Emergence of a new self-replicator from a dynamic combinatorial library requires a specific pre-existing replicator

Citation for published version (APA): Altay, Y., Tezcan, M., & Otto, S. (2017). Emergence of a new self-replicator from a dynamic combinatorial library requires a specific pre-existing replicator. Journal of the American Chemical Society, 139(39), 13612-13615. https://doi.org/10.1021/jacs.7b07346

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Emergence of a New Self-Replicator from a Dynamic Combinatorial Library Requires a Specific Pre-Existing Replicator Yigit Altay, Meniz Tezcan, and Sijbren Otto* Centre for Systems Chemistry, Stratingh Institute, Nijenborgh 4, 9747 AG Groningen, The Netherlands

*S Supporting Information

combinatorial libraries (DCLs) made from building-blocks ABSTRACT: Our knowledge regarding the early steps in featuring two thiol groups for reversible disulfide chemistry13b the formation of evolvable life and what constitutes the and a peptide that is predisposed to β-sheet formation by virtue minimal molecular basis of life remains far from complete. of alternating hydrophobic and hydrophilic amino-acid The recent emergence of systems chemistry reinvigorated residues. In solution, reaction between these building blocks the investigation of systems of self-replicating molecules to and oxygen from the air yields a DCL that consists of a mixture address these questions. Most of these studies focus on of disulfide macrocycles of different ring sizes (Scheme 1). If single replicators and the effects of replicators on the one of the macrocycles can stabilize itself through self-assembly, emergence of other replicators remains under-investigated. the product distribution shifts toward this compound at the Here we show the cross-catalyzed emergence of a novel expense of the other compounds in the library. Assembly self-replicator from a dynamic combinatorial library made occurs through a nucleation−growth mechanism which allows from a threonine containing peptide building block, which, exponential replication to be achieved: growing fibers break by itself, only forms trimers and tetramers that do not into fragments by mechanical agitation (i.e., stirring or ff replicate. Upon seeding of this library with di erent shaking), which increases the number of ends from where the ff replicators of di erent macrocycle size (hexamers and fibers grow.13b octamers), we observed the emergence of hexamer In most of our previous studies,13d the emergence of replicator consisting of six units of the threonine peptide replicators occurred spontaneously. In an intriguing recent only when it is seeded with an octamer replicator study,13a we showed that assembly driven self-replication could containing eight units of a serine building block. These fi also be triggered by a template that raises the concentration of results reveal for the rst time how a new replicator can the potential replicator above its critical aggregation concen- emerge in a process that relies critically on the assistance tration. However, until now, the role of existing replicators on by another replicator through cross-catalysis and that the emergence of new ones has received little attention. Yet, replicator composition is history dependent. cross-catalysis may be a powerful mechanism for the diversification and evolution of replicators and the development of replicator “ecosystems”.14 ow life originated and how life may be synthesized de We now report a system in which autonomous replicator H novo are among the grand challenges in contemporary emergence is not observed, but where replicator emergence science. Research in these areas has focused on the requires cross-catalysis by another pre-existing replicator. Downloaded via TU EINDHOVEN on November 21, 2018 at 14:30:32 (UTC). biomolecules essential to current life (proteins, RNA and Cross-catalyzed emergence was mediated only by a replicator DNA) or on the bottom-up construction of chemical systems with a specific ring size and peptide sequence. We also show that mimic the essential characteristics of life. Over the last that replication is strongly dependent on the sample history 1 See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles. decades, the fields of systems chemistry, and dynamic marking an important step in the direction of self-replicator combinatorial chemistry2 in particular, have developed evolution. synthetic systems that capture some of the essential character- In the course of a systematic investigation of the effect of the istics of life: compartmentalization,3 reaction networks structure of the peptide building blocks with the general (addressing the issue of metabolism)4 and systems featuring architecture shown in Scheme 1 on the formation of replicators, self- and cross-replicating molecules.5 we encountered unexpected behavior upon using building block In this study, we will focus on self-replication. Until now, 1. In agitated DCLs made from 2−5, individually, self- relatively few replicators have been reported. Following replicators emerged spontaneously in all cases. Yet in similar pioneering work by von Kiedrowski,6 completely synthetic experiments starting from building block 1, we did not observe replicators were developed by Rebek7 and Philp.8 Joyce, any replicators. Using our standard protocol, we rapidly Lehman and Szostak developed systems of replicating RNAs9 oxidized a solution of 1 (3.8 mM in 50 mM borate buffer whereas Chmielewski,10 Ghadiri,11 Ashkenasy12 and us13 pH 8.1) to 80% (conversion of thiols into disulfides) using focused on peptide-based replicators. Though in the majority sodium perborate solution (80 mM), followed by slower of these studies replicators were designed in full structural further oxidation mediated by oxygen present in the air. detail, we explored how replicators emerged from complex mixtures where the structure of the emerging replicator was not Received: July 14, 2017 predetermined. For this purpose, we developed dynamic Published: September 14, 2017

© 2017 American Chemical Society 13612 DOI: 10.1021/jacs.7b07346 J. Am. Chem. Soc. 2017, 139, 13612−13615 Journal of the American Chemical Society Communication

Scheme 1. (a) A dynamic Combinatorial Library of Differently Sized Macrocyclic Disulfides Is Formed upon Oxidation of a Threonine Containing Peptide Functionalized Dithiol; (b) Selective Formation of Replicator 16 upon Cross-Seeding; (c) Schematic Representation of the Tentative Mechanism through Which Replicator 48 Gives Rise to Replicator 16

Figure 1. Kinetic profile of a dynamic combinatorial library made from building block 1 (3.8 mM in 50 mM borate buffer, pH 8.1) stirred at 1200 rpm and (a) kept under ambient conditions, (b) 80% oxidized and kept under an inert atmosphere; kinetic profile of a dynamic combinatorial library made from an 80% oxidized solution of building ff block 1 (3.8 mM in 50 mM borate bu er, pH 8.1) seeded with (c) 48 (cross-seeding), (d) 16 (self-seeding), stirred at 1200 rpm.

macrocycles (see SI Figure S2) with one notable exception: only cross-seeding with 48 induced the formation of a new macrocycle (16, Figure 1c). The sigmoidal growth of 16 is consistent with self-replication. fi To con rm that 16 is a self-replicator, we added it as a seed to a DCL made from 1. Figure 1d shows that 16 grows rapidly upon seeding and accounts for 72% of the library material within 1 day (see SI Figure S5 for results of seeding with smaller amounts of seed).15 Note that the lag phase, that was observed in the library to which 48 was added, diminished when 16 was used as a seed instead of 48. The presence of this lag fi phase suggests that growth of 16 occurs on the ends of bers of 48, but it is a relatively rare event and that fragmentation of these nuclei of 16 (induced by 48) into secondary nuclei is ffi required before replication of 16 becomes e cient (see Scheme Solutions were stirred at 1200 rpm or left nonagitated. The 1c). This interpretation was further supported by the fact that fi kinetic pro les of these libraries, monitored by UPLC-MS, seeding a library made from 1 with 48 in the absence of show the formation trimers (13) and tetramers (14), neither of mechanical agitation dramatically slowed down the rate of which self-assembles or self-replicates (Figure 1a; see SI Figure replication of 16 (Figure S3). Detailed analysis of the early stage S1 for the nonagitated sample). The absence of any replicator of the growth of 16 seeded by 48 (Figure S4) did not reveal any might be explained with the library reaching complete oxidation mixed macrocycles containing both building blocks, lending before any replicator had the chance to emerge (disulfide further support for the proposed mechanism. exchange requires a catalytic amount of thiol). In order to The structure of the assemblies formed by 16 was prevent freezing the library by complete oxidation, we repeated characterized by transmission electron microscopy (TEM), the experiment at a constant oxidation level of 80% and circular dichroism (CD) spectroscopy and thioflavin T monitored the composition of the small DCL over the course fluorescence assays (Figure 2). Negative staining TEM of one month under an inert atmosphere. Also in this micrographs of a sample dominated by 16 revealed laterally fi experiment, 13 and 14 dominated the library and no replicator associated bers that were approximately 100 nm long (Figure emergence was observed (Figure 1b). 2c). In contrast, libraries made from 1 containing mostly cyclic We then investigated whether the formation of replicators trimers and tetramers did not show any ordered or disordered made from 1 could be induced by cross-seeding with preformed aggregates. The CD spectrum of samples dominated by 16 replicators made from building blocks 2, 3, 4 or 5. Thus, 10 mol showed positive helicity at 196 nm and negative helicity at 218 β 16 %of26, 36, 46, 48, 58, prepared following published nm, indicative of a -sheet structure (Figure 2a). We protocols,13c,d was added to stirred DCLs made from 1 and observed only random coil secondary structure for all other the compositions of the mixtures were monitored over time. libraries made from 1 that were dominated by trimers and These experiments failed to induce the formation of new tetramers. Thioflavin T fluorescence measurements17 also

13613 DOI: 10.1021/jacs.7b07346 J. Am. Chem. Soc. 2017, 139, 13612−13615 Journal of the American Chemical Society Communication

Figure 3. Kinetic profile of a nonagitated dynamic combinatorial library made from building block 4 (3.8 mM in 50 mM borate buffer, pH 8.1) 80% oxidized (a) nonseeded (b) after addition of 10 mol fi percent of preformed 16 as seed. (c) Kinetic pro le of a library made Figure 2. (a) CD spectra and (b) maximum thioflavin T fluorescence by mixing peptide 1 and peptide 4 (3.42 mM in 1 and 0.38 mM in 4) emission intensity (at 492 nm) of nonseeded and seeded (10 mol %) in borate buffer (pH 8.1, 50 mM) to form a 3.8 mM library. The libraries made from building block 1 (3.8 mM in 50 mM borate buffer, library was then oxidized to 80% with freshly prepared perborate pH 8.1): i, stirred; ii, inert atm.; iii, nonagitated; iv, seeded with 48; v, solution (80 mM) and stirred at 1200 rpm under an inert atmosphere. seeded with 58; vi, seeded with 26; vii, seeded with 36; viii, seeded with 46. (c) TEM micrographs of the library corresponding to Figure 1c. DCL with the same overall building block composition as the Scale bars are 100 nm. one shown in Figure 3b (3.42 mM in 1 and 0.38 mM in 4) but now mixed these building blocks at the start of the experiment. fi β fi 18 con rmed a -sheet amyloid- bril-like structure of 16, evident We monitored the sample over a period of 40 days but did not from a more than 50-fold increase in emission intensity, detect the emergence of replicators 16 or 48 (Figure 3c). This whereas all libraries dominated by 13 and 14 showed a 3-fold experiment shows that the history of the sample is a decisive increase at most (Figure 2b). factor in determining replicator presence or absence, just like In order to investigate whether cross-catalysis between the 16 the evolutionary history dictates the species composition in and 48 replicators is reciprocal, libraries were made from current life. Though history-dependence is a widespread ff 19 20 building block 4 and the e ect of seeding by 16 was probed. phenomenon in materials science and protein folding, it Mechanical agitation was not applied, as agitation induces the had not yet been reported to dictate the nature of self- autonomous formation of the 48 replicator. We observed the replicating molecules. Moreover, the history dependence emergence of 48 and 46 alongside some mixed hexamers observed here involves the interaction history between containing both building blocks 1 and 4 in the library seeded molecules in the mixture, and not merely the history in terms with 16, whereas the nonseeded control only led to the of physical properties, such as pH or temperature. formation of nonassembling 43 and 44 macrocycles (Figure In conclusion, we have shown how the emergence of a new 3a,b). Thus, cross-catalysis appears to be reciprocal, albeit not replicator based on threonine-containing building block 1 relies fi completely symmetrical: 48 induces the formation of 16 but not on the presence of a speci c pre-existing replicator containing 18 whereas 46 does not give rise to any replicators based on 1, serine residues. Cross-catalysis between replicators (and within the time frame of our experiments. Conversely, 16 therefore replicator mutation) in this system was found to be fi induces the formation of both 46 and 48. These perhaps remarkably speci c, as structurally closely related replicators nonintuitive cross-catalytic effects prompted us to explore their failed to show the same effect. Our results also constitute an origin. The fact that replicators based on the more hydrophobic important first step in the development of abiotic systems of building block 1 are hexamers, whereas those based on the replicators in the direction of primitive life. While in previous more hydrophilic 4 are predominantly octamers fits with the work the presence of the building blocks of a replicator was general trend that we reported previously:13d more hydro- typically sufficient for replicators to emerge (after some lag phobic building blocks allow for self-assembly and concomitant phase), in contemporary life, new species only derive from pre- replication already at a smaller ring size. What remains puzzling existing ones (for as far as we know, no new life forms emerge is why the replication by 16 is only triggered by 48 and not by solely from abiotic materials). Thus, in current life, the species any other of our established replicators. We suspect that this present at any given time point reflect not only the available could be due to specific interactions involving the OH-groups resources but also the evolutionary history of the various of the serine residue in 4. This hypothesis is supported by the biological species. Our results represent a first step in the observation that octamers of alanine containing building block transition from a regime where replicator abundance is 5, thus lacking these OH groups, do not induce the formation governed by building block availability to one where pre- of any replicators based on 1. existing replicators control the new replicator population. So, Finally, we investigated to what extent the replicator instead of replicator distributions being defined solely by the distribution is dependent on sample history. We prepared a present conditions, the systems (evolutionary) history is now

13614 DOI: 10.1021/jacs.7b07346 J. Am. Chem. Soc. 2017, 139, 13612−13615 Journal of the American Chemical Society Communication also becoming an essential controlling factor. Such history- (7) (a) Tjivikua, T.; Ballester, P.; Rebek, J. J. Am. Chem. Soc. 1990, dependent systems of self-replicators represent an important 112, 1249−1250. (b) Pieters, R.; Huc, I.; Rebek, J. Angew. Chem., Int. − strategic direction in research on the origin of life and the Ed. Engl. 1994, 33, 1579 1581. development of de novo life. (8) (a) Kosikova, T.; Mackenzie, H.; Philp, D. Chem. - Eur. J. 2016, 22, 1831−1839. (b) del Amo, V.; Philp, D. Chem. - Eur. J. 2010, 16, − ■ ASSOCIATED CONTENT 13304 13318. (c) Sadownik, J. W.; Philp, D. Angew. Chem., Int. Ed. 2008, 47, 9965−9970. (d) Vidonne, A.; Philp, D. Eur. J. Org. Chem. *S Supporting Information 2009, 2009, 593−610. (e) Bottero, I.; Huck, J.; Kosikova, T.; Philp, D. The Supporting Information is available free of charge on the J. Am. Chem. Soc. 2016, 138, 6723−6726. ACS Publications website at DOI: 10.1021/jacs.7b07346. (9) (a) Paul, N.; Joyce, G. Curr. Opin. Chem. Biol. 2004, 8, 634−639. (b) Hayden, E. J.; von Kiedrowski, G.; Lehman, N. Angew. Chem., Int. UPLC and LC-MS methods and data, kinetic data for all Ed. 2008, 47, 8424−8428. (c) Szostak, J. W. J. Biomol. Struct. Dyn. libraries with and without seeding, CD spectra, ThT 2013, 31,2−3. (d) Jia, T. Z.; Fahrenbach, A. C.; Kamat, N. P.; fluorescence experiments for the DCLs made from Adamala, K. P.; Szostak, J. W. Nat. Chem. 2016, 8, 915−921. peptides 1−5, and TEM micrographs for DCLs made (10) Yao, S.; Ghosh, I.; Zutshi, R.; Chmielewski, J. Nature 1998, 396, from peptides 1−5 (PDF) 447−450. (11) (a) Ura, Y.; Beierle, J. M.; Leman, L. J.; Orgel, L. E.; Ghadiri, M. − ■ AUTHOR INFORMATION R. Science 2009, 325,73 77. (b) Lee, D.; Severin, K.; Yokobayashi, Y.; Ghadiri, M. Nature 1997, 390, 591−594. (c) Lee, D.; Granja, J.; Corresponding Author Martinez, J.; Severin, K.; Ghadiri, M. Nature 19