DOI: 10.1038/NCHEM.2419 Diversification of Self-Replicating Molecules Jan W. Sadownik, Elio Mattia, Piotr Nowak, Sijbren Otto* University of Groningen, Center for Systems Chemistry, Stratingh Institute for Chemistry, Nijenborgh 4, 9747 AG Groningen, The Netherlands. *E-mail corresponding author: s.otto@rug.nl Contents: 1. Materials and methods S2 2. UPLC analysis S2 3. Mass spectrometry analysis of the compounds within the studied DCLs. S3 4. Preparation of DCLs for diversification experiments. S16 5. Repeats of the diversification experiment. S19 6. Preparation of DCL A and seeding experiments. S23 7. Reduction and re-equilibration of a DCL consisting of sets I and II. S27 8. Seeding of DCL made from 1 and 2 with (1) 6. S27 9. Replication reaction of building block 1 in isolation. S28 10. Replication reaction of building block 2 in isolation. S29 11. Influence of mechanical agitation on the emergence of set II. S29 NATURE CHEMISTRY www.nature.com/naturechemistry 1
1. Materials and methods Peptide building blocks 1 and 2 were synthesized by Cambridge Peptides Ltd (Birmingham, UK) from 3,5-bis(tritylthio)benzoic acid, which was prepared via a previously reported procedure (30). The buffer was prepared from anhydrous borax (Fluka) and boric acid (Merck Chemicals) dissolved in doubly distilled water from in-house double distillation facilities. Sodium perborate used for the oxidation of the thiols and dithiothreitol (DTT) used for reducing the libraries were purchased from Sigma Aldrich. Acetonitrile (ULC/MS grade) and water (ULC/MS grade) were obtained from Biosolve BV. Trifluoroacetic acid was purchased from Sigma Aldrich. Libraries were prepared in clear HPLC glass vials (12 32 mm) closed with Teflon-lined snap caps purchased from Jaytee. Library solutions were stirred using Teflon coated micro-stirrer bars (2 2 5 mm) obtained from VWR. Samples were stirred on an Heidolph MR Hi-Mix D magnetic stirrer at 1200 rpm. 2. UPLC analysis UPLC analyses were performed on Waters Acquity UPLC I-class or H-class systems equipped with a PDA detector. All analyses were performed using a reversed-phase UPLC column (Aeris Peptide 1.7 µm XB-C18 150 2.10 mm, purchased from Phenomenex). UV absorbance was monitored at 254 nm. Column temperature was kept at 35 C. UPLC-MS was performed using a Waters Acquity UPLC H-class system coupled to a Waters Xevo- G2 TOF. The mass spectrometer was operated in the positive electrospray ionization mode. Capillary, sampling cone, and extraction cone voltages were kept at 2.5 kv, 30 V, and 4 V, respectively. Source and desolvation temperatures were set at 150 C and 500 C, respectively. Nitrogen was used as both cone (5 L/h) and desolvation gas (500 L/h). Injection volume: 5 µl of a library subjected to a 1:20 dilution in doubly distilled water. Method: Eluent flow: 0.3 ml/min; eluent A: UPLC grade water (0.1 v% trifluoroacetic acid); eluent B: UPLC grade acetonitrile (0.1 v% trifluoroacetic acid): time (min) %A %B 0,0 90,0 10,0 1,0 90,0 10,0 1,3 75,0 25,0 3,0 72,0 28,0 11,0 60,0 40,0 11,5 5,0 95,0 12,0 5,0 95,0 12,5 90,0 10,0 17,0 90,0 10,0 Table S1. UPLC method for the analysis of DCLs made from 1 and 2. NATURE CHEMISTRY www.nature.com/naturechemistry 2
3. Mass spectrometry analysis of the compounds within the studied DCLs. Figure S1. Mass spectrum of 1 (retention time 6.6 min) from the LC-MS analysis of a DCL made from 1 and 2. m/z calculated: 760.35 [M+H] 1+, 380.68 [M+2H] 2+ ; m/z observed: 760.32 [M+H] 1+, 380.65 [M+2H] 2+. Figure S2. Mass spectrum of 2 (retention time 4.5 min) from the LC-MS analysis of a DCL made from 1 and 2. m/z calculated: 700.32 [M+H] 1+, 350.66 [M+2H] 2+ ; m/z observed: 700.29 [M+H] 1+, 350.64 [M+2H] 2+. NATURE CHEMISTRY www.nature.com/naturechemistry 3
Figure S3. Mass spectrum of (2) 2 (retention time 5.6 min) from the LC-MS analysis of a DCL made from 1 and 2. m/z calculated: 1397.61 [M+H] 1+, 699.31 [M+2H] 2+, 466.54 [M+3H] 3+ ; m/z observed: 1397.59 [M+H] 1+, 699.28 [M+2H] 2+, 466.51 [M+3H] 3+. Figure S4. Mass spectrum of (1)(2) (retention time 7.3 min) from the LC-MS analysis of a DCL made from 1 and 2. m/z calculated: 1458.65 [(M+1)+H] 1+, 729.32 [M+2H] 2+, 486.55 [M+3H] 3+ ; m/z observed: 1458.61 [(M+1)+H] 1+, 729.29 [M+2H] 2+, 486.52 [M+3H] 3+. NATURE CHEMISTRY www.nature.com/naturechemistry 4
Figure S5. Mass spectrum of (1) 3 (retention time 9.9 min) from the LC-MS analysis of a DCL made from 1 and 2. m/z calculated: 1137.50 [(M+1)+2H] 2+, 759.01 [(M+2)+3H] 3+, 569.25 [(M+1)+4H] 4+, 455.61 [(M+1)+5H] 5+ ; m/z observed: 1137.48 [(M+1)+2H] 2+, 758.98 [(M+2)+3H] 3+, 569.23 [(M+1)+4H] 4+, 455.58 [(M+1)+5H] 5+. Figure S6. Mass spectrum of (1) 2 (2) (retention time 8.8 min) from the LC-MS analysis of a DCL made from 1 and 2. m/z calculated: 1107.48 [(M+1)+2H] 2+, 738.99 [(M+2)+3H] 3+, 554.25 [(M+1)+4H] 4+ ; m/z observed: 1107.45 [(M+1)+2H] 2+, 738.97 [(M+2)+3H] 3+, 554.22 [(M+1)+4H] 4+. NATURE CHEMISTRY www.nature.com/naturechemistry 5
Figure S7. Mass spectrum of (1)(2) 2 (retention time 7.5 min) from the LC-MS analysis of a DCL made from 1 and 2. m/z calculated: 1077.46 [(M+1)+2H] 2+, 718.98 [(M+2)+3H] 3+, 539.24 [(M+1)+4H] 4+ ; m/z observed: 1077.43 [(M+1)+2H] 2+, 718.95 [(M+2)+3H] 3+, 539.21 [(M+1)+4H] 4+. Figure S8. Mass spectrum of (2) 3 (retention time 6.3 min) from the LC-MS analysis of a DCL made from 1 and 2. m/z calculated: 1047.45 [(M+1)+2H] 2+, 698.97 [(M+2)+3H] 3+, 524.23 [M+4H] 4+ ; m/z observed: 1047.42 [(M+1)+2H] 2+, 698.94 [(M+2)+3H] 3+, 524.20 [M+4H] 4+. NATURE CHEMISTRY www.nature.com/naturechemistry 6
Figure S9. Mass spectrum of (1) 4 (retention time 8.6 min) from the LC-MS analysis of a DCL made from 1 and 2. m/z calculated: 1516.67 [(M+2)+2H] 2+, 1011.45 [(M+2)+3H] 3+, 758.59 [(M+1)+4H] 4+ ; m/z observed: 1516.64 [(M+2)+2H] 2+, 1011.42 [(M+2)+3H] 3+, 758.56 [(M+1)+4H] 4+. Figure S10. Mass spectrum of (1) 3 (2) 1 (retention time 7.7 min) from the LC-MS analysis of a DCL made from 1 and 2. m/z calculated: 1486.65 [(M+2)+2H] 2+, 991.44 [(M+2)+3H] 3+, 743.58 [(M+1)+4H] 4+, 595.06 [(M+1)+5H] 5+ ; m/z observed: 1486.62 [(M+2)+2H] 2+, 991.41 [(M+2)+3H] 3+, 743.55 [(M+1)+4H] 4+ 595.04 [(M+1)+5H] 5+. NATURE CHEMISTRY www.nature.com/naturechemistry 7
Figure S11. Mass spectrum of (1) 2 (2) 2 (retention time 6.7 min) from the LC-MS analysis of a DCL made from 1 and 2. m/z calculated: 1456.63[(M+2)+2H] 2+, 971.42 [(M+2)+3H] 3+, 728.82 [(M+2)+4H] 4+ ; m/z observed: 1456.59 [(M+2)+2H] 2+, 971.39 [(M+2)+3H] 3+, 728.78 [(M+2)+4H] 4+. Figure S12. Mass spectrum of (1) 1 (2) 3 (retention time 5.7 min) from the LC-MS analysis of a DCL made from 1 and 2. m/z calculated: 1426.61 [(M+2)+2H] 2+, 951.41 [(M+2)+3H] 3+, 713.81 [(M+2)+4H] 4+, 571.05 [(M+1)+5H] 5+ ; m/z observed: 1426.59 [(M+2)+2H] 2+, 951.38 [(M+2)+3H] 3+, 713.78 [(M+2)+4H] 4+, 571.03 [(M+1)+5H] 5+. NATURE CHEMISTRY www.nature.com/naturechemistry 8
Figure S13. Mass spectrum of (2) 4 (retention time 4.8 min) from the LC-MS analysis of a DCL made from 1 and 2. m/z calculated: 1396.59 [(M+2)+2H] 2+, 931.40 [(M+2)+3H] 3+, 698.80 [(M+2)+4H] 4+ ; m/z observed: 1396.55 [(M+2)+2H] 2+, 931.37 [(M+2)+3H] 3+, 698.78 [(M+2)+4H] 4+. Figure S14. Mass spectrum of (1) 6 (retention time 9.1 min) from the LC-MS analysis of a DCL made from 1 and 2. m/z calculated: 1516.67 [(M+3)+3H] 3+, 1137.75 [(M+3)+4H] 4+, 910.80 [(M+5)+5H] 5+, 759.00 [(M+4)+6H] 6+ ; m/z observed: 1516.62 [(M+3)+3H] 3+, 1137.72 [(M+3)+4H] 4+, 910.77 [(M+5)+5H] 5+, 758.99 [(M+4)+6H] 6+. NATURE CHEMISTRY www.nature.com/naturechemistry 9
Figure S15. Mass spectrum of (1) 5 (2) 1 (retention time 8.5 min) from the LC-MS analysis of a DCL made from 1 and 2. m/z calculated: 1496.66 [(M+3)+3H] 3+, 1122.74 [(M+3)+4H] 4+, 898.60 [(M+3)+5H] 5+, 749.00 [(M+4)+6H] 6+ ; m/z observed: 1496.61 [(M+3)+3H] 3+, 1122.72 [(M+3)+4H] 4+, 898.56 [(M+3)+5H] 5+, 749.96 [(M+4)+6H] 6+. Figure S16. Mass spectrum of (1) 4 (2) 2 (retention time 7.9 min) from the LC-MS analysis of a DCL made from 1 and 2. m/z calculated: 1476.65 [(M+3)+3H] 3+, 1107.73 [(M+3)+4H] 4+, 886.59 [(M+3)+5H] 5+, 738.99 [(M+4)+6H] 6+ ; m/z observed: 1476.61 [(M+3)+3H] 3+, 1107.70[(M+3)+4H] 4+, 886.55 [(M+3)+5H] 5+, 738.96 [(M+4)+6H] 6+. NATURE CHEMISTRY www.nature.com/naturechemistry 10
Figure S17. Mass spectrum of (1) 3 (2) 3 (retention time 7.3 min) from the LC-MS analysis of a DCL made from 1 and 2. m/z calculated: 1456.63 [(M+3)+3H] 3+, 1092.72 [(M+3)+4H] 4+, 874.38 [(M+4)+5H] 5+, 782.82 [(M+3)+6H] 6+ ; m/z observed: 1456.59 [(M+3)+3H] 3+, 1092.70 [(M+3)+4H] 4+, 874.35 [(M+4)+5H] 5+, 782.79 [(M+3)+6H] 6+. Figure S18. Mass spectrum of (1) 2 (2) 4 (retention time 6.6 min) from the LC-MS analysis of a DCL made from 1 and 2. m/z calculated: 1436.62 [(M+3)+3H] 3+, 1077.71 [(M+3)+4H] 4+, 862.37 [(M+3)+5H] 5+ ; m/z observed: 1436.58 [(M+3)+3H] 3+, 1077.68 [(M+3)+4H] 4+, 862.35 [(M+3)+5H] 5+. NATURE CHEMISTRY www.nature.com/naturechemistry 11
Figure S19. Mass spectrum of (1) 1 (2) 5 (retention time 5.9 min) from the LC-MS analysis of a DCL made from 1 and 2. m/z calculated: 1416.61 [(M+3)+3H] 3+, 1062.71 [(M+3)+4H] 4+, 850.77 [(M+3)+5H] 5+, 708.97 [M+6H] 6+ ; m/z observed 1416.58 [(M+3)+3H] 3+, 1062.67 [(M+3)+4H] 4+, 850.73 [(M+3)+5H] 5+, 708.95 [M+6H] 6+. Figure S20. Mass spectrum of (2) 6 (retention time 5.1 min) from the LC-MS analysis of a DCL made from 1 and 2. m/z calculated: 1396.59 [(M+4)+3H] 3+, 1047.70 [(M+3)+4H] 4+, 838.76 [(M+5)+5H] 5+, m/z observed 1396.56 [(M+4)+3H] 3+, 1047.68 [(M+3)+4H] 4+, 838.72 [(M+5)+5H] 5+. NATURE CHEMISTRY www.nature.com/naturechemistry 12
Figure S21. Mass spectrum of (2) 8 (retention time 4.4 min) from the LC-MS analysis of a DCL made from 1 and 2 seeded with (2) 8. m/z calculated: 1861.46 [(M+3)+3H] 3+, 1396.59 [(M+4)+4H] 4+, 1117.68 [(M+6)+5H] 5+, 931.40 [(M+4)+6H] 6+ ; m/z observed: 1861.36 [(M+3)+3H] 3+, 1396.55 [(M+4)+4H] 4+, 1117.65 [(M+6)+5H] 5+, 931.35 [(M+4)+6H] 6+. Figure S22. Mass spectrum of (1)(2) 7 (retention time 4.9 min) from the LC-MS analysis of a DCL made from 1 and 2 seeded with (2) 8. m/z calculated: 1881.47 [(M+3)+3H] 3+, 1411.85 [(M+1)+4H] 4+, 1129.68 [(M+3)+5H] 5+, 941.07 [(M+2)+6H] 6+, 806.92 [(M+3)+7H] 7+ ; m/z observed: 1881.40 [(M+3)+3H] 3+, 1411.80 [(M+1)+4H] 4+, 1129.66 [(M+3)+5H] 5+, 941.04 [(M+2)+6H] 6+, 806.90 [(M+3)+7H] 7+. NATURE CHEMISTRY www.nature.com/naturechemistry 13
Figure S23. Mass spectrum of (1) 2 (2) 6 (retention time 5.4 min) from the LC-MS analysis of a DCL made from 1 and 2 seeded with (2) 8. m/z calculated: 1901.82 [(M+4)+3H] 3+, 1426.86 [(M+3)+4H] 4+, 1141.69 [(M+5)+5H] 5+, 951.41 [(M+4)+6H] 6+ ; m/z observed: 1901.72 [(M+4)+3H] 3+, 1426.81 [(M+3)+4H] 4+, 1141.66 [(M+5)+5H] 5+, 951.38 [(M+4)+6H] 6+. Figure S24. Mass spectrum of (1) 3 (2) 5 (retention time 6.0 min) from the LC-MS analysis of a DCL made from 1 and 2 seeded with (2) 8. m/z calculated: 1921.83[(M+1)+3H] 3+, 1441.62 [(M+4)+4H] 4+, 1153.50 [(M+4)+5H] 5+, 961.25 [(M+3)+6H] 6+ ; m/z observed: 1921.69[(M+1)+3H] 3+, 1441.58 [(M+4)+4H] 4+, 1153.46 [(M+4)+5H] 5+, 961.22 [(M+3)+6H] 6+. NATURE CHEMISTRY www.nature.com/naturechemistry 14
Figure S25. Mass spectrum of (1) 4 (2) 4 (retention time 6.5 min) from the LC-MS analysis of a DCL made from 1 and 2 seeded with (2) 8. m/z calculated: 1941.84 [(M+4)+3H] 3+, 1456.88 [(M+5)+4H] 4+, 1165.51 [(M+4)+5H] 5+, 971.42 [(M+4)+6H] 6+ ; m/z observed: 1941.75 [(M+4)+3H] 3+, 1456.84 [(M+5)+4H] 4+, 1165.47 [(M+4)+5H] 5+, 971.39 [(M+4)+6H] 6+. Figure S26. Mass spectrum of (1) 5 (2) 3 (retention time 7.0 min) from the LC-MS analysis of a DCL made from 1 and 2 seeded with (2) 8. m/z calculated: 1961.85 [(M+5)+3H] 3+, 1471.64 [(M+4)+4H] 4+, 1177.51 [(M+4)+5H] 5+, 981.60 [(M+5)+6H] 6+ ; m/z observed: 1961.73 [(M+5)+3H] 3+, 1471.60 [(M+4)+4H] 4+, 1177.49 [(M+4)+5H] 5+, 981.55 [(M+5)+6H] 6+. NATURE CHEMISTRY www.nature.com/naturechemistry 15
4. Preparation of DCLs for diversification experiments. DCLs displaying diversification behaviour were prepared by dissolving building block 1 (1.5 mg) and 2 (1.4 mg) in 400 µl sodium borate buffer (50 mm, ph 8.0) giving a total concentration of monomers of 7.6 mm in 1:1 molar ratio. The solutions were left stirring and were analysed daily until all replicators emerged from the libraries (Figure S27). Examples of UPLC chromatograms after 2, 5 and 35 days are given in Figures S28 to S30. A DCL prepared in the same way was also seeded with (2) 8 which was obtained from a stirred solution of building block 2 (Figure S43). 80 µl of 3.8 mm solution of (2) 8 in sodium borate buffer (50 mm, ph 8.0) was added to 400 µl of the library. The solution was left stirring and was analysed daily (Figure S31) until all set of replicators emerged from the libraries (Figure S32). Figure S27. Change in product distribution with time of a DCL made from building blocks 1 and 2 (3.8 mm each in 50 mm, ph 8.0 aqueous borate buffer) showing the emergence of sets I (red) and II (blue) after 3 and 10 days, respectively. The grey lines represent trimer and tetramer macrocycles. NATURE CHEMISTRY www.nature.com/naturechemistry 16
2 (1)(2) 3 (2) 4 (2) 2 1 [(2) 3 overlaps] (1) 2(2) 2 (1)(2) (1)(2) 2 (1) 3(2) (1) 2(2) (1) 4 (1) 3 Artifact due to column wash Figure S28. UPLC chromatogram (monitored at 254 nm) of a DCL made from building blocks 1 and 2 (1:1 molar ratio, 7.6 mm) after 2 days of stirring. 2 (2) 4 (1) 5(2) (1)(2) 3 (1) 4(2) 2 (2) 2 (1) 3(2) 3 (2) 3 (1) 6 (1) 2(2) 2 Artifact due to column wash (1) 2(2) Figure S29. UPLC chromatogram (monitored at 254 nm) of a DCL made from building blocks 1 and 2 (1:1 molar ratio, 7.6 mm) after 5 days of stirring after the emergence of set I and before the emergence of set II. NATURE CHEMISTRY www.nature.com/naturechemistry 17
(1) 5(2) (1) 4(2) 2 (1) 5(2) (2) 6 (1) 4(2) 2 (1) 3(2) 3 (1) 6 Artifact due to column wash Figure S30. UPLC chromatogram (monitored at 254 nm) of a DCL made from building blocks 1 and 2 (1:1 molar ratio, 7.6 mm) after 35 days of stirring after the emergence of set I and set II. Figure S31. Change in product distribution with time of a DCL made from building blocks 1 and 2 (3.8 mm each in 50 mm, ph 8.0 borate buffer) seeded with 10 mol% of (2) 8 at t=0 showing the emergence of sets I (red) and III (cyan). NATURE CHEMISTRY www.nature.com/naturechemistry 18
(1) 2(2) 6 (1) 3(2) 5 (1) 4(2) 4 (1) 4(2) 2 (1) 5(2) (1)(2) 7 (2) 8 (1) 3(2) 3 Artifact due to column wash (1) 6 Figure S32. UPLC chromatogram (monitored at 254 nm) of a DCL made from building blocks 1 and 2 (1:1 molar ratio, 7.6 mm) seeded with (2) 8 after thirty five days of stirring after the emergence of set I and set III. 5. Repeats of the diversification experiment. The library setup and analysis as described in section 4 was repeated 12 times. Figure S33 shows the results plotting the change of hexamer concentrations in time (left plot) with other species omitted for clarity and the final distribution of hexamers within the library (right plot). Qualitatively similar results were observed in ten out of twelve repeats of the experiment (Figure S33 entries A-J). In these cases the emergence of set I (red) was separated in time from the emergence of set II (blue). Although the rates of formation were not always the same the final bimodal distribution of macrocycle composition is very similar. For entries K and L all macrocycles emerge almost simultaneously; in case of K after 3 days and in case of L after 10 days. In these cases the final distribution of hexamers shows no obvious separation into replicator sets. NATURE CHEMISTRY www.nature.com/naturechemistry 19
A B C D NATURE CHEMISTRY www.nature.com/naturechemistry 20
E F G H NATURE CHEMISTRY www.nature.com/naturechemistry 21
I J K L Figure S33. Plots describing the repeats of the diversification experiment of Figure 2. The hexamers are labeled: NATURE CHEMISTRY www.nature.com/naturechemistry 22
6. Preparation of DCL A and seeding experiments. DCL A was prepared by dissolving building block 1 (0.4 mg) and 2 (3.6 mg) in 2 ml sodium borate buffer (50 mm, ph 8.0) giving a total concentration of monomers 2 mm in 1:10 molar ratio. Subsequently 90 mol% of the mixture was oxidized by adding 36 µl of 50 mm sodium perborate solution in doubly distilled water (Figure S34). (2) 4 (2) 3 (1)(2) 3 (1)(2) 2 Figure S34. UPLC chromatogram (monitored at 254 nm) of a DCL A made from building blocks 1 and 2 (1:10 molar ratio, 2 mm) after oxidation. NATURE CHEMISTRY www.nature.com/naturechemistry 23
(2) 4 (1)(2) 7 (2) 8 (1) 2(2) 6 (2) 3 (1)(2) 2 Figure S35. UPLC chromatogram (monitored at 254 nm) of the unseeded library after 2 weeks. Libraries termed seed X, Y and Z were prepared by dissolving appropriate ratios of building blocks 1 and 2 in sodium borate buffer (50 mm, ph 8.0) and letting the libraries be oxidized by air while stirring until the library consisted of only hexamers. Seed X: 3.8 mm solution made from 1 (Figure S36). Seed Y: solution made from 3.45 mm of 1 and 0.35 mm of 2 (10:1 molar ratio) (Figure S37). Seed Z: solution made from 2.85 mm of 1 and 0.95 mm of 2 (3:1 molar ratio) (Figure S38). NATURE CHEMISTRY www.nature.com/naturechemistry 24
(1) 6 Figure S36. UPLC chromatogram (monitored at 254 nm) of seed X consisting of only (1) 6. (1) 6 (1) 5(2) (1) 4(2) 2 Figure S37. UPLC chromatogram (monitored at 254 nm) of seed Y consisting of (1) 6, (1) 5 (2) and (1) 4 (2) 2 in 22:6:1 molar ratio, respectively. NATURE CHEMISTRY www.nature.com/naturechemistry 25
(1) 5(2) (1) 4(2) 2 (1) 6 (1) 3(2) 3 (1) 2(2) 4 Figure S38. UPLC chromatogram (monitored at 254 nm) of seed Z consisting of (1) 6, (1) 5 (2), (1) 4 (2) 2, (1) 3 (2) 3 and (1) 2 (2) 4 in 17:24:18:6:1 molar ratio. DCL A was split into three samples of 500 µl and to each sample 40 µl of the appropriate seed X, Y or Z was added. In the case where Z was the seed we observed that the concentration of the hexamers in set I (Fig. S39 red) remains approximately constant while the intermediate species (1) 3 (2) 3 and the hexamers of set II emerge. (Figure S39). 35 30 relative peak area % 25 20 15 10 5 S6 S5F1 S4F2 S3F3 S2F4 S1F5 F6 0 0 5 10 15 20 time / days Figure S39. Change in product distribution with time of DCL A (see main text) which was seeded with Z. While set I (red) remains approximately constant we observe the emergence of set II (blue). NATURE CHEMISTRY www.nature.com/naturechemistry 26
7. Reduction and re-equilibration of a DCL consisting of sets I and II. A DCL which showed diversification behaviour displayed a bimodal distribution of the different hexamers within the sets. After 35 days all the thiols in solution were oxidized to disulfides and we did not observe further disulfide exchange and no changes in the distribution of library members over the course of 14 days. In order to facilitate further exchange the library (400 µl, 7.6 mm) was reduced with dithiothreitol (DTT) solution (18 µl, 50 mm). In order to prevent re-oxidation the solution was degassed and left stirring in a glovebox in the absence of oxygen. After 30 days the sample was analysed revealing that disulfide exchange between the library members led to a statistical distribution of building blocks within the hexameric set of macrocycles with (1) 3 (2) 3 being the dominant product (Figure S40). (1) 3(2) 3 (1) 2(2) 4 (1) 4(2) 2 (1) 5(2) (1)(2) 5 (1) 6 (2) 6 Figure S40. UPLC chromatogram (monitored at 254 nm) of a library with a statistical distribution of mixed hexamers, obtained upon re-equilibration of a mixture of replicator sets I and II. 8. Seeding of DCL made from 1 and 2 with (1) 6. UPLC-MS analysis does not allow one to assess the composition of stacks of macrocycles. In principle, (1) 6, (1) 5 (2), (1) 4 (2) 2 and (1) 3 (2) 3 could all form separate stacks. However, this arrangement is highly unlikely, given that the different hexamers all emerge and grow in parallel, with similar kinetics, suggesting cross-catalysis between macrocycles of different composition. Furthermore, seeding a mixture of trimers and tetramers made from equimolar amounts of 1 and 2 with (1) 6 promoted the growth of the different 1-rich hexamer isomers to comparable extents (Figure S41). If (1) 6 were to form separate stacks then such seeding experiment should have led to the selective growth of (1) 6. This experiment also indicates that (1) 6 is able to promote the formation of mutant replicators that incorporate one or more units of building block 2. A solution of building blocks 1 and 2 (200 µl, 7.6 mm) in sodium borate buffer (50 mm, ph 8.0) was oxidized 75 mol% using sodium perborate (50 mm, 22.8 µl). 12.5 mol% of (1) 6 (50 µl, 3.8 mm) was added and the solution was left stirring overnight. Since (1) 6 is a replicator we expected the concentration of this species to grow together with the related mutants of set I. The UPLC analysis revealed that one day after seeding (1) 6 accounted for 15.7 % of the library. (1) 5 (2) 1 also grew to 15.7 %, (1) 4 (2) 2 to 18 % and (1) 3 (2) 3 to 10.9 %. Also smaller amounts of (2)-rich hexamers were present: NATURE CHEMISTRY www.nature.com/naturechemistry 27
(1) 2 (2) 4 4.1 % and (1) 1 (2) 5 0.7%. In the subsequent 2 days the amount hexamers from (1) 6 to (1) 4 (2) 2 remained constant and the concentration of (1) 3 (2) 3 as well as the mutants within set II increased until only hexamers were present in solution (Figure S41). relative peak area % 20 18 16 14 12 10 8 6 4 2 0 (2) 1 6 (1) 2 1(2) (1) 5 (1) 2(2) 3 1(2) 4 5 (1) 3(2) 4 3 (1) 4(2) 5 (1) 5(2) 2 6 1 (1) 7 6 Series1 1 day after seeding 2 days after seeding Series3 3 days after seeding Figure S41. Distribution of hexamers over three days after seeding an oxidized library of 1 and 2 with (1) 6. 9. Replication reaction of building block 1 in isolation. A DCL prepared with 1.5 mg of 1 (3.8 mm) in 400 µl sodium borate buffer (50 mm, ph 8.0) when left stirring results in selective formation of (1) 6 replicator (Figure S42). Figure S42. Change in product distribution with time of a DCL made from building block 1. Data reproduced from ref. 31. NATURE CHEMISTRY www.nature.com/naturechemistry 28
10. Replication reaction of building block 2 in isolation. A DCL prepared with 1.4 mg of 2 (3.8 mm) in 400 µl sodium borate buffer (50 mm, ph 8.0) when left stirring results in selective formation of (1) 8 replicator (Figure S43). Figure S43. Change in product distribution with time of a DCL made from building block 2. Data reproduced form ref. 31. 11. Influence of mechanical agitation on the emergence of set II. A DCL was prepared by dissolving building block 1 (1.5 mg) and 2 (1.4 mg) in 400 µl sodium borate buffer (50 mm, ph 8.0) giving a total concentration of monomers of 7.6 mm in 1:1 molar ratio. The solution was left stirring at 1200 rpm for 3 days to the point where set I emerged. At this point the solution was split into two vials: one was stirred while the other was kept without agitation. The vials were monitored after two weeks. The one which was stirred consisted of all possible hexamer mutants (Figure S44) whereas the non-agitated sample showed only set I and no emergence of set II (Figure S45). NATURE CHEMISTRY www.nature.com/naturechemistry 29
(1) 2(2) 4 (1) 3(2) 3 (1) 4(2) 2 (1) 1(2) 5 (1) 5(2) 1 (1) 6 (2) 6 Figure S44. UPLC chromatogram (monitored at 254 nm) of a library which was stirred continuously. Also after the emergence of set I. (1) 5(2) 1 (1) 4(2) 2 (2) 4 (1)(2) 3 (1)(2) 2 (1) 6 (1) 3(2) 3 (2) 3 (1) 2(2) 2 Figure S45. UPLC chromatogram (monitored at 254 nm) of a library for which stirring was stopped after the emergence of set I. NATURE CHEMISTRY www.nature.com/naturechemistry 30