NS109 - Research paper
Nanoscience master program
Towards Darwinian Evolution in Non-Enzymatic
Self-Replicating Molecular Systems
H. Duim*1
Abstract
In this review systems of self-replicating molecules will be discussed in the context of the origin of life.
One of the important aspects of life is the ability to reproduce and evolve continuously. In this review
we will consider what is needed to achieve Darwinian evolution in self-replicating molecules and how
this links to the origin of an RNA world from some basic prebiotic chemical building blocks. It will be
shown that in vitro evolution of self-replicating molecules in the presence of a catalyzing enzyme is
readily achieved, although it still remains difficult to achieve unbounded evolution of non-enzymatic
self-reproducing molecules. Understanding of the evolution of such non-enzymatic systems would
provide a better understanding of how the RNA world could have emerged.
Keywords
Origin of Life — Autocatalysis —Non-enzymatic self-replication — In vitro evolution — RNA world
1 Email:
[email protected]
*Supervisor: Prof. S. Otto, University of Groningen
Introduction
Contents
1
Introduction
2
Replicating systems
2
1.1 Minimal self-replicating system . . . . . . . . . . 3
1.2 Reciprocal self-replicating system . . . . . . . 3
1.3 Reaction kinetics . . . . . . . . . . . . . . . . . . . . . 3
1.4 Self-replication in action . . . . . . . . . . . . . . . 5
2
Requirements for Darwinian evolution
7
2.1 Amplification . . . . . . . . . . . . . . . . . . . . . . . . 7
2.2 Mutation . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
2.3 Selection . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
3
In vitro evolution
8
3.1 RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
3.2 Cross-catalyzing RNA . . . . . . . . . . . . . . . . . 9
3.3 Cooperative catalytic system . . . . . . . . . . 11
3.4 Cross-catalyzing peptides . . . . . . . . . . . . . 13
4
Conclusions and perspectives
14
Acknowledgments
16
References
16
Our current understanding of the origin of life, points
to the existence of a RNA world which has acted as
precursor of the RNA-DNA-protein world in which we
are living. In this RNA world, RNA molecules act
both as the carriers of genetic information and catalyst
of their own replication. It is proposed that ribonucleotides were formed from prebiotic chemicals that
were present in the prebiotic soup. Such reactions have
indeed successfully been demonstrated in the laboratory by different groups.2, 3 These nucleotides, when
activated, could then assemble into larger structures to
form random and functional oligonucleotides (RNA).
These RNA molecules could in turn be copied via a nonenzymatic copying process in which the RNA molecule
acts as a template for the formation of its complementary strand. Natural selection would then lead to the
amplification of functional RNA molecules and the extinction of less fit molecular species. If simultaneously
a compartmentalization process took place, functional
RNA molecules might have accumulated in some type
of protocells, leading to RNA-based organism and the
RNA world. We can still find the relics of this RNA
world as the genome of viral species and as an important
contributor in the translation of our DNA. This pathway
Towards Darwinian Evolution in Non-Enzymatic Self-Replicating Molecular Systems — 2/18
Figure 1. Chemical pathway from prebiotic chemical building blocks to the RNA world. Step (1) involves the formation of
nucleotides. When activated these nucleotides can assemble (2) to form short sequences of nucleotides called oligonucleotides.
These oligonucleotides can either be functional or non-functional. These oligonucleotides, or RNA molecules, can then replicate
(3) via template-directed polymerization of nucleotides Figure taken from ref.1
from the prebiotic building blocks to the RNA world is
schematically depicted in Figure 1. While the evolutionary capabilities of RNA molecules have already been
established by Spiegelman in the 1960’s and has been
expanded in later years, it still remains difficult to obtain multiple cycles of non-enzymatic RNA replication
in the laboratory. This review will not be concerned
with compartmentalization or metabolic requirements
for the emergence of life in too much detail, but will
focus on the study of (non-enzymatic) self-replication
and evolution of such systems.
The most important feature of the RNA world is
the capability of RNA to create copies of its own structure. The capability of self-replication is imperative
for evolution to occur, and hence for the emergence of
life. Generally there are three different approaches to
achieve replication of RNA in the lab.4 The first of these
is the template-directed replication polymerization that
was previously mentioned, in which a RNA molecule
acts as a template that directs nucleotides to the formation of a complementary strand. A second approach
makes use of a general RNA polymerase ribozyme that
catalyzes the replication process. While this ribozyme
is not a protein and can indeed exist in a RNA world,
it should be noted that such a polymerase would consist of a relatively long sequence of nucleotides and is
therefore unlikely to occur in the early stages of prebiotic chemistry. Instead, such a polymerase ribozyme
would itself be a product of an evolutionary process, a
fact that should be kept in mind when considering evolution experiments involving such structures. A third
approach utilizes RNA molecules that in itself are not
autocatalytic, but can become autocatalytic in combina-
tion with other RNA molecules, leading to a mutually
autocatalytic system.
This review aims to cover the basic principles of
non-enzymatic self-replicating systems and will discuss
some examples of self-replicating molecules that were
synthesized in the lab. In a subsequent section basic
requirements for evolution will be discussed, followed
by recent developments of in vitro evolution experiments. Throughout the review the importance of selfreplication and evolution in the origin of life will be
emphasized.
1. Replicating systems
The most instructive self-replicating system to consider
is probably that of DNA. A DNA molecule consists of
two strands of nucleotides that are intertwined to form
a double helix. During the replication process of DNA,
each of these strands acts as a template for the formation of a complementary strand. In this way an exact
copy of the original structure of DNA is formed and
the DNA has successfully replicated itself. The replication of DNA however is a complex process mediated by
enzymes such as DNA polymerase and topoisomerase.
To understand the origin of life it would be very interesting indeed to achieve molecular replication without
the need of enzymes, since enzymes themselves must
be products of an evolutionary process and can thus
not explain the emergence of living systems from basic
chemical building blocks. The following section will
treat some basics of self-replicating systems, closely
following Philp and Vidonne5 and Von Kiedrowski et
al.6
Towards Darwinian Evolution in Non-Enzymatic Self-Replicating Molecular Systems — 3/18
Figure 2. Scheme depicting the minimal system for selfreplication. Building blocks A and B can bind to form either
template T or its inactive counterpart Tinactive . The formation
of template T directs A and B to a configuration in which they
are in close proximity, leading to the formation of the [T·T]
complex. Dissociation of this complex leads to replication of
the initial template molecule. Figure taken from ref.5
1.1 Minimal self-replicating system
The simplest form of self-replicating systems is that in
which the replicator acts as a catalyst for its own formation from a set of basic building blocks. This simplest
form of a self-replicating system is depicted in Figure
2 and is called a minimal self-replicating system. An
essential requirement for a minimal replicating system
is that molecules A and B are complementary to each
other so that they are able to bind to each other via a
reversible interaction. Figure 2 shows three different
channels in a minimal replicating system. Building
blocks A and B can react via the bimolecular reaction
pathway, to form the template T. In the second pathway
- binary complex formation - A and B first bind together
reversibly through non-covalent interactions to form a
complex [A·B]. This complex however will readily undergo a covalent reaction due to the increased effective
molarity that A and B will experience, leading to an inactive template Tinactive which is folded back onto itself.
The final pathway in the minimal replication system
is the autocatalytic cycle. In this cycle, the building
blocks A and B bind reversibly to the complementary
recognition sites on the template molecule T. This arrangement brings molecules A and B in close proximity
to each other, leading to an increased effective molarity
and enhanced bond formation. When A and B indeed
ligate to each other, a [T·T] complex is formed, which
can then dissociate to yield two identical T molecules.
The autocatalytic cycle thus leads to a replication of the
original template molecule.
Initially, when there are virtually no template molecules
in the mixture but only building blocks A and B, the bi-
molecular and binary complex pathways leading to the
formation of T and Tinactive will be dominant. Clearly,
the inactive template can not lead to autocatalysis and
is therefore parasitic to the self-replication process and
should be minimized if possible. Upon formation of
T, the autocatalytic pathway will become increasingly
important. A requirement for effective autocatalysis
however is the dissociation of the [T·T] into two individual template molecules. If this complex would not
dissociate, the newly formed template molecule would
not lead to further enhancement of the reaction rate, effectively staggering the autocatalytic cycle. This effect
is called product inhibition and is an important limiting
factor in many synthetic replicator systems and should
again be reduced as much as possible for reasons that
will become apparent in a later section of this review.
1.2 Reciprocal self-replicating system
A more complicated situation arises when the template molecules under consideration are no longer selfcomplementary, but instead are complimentary to a second template molecule. In fact, the previously mentioned replication of DNA is an excellent example of
such a reciprocal self-replicating system. One strand of
the double helix acts as a template for the formation of
the other complementary strand and vice versa. Figure
3 shows a schematic representation of a reciprocal replicating system, it consists of two catalytic cycles which
both lead to the same template duplex [TCD ·TEF ]. Instead of two building blocks the reciprocal system has
four basic building blocks labeled C, D, E and F. Building blocks C and D for instance, can react to form the
template TCD which catalyzes the formation of the complementary template TEF from building blocks E and F.
Similarly the TEF template can promote the formation
of the TCD template.
1.3 Reaction kinetics
When considering a mixture containing only building
blocks A and B, the formation of template molecules T
can initially only take place via the bimolecular reaction
pathway (Figure 2). The bimolecular reaction is a relatively slow reaction, since it involves the spontaneous
formation of a covalent bond between the two reactants.
However, if a sufficiently large amount of the template
molecules is formed, the autocatalytic cycle will play
an increasingly dominant role. Because the catalytic
cycle leads to a doubling of the template molecules after
each run, an exponential increase in the concentration of
Towards Darwinian Evolution in Non-Enzymatic Self-Replicating Molecular Systems — 4/18
Figure 3. A cross-catalytic replication scheme in which
the formation of one template stimulates the formation of a
different, complementary template. Figure taken from ref.5
the reaction product would be expected. Naturally, this
exponential increase can not continue indefinitely and
will slow down as the concentration of available building blocks decreases. In summary, this would mean
that for an idealized minimal self-replicating system the
concentration of the reaction product T would show an
S-like or sigmoidal shape. This behavior is depicted in
Figure 4 (b), in which the reaction rate is depicted by
the dashed line and the concentration of the product of
the reaction is depicted by the solid line.
The general rate equation for a minimal self-replicating
system should have the form of Equation 1.6 In which
c is the concentration of the template, αcP is the autocatalytic term and b is the non-autocatalytic term.
dc
= αc p + b
(1)
dt
For 0 < p < 1 the autocatalytic term describes a subexponential growth, whereas p = 1 corresponds to true
exponential growth in the system.6 From this equation
the functional form of the template concentration can
easily be obtained by integration, leading to Equation
2 for parabolic growth (p = 12 ) and Equation 3 for the
case of exponential growth.
1
t
c(t) = (c02 + α )2
2
(2)
c(t) = c0 eαt
(3)
Figure 4. Typical concentration profiles of self-reproducing
systems without any non-autocatalytic contributions. (a)
When the efficiency of the autocatalytic cycle is limited by
product inhibition parabolic growth is observed p = 12 . (b)
When instead product inhibition is absent exponential growth
occurs p = 1. Figure taken from ref.5
In reality however, exponential growth in synthetic
replicator systems is challenging to achieve. Firstly,
there is the binary complex formation which just like
the autocatalytic cycle leads to an acceleration of the
template formation. However, the resulting inactive template Tinactive does not allow for any further molecular
recognition and will not partake in further catalysis of
the reaction. As mentioned before, the binary complex
pathway will thus hinder the self-replication process
and will lead to a deviation from exponential growth. A
synthetic chemist who aims to achieve an efficient selfreplicating system should therefore strive to decrease
the contribution of the binary complex pathway or, if
possible, eliminate it altogether.
If the chemist indeed succeeds in fully eradicating
the binary complex formation, he would in our model
end up with a purely autocatalytic system. Nonetheless,
perhaps much to his surprise, the system would most
likely still exhibit subexponential growth. To understand why this is so one needs to consider the effect of
the dissociation rate of the [T·T] complex on the efficiency of the autocatalytic reaction. For the reaction the
be efficient, the complex should dissociate quite readily
in order to obtain two free template molecules which
Towards Darwinian Evolution in Non-Enzymatic Self-Replicating Molecular Systems — 5/18
can then again undergo autocatalysis. This requires
the [T·T] complex to be thermodynamically less stable
than the trimer [A·B·T]. If the template complex is too
stable, the template molecules will not dissociate and
the reaction rate will be severely limited by this product
inhibition. Note that such a strongly inhibited system
would still classify as a self-replicating system since
it manages to produce a copy of the original template,
but is not considered to be autocatalytic since it fails to
reproduce significant amounts of template molecules.
A system in which product inhibition determines
the overall reaction rate will deviate from reaction order
p = 1 and the concentration profile will show a subexponential growth. In the case of p = 0.5 the system
is said to obey the square root law of autocatalytic systems, as can be understood from the form of Equation
2. Figure 4 a shows a typical concentration profile for
an autocatalytic systems that shows parabolic growth.
1.4 Self-replication in action
Pioneering work in the field of non-enzymatic selfreplication has been performed by the groups of Von
Kiedrowksi7 and Orgel. The latter of these were the first
to report on a template-directed self-replicating oligonucleotide.8 However, it was later realized that this system
did not represent a minimal self-replicating system but
instead involved a polycondensation of activated nucleotides.9, 10 Only a few years later, Von Kiedrowski
etal. were the first to report multiple cycles of selfreplication in a system of complementary nucleotidebased oligomers. To achieve template-directed selfreplication without the aid of enzymes, they used two
trinucleotides. Upon activation, these trinucleotides can
condense to form a hexamer template molecule T , depicted in Figure 5, which catalyzes its own formation.
The autocatalytic nature of the reaction was proven by
adding small amounts of preformed template molecules
to the reaction mixture, which resulted in a parabolic
reaction rate of p = 12 . Exponential growth in this system is not obtained due to the high stability of the [T·T]
dimer, leading to product inhibition. Although the efficiency of the reported autocatalytic cycle is rather low,
it still was a clear demonstration of a template-directed
self-replicating system and Von Kiedrowski did not fail
to recognize the potential of natural selection in such
systems.
Later research focused on overcoming the product
inhibition problem in order to obtain exponential instead of parabolic growth of the template molecules.
Figure 5. The first oligonucleotide capable of templatedirected self-replicating without the need of enzymes. The
depicted hexamer template T is formed from two trimer building block and catalyzes its own formation. Self-reproduction
of this molecule was shown to result in parabolic growth of
the template concentration. Figure taken from ref.7
Exponential growth of the replicating species is a vital
property for Darwinian evolution and is thus necessary
for the emergence of an RNA world from prebiotic
building blocks.
A very successful approach to overcoming product
inhibition involves the immobilization of the template
molecules by fixing them onto a solid support. On first
sight this might seem like a rather artificial approach
that provides no real link to prebiotic conditions. However, evidence has been found that surfaces of mineral
complexes might have played a major role in catalyzing
prebiotic replication processes.12, 13 Kiedrowski etal.
were able to demonstrate exponential growth of oligonucleotides using a method that they gave the eloquent
anagram; SPREAD (Surface-Promoted Replication and
Exponential Amplification of DNA analogues).11 In the
SPREAD technique an oligonucleotide template strand
is immobilized via an irreversible interaction with a
solid support. A complementary strand is then produced
via the template-directed binding of free nucleotides
from the solution. The copied strand is released from
the template and is in turn itself immobilized on a solid
support preventing product inhibition via the formation
of stable template dimers. Like Kiedrowski and coworkers rightfully notice, this system allows for evolutionary
Towards Darwinian Evolution in Non-Enzymatic Self-Replicating Molecular Systems — 6/18
Figure 8. Self-replication of a helical peptide. Molecular
recognition causes the small peptide fragments to assemble
on the template peptide in order to form a stable coiledcoil structure. Formation of a covalent bond gives rise to a
template-duplex in a coiled-coil configuration. Depending
on the length and stability of the coiled-coil, the duplex
dissociates in the original template and a copy. Figure taken
from ref.17
Figure 6. Replication scheme involving the SPREAD technique which prevents product inhibition. (1) A template
molecule is immobilized on a solid support and (2, 3) a complementary copy is produced by template-directed replication.
Finally the copied strand is (4) released from the template
and is in turn immobilized. Figure taken from ref.11
Figure 7. Figure showing (A) a coiled coil motif due to
hydrophobic interactions between hydrophobic amino acids
in the individual helices. (B) Helical wheel diagram showing
how the hydrophobic amino acids situated on the a and d sites
can interact with each other to form the coiled coil. Figure
taken from ref.14
processes to take place. Moreover, such immobilized
systems are proposed to be even capable of amplification of mutations. The introduction of mutations would
lead to a weaker base pairing between the template
molecule and its copy, thus increasing the efficiency of
the separation of the template duplex.11
Considering the proposed abundance of aminoacids,
it is natural to assume the presence of peptides and
oligopeptides under prebiotic conditions. However, initially only very short peptides were produced in experiments under prebiotic conditions, eliminating them as
a potential precursor of life. When forming α-helices
however, longer polypeptides can be stabilized by the
formation of coiled-coil motifs as in Figure 7. If every
a and d position of each individual helix is occupied by
a hydrophobic amino acid, the helices can intertwine
and bury their hydrophobic side groups into each other.
This hydrophobic interaction that drives the formation
of coiled-coil motifs can be further enhanced by electrostatic interactions between amino acids residing on
the c and g positions of the α-helices.
Ghadiri etal. showed that such coiled-coil peptides
were capable of self-replication.15 As depicted in Figure 8, helical polypeptides can act as a template for
shorter peptide fragments by means of molecular recognition. The peptide building blocks again are ligated,
resulting in the formation of a template duplex with a
coiled-coil motif. When separated from the original
template, a copy of the template is obtained. Initially
these replicating systems were reported to show only
parabolic growth, because of the very high stability of
the coiled-coil structure leading to product inhibition.15
This problem was later solved by Isaac and coworkers
by reducing the length of the template molecule, which
led to a decreased stability of the template duplex. Using this approach they obtained near exponential growth
of the template concentration of p = 0, 91.16, 17
The above examples all illustrate that, while not
trivial, it is indeed possible to obtain self-replicating
behavior in the absence of enzymes. Although certain
constraints have to be imposed on the system in order
to obtain efficient autocatalysis, for instance immobilization of the template molecule or compartmentalization of the reactants, it is likely that such systems have
played a major role in the emergence of RNA molecules
form basic prebiotic building blocks. While this marks
a significant contribution to our understanding of the
early stages of prebiotic life (Figure 1) it cannot directly
explain how more complex structures like all different types of ribozymes could have formed in the RNA
Towards Darwinian Evolution in Non-Enzymatic Self-Replicating Molecular Systems — 7/18
world. To understand how simple oligonucleotides can
eventually give rise to increasingly complex molecules
we will turn to the topic of Darwinian evolution in
molecular systems.
2. Requirements for Darwinian evolution
One of the most remarkable and key features of life
is the fact that it has a strong tendency to diversify
and increase in complexity. Whereas life once must
have started out as a cellular life form, it has diversified
into a vast variety of species∗ ranging from aquatic
to airborne species. The mechanisms governing this
diversification in biological systems were first described
by Darwin in his famous work On the Origin of Species,
but are still not understood in full detail.18 It was only
in the 1960’s that Spiegelmann extended the scope of
Darwinian evolution to chemical systems by studying
the evolution of RNA-complexes.19 Understanding of
how species can evolve on a molecular scale might
eventually also provide better understanding of how
species arise in nature.
Darwinian evolution in a chemical system involves
three different processes that are summarized in Figure 9. First the parent molecule is replicated (amplified) to yield a large number of copies, which can be
achieved with an autocatalytic cycle as previously described. A requirement for Darwinian evolution in a
self-replicating system is that the concentration profile is sigmoidal, meaning that an exponential growth
(p = 1) is a prerequisite.16, 20, 21 Mutation involves the
emergence of a difference between the parent template
and its copies. While for a simple system the copying
process might be relatively flawless, for more complicated structures occasional errors might occur during
the copying process. If too many errors occur in the
replication process, the structure of the parent template
might be lost entirely and the hereditary information
is not passed on: the so-called error catastrophe.22, 23
Small mutations however might actually be advantageous for the system in the sense that the newly formed
copies are more stable or might prevail under a change
of environment.
2.1 Amplification
During amplification a large number of copies of the
parent molecule is made via a replication process. In
∗ Recent
studies estimate that the total number of species on
earth lies around 8 million, of which a large number still remains
undiscovered until this very day.
Figure 9. Three processes involved in Darwinian evolution.
Species must be amplified to obtain a large population. During this amplification mutations might occur, which leads to
natural or artificial selection. Figure taken from ref.24
animals or other lifeforms it is quite clear that reproduction leads to a transfer of genetic information from the
parent to the offspring. Not only are the vital structures
of the organism transferred but also some peculiarities
like a specific eye color or a hereditary disease is passed
on to the next generation. The transfer of information in
replicating molecules may be a bit less obvious, but if
one considers a polymer with a specific sequence of subunits, it is clear that some form of genetic information is
transferred if the copies have the exact same sequence
as the parent molecule.
The survival of a (molecular) species under certain environmental conditions depends on the rate of
amplification and the rate of decomposition. If a molecular species decomposes at a higher rate than that it is
amplified, the species will become extinct. If, on the
other hand, the sequence and structure of the molecule
is such that amplification dominates the decomposition, the species is well-adapted to its environment and
will persist under the external conditions. The tradeoff between the rate of amplification and the rate of
decomposition can be captured by a third parameter;
the rate of accumulation. A high positive rate of accumulation means that the species is thriving in that
environment, while a negative rate means that it will
perish over time.25
2.2 Mutation
The environmental conditions of a molecular species
however will not be in a steady-state. Instead, the environment is continuously evolving. Consider for instance
changes in temperature throughout the year or day, different amounts of light and moisture to which a system
Towards Darwinian Evolution in Non-Enzymatic Self-Replicating Molecular Systems — 8/18
will inevitably be subjected. A species that is very welladapted to a certain environment, might not persist at
a later time when the environmental conditions have
changed. In fact, if it were not for the presence of small
mutations in the genetic information, a species would
be very fragile. Small errors in the replication process
lead to mutations of the genome, this can for instance
manifest itself as the insertion, omission or replacement
of a subunit of the molecular sequence that is copied. If
the number of mutations is only small compared to the
length of the molecule, hereditary information is still
largely preserved. Moreover, the mutants may be better
adapted to a change in environmental conditions than
their parents. If the mutants indeed have a higher rate
of accumulation than their parents, they will eventually
overtake their parents and will become the new dominant species. Eigen et al. noted however that the case
is somewhat more complicated than a single mutant
species overtaking another species. They introduced
the concept of so-called quasi-species as an analogue to
conventional species in biology. A quasi-species consists of a master sequence with a dynamic distribution
of closely related mutants. This concept captures the
fact that for relatively high mutation rates not a single
fittest type of species, but rather a distribution of closely
related mutants survives. The mutants in this asymmetric distribution around a master sequence all replicate
at a different rate, which leads to the production of
further mutants.10, 22 Such quasispecies behavior was
recently reported in an in vitro evolution experiment
with replicating RNA species.26
There must of course be a constraint on the number
of mutations that can occur without losing too much
hereditary information from the parent molecules. In
the same work, Eigen showed that there must be an
inverse relation between the length of the genome and
the mutation rate. That is, the replication process of a
long molecule should have a much higher fidelity than
that of a smaller molecule. If the rate at which errors in
the replication occur exceeds a certain error threshold,
the genetic information will disintegrate and the species
will go extinct.22, 27 In fact, the reason that viruses are so
good in adapting to different environments and always
seem to be one step ahead of the defense mechanisms
of the host is because the replication process of the
viral genome operates very close to the error threshold,
allowing for as many mutations as possible without the
loss of genetic information27, 28
2.3 Selection
Natural selection operates on the phenotype, i.e., the
observable traits of a species. An individual that is better adapted to its environment is more likely to survive
then one that is less adapted. For instance, a white polar
rabbit has a much higher chance of survival in an arctic
environment than a brown polar rabbit. This higher
survival rate will lead to a larger amount of offspring
for that type of individual, favoring their presence in the
population. The phenomenon of natural selection also
operates at the molecular level. In a system of replicating molecules that are competing for the same building
block, the molecules that have the highest replication
rate will come to dominate others. The governing selection criterion in such a system is thus the capability of
efficient self-replication.
It is also possible to perform artificial selection on
molecules, similar to breeding of animal species. Desirable traits are in this case selected by the experimenter,
for example the ability to bind a certain ligand or to act
as a catalyst in a chemical reaction.25 However, in these
cases evolution is directed by the researcher and will
not lead to spontaneous diversification of the system
as would be typical for life. In order to obtain a form
of life from a molecular system, it should be able to
grow increasingly complex and diverse. Systems that
undergo such undirected diversification can in the end
give rise to ecosystems full of complex organisms or
structures.29 Note that these organisms then would all
be part of an evolving ecosystem, and a proper and complete description of life should therefore not only be
on the individual level but also on the level of entire
ecosystems.30
3. In vitro evolution
The first experiment that was concerned with Darwinian
type evolution in a chemical system was performed by
Spiegelman et al. in 1967.19 RNA replicase and a small
input of genomic RNA were successfully isolated from
a bacteriophage called Qβ . The RNA molecules in this
system are replicated by an RNA replicase molecule.
By successive rounds of amplification and selection,
selection pressure was introduced to the system by favoring fast reproducing entities of the genomic RNA.
Since smaller sequences are able to replicate at a higher
rate then longer sequences, shortened mutant species
are favored over longer sequences. This eventually led
to a strong decrease in the genome size of the RNA
Towards Darwinian Evolution in Non-Enzymatic Self-Replicating Molecular Systems — 9/18
molecules. However, this result is not as trivial as it
may seem on first sight since it is of vital importance
that the mutant species do not lose their ability to reproduce, indicating that only specific parts of the genome
that are not needed for recognition by the polymerase
were deleted. Although the RNA molecules involved
are not strictly self-replicating but are in fact replicated
by the RNA replicase, the study still marks a starting
point in the field of in vitro evolution. In the following
sections some recent advancements towards the synthesis of de novo life will be discussed.
3.1 RNA
Owing to the importance of RNA in viral species and
the origin of life, evolution experiments are most often performed using RNA molecules or closely related
derivatives. In fact, it is nowadays possible to perform
natural selection on oligonucleotides by iterative amplification and selection processes using a technique called
systematic evolution of ligands by exponential enrichment, or SELEX. In SELEX, a library of DNA and
RNA sequences is exposed to for instance a certain target. In multiple selection rounds the binding species are
selected and amplified, while the non-binding DNA and
RNA molecules are disposed of. In this way molecules
can be easily selected on their ability to bind to a specific target.31–33
However, these in vitro evolution experiments all
exploit RNA-based enzymes (ribozymes) or proteins in
their replication process to obtain exponential growth.
A minimal form of such a catalyzing ribozyme would
have a length of around 200 nucleotides, which means
that it is a relatively long and complex molecule. While
ribozymes supposedly have played an important role in
the RNA world, it does not seem very likely that these
complex ribozymes were present in the very first stages
of the RNA world. Efforts have been made to obtain in
vitro evolution of RNA in the absence of any enzymes.
Unfortunately, the demonstration of multiple cycles of
non-enzymatic RNA replication in a test tube is troubled
by a few factors. First of all a non-enzymatic replication
mechanism would result in a random linkage of the
nucleotides, giving rise to a distribution of 3’5’ and 2’5’
linkages as depicted in Figure 10. In current biological
systems however the backbone is homogeneous instead
of heterogeneous and only the 3’5’ linkage is observed.1
Apart from this, it is observed that the RNA duplex that
is formed upon replication is quite stable and can have
dissociation temperatures as high as 90 ◦ C.1, 13 Without
Figure 10. Different types of backbone linkage in an RNA
molecule. Individual nucleotides can either bind to the 3’ or
2’ site of another nucleotide. Fig taken from ref.1
an enzyme that separates the newly created strands, this
stability would lead to product inhibition, staggering
the self-replication process.
3.2 Cross-catalyzing RNA
To avoid the need for catalyzing ribozymes, it is proposed that cross-catalytic cycles may have played an
important role in the primary stages of the RNA world.
Joyce et al.34 showed that a system of two RNA enzymes can catalyze each others synthesis from a mixture
of four different building blocks via template-directed
replication. The RNA ligase molecule E can bind two
oligonucleotide building blocks A’ and B’ and promote
their ligation to form the ligase E’. The newly formed
ligase E’ can then in turn promote the formation of E,
as depicted in Figure 11 a. This cross-catalytic reaction however occurs at a very slow rate. In order to
enhance this rate, enzymatic in vitro evolution of the
RNA molecules was performed in order to obtain a set
of fast replicating species.
It was found from in vitro evolution experiments
that the introduction of G·U base pairs close to the site
of ligation leads to enhanced cross-catalytic activity.
Figure 11 b shows the sequence and secondary structure of the A·B·E’ complex. The site of ligation is
indicated by the curved arrow and the G·U pairs that
are depicted in a solid box induce a wobble in the sequence that results in the enhanced catalytic behavior.
Towards Darwinian Evolution in Non-Enzymatic Self-Replicating Molecular Systems — 10/18
Figure 11. (a) Cross-catalyzed replication of template molecules E and E’ from their building blocks A(’) and B(’) . (b)
Secondary structure of the template duplex. The curved arrow denotes the site of ligation of the building blocks, the dashed
boxes include the sequences to be mutated and the solid lines indicate the sites of the G·U base pairs inducing the wobble
that enhances catalytic activity. (c) The altered sequences of the 12 different template moluces, the E’ molecules have
complementary sequences in the base pairing part (horizontal) and identical sequences in the catalytic part (vertical). Dark
circles denote the differences relative to E1. Figure taken from ref.34
When this wobble is installed in both enzymes of the
cross-catalytic set, exponential growth of the system can
be achieved over multiple cycles. Since this necessity
for evolution is now satisfied, Darwinian evolution in a
cross-catalytic system lies within reach. To study this,
Joyce and his team prepared 12 pairs of cross-catalytic
enzymes and their corresponding building blocks, that
have alteration in parts of the sequence denoted by the
dashed line in Figure 11 b. The different pairs are denoted as E1 to E12 and are shown in Figure 11 c. It is
important to note that mutations between enzymes are
such that the stability of the ligase duplex due to base
pairing is not altered, but only the catalytic activity and
replication rate are affected. All these cross-replicating
enzymes were shown to have good amplification, with
the E1 pair showing the highest rate of amplification.
A serial transfer experiment was performed on a
mixture that contained the 12 different enzyme pairs
and its corresponding 48 building blocks (A1, A1’, B1,
and B1’ for pair E1 for example). In such a serial transfer experiment a small percentage, in this case 5%, is
transferred to a new reaction mixture after an amplification round took place. By doing this for multiple
rounds, large amplification factors can be achieved. For
this experiment it is important to note that A1 does not
necessarily have to be ligated to B1, but that it in fact
can ligate to any of the other B-type building blocks
although they are mismatched to the template. This
freedom of recombination leads to 132 possible combinations of building blocks. After 20 successive transfers
a 1025 -fold amplification was reached. A sample of
100 of these clones contained only 7 non-recombinant
clones, whereas the rest were all ligated to building
blocks that were not their original partners. Figure 12
Towards Darwinian Evolution in Non-Enzymatic Self-Replicating Molecular Systems — 11/18
Figure 12. Distribution of the species present in the reaction
mixture after 20 transfers. E and E’ molecules are represented by the dark and light shaded bars respectively. Note
how certain species have come to dominate the population,
particularly A5B3. Moreover, only 7 molecules were found
to be paired to their original partner (corresponding to the
shaded diagonal). Figure taken from ref.34
shows the distribution of different E (dark columns) and
E’ (light columns) enzymes in the final sample. This
result shows how fitter molecular species can come to
dominate the population after several rounds of amplification. Fitness of the molecules depends in this case on
their ability to perform cross-catalytic replication with
another molecules.
This study by Joyce et al. demonstrates how survival of the fittest can lead to certain molecules dominating a population in a cross-catalytic replication process. However, the evolution of this system lacks openendedness in the sense that it can not lead to complexification of the species and there is an upper bound to the
final diversity of the species. The number of building
blocks that is provided to the system predetermines the
total diversity of the final species, in this case 12 × 12
different possible species.
3.3 Cooperative catalytic system
The concept of such a cross-replicating system can be
readily extended to higher order systems, involving
three, four or even more members. Eventually one
could envision an entire network of cross-replicating
molecules. Lehman et al.35 showed that a mixture
of relatively short RNA segments can self-assemble to
form self-replicating ribozymes. These ribozymes in
turn gave rise to spontaneous formation of cooperative
networks, that were shown to grow faster then the autocatalytic replication rate of the individual ribozymes.
Moreover, cooperative systems are generally more stable towards parasites then self-replicators and are able
to gain in complexity.4, 35
In the study a ribozyme of around 200 nucleotides
called Azoarcus was used. This ribozyme consist of
four different RNA strands that can self-assemble covalently in an autocatalytic manner, as depicted in Figure
13 a. The effectiveness of this self-replication process
depends on the ability of the internal guide strand (IGS)
to recognize the three target sites. To form a cooperative set, the ribozymes were fragmented in two different
pieces in three different ways, creating three different
pairs I1, I2 and I3 which are shown encircled in Figure 13 b. Furthermore, the target and IGS sequences
were altered such that autocatalytic self-replication is
minimized. The sequence was however chosen such
that the IGS of one pair is matched to the target sites
of the next pair. In this way one ribozyme, say E1, can
catalyze the formation of the next ribozyme, E2, from
its non-covalently bound building blocks I2. This ribozyme can in turn catalyze the formation of E3 from
its building block and finally, to close the cycle, E3
can catalyze the formation of the E1 ribozyme. This
cooperative system is depicted in Figure 13 b and it
was observed that a mixture containing all three pairs
resulted in a much higher yield (a factor 125) than the
isolated pairs, proving that the system replicates in a
cooperative manner.
Interestingly, it was shown that in isolation the autocatalytic replicators dominate the mixture, whereas in
a mixture of all different components the cooperative
network grows much faster than the selfishly replicating molecules. However, this result was obtained using
deliberately designed pairs with specific targets. Things
get a lot more fascinating when one of the nucleotides
of the IGS (M) and target sites (N) is randomized, creating a mixture of 48 matched and unmatched pairs
in total, as schematically depicted in Figure 14 a. After incubation of all six sets of Figure 14 a for several
hours, all of these 48 possible sequences were indeed
found in the mixture. The most remarkable and vital
point reported on by Lehman and his coworkers however, is the observation that initially the replication is
dominated by small two-membered cooperative cycles
and even autocatalytic cycles in which N and M are
complimentary. This initial rise of the autocatalytic
replicators is depicted in Figure 14 b by the dashed line
with crosses, the contribution of the two-membered cycles is depicted by the dashed lines with dots (depicted
Towards Darwinian Evolution in Non-Enzymatic Self-Replicating Molecular Systems — 12/18
Figure 13. (a) Secondary structure of the Azoarcus ribozyme consisting of four different strands of RNA, W, X, Y, and Z.
Self-replication is mediated by recognition of the target sites by the IGS (grey boxes), leading to ligation of the strands at certain
sites, denoted by dots; •. The dashed line indicates the catalytic core of the resulting ribozyme. (b) Diagram of a cooperative
replicating system. The formation of the covalent ribozyme E3 from the non-covalent I3 complex is catalyzed by E2 ribozyme.
The formation of this E2 is in turn catalyzed by E1, which is catalyzed by E3, resulting in a cyclic dependence. Numbers above
the arrows denote the advantage of cooperativity. Figure taken from ref.35
value ×10). At later times a transition to the more complex three-membered cylces was observed as witnessed
by the rise of the solid line (×10, 000). After 8 hours, it
was observed that replication occurs almost exclusively
via cooperative cycles and that all genotypes contribute
increasingly with time. This result shows how an initially autocatalytic cycle can give rise to increasingly
complex systems of cooperative replication over time.
To better mimic prebiotic conditions in which iterations over multiple generations would have occured, a
serial transfer experiment using the same set of replicators was also performed. In this experiment an aliquot
of the reaction mixture is transferred to a new flask
with building blocks every hour, so that the more stable
and fast replicating molecules and networks are favored.
Again a transition from autocataltyic cycles to more
complex systems was observed, but this time only a
select number of genotypes was present in the final
mixture in significant amounts.
Such cooperative systems are capable of complexification and natural selection and can therefore be of
importance in bridging the gap between replication of
simple short RNA molecules from nucleotide building
blocks and the formation of more complex ribozymes.
Figure 14. (a) The different combinations of IGS strands,
tags and break junction give rise to a total of 48 different pairs.
(b) Graph showing the frequency of autocatalytic replicators
(dashed, crosses), the two-membered cycles (dashed, dots;
×10 depicted value) and the three-membered cycles (solid,
×10, 000) over time. Not the emergence of the more complex
three-membered cycles at later times.
Towards Darwinian Evolution in Non-Enzymatic Self-Replicating Molecular Systems — 13/18
The observed cooperative behavior relies on recognition strands and tags, so that it will only play a role for
the assembly of intermediated sized oligonucleotides.
Small oligonucleotides would likely still replicate more
efficiently via auto or cross catalytic cycles. At a certain length scale the formation of cooperative systems
becomes favorable and these mechanisms might take
over the replication process, allowing for complexification and diversification of the system. However, since
the replication of each member is dependent on one or
more other members of the system, the members should
be in close proximity to each other in order to obtain
a stable system. This requires heterogeneity of the reaction mixture, which is of course readily achieved in
the laboratory but is not so prevalent under prebiotic
conditions. In order to fulfill this heterogeneous condition locally, a specialized compartmentalization should
act in concert with the cooperative replication system.
How such compartmentalization might occur is another
topic entirely, but it was proposed that compartmentalization can actually aid Darwinian evolution of replicase
molecules.36, 37
3.4 Cross-catalyzing peptides
It should also be considered that it is possible that there
was a predecessor to RNA that actually bears little to no
resemblence to RNA, yet is capable of self-replication
and information storage.38 The transition from such a
system to the RNA world is however much harder to
envision than the transition from a system that is closely
related to RNA, since information must be successfully
transferred between the two systems to ensure genetic
continuity.
Recently, Otto et al. have demonstrated a selfreplicating system capable of diversification using a
systems chemistry approach.39 Following their successful design of an exponentially growing self-replicating
system,40 they use two building blocks, 1 and 2, to form
a dynamic combinational library (DCL) of molecules.
These building blocks consist of an aromatic core that
is functionalized with two thiol groups and a peptide
chain (Figure 16 a). Building block 1 and 2 are very
closely related to each other and differ only in the residual group on the peptide chain. These peptide building
blocks can then be oxidized to form macrocycles of different sizes as depicted in Figure 16 b. The design of the
peptide chains is such that self-assembly of the chains
into parallel β -sheets is promoted, which in turn leads
to the formation of stacks of macrocycles as shown in
Figure 15. Plot showing the relative concentrations of set I
(red), set II (blue) and the link between them; set III (purple).
Note how at first only set I is present in the mixture, while at
some point this set gives rise to the descendant set II. Figure
taken from ref.39
Figure 16 c. Growth of these stacks occurs exclusively
via the ends of the fibers and it is therefor not surprising that the reaction rate is strongly dependent on the
amount of fibers present in the mixture. As soon as a
fiber reaches a critical length it can fragment when mechanically agitated, for instance by stirring or shaking
the mixture. When fragmentation occurs, the number
of available fiber ends is doubled, leading to an exponential self-replication.
In a previous work by the same group it was already
shown that the less hydrophobic building block 2 tends
to form larger octameric macrocycles than the more
hydrophobic building block 1 which forms hexamers.40
This is reasonable, since a weaker hydrophobic interaction would need more individual interactions in order
to achieve the same stability as a more hydrophobic
counterpart.
By using a mixture containing two different building blocks instead of one, the replicators can potentially
undergo mutation by incorporating a different building
block into their macrocycles. A mixture with equal
concentrations of both building blocks was prepared
and monitored over the course of 35 days. Initially a
complex mixture of four different trimers and five different tetramers was observed, which were attributed to
the disulfide exchange of the oxidized building blocks.
After some days however, a set of hexamers which was
enriched in building block 1 arose in the mixture. This
set was labeled set I and corresponds to the red line in
Figure 15. As the emergence of set I depletes the mixture from building block 1 the environmental conditions
are essentially changed up to the point where a second
set of hexamers arises which is rich in building block 2.
Towards Darwinian Evolution in Non-Enzymatic Self-Replicating Molecular Systems — 14/18
Under normal conditions this second set would be
unfavorable since it involves a large amount of building
block 2, which is less hydrophilic than 1 and tends
to form octameric instead of hexameric cycles. This
behavior can be explained by noting that there is an
ancestral relationship between set I and set II, in which
set I is the ancestor of set II. When macrocycles that
are rich in building block 2 are exposed at the fiber
ends of set I, they act as a template for the formation of
members of set II. Indeed, significant amounts of set II
members only form when a seed of set I is present that
contains 2-enriched members. Set I is therefore able to
transfer information about its macrocyclic size to set II.
This study shows how replicators that compete for
the same building blocks diversify to give rise to two
sets of replicators, that both require the same building
blocks but not to an equal extent. A rough macroscopic
analogue to this is the famous example of the finches
that Darwin observed on the Galapagos islands. As
the finches competed for food, they started to diversify and fill different food niches eventually giving rise
to entirely different species. The experiment showed
evolutionary behavior with ancestral behavior, in the
sense that the system was able to diversify and grow
in complexity on its own account and that information was passed on between two successive generations.
Although this system does not directly relate to the
emergence of complicated RNA structures from simple
oligonucleotides, it provides additional insight into Darwinian evolution in non-enzymatic replicating systems
and marks another important step towards the realization of synthetic life.
4. Conclusions and perspectives
It has been shown that in vitro synthesized molecular
systems are capable of undergoing Darwinian evolution.
However, it still remains difficult to obtain Darwinian
evolution in systems that do not require catalyzing enzymes in their replication process. The absence of evolution in simple self-replicating molecules is mainly
due to the fact that it is difficult to achieve exponential
growth of the replicator. Factors limiting the efficiency
of the self-replication process are the presence nonautocatalytic pathways and product inhibition. Methods
to avoid product inhibition like the SPREAD technique
and the destabilization of template-duplexes have been
developed, but still only a small number of evolution
experiments were performed in the absence of enzymes.
A good way to obtain exponential growth of the
replicator is introducing a catalyzing enzyme in the
system. Darwinian evolution in enzyme-catalyzed selfreplicating systems have been widely reported and provides good insights into how our current RNA-DNAProtein world might have evolved from an RNA-world.
How this RNA-world could have emerged from prebiotic building blocks in the absence of enzymes remains
an unsolved problem up to date, and efforts are made
to better understand evolution in non-enzymatic selfreplicating systems. It should also be noted that the
replication of relatively long RNA sequences without
the aid of a sophisticated enzyme is problematic, since
the non-enzymatic replication process has a relatively
low fidelity, limiting the length of RNA that can be
copied without loss of information. Since the assembly
and subsequent replication of RNA sequences in the
absence of enzymes is troublesome, the possibility of
other information carrying polymers as precursors to
RNA should be considered as a feasible option. However, since such proposed molecules are not found in
nature their existence remain very speculative and is
only legitimate because their building blocks are expected to be present in the prebiotic soup.
Promising steps towards in vitro evolution without
enzymes have been made using cross-catalytic cycles
and system approaches, instead of autocatalytic replicators. Such systems have been shown to be capable of
growing in complexity and allowing for diversification
of the member species. However, evolution is such system is often in some sense directed by the experimenter
by supplying certain building blocks, introducing specific mutations or applying a selection pressure for a
Towards Darwinian Evolution in Non-Enzymatic Self-Replicating Molecular Systems — 15/18
Figure 16. Figure depicting (a) building blocks consisting of a peptide attached to an aromatic ring. Building blocks 1 and 2
differ only in the nature of their side groups. (b) The building blocks can form macrocycles of different sizes upon oxidation,
which are in equilibrium with eachother. (c) Full scheme showing how building blocks oxidize to form macrocycles that in turn
form stacks due to β -sheet formation. Stacks grow from their fiber ends and fragment upon aggitation, leading to more fiber
ends and a faster growth. Figure taken from ref.39
Towards Darwinian Evolution in Non-Enzymatic Self-Replicating Molecular Systems — 16/18
certain functionality of the final products. These system
approaches towards achieving open-ended Darwinian
evolution appear to be very promising and will likely
play an important role in unraveling the mechanisms
that governed the pre or early RNA world. Further
studies should be aimed at obtaining and understanding
undirected evolution processes, since these have not
only played an important role in the emergence of life
but also govern the evolution of macroscopic species.
It must also be noted that the capability of selfreplication in itself does not yet constitute life. In order
to construct a protocell that fulfills the minimal requirements for life, one does not only need a system that
is capable of self-replication but one that also shows
metabolic activity and is isolated from its environment.
As we have seen, there is still no full understanding of
how the RNA world could have come into being and
there are still open question to be answered in this field.
At the same time it is not clear how compartmentalization and the emergence of metabolism could have taken
place. The construction of a protocell from scratch is
therefore the ultimate goal for the synthesis of life. This
would however require a very thorough understanding
of life and its origins and we can only speculate whether
this goal will ever be achieved or not.
Acknowledgments
I would like to thank Prof. Sijbren Otto for his guidance
and advice in the writing of this paper and for bringing
this fascinating topic to my attention. I would also like
to thank Prof. Ryan Chiechi for his inspiring lectures
on the art of scientific writing and literature searching,
which helped me considerably during the writing of this
review.
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