University of Groningen
Novel peptide replicators from dynamic combinatorial libraries
Altay, Yigit
DOI:
10.33612/diss.90041906
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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 5
Sustaining a Distribution of Replicators
Out of Equilibrium
“Rules are foolish, arbitrary, mindless things that raise you quickly to a level of acceptable mediocrity, then, prevent you from progressing further.”
Bruce Barnbaum
Abstract
Erwin Schr¨odinger, in his inspirational book “What is Life”, posed the ques-tion “How can the events in space and time which take place within the spatial boundary of a living organism be accounted for by physics and chemistry?”. Life itself does not operate under thermodynamic equilibrium but constantly changes with time due to external (energy or matter) input and destructive mechanisms. So, we might come a little closer to the answer on this important and very much discussed question by studying out-of-equilibrium chemical systems. In this chapter, we study mixed-building-block systems, where the continuous sup-ply of building blocks and simultaneously continuous removal of replicators from the system is enabled. Following successful optimization of the experimental setup and the solution preparation, further optimizations are done on the rate of infusion and withdrawal in order to tackle the challenge of reaching a steady-state distribution of replicators. We showed that mutant replicators made from a mixture of building blocks reach a steady state and can sustain themselves over a few turnovers. These results represent an important milestone for quasi-speciation in self-replicators emerging from complex molecular networks.
228 5. Sustaining a Distribution of Replicators Out of Equilibrium
5.1
Introduction
L
ife is one of the greatest mysteries to be solved. How it originates and propagates remains poorly understood.1–4Although there is no consensus on the definitionof life, there are indispensable requirements to call something alive. Among the three fundamental elements for life (compartmentalization,5–8metabolism9–12and replica-tion13–16), replication is the most essential element for the emergence of new species
and thus for evolution. One might argue that all three elements are equally essential; but without replication, it is clear that life cannot be inherited and thus it cannot propagate. In order to achieve Darwinian evolution, the following four requirements need to be fulfilled:17 (1) entities must reproduce to form a new generation
(re-production); (2) the offsprings must tend to resemble their parents (heredity); (3) there should be a genetic diversity in individual characteristics among the members of the population (variation); and lastly (4) there must be a variation of individual reproductive success of organisms with respect to their environment depending on heritable characteristics (fitness).
Figure 5.1: Schematic representation of emergence of a distribution of replicators from a two-building-block system.
Simple networks of self-replicating molecules14,16,18–25 do not meet all these
re-quirements, mainly because in most cases there is only one type of molecule that is able to reproduce. The inspiring work by Sadownik et al.26 shows that the addition
of a second building block can lead to emergence of a distribution of replicators in which autocatalysis and cross-catalysis is enabled. However, the replicators produced in Sadownik’s work cannot be called quasi-species yet before the ability of such a dis-tribution of replicators to sustain itself in an out-of-equilibrium regime is proven.
5.2. Results and Discussion 229
In the definition described by Eigen27 and Schuster28 a quasi-species is a mutant
distribution centered around an optimal replicator that can persist in an environ-ment where error-prone replication and destruction of replicators occur at the same time.28–30 In the quasi-species model, evolution acts on the distribution.
In this chapter, we introduce two building block replicator systems made from widely studied peptides 1, 2 and 3 (Figure 5.1) that are provided with a constant addition of building blocks (infusion) and a constant removal of part of the reaction mixture (withdrawal). The infusion of new building blocks provides the replicators with enough “food” to keep replicating and withdrawal fulfills the role of replicator destruction by its physical removal from the system. After complete characterization of the systems and optimization of the experimental setup, we were able to show that the 1-2 mixed building block system can sustain itself in the out-of-equilibrium flow regime and thus can be called a quasi-species.
5.2
Results and Discussion
5.2.1
Emergence of a family of replicators in
two-building-block systems
We set up three sets of libraries (total concentration of 3.8 mM, equimolar in individ-ual peptides) with the following combinations of the peptides: 1-2, 1-3 and 2-3. All libraries were set up in 50 mM phosphate-buffered saline (PBS) containing 150 mM NaCl having pH 8.2 to increase the rate of replication as described in Chapter 2 and left agitated for 7 days under slow air oxidation. Individual libraries of these peptides form hexamers (16) and octamers (28and 38). When the building blocks are mixed,
1-2 and 1-3 forms a family of hexamer replicators and 2-3 forms a family of octamer replicators (Figure 5.2a-c). In all the mixed libraries that contain peptide 1, the ring size of the mutant replicators are dictated by peptide 1. This is likely because of the more hydrophobic nature of the phenylalanine moiety in this particular peptide.
Sadownik et al.26 described the self-replication behavior of 1-2 hexamers.
How-ever, the complete characterization of the amyloid-like structure of the replicators was not performed until now. In Figure 5.2d-e, we confirmed the amyloid type β-sheet structure of these assemblies using circular dichroism (CD) and thioflavin T (ThT) fluorescence assays.
228 5. Sustaining a Distribution of Replicators Out of Equilibrium
5.1
Introduction
L
ife is one of the greatest mysteries to be solved. How it originates and propagates remains poorly understood.1–4Although there is no consensus on the definitionof life, there are indispensable requirements to call something alive. Among the three fundamental elements for life (compartmentalization,5–8metabolism9–12and replica-tion13–16), replication is the most essential element for the emergence of new species
and thus for evolution. One might argue that all three elements are equally essential; but without replication, it is clear that life cannot be inherited and thus it cannot propagate. In order to achieve Darwinian evolution, the following four requirements need to be fulfilled:17 (1) entities must reproduce to form a new generation
(re-production); (2) the offsprings must tend to resemble their parents (heredity); (3) there should be a genetic diversity in individual characteristics among the members of the population (variation); and lastly (4) there must be a variation of individual reproductive success of organisms with respect to their environment depending on heritable characteristics (fitness).
Figure 5.1: Schematic representation of emergence of a distribution of replicators from a two-building-block system.
Simple networks of self-replicating molecules14,16,18–25 do not meet all these
re-quirements, mainly because in most cases there is only one type of molecule that is able to reproduce. The inspiring work by Sadownik et al.26 shows that the addition
of a second building block can lead to emergence of a distribution of replicators in which autocatalysis and cross-catalysis is enabled. However, the replicators produced in Sadownik’s work cannot be called quasi-species yet before the ability of such a dis-tribution of replicators to sustain itself in an out-of-equilibrium regime is proven.
5.2. Results and Discussion 229
In the definition described by Eigen27 and Schuster28 a quasi-species is a mutant
distribution centered around an optimal replicator that can persist in an environ-ment where error-prone replication and destruction of replicators occur at the same time.28–30 In the quasi-species model, evolution acts on the distribution.
In this chapter, we introduce two building block replicator systems made from widely studied peptides 1, 2 and 3 (Figure 5.1) that are provided with a constant addition of building blocks (infusion) and a constant removal of part of the reaction mixture (withdrawal). The infusion of new building blocks provides the replicators with enough “food” to keep replicating and withdrawal fulfills the role of replicator destruction by its physical removal from the system. After complete characterization of the systems and optimization of the experimental setup, we were able to show that the 1-2 mixed building block system can sustain itself in the out-of-equilibrium flow regime and thus can be called a quasi-species.
5.2
Results and Discussion
5.2.1
Emergence of a family of replicators in
two-building-block systems
We set up three sets of libraries (total concentration of 3.8 mM, equimolar in individ-ual peptides) with the following combinations of the peptides: 1-2, 1-3 and 2-3. All libraries were set up in 50 mM phosphate-buffered saline (PBS) containing 150 mM NaCl having pH 8.2 to increase the rate of replication as described in Chapter 2 and left agitated for 7 days under slow air oxidation. Individual libraries of these peptides form hexamers (16) and octamers (28and 38). When the building blocks are mixed,
1-2 and 1-3 forms a family of hexamer replicators and 2-3 forms a family of octamer replicators (Figure 5.2a-c). In all the mixed libraries that contain peptide 1, the ring size of the mutant replicators are dictated by peptide 1. This is likely because of the more hydrophobic nature of the phenylalanine moiety in this particular peptide.
Sadownik et al.26 described the self-replication behavior of 1-2 hexamers.
How-ever, the complete characterization of the amyloid-like structure of the replicators was not performed until now. In Figure 5.2d-e, we confirmed the amyloid type β-sheet structure of these assemblies using circular dichroism (CD) and thioflavin T (ThT) fluorescence assays.
230 5. Sustaining a Distribution of Replicators Out of Equilibrium
Figure 5.2: UPLC traces (monitored at 254 nm) of DCLs (0.76 mM in 50 mM PBS, pH 8.2) made from a) 1-2 b) 1-3 and c)2-3. d) CD spectra of DCLs made from peptide 1-2 (blue line), 1-3 (green line) and 2-3 (yellow line). e) Normalized thioflavin T (ThT) fluorescence emission of 1-2 (blue line), 1-3 (green line) and 2-3 (yellow line).
Additionally, in Figure 5.3, we show the formation of laterally associated fibers as a result of the self-assembly of these replicators by transmission electron microscopy (TEM).
5.2. Results and Discussion 231
Figure 5.3: Transmission electron micrographs of a) 1-2, b) 1-3 and c) 2-3. Images on the second row are taken at higher magnification of the corresponding samples on the first row.
5.2.2
Optimization of the Experimental Setup for Out of
Equi-librium Self-Replication
The experimental setup consists of a stirring plate where the DCLs are constantly agitated and two syringe pumps: the one is for infusion of the “food”, the other one is for withdrawal of a portion of the entire solution. The food solution is a DCL that is pre-oxidized to a certain oxidation level (80 mol% with respect to monomers) containing monomers, trimers and tetramers. Preliminary experiments showed that the oxidation level of the food solution is not constant during the course of the exper-iment which is about 2-3 weeks. Since the food oxidation level changes, there might be consequences that affects the outcome and so this variability should be eliminated. For this purpose, several modifications were introduced to keep the oxidation level of the food constant, at least, within each cycle (i.e. the time between the change of the food, approximately 4 days). These include modifications of the buffer prepa-ration, modifications involving the infusion syringe and modifications of the entire experimental setup.
232 5. Sustaining a Distribution of Replicators Out of Equilibrium
Modifications of the Buffer Preparation: In a preliminary experiment, food solu-tions were prepared under ambient condisolu-tions with freshly prepared buffer solusolu-tions that were degassed with nitrogen gas for 20 min prior to use. Since the food oxidized completely just after a day, the degassing time was increased to 2 hours. However, no significant improvement was observed. Then we decided to use freeze-pump-thaw (FPT) cycling to eliminate the oxygen present in the buffer solutions. After the FPT cycle is complete, buffer solutions are transferred to a glovebox and the food solutions were prepared under an inert atmosphere. Figure 5.4 shows that the oxidation level of the food does not change over 28 days by more than 10% for a library that is set up in FPT cycled buffer solution.
Figure 5.4: UPLC traces (monitored at 254 nm) of a food solution prepared in a FPT cycled borate buffer (50 mM, pH 8.2) and kept under inert atmosphere. Day 0 is shown in black line and Day 28 shown in red line.
Modifications Involving the Infusion Syringe: In the preliminary experiments done by Dr. Sadownik, plastic syringes were used, but after improvements on the buffer preparation, they were replaced by Hamilton syringes as they are more robust and work better in a syringe pump.
The Hamilton Company guarantees that the syringes are gas-tight up to 24 hours. However, syringes are not tested for a longer period of time and Hamilton does not guarantee that oxygen in the air would not leak into the syringes. We reasoned that the screw-head of the syringes are a likely source of such leakage. So we ran a set of experiments using different types of materials to seal the screw-head of the syringe. Conditions and corresponding results that are listed in Table 5.1 show that the attempts to seal the infusion syringe failed if the experiment is run under ambient conditions. In contrast all the experiments done under an inert atmosphere showed minor changes in the oxidation level over a week. In one particular experiment (No. 6 in the Table 5.1), the syringe was first placed under ambient conditions and we observed 6.7% further oxidation in one day. Then the syringe was placed under an inert atmosphere and 1.8% further oxidation was observed in a week (see Figure
5.2. Results and Discussion 233
No. Condition Syringe
Volume Sealent
Change in the
oxidation level Duration 1-2 1-3
1 ambient 1 mL - 20.3% 28.8% 14 days 2 ambient 1 mL Teflon⃝R 15.7% 21.2% 13 days
3 ambient 1 mL Parafilm⃝R 18.2% 14.9% 12 days
4 ambient 5 mL Parafilm⃝R
5.00% 28.8% 11 days 5 ambient 1 mL Sugru 11.4% 21.1% 5 days 6* inert 1 mL Teflon⃝R - 1.8% 5 days
7 inert 1 mL - 2.4% 1.1% 7 days
8 inert 5 mL Parafilm⃝R 3.1% -2.0% 7 days
Table 5.1: The change of the oxidation level in infusion syringe with the corresponding manipulations. *No. 6 is started in ambient conditions and then placed under inert atmo-sphere. Change of the oxidation level for that sample is calculated from the point where it is placed under inert atmosphere. Relevant plots can be seen in the Figures 5.20, 5.21 and 5.22.
5.21). This result suggests that the oxygen that oxidizes the food is leaking into the syringe and the contribution from oxygen that might be dissolved in the buffer solution is insignificant.
Modifications of the Experimental Setup: Considering the results above, we de-cided to place the entire setup under an inert atmosphere. However, due to large size of the setup and considering that more than one person would want to run experi-ments using similar setups, it is not feasible to dedicate a glovebox to every single flow experiment. So we decided to use an Atmosbag, an inflatable polyethylene glove-box purchased from Sigma Aldrich, under constant flow of N2 in order to provide
overpressure at all times.
Experiments done in the Atmosbag showed that the oxidation level can be kept relatively constant (within 5-10%) over 4 days (See SI Figure 5.29, 5.35, and 5.39). As the food solutions were refilled at every 4 days (when 1 mL syringes are used), this setup was good enough to proceed with confidence on the oxidation level. In principle, under a completely inert atmosphere, the same setup can be used without the need of changing the food solution over the period of entire experiment if larger volume syringes are used.
5.2.3
Optimization of the Flow Rate
The rate of replication has to be larger or equal to the rate of destruction (i.e. outflow) in order for the system to sustain itself when provided with a constant
234 5. Sustaining a Distribution of Replicators Out of Equilibrium
Figure 5.5: Schematic representation of the experimental setup placed within the Atmos-bag.
supply of food. Adjustment of the infusion and withdrawal rate was required to reach a steady state with non-zero replicator concentration. In all experiments, the rate of infusion was kept identical to the rate of withdrawal and experiments were conducted under ambient conditions (i.e. without the Atmosbag).
We ran the replicates of mixed libraries made by 1-2, 1-3 and 2-3 at flow rates of 5µL/h, 10 µL/h and 20 µL/h (Figure 5.6). When we used 5 µL/h flow rate, we ob-served that at the end of almost 2 turnovers, the replicator distribution was sustained. However, it does not reach a distribution that is different than the one at thermo-dynamic equilibrium. Out-of-equilibrium assemblies require a continuous supply of energy and can only be sustained as long as energy is provided. When the energy supply is stopped, the system would revert to the thermodynamic minimum state. In the experiments with 10µL/h flow rate, we observed that all the replicators were successfully sustained at the end of 3 turnovers. While we observed a shift towards the 1 rich mutants in the distributions of 1-2 and 1-3 hexamer replicators, we did not observe any significant change in the distribution of 2-3 octamer replicators. Lastly for the case of 20µL/h flow rate, we observed a significant decrease in the overall replicator percentage in all the libraries at the end of 7 turnovers. The distribution of the hexamer replicators in 1-2 and 1-3 shifted even further towards the 1 rich replicators while the 2-3 octamer replicators were essentially extinct.
5.2. Results and Discussion 235
Figure 5.6: Initial (blue bars) and final (red bars) distributions of replicators formed in the DCLs made from 1-2, 1-3 and 2-3 (0.76 mM in 50 mM borate buffer, pH 8.2) at different flow rates.
Under out-of-equilibrium flow conditions, the 1-2 libraries showed the most sig-nificant change in the library distribution. Additionally, the optimum flow rate was found to be 10µL/h. Smaller flow rates could also be suitable but we could not continue experiments more than 2 turnovers (around 2 weeks) as the experimental conditions were not allowing to do so at that time. Considering these findings, we decided to continue with the 1-2 mixed libraries using a 10µL/h flow rate in our experiments.
234 5. Sustaining a Distribution of Replicators Out of Equilibrium
Figure 5.5: Schematic representation of the experimental setup placed within the Atmos-bag.
supply of food. Adjustment of the infusion and withdrawal rate was required to reach a steady state with non-zero replicator concentration. In all experiments, the rate of infusion was kept identical to the rate of withdrawal and experiments were conducted under ambient conditions (i.e. without the Atmosbag).
We ran the replicates of mixed libraries made by 1-2, 1-3 and 2-3 at flow rates of 5µL/h, 10 µL/h and 20 µL/h (Figure 5.6). When we used 5 µL/h flow rate, we ob-served that at the end of almost 2 turnovers, the replicator distribution was sustained. However, it does not reach a distribution that is different than the one at thermo-dynamic equilibrium. Out-of-equilibrium assemblies require a continuous supply of energy and can only be sustained as long as energy is provided. When the energy supply is stopped, the system would revert to the thermodynamic minimum state. In the experiments with 10µL/h flow rate, we observed that all the replicators were successfully sustained at the end of 3 turnovers. While we observed a shift towards the 1 rich mutants in the distributions of 1-2 and 1-3 hexamer replicators, we did not observe any significant change in the distribution of 2-3 octamer replicators. Lastly for the case of 20µL/h flow rate, we observed a significant decrease in the overall replicator percentage in all the libraries at the end of 7 turnovers. The distribution of the hexamer replicators in 1-2 and 1-3 shifted even further towards the 1 rich replicators while the 2-3 octamer replicators were essentially extinct.
5.2. Results and Discussion 235
Figure 5.6: Initial (blue bars) and final (red bars) distributions of replicators formed in the DCLs made from 1-2, 1-3 and 2-3 (0.76 mM in 50 mM borate buffer, pH 8.2) at different flow rates.
Under out-of-equilibrium flow conditions, the 1-2 libraries showed the most sig-nificant change in the library distribution. Additionally, the optimum flow rate was found to be 10µL/h. Smaller flow rates could also be suitable but we could not continue experiments more than 2 turnovers (around 2 weeks) as the experimental conditions were not allowing to do so at that time. Considering these findings, we decided to continue with the 1-2 mixed libraries using a 10µL/h flow rate in our experiments.
236 5. Sustaining a Distribution of Replicators Out of Equilibrium
5.2.4
Sustaining a Distribution of Replicators Out of
Equilib-rium
Sustaining the replicator distribution in an out-of equilibrium regime is the main chal-lenge in confirming quasi-speciation of the mutant replicators. Preliminary experi-ments showed that there is a significant variation in the outcome as the concentration of the inflowing matter and the replicators in the reaction vial changes. To probe that effect and finally find an optimum concentration that leads to establishment of a steady state in replicator distribution, we set up libraries at different concentrations and varied the concentration of food solutions. In contrast to the experiments done for the flow rate optimization, starting solutions in the reaction vial contained only replicator 16instead of the entire hexamer replicator family of 1-2.
No. Condition Reaction Vial Content Infusion Syringe Content
Food Replicators Food
1 Flow - 0.76 mM 0.76 mM 2 Flow - 0.76 mM 1.52 mM 3 Flow - 1.52 mM 1.52 mM 4 Flow - - 1.52 mM 5 Flow 1.52 mM - 1.52 mM 6 Batch 1.52 mM -
-Table 5.2: List of experiments done for the concentration optimization. “Food” refers to a pre-oxidized (80 mol % w.r.t. monomers) DCL that contains almost exclusively monomers, trimers and tetramers. “Replicators” refers to a fully oxidized library of 1 that contains more than 95% 16. All the libraries in the reaction vial and infusion syringe were prepared in 50
mM borate buffer, pH 8.2.
Experiments at various concentrations showed that there is also a concentration dependence in these systems. When low equimolar concentrations are used (Table 5.2, No.1 and Figure 5.7a), the system appears to reach a steady state on day 6 and remains stable for at least 2 days. However, then non-assembling trimers and tetramers start to accumulate and percentage of hexamer replicators drops. This shows that rate of replication is not as fast as the destruction in the system and that after 8 days, replicators start to go extinct. Increasing the concentration of the food (Table 5.2, No.2 and Figure 5.7b) only increases the rate of trimer/ tetramer accumulation and hampers the replication significantly.
5.2. Results and Discussion 237
Figure 5.7: Kinetic profiles of hexamers, tetramers and trimers in the DLCs corresponding to libraries a) No.1 and b) No.2 in Table 5.2 made from mixed libraries of 1-2.
As we increased the concentration in both food and the reaction vial (Table 5.2, No.3 and Figure 5.8) we observed that the steady state is established after 4 turnovers (14 days) and stays stable over more than 5 turnovers (20 days) although at a very low replicator concentration. This result is in line with the simulations that are done by Randy Olson and Bjørn Østman31on the visualization of fitness landscapes: The
replicator population stays on highest peak in the energy landscape at low mutation rate (i.e. the starting point of the experiment where the hexamer replicator 16is the
fittest) and as the mutation rate increases (i.e. inflow of the food solutions containing mixed building blocks) population evolves of the highest peak and reaches a higher average fitness.
One interesting point is that we can detect only 1521 and 16 at the final
compo-sition. The library ends up in a few 1-rich replicators rather than a shifted Gaussian distribution of 1 rich replicators that we obtained before. This might be a result of the starting solution. Although we thought that the system should behave like a state function, results showed that the starting composition actually makes a difference, possibly because of the initial interactions.
Control experiments, where we infuse food into borate buffer that does not contain any of the building blocks (Table 5.2, No.4) or into a food solution (Table 5.2, No.5),
236 5. Sustaining a Distribution of Replicators Out of Equilibrium
5.2.4
Sustaining a Distribution of Replicators Out of
Equilib-rium
Sustaining the replicator distribution in an out-of equilibrium regime is the main chal-lenge in confirming quasi-speciation of the mutant replicators. Preliminary experi-ments showed that there is a significant variation in the outcome as the concentration of the inflowing matter and the replicators in the reaction vial changes. To probe that effect and finally find an optimum concentration that leads to establishment of a steady state in replicator distribution, we set up libraries at different concentrations and varied the concentration of food solutions. In contrast to the experiments done for the flow rate optimization, starting solutions in the reaction vial contained only replicator 16instead of the entire hexamer replicator family of 1-2.
No. Condition Reaction Vial Content Infusion Syringe Content
Food Replicators Food
1 Flow - 0.76 mM 0.76 mM 2 Flow - 0.76 mM 1.52 mM 3 Flow - 1.52 mM 1.52 mM 4 Flow - - 1.52 mM 5 Flow 1.52 mM - 1.52 mM 6 Batch 1.52 mM -
-Table 5.2: List of experiments done for the concentration optimization. “Food” refers to a pre-oxidized (80 mol % w.r.t. monomers) DCL that contains almost exclusively monomers, trimers and tetramers. “Replicators” refers to a fully oxidized library of 1 that contains more than 95% 16. All the libraries in the reaction vial and infusion syringe were prepared in 50
mM borate buffer, pH 8.2.
Experiments at various concentrations showed that there is also a concentration dependence in these systems. When low equimolar concentrations are used (Table 5.2, No.1 and Figure 5.7a), the system appears to reach a steady state on day 6 and remains stable for at least 2 days. However, then non-assembling trimers and tetramers start to accumulate and percentage of hexamer replicators drops. This shows that rate of replication is not as fast as the destruction in the system and that after 8 days, replicators start to go extinct. Increasing the concentration of the food (Table 5.2, No.2 and Figure 5.7b) only increases the rate of trimer/ tetramer accumulation and hampers the replication significantly.
5.2. Results and Discussion 237
Figure 5.7: Kinetic profiles of hexamers, tetramers and trimers in the DLCs corresponding to libraries a) No.1 and b) No.2 in Table 5.2 made from mixed libraries of 1-2.
As we increased the concentration in both food and the reaction vial (Table 5.2, No.3 and Figure 5.8) we observed that the steady state is established after 4 turnovers (14 days) and stays stable over more than 5 turnovers (20 days) although at a very low replicator concentration. This result is in line with the simulations that are done by Randy Olson and Bjørn Østman31on the visualization of fitness landscapes: The
replicator population stays on highest peak in the energy landscape at low mutation rate (i.e. the starting point of the experiment where the hexamer replicator 16is the
fittest) and as the mutation rate increases (i.e. inflow of the food solutions containing mixed building blocks) population evolves of the highest peak and reaches a higher average fitness.
One interesting point is that we can detect only 1521and 16at the final
compo-sition. The library ends up in a few 1-rich replicators rather than a shifted Gaussian distribution of 1 rich replicators that we obtained before. This might be a result of the starting solution. Although we thought that the system should behave like a state function, results showed that the starting composition actually makes a difference, possibly because of the initial interactions.
Control experiments, where we infuse food into borate buffer that does not contain any of the building blocks (Table 5.2, No.4) or into a food solution (Table 5.2, No.5),
238 5. Sustaining a Distribution of Replicators Out of Equilibrium shows emergence of hexamer replicators but only in a minor amount (not more then 2.5 % of total library material). The batch experiment also showed no emergence of hexamer replicators of 1-2. This is possibly due to because high pre-oxidation level (80 %) of the starting solution and the low stirring rate (350 rpm).
Figure 5.8: Initial (blue bars) and final (red bars) distributions of replicators formed in the DCLs made from 1-2, 1-3 and 2-3 (0.76 mM in 50 mM borate buffer, pH 8.2) at different flow rates.
5.2.5
Limitations
The main limitation is the very high quantitative variability due to the inherent sen-sitivity of the system. If several identical replicates of each experiment are prepared and run at the same time, this variability is reduced significantly. However, if the same experiment was repeated at a different time, minor difference in the prepara-tion of the soluprepara-tions, building block composiprepara-tions, initial oxidaprepara-tion levels or ambient temperature affect the outcome.
We now believe that a robust system which is highly reproducible needs to fulfill the following requirements:
• The system should be under an inert atmosphere at all times. Possibly a glovebox should be dedicated for flow experiments.
• The building block composition should be identical in each experiment. Mis-handling of the building blocks, or the introduction of any contamination should be eliminated. The building blocks should be stored in a freezer under an inert atmosphere.
• Reaction vials should be sealed to minimize losses due to evaporation.
• The system should be kept at constant temperature at all times. Temperature fluctuations during day and night or summer and winter affect the results.
5.3. Conclusions 239
• Samples should be analyzed immediately after preparation. Queuing the sam-ples should be avoided. (Running a UPLC sample over and over again for 2 hours did not reveal any considerable change in the composition of the sample. So this is not as crucial as others.)
• Analysis of the UPLC chromatograms should be automated after optimization for a certain mixed library.
5.3
Conclusions
To sum up, we have characterized three systems of mixed-building-block replicators in detail. CD, ThT fluorescence assays and TEM confirmed that the replicator fam-ilies of 1-2, 1-3 and 2-3 exhibit β-sheet structure in amyloid-like fibrils. Second, optimization of the experimental setup and buffer preparation resulted in a protocol in which the oxidation level of the food solutions remains constant during the ex-periments. And lastly, among all these mixed libraries, replicators made from 1-2 exhibited a significant shift towards a non-Gaussian distribution after a few turnovers. More importantly, this distribution can be sustained over 5 turnovers which indicates that the steady state is established out of equilibrium.
These results mark a milestone towards creating quasi-species of hexamer repli-cators based on the 1-2 mixed building block system. Now, the behavior of these replicators in response to a change in the environment should be investigated to probe the adaptive nature of the system. Additionally further experiments should be carried out to check the extent to the original replicator distribution which is re-established upon disconnecting the system from flow. Overall, our results demon-strate that incorporation of replication, exchange and mutation, enables the study of quasi-speciation in a fully synthetic system. This will open up new possibilities in synthetic self-replicator research and will allow access to life-like properties that are governed by evolutionary processes such as mutation, adaptation, fitness and variation.
5.4
Acknowledgements
Dr. J. Sadownik is recognised for the preliminary experiments and M. Altay is acknowledged for negative staining TEM imaging and the exhaustive discussions on the results. Dr. C. G. Pappas is gratefully acknowledged for proof-reading this chapter and for giving precious feedback.
238 5. Sustaining a Distribution of Replicators Out of Equilibrium shows emergence of hexamer replicators but only in a minor amount (not more then 2.5 % of total library material). The batch experiment also showed no emergence of hexamer replicators of 1-2. This is possibly due to because high pre-oxidation level (80 %) of the starting solution and the low stirring rate (350 rpm).
Figure 5.8: Initial (blue bars) and final (red bars) distributions of replicators formed in the DCLs made from 1-2, 1-3 and 2-3 (0.76 mM in 50 mM borate buffer, pH 8.2) at different flow rates.
5.2.5
Limitations
The main limitation is the very high quantitative variability due to the inherent sen-sitivity of the system. If several identical replicates of each experiment are prepared and run at the same time, this variability is reduced significantly. However, if the same experiment was repeated at a different time, minor difference in the prepara-tion of the soluprepara-tions, building block composiprepara-tions, initial oxidaprepara-tion levels or ambient temperature affect the outcome.
We now believe that a robust system which is highly reproducible needs to fulfill the following requirements:
• The system should be under an inert atmosphere at all times. Possibly a glovebox should be dedicated for flow experiments.
• The building block composition should be identical in each experiment. Mis-handling of the building blocks, or the introduction of any contamination should be eliminated. The building blocks should be stored in a freezer under an inert atmosphere.
• Reaction vials should be sealed to minimize losses due to evaporation.
• The system should be kept at constant temperature at all times. Temperature fluctuations during day and night or summer and winter affect the results.
5.3. Conclusions 239
• Samples should be analyzed immediately after preparation. Queuing the sam-ples should be avoided. (Running a UPLC sample over and over again for 2 hours did not reveal any considerable change in the composition of the sample. So this is not as crucial as others.)
• Analysis of the UPLC chromatograms should be automated after optimization for a certain mixed library.
5.3
Conclusions
To sum up, we have characterized three systems of mixed-building-block replicators in detail. CD, ThT fluorescence assays and TEM confirmed that the replicator fam-ilies of 1-2, 1-3 and 2-3 exhibit β-sheet structure in amyloid-like fibrils. Second, optimization of the experimental setup and buffer preparation resulted in a protocol in which the oxidation level of the food solutions remains constant during the ex-periments. And lastly, among all these mixed libraries, replicators made from 1-2 exhibited a significant shift towards a non-Gaussian distribution after a few turnovers. More importantly, this distribution can be sustained over 5 turnovers which indicates that the steady state is established out of equilibrium.
These results mark a milestone towards creating quasi-species of hexamer repli-cators based on the 1-2 mixed building block system. Now, the behavior of these replicators in response to a change in the environment should be investigated to probe the adaptive nature of the system. Additionally further experiments should be carried out to check the extent to the original replicator distribution which is re-established upon disconnecting the system from flow. Overall, our results demon-strate that incorporation of replication, exchange and mutation, enables the study of quasi-speciation in a fully synthetic system. This will open up new possibilities in synthetic self-replicator research and will allow access to life-like properties that are governed by evolutionary processes such as mutation, adaptation, fitness and variation.
5.4
Acknowledgements
Dr. J. Sadownik is recognised for the preliminary experiments and M. Altay is acknowledged for negative staining TEM imaging and the exhaustive discussions on the results. Dr. C. G. Pappas is gratefully acknowledged for proof-reading this chapter and for giving precious feedback.
240 5. Sustaining a Distribution of Replicators Out of Equilibrium
5.5
Materials and Methods
5.5.1
Materials
UPLC grade water was used in all experiments. Disodium phosphate (Sigma-Aldrich), monosodium phosphate (Sigma-Aldrich), boric anhydride (Sigma-Aldrich), sodium chloride (Merck Chemicals) and sodium hydroxide (Merck Chemicals) were utilized for buffer preparation and pH adjustments. For UPLC measurements, UPLC grade acetonitrile, water and trifluoroacetic acid were purchased from Biosolve BV.
Reagents were purchased from Sigma-Aldrich, Merck, Fluka, or Acros and used as received. The Atmosbag (51ˆ58 cm, an inflatable polyethylene glove box alternative) was purchased from Sigma-Aldrich.
5.5.2
Peptide Synthesis
The peptides 1, 2, and 3 were synthesized by Cambridge Peptides Ltd. (Birming-ham, U.K.) by coupling 3,5-bis(tritylthio)-benzoic acid, which was synthesized via a previously reported procedure.32
All peptides had purity higher than 95%. Impurities were mainly resulting from the formation of disulfides (i.e. dimers, cyclic trimers etc.)
5.5.3
Library Preparation and Sampling
Food Solutions: Method I :
Borate buffer was degassed for an hour with nitrogen prior to use. Equimolar ([1] = 0.76 mM, [2] = 0.76 mM) mixtures of building blocks were prepared (600µL) and partially oxidized with 15µL perborate solution (30 mM in water).
Method II :
Borate buffer (250 mL, 50 mM, pH 8.2) was degassed using freeze-pump-thaw cy-cling and transferred to a glovebox. Building blocks and sodium perborate tetrahy-drate was weighted in ambient conditions and transferred to the glovebox in the powder form. Equimolar mixtures of building blocks were prepared with a final con-centration of 0.76 mM or 1.52 mM. Then libraries were partially oxidized (80 mol% with respect to the monomers) using freshly prepared perborate solution (50 mM). Libraries were agitated at 1200 rpm for 15 min and left non-agitated for a week to equilibrate prior to use.
5.5. Materials and Methods 241
Replicator Stock Solutions: Preparation of 16 Stock Solution:
Libraries were set up at a concentration of 3.8 mM in building block in phosphate-buffered saline (PBS) (50 mM, with 150 mM NaCl, pH 8.2). The volume of each library was at least 500µL. All libraries were equilibrated in an HPLC vial (12ˆ32 mm) with a Teflon-coated screw cap. All HPLC vials were equipped with a cylindrical stirrer bar (2ˆ 5 mm, Teflon coated, purchased from VWR) and were stirred at 1200 rpm using an IKA RCT basic hot plate stirrer at room temperature.
Preparation of Stock Solutions of Mixed Replicators:
For the flow experiments, starting solutions were prepared by mixing the two building blocks (in a 1:1 mol ratio) in PBS (50 mM, 150mM NaCl, pH 8.2) in 3.8 mM final concentration. Libraries were agitated at 1200 rpm using an IKA RCT basic hot plate stirrer and equilibrated for a week. Replicator formation was confirmed by UPLC and UPLC-MS analysis.
Flow Experiments: Set-up I :
Stock solutions were diluted to 0.76 mM using borate buffer (50 mM, pH 8.2). Library volume for all the flow experiments was 750µL. Gas tight syringes (500 µL) were filled with appropriate food solutions. For outflow, 1 mL gas tight syringes were used. In order to eliminate the air in the dead volume of the syringes, a small amount of buffer is withdrawn prior to the experiment. Stirring speed is kept at 300-400 rpm to minimize splash.
Set-up II :
An Atmosbag was equipped with a stirring plate and two flow-pumps as shown in Figure 5.5. Inlets and outlets were sealed with a combination of Teflon tape, parafilm and duct tape. Stock solutions were diluted to 0.76 mM or 1.52 mM using borate buffer (50 mM, pH 8.2). Library volume for all the flow experiments was 750µL. Gas tight syringes (1 mL) were filled with appropriate food solutions and placed in the infusion pump. The withdrawal pump is equipped with 1 mL gas tight syringes. In order to eliminate the air in the dead volume of the syringes in the withdrawal pumps, a small amount of buffer is withdrawn prior to the experiment. Stirring speed kept at 350 rpm to minimize splash. Flow rates are identical for infusion and withdrawal syringes.
Sampling:
Aliquots of 10µL of each sample was first diluted with 10 µL DMF and then diluted with 30µL UPLC grade water prior to UPLC or LC-MS analysis.
240 5. Sustaining a Distribution of Replicators Out of Equilibrium
5.5
Materials and Methods
5.5.1
Materials
UPLC grade water was used in all experiments. Disodium phosphate (Sigma-Aldrich), monosodium phosphate (Sigma-Aldrich), boric anhydride (Sigma-Aldrich), sodium chloride (Merck Chemicals) and sodium hydroxide (Merck Chemicals) were utilized for buffer preparation and pH adjustments. For UPLC measurements, UPLC grade acetonitrile, water and trifluoroacetic acid were purchased from Biosolve BV.
Reagents were purchased from Sigma-Aldrich, Merck, Fluka, or Acros and used as received. The Atmosbag (51ˆ58 cm, an inflatable polyethylene glove box alternative) was purchased from Sigma-Aldrich.
5.5.2
Peptide Synthesis
The peptides 1, 2, and 3 were synthesized by Cambridge Peptides Ltd. (Birming-ham, U.K.) by coupling 3,5-bis(tritylthio)-benzoic acid, which was synthesized via a previously reported procedure.32
All peptides had purity higher than 95%. Impurities were mainly resulting from the formation of disulfides (i.e. dimers, cyclic trimers etc.)
5.5.3
Library Preparation and Sampling
Food Solutions: Method I :
Borate buffer was degassed for an hour with nitrogen prior to use. Equimolar ([1] = 0.76 mM, [2] = 0.76 mM) mixtures of building blocks were prepared (600µL) and partially oxidized with 15µL perborate solution (30 mM in water).
Method II :
Borate buffer (250 mL, 50 mM, pH 8.2) was degassed using freeze-pump-thaw cy-cling and transferred to a glovebox. Building blocks and sodium perborate tetrahy-drate was weighted in ambient conditions and transferred to the glovebox in the powder form. Equimolar mixtures of building blocks were prepared with a final con-centration of 0.76 mM or 1.52 mM. Then libraries were partially oxidized (80 mol% with respect to the monomers) using freshly prepared perborate solution (50 mM). Libraries were agitated at 1200 rpm for 15 min and left non-agitated for a week to equilibrate prior to use.
5.5. Materials and Methods 241
Replicator Stock Solutions: Preparation of 16 Stock Solution:
Libraries were set up at a concentration of 3.8 mM in building block in phosphate-buffered saline (PBS) (50 mM, with 150 mM NaCl, pH 8.2). The volume of each library was at least 500µL. All libraries were equilibrated in an HPLC vial (12ˆ32 mm) with a Teflon-coated screw cap. All HPLC vials were equipped with a cylindrical stirrer bar (2ˆ 5 mm, Teflon coated, purchased from VWR) and were stirred at 1200 rpm using an IKA RCT basic hot plate stirrer at room temperature.
Preparation of Stock Solutions of Mixed Replicators:
For the flow experiments, starting solutions were prepared by mixing the two building blocks (in a 1:1 mol ratio) in PBS (50 mM, 150mM NaCl, pH 8.2) in 3.8 mM final concentration. Libraries were agitated at 1200 rpm using an IKA RCT basic hot plate stirrer and equilibrated for a week. Replicator formation was confirmed by UPLC and UPLC-MS analysis.
Flow Experiments: Set-up I :
Stock solutions were diluted to 0.76 mM using borate buffer (50 mM, pH 8.2). Library volume for all the flow experiments was 750µL. Gas tight syringes (500 µL) were filled with appropriate food solutions. For outflow, 1 mL gas tight syringes were used. In order to eliminate the air in the dead volume of the syringes, a small amount of buffer is withdrawn prior to the experiment. Stirring speed is kept at 300-400 rpm to minimize splash.
Set-up II :
An Atmosbag was equipped with a stirring plate and two flow-pumps as shown in Figure 5.5. Inlets and outlets were sealed with a combination of Teflon tape, parafilm and duct tape. Stock solutions were diluted to 0.76 mM or 1.52 mM using borate buffer (50 mM, pH 8.2). Library volume for all the flow experiments was 750µL. Gas tight syringes (1 mL) were filled with appropriate food solutions and placed in the infusion pump. The withdrawal pump is equipped with 1 mL gas tight syringes. In order to eliminate the air in the dead volume of the syringes in the withdrawal pumps, a small amount of buffer is withdrawn prior to the experiment. Stirring speed kept at 350 rpm to minimize splash. Flow rates are identical for infusion and withdrawal syringes.
Sampling:
Aliquots of 10µL of each sample was first diluted with 10 µL DMF and then diluted with 30µL UPLC grade water prior to UPLC or LC-MS analysis.
242 5. Sustaining a Distribution of Replicators Out of Equilibrium
5.5.4
Circular Dichroism (CD)
Samples were prepared by diluting libraries to 0.24 mM (with respect to total building block concentration). All spectra were recorded in a Jasco J-715 CD spectrophotome-ter using HELMA quartz cuvettes with path length of 0.1 cm. Continuous spectra were obtained from 190 nm to 300 nm at room temperature. All reported spectra were an average of three scans recorded at a scan rate of 200 nm/min with 2-nm step interval and measured in millidegrees. Solvent spectra were subtracted from all spectra.
5.5.5
Thioflavin T (ThT) Fluorescence Assay
A ThT stock solution (2.2 mM) was prepared in 10 mL phosphate buffer (50 mM phosphate, 150 mM NaCl, pH 8.2) and filtered through a 0.2µm syringe filter. On the day of analysis, 50µL of the stock solution was diluted into 5 mL phosphate buffer (50 mM phosphate, 150 mM NaCl, pH 8.2) to generate the working solution of 22µm. The fluorescence intensity of 450µL ThT solution was measured by excitation at 440 nm (slit width 5 nm) and emission between 480-700 nm (slit width 5 nm), averaging 3 accumulations. An aliquot of 80µL of peptide solution (100 µm in borate buffer) was added to the HELMA 10ˆ2 mm quartz cuvette, incubated for 2 min, and the intensity was measured over 3 accumulations. All fluorescence measurements were recorded on a JASCO FP6200 fluorimeter equipped with a 480 nm high-pass cut-off filter on the emission channel to avoid high order diffractions originating from the excitation wavelength.
5.5.6
Transmission Electron Microscopy (TEM) Analysis
All images were obtained after negatively staining the sample on the grid. Samples were diluted 20-fold with water prior to deposition. A small aliquot (5 mL) of diluted sample was deposited on a 400-mesh copper grid covered with a thin carbon film (Agar Scientific). After 30 s of deposition, the droplet was gently blotted on filter paper. The sample was stained twice (4 mL each time) with a 2% uranyl acetate solution and blotted again on filter paper after deposition (30 s each time). The grids were observed on a Philips CM12 transmission electron microscope operating at 120 kV. All images were recorded on a slow scan CCD camera.
5.6
UPLC and LC-MS analyses
UPLC analyses were performed on a Waters Acquity UPLC I-class system equipped with a PDA detector. A reversed-phase UPLC column (Aeris 1.7µm XB-C18 150 ˆ
5.6. UPLC and LC-MS analyses 243
2.10 mm, purchased from Phenomenex) was used for the analyses of all samples, while UV absorbance was monitored at 254 nm. The column temperature was equilibrated at 35˝C prior to injections.
UPLC-MS analyses were performed using a Waters Acquity UPLC H-class system coupled to a Waters Xevo-G2 TOF. The mass spectrometer was operated under the following conditions: electrospray ionization mode, positive; capillary voltage, 2.5 kV; sampling cone voltage, 30 V; extraction cone voltage, 4.0 V. Samples were prepared by dissolving 10µL of a 3.8 mM library in 10 µL DMF and then further diluting them by 30µL UPLC grade water. The operating parameters were the following: eluent flow rate 0.3 mL/min; eluent A, UPLC grade water with 0.1 v% trifluoroacetic acid; eluent B, UPLC grade acetonitrile with 0.1 v% trifluoroacetic acid.
5.6.1
UPLC Methods
Libraries were analyzed using the following method (linear gradient):
Time, min. A% B% 0.00 90 10 1.00 90 10 1.30 75 25 3.00 72 28 11.00 60 40 11.50 5 95 12.00 5 95 12.50 90 10 15.00 90 10
Table 5.3: Elution profile used for the analysis of the mixed building block DCLs made from 1-2 and 1-3.
242 5. Sustaining a Distribution of Replicators Out of Equilibrium
5.5.4
Circular Dichroism (CD)
Samples were prepared by diluting libraries to 0.24 mM (with respect to total building block concentration). All spectra were recorded in a Jasco J-715 CD spectrophotome-ter using HELMA quartz cuvettes with path length of 0.1 cm. Continuous spectra were obtained from 190 nm to 300 nm at room temperature. All reported spectra were an average of three scans recorded at a scan rate of 200 nm/min with 2-nm step interval and measured in millidegrees. Solvent spectra were subtracted from all spectra.
5.5.5
Thioflavin T (ThT) Fluorescence Assay
A ThT stock solution (2.2 mM) was prepared in 10 mL phosphate buffer (50 mM phosphate, 150 mM NaCl, pH 8.2) and filtered through a 0.2µm syringe filter. On the day of analysis, 50µL of the stock solution was diluted into 5 mL phosphate buffer (50 mM phosphate, 150 mM NaCl, pH 8.2) to generate the working solution of 22µm. The fluorescence intensity of 450µL ThT solution was measured by excitation at 440 nm (slit width 5 nm) and emission between 480-700 nm (slit width 5 nm), averaging 3 accumulations. An aliquot of 80µL of peptide solution (100 µm in borate buffer) was added to the HELMA 10ˆ2 mm quartz cuvette, incubated for 2 min, and the intensity was measured over 3 accumulations. All fluorescence measurements were recorded on a JASCO FP6200 fluorimeter equipped with a 480 nm high-pass cut-off filter on the emission channel to avoid high order diffractions originating from the excitation wavelength.
5.5.6
Transmission Electron Microscopy (TEM) Analysis
All images were obtained after negatively staining the sample on the grid. Samples were diluted 20-fold with water prior to deposition. A small aliquot (5 mL) of diluted sample was deposited on a 400-mesh copper grid covered with a thin carbon film (Agar Scientific). After 30 s of deposition, the droplet was gently blotted on filter paper. The sample was stained twice (4 mL each time) with a 2% uranyl acetate solution and blotted again on filter paper after deposition (30 s each time). The grids were observed on a Philips CM12 transmission electron microscope operating at 120 kV. All images were recorded on a slow scan CCD camera.
5.6
UPLC and LC-MS analyses
UPLC analyses were performed on a Waters Acquity UPLC I-class system equipped with a PDA detector. A reversed-phase UPLC column (Aeris 1.7µm XB-C18 150 ˆ
5.6. UPLC and LC-MS analyses 243
2.10 mm, purchased from Phenomenex) was used for the analyses of all samples, while UV absorbance was monitored at 254 nm. The column temperature was equilibrated at 35˝C prior to injections.
UPLC-MS analyses were performed using a Waters Acquity UPLC H-class system coupled to a Waters Xevo-G2 TOF. The mass spectrometer was operated under the following conditions: electrospray ionization mode, positive; capillary voltage, 2.5 kV; sampling cone voltage, 30 V; extraction cone voltage, 4.0 V. Samples were prepared by dissolving 10µL of a 3.8 mM library in 10 µL DMF and then further diluting them by 30µL UPLC grade water. The operating parameters were the following: eluent flow rate 0.3 mL/min; eluent A, UPLC grade water with 0.1 v% trifluoroacetic acid; eluent B, UPLC grade acetonitrile with 0.1 v% trifluoroacetic acid.
5.6.1
UPLC Methods
Libraries were analyzed using the following method (linear gradient):
Time, min. A% B% 0.00 90 10 1.00 90 10 1.30 75 25 3.00 72 28 11.00 60 40 11.50 5 95 12.00 5 95 12.50 90 10 15.00 90 10
Table 5.3: Elution profile used for the analysis of the mixed building block DCLs made from 1-2 and 1-3.
244 5. Sustaining a Distribution of Replicators Out of Equilibrium Time, min. A% B% 0.00 90 10 1.00 90 10 1.30 78 22 11.00 68 32 16.00 59 41 16.50 5 95 17.00 5 95 17.50 90 10 20.00 90 10
Table 5.4: Elution profile used for analysis of the mixed building block DCLs made from 2-3.
5.7
Kinetic Profiles
Dynamic combinatorial libraries made from mixed building blocks that are operated out-of-equilibrium, like similar replicating systems we investigated before, are prone to considerable quantitative variability that is mostly likely a result of the inherent sensitivity of the kinetics of exponentially growing systems to small perturbations. In Section 5.7.3, we present the repeats of the same experiment instead of averaging the data and plotting with error bars.
5.7. Kinetic Profiles 245
5.7.1
Different Flow Rates
1. Flow rate 5 µL/h
Figure 5.9: Kinetic profiles showing the replicator distribution of mixed libraries of a) 1-2 and b) 1-3 under out-of-equilibrium conditions. Plots in the second row shows the initial and final distributions presented in the first row. Flow rate, 5µL/h; library volume, 750 µL; library concentration, 0.76 mM; food concentration, 0.76 mM; oxidation level of the food, 80%; turnover time, 150 h.
246 5. Sustaining a Distribution of Replicators Out of Equilibrium
Figure 5.10: UPLC traces (monitored at 254 nm) of mixed libraries of 1-2 corresponding to Figure 5.9a.
Figure 5.11: UPLC traces (monitored at 254 nm) of mixed libraries of 1-3 corresponding to Figure 5.9b.
5.7. Kinetic Profiles 247
2. Flow rate 10 µL/h
Figure 5.12: Kinetic profiles showing the replicator distribution of mixed libraries of a) 1-2, b) 1-3 and c) 2-3 under out-of-equilibrium conditions. Plots in the second row shows the initial and final distributions presented in the first row. Flow rate, 10µL/h; library volume, 750µL; library concentration, 0.76 mM; food concentration, 0.76 mM; oxidation level of the food, 80%; turnover time, 75 h.
248 5. Sustaining a Distribution of Replicators Out of Equilibrium
Figure 5.13: UPLC traces (monitored at 254 nm) of mixed libraries of 1-2 corresponding to Figure 5.12a.
Figure 5.14: UPLC traces (monitored at 254 nm) of mixed libraries of 1-3 corresponding to Figure 5.12b.
5.7. Kinetic Profiles 249
Figure 5.15: UPLC traces (monitored at 254 nm) of mixed libraries of 2-3 corresponding to Figure 5.12c.
250 5. Sustaining a Distribution of Replicators Out of Equilibrium 3. Flow rate 20 µL/h
Figure 5.16: Kinetic profiles showing the replicator distribution of mixed libraries of a) 1-2, b) 1-3 and c) 2-3 under out-of-equilibrium conditions. Plots in the second row shows the initial and final distributions presented in the first row.Flow rate, 20µL/h; library volume, 750µL; library concentration, 0.76 mM; food concentration, 0.76 mM; oxidation level of the food, 80%; turnover time, 37.5 h.
5.7. Kinetic Profiles 251
Figure 5.17: UPLC trace (monitored at 254 nm) of mixed libraries of 1-2 corresponding to Figure 5.16a.
Figure 5.18: UPLC trace (monitored at 254 nm) of mixed libraries of 1-3 corresponding to Figure 5.16b.
250 5. Sustaining a Distribution of Replicators Out of Equilibrium 3. Flow rate 20 µL/h
Figure 5.16: Kinetic profiles showing the replicator distribution of mixed libraries of a) 1-2, b) 1-3 and c) 2-3 under out-of-equilibrium conditions. Plots in the second row shows the initial and final distributions presented in the first row.Flow rate, 20µL/h; library volume, 750µL; library concentration, 0.76 mM; food concentration, 0.76 mM; oxidation level of the food, 80%; turnover time, 37.5 h.
5.7. Kinetic Profiles 251
Figure 5.17: UPLC trace (monitored at 254 nm) of mixed libraries of 1-2 corresponding to Figure 5.16a.
Figure 5.18: UPLC trace (monitored at 254 nm) of mixed libraries of 1-3 corresponding to Figure 5.16b.
252 5. Sustaining a Distribution of Replicators Out of Equilibrium
Figure 5.19: UPLC trace (monitored at 254 nm) of mixed libraries of 2-3 corresponding to Figure 5.16c.
5.7.2
Modifications of the Infusion Syringe
Optimization on the infusion syringes are done to reach a stable oxidation level during the experiment. In all experiments, one set of 1-2 and 1-3 food was prepared in the glovebox with a target oxidation level of 70 mol %. Hamilton gas tight syringes used in all experiments with different manipulations on the screw-head which is the most likely leakage point.
5.7. Kinetic Profiles 253
Figure 5.20: a) Syringe volume, 1 mL; no sealing b) syringe volume, 1 mL; sealed with Teflon; c) syringe volume, 1 mL; sealed with parafilm; d) syringe volume, 5 mL; sealed with parafilm; e) syringe volume, 1 mL; sealed with Sugru. Plots labeled with (i) represent changes in 11 and 21 in a 1-2 mixed library, (ii) represent changes in 11 and 31 in a 1-3 mixed library and (iii) represent changes in the total monomer composition in both of the mixed libraries.
254 5. Sustaining a Distribution of Replicators Out of Equilibrium
Figure 5.21: Change in the (a) individual monomer composition and (b) overall monomer composition over time in the infusion-syringe (volume, 1 mL; sealed with Teflon tape) containing mixed 1-3 food. On day 1, the syringe was placed in an inert atmosphere to find the source of oxygen.
Figure 5.22: Change in the monomer composition in a syringe kept under inert atmosphere of a) volume, 1 mL; no sealing; i) 1-2 mixed library, ii) 1-3 mixed library and iii) overall change in both systems, b) volume, 5 mL; sealed with parafilm; i) 1-2 mixed library, ii) 1-3 mixed library and iii) overall change in both systems. c) Change in the monomer composition in a syringe kept i) under inert atmosphere, ii) under ambient conditions and iii) comparison of total monomer levels between inert and ambient conditions.
5.7. Kinetic Profiles 255
5.7.3
Optimization of the Concentration of Reaction Vial and
Food
1. Infusion of 0.76 mM food into 0.76 mM replicator library
Figure 5.23: Kinetic profiles showing the distribution of a) cyclic hexamer replicators, b) cyclic tetramers and c) cyclic trimers made from mixed libraries of 1-2. Plots in the second row shows the initial and final distributions presented in the first row. Flow rate, 10µL/h; library volume, 750 µL; library concentration, 0.76 mM; food concentration, 0.76 mM; oxidation level of the food, 80%; turnover time, 75 h.
256 5. Sustaining a Distribution of Replicators Out of Equilibrium
Figure 5.24: a) UPLC traces (monitored at 254 nm) of mixed libraries of 1-2 (correspond-ing to Figure 5.23). Flow rate, 10µL/h; library volume, 750 µL; library concentration, 0.76 mM; food concentration, 0.76 mM; oxidation level of the food, 80%; turnover time, 75 h. b) UPLC trace (monitored at 254 nm) of mixed libraries of 1-2 on day 14 (corresponding to Figure 5.24a) showing the identified peaks.
5.7. Kinetic Profiles 257
2. Infusion of 1.52 mM food into 0.76 mM replicator library
Figure 5.25: (a) and (b) represent repeats of same experiment where (i) shows the kinetic profiles with the distribution of hexamer replicators made from 1-2, (ii) shows a magnifi-cation of the corresponding data for the mixed hexamers, (iii) is the bar graph showing the initial and final library composition in terms of cyclic hexamers ploted in (i) and (ii). Flow rate, 10µL/h; library volume, 750 µL; library concentration, 0.76 mM; food concentration, 1.52 mM; oxidation level of the food, 80%; turnover time, 75 h.
256 5. Sustaining a Distribution of Replicators Out of Equilibrium
Figure 5.24: a) UPLC traces (monitored at 254 nm) of mixed libraries of 1-2 (correspond-ing to Figure 5.23). Flow rate, 10µL/h; library volume, 750 µL; library concentration, 0.76 mM; food concentration, 0.76 mM; oxidation level of the food, 80%; turnover time, 75 h. b) UPLC trace (monitored at 254 nm) of mixed libraries of 1-2 on day 14 (corresponding to Figure 5.24a) showing the identified peaks.
5.7. Kinetic Profiles 257
2. Infusion of 1.52 mM food into 0.76 mM replicator library
Figure 5.25: (a) and (b) represent repeats of same experiment where (i) shows the kinetic profiles with the distribution of hexamer replicators made from 1-2, (ii) shows a magnifi-cation of the corresponding data for the mixed hexamers, (iii) is the bar graph showing the initial and final library composition in terms of cyclic hexamers ploted in (i) and (ii). Flow rate, 10µL/h; library volume, 750 µL; library concentration, 0.76 mM; food concentration, 1.52 mM; oxidation level of the food, 80%; turnover time, 75 h.
258 5. Sustaining a Distribution of Replicators Out of Equilibrium
Figure 5.26: Kinetic profiles showing the distribution of cyclic tetramer replicators made from 1-2. a) and b) show repeats of the same experiment. Plots in the second row show the initial and final distributions presented in the first row. Flow rate, 10µL/h; library volume, 750µL; library concentration, 0.76 mM; food concentration, 1.52 mM; oxidation level of the food, 80%; turnover time, 75 h.
Figure 5.27: Kinetic profiles showing the distribution of cyclic trimer replicators made from 1-2. a) and b) show repeats of the same experiment. Plots in the second row show the initial and final distributions presented in the first row. Flow rate, 10µL/h; library volume, 750µL; library concentration, 0.76 mM; food concentration, 1.52 mM; oxidation level of the food, 80%; turnover time, 75 h.
5.7. Kinetic Profiles 259
Figure 5.28: a) UPLC traces (monitored at 254 nm) of mixed libraries of 1-2. Flow rate, 10µL/h; library volume, 750 µL; library concentration, 0.76 mM; food concentration, 1.52 mM; oxidation level of the food, 80%; turnover time, 75 h. b) UPLC trace (monitored at 254 nm) of mixed libraries of 1-2 at day 17 (corresponding to Figure 5.28a) showing the identified peaks.
260 5. Sustaining a Distribution of Replicators Out of Equilibrium
Figure 5.29: UPLC traces (monitored at 254 nm) of the food (corresponding to the exper-iment in Figure 5.28) for the flow experexper-iment on mixed libraries of 1-2. Chromatograms of food solution are recorded at the beginning (black lines) and at the end (red lines) of each cycle of 1.0 mL addition (approximately 4 days).
3. Infusion of 1.52 mM food into 1.52 mM replicator library
Figure 5.30: Kinetic profiles showing the total library distribution of mixed libraries of 1-2. a), b) and c) show repeats of the same experiment. Flow rate, 10µL/h; library volume, 750µL; library concentration, 1.52 mM; food concentration, 1.52 mM; oxidation level of the food, 80%; turnover time, 75 h.
5.7. Kinetic Profiles 261
Figure 5.31: Kinetic profiles showing the distribution of hexamer replicators made from 1-2. a), b) and c) show repeats of the same experiment. Plots in the second row show the initial and final distributions presented in the first row. Flow rate, 10µL/h; library volume, 750µL; library concentration, 1.52 mM; food concentration, 1.52 mM; oxidation level of the food, 80%; turnover time, 75 h.
Figure 5.32: Kinetic profiles showing the distribution of cyclic tetramers replicators made from 1-2. a), b) and c) show s the repeats of the same experiment. Plots in the second row show the initial and final distributions presented in the first row. Flow rate, 10µL/h; library volume, 750µL; library concentration, 1.52 mM; food concentration, 1.52 mM; oxidation level of the food, 80%; turnover time, 75 h.
262 5. Sustaining a Distribution of Replicators Out of Equilibrium
Figure 5.33: Kinetic profiles showing the distribution of cyclic trimers replicators made from 1-2. a), b) and c) show repeats of the same experiment. Plots in the second row show the initial and final distributions presented in the first row. Flow rate, 10µL/h; library volume, 750µL; library concentration, 1.52 mM; food concentration, 1.52 mM; oxidation level of the food, 80%; turnover time, 75 h.
5.7. Kinetic Profiles 263
Figure 5.34: a) UPLC traces (monitored at 254 nm) of mixed libraries of 1-2. Flow rate, 10µL/h; library volume, 750 µL; library concentration, 1.52 mM; food concentration, 1.52 mM; oxidation level of the food, 80%; turnover time, 75 h. b) UPLC trace (monitored at 254 nm) of mixed libraries of 1-2 at day 34 (corresponding to Figure 5.34a) showing the identified peaks.
262 5. Sustaining a Distribution of Replicators Out of Equilibrium
Figure 5.33: Kinetic profiles showing the distribution of cyclic trimers replicators made from 1-2. a), b) and c) show repeats of the same experiment. Plots in the second row show the initial and final distributions presented in the first row. Flow rate, 10µL/h; library volume, 750µL; library concentration, 1.52 mM; food concentration, 1.52 mM; oxidation level of the food, 80%; turnover time, 75 h.
5.7. Kinetic Profiles 263
Figure 5.34: a) UPLC traces (monitored at 254 nm) of mixed libraries of 1-2. Flow rate, 10µL/h; library volume, 750 µL; library concentration, 1.52 mM; food concentration, 1.52 mM; oxidation level of the food, 80%; turnover time, 75 h. b) UPLC trace (monitored at 254 nm) of mixed libraries of 1-2 at day 34 (corresponding to Figure 5.34a) showing the identified peaks.
264 5. Sustaining a Distribution of Replicators Out of Equilibrium
Figure 5.35: UPLC traces (monitored at 254 nm) of the food (corresponding to the ex-periment in Figure 5.34) used in the infusion syringe for the flow exex-periment containing the mixed library of 1-2. Chromatograms of food solution are recorded at the beginning (black lines) and at the end (red lines) of each cycle of 1.0 mL addition (approximately 4 days).
5.7. Kinetic Profiles 265
4. Infusion of 1.52 mM food into borate buffer
Figure 5.36: a) Kinetic profile showing the distribution of hexamer replicators made from 1-2, b) zoomed in kinetic profile of (a) and c) initial and final distribution of hexamer repli-cators depicted in (a). Flow rate, 10µL/h; library volume, 750 µL; library concentration, 0 mM; food concentration, 1.52 mM; oxidation level of the food, 80%; turnover time, 75 h.
Figure 5.37: Kinetic profiles showing the distribution of a) cyclic tetramers and b) cyclic trimers made from 1-2. Plots in the second row show the initial and final distributions presented in the first row. Flow rate, 10µL/h; library volume, 750 µL; library concentration, 0 mM; food concentration, 1.52 mM; oxidation level of the food, 80%; turnover time, 75 h.
266 5. Sustaining a Distribution of Replicators Out of Equilibrium
Figure 5.38: a) UPLC traces (monitored at 254 nm) of mixed libraries of 1-2. Flow rate, 10µL/h; library volume, 750 µL; library concentration, 0 mM; food concentration, 1.52 mM; oxidation level of the food, 80%; turnover time, 75 h. b) UPLC trace (monitored at 254 nm) of mixed libraries of 1-2 at day 14 (corresponding to Figure 5.38a) showing the identified peaks.
5.7. Kinetic Profiles 267
Figure 5.39: UPLC traces (monitored at 254 nm) of the food (corresponding to the experi-ment in Figure 5.38) for the flow experiexperi-ment on the mixed libraries of 1-2. Chromatograms of the food solution are recorded at the beginning (black lines) and at the end (red lines) of each cycle of 1.0 mL addition (approximately 4 days).
5. Infusion of 1.52 mM food into 1.52 mM food library
Figure 5.40: a) Kinetic profiles showing the total library distribution of mixed libraries of 1-2. Flow rate, 10µL/h; library volume, 750 µL; library concentration, 1.52 mM; food concentration, 1.52 mM; oxidation level of the food, 80%; turnover time, 75 h. Library and the food compositions are identical at the beginning. Panels b) and c) show repeats of the same experiment.
268 5. Sustaining a Distribution of Replicators Out of Equilibrium
Figure 5.41: a) Kinetic profiles showing the hexamer distribution of mixed libraries of 1-2. Flow rate, 10µL/h; library volume, 750 µL; library concentration, 1.52 mM; food concentration, 1.52 mM; oxidation level of the food, 80%; turnover time, 75 h. Library and the food compositions are identical at the beginning. Panels b) and c) show repeats of the same experiment.
Figure 5.42: a) Kinetic profiles showing the cyclic tetramer distribution of mixed libraries of 1-2. Flow rate, 10µL/h; library volume, 750 µL; library concentration, 1.52 mM; food concentration, 1.52 mM; oxidation level of the food, 80%; turnover time, 75 h. Library and the food compositions are identical at the beginning. Panels b) and c) show repeats of the same experiment.
5.7. Kinetic Profiles 269
Figure 5.43: a) Kinetic profiles showing the cyclic trimers in the library distributions of mixed libraries of 1-2. Flow rate, 10µL/h; library volume, 750 µL; library concentration, 1.52 mM; food concentration, 1.52 mM; oxidation level of the food, 80%; turnover time, 75 h. Library and the food compositions are identical at the beginning. Panels b) and c) show repeats of the same experiment.
Figure 5.44: UPLC traces (monitored at 254 nm) of the food (corresponding to the ex-periment in Figure 5.45) used in the infusion syringe for the flow exex-periment on the mixed library of 1-2. Chromatograms of food solution are recorded at the beginning (black lines) and at the end (red lines) of each cycle of 1.0 mL addition (approximately 4 days).
