University of Groningen
Novel peptide replicators from dynamic combinatorial libraries
Altay, Yigit
DOI:
10.33612/diss.90041906
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Publication date: 2019
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Altay, Y. (2019). Novel peptide replicators from dynamic combinatorial libraries. University of Groningen. https://doi.org/10.33612/diss.90041906
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Chapter 7
312 7. Conclusion and Perspectives
L
ife is the most complex chemical system that is governed by the rules of physics and developed throughDarwinian evolution. In order to mimic life-like features or, more fundamentally, create life-like forms from scratch, systems chemistry is the ultimate tool as it combines the bottom-up approach of chemistry with the complexity typically associated with biology. In the framework of systems chemistry, complex molecular networks may help us to understand and eventually integrate the three main characteristics of life: compartmentalization, metabolism and replication. All four fundamental elements of Darwinian evolution (i.e. heredity, variation, fitness and adaption) rely on replication making it an indispensable element of life. That is why this entire thesis is dedicated to replicating systems and their emergent properties.Self-replicating peptides emerging from dynamic combinatorial libraries have been extensively studied in our group. In the last 10 years, the mechanism of replica-tion1and the mechanism of building block exchange2have been revealed using both
bench-top experiments and computer simulations.3But still, it is extremely difficult
to design a peptide that will form a replicator which has a predetermined size and to incorporate well-defined information in them. To uncover the selection criteria for the emergence of peptide-based replicators in dynamic combinatorial libraries, we have extended our portfolio with thirteen novel peptides with various modifications in the structure and analyzed the library behaviors and emergent properties under different conditions in Chapter 2. The chapter covers a wide range of parameter space: sequence variations, structural modifications, environmental effects with in-corporation of molecular dynamic simulations. These results revealed that there is probably a size limit on macrocycles that can form stable self-assembled structures. These results extend our knowledge on the relation between building block structure and the emergence of replicators and show the limits of our system to some extent. However it should be noted that other variables, which remain unknown to day, might play a role to stabilize larger macrocycles and thus might be capable of extending the limits. So, this chapter stands as another milestone in understanding the emergence of peptide self-replicators from dynamic combinatorial libraries.
For a chemist having a dream of creating life in a laboratory, it is obvious that single replicator systems cannot go beyond an exercise in the basics. In an ecosystem, all species are dependent on others. Interactions of different species can result in different types of symbiotic relationships, namely; mutualism, commensalism and parasitism. The motivation behind Chapter 3 and Chapter 4 was to investigate such symbiotic relationships between replicators that are made from different building blocks. Alongside this, we have also discovered a fully parasitic system using a similar system.4 These systems not only provide information on how different replicators
interact with each other, but also enables the development of new strategies and pathways to access replicators that do not emerge spontaneously.
313 Life is a collection of out-of-equilibrium processes that work simultaneously in harmony. That is why Chapter 5 focuses on the self-replication that operates out of equilibrium by enabling replication and destruction pathways to occur simulta-neously. We achieved very promising results towards creating replicator families as “quasi-species”, but, adaptive behavior of them is yet to be demonstrated. We believe that out-of-equilibrium conditions will bring more unpredictable life-like features to synthetic replicator systems as small perturbations will likely have bigger impact than in an equilibrium regime.
As we have stated before, self-replicating peptide systems are prone to consider-able quantitative variability due to the inherent sensitivity to small perturbations. So, several repeats of an experiment should be run to prevent drawing wrong conclu-sions based on a single experiment. Additionally, when addressing stochastic effects, it becomes a necessity to run statistical analysis on a large number of repeats. In such cases, the chromatographic analysis, which forms the basis of the daily analysis routine in our lab, becomes a bottle-neck, as each sample run takes 15 minutes on average. To improve the timescale of daily routine analysis of our systems, we col-laborated with the Margulies group and showed in Chapter 6 that a combinatorial fluorescent sensor can be implemented in our system. Obtaining the compositional information as an optical read-out significantly reduces the analysis time. We hope that this combinatorial sensor will become a daily routine in labs and will enable the analysis of large numbers of DCLs in a significantly shorter time.
The establishment of supramolecular chemistry followed by systems chemistry have extended scientists’ horizons. Chemistry moved forward from isolated pure compounds to complex molecular systems. Self-replicating systems that are made out of single building blocks have been studied extensively and now the focus should shift to the replicator interactions (i.e. cross-catalysis). On the short term, any progress on the development of dissipative systems of co-existing and cooperating replicators would dramatically increase the possibilities of creating life-like features in the lab. On the long term, development of a compartment that is sustained by (relatively) simple chemical reactions and that can be integrated into a system that can undergo self-replication and Darwinian evolution is required to make the scientists dream of creating life de novo come true.
312 7. Conclusion and Perspectives
L
ife is the most complex chemical system that is governed by the rules of physics and developed throughDarwinian evolution. In order to mimic life-like features or, more fundamentally, create life-like forms from scratch, systems chemistry is the ultimate tool as it combines the bottom-up approach of chemistry with the complexity typically associated with biology. In the framework of systems chemistry, complex molecular networks may help us to understand and eventually integrate the three main characteristics of life: compartmentalization, metabolism and replication. All four fundamental elements of Darwinian evolution (i.e. heredity, variation, fitness and adaption) rely on replication making it an indispensable element of life. That is why this entire thesis is dedicated to replicating systems and their emergent properties.Self-replicating peptides emerging from dynamic combinatorial libraries have been extensively studied in our group. In the last 10 years, the mechanism of replica-tion1and the mechanism of building block exchange2have been revealed using both
bench-top experiments and computer simulations.3But still, it is extremely difficult
to design a peptide that will form a replicator which has a predetermined size and to incorporate well-defined information in them. To uncover the selection criteria for the emergence of peptide-based replicators in dynamic combinatorial libraries, we have extended our portfolio with thirteen novel peptides with various modifications in the structure and analyzed the library behaviors and emergent properties under different conditions in Chapter 2. The chapter covers a wide range of parameter space: sequence variations, structural modifications, environmental effects with in-corporation of molecular dynamic simulations. These results revealed that there is probably a size limit on macrocycles that can form stable self-assembled structures. These results extend our knowledge on the relation between building block structure and the emergence of replicators and show the limits of our system to some extent. However it should be noted that other variables, which remain unknown to day, might play a role to stabilize larger macrocycles and thus might be capable of extending the limits. So, this chapter stands as another milestone in understanding the emergence of peptide self-replicators from dynamic combinatorial libraries.
For a chemist having a dream of creating life in a laboratory, it is obvious that single replicator systems cannot go beyond an exercise in the basics. In an ecosystem, all species are dependent on others. Interactions of different species can result in different types of symbiotic relationships, namely; mutualism, commensalism and parasitism. The motivation behind Chapter 3 and Chapter 4 was to investigate such symbiotic relationships between replicators that are made from different building blocks. Alongside this, we have also discovered a fully parasitic system using a similar system.4 These systems not only provide information on how different replicators
interact with each other, but also enables the development of new strategies and pathways to access replicators that do not emerge spontaneously.
313 Life is a collection of out-of-equilibrium processes that work simultaneously in harmony. That is why Chapter 5 focuses on the self-replication that operates out of equilibrium by enabling replication and destruction pathways to occur simulta-neously. We achieved very promising results towards creating replicator families as “quasi-species”, but, adaptive behavior of them is yet to be demonstrated. We believe that out-of-equilibrium conditions will bring more unpredictable life-like features to synthetic replicator systems as small perturbations will likely have bigger impact than in an equilibrium regime.
As we have stated before, self-replicating peptide systems are prone to consider-able quantitative variability due to the inherent sensitivity to small perturbations. So, several repeats of an experiment should be run to prevent drawing wrong conclu-sions based on a single experiment. Additionally, when addressing stochastic effects, it becomes a necessity to run statistical analysis on a large number of repeats. In such cases, the chromatographic analysis, which forms the basis of the daily analysis routine in our lab, becomes a bottle-neck, as each sample run takes 15 minutes on average. To improve the timescale of daily routine analysis of our systems, we col-laborated with the Margulies group and showed in Chapter 6 that a combinatorial fluorescent sensor can be implemented in our system. Obtaining the compositional information as an optical read-out significantly reduces the analysis time. We hope that this combinatorial sensor will become a daily routine in labs and will enable the analysis of large numbers of DCLs in a significantly shorter time.
The establishment of supramolecular chemistry followed by systems chemistry have extended scientists’ horizons. Chemistry moved forward from isolated pure compounds to complex molecular systems. Self-replicating systems that are made out of single building blocks have been studied extensively and now the focus should shift to the replicator interactions (i.e. cross-catalysis). On the short term, any progress on the development of dissipative systems of co-existing and cooperating replicators would dramatically increase the possibilities of creating life-like features in the lab. On the long term, development of a compartment that is sustained by (relatively) simple chemical reactions and that can be integrated into a system that can undergo self-replication and Darwinian evolution is required to make the scientists dream of creating life de novo come true.
7.1. References 315
7.1
References
[1] Colomb-Delsuc, M.; Mattia, E.; Sad-ownik, J. W.; Otto, S. Nat. Commun. 2015, 6, 7427.
[2] Mattia, E.; Pal, A.; Leonetti, G.; Otto, S. Synlett 2017, 28, 103-107. [3] Frederix, P. W. J. M.; Ide, J.;
Al-tay, Y.; Schaeffer, G.; Surin, M.; Beljonne, D.; Bondarenko, A. S.; Jansen, T. L. C.; Otto, S.; Mar-rink, S. J. ACS Nano 2017, 11, 7858-7868.
[4] Altay, M.; Altay, Y.; Otto, S. Angew. Chem. Int. Ed. 2018, 139, 10564-10568.
7.1. References 315
7.1
References
[1] Colomb-Delsuc, M.; Mattia, E.; Sad-ownik, J. W.; Otto, S. Nat. Commun. 2015, 6, 7427.
[2] Mattia, E.; Pal, A.; Leonetti, G.; Otto, S. Synlett 2017, 28, 103-107. [3] Frederix, P. W. J. M.; Ide, J.;
Al-tay, Y.; Schaeffer, G.; Surin, M.; Beljonne, D.; Bondarenko, A. S.; Jansen, T. L. C.; Otto, S.; Mar-rink, S. J. ACS Nano 2017, 11, 7858-7868.
[4] Altay, M.; Altay, Y.; Otto, S. Angew. Chem. Int. Ed. 2018, 139, 10564-10568.
Summary
This thesis focuses on the emergence of novel peptide self-replicators from dynamic combinatorial libraries. Thiol functionalised peptide building blocks, first reported by our group in 2010, form the basis of all chapters. The work performed here has two main objectives: First, to deepen the insights obtainable from self-replicating systems and second, to use peptide building blocks to mimic life-like features such as replication. Alongside this, we developed novel strategies to access novel replicators and to dictate their properties.
Chapter 1 provides a short overview of systems chemistry and dynamic combi-natorial chemistry as a tool for de novo life research.
In Chapter 2 we have found that the amino acids that are close to the C-terminus play a critical role in replicator ring size selection and self-assembly. Molecular dy-namic simulations revealed that an increase in macrocycle size results in a more glob-ular shape with a more shielded core. Beyond 8-membered macrocycles the complex interactions at the core may prevent stacking of another macrocycle. So it is unlikely to have a replicator larger than octamer with the current design of our building blocks. The results also show that the size of the replicator is not solely determined by peptide hydrophobicity. Inter- and intra-molecular interactions can be strongly altered by a change of a single amino acid to the point of prohibiting replication altogether. We have also found that not only the amino acids that are close to the C-terminus, but also the charge on the C-terminus itself is important for the system to self-assemble. Molecular dynamic simulations support our findings (removal of the C-terminus charge hampers self-assembly) and revealed the contribution of the C-terminus charge to salt-bridge formation and its effects on inter-sheet distances. We have also showed that a high ionic strength can speed up the nucleation process and alter the morphology of the resulting fibers.
