Existing Self-Replicators Can Direct the Emergence of New Ones
Altay, Yigit; Altay, Meniz; Otto, Sijbren
Published in:
Chemistry
DOI:
10.1002/chem.201803027
IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from
it. Please check the document version below.
Document Version
Final author's version (accepted by publisher, after peer review)
Publication date:
2018
Link to publication in University of Groningen/UMCG research database
Citation for published version (APA):
Altay, Y., Altay, M., & Otto, S. (2018). Existing Self-Replicators Can Direct the Emergence of New Ones.
Chemistry, 24(46), 11911-11915. https://doi.org/10.1002/chem.201803027
Copyright
Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).
Take-down policy
If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.
Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum.
Yigit Altay,
[a]Meniz Altay,
[a]and Sijbren Otto*
[a]Abstract: The study of the interplay between different
self-replicating molecules constitutes an important new phase in the synthesis of life and in unravelling the origin of life. Here we show how existing replicators can direct the nature of a newly formed replicator. Starting from the same building block, 6-ring replicators formed when the mixture was exposed to pre-existing 6-membered replicators, while pre-formed 8-membered replicators funneled the building block into 8-ring replicators. Not only ring size, but also the mode of assembly of the rings into stacks was inherited from the pre-existing replicators. These results show that the nature of self-replicating molecules can be strongly influenced by the interplay between different self-replicators, overriding preferences innate to the structure of the building block.
The process through which chemistry can transition into biology remains shrouded in mystery, yet represents one of the grand challenges in contemporary science.[1 ] At some stage in the
origin of life and in the process of synthesizing life de-novo, the ability to replicate needs to emerge. A good understanding of the requirements for self-replication now exists[ 2 ] and several chemical self-replicating systems have been reported.[2,3 ] Also the emergence of self-replicators from relatively complex mixtures (in particular dynamic combinatorial libraries, DCLs[4]) has been described by Philp[ 5 ] and by us.[ 6 ] We previously developed DCLs using building blocks equipped with two thiol groups that can oxidize to form a disulfide macrocycles which continuously exchange building blocks through reversible disulfide exchange reactions. These building blocks were equipped with short peptide chains, containing alternating hydrophilic and hydrophobic amino acids, predisposing them to the formation of β-sheet assemblies. Beyond a critical macrocycle size (which depends on the peptide sequence)[6g] macrocycles can assemble into stacks, held together by β-sheets. This assembly process stabilizes the macrocycles that assemble and leads to the autocatalytic formation of more of these assembling macrocycles (i.e. self-replication) through a nucleation-growth mechanism. Exponential replication can be enabled by mechanical agitation that causes growing fibers to fragment, thereby increasing the number of fiber ends from which the fibers grow.[6d] Until now, most efforts have focused on systems in which only a single replicator emerges and persists.
The next phase in the development of such inanimate systems towards life involves the evolution of replicators[2a,7] and,
subsequently, replicator communities.[3b,c,5,6a,c,7, 8 ] In this new
phase new questions arise, including: How do replicators interact and what are the consequences of such interactions?
And how do existing replicators affect the emergence of new ones? While answers to the former question can be informed by knowledge from contemporary biology, where much is known about how species interact, the latter question has no known counterpart in biology. In current life, all new species derive from existing ones and no new life seems to emerge from scratch. Yet in the early stages of biogenesis replicator emergence is likely to have been much more common.
Herein we describe that existing self-replicators can steer the emergence of new ones, resulting in self-replicators that are different from those that would have emerged in the absence of pre-existing replicators. Thus, replicator composition is not merely dictated by the availability of specific precursors, but becomes dependent on the history of the sample and interactions with pre-existing replicators.
Scheme 1. (a) Structures of the dithiol building blocks. (b) Oxidation of the
tyrosine containing building block leads to formation of a dynamic combinatorial library of differently sized macrocyclic disulfides. (c) Selective formation of 16 or 18 upon cross-seeding with other pre-formed replicators
made from building blocks 2-6 and schematic representation of a tentative mechanism through which these pre-existing replicators direct the formation of the new replicator 16 and 18.
[a] Y. Altay, M. Altay and Prof. Dr. S. Otto.
University of Groningen, Centre for Systems Chemistry, Stratingh Institute, Nijenborgh 4, 9747 AG, Groningen, The
Netherlands E-mail: s.otto@rug.nl
In the course of our work on the emergence of replicators from DCLs[6] we prepared building block 1 (Scheme 1a), differing from our previously reported building blocks by featuring a tyrosine residue. We prepared DCLs by oxidizing 1 at 3.8 mM concentration in borate buffer (50 mM, pH 8.2). Depending on the speed of oxidation and the mode of agitation the product distribution differs markedly. When slowly oxidizing a stirred solution by exposing it to oxygen from the air, the cyclic trimer 13
is the dominant product (Figure 1a). Repeating this experiment in the absence of agitation produced a mixture of trimer and tetramer macrocycles (Figure 1b). Oxidizing the solution rapidly to 80% using perborate and subsequently placing it under an inert atmosphere also produced a mixture of 13 and 14 (Figure
1c). However, when this experiment was repeated but now the sample was exposed to air after being oxidized with perborate, cyclic octamer was formed (Figure 1d). This behavior was found to be qualitatively reproducible (see Figure S1).
For the samples that produced mixtures of 13 and 14 we
did not detect any self-assembled structures by TEM analysis. However, for the experiments of Figure 1a and d, producing mainly 13 or 18, respectively, TEM analysis revealed the
presence of fibers (Figure S72). Seeding experiments confirmed that 18 is able to self-replicate (vide supra). Similar experiments
on 13 were inconclusive, as the rate of emerge of 13 is limited by
the rate of oxidation of 1 (cf. Figure 1a).
Figure 1. Kinetic profiles9
(monitored at 254 nm) of libraries made from peptide 1 (3.8 mM in 50 mM borate buffer, pH 8.2) (a) stirred at 1200 rpm in air, (b) left non-agitated and exposed to air, (c) oxidized to 80% by perborate and stirred at 1200 rpm under inert atmosphere, (d) oxidized to 80% by perborate and stirred at 1200 rpm while exposed to air. Data for the repeats of these experiments are shown in Figure S1.
The results above show that the behavior of DCLs made from 1 is unusually sensitive to small changes in the experimental conditions and that the different products that are formed are separated by relatively high activation energy barriers. The relatively high plasticity of this system (even some hexamer can be formed transiently – cf. Figure 1a) makes it an ideal
candidate to probe the extent to which replicator emergence can directed by introducing other replicators.
Figure 2. Kinetic profiles (monitored at 254 nm) of replicator growth for
libraries of peptide 1 (3.8 mM in 50 mM borate buffer, pH 8.2. Samples were oxidized to 80 mol% with respect to monomer (with a 80 mM perborate solution) and then exposed to 10 mol% of (a) 26, (b) 36, (c) 46, (d) 56, (e) 58, (f) 68.
Thus, we exposed freshly prepared DCLs made from building block 1 to a series of different replicators: 26, 36, 46, 56,
58 and 68 (10 mol%) which we prepared following previously
described procedures.[6g] Remarkably, all DCLs to which hexamer replicators (26, 36, 46 and 56) were added exhibited
product distributions dominated by hexamer 16 (Figure 2a-d).
Furthermore, all libraries seeded with octamer replicators (58
and 68) formed 18 dominated libraries (Figure 1e-f). In some
cases also mixed macrocycles were observed (1n26-n in the
presence of 26 and 1n58-n in the presence of 58). Thus,
pre-existing replicators control the ring size of the new replicators made from building block 1, in some cases overriding the inherent preference of 1 to produce octamer replicator.
Figure 3. Kinetic profiles (monitored at 254 nm) of replicator growth for
libraries made from building block 1 (3.8 mM in 50 mM borate buffer, pH 8.2). Samples are pre-oxidized to 80 mol% with respect to monomer (with a 80 mM perborate solution), non-seeded (blue) or seeded (red) on day 0 with 10 mol% of (a) 18 obtained from the DCL corresponding to Figure 2f and (b) 16 obtained
from the DCL corresponding to Figure 2a. For clarity only the data of the relevant macrocycle size is shown .
In order to confirm that 16 and 18 are self-replicators, seeding
experiment were performed using the seeds obtained from the libraries corresponding to Figure 2a and 2f. When a freshly prepared library of peptide 1 is seeded with 10 mol% seed of 18
or 16, we observed rapid growth of the corresponding
macrocycles (Figure 3), relative to the non-seeded control (blue lines), confirming that 18 and 16 are indeed able to self-replicate.
Figure 4. CD spectra of samples dominated by (a) 16 and (b) 18 and (c)
normalized maximum thioflavin T fluorescence emission intensity (at 492 nm) of non-seeded or seeded (10 mol %) DCLs made from peptide 1 (3.8 mM in 50 mM borate buffer pH 8.2): i, non-agitated; ii, stirred at 1200 rpm and kept under a nitrogen atmosphere; iii, stirred at 1200 rpm in the presence of air; iv, seeded with 46; v, seeded with 56; vi, seeded with 36; vii, seeded with 26; viii,
oxidized to 80% using perborate and stirred at 1200 rpm; ix, seeded with 58; x,
seeded with 68.
The structures of the newly formed 16 and 18 replicators
were characterized by circular dichroism (CD) spectroscopy, thioflavin T fluorescence assays, TEM and IR spectroscopy. The CD spectra of the samples dominated by 16 showed a positive
helicity at around 190 nm and a negative helicity at 210 nm (Figure 4a). These bands appear at wavelengths that are somewhat smaller than those typical for β-sheets. According to Pribic et al.[ 10 ] such shifted signals may arise in tyrosine containing peptides due to 𝜋-𝜋* transitions that complicate the far UV region of the CD spectrum and are still in agreement with a β-sheet structure. The three libraries that produced 18
replicators showed three rather different signatures in their CD spectra (Figure 4b) which we tentatively assignedto parallel β-sheet (for the sample in which 18 emerged autonomously),
anti-parallel β-sheet (when the formation of 18 was triggered by 58)
and mixed (parallel and anti-parallel) β-sheet structures (when the formation of 18 was triggered by 68). Thioflavin T assays
showed an at least 40 times increase in emission for the 16
containing sample and an at least 20 times increase for the 18
containing samples, compared to samples in the absence of replicator, which supports amyloid type β-sheet structures for all samples.
Negative staining transmission electron microscopy (TEM) revealed bundles of fibers having a right-handed helicity for all
samples dominated by 16 (Figure 5a-d and Figure S73). For the
samples of 18 that showed parallel β-sheets we observed single
fibers with a width of ~4.5 nm. In contrast, for samples of 18 that
showed anti-parallel β-sheets we observed a high degree of lateral association of the fibers. In the sample of 18 that showed
mixed β-sheets we observed laterally associated fibers along with single fibers. Thus, it appears that the lateral association of the fibers occurs through antiparallel β-sheet formation.
Figure 5. Transmission electron microscopy images of DCLs made from 1
seeded with (a) 26; (b) 36; (c) 46; (d) 56; (e) 58 and (f) 68.
We further characterized the different assemblies of 16 and
18 by IR spectroscopy (Figure 6). The frequency of the C=O
bands are in the range expected for β-sheet assemblies.[11]
Only for the sample of 18 where we suspect the formation of
antiparallel β-sheets and which exhibited extensive lateral association of the fibers, we observed an additional band at 1615 cm-1, which is associated with the phenyl ring of tyrosine.
The fact that this band is only observed for the 18 sample that
shows extensive laterally associated fibers suggests that in this sample those phenols are in a an environment that differs from the one in a non-associated fiber, which would be in agreement with the postulated occurrence of parallel and antiparallel β-sheet assemblies.
Taken together these results suggest that replicator 18 can
exhibit different modes of assembly: one in which its fibers show lateral association through anti-parallel β-sheet formation and one in which such interactions are absent. Remarkably, this mode of assembly is dictated by the replicator that triggered its emergence. Thus, not only the information regarding ring size is transferred, but also information regarding the mode of assembly of these rings. Seeding experiments showed that both forms of information are to some extent hereditable: seeding a DCL made from 1 with a sample of 18 that showed laterally
associated fibers induced the formation of more 18 that also
showed fiber bundles, while seeding a similar DCL with a sample of 18 that showed no fiber bundling induced more 18 that
existed as non-associated fibers (See SI Figure S74) .
Figure 6. ATR-IR spectra of different replicators obtained from DCLs made
from peptide 1 (3.8 mM in 50 mM borate buffer pH 8.2): vii, seeded with 26; viii, oxidized to 80% using perborate and stirred at 1200 rpm in the presence
of air; ix, seeded with 58. Assignment: TFA at 1675 cm-1; amide I band (C=O)
at 1631 cm-1; C-C ring stretching at 1615 cm-1; amide II band (C-N) at 1537 cm-1; C-C ring stretching at 1515 cm-1.
Finally, we investigated the influence of the sample history on replicator composition. We prepared DCLs having the same building block compositions as those shown in Figures 2a-f, but now the building blocks were mixed prior to oxidation. All of the DCLs produced 13 and 14 along with mixed trimers and
tetramers (See SI Figure S45-68), but no significant amount of any replicator. These data show that the history of the sample is an essential factor determining not only the nature, but also the presence or absence of replicators.
In summary, our results show that both the molecular structure (ring size) and the mode of assembly (parallel or antiparallel β-sheets and degree of fiber bundling) of newly emerging replicators can be controlled by pre-existing replicators. The interactions between replicators can override the preference for a particular structure and ring size innate to the structure of the building blocks of the replicator. While involving similar (but not identical) replicators to the ones reported in a previous study,[6a] our current system shows behavior that is exactly opposite to that reported previously. In the previous work we showed how one specific pre-existing replicators can help the
formation of one other specific replicator, that does not readily form by itself. This behavior was only observed for these two specific replicators and the structure of the newly formed replicator appeared to be invariable. Our current results shows that replicators, rather than having an apparently predetermined structure, can also be highly plastic and respond to the presence of any of a range of pre-existing replicators by adopting the ring size of the specific replicator it was exposed to. As a consequence the composition and nature of the system of replicators reflects sample history and inter-replicator interactions, which are both key prerequisites for Darwinian evolution.
Acknowledgements
We are grateful for support from the ERC, NWO, COST Action CM1304 and the Ministry of Education, Culture and Science (Gravitation program 024.001.035).
Keywords: self-replication • dynamic combinatorial chemistry • de-novo life • origin of life • systems chemistry
[1] a) J. W. Szostak, Angew. Chem. Int. Ed. 2017, 56, 11037-11043; b) K. Ruiz-Mirazo, C. Briones, A. de la Escosura, Chem. Rev. 2014, 114, 285-366; b) A. Pross, What Is Life?: How chemistry becomes biology. Oxford University Press, Oxford, 2012.
[2] For reviews, see: a) H. Duim, S. Otto, Beilstein J. Org. Chem. 2017, 13, 1189–1203; b) T. Kosikovaa, D. Philp, Chem. Soc. Rev. 2017, 46, 7274-7305; c) G. Clixby, L. Twyman, Org. Biomol. Chem. 2016, 14, 4170-4184; d) N. Wagner, G. Ashkenasy, Isr. J. Chem. 2015, 55, 880-890; e) A. J. Bissette, S. P. Fletcher, Angew. Chem. Int. Ed. 2013, 52, 12800-12826; f) V. Patzke, G. von Kiedrowski, ARKIVOC, 2007, 5, 293-310.
[3] For recent examples, see: a) T. S. Jayathilaka, N. Lehman, Chem. Bio.
Chem. 2018, 19, 217-220; b) J. W. Sadownik, T. Kosikova, D. Philp, J. Am. Chem. Soc. 2017, 139, 17565-17573; c) T. Kosikova, D. Philp, J. Am. Chem. Soc. 2017, 139, 12579-12590; d) c) J. Nanda, B. Rubinov,
D. Ivnitski, R. Mukherjee, E. Shtelman, Y. Motro,Y. Miller, N. Wagner, R. C. Luria, G. Ashkenasy, Nature Commun. 2017, 8, 434; e) S. M. Morrow, A. J. Bissette, S. P. Fletcher, Tetrahedron 2017, 73, 5005– 5010; f) D. Komaromy, M. Tezcan, G. Schaeffer, I. Maric, S. Otto,
Angew. Chem. Int. Ed. 2017, 56, 14658-14662.
[4] For reviews, see: a) O. S. Miljanic, Chem. 2017, 2, 502−524; b) Y. Jin, C. Yu, R. J. Denman, W. Zhang, Chem. Soc. Rev. 2013, 42, 6634−6654; c) F. B. L. Cougnon, J. K. M. Sanders, Acc. Chem. Res.
2012, 45, 2211−2221; d) E. Moulin, G. Cormos, N. Giuseppone, Chem.
Soc. Rev. 2012, 41, 1031−1049; e) S. Ladame, Org. Biomol. Chem.
2008, 6, 219−226; f) P. T. Corbett, J. Leclaire, L. Vial, K. R. West, J.-L. Wietor, J. K. M. Sanders, S. Otto, Chem. Rev. 2006, 106, 3652−3711. [5] a) T. Kosikova, H. Mackenzie, D. Philp, Chem. Eur. J. 2016, 22,
1831-1839; b) J. Sadownik, D. Philp, Org. Biomol. Chem. 2015, 13, 10392-10401; c) V. del Amo, D. Philp, Chem. Eur. J. 2010, 16, 13304-13318; d) V. del Amo, A. Slawin, M. Z. Alexandra, D. Philp, Org. Lett. 2008, 10, 4589-4592.
[6] a) Y. Altay, M. Tezcan, S. Otto, J. Am. Chem. Soc. 2017, 139, 13612-13615; b) E. Mattia, A. Pal, G. Leonetti, S. Otto, Synlett 2017, 28, 103-107; c) J. W. Sadownik, E. Mattia, P. Nowak, S. Otto, Nat. Chem. 2016, 8, 264-269; d) M. Colomb-Delsuc, E. Mattia, J. W. Sadownik, S. Otto,
Nat. Commun. 2015, 6, 7427; e) G. Leonetti, S. Otto, J. Am. Chem. Soc. 2015, 137, 2067-2072; f) P. Nowak, M. Colomb-Delsuc, S. Otto,
J. Li, J. Am. Chem. Soc. 2015, 137, 10965–10969; g) M. Malakoutikhah, J. Peyralans, M. Colomb-Delsuc, H. Fanlo-Virgós, M. Stuart, S. Otto, J. Am. Chem. Soc. 2013, 135, 18406-18417; h) J. M. A. Carnall, C. A. Waudby, A. M. Belenguer, M. C. A. Stuart, J. J.-P. Peyralans, S. Otto, Science 2010, 327, 1502-1506.
[7] a) P. G. Higgs, N. Lehman, Nat. Rev. Genet. 2015, 16, 7-17;b) N. Vaidya, M. L. Manapat, I. A. Chen, R. Xulvi-Brunet, E. J. Hayden, N. Lehman, Nature 2012, 491, 72-77.
[8] a) J. A. M. Yeates, C. Hilbe, M. Zwick, M. A. Nowak, N. Lehman, Proc.
Natl. Acad. Sci. U. S. A. 2016, 113, 5030-5035; b) Z. Dadon, N.
Wagner, S. Alasibi, M. Samiappan, R. Mukherjee, G. Ashkenasy, Chem.
Eur. J. 2015, 21, 648-654; c) P. Nghe, W. Hordijk, S. A. Kauffman, S. I.
Walker, F. J. Schmidt, H. Kemble, J. A. M. Yeates, N. Lehman, Mol.
Biosyst. 2015, 11, 3206-3217; d) Z. Dadon, N. Wagner, G. Ashkenasy,
Angew. Chem. Int. Ed. 2008, 47, 6128; e) D. Lee, K. Severin, M.
Ghadiri, Curr. Opin. Chem. Biol. 1997, 1, 491-496.
[9]
Compounds are quantified in terms of the percentage of the total peak area in the chromatogram. This total peak area is comparable for mixtures with very different macrocycle compositions, indicating that the molar absorptivity of the building block unit is independent of the macrocycle in which it resides. Thus, peak areas correlate directly with macrocycle concentrations (expressed in units of building block). [10] R. Pribic, I. Vanstokkum, D. Chapman, P. Haris, M. Bloemendal, Anal.
Biochem. 1993, 214, 366-378.
Entry for the Table of Contents
COMMUNICATION
We survey how existing replicators can affect the emergence of new ones in a dynamic combinatorial library made from a tyrosine containing building block. Seeding with pre-existing replicators of different sizes (hexamer or octamer) directs the ring size of the tyrosine-containing replicator.
Yigit Altay, Meniz Altay, Sijbren Otto*
Page No. – Page No.
How Existing Replicators Direct the Emergence of New Ones
