1. The Effect of RNA Structure on Nonenzymatic Template-Directed
Polymerization in the RNA World
A thesis presented by
Nikola A. Ivica
for the partial fulfillment
of the requirements
for the degree with honors of Bachelor of Arts
in the field of Chemical and Physical Biology
Harvard University
Cambridge, Massachusetts
March 2012

2. 2
ACKNOWLEDGMENTS
I would like to thank Dr. Irene A. Chen for her mentorship and advising. I am thankful to
Dr. Benedikt Obermayer and Professor Ulrich Gerland for help with theoretical predictions,
discussions and advice. My thanks also go to Dr. Sudha Rajamani and other members of the
Irene Chen Lab for their comments, advice on experimental models, and help during entire time
spent in the lab. I would also like to thank Dr. Bodo Stern and Dr. Thomas Torello for comments,
and Professor Erin O’Shea for use of equipment. Finally, I want to thank my family and friends
for their encouragement and support. This research was funded by the Harvard Research College
Program and the 2011 summer undergraduate research fellowship from Harvard Origins of Life
Initiative.

3. 3
LIST OF CONTRIBUTIONS
The project was conceived by Dr. Irene A. Chen following her work on the error
‘catastrophe’ in nonenzymatic template-directed replication of RNA and other projects
concerning the RNA world and origins of life. The project was jointly designed by Nikola A.
Ivica and Dr. Irene A. Chen. The experimental data on nonenzymatic polymerization reactions
and hybridization percentages was obtained independently by Nikola A. Ivica. The design of
template sequences was jointly done by Dr. Irene A. Chen, Dr. Benedikt Obermayer, and Nikola
A. Ivica. The theoretical predictions of template structural parameters were done by Dr. Benedikt
Obermayer. The ‘asymmetry hypothesis’ was jointly developed by Dr. Benedikt Obermayer,
Professor Ulrich Gerland, and Dr. Irene A. Chen. Data and results were interpreted by Nikola A.
Ivica with assistance and guidance from Dr. Irene A. Chen and Dr. Benedikt Obermayer.

4. 4
ABSTRACT
The RNA world hypothesis states that the modern living systems based on DNA and
proteins were preceded by living systems based solely on RNA. The replication of genetic
material was believed to be driven by nonenzymatic template-directed polymerization of RNA.
We show that the structural stability of template strand hinders the process of nonenzymatic
template-directed primer extension. Furthermore, we analyze the problem of ribozyme
emergence and show that there are structurally unstable RNA sequences that can act as template
strands and give rise to structurally stable sequences that can potentially act as a ribozymes.

5. 5
TABLE OF CONTENTS
List of Figures/Tables 6
Abbreviations and Terminology 7
Introduction 8
Results and Discussion 15
Sequence Design 15
Formation of the Template-Primer Complex 21
Correlating Rates of Primer Extension with Predicted Structural Parameters 24
Asymmetric Sequences 29
Conclusion 34
Materials and Methods 36
References 38
Appendix 43

6. 6
LIST OF FIGURES/TABLES
Figure 1. Nonenzymatic template directed polymerization. 13
Figure 2. Asymmetric sequences and structural stability. 14
Figure 3. Template sequence design. 17
Figure 4. Predicted secondary structures of template and primer strands in 18
equilibrium with template-primer complex.
Table 1. Predicted structural parameters. 20
Figure 5. Template-primer heterodimer complex native gel. 22
Figure 6. Theoretical and experimental values of hybridization percentage. 23
Figure 7. Experimental approach to determining the rate of primer extension. 26
Figure 8. The primer extension reaction. 27
Figure 9. Correlation plots. 28
Figure 10. Histogram of ensemble minimum free energies. 31
Figure 11. Heat map histogram for G-U base pairing. 32
Figure 12. Testing asymmetric sequences. 33
Table 1A. Numerical values for the rate of primer extension. 45
Table 2A. Numerical values for hybridization percentage. 46

7. 7
ABBREVIATIONS AND TERMINOLOGY
Guanosine- G
Cytosine- C
Uracil- U
Adenosine- A
Guanosine 5’-phosphorimidazolide- ImpG
Guanosine triphosphate- GTP
Ribonucleic acid- RNA
Deoxyribonucleic acid- DNA
Watson-Crick base pairs are G-C and/or A-U nucleotides on different strands of RNA forming
multiple hydrogen bonds.
In nonenzymatic template-directed polymerization of nucleic acids the template strand is the
strand which catalyzes the polymerization of the primer strand.
N-mer is an oligonucleotide containing n nucleotides (e.g. 46mer).

8. 8
The Effect of RNA Structure on Nonenzymatic Template-Directed
Replication in the RNA World
Introduction
The beginning of the twentieth century has brought for the first time a serious scientific
discussion on the chemical origin of life (Oparin 1938; Urey 1952). The first experimental
attempt to verify some of these theories came in 1953 when Stanley Miller published his famous
work on the production of several biologically relevant amino acids under hypothetical prebiotic
conditions (Miller 1953). At the same time scientists were acquiring new knowledge about the
structural properties of DNA and proteins using X-ray crystallography, which helped develop
new theories about the origins of life based primarily on the biochemistry of current living
systems (Franklin, Gosling 1953; Watson, Crick 1953; Kendrew et al. 1958). The novel
understanding of cellular functioning and replication was the template for a widely accepted
theory that life requires molecular dichotomy in molecules for information storage and molecules
that act as catalysts of various kinetically slow reactions. A popular idea of the first self-
replicating system was a system consisting of RNA as an information carrier, and proteins for
catalysis of naturally unfavorable reactions (Eigen et al. 1981). However, this theory inevitably
led to a chicken-and-egg problem in that it was impossible to tell how the system originated-
RNA could not originate without proteins and vice versa. Francis Crick, Leslie Orgel, and Carl
Woese proposed already in 1968 that the stable structure of tRNA indicates that RNA can have a
catalytic function besides its known function of storing genetic material in messenger RNA or as
an entire genome in RNA viruses (Crick 1966; Woese 1966; Orgel, Crick 1993). However, the
experimental confirmation of RNA catalytic activity happened almost two decades later with the

9. 9
discovery of the first ribozymes. Thomas Cech reported in 1986 that Tetrahymena ribosomal
RNA catalyzes its own splicing, while Frank Westheimer reported in the same year that RNA
molecules of E. coli function as ribonucleases (Zaug, Cech 1986; Westheimer 1986). Both
ribozymes must also catalyze the reverse reactions- ligation and transphosphorylation- which
indicated that ribozymes can unify the dichotomy of information storage and reaction catalysis.
The hypothetical biosphere in which RNA replaces the roles of DNA and protein enzymes in the
modern cells is called the ‘RNA world’ (Gilbert 1986). Today, the RNA world seems as a very
plausible scenario for the early stage of life as scientists have discovered many new ribozymes,
function of RNA primers in DNA replication, RNA precursors for the synthesis of DNA, and
ancient cofactors that contain ribonucleotide motifs. All these properties that are conserved
throughout modern biology seem to be relics of RNA based biochemistry (Itoh, Tomizawa 1980;
Lazcano et al. 1988; Meyers et al. 2008; Ruvkun 2008). Moreover, elucidation of the ribosome
structure which revealed that its RNA component is responsible for catalyzing peptide bond
formation is perhaps the strongest evidence in favor of the RNA world hypothesis
(Ramakrishnan 2002).
The synthesis of a living system similar to the one that existed in the RNA world would not
only be significant for our understanding of the likely origin of life, but would also help us
understand better capabilities and functions of RNA in modern organisms. Moreover, a relatively
simple, self-replicating and evolving system would reveal the fundamental organizing principles
of chemistry and biology which are hidden by the complexity of present life forms (Muller 2006).
Recent studies of the RNA world and efforts to design a self-replicating system based on RNA
and RNA-like polymers have focused on nonenzymatic template-directed polymerization of
nucleic acids and RNA replicase engineering (Szostak et al. 2001). So far, both approaches have

10. 10
shown considerable success, especially the design of ribozymes capable of undergoing cross-
catalytic replication, albeit lacking the ability to bring about inventive Darwinian evolution (Kim,
Joyce 2004; Joyce 2009). Engineering of an RNA replicase- a ribozyme that can replicate its
own sequence by acting both as a template for information storage and an RNA polymerase- has
produced ribozymes that are sequence-specific and unable to polymerize RNA strands of their
own length (Wochner, Attwater et al. 2011). While the RNA world requires a wide spectrum of
ribozyme activities, the suspiciously long time to develop a ribozyme-based self-replicating
system indicates that it is very unlikely that such system emerged early on. The more likely
scenario is that replication in the RNA world was based on nonenzymatic template-directed
polymerization of RNA.
Research groups of Leslie Orgel and Jack Szostak have made considerable progress in
demonstrating that short RNA templates can be copied in the presence of suitable primer and
activated nucleotide substrates, without the use of any external catalysts (Acevedo, Orgel 1987;
Orgel 2004; Schrum et al. 2009). The templating ability is a special characteristic of nucleic
acids which besides enabling the process of copying, also increases the complexity of the
sequences through mutations. Several groups have shown recently that all four canonical
nucleotides can be incorporated into the copied sequence, and that activated ribonucleotides
adopt optimal conformation for the transphosphorylation reaction (Deck, Jauker et al. 2011;
Zhang et al. 2012). Furthermore, research groups investigating possible prebiotic chemical
pathways discovered that activated nucleotides, as well as short RNA polymers can be
synthesized without the aid of the modern biosynthetic machinery, which is an important
precondition for the template-directed replication (Ertem et al. 1998; Powner et al. 2009). Figure
1 is a diagram of a general template-directed polymerization of nucleic acids. The diagram

11. 11
shows a three-step dissection of a single-nucleotide primer extension (multiple extension
reactions would constitute a polymerization process). The first step of the extension process is
the hybridization of partially complementary template and primer strands to form a template-
primer complex. The second step is the binding of the activated nucleotide to the template strand
one position upstream of the bound primer strand, to form a pre-reaction complex. The final step
of the extension process is a nucleophillic attack of the primer strand 3’-hydroxide, yielding the
extended primer and the activated nucleotide’s leaving group as a product.
An important aspect of the template-directed polymerization reaction that has so far been
overlooked is the effect of RNA 3-D folding conformation on the rate of primer extension. The
process of RNA folding which determines its structure and function, is driven by base-pairing
and stacking, ion-mediated electrostatics, thermally driven chain fluctuation, and other
noncanonical interactions (Chen 2008). The structure of RNA is primarily determined by its
sequence content, and it is known that more complex sequences, such as most ribozymes, form
stable structures with low minimum folding energies (Clote et al. 2005). We hypothesize that
RNA sequences which fold into a stable structure cannot be copied via nonenzymatic template-
directed polymerization. This handicap of stable RNA sequences can be explained in two ways.
First, a stable template sequence will not form a template-primer complex because its
intramolecular base-pairing interactions can outcompete the intermolecular base-pairing
interactions between the template and the primer strand. The extension of the primer is therefore
stopped at the first step. The second reason is that even if the template-primer complex forms,
the overhang part of the template sequence may form a stable structure and sterically block the
binding of the activated nucleotide, stopping the reaction at the second step. We show that this
phenomenon indeed occurs. By correlating the rate of primer extension with different structural

12. 12
parameters of the template strand we were able to prove not only that stable sequences cannot
catalyze their copying, but also that the rate of primer extension can be roughly predicted from
the knowledge of the template sequence.
Our findings pose a serious problem for the RNA world. The inability of stable sequences
to be copied via nonenzymatic template-directed polymerization means that there was no way for
stable ribozymes to be copied and replicated. We solve this problem by introducing the concept
of asymmetric sequences. A simple way to understand the concept is to look at perfect RNA
hairpins. Perfect hairpins are a well characterized example of stable sequences and are the
building blocks of secondary RNA structure (Varani et al. 1991). Figure 2 shows a diagram of
several RNA hairpins. It is important to note that without G-U base-pairing, a general perfect
hairpin sequence S has the same sequence as its complement S’. Therefore, without G-U base-
pairing a perfect hairpin will have the same structural stability as its complement sequence. On
the other hand, if the G-U base pairs are allowed to form, a perfect hairpin will not be as stable as
its complementary sequence. Because the symmetry in sequence stability between complements
is violated, we term such complementary sequences asymmetric. We show theoretically that
sequences with increased percentage of G and U, exhibit asymmetric properties. Moreover, we
confirm experimentally our results and show that there are sequences such that one can function
as a good template in template-directed polymerization, while its complement can form a very
stable fold and potentially function as a ribozyme. The stable sequences in the RNA world could
therefore emerge as a consequence of asymmetric sequence properties.

13. 13
Figure 1. Nonenzymatic template-directed polymerization.
A single nucleotide primer extension can be dissected into three steps. (1) Upon mixing, the
template strand (blue) and the complementary primer strand (red) form a template-primer
complex. (2) Addition of activated nucleotide results in the formation of the pre-reaction
complex in which the activated nucleotide binds to the unpaired complementary base on the
template strand one position upstream of the primer strand. (3) The 3’-OH group on the primer
strand attacks the activated nucleotide yielding the extended primer product and a leaving group.
Dashed lines represent Watson-Crick hydrogen bonding.

14. 14
Figure 2. Asymmetric sequences and structural stability.
On the left (red), the two complementary perfect hairpins S and S’ are represented. S and S’
sequences are equivalent in sequence since G-U base pairs are not allowed. On the right (blue),
the perfect hairpin A gives rise to complementary hairpin A’ which is structurally less stable.
The asymmetry in structural stability between A and A’ arises from the G-U base pairing.
Dashed lines represent Watson-Crick or G-U hydrogen bonding.
A A’S S’
Symmetric complementary
sequences
(G-U pairs not allowed)
Asymmetric complementary
sequences
(G-U pairs allowed)

15. 15
Results and Discussion
Sequence Design
In order to investigate the effect of RNA structure on the rate of primer extension in
nonenzymatic template-directed polymerization, we designed template sequences based on the
secondary structure prediction. The energies involved in the formation of RNA secondary
structure are larger than those involved in tertiary interactions, so our structural predictions can
be accurately approximated by predicting only the secondary structure of RNA (Tinoco,
Bustamante 1999). The Vienna RNA Package is a comprehensive collection of tools that offers
algorithms for RNA folding and comparison, and prediction of RNA-RNA interactions
(Hofacker 2003; Gruber et al. 2008). One of the core Vienna programs is RNAfold which can be
used to predict the minimum free energy (MFE) secondary structure of single RNA sequences
(Hofacker et al. 1994). We used RNAfold to obtain MFE values for template sequences, as a
quantitative measure of their structure- a very stable RNA structure is characterized by low (high
negative) MFE value and vice versa. We were expecting that templates with different MFE
values will have different rate of nonenzymatic template-directed primer extension. RNAcofold
program computes the hybridization energy and base-pairing pattern of two RNA sequences
(Mathews et al. 1999; Bernhart et al. 2006). We used RNAcofold to obtain additional structural
parameters that have effect on the rate of primer extension. Template-primer hybridization
energy is the energy of binding between the template and the primer strand. Template-
hybridization energy can be used to obtain equilibrium constants for template and primer
homodimer, monomer, and template-primer heterodimer states which exist in the solution.
Furthermore, for given a concentration of template and primer we can predict the amount of each
state existing in the solution. We define hybridization percentage as:

16. 16
XTP (1)
2XPP+XTP+XP
where XTP is the amount of template-primer heterodimer, XPP the amount of primer homodimer,
and XP the amount of primer monomer in the solution for equal concentration of template and
primer strands.
We used the Vienna RNA Package to design thirteen template sequences (Sequences I-
XIII) that are partially complementary to a single existing primer sequence (for exact sequences
see Appendix). The template sequences are 46 nucleotides long with the exception of Sequence I
which is a 56mer, and all bind the 20mer primer sequence along the 3’-end (Figure 3). The
primer is extended by an activated G nucleotide (ImpG) which binds to the C of the template
strand one position upstream of the primer 3’-end. We selected the template sequences that have
a spectrum of predicted MFE values, ranging from -58 to -10 kcal/mol. The sequences with low
MFE also have low values of hybridization percentage and high template-primer binding
energies. This is expected since template-primer heterodimer competes with folded monomers
and homodimers in the solution. Templates with low MFE form stable monomer structures
shifting the equilibrium away from the template-primer heterodimer state. Besides template MFE,
hybridization percentage and template-primer binding energy, we calculated the fourth structural
parameter termed pairing probability. Pairing probability is the probability that a template is
found in a template-primer heterodimer state and that the cytosine nucleotide of the template
strand which base-pairs the activated nucleotide is not base-paired to any other nucleotide, so it
is not internally blocked. Pairing probability predicts for what fraction of the time the template
cytosine will be open to bind the activated nucleotide and catalyze the primer extension. All
predicted structural parameters are shown in Table 1. In addition, Figure 4 shows secondary
structures of template, primer, and template-primer complex predicted by the Vienna Package.

17. 17
Figure 3. Template sequence design.
Template sequence I is a 56mer and binds the 20mer primer strand along its entire length. All
other template sequences (II-XIII) are 46mers and bind the primer strand with 15 base pairs,
leaving a 5 nucleotide overhang. Also shown is the activated nucleotide (ImpG) binding to the
cytosine of template strand. For exact primer and template sequences see Appendix.

18. 18
Figure 4. Predicted secondary structures of template (blue), and primer strands (red), in
equilibrium with template-primer complex.
Sequence I Sequence II
Sequence III Sequence IV
Sequence V Sequence VI

19. 19
Sequence VII Sequence VIII
Sequence IX Sequence X
Sequence XI Sequence XII
Sequence XIII

20. 20
Template
Sequence
Template
MFE
Template-Primer
Binding Energy
Hybridization
Percentage
Pairing
Probability
(kcal/mol) (kcal/mol) (%) (%)
I -58.9064 0.5651 00.00 00.00
II -45.4342 3.3690 00.00 00.00
III -38.4583 -1.3171 00.00 00.00
IV -32.4522 -8.8373 56.70 00.00
V -26.1618 -9.7032 77.59 00.02
VI -15.3438 -18.8263 99.99 71.31
VII -13.3639 -19.7644 100.0 99.03
VIII -15.1654 -20.0085 100.0 98.07
IX -14.1313 -12.5356 97.76 95.00
X -26.1222 -8.7811 57.61 51.91
XI -13.7888 -16.0329 93.29 55.40
XII -12.2066 -19.0098 99.99 27.44
XIII -10.9408 -19.6699 100.0 90.36
Table 1.
Table representing predicted structural parameters using the Vienna RNA Package. The first
column represents a specific template sequence. The second column represents values for the
minimum free energy (MFE) of a template strand. The third column represents the energy of
template-primer binding interaction. The fourth column represents the percentage of template-
primer heterodimer in the solution. The fifth column represents the pairing probability of the
template.

21. 21
Formation of the Template-Primer Complex
The first step of template-directed RNA polymerization is the formation of template-
primer heterodimer complex. The heterodimer is held together by Watson-Crick base pairing,
and in the solution exists in equilibrium with template monomer, primer monomer, and template-
template, and primer-primer homodimers. We were able to experimentally determine the
percentage of template-primer heterodimer (hybridization percentage) for different template
sequences, by mixing the fluorescently labeled primer and template sequence in a 1:1 mixture
and running it on a native gel (Figure 5). The bands on the gel represent only fluorescently
labeled primer. Free primer runs faster on the native gel than the template-primer complex, so
the two are separated by size, and the percentage of the bound primer is calculated by taking the
ratio of the entire gel column area (template-primer complex and primer monomer) to the area
above the bottom band (template-primer complex only). The experimental values obtained are
similar to the predicted theoretical values for hybridization percentage using (Figure 6). This is
an important confirmation of predicted values because the template-primer binding energy, as
well as the pairing probability values are based on the same parameters which determine the
hybridization percentage. The two sequences which show the greatest deviation are I and XII.
The wrong theoretical predictions are likely the result of complex interactions between different
RNA molecules which the prediction software does not taken into account. For exact numerical
results see Appendix.

22. 22
Figure 5. Template-primer heterodimer complex native gel.
Native gel representing the amount of template-primer heterodimer complex formed for different
template sequences. The control band is a fluorescently labeled primer alone. Other bands I-XIII
contain the labeled primer and a corresponding template strand in a 1:1 ratio.
Primer control
I II III IV V VI VII VIII IX X XI XII XIII

23. 23
Figure 6. Theoretical and experimental values of hybridization percentage.
The plot shows experimentally determined (red diamonds) and theoretically predicted
(black circles) values of template-primer complex formation. The error bars are standard
deviation from the mean, based on two experimental results. For exact numerical results
see Appendix.

24. 24
Correlating Rates of Primer Extension with Predicted Structural Parameters
We have determined the rates of primer extension for different template sequences. The
reaction mechanism and experimental approach are depicted in Figure 7. The 3’-end of the
primer sequence was modified to have a more basic 3’-amino group instead of the naturally
occurring 3’-hydroxyl in order to increase the rate of extensions so that it is analytically tractable
within 24 hours. In all reactions we used ImpG as activated mononucleotide for the extension of
the primer strand rather than the naturally occurring GTP which is kinetically more stable and
hydrolyzes too slow for the extension to be observed. Finally, the primer sequence was
fluorescently labeled at the 5’-end with Cy3 for analysis with polyacrylamide gel electrophoresis.
The polyacrylamide gel depicting the reaction progress and the plot of the reaction progress are
shown in Figure 8. The exponential rise to maximum of the reaction progress occurs because the
activated nucleotide gets hydrolyzed in the solution, and in addition it is known that guanosine
monophosphate, which is the product of ImpG hydrolysis, inhibits the rate of primer extension
(Deck, Jauker 2011). In addition, the hydrolysis of ImpG prevents the reaction from going to
completion. The rate of the reaction is determined by linear approximation using the first several
time points. The rates of primer extension for different template sequences vary from 0.0587 hr-1
to 4.89 hr-1
(Figure 9, for exact rates see Appendix). The rate of primer extension without the
presence of a template strand is 0.0365 hr-1
(Appendix).
In order to understand what structural parameter of the template sequence is responsible
for the observed differences in the rate of primer extension we plotted the observed rate values
against the values for different structural parameters (Figure 9). The MFE of the template,
template-primer binding energy and the amount of hybridization are all necessary but not
sufficient conditions for the high rate of primer extension. Sequences XI, XII, and XIII are

25. 25
relatively unstructured and have high template MFE values (-13.78 kcal/mol, -12.20 kcal/mol
and -10.94 kcal/mol respectively), but much lower rates of primer extension than sequence VIII
which is more structured and with somewhat lower template MFE of -15.16 kcal/mol. Similarly,
sequences VI, XII and XIII have template-binding energies -18.83 kcal/mol, -19.01 kcal/mol
and -19.67 kcal/mol respectively, which are similar to sequences VII and VIII (-19.76 kcal/mol,
and -20.01 kcal/mol respectively) but much lower rates of primer extension. Finally, sequence
VI has the same hybridization percentage as sequences VII and VIII, however sequence VI has
the rate of primer extension 0.32 hr-1
while sequences VII and VIII have rates of primer
extension 4.00 hr-1
and 4.89 hr-1
, respectively. These properties of template MFE, template-
primer binding energy, and hybridization percentage can be qualitatively seen on the graphs in
Figure 9 where there appears to be a trend that less structured template sequences have higher
rates of primer extension, but this trend is not definite. The pairing probability shows a smooth
exponential correlation with the rate of primer extension. This is the only structural parameter
that takes into account the probability that the activated nucleotide binds the template sequence.
The strong correlation between the pairing probability and the rate of primer extension indicates
that even if primer–template complex forms, the extension event might not occur. Therefore,
only sequences with high pairing probability also show high rate of primer extension.

26. 26
Figure 7. Experimental approach to determining the rate of primer extension.
The template strand (blue) and the primer strand (red) are complementary in sequence, and every
template strand has a cytosine base upstream of the 3’-end of the primer strand. The 3’-end of the
primer strand is modified so that it has a 3’-amino group, and is fluorescently labeled at its 5’-
end (not shown in this figure). The activated nucleotide used is ImpG (black).

27. 27
Figure 8. The primer extension reaction.
The gel above shows the extension of labeled primer at certain time points. The primer [n] is the
starting 20mer primer previously described. Upon mixing the primer with a template sequence
and ImpG the extended primer [n+1] begins to appear. The [n] and [n+1] primers were separated
by polyacrilamide gel electrophoresis. The plot bellow shows the ratio of the amount of [n+1] to
[n] at specific time points.
Primer [n] Extended primer [n+1]

28. 28
Figure 9. Rate of primer extension for different template strands and correlation with different
structural parameters. The uppermost column plot shows the rate of primer extension for
different template strands. The four plots bellow show the correlation of the rate of primer
extension with different structural parameters previously described.

29. 29
Asymmetric Sequences
The obtained rates for primer extension indicate that a well folded ribozyme sequence
would be hard to either copy or replicate. Therefore, our aim is to find unstably folded sequences
whose complements have stable folds. The nonenzymatic template-directed copying of such
sequences would produce stable sequence which can potentially act as ribozymes. We term
complementary sequences with different structural stabilities asymmetric sequences. Using the
Vienna RNA Package we folded 500000 random 35mers with equal representation of all four
nucleotides. The energy histogram in Figure 10 shows the MFEs of folded sequences. The
histogram is identical for the sequences and their complements. The mean MFE of the folded
sequences is Ê= -6.12 kcal/mol with standard deviation σ= 2.85 kcal/mol. We define stable
sequences as those with MFE < Ê-σ and unstable sequences as those with MFE > Ê+σ. The
sequences which we are looking for are stable sequences with unstable complement sequences.
There are only 0.6% of such sequences indicating that for a random sequence pool, stable
sequences tend to have stable complements.
As explained in introduction, we assume that asymmetric sequences arise as a result of
G-U base pairing. We decided to compare the MFE values of the sequences with and without
allowing for G-U base pairs. Figure 11 shows a heat map energy histogram for these two
scenarios. Sequences with G-U base pairs are on average more stable than the sequences without
it, indicating that folding asymmetry results from G-U base pairs. Furthermore, we investigated
whether any bias in the base composition of sequences promotes sequence asymmetry. We found
that sequences with base composition 0% A, 25% C, 50% G, and 25% U have Ê= -12.08
kcal/mol while there complements have ÊC= -7.06 kcal/mol. This is a reasonable finding because
the lower amount of A promotes G-U base pairing. We conclude that asymmetric sequence

30. 30
properties arise with sequences that have high percentage of G,U bases and low percentage of A
base.
To test our hypothesis we designed a template sequence with low MFE (stable fold)
which has a complementary sequence with high MFE (unstable fold). As a control we used two
complementary sequences with similar folding energies. Figure 12 shows the asymmetric
sequences, their secondary structures, and numerical values for structural parameters, and rates
of primer extension. The stable fold sequence is by base content 39% G, 37% U, 15% C, and 9%
A, while its complementary unstable fold is 15% G, 9% U, 39% C, and 37% A. The two
asymmetric sequences differ considerably in the template MFE values and pairing probabilities,
while their template-primer binding energies and hybridization percentage are very similar.
Finally, as we predicted, unstable fold has about two orders of magnitude higher rate of primer
extension than stable fold sequence, which means that even tough stable fold cannot be copied
it can still be produced by copying of unstable fold.

31. 31
Figure 10. Histogram of ensemble minimum free energies.
The histogram shows MFE values of random unbiased 35mers. The vertical axis represents the
percentage of sequences with a given MFE. The mean free energy is Ê= -6.12 kcal/mol with
standard deviation σ= 2.85 kcal/mol. About 0.6% of all sequences have a large difference
between their folding energy and the energy of the complement, defined as E < Ê-σ (green) and
EC > Ê+σ (blue).
Ê ± σ
Stable sequences
Unstable
sequences
P (%)
E (kcal/mol)
(kcal/mol)

32. 32
Figure 11. Heat map histogram for G-U base pairing.
The figures are two dimensional histograms of free energies E for random 35mers and the
energies EC of their complements for two base-pairing scenarios: on the left G-U pairs are not
included, whereas on the right G-U base pairing is included. The histogram color represents the
percentage of sequences with certain E and EC: blue and purple represent relatively low
percentage, while red and yellow represent relatively high percentage. We observe that
sequences with G-U base pairs are more likely to form asymmetric sequences than sequences
without G-U base pairs.
G-U base pairs not allowed G-U base pairs allowed
E (kcal/mol) E (kcal/mol)
EC
(kcal/mol)
EC
(kcal/mol)

33. 33
Template
Sequence
Template
MFE
Template-Primer
Binding Energy
Hybridization
Percentage
Pairing
Probability
Rate of Primer
Extension
(kcal/mol) (kcal/mol) (%) (%) (hr-1
)
Stable fold -25.5894 -20.8668 100.0 32.00 0.0713
Unstable fold -11.2335 -24.3818 97.76 90.39 6.7187
Control -26.1618 -9.7032 78.51 00.02 0.2267
Control complement -25.5617 -8.1089 18.14 00.10 0.1671
Figure 12. Testing asymmetric sequences.
The figure above shows stable fold and complementary unstable fold, and their predicted
secondary structures. The table provides values for different structural parameters and the rate of
primer extension.
Asymmetric Sequences
Stable fold Unstable fold

34. 34
Conclusion
The goal of our research was to better understand the fundamental principles that govern
replication and evolution in the RNA world. We show that the rate of template-directed
polymerization of RNA depends on the structure of the template strand. The structural
parameters that govern template-primer interaction such as MFE, hybridization percentage, and
template-primer binding energy, are all necessary requirements for ensuring primer extension but
are not sufficient. One possible explanation is that activated nucleotides can be sterically blocked
away from the template strand even though the template-primer complex is formed. The
structural parameter which takes into account the probability that the activated nucleotide will
bind the template strand is the ‘pairing probability’ and it has a very smooth exponential
correlation with the rate of primer extension. Pairing probability depends both on the probability
that template-primer complex will form and that the pairing template base will be unpaired.
These two probabilities are themselves correlated, which explains why there is an exponential
rather than linear correlation between pairing probability and the rate of primer extension.
As already mentioned, our findings indicate that well folded RNA sequences such as
ribozymes cannot be directly copied or replicated via nonenzymatic template-directed
polymerization. Figure 9 shows that template sequences with MFE less than -20 kcal/mol are
essentially not catalyzing the rate of primer extension. Such sequences would therefore not be
copied in the RNA world. We found that this problem can be circumvented by introducing the
concept of asymmetric sequences. Asymmetric sequences are complementary sequences which
have different structural stabilities. Such asymmetric properties arise from G-U base pairs, and
we show that sequences with base content that promotes G-U base pairing such as increased G,U

35. 35
percentage, and lower percentage of A. Such sequences exhibit asymmetric properties and have
up to 100 times different rates of primer extension between the complements.
Our findings have several important implications. Concerning the RNA world, our results
suggest that the early RNA sequences must have been structurally unstable, because only those
sequences would be able to replicate. Some groups have already suggested that early RNA
sequences were indeed short, low in diversity and information content, and therefore structurally
unstable (Derr et al. 2012). Further, the increase in sequence complexity had a large penalty in
the efficiency of replication, since more complex sequences are more structurally stable. Possibly,
the only way that complex and stable sequences could emerge and act as ribozymes was via
asymmetric sequences. As we have shown, the emergence of asymmetric sequences is simply a
consequence of G-U base pairing in RNA. Finally, our results have clear implications for the
design of a self-replicating system based on nonenzymatic template-directed replication, as we
have proved that using structural parameters it is possible to predict what rate of primer
extension a template sequence will have.

36. 36
Materials and Methods
Synthesis of ImpG
Guanosine 5‘-phosphorimidazolide was synthesized based on a previously published
protocol, by GL Synthesis Inc. in Worcester, MA (Rajamani et al. 2010; Lohrmann and Orgel
1978; Prabahar et al. 1994). The purity of ImpG was verified by mass spectrometry and HPLC as
previously descrbied (Rajamani et al. 2010) and were found to be >93% pure.
Oligonucleotides for nonenzymatic polymerization
The fluorescently labeled RNA primer was synthesized using reverse synthesis in the W.
M. Keck Biotechnology Resource Laboratory at Yale University in New Haven, CT. The
synthesis used 3’-O-tritylamino-N6-benzoyl- 2’,3’-dideoxyguanosine-5’-cyanoethyl
phosphoramidite at the 3’-terminus and was labeled with Cy3 at the 5’-terminus. The primer
was PAGE-purified and its mass was verified by MALDI-TOF (data not shown). RNA template
sequences were synthesized by Dharmacon in Lafayette, CO and RNA excess primer was from
UCDNA Services in Calgary, AB, Canada.
Nonenzymatic Template-Directed Polymerization
The primer extension reactions were done at room temperature. Fluorescently labeled
primer (final concentration 1.5µM) and template strand (final concentration 1.5µM) were mixed
in a solution containing Tris HCl (final concentration 100mM) and NaCl (final concentration
200mM). The solution was heated up to 95ºC for 5 min and then slowly cooled down to room
temperature to ensure the proper formation of template-primer complex. ImpG was added to

37. 37
final concentration of 11.51mM. The reaction was stopped at different time points by taking out
1µL of reaction mixture and adding it to 4µL of 8M urea solution. In order to prevent binding of
the labeled primer to the template strand in the gel we added 40X unlabeled RNA primer. The
reaction was analyzed by running the reaction mixture on 20% polyacrilamide urea gel. The gel
was scanned using GE (Amersham) Typhoon Trio Imager at Green (532nm) wavelength. The
band intensities were analyzed using ImageQuant and SigmaPlot software tools.
Determining Hybridization Percentage
Fluorescently labeled primer (final concentration 1.5µM) was mixed with a template strand (final
concentration 1.5µM) in a solution containing Tris HCl (final concentration 100mM) and NaCl
(final concentration 200mM). The solution was heated up to 95ºC for 5 min and then slowly
cooled down to room temperature to ensure the proper formation of template-primer complex.
The solution was then run on a nondenaturing polyacrilamide gel at 4 ºC. The loading buffer
used was 80% glycerol:20% water, 100mM Tris HCl, 50mM EDTA. The nondenaturing gel was
made by mixing 32mL non-urea ProtoGel (National Diagnostic Inc.), 4mL of 10X TBE buffer,
4mL of water, 16µL of tetramethylmethylenediamine, and 350µL of 10% ammonium persulfate.
The gel was scanned using GE (Amersham) Typhoon Trio Imager at Green (532nm) wavelength.
The band intensities were analyzed using ImageQuant and SigmaPlot software tools.

38. 38
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43. 43
APPENDIX
Oligonucleotide Sequences
Primer sequence for templates I-XIII: 5’ Cy3 –GGG AUU AAU ACG ACU CAC U-NH2
Primer sequence for stable fold template: 5’ Cy3- AGG CCC AGU CCA AUC G- NH2
Primer sequence for unstable fold template: 5’ Cy3- GGC GAG UUC UUU UUG- NH2
Primer sequence for control template: 5’ Cy3 –GGG AUU AAU ACG ACU CAC U-NH2
Primer sequence for control complement template: 5’ Cy3- UAA UAA UUA CCA CUG- NH2
Template Sequence I: 5’ –GGG AUU AAU ACG ACU CAC UGG AGA UCA AGU GAU CUC
CAG UGA GUC GUA UUA AUC CC
Template Sequence II: 5’ –UAA UAC GAC UCA CUG GAG AUC AAG UGA UCU CCA
GUG AGU CGU AUU A
Template Sequence III: 5’ –UAA UAC GAC UAA CUG GAG AUC AAG UGA UCU CCA
GUG AGU CGU AUU A
Template Sequence IV: 5’ –UAA UAC GAG AGA CUG GAG AUC AAG UGA UCU CCA
GUG AGU CGU AUU A
Template Sequence V: 5’ –UAA UAA UUA CCA CUG GAG AUG AAG UGA UCU CCA
GUG AGU CGU AUU A
Template Sequence VI: 5’ –UAA UAC CUG AGA CUG AAG AUC AAG UCA UCU CCA
GUG AGU CGU AUU A
Template Sequence VII: 5’ –UAC CCU CGU UCU AGG ACG AAU AAU AUU UGG CCA
GUG AGU CGU AUU A
Template Sequence VIII: 5’ –ACC GGC CUG CCG AUU CCG GAU UUC CCA UCU CCA
GUG AGU CGU AUU A
Template Sequence IX: 5’ -UAU GCG GCA AAU UCA CUC UAC ACU CAU CUA CCA
GUG AGU CGU AUU A
Template Sequence X: 5’ -CUC AAU ACA GAC UCG UGG UUG AGU GUA CAG CCA
GUG AGU CGU AUU A
Template Sequence XI: 5’ -UAC AUU GCA UAC AAA UCG AUC AGG GGC GCG CCA
GUG AGU CGU AUU A
Template Sequence XII: 5’ -UAA UUC CUG AGA CUG AUG AUC AAG UUA ACU CCA
GUG AGU CGU AUU A

44. 44
Template Sequence XIII: 5’ -UAA GAC CUA AGA CAG AAG AUC ACG UCA UCU CCA
GUG AGU CGU AUUA
Stable fold template: 5’ -GGC GAG UUC UUU UUG GGU UGU UGU CGA CUC CGA UUG
GAC UGG GCC U
Unstable fold template: 5’ -AGG CCC AGU CCA AUC GGA GUC GAC AAC AAC CCA
AAA AGA ACU CGC C
Control template: 5’ -UAA UAA UUA CCA CUG GAG AUG AAG UGA UCU CCA GUG
AGU CGU AUU A
Control complement template: 5’ -UAA UAC GAC UCA CUG GAG AUC ACU UCA UCU
CCA GUG GUA AUU AUU A

45. 45
Table 1A. Numerical values for the rate of primer extension. Shown are values for two
independent experiments, standard deviation and the mean value. The units are hr-1
.
Rate of primer sequence for stable fold in the absence of stable fold template: 0.0478 hr-1
Rate of primer sequence for unstable fold in the absence of unstable fold template: 0.0393 hr-1
Rate of primer sequence for control in the absence of control template: 0.0365 hr-1
Rate of primer sequence for control complement in the absence of control complement
template: 0.0941 hr-1
Template I II III IV V VI VII
Exp. 1 0.1942 0.1969 0.0368 0.1373 0.2492 0.3438 4.066
Exp. 2 0.2194 0.1284 0.1805 0.1149 0.2042 0.2936 3.9411
St. dev. 0.01782 0.04844 0.10161 0.01584 0.03182 0.0355 0.08832
Mean 0.2068 0.16265 0.10865 0.1261 0.2267 0.3187 4.00355
Template VIII IX X XI XII XIII none
Exp 1 4.6038 1.8434 0.9028 0.2047 0.0224 1.0559 0.0501
Exp 2 5.185 2.0756 0.8054 0.3208 0.095 1.1083 0.0229
St. dev. 0.41097 0.16419 0.06887 0.0821 0.05134 0.03705 0.01923
Mean 4.8944 1.9595 0.8541 0.26275 0.0587 1.0821 0.0365
Template
Stable
fold
Unstable
fold
Control
Control
complement
Exp 1 0.0446 5.6336 0.2492 0.1946
Exp 2 0.0981 7.8037 0.2042 0.1395
St. dev. 0.03783 1.53449 0.03182 0.03896
Mean 0.07135 6.71865 0.2267 0.16705

46. 46
Table 2A. Numerical values for hybridization percentage. Shown are values for two independent
experiments, standard deviation, mean value, and the value of theoretical prediction.
Template I II III IV V VI VII
Exp. 1 59.06 7.17 8.79 43.78 80.02 97.87 96.86
Exp. 2 44.69 1.74 7.3 41.64 77 98.29 98.43
St. dev. 10.1611 3.83959 1.05359 1.51321 2.13546 0.29698 1.11016
Mean 51.875 4.455 8.045 42.71 78.51 98.08 97.645
Theory 0 0 0 56.7 77.59 99.99 100
Template VIII IX X XI XII XIII
Exp. 1 98.17 93.11 50.99 89.12 1.42 94.45
Exp. 2 98.58 90.27 41.83 88.52 6.93 93.03
St. dev. 0.28991 2.00818 6.4771 0.42426 3.89616 1.00409
Mean 98.375 91.69 46.41 88.82 4.175 93.74
Theory 100 97.76 57.61 93.29 99.99 100
Template
Stable
fold
Unstable
fold Control
Control
complement
Exp. 1 100 97.93 80.02 18.35
Exp. 2 100 97.6 77 17.94
St. dev. 0 0.23335 2.135462 0.28991
Mean 100 97.765 78.51 18.145
Theory 100 100 77.59 35.9