Protocells and RNA Self-Replication
Gerald F. Joyce1
and Jack W. Szostak2
1
The Salk Institute for Biological Studies, La Jolla, California 92037
2
Howard Hughes Medical Institute and Department of Molecular Biology, Massachusetts General Hospital, Boston,
Massachusetts 02114
Correspondence: gjoyce@salk.edu; szostak@molbio.mgh.harvard.edu
SUMMARY
The general notion of an “RNA world” is that, in the early development of life on the Earth,
genetic continuity was assured by the replication of RNA, and RNA molecules were the chief
agents of catalytic function. Assuming that all of the components of RNA were available in
some prebiotic locale, these components could have assembled into activated nucleotides that
condensed to form RNA polymers, setting the stage for the chemical replication of polynucleotides
through RNA-templated RNA polymerization. If a sufficient diversity of RNAs could be
copied with reasonable rate and fidelity, then Darwinian evolution would begin with RNAs
that facilitated their own reproduction enjoying a selective advantage. The concept of a “protocell”
refers to a compartment where replication of the primitive genetic material took place
and where primitive catalysts gave rise to products that accumulated locally for the benefit of
the replicating cellular entity. Replication of both the protocell and its encapsulated genetic
material would have enabled natural selection to operate based on the differential fitness of
competing cellular entities, ultimately giving rise to modern cellular life.
Outline
1 Introduction
2 Compartmentalized systems
3 Nature of the first genetic material
4 Chemical RNA replication systems
5 RNA-catalyzed replication systems
6 Compartmented genetics
7 Summary
References
Editors: Thomas R. Cech, Joan A. Steitz, and John F. Atkins
Additional Perspectives on RNA Worlds available at www.cshperspectives.org
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1 INTRODUCTION
The first cells must have been simple in comparison with
modern highly evolved cells and therefore are often referred
to as “protocells.” Protocells must have been simple enough
to self-assemble spontaneously in a chemically rich environment
under appropriate physical conditions but sufficiently
complex that they were poised to evolve to greater
complexity, ultimately giving rise to all of modern biology
(Szostak et al. 2001). What kind of cell structure and composition
would have been consistent with the dual requirements
of original simplicity and future complexity?
Given that all modern cells are bounded by a semipermeable
cell membrane, it is reasonable to postulate
that protocells were also membrane-bound structures, albeit
with very different membranes than modern cells
(Deamer 1997). Similarly, all modern cells contain diverse
RNA molecules that encode information and fulfill many
biochemical functions. The central role of RNA in modern
cellular biochemistry, and especially the role of the large
ribosomal RNA as the catalyst of protein synthesis (Steitz
and Moore 2003), provides a powerful argument in support
of RNA as the dominant genetic and functional biopolymer
at an early stage in the evolution of life. Whether RNA,
a close relative of RNA, or some much simpler genetic
material formed the original basis of protocell heredity remains
controversial. However, recent advances in prebiotic
chemistry suggest that RNA itself may have emerged directly
from the chemistryof theyoung Earth and may therefore
have been the genetic polymer of the first protocells
(Sutherland 2016).
Many aspects of modern biology are universal, including
the translation apparatus and instructed protein synthesis,
enzyme-catalyzed metabolism, and protein-mediated membrane
transport. However, all of these complex processes
are the result of extensive evolution and therefore could
not have been present in protocells. In contrast, short
noncoded peptides and a wide range of other products of
prebiotic chemistry likely were present and may have played
important roles in the complex environment required to
nurture the assembly, growth, and division of protocells.
Protocells may be viewed as the products of a series of
self-assembly processes that took place in specific early
Earth environments that provided the necessary chemical
building blocks and sources of energy (Fig. 1). The classical
bilayer membrane structure results from the spontaneous
self-assembly of separate amphiphilic molecules into a lower
energy aggregate state. The self-assembly of RNA is
more complex, but given suitably activated ribonucleotides,
RNA polymers can form spontaneouslyon mineral surfaces
Figure 1. Schematic diagram of a protocell. Protocells bounded by multiple bilayer membranes composed of simple
amphiphilic molecules would have been permeable to nucleotides and metal ions complexed with citrate or other
ligands. Larger genomic and functional RNA molecules would have been trapped within the protocell interior.
Protocell replication would involve growth and division of the membrane boundary as well as replication of the
genomic RNA.
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(Ferris et al. 1996) or simply upon freezing because of the
concentration of ribonucleotides in the eutectic phase
between ice crystals (Kanavarioti et al. 2001).
As membrane sheets form, the high energy of the membrane
edge drives closure into vesicles, which will trap
molecules such as RNA that are present in the surrounding
solution. Seen in this way, the assembly of protocells appears
as a straightforward and perhaps inevitable process,
given the right environment. The real problem is not protocell
formation, but protocell replication, including growth
and division of the protocell membrane and replication
of the encapsulated genetic material. Furthermore, these
processes must be mutually compatible, and the sources
of energy required to drive protocell growth and division
must also be compatible with the integrity of the protocell
components. The nature of these replication processes and
the way that protocell replication led to the evolution of
functional RNAs and thus to the complexity of the RNA
world is the subject of this review.
2 COMPARTMENTALIZED SYSTEMS
What evolutionary advantages does compartmentalization
provide compared with the replication of informational
molecules in solution? Simple processes of natural selection
can occur in solution. Even without replication, chemical
and physical factors will control and alter the sequencesthat
are generated and persist (Chen and Nowak 2012). In an
environment that allows for nonenzymatic replication,
sequences that are more efficiently replicated will have a
selective advantage. Repeated cycles of replication would
therefore be expected to shape the structure of a population
by influencing base composition, template length, secondary
structure, and other properties. But what about the
evolution of functional properties such as molecular
recognition and catalysis? Numerous in vitro selection experiments
have shown that, even without compartmentalization,
RNAs can be obtained that bind a ligand or catalyze
a self-modification reaction.
In nature, selective pressures might favor binding to
mineral surfaces, for example, to prevent RNAs from being
washed out of a favorable local environment. However, for
more complex functions, such as RNA-catalyzed replicative
or metabolic reactions, some form of compartmentalization
becomes essential. Consider an RNA that catalyzes formation
of a metabolite that contributes to the replication of
that RNA, for example, an activated nucleotide. In solution,
the products of that reaction will diffuse away from the
ribozyme that generated them, thus failing to benefit the
RNA itself and perhaps benefiting other neighboring
RNAs. A similar argument can be made with respect to
an RNA replicase, which in free solution might replicate
other surrounding RNAs, thus failing to benefit itself. Compartmentalization
enables the positive feedback required
for Darwinian evolution by keeping useful products close
to the catalysts that generated them and by keeping molecules
related by descent physically closer, on average, compared
with more distantly related molecules.
Compartmentalization is also essential to prevent system
crashes caused by the evolution of parasites. The activity
of a ribozyme RNA replicase, or indeed any ribozyme,
relies on its folded structure, but the formation of a stable
folded structure is at odds with the properties of an ideal
template, which is unfolded and readily available to be
copied. In free solution, parasitic RNAs that are better templates
will increase at the expense of functional replicase
RNAs, leading to a population crash. In a population of
small compartments, however, the emergence of a parasitic
sequence in one compartment will not affect the population
as a whole, even if it dooms the descendants of that compartment.
Experimentally, even transient compartmentalization
is sufficient to prevent extinction caused by parasites
(Bansho et al. 2016; Matsumura et al. 2016). These considerations
imply that compartmentalization is an essential
step on the path to biology. But of the many ways that
compartmentalization can be achieved, which is most relevant
to the origin of life?
2.1 Pros and Cons of Membrane Systems
The formation of compartments through the assembly
of bilayer membranes has a major conceptual advantage
over other types of compartments, namely, its continuity
with known biology. If the first protocells were membranebound
compartments that could grow and divide, together
with the replication of their encapsulated genetic material,
then there could be a continuous line of descent from the
first protocells to all subsequent cellular biology. Other
forms of compartmentalization would have had to undergo
a transition to membrane-based compartments. Such a
transition would have become increasingly difficult as the
system of replicating and functional RNAs became more
highly adapted to any prior form of compartmentalization.
For example, compartments that are more open to the environment
might enable the evolution of ribozymesthat use
large, polar, or charged substrates that would become inaccessible
during a transition to membrane encapsulation.
There are, however, several conceptual challenges with a
membrane-based protocell structure. Chief among these
is that bilayer membranes are a barrier to free access to
the substrates needed for genome replication, most notably
charged nucleotides. Modern cell membranes, composed of
phospholipids, sterols, and other complex lipids, are nearly
complete barriersto the permeation of nucleotides and even
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divalent cations such as Mg2+
(Deamer 1997). Because
primitive cells would have lacked the sophisticated transport
machinery that controls molecular trafficking in modern
cells, protocells must have been bounded by simpler,
more permeable membranes. Membranes formed from
single-chain amphiphiles, such as fatty acids and their glycerol
esters as well as fatty alcohols and related compounds,
tend to be much more permeable than membranes composed
of two-chain lipids. Shorter alkyl chains also contribute
to increased permeability but require a higher critical
concentration for bilayer membrane stability.
The prebiotic synthesis of membrane-forming amphiphilic
molecules is poorly studied. Highly unsaturated carbon
chains are abundant in interstellar molecular clouds
(Duley and Hu 2009), and these molecules may be among
the source materials for the heterogeneous collections
of amphiphiles present in organic-rich meteorites. Lipids
have been extracted from carbonaceous chondrites, such as
Murchison, and have been shown to assemble into vesiclelike
compartments (Deamer 1985). Whether linear fatty
acids could be synthesized by robust chemical pathways
on the early Earth is less clear. The best studied pathways
involve Fischer–Tropsch-type chemistry, in which carbon
monoxide and hydrogen react under high temperature
and pressure on the surface of transition metal minerals
to generate terminally oxygenated, saturated linear chains
(Rushdi and Simoneit 2001). However, the relevance of this
chemistry to early Earth conditions is debated. Such reactions
tend to give chain length distributions that fall off
exponentially with increasing length, suggesting that the
resulting membranes would be dominated by short-chain
fatty acids and alcohols, but stabilized by a small fraction of
long-chain species (Budin et al. 2014).
Protocell membranes must be compatible with the
physical and chemical conditions necessary for RNA chemistry.
Because fatty acid–based membranes, nonenzymatic
RNA copying, and ribozyme activity all tend to be optimal
at moderate salt concentrations and neutral to mildly
alkaline pH, this requirement might not seem to be a
major concern. However, nonenzymatic copying of RNA
templates requires very high concentrations of divalent
cations, typically Mg2+
, and most ribozymes require high
concentrations of Mg2+
for optimal activity. In contrast,
fatty acid–based membranes become unstable in ≥4 mM
Mg2+
, indicating an apparent incompatibility between the
two systems.
One potential resolution of this conflict involves chelation
of Mg2+
by citrate, which protects membranes from the
disruptive effects of free Mg2+
while allowing nonenzymatic
RNA copying chemistry to proceed with only a moderate
reduction of activity (Adamala and Szostak 2013a). As an
added benefit, the Mg2+
-catalyzed degradation of RNA is
prevented by citrate. However, it is unlikely that sufficient
citrate was present in the prebiotic environment, and thus
alternatives must be sought. A short acidic peptide in
the active site of cellular RNA polymerase binds Mg2+
and positions it for catalysis (Zhang et al. 1999; Cramer
et al. 2001). Could acidic peptides synthesized prebiotically
have played a similar role in early RNA copying? The condensation
of acidic amino acids is catalyzed by positively
charged mineral surfaces (Ferris et al. 1996), suggesting that
such peptides might have been available on the early Earth.
However, catalysis of templated RNA polymerization by
acidic peptides has not been shown. Alternatively, RNA
copying strategies that do not require a high concentration
of divalent cations would circumvent the problem.
2.2 Alternatives to Membrane Systems
A fundamental requirement for the evolution of complex
adaptive traits is to restrict the free diffusion of genomic
and functional polymers and their metabolic products so
that mutations that lead to improved activity have the opportunity
to benefit the mutant more than nearby wild-type
individuals. In addition, compartmentalization is essential
to prevent a fatal increase in parasitic sequences. Although
these requirements could be met by a system of growing
and dividing vesicles, other mechanisms of spatial isolation
could, in principle, provide a solution. Such alternatives
include porous rocks, particulate mineral surfaces, noncovalent
assemblies of oligonucleotides, emulsion droplets,
and phase-separated droplets, including coacervates.
The porous rocks seen in hydrothermal vents have long
been proposed as a possible site for the origin of life (Lane
and Martin 2012). As the heated vent fluids cool, dissolved
minerals form precipitates and build up a network of interconnected
microscale channels. Transverse temperature
gradients across such channels have been proposed to
support a combination of thermophoresis and convection,
resulting in very strong concentration gradients (Baaske
et al. 2007).
Laboratory experiments using glass capillaries as analogs
of porous rock channels have shown the simultaneous
concentration of dilute fatty acids and DNA, leading to the
assembly of vesicles with encapsulated DNA near the bottom
of the capillary tube (Budin et al. 2009). Subsequent
experiments have shown that temperature gradients across
narrow channels can concentrate oligonucleotides, facilitating
the assembly of longer DNA strands (Mast et al. 2013).
Concentration as a result of thermophoresis can also preferentially
enrich longer products relative to shorter ones,
providing a means to overcome the replication bias that
favors shorter templates (Kreysing et al. 2015). Because
thermophoresis is strongly inhibited by high salt concenG.F.
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trations, this concentration mechanism would only be operative
in freshwater hydrothermal vents, such as those currently
found in Lake Yellowstone (Inskeep et al. 2015). How
nucleic acids that evolved within rock compartments would
transition to membrane compartments is not clear. Nevertheless,
the remarkable concentration effects achieved by
thermophoresis deserve further study.
Another scenario for spatial isolation involves the
assembly and replication of RNA on mineral surfaces.
The ability of montmorillonite clay to catalyze the polymerization
of activated mononucleotides is well known (Ferris
et al. 1996; Ferris 2002). The resulting RNAs are tightly
bound to the mineral surface, suggesting that mineral particles
might function like compartments, with each particle
bearing a spatially isolated population of RNA molecules. If
RNAs could replicate while remaining at least partially
bound to the mineral surface, the system could in principle
support the Darwinian evolution of functional RNAs. Although
some early work supported the idea that RNA templates
can be copied while adsorbed onto a mineral surface
(Gibbs et al. 1980; Schwartz and Orgel 1985), further work
is needed to confirm the template-directed synthesis of
mineral-immobilized RNA.
Compartments based on liquid–liquid phase separation,
including coacervate droplets, are a third alternative
to membrane vesicles. The strong electrostatic interactions
between polyanionic nucleic acids and various polycations
leads to the formation of complex coacervates, which, depending
on their composition and the ambient conditions,
may remain as liquid droplets. The contents of these droplets
are highly concentrated relative to the bulk solution,
and there is little barrier to the uptake of nutrients from
the environment into the coacervate droplets. However,
phase-separated droplets are subject to Ostwald ripening,
leading to droplet growth, and to fusion events, leading to
coalescence. Fusion events can be blocked by a surfactant
coating, but this would eliminate the presumed advantage
of enhanced accessto Mg2+
andothercharged species (Tang
et al. 2014; Aumiller et al. 2016). Furthermore, in some coacervates,
the RNA components exchange rapidly between
droplets, so there may not be the degree of isolation required
for Darwinian behavior (Jia et al. 2014). Further study is
needed to determine whether phase-separated droplets
can act as isolated compartments and whether RNA replication
can be performed in aqueous two-phase systems.
Emulsion droplets provide a fourth alternative to membrane
vesicles. The local accumulation of hydrocarbon oils
on the early Earth is not implausible. Given sufficient turbulent
mixing and the presence of stabilizing surfactants,
water-in-oil emulsion droplets would be likely to form.
Such droplets could grow through fusion or Ostwald ripening,
followed byshear-induced division, leading to acycle of
growth and division. Although attractive as a simple model
of protocell growth and division, the limited accessibility of
aqueous emulsion droplets to sources of nutrients would be
a major problem. However, even if emulsion droplets are
not directly relevant to origin of life scenarios, they provide
an interesting model system for the laboratory study of
evolutionary phenomena (Ichihashi et al. 2013).
Finally, nucleic acids might provide the basis for their
own compartmentalization. The growing field of DNA
(and RNA) origami provides many examples of compartments
assembled from nucleic acids (for reviews, see
Pinheiro et al. 2011; Hong et al. 2017). An early example
was the assembly of an octahedral structure from a defined
single-stranded DNA (Shih et al. 2004). In principle, genomic
strands that replicate within such a compartment could
encode the compartment itself. There are examples of DNA
origami that undergo self-replication based on growth and
division processes analogous to those of membrane systems
(Schulman et al. 2012). Thus far, however, the properties
of encapsulation and replication have not been combined
within a single system.
2.3 Pathways for Vesicle Division
Early work by Luisi and colleagues explored processes
that could potentially lead to the growth and division of
micelles and vesicles. By encapsulating ferritin particles in
preformed 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphatidylcholine
(POPC) vesicles, electron cryomicroscopy evidence
was obtained suggesting that the addition of oleate
to POPC vesicles led to vesicle growth and subsequent division
(Berclaz et al. 2001). Experiments using fluorescent
markers of membrane area and vesicle content confirmed
that oleate addition to preformed vesicles led to growth, but
division was not observed unless the vesicles were forced
through small pores (Hanczyc et al. 2003). However, the
addition of excess alkaline oleate micelles to preformed oleate
vesicles, buffered at a lower pH, led to an increase in
vesicle number. Strikingly, the size of the newly formed
vesicles was close to that of the original preformed vesicles
(Rasi et al. 2004).
More recently, vesicle growth has been observed directly
by video microscopy, following the addition of oleate to
larger multilamellar vesicles ∼4 µm in diameter (Zhu
and Szostak 2009). Surprisingly, vesicle growth proceeded
through the extrusion of a thin filament from the outermost
bilayer. Over time, this filament grew in length, and equilibration
with inner bilayers resulted in a filamentous multilamellar
vesicle under conditions in which surface area
growth is much faster than volume growth owing to osmotic
constraints. Filamentous vesicles are subject to a pearling
instability that likely contributes to the ease with which they
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are fragmented under mild shear stress. Gentle agitation is
sufficient to bring about vesicle division without loss of the
vesicle contents.
Alternatively, in an environment rich in organic thiols,
photochemical oxidative processes that lead to disulfide
formation can induce extreme pearling in such vesicles,
resulting in spontaneous division into a large number of
small daughter vesicles (Zhu et al. 2012). In contrast to
the behaviorof multilamellar vesicles, large unilamellar vesicles
are fragile and tend to rupture with extensive loss of
contents under shear stress. These features combine to
make multilamellar vesicles and filamentous growth attractive
as a means of simple, environmentally driven cycles
of growth and division, requiring only episodic delivery of
additional amphiphiles and a moderately turbulent envi-
ronment.
Competition between vesicles for limiting membrane
components could provide a simple scenario for protocell
growth that obviates the need for periodic input of new
amphiphilic material. If some vesicles in a population are
able to grow by obtaining membrane components from
surrounding vesicles, many generations of growth and
division could occur within a given locale. Furthermore,
the resulting competition for resources could drive natural
selection and thus Darwinian evolution.
The first experimental demonstration of competition
between protocells arose from studies of osmotically driven
growth (Chen et al. 2004). Vesicles with a high concentration
of encapsulated RNA are osmotically swollen and the
membrane is under tension. This high-energy state can be
relaxed by the incorporation of additional fatty acid molecules
into the membrane. Because single-chain amphiphiles
exchange rapidly between vesicles, fatty acid vesicles containing
RNA grow in the presence of empty (relaxed) fatty
acid vesicles. This simple physical mechanism could lead to
a coupling between RNA replication and membrane growth
and to competition between protocells. However, it is energetically
expensive to cause osmotically swollen vesicles
to divide, making it difficult to couple competitive growth
to a faster overall cell cycle.
Subsequent studies revealed that a small fraction of
phospholipid in a largely fatty acid membrane can also
lead to competitive growth (Budin and Szostak 2011). In
this case, competition results from the slower dissociation
of fatty acid molecules from membranes that contain
phospholipids, which results in growth of phospholipidcontaining
vesicles at the expense of vesicles that do not
contain (or contain less) phospholipid. Thus, any vesicle
that also contained a heritable catalyst of phospholipid synthesis
would experience a strong selective advantage. Other
molecules that have a similar effect in slowing fatty acid
dissociation, such as hydrophobic peptides, also confer a
competitive growth advantage, implying that a heritable
RNA that catalyzes the synthesis of hydrophobic peptides
could also provide a selective advantage through competition
at the cellular level (Adamala and Szostak 2013b).
An alternative to protocell growth through molecular
accretion is stepwise growth through vesicle fusion. Cycles
of fusion and division could allow replicating RNAs to
spread throughout a population of vesicles, with strong selection
for replication efficiency. Under this scenario,
selection for increased efficiency of RNA-catalyzed RNA
replication would come first, followed by the evolution of
additional ribozymes because of the competition between
protocells. Wet/dry cycles transform vesicles into lamellar
stacks that upon rehydration again form vesicles. Whether
wet/dry cycles could lead to the spread of replicating RNAs
through a vesicle population remains unclear. Further
studies of the fate of encapsulated RNAs following wet/
dry cycles may shed light on the potential role of such processes
in early RNA evolution.
3 NATURE OF THE FIRST GENETIC MATERIAL
3.1 RNA as the First Genetic Material
The prebiotic synthesis of nucleotides could have occurred
in several ways. The simplest, conceptually, would be to
synthesize a nucleoside base, couple it to ribose, and finally
to phosphorylate the resulting nucleoside. Recent studies,
however, suggest that other routes may be more feasible,
most notably those that involve the coassembly of the base
and sugar phosphate.
Many sugars, including the four pentoses, react readily
with cyanamide to form stable bicyclic amino-oxazolines
(Fig. 2A) (Sanchez and Orgel 1970). The free sugars are
unstable (Larralde et al. 1995) and cannot be synthesized
directly in the presence of cyanamide. However, the sugars
could be synthesized in one locale through the “formose”
reaction (Mizuno and Weiss 1974; Müller et al. 1990;
Ricardo et al. 2004; Kim et al. 2011), then combined with
cyanamide in a different locale to give the sugar aminooxazolines.
When this reaction is performed with acomplex
mixture of sugars, the ribose derivative strikingly crystallizes
from aqueous solution, segregating it from the other
sugars (Springsteen and Joyce 2004).
Sutherland and colleagues (Ingar et al. 2003; Anastasi
et al. 2007; Powner et al. 2009) avoided the complication
of the separate synthesis of sugars by starting simply
with glycolaldehyde and cyanamide, which in the presence
of 1 M phosphate at neutral pH gives 2-amino-oxazole an
excellent yield (Fig. 2B). The phosphate both buffers and
catalyzes the reaction, directing glycolaldehyde toward
2-amino-oxazole, rather than a complex mixture of prodG.F.
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ucts. Glyceraldehyde is then added, resulting in formation
of the various pentose amino-oxazolines, including the
arabinose compound. Arabinose amino-oxazoline in turn
can react with cyanoacetylene, also in phosphate buffer, to
form cytosine 2′
,3′
-cyclic phosphate as the major product.
This approach still requires sequential reactions. However,
glycolaldehyde and glyceraldehyde can react with 2-aminothiazole
to give the corresponding aminals, which selectively
precipitate to give crystalline reservoirs of these key
intermediates (Islam et al. 2017).
A potential route from arabinose amino-oxazoline to
pyrimidine nucleotides first was explored nearly 40 years
ago (Tapiero and Nagyvary 1971) and has been reexamined
in light of alternative routes to the pentose amino-oxazolines
(Ingar et al. 2003). Like arabinose, arabinose 3-phosphate
reacts with cyanamide to give the corresponding
amino-oxazoline (Fig. 2C). This in turn reacts with cyanoacetylene
to form a tricyclic intermediate that hydrolyzes to
produce a mixture of cytosine arabinoside-3′
-phosphate and
cytosine 2′
,3′
-cyclic phosphate. Ribose amino-oxazoline
A
O
HO
OH O
OH
H
H
OH OH
OH
OHO
2C NHN
C
2OH
H2
O
HO NO
N
O
P O–
O–
O
OH
HO
HO O
O
P O–
O–
O
O
HO
O O
OH
H
H
OH
P O–
O–
O
O
HO NO
N
NH
O
P O–
O–
O
O
HO N
N
N
O
O
P
O
O O–
H2
H
O
O
O N
N
NH2
O
OH
P O–
O–
O
C CN CH
+
2C NHN
O
HO
N
OH O
NH2
+
B
O
HO NO
OH
NH2
NH2
O
HO
N
OH
HO OH
O
O
NH2
O N
2C NHN
H2PO4 (pH 7.0)
– H2O
–
–
NH2
O
HO NO
OH
NH2
O
HO NO
N
NH
OH
O
HO N
N
O
O
P
O
O O–
C CN CH
H2PO4 (pH 6.5) –
H2PO4 (pH 6.5)
H2PO4 (pH 7.0)–
HO O
Figure 2. Potential prebiotic synthesis of pyrimidine nucleosides. (A) Reaction of ribose with cyanamide to form a
bicyclic product, with cyanamide joined at both the anomeric carbon and 2-hydroxyl. (B) Reaction of glycolaldehyde
with cyanamide in neutral phosphate buffer, followed by addition of glyceraldehyde, to form ribose and arabinose
amino-oxazoline (and lesser amounts of the xylose and lyxose compounds). Arabinose amino-oxazoline then reacts
with cyanoacetylene to give cytosine 2′
,3′
-cyclic phosphate as the major product. (C) Analogous reaction of arabinose-3-phosphate
to form a bicyclic product, which then reacts with cyanoacetylene to form a tricyclic intermediate
that hydrolyzes to give a mixture of cytosine arabinoside-3′
-phosphate and cytosine 2′
,3′
-cyclic phosphate.
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(RAO) also reacts with cyanoacetylene, generating the
2,2′
-anhydro-ribonucleoside, but in this case as the αanomer.
However, ring opening of this compound with
sulfide generates α-2-thio-C, which readily photoanomerizes
to give the β-anomer, which in turn desulfurizes to
give C or deaminates to give 2-thio-U and U (Fig. 3) (Xu
et al. 2017).
A similar approach has been applied to the chemoselective
assembly of purine nucleotide precursors (Powner et al.
2010). It has long been known for the purines, but not the
pyrimidines, that simply heating the nucleobase with ribose
or ribose-phosphate results in the synthesis of the corresponding
ribonucleoside or ribonucleotide, but in low yield
(Fuller et al. 1972). Two alternative approaches that have
been explored recently involve either ribosylation of formamidopyrimidine,
which is selective for the N9 position, but
gives a mixture of α/β-furanosides and pyranosides (Becker
et al. 2016), and a route from 2-thiooxazole to the pentose
amino-oxazolidinone thiones and ultimately to the corresponding
8-oxopurine nucleotides (Stairs et al. 2017).
The story remains incomplete, however, because these
syntheses still require temporally separated reactions using
high concentrations of just the right reactants and would
be disrupted by the presence of other closely related compounds.
The reactions channel material toward the desired
products, but other fractionation processes are required
to provide the correct starting materials at the requisite
time and place. In addition to the selective crystallization
processes described above, it has been proposed that precipitation
of ferrocyanide salts could generate a concentrated
reservoir of starting materials that can be liberated by
geothermal activity (Patel et al. 2015). Other potential fractionation
processes, involving either selective synthesis or
selective degradation, are actively being pursued.
Even chemical fractionation could not achieve on a
macroscopic scale one desirable separation, the resolution
of D-ribonucleotides from their L-enantiomers. This is a
serious problem because experiments on the nonenzymatic
template-directed polymerization of imidazole-activated
mononucleotides suggest that the polymerization of the
D-enantiomer is strongly inhibited by the L-enantiomer
(Joyce et al. 1984). This difficulty may not be insuperable;
perhaps with a different mode of phosphate activation,
the inhibition would be less severe. However, enantiomeric
cross-inhibition is certainly a serious problem if life arose in
a racemic environment.
It is possible that the locale for life’s origins was not
racemic, although the global chemical environment contained
nearly equal amounts of each pair of stereoisomers.
There likely were biases in the inventory of compounds delivered
to the Earth by comets and meteorites (Cronin and
Pizzarello 1997; Engel and Macko 1997; Pizzarello et al. 2003;
Glavin and Dworkin 2009). These in turn could have biased
terrestrial syntheses, although the level of enantiomeric enrichment
generally declines with successive chemical reactions.
A special exception involves a remarkable set of
reactions and fractionation processes that amplify a slight
chiral imbalance, even to the level of local homochirality
(Kondepudietal.1990;Soaietal.1995;Viedma2005;Kluss-
mannetal.2006;Noorduinetal.2008;Viedmaetal.2008).As
discussed elsewhere in this collection (Blackmond 2018),
these systems have in common both a catalytic process for
O
O
N
NH2
NH2
HO HO
HCCCN
HS–
HO
OHHO HO
HO
O
NH2
HO
O
N
N
+
O N
N
S
O N
OH
N
S
HO
NH2
Crystalline RAO
β-Anomer of 2-thio-C
hν
α-Anomer of 2-thio-C
Figure 3. Potential prebiotic synthesis of 2-thio-C starting from ribose amino-oxazoline (RAO). RAO, which is
generated by the reaction of glyceraldehyde with 2-amino-oxazole, crystallizes readily, leading to its accumulation as
a reservoir of purified material. Subsequent reaction with cyanoacetylene generates an anhydronucleoside intermediate,
which upon attack by hydrosulfide gives the α-anomer of 2-thio-C. Exposure to ultraviolet light results in
photoanomerization to give the β-anomer of 2-thio-C, which then desulfurizes to form cytosine.
G.F. Joyce and J.W. Szostak
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amplification of same-handed molecules and an inhibition
process for suppression of opposite-handed molecules.
3.2 Potential Alternatives to RNA
The problems that arise when one triesto understand how an
RNAworld could have arisen de novo on the primitive Earth
are sufficiently challenging that one must consider other possibilities.
What kind of alternative genetic systems might have
preceded the RNA world? How could they have “invented”
the RNA world? These topics have generated a good deal of
speculative interest and some relevant experimental data.
Eschenmoserand colleagues undertook a systematic study
of the properties of analogs of nucleic acids in which ribose is
replacedbysomeothersugar,orinwhichthefuranoseformof
ribose is replaced by the pyranose form (Fig. 4) (Eschenmoser
1999). Strikingly, polynucleotides based on the pyranosyl analog
of ribose (p-RNA) form Watson–Crick-paired double
helices that are more stable than RNA, and p-RNAs are less
likely than the corresponding RNAs to form multiple-strand
competing structures (Pitsch et al. 1993, 1995, 2003). Furthermore,
the helices twist much more gradually than those of
standard nucleic acids, which should make it easier to separate
strands of p-RNA during replication. p-RNA appears to be an
excellent choice as a genetic system; in some ways it seems an
improvement compared with the standard nucleic acids.
However, p-RNA does not interact with normal RNA to
form base-paired double helices.
Most double-helical structures reported in the literature
are characterized by a backbone with a six-atom repeat.
Eschenmoser and colleagues made the surprising discovery
that an RNA-like structure based on threose nucleotide
analogs (TNA; Fig. 4C), although it involves a five-atom
repeat, can still form a stable double-helical structure with
standard RNA (Schöning et al. 2000). This provides an
example of a pairing system based on a sugar that could
be formed more readily than ribose. Tetroses are the unique
products of the dimerization of glycolaldehyde, whereas
pentoses are formed along with tetroses and hexoses from
glycolaldehyde and glyceraldehyde. A structural simplification
of Eschenmoser’s TNA has been achieved by Meggers
and colleagues (Zhang et al. 2005). They replaced threose by
its open chain analog, glycol, in the backbone of TNA,
resulting in glycol nucleic acid (GNA; Fig. 4D). Complementary
oligomers of GNA form antiparallel, double helices
with surprisingly high duplex stabilities. The activated
monomer of GNA is susceptible to intramolecular cyclization,
but this might be overcome by using dimeric or short
oligomeric building blocks.
These and similar studies suggest that there are many
ways of linking together the nucleotide bases into chains
that are capable of forming base-paired double helices. It is
not clear that it is much easier to synthesize the monomers
of p-RNA, TNA, or GNA than to synthesize the standard
nucleotides. However, it is possible that a base-paired structure
of this kind will be discovered that can be synthesized
readily under prebiotic conditions. It also may be fruitful to
explore a broader range of potential precursors to RNA,
changing the recognition elements as well as the backbone.
A strong candidate for the first genetic material would be
any informational macromolecule that is replicable in a
sequence-general manner and derives from compounds
that would have been abundant on the primitive Earth,
and preferably has the ability to cross-pair with RNA.
4 CHEMICAL RNA REPLICATION SYSTEMS
4.1 Templated Polymerization of Mononucleotides
The nonenzymatic template-directed polymerization of activated
nucleotides has been studied for decades as a model
O
HO
O
–
O
O
P
O
O
N
O
HO
O–
O
O
P
O
N
O–
O
O
P
O
N
O
HO
A B D
O
PO O–
O
N
O
PO O–
O
N
O
PO O–
O
N
O
C
O
O
PO O–
O
N
O
O
PO O–
O
N
O
O
PO O–
O
N
O
O
OHO
O
N
O
OHO
P
O
O–
O
P OO
O–
N
O
OH
OP
O
O–
O N
Figure 4. The structures of (A) RNA; (B) p-RNA; (C) TNA; and (D) GNA.
Protocells and RNA Self-Replication
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for the emergence of replicating informational systems
during the origin of life. Rather than recount the detailed
history of this field, which has been extensively reviewed
(Joyce 2002; Orgel 2004), this review focuses on recent
developments that have led to significant improvements
in the extent and generality of RNA-templated synthesis
of RNA, concluding with a discussion of the remaining
barriers to the development of prebiotically plausible replication
chemistry.
Template-directed primer extension with imidazoleactivated
monomers is attractive as a prebiotic means
of copying RNA templates, in part for its similarity to
the universal biological processes of enzymatic RNA and
DNA replication. Since the discovery in 1981 that 2-methylimidazole
(2MeI) is an especially good nucleotide activating
agent (Inoue and Orgel 1981, 1983), nucleoside
5′
-phosphoro-2-methylimidazolides have been widely used
for primer extension experiments. The mechanism of the
reaction between a growing primer chain and an incoming
imidazole-activated monomer was assumed to parallel that
of enzymatic primer extension, which proceeds via in-line
SN2 nucleophilic substitution. However, the very slow
rate at which the last nucleotide of a template is copied
relative to internal sites (Wu and Orgel 1992a), suggested
that other factors influenced the reaction. Indeed, the same
investigators showed that the reaction of the primer with an
activated nucleotide is catalyzed by a downstream, activated
nucleotide that is base-paired to the template at the primer
+2 position. Furthermore, the identity of the leaving group
on the 5′
-phosphate of the downstream nucleotide has a
large effect on the rate of the reaction between the primer
and the adjacent monomer (Wu and Orgel 1992b). Only
recently has the mechanism behind these effects become
clear.
In the course of comparing the rates of oligonucleotide
ligation and primer extension, Prywes and Szostak rediscovered
the catalytic effect of a downstream, activated
nucleotide and showed that this effect is much stronger
for an oligonucleotide than a mononucleotide at the downstream
position (Prywes et al. 2016). Initially, catalysis was
thought to result from noncovalent interactions between
the leaving groups of adjacent activated nucleotides, but
subsequent experiments suggested that the relevant interaction
was covalent and involved the off-template formation
of a reactive intermediate. Nuclear magnetic resonance
experiments identified this intermediate as a 5′
,5′
-imidazolium-bridged
dinucleotide, generated by an attack of the
nucleophilic nitrogen of an unprotonated phosphoroimidazolide
on the phosphate of a protonated imidazolide (Fig.
5). The template specificity of primer extension suggested
that the intermediate could bind to the template through
two adjacent Watson–Crick base pairs (Walton and Szostak
2016). The Richert laboratory also noted the formation of an
imidazolium-bridged dinucleotide and its high reactivity in
primer extension reactions (Kervio et al. 2016). More recent
kinetic studies of primer extension with 2-aminoimidazole(2AI)
activated monomers showed that all detectable primer
extension occurs via the 2-aminoimidazolium intermediate
and not through the reaction of a primer with an
activated monomer (Walton and Szostak 2017).
O
OH
O
NH
NN
N
O
NH2
N
H
N
P
O–
OH
O
OH
O
NH
NN
N
O
NH2
N
N
P
O–
OH
O
+
N
H
N
O
OH
O
NH
NN
N
O
NH2P
OH
O
OH
O
NH
NN
N
O
NH2
N
N
P
O–
OH
O
O
+
O
O–
+
Figure 5. Formation of a 5′
,5′
-linked imidazolium-bridged dinucleotide, which is the reactive intermediate in
template-directed polymerization of imidazole-activated nucleotides.
G.F. Joyce and J.W. Szostak
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4.2 Alternative Means of Monomer Activation
The Richert laboratory has explored a wide range of phosphate-activating
groups in an effort to improve the rate
and generality of primer extension chemistry. One of the
problems with nonenzymatic primer extension is that the
3′
-hydroxyl of the primer must be deprotonated during
nucleophilic attack on the phosphate of the incoming
monomer. Conversely, an imidazole-based activating group
must be protonated to function as a leaving group, and
this typically requires a lower pH because the pKa of this
group is in the range of 6–8. Therefore, a leaving group that
departs as an anion (with low pKa) could provide a much
faster reaction rate at elevated pH. 1-Hydroxy-7-azabenzotriazole
(HOAt) functions in this manner and results in
rapid primer extension attributable to direct reaction of
the primer with the HOAt-activated monomer (Kervio
et al. 2016).
A related approach is to use an N-alkyl-imidazole as
an activating agent because it can leave as a neutral species
that does not require protonation. Following this approach,
the Richert laboratory used N-ethyl-imidazole (NEI) as an
organocatalyst to facilitate in situ nucleotide activation with
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC).
The nucleoside 5′
-phosphate reacts first with EDC to
form a reactive isourea intermediate, then displacement
by NEI yields NEI-activated monomers that can then react
with the primer (Jauker et al. 2015). Although the highly
reactive activated nucleotides are labile to hydrolysis at
elevated pH, the continuous addition of EDC could in
principle lead to continuing primer extension. An efficient
and prebiotically plausible means of performing nucleotide
activation and primer extension in the same reaction
mixture would greatly simplify the conditions required for
protocell replication.
In view of the catalytic effect on primer extension by an
activated downstream nucleotide, a series of substituted
imidazoles were tested for their influence on the rate of
reaction, in all cases with 2MeI-activated guanylate as the
incoming monomer. The magnitude of the catalytic effect
correlated positively with the pKa of the imidazole and with
a smaller size of the imidazole substituent. 2AI has both
of these properties and proved to be a superior activating
group (Li et al. 2017). This effect is now understood to
reflect, at least in part, the greater stability of the 2-aminoimidazolium-bridged
dinucleotide intermediate compared
with the relatively unstable 2-methylimidazolium intermediate.
This allows the 2AI intermediate to build up to higher
levels, significantly enhancing the rate and extent of primer
extension (Walton and Szostak 2017). The potential prebiotic
synthesis of 2AI is intriguing because it can be generated
in the same reaction mixture as the related heterocycle
2-aminooxazole, a precursor of the pyrimidine nucleotides
(Fahrenbach et al. 2017).
4.3 Alternative Modes of Strand Elongation
Primer extension with activated monomers is a biologically
inspired mode of template copying. In principle, however,
template copying could also be accomplished by the ligation
of activated oligonucleotides. Unfortunately, nonenzymatic
RNA ligation reactions tend to have low yield and poor
fidelity. An interesting exception comes from a study of
template-directed ligation of DNA oligonucleotides mediated
by cyanogen bromide (James and Ellington 1997).
Although still low yielding, when random hexamers were
annealed to a defined template, most of the ligation products
had perfect complementarity to the template. This
study suggests that oligonucleotide ligation could play a
significant role in nonenzymatic RNA-templated synthesis,
if more efficient ligation chemistry could be defined.
The ligation of oligonucleotides activated at the 2′
(3′
)
end has traditionally been hampered by the fact that activation
of a 2′
- or 3′
-phosphate, for example, by reaction
with cyanoimidazole or EDC, leads to rapid formation of
a 2′
,3′
-cyclic phosphate, which is only weakly activated and
yields predominately 2′
,5′
-linked ligation products. This
outcome can be partially avoided if the 3′
-phosphate is
first acetylated by reaction with N-acetyl-imidazole or other
acetylating reagents, in which case the acetyl group is preferentially
transferred to the 2′
-hydroxyl (Bowleret al. 2013).
Subsequent activation of the 3′
-phosphate allows for ligation
and formation of a 3′
,5′
-phosphodiester. In a final step,
the 2′
-hydroxyl is regenerated by slow hydrolytic loss of the
acetyl group. This process can be iterated to replace labile
2′
,5′
linkages, which lead to strand cleavage by transesterification,
with a progressively higher proportion of the more
stable 3′
,5′
linkages (Mariani and Sutherland 2017).
Surprisingly, the rate of ligation of a primer to a
5′
-imidazole-activated oligonucleotide is very slow compared
with the rate of primer extension with similarly activated
monomers (Prywes et al. 2016). This is now explained
by the fact that primer extension with monomers proceeds
through the highly reactive imidazolium-bridged dinucleotide
intermediate discussed above (Walton and Szostak
2017). Because the ligation reaction is slow, the final yield
is determined by competition with hydrolysis of the activated
phosphate. This suggests that chemistry that would reactivate
hydrolyzed substrates, such as the combination of
EDC and an N-alkyl imidazole explored by the Richert laboratory(Jaukeret
al.2015),couldleadtoa highligationyield.
Because oligonucleotides can have a long residence time
on the template, a slow rate of ligation is not necessarily a
problem, so long as the reaction can go to completion. An
Protocells and RNA Self-Replication
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intriguing possibility is that a hybrid process, in which
short oligonucleotides act as primers at multiple sites on a
template, followed by gap filling by primer extension with
monomers, and a final stage of oligonucleotide ligation,
could result in effective copying of long RNA templates
(Szostak 2011). Such a hybrid process would avoid some
of the difficulties inherent to template copying solely by
ligation of oligonucleotide substrates, such as the formation
of gaps or overhangs.
4.4 Fidelity and the Error Threshold
The concept of an error threshold, that is, an upper limit to
the frequency of copying errors that can be tolerated by a
replicating macromolecule, was first introduced by Eigen
(1971). Eigen’s model envisaged a population of replicating
polynucleotides that draw on a limited supply of activated
mononucleotides to produce additional copies of themselves.
In this model, the rate of synthesis of new copies of
a particular replicating RNA is proportional to its concentration,
resulting in autocatalytic growth. The net rate of
production is the difference between the rate of formation
of error-free copies and the rate of decomposition of existing
copies of the RNA. For an advantageous RNA to outgrow
its competitors, its net rate of production must exceed
the mean rate of production of all other RNAs in the population.
Only the error-free copies of the advantageous
RNA contribute to its net rate of production, but all the
copies of the other RNAs contribute to their collective
production. Thus, the relative advantage enjoyed by the
advantageous individual compared with the rest of the
population (often referred to as the “superiority” of the advantageous
individual) must exceed the probability of producing
an error copy of that advantageous individual.
The proportion of copies of an RNA that are error free is
determined by the fidelity of the component condensation
reactions that are required to produce a complete copy. For
simplicity, considera self-replicating RNA that is formed by
n condensation reactions, each having mean fidelity q. The
probability of obtaining a completely error-free copy is given
by qn
, which is the product of the fidelity of the component
condensation reactions. If an advantageous individual
is to outgrow its competitors, qn
must exceed the superiority,
s, of that individual. Expressed in terms of the numberof
reactions required to produce the advantageous individual,
n < |ln s| / |ln q|.
For s > 1 and q > 0.9, this equation simplifies to
n < ln s / (1 – q).
This isthe “error threshold,” which describesthe inverse
relationship between the fidelity of replication, q, and the
maximum allowable number of component condensation
reactions, n. The maximum number of component reactions
is highly sensitive to the fidelity of replication but
depends only weakly on the superiority of the advantageous
individual. For a self-replicating RNA that is formed by the
template-directed condensation of activated mononucleotides,
a total of 2n – 2 condensation reactions are required
to produce a complete copy. This takes into account the
synthesis of both complementary strands.
When chemical replication was first established, fidelity
was likely to be poor and there would have been strong
selection pressure favoring improvement of fidelity. As
fidelity improves, a larger genome can be maintained.
This allows exploration of a larger number of possible sequences,
some of which may lead to further improvement
in fidelity, which in turn allows a still larger genome size,
and so on. If one assumes that the first genomes encoded
a small but relatively efficient ribozyme, for which the
identity of roughly 25 nucleotides must be specified, then
a fidelity of ∼0.98 would be required to enable inheritance
of the corresponding information.
An analysis of error rates in the RNA-templated polymerization
of imidazole-activated nucleotides indicated
that most errors result from G•U wobble pairing (Rajamani
et al. 2010). Calculated overall error rates from that study
were 5%–15%, which would severely limit the size of a
primitive genome. However, primer extension following a
mismatch was generally very slow, depending on the specific
mismatch. This stalling effect slows template copying,
but as a consequence, the first copies to be completed are
more likely to be accurate compared with those completed
at later times. If full-length copies can serve as templates
for the next round of copying as soon as they are completed,
then there will be enrichment for accurate copies during the
course of exponential amplification.
Template copying fidelity can be improved by the use
of nucleotide analogs. The weak base-pairing of U with A
allows for the misincorporation of C opposite A, and the
similar strength of the U•A and U•G base pairs allows
for the misincorporation of U opposite G. Remarkably,
replacing U with 2-thio-U reduces both of these sources
of errors because the 2-thio-U•A base pair is almost as
strong as a C•G base pair. Template copying experiments
with 2MeI-activated monomers suggest that this substitution
reduces wobble-pairing errors from 4%–5% to ∼2% at
template G positions, suggesting that an overall error rate of
1%–2% may be possible (Heuberger et al. 2015).
Replacement of U by 2-thio-U also results in faster and
more efficient copying of templates that contain A residues.
The combined effect of faster and more accurate copying
makes the 2-thio substitution attractive and raises the
question of the prebiotic plausibility of 2-thio-U. Recent
G.F. Joyce and J.W. Szostak
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advances in the prebiotic chemistry of pyrimidine nucleotides
suggest that a mixture of U and 2-thio-U might
be obtained through the synthesis of 2-thio-C, followed
by spontaneous deamination (Fig. 5) (Xu et al. 2017). The
2-thio-U then would be preferentially incorporated into
RNA. Although speculative at this point, the hypothesis
that primordial RNA contained 2-thio-U rather than U is
worth considering.
4.5 Complete Cycles of Replication
The inheritance of RNA-based genetic information requires
cycles of both plus- and minus-strand synthesis. Because
the product of template copying is a double-stranded
RNA, there must be some means of either strand separation
or strand displacement synthesis. Transient temperature
fluctuations could lead to thermal strand separation, but
long RNA duplexes (≥30 base pairs) are difficult to
denature thermally. Many environmental factors affect
the melting temperature of RNA duplexes and could facilitate
thermal strand separation, such as low ionic strength or
the presence of high concentrations of formamide or urea.
A more difficult problem is that strand reannealing is
very fast relative to the time scale of nonenzymatic template
copying. Strand reannealing can be slowed by dilution but
achieving a reannealing time of hours would require strand
concentrations in the picomolar range, which likely would
be too low to maintain RNA replication within a protocell.
Thus, environmental conditions that might drastically slow
reannealing kinetics are of great interest. Recent work from
Hud and colleagues has shown that the combination of
template secondary structure and high solvent viscosity
can significantly slow the reannealing of long templates
(300–500 nucleotides), while still allowing shorter oligonucleotides
(∼30 nucleotides) to diffuse rapidly and anneal
to complementary sites on the template where ligation
can occur (He et al. 2017). Whether this approach can be
extended to shorter templates and copying with monomers
or short oligonucleotides remains to be seen.
Recently, studies of liquid–liquid phase separation suggest
that this phenomenon might be relevant to protocell
structure and function (Koga et al. 2011; Frankel et al.
2016). The high viscosity of phase-separated droplets could
slow reannealing, whereas the colocalization of RNA templates
and substrates could facilitate template copying
(Aumiller et al. 2016). The recent observation of phaseseparated
droplets containing only RNA is intriguing in
this regard (Jain and Vale 2017). These droplets form
when the RNAs contain certain triplet repeats, which enable
the formation of a highly branched interconnected
network. The droplets seem to be liquid-like upon initial
phase separation, but rapidly mature to a gel-like state. It is
possible that template strands trapped within such a matrix
are unable to diffuse and therefore reanneal but could still
be copied by short oligonucleotide or monomer substrates.
Any physical means of immobilizing RNA strands after
strand separation could prevent reannealing, so binding
of RNA to surfaces such as membranes, mineral particles,
or self-assembling peptide filaments could potentially facilitate
replication.
The alternative solution of strand displacement synthesis
is appealing because this is the strategy that is universally
used in biology. However, biological strand displacement
relies on helicases and/or polymerases that can transduce
some of the chemical energy used to drive primer extension
into the mechanical energy needed for strand separation. It
is unclear how this could be achieved in a prebiotic setting
devoid of enzymes.
4.6 Current Status of Laboratory Protocell
Replication Systems
The experimental realization of a complete protocell model
will require replication of mixed-sequence templates long
enough to encode functional ribozymes within replicating
vesicles. This goal has been gradually approached over
the past decade. The first demonstration of nonenzymatic
template copying within fatty acid vesicles was achieved
by copying an RNA template with 5′
-activated 2′
-aminonucleotides.
This model had the advantage of rapid primer
extension in the absence of Mg2+
(Mansy et al. 2008). Five
years later, chelation of Mg2+
with citrate was found to
protect fatty acid membranes, while still allowing templated
RNA polymerization to proceed. Addition of citrate enabled
the copying of an RNA homopolymer template inside
fatty acid vesicles. Subsequently, the use of the 2AI leaving
group combined with activated downstream trinucleotides
enabled the efficient copying of mixed-sequence templates
containing up to seven nucleotides. This approach was
recently adapted to template copying within vesicles by
increasing the permeability of fatty acid membranes using
Mg2+
-citrate. As a result, it has been possible to copy short
RNA templates inside fatty acid vesicles following the
addition of activated monomers and trimers to the outside
of the vesicles (O’Flaherty et al. 2018).
5 RNA-CATALYZED REPLICATION SYSTEMS
5.1 The Replicase Model
The notion of the RNA world places emphasis on an RNA
molecule that catalyzes its own replication. Such a molecule
must function as an RNA-dependent RNA polymerase,
acting on itself (or copies of itself) to produce complemenProtocells
and RNA Self-Replication
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tary RNAs, and acting on the complementary RNAs to
produce additional copies of itself. The efficiency and fidelity
of this process must be sufficient to produce viable
“progeny” RNA molecules at a rate that exceeds the rate
of decomposition of the “parents.” Beyond these requirements,
the details of the replication process are not highly
constrained.
The RNA-first view of the origin of life assumes that
a supply of activated nucleotides was available through
some set of abiotic processes. Furthermore, it assumes
that a means existed to convert the activated nucleotides
to an ensemble of random sequence polynucleotides, a subset
of which had the ability to undergo nonenzymatic
replication. From this population of replicating RNAs, particular
RNAs would have gained in relative abundance if
they enjoyed a selective advantage because of a higher net
rate of production. There would have been selection for the
more favorable template sequences and for RNAs that promote
the replication process. The most rudimentary form
of replicase function would involve assistance of chemical
replication to improve that rate, accuracy, or sequence generality
of RNA synthesis. With ongoing selective pressure,
this chemical replication process would become supplanted
by a biochemical replication process. The latter likely used a
less-reactive, but energetically favorable, activating group
on the mononucleotides, such as a polyphosphate, so that
the RNA-catalyzed reaction dominated over spontaneous
polymerization.
Although it is unclear how the first RNA replicase ribozyme
arose, it is not difficult to imagine how such a molecule,
once developed, would function. The polymerization
of activated nucleotides proceeds via nucleophilic attack
by the 3′
-hydroxyl of a template-bound oligonucleotide at
the α-phosphorus of an adjacent template-bound nucleotide
derivative. This reaction could be assisted by favorable
orientation of the reactive groups, deprotonation of the
nucleophilic 3′
-hydroxyl, stabilization of the trigonalbipyramidal
transition state, or charge neutralization of
the leaving group. All of these tasks might be performed
by RNA (Narlikar and Herschlag 1997; Emilsson et al.
2003), acting either alone (Strobel and Ortoleva-Donnelly
1999) or with the help of a suitably positioned metal cation
or other cofactor (Shan et al. 1999, 2001).
5.2 From Ligase to Polymerase
The possibility that an RNA replicase ribozyme could have
existed has been made abundantly clear by work involving
ribozymes that have been developed through in vitro
evolution (Bartel and Szostak 1993; Ekland et al. 1995;
Ekland and Bartel 1996; Jaeger et al. 1999; Robertson and
Ellington 1999; Johnston et al. 2001; Rogers and Joyce 2001;
McGinness and Joyce 2002; Ikawa et al. 2004; Wochner
et al. 2011; Sczepanski and Joyce 2014; Horning and Joyce
2016). Bartel and Szostak (1993), for example, began with
a large population of random sequence RNAs and evolved
the “class I” RNA ligase ribozyme, an optimized version
of which is about 100 nucleotides in length and catalyzes
the joining of two template-bound oligonucleotides. Condensation
occurs between the 3′
-hydroxyl of one oligonucleotide
and the 5′
-triphosphate of another, forming
a 3′
,5′
-phosphodiester linkage and releasing inorganic pyrophosphate.
This reaction is classified as ligation owing to
the nature of the oligonucleotide substrates but involves the
same chemical transformation as is catalyzed by modern
RNA polymerase enzymes.
X-ray crystal structures of two RNA ligase ribozymes,
the L1 and class I ligases, provide a glimpse into mechanistic
strategies a ribozyme could use to catalyze RNA polymerization
(Fig. 6) (Robertson and Scott 2007; Shechner et al.
2009). Both ligases are dependent on Mg2+
ions for their
activity. The L1 structure (Fig. 6A) shows a bound metal ion
in the active site, coordinated by three nonbridging phosphate
oxygens, one of which belongs to the newly formed
phosphodiester linkage. This Mg2+
ion is favorably positioned
to help neutralize the increased negative charge of
the transition state and, potentially, to activate the 3′
-hydroxyl
nucleophile and to help orient the α-phosphate for a
more optimal in-line alignment. In the class I structure (Fig.
6B), no catalytic metal ion is seen in the vicinityof the active
site, although there is what appears to be an empty metalbinding
site formed by two nonbridging phosphate oxygens,
positioned directly opposite the ligation junction in
a manner similar to that observed for the L1 ligase. These
structures point to a universal catalytic strategy, very similar
to that used by modern protein-based RNA polymerases.
Subsequent to its isolation as a ligase, the class I
ribozyme was shown to catalyze a polymerization reaction
in which the 5′
-triphosphate-bearing oligonucleotide is replaced
by one or more nucleoside 5′
-triphosphates (NTPs)
(Ekland and Bartel 1996). This reaction proceeds with high
fidelity (q = 0.92), although the reaction rate drops sharply
with successive nucleotide additions.
Bartel and colleagues performed further in vitro evolution
experiments to convert the class I ligase to a bona fide
RNA polymerase that operates on a separate RNA template
(Johnston et al. 2001). To the 3′
end of the class I ligase, they
added 76 random sequence nucleotidesthat were evolved to
form an accessory domain that assists in the polymerization
of template-bound NTPs. The polymerization reaction is
applicable to a variety of template sequences, and for wellbehaved
sequences proceeds with an average fidelity of
0.97. This would be sufficient to support a genome length
of 20–30 nucleotides, although the ribozyme itself contains
G.F. Joyce and J.W. Szostak
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about 190 nucleotides. The ribozyme has a catalytic rate
for NTP addition of at least 1.5 min−1
, but its Km is so
high that, even in the presence of micromolar concentrations
of oligonucleotides and millimolar concentrations of
NTPs, it requires about 2 h to complete each NTP addition
(Lawrence and Bartel 2003). The ribozyme operates best
under conditions of high Mg2+
concentration but becomes
degraded under those conditions after 24 h, by which time
it has added no more than 14 NTPs (Johnston et al. 2001).
Further evolutionary optimization of the class I polymerase
ribozyme has led to substantial improvement of its
biochemical properties. By directly selecting for extension
of an external primer on a separate template, Zaher and
Unrau (2007) were able to improve the maximum length
of template-dependent polymerization to >20 nucleotides,
at a rate approximately threefold faster than that of the
parent for the first nine monomer additions and up to 75fold
faster for additions beyond 10 nucleotides. In addition,
this ribozyme variant displays significantly improved fidelity,
particularly with respect to discrimination against G•U
wobble pairs. This improved fidelity appears to be the underlying
source for the improvements in the maximum
length of extension and the rate of polymerization.
5.3 From Polymerase to Replicase
Using a sophisticated emulsion-based in vitro selection
technique, the activity of the class I polymerase was greatly
improved so that it can synthesize long RNA products, in
some cases exceeding 100 nucleotides in length (Wochner
et al. 2011). The improved ribozyme contains a 5′
-terminal
“processivity tag” that binds to a complementary region
of the template, thus overcoming the problem of a high
Km through pseudointramolecularity. It continues to
show a good average fidelity of 0.97. When the reaction is
performed in the eutectic phase of water ice, even longer
polymers can be obtained because of the increased concentration
of reactants and reduced rate of RNA degradation
under these near-frozen conditions (Attwater et al.
2013). However, the optimized polymerase strongly prefers
cytidine-rich templates that lack any secondary structure,
which precludes the synthesis of most functional RNAs.
The class I polymerase was further optimized by selecting
for its ability to react quickly and to copy “difficult”
templates, including those that are cytidine-poor or contain
regions of stable secondary structure (Horning and Joyce
2016). This resulted in a polymerase ribozyme that requires
less than a minute to complete each NTP addition and can
synthesize a variety of complex structured RNAs. It still
struggles with highly structured templates or runs of consecutive
template A residues. It also has a reduced fidelity of
0.92, primarily because of an increased frequency of G•U
wobble mutations. Nonetheless, because of its much greater
sequence generality, this optimized polymerase can catalyze
the reciprocal synthesis of both an RNA and its complement,
enabling the exponential amplification of short RNA
templates in an all-RNA form of the polymerase chain
reaction.
A B
Figure 6. X-ray crystal structure of the (A) L1 ligase, and (B) class I ligase ribozymes. Insets show the putative
magnesium ion-binding sites at the respective ligation junctions. The structures are rendered in rainbow continuum,
with the 5′
-triphosphate-bearing end of the ribozyme colored violet and the 3′
-hydroxyl-bearing end of the substrate
colored red. The phosphate at the ligation junction is shown in white, and the proximate magnesium ion (modeled for
the class I ligase) is shown as a yellow sphere, with dashed lines indicating coordination contacts.
Protocells and RNA Self-Replication
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This most highly optimized class I polymerase also
has the ability to synthesize DNA polymers on an RNA
template (Samanta and Joyce 2017). The ribozyme can incorporate
all four deoxynucleoside 5′
-triphosphates at a rate
of about one addition per minute and can generate products
containing up to 32 deoxynucleotides. Reverse transcriptase
activity likely was necessary for the transition from
RNA to DNA genomes during the early history of life on
Earth. The fact that an RNA polymerase ribozyme also can
function as a reverse transcriptase suggests that a bridge
may have existed between RNA and DNA as a selective
adaptation of the RNA world. DNA is much more stable
compared with RNA and thus provides a larger and more
secure repository for genetic information.
Generalized RNA-catalyzed RNA replication has not
yet been achieved and it is not possible for the class I polymerase
ribozyme to synthesize additional copies of itself.
The most advanced form of the polymerase cannot replicate
long RNA sequences because attemptsto do so are thwarted
by the emergence of shorter ampliconsthat are copied more
efficiently. There must be a selective advantage in maintaining
the full-length amplicon and that advantage must
exceed the probability of producing an error copy. It is
reasonable to expect that a general RNA replicase will eventually
be developed in the laboratory, thus providing a
working model of RNA-based life. However, like the search
for life elsewhere in the universe, such expectations must be
met through direct observation rather than speculation.
Despite falling short of the goal of generalized RNAcatalyzed
RNA replication, there is one example of an RNA
enzyme that catalyzes its own amplification and can undergo
limited Darwinian evolution. This replication system
uses a pair of ligase ribozymes that each catalyze the formation
of the other, using a mixture of four different substrate
oligonucleotides (Lincoln and Joyce 2009). In reaction mixtures
containing only these RNA substrates, MgCl2, and
buffer, a small starting number of ribozymes gives rise to
many additional ribozymes through RNA-catalyzed exponential
amplification. The replication process can be sustained
indefinitely by replenishing the supply of substrates,
in one case achieving an overall amplification factor of
10100
-fold in 37.5 h (Robertson and Joyce 2014).
6 COMPARTMENTED GENETICS
6.1 Integration of Genetic and Compartment
Systems
Assuming that the chemistry of RNA replication can be
fully realized in a manner compatible with protocell replication,
what other challenges must be addressed for the
system to be capable of undergoing Darwinian evolution?
Because functional RNAs are likely to provide significant
selective advantage, it is helpful to understand the conditions
necessary to obtain robust ribozyme function within
model protocells. Such studies have generally used simple
ribozymes, such as the self-cleaving hammerhead ribozyme,
for which activity within fatty acid–based vesicles
has been shown (Chen et al. 2005). Because most ribozymes
require high concentrations of Mg2+
for optimal activity,
it is necessary to use a lipid composition that maintains
membrane stability in the presence of Mg2+
. Alternatively,
it is possible that the first ribozymes did not require high
concentrations of Mg2+
, instead relying on monovalent
cations or other cofactors.
New questions arisewhen considering the integration of
replicating nucleic acids with replicating vesicles. For example,
what mechanisms might serve to keep the membrane
and RNA systems in balance so that neither out-replicates
the other? One potential answer to this problem of
homeostasis is the presence of short oligonucleotides that
are complementary to a replicase ribozyme, generated as
either replication intermediates or degradation fragments,
that inactivate the replicase when present in high concentrations.
Upon dilution, as would occur upon vesicle growth
(Engelhart et al. 2016), this inhibition would be relieved and
RNA replication could continue.
6.2 Membrane Permeability, Trafficking,
and the Origins of Metabolism
Assuming that the first protocells were heterotrophic, the
protocell membranes must have been permeable to nutrients
in the external environment. Fatty acid–based membranes
show high permeability to ions, including divalent
cations, as well as to larger polar and charged molecules,
such as nucleotides. Environmental factors, including elevated
temperature and the presence of divalent cations, can
further enhance membrane permeability. High membrane
permeability is likely to be necessary for protocell growth
but is not a demanding physical requirement. However,
high permeability can also limit the ability of a protocell
to evolve metabolic functions because any metabolites synthesized
within the protocell could leak out and be lost to
the environment or captured by competing protocells. One
possibility is that protocells did not evolve a metabolism
until membrane permeability had decreased, perhaps
driven by increasing phospholipid content resulting from
the selective growth advantage provided by those phospholipids
(Budin and Szostak 2011). Many compounds of
biological metabolism are charged carboxylates or phosphate
esters, which may reflect the evolution of metabolism
at a time when cellular membranes were more permeable
than in modern cells.
G.F. Joyce and J.W. Szostak
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6.3 Remaining Challenges
The experiments described above bring the field tantalizingly
close to a replicating and evolving protocell, but
several additional problems must be solved to reach that
goal. Divalent metal cations appear to be essential for
efficient RNA copying, but the poor affinity of the catalytic
metal for the reaction center means that very high concentrations
of these ions are required, which causes problems
for both the RNA (degradation, hydrolysis of activated
monomers) and for the fatty acid–based membranes.
RNA polymerase enzymes solve these problems by binding
and precisely positioning the metal ion for catalysis
(Zhang et al. 1999; Cramer et al. 2001; Robertson and Scott
2007; Shechner et al. 2009). A prebiotically plausible
means of achieving effective metal ion catalysis at low
ambient concentration would greatly simplify the development
of model protocells. Another key problem is the
mobilization of chemical energy to activate (and reactivate)
nucleotides for polymerization in a specific manner
and under mild conditions. It also is not clear how to
achieve multiple generations of nucleic acid replication
within the context of a protocell. If and only if all of these
challenges can be met will it be possible to synthesize an
evolving protocell.
6.4 Implications for Synthetic Biology
and Biotechnology
The main purpose of research into both nonenzymatic and
RNA-catalyzed RNA replication is to discern plausible
pathways leading from chemistry to biology, and thus to
provide potential explanations for the origin of life on
Earth. However, the development of replicating protocells
also will provide new approaches for the compartmentalized
evolution of novel functional RNAs. One of the longstanding
limitations of conventional in vitro evolution
methods is that selection depends on self-modification of
the catalytic RNA and thus does not enrich for high catalytic
turnover. Replicating protocells may provide a new way
to obtain efficient ribozymes, so long as their function can
be linked to the selective advantage of the protocell.
Many research groups are involved in the construction
of artificial cells, with the goal of reconstituting some (orall)
of the complex subsystems of microbial life. Most such
systems therefore include the protein synthesis machinery,
as either a crude extract or a reconstituted translation system.
Reconstituting a fully functional and replicating cell
from purified biological components is a major challenge
and has inspired efforts to simplify aspects of cellular metabolism
that seem gratuitous. For example, many chemical
modifications of ribosomal RNA (rRNA) are essential for
ribosome assembly, but recently Ichihashi and colleagues
were able to evolve a variant small subunit rRNA that lacks
modifications and can still be efficiently assembled into
ribosomes (Murase et al. 2018). If this approach can be
extended to the large subunit rRNA, ribosome assembly
will be greatly simplified. Similar approaches are aimed at
evolving a simpler ribosome, comparable with evolutionary
intermediates in the path from the first peptidyl transferase
ribozyme to the modern ribosome. Such approaches may
contribute to the development of a comprehensive picture
of possible paths from simple protocells to complex RNA
world cells, and ultimately to the evolution of modern cells
that contain DNA genomes, instructed protein synthesis,
and a complex metabolism.
7 SUMMARY
The constraints that must have been met to originate a selfsustained
evolving system are reasonably well understood.
One can sketch out a logical order of events, beginning with
prebiotic chemistry and ending with protocellular life.
However, it must be said that many details of this process
remain obscure, and even for reactions that have been convincingly
shown in the laboratory, it is difficult to know
whether those same reactions were operable in the historical
origins of life.
The presumed RNA world should be viewed as a milestone,
a plateau in the early history of life on Earth. So, too,
the concept of an RNA world has been a milestone in the
scientific study of life’s origins. Although this concept does
not fully explain how life originated, it has helped to guide
scientific thinking and has served to focus experimental
efforts. Further progress will depend primarily on new experimental
results as chemists, biochemists, and molecular
biologists work together to address problems concerning
molecular replication, ribozyme enzymology, and RNAbased
cellular processes.
ACKNOWLEDGMENTS
J.W.S. is an Investigator of the Howard Hughes Medical
Institute. This work was supported by research grants
287624 (G.F.J.) and 290363 (J.W.S.) from the Simons
Foundation, research grants 80NSSC17K0462 (G.F.J.) and
NNX15AL18G (J.W.S.) from the National Aeronautics and
Space Administration (NASA), and research grant CHE-
1607034 (J.W.S.) from the National Science Foundation
(NSF). Some portions of the text were published in prior
editions of The RNA World. The contributions of Leslie
Orgel to those prior versions and to the scientific literature
of the origins of life are gratefully acknowledged.
Protocells and RNA Self-Replication
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G.F. Joyce and J.W. Szostak
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2018; doi: 10.1101/cshperspect.a034801Cold Spring Harb Perspect Biol
Gerald F. Joyce and Jack W. Szostak
Protocells and RNA Self-Replication
Subject Collection RNA Worlds
Transcriptase Enzymes
Single-Molecule Analysis of Reverse
Linnea I. Jansson and Michael D. Stone Insights into Eukaryotic Translation
Complexity: Ribosomal Structures Provide
Extensions, Extra Factors, and Extreme
Melanie Weisser and Nenad Ban
Structural Biology of Telomerase
Yaqiang Wang, Lukas Susac and Juli Feigon Regulation
CRISPR Tools for Systematic Studies of RNA
Gootenberg, et al.
Jesse Engreitz, Omar Abudayyeh, Jonathan
with Transcription
Nascent RNA and the Coordination of Splicing
Karla M. Neugebauer
Relating Structure and Dynamics in RNA Biology
al.
Kevin P. Larsen, Junhong Choi, Arjun Prabhakar, et
Complexes
RNA−Integrative Structural Biology of Protein
Magnetic Resonance (NMR) Spectroscopy for
Combining Mass Spectrometry (MS) and Nuclear
Allain
Alexander Leitner, Georg Dorn and Frédéric H.-T.
Synthetic Genetics
Beyond DNA and RNA: The Expanding Toolbox of
Holliger
Alexander I. Taylor, Gillian Houlihan and Philipp
of mRNA
Nucleotides That Comprise the Epitranscriptome
Discovering and Mapping the Modified
Bastian Linder and Samie R. Jaffrey
Lessons from Yeast
Structural Basis of Nuclear pre-mRNA Splicing:
Nagai
Clemens Plaschka, Andrew J. Newman and Kiyoshi
Imaging
Illuminating Genomic Dark Matter with RNA
Arjun Raj and John L. Rinn Microscope
under the Single-Molecule Fluorescence
Coming Together: RNAs and Proteins Assemble
Ameya P. Jalihal, Paul E. Lund and Nils G. Walter
From Retroelements to Research Tools
Group II Intron RNPs and Reverse Transcriptases:
Marlene Belfort and Alan M. Lambowitz
Mapping the dsRNA World
Daniel P. Reich and Brenda L. Bass
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The Origin of Biological Homochirality
Donna G. Blackmond Splicing in Higher Eukaryotes
Structural Insights into Nuclear pre-mRNA
Berthold Kastner, Cindy L. Will, Holger Stark, et al.
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