapiro, R. 1999. Prebiotic cytosine synthesis: a critical analysis and
implications for the origin of life. Proc Natl Acad Sci USA 96:4396-
Shapiro, R. 2002. Comments on ‘concentration by evaporation and
the prebiotic synthesis of cytosine.’ Orig Life Evol Biosph 32:275–
Sleeper, H.L. and Orgel, L.E. 1979. The catalysis of nucleotide poly-
merization by compounds of divalent lead. J Mol Evol 12:357–
formose reaction. React Kinet Catal Lett 14:119–124.
Steitz, T.A. and Moore, P.B. 2003. RNA, the first macromolecular cat-
alyst: the ribosome is a ribozyme. Trends Biochem Sci 28:411–418.
Sulston, J., Lohrmann, R., Orgel, L.E., and Miles, H.T. 1968a. Nonen-
Proc Natl Acad Sci USA 59:726–733.
Sulston, J., Lohrmann, R., Orgel, L.E., and Miles, H.T. 1968b. Speci-
Natl Acad Sci USA 60:409–415.
Sulston, J., Lohrmann, R., Orgel, L.E., Schneider-Bernloehr, H.,
Weimann, B.J., and Miles, H.T. 1969. Non-enzymic oligonucleotide
synthesis on a polycytidylate template. J Mol Biol 40:227–234.
Tapiero, C.M. and Nagyvary, J. 1971. Prebiotic formation of cytidine
nucleotides. Nature 231:42–43.
Tsuhako, M., Fujimoto, M., Ohashi, S.A., Nariai, H., and
Motooka, I. 1984. Phosphorylation of nucleosides with sodium
cyclo-triphosphate. Bull Chem Soc Japan 57:3274–3280.
Unrau, P.J. and Bartel, D.P. 1998. RNA-catalysed nucleotide synthesis.
Voet, A.B. and Schwartz, A.W. 1983. Prebiotic adenine synthesis from
HCN-evidence for a newly discovered major pathway. Biorg Chem
Wachtershauser, G. 1988. Before enzymes and templates: theory of
surface metabolism. Microbiol Rev 52:452–484.
PREBIOTIC CHEMISTRY AND ORIGIN OF RNA WORLD123
Wakamatsu, H., Yamada, Y., Saito, T., Kumashiro, I., and Takenishi, T.
1966. Synthesis of adenine by oligomerization of hydrogen cyanide.
J Org Chem 31:2035–2036.
Walde, P., Wick, R., Fresta, M., Mangone, A., and Luisi, P.L. 1994.
Autopoietic self-reproduction of fatty acid vesicles. J Am Chem Soc
Wang, K.J. and Ferris, J.P. 2001. Effect of inhibitors on the montmo-
rillonite clay-catalyzed formation of RNA: studies on the reaction
pathway. Orig Life Evol Biosph 31:381–402.
White, H.B., 3rd. 1976. Coenzymes as fossils of an earlier metabolic
state. J Mol Evol 7:101–104.
Wilson, D.S. and Szostak, J.W. 1999. In vitro selection of functional
nucleic acids. Annu Rev Biochem 68:611–647.
Woese, C. 1967. The Genetic Code, the Molecular Basis for Genetic
Expression. New York: Harper and Row.
und chemie 88:253–256.
sis on oligodeoxycytidylate sequences in hairpin oligonucleotides.
J Am Chem Soc 114:317–322.
Wu, T. and Orgel, L.E. 1992b. Nonenzymatic template-directed syn-
thesis on hairpin oligonucleotides. 2. Templates containing cytidine
and guanosine residues. J Am Chem Soc 114:5496–5501.
Wu, T. and Orgel, L.E. 1992c. Nonenzymatic template-directed syn-
uridine residues. J Am Chem Soc 114:7963–7969.
Yamagata, Y. 1999. Prebiotic formation of ADP and ATP from AMP,
calcium phosphates and cyanate in aqueous solution. Orig Life Evol
Yamagata, Y., Inoue, H., and Inomata, K. 1995. Specific effect of mag-
phosphate in aqueous solution. Orig Life Evol Biosph 25:47–52.
Yamagata, Y., Watanabe, H., Saitoh, M., and Namba, T. 1991. Volcanic
with DNA in template-directed synthesis. Helv Chim Acta 83:1678–
Zolotov, M.Y., Seewald, J.S., and McCallum, T.M. 2001. Experimen-
tal investigation of aqueous carbon monoxide reactivity under hy-
drothermal condition. In Eleventh Annual V. M. Goldschmidt Con-
ference. Hot Springs, VA.
Zubay, G. 1998. Studies on the lead-catalyzed synthesis of aldopen-
toses. Orig Life Evol Biosph 28:13–26.
Zubay, G. and Mui, T. 2001. Prebiotic synthesis of nucleotides. Orig
Life Evol Biosph 31:87–102.