Periodic Reporting for period 1 - SRCV (Molecular Device for Optical Monitoring of Self-Replication in Compartments)
Reporting period: 2020-04-01 to 2022-03-31
Self-replicating systems play an important role in research on the synthesis and origin of life. Monitoring of these systems has mostly relied on techniques such as NMR or chromatography, which are limited in throughput and demanding when monitoring replication in real time. To circumvent these problems, We have demonstrated the use of a pattern-generating fluorescent molecular probe for straightforwardly detecting, discriminating between, and real-time tracking of peptide-based self-replicators. In systems dominated by a single replicator the sensor is able to discriminate between replicators with different macrocycles sizes (e.g. hexamers, octamers, pentamers) and amino-acid composition. It is also able to differentiate macrocycles with the same size but subtly different peptide sequences. Furthermore, the conversion of building block into replicator could be monitored qualitatively in situ and in real time without interfering with the replication process. The probe was also found to respond differently to different aggregates formed by the building block and the replicator precursors, indicating that its discriminating ability extends beyond the fibrous aggregates for which it was designed. This new technology for the optical analysis of self-replicating systems opens the door for continuous monitoring of parallel experiments in high-throughput ways in small volumes (i.e. in microdroplets or other protocell environments), enabling, for example, the study of stochastic effects, which may prove important in the emergence and evolution of new forms of life.
We have developed a unimolecular combinatorial fluorescent molecular device (also called ID-probe, sensor, probe) and study the probe binding to replicators by means of a fluorescence assay to obtain a fingerprint (optical pattern) and corresponding PCA map of different full-grown replicators in bulk. We first synthesized a family of dithiol-containing peptides. Their self-replication properties were analyzed by UPLC. Also, parallelly, we designed and synthesized an ID-probe to study the self-replicating systems made from dithiol-containing peptides. We showed that pattern-generating probe or ID-probe can be used to discriminate between building blocks, intermediates and final replicators prepared separately. We subsequently showed that the sensor can discriminate between replicators of subtly different chemical nature (for example, by replacing a single amino acid in the peptide chain). Then, we also showed that the sensor can optically monitor the growth of the self-replicators in bulk solution in real-time. As a first test of the suitability of the ID-probe to track the emergence and growth of replicators in real time, where the molar ratio of precursors and replicators dynamically changes, the emission of sensor was recorded upon exposure to mixtures containing different molar ratios of separately prepared replicators and precursors and the mixtures represent different stages of replicator emergence. PCA of the resulting data shows that sensor can discriminate between different molar ratios of precursors and replicators.
Encouraged by these results we investigated whether ID-probe could be used to track the spontaneous emergence of replicators in situ and in real time. So, we co-incubated building block and sensor and followed the changes in the emission of the ID-probe over time. To confirm that the presence of the ID-probe does not affect the dynamic formation of the different replicators in the mixtures we analyzed the replication process and its kinetics by previously established techniques (UPLC-MS, TEM). The UPLC-based kinetic profile obtained in the presence of sensor is comparable to one acquired without the ID-probe. The small differences between the data in these two figures is similar in magnitude to the differences we typically observe in the emergence of replicators in experiments conducted at different times. TEM images revealed that similar fibers were obtained in the absence and presence of the probe. Analyzing the patterns generated by sensor over time by PCA shows that the ID-probe allows for qualitatively tracking of the growth of replicators real-time in-situ. Control experiment in the absence of replicators revealed that the emission of sensor remained unchanged over time, confirming that the observed changes in fluorescence patterns resulted from changes in the composition of the mixture. In principle one could envisage that the probe may also find use for quantitative kinetic studies, but this will require enhancing the accuracy with which it reports on small differences in composition. Then, we targeted to develop the methodology for placing replicators in confinement. Our aim is to achieve replicator-loaded coacervate droplets. We have developed coacervates by mixing polydiallyldimethylammonium chloride (PDADMAC) and poly(acrylic acid) (PAA). The formation of coacervate was confirm by optical microscopy. In the next step, as a preliminary study we have separately prepared precursors and replicators and mixed these with polydiallyldimethylammonium chloride (PDADMAC) and poly(acrylic acid) (PAA).
The fluorescence of the different samples was recorded and the emission pattern obtained subjected to PCA analysis. The PCA plot indicates that the sensor can discriminate between these materials when they reside inside coacervates. Confocal images showed that sensor partitioned inside the coacervate droplets. However, to exactly quantify the amount of sensor as well as replicator present inside and outside of the coacervates and to real-time monitoring of the emergence and growth of self-replicators inside compartments will require additional work and more time.
Very recently, one publication describing an optical probe for real-time monitoring of self-replicator emergence and distinguishing between replicators has been published in Journal of the American Chemical Society (https://pubs.acs.org/doi/10.1021/jacs.1c11594?ref=PDF) which has an impact factor (IF) of 15.41. This publication attracted a lot of attention and was selected by the American Chemical Society (ACS) editors to be highlighted as a spotlight.
Encouraged by these results we investigated whether ID-probe could be used to track the spontaneous emergence of replicators in situ and in real time. So, we co-incubated building block and sensor and followed the changes in the emission of the ID-probe over time. To confirm that the presence of the ID-probe does not affect the dynamic formation of the different replicators in the mixtures we analyzed the replication process and its kinetics by previously established techniques (UPLC-MS, TEM). The UPLC-based kinetic profile obtained in the presence of sensor is comparable to one acquired without the ID-probe. The small differences between the data in these two figures is similar in magnitude to the differences we typically observe in the emergence of replicators in experiments conducted at different times. TEM images revealed that similar fibers were obtained in the absence and presence of the probe. Analyzing the patterns generated by sensor over time by PCA shows that the ID-probe allows for qualitatively tracking of the growth of replicators real-time in-situ. Control experiment in the absence of replicators revealed that the emission of sensor remained unchanged over time, confirming that the observed changes in fluorescence patterns resulted from changes in the composition of the mixture. In principle one could envisage that the probe may also find use for quantitative kinetic studies, but this will require enhancing the accuracy with which it reports on small differences in composition. Then, we targeted to develop the methodology for placing replicators in confinement. Our aim is to achieve replicator-loaded coacervate droplets. We have developed coacervates by mixing polydiallyldimethylammonium chloride (PDADMAC) and poly(acrylic acid) (PAA). The formation of coacervate was confirm by optical microscopy. In the next step, as a preliminary study we have separately prepared precursors and replicators and mixed these with polydiallyldimethylammonium chloride (PDADMAC) and poly(acrylic acid) (PAA).
The fluorescence of the different samples was recorded and the emission pattern obtained subjected to PCA analysis. The PCA plot indicates that the sensor can discriminate between these materials when they reside inside coacervates. Confocal images showed that sensor partitioned inside the coacervate droplets. However, to exactly quantify the amount of sensor as well as replicator present inside and outside of the coacervates and to real-time monitoring of the emergence and growth of self-replicators inside compartments will require additional work and more time.
Very recently, one publication describing an optical probe for real-time monitoring of self-replicator emergence and distinguishing between replicators has been published in Journal of the American Chemical Society (https://pubs.acs.org/doi/10.1021/jacs.1c11594?ref=PDF) which has an impact factor (IF) of 15.41. This publication attracted a lot of attention and was selected by the American Chemical Society (ACS) editors to be highlighted as a spotlight.
The new technology for the optical analysis of self-replicating systems opens the door for continuous monitoring of parallel experiments in high-throughput ways in small volumes (i.e. in microdroplets or other protocell environments), enabling, for example, the study of stochastic effects, which may prove important in the emergence and evolution of new forms of life.