Controlled mutation in the replication of synthetic oligomers

Replication of sequence information with mutation is the molecular basis for the evolution of functional biopolymers. Covalent template-directed synthesis has been used to replicate sequence information in synthetic oligomers, and the covalent base-pairs used in these systems provide an opportunity to manipulate the outcome of the information transfer process through the use of traceless linkers. Two new types of covalent base-pair have been used to introduce mutation in the replication of an oligotriazole, where information is encoded as the sequence of benzoic acid and phenol monomer units. When a benzoic acid–benzoic acid base-pairing system was used, a direct copy of a benzoic acid homo-oligomer template was obtained. When a phenol–benzoic acid base-pairing system was used, a reciprocal copy, the phenol homo-oligomer, was obtained. The two base-pairing systems are isosteric, so they can be used interchangeably, allowing direct and reciprocal copying to take place simultaneously on the same template strand. As a result, it was possible to introduce mutations in the replication process by spiking the monomer used for direct copying with the monomer used for reciprocal copying. The mutation rate is determined precisely by the relative proportions of the two monomers. The ability to introduce mutation at a controlled rate is a key step in the development of synthetic systems capable of evolution, which requires replication with variation.


Covalent template-directed mutation of chemical information encoded in template 1.
Scheme S5 summarizes the process for the covalent template-directed mutation of chemical information encoded in template 1. This process encompasses four steps: 1) Monomer attachment: Template 1 was loaded with different proportions of 1-mers 4 and 7 via ester coupling using EDC/DMAP as coupling reagents. This step determines the mutation rate of the process, as the obtained preZIP intermediates are enriched in phenol (X=O; Y=CO) or benzoic acid (X=CO; Y=O) 1-mers according to the initial ratio of 1-mers 4 and 7 used.
2) ZIP: CuAAC intramolecular reaction between the reactive groups of the attached 1-mers leads to the formation of the corresponding duplexes. The reaction is carried out in the presence of an excess of a capping azide (tert-butylbenzyl azide), which reacts with the pendant alkyne group of the duplexes preventing cyclization and intermolecular oligomerization through the reactive chain ends after intramolecular reaction. S5,S6 As the backbone is directional, there are two possible arrangements of the duplexes: parallel and antiparallel in regard to the backbone direction (the duplexes shown in Scheme S5 shows the antiparallel arrangement).
3) Capping: Phenyl propargyl ether was used to cap the pendant azide groups in the obtained duplexes 4) Cleavage: Basic hydrolysis of the ester groups promote the release of the biphenyl traceless linkers. The template 1 is regenerated along with the 3-mer oligomer sequences 8-15. The proportion of this oligomer sequences depends on the mutation rate determined in the attach step.

Scheme S5.
Step 1: Monomer attachment. Scheme S6 shows the synthetic approach for the attachment of 1-mers to the template. Five different experiments were performed using a different ratio of 1-mers 4 and 7.
Template 1, 1-mers 4 and 7, EDC and DMAP were mixed in a round-bottom flask and, under N2, CH2Cl2 was added. The reaction was stirred overnight at room temperature. The solution was diluted with EtOAc and washed with 0.1 M HCl soln. (3x), H2O (1x) and brine. The organic phase was dried with MgSO4 and concentrated. The crude material was purified by flash column chromatography on silica gel (gradient from 0% to 60% of EtOAc in Pet. Ether and then gradient from 0% to 4% of MeOH in CH2Cl2) to give the corresponding pre-ZIP intermediates.
PreZIP intermediate: pre-ZIP S9 (0.022 g, 72%) as a white foam. Fig. S2 shows the UPLC traces of the starting template, reaction crude and pure pre-ZIP S9. Full characterization of pre-ZIP S9 appears in the next page.

Determination of mutator population in preZIP intermediates
1 H NMR was used to quantify the population of mutator in the preZIP intermediates from the 1-mer attach experiments 1-5. Fig. S7 shows the stacked 1 H NMR for these preZIP intermediates, with expansions of the regions corresponding to the alkyne CH, methylene groups and the aromatic protons. Some signals can be clearly assigned to the benzoic acid (blue) and phenol (red) 1-mer residues attached to the template.

S52
The alkyne protons for the preZIP intermediates from experiments 2-4 were used to quantify the population of mutator (phenol 1-mer). Deconvolution of these peaks were performed using the Global Spectral Deconvolution (GSD) algorithm provided by MestreNova 10. Fig. S8 shows the fitting model, individual functions and residuals obtained in experiments 2-4. Table S1 shows the areas of the individual curves and the population of mutator extracted from the fitted NMR curves, which are the values used in the x axis in Fig. 8 in the text.

Scheme S7.
! General procedure for ZIP reaction.
A solution of 1-(azidomethyl)-4-tert-butylbenzene S7 in dry and degassed THF (1 mL) was added to a solution of pre-ZIP intermediates in dry and degassed THF under N2 atmosphere. Cu(CH3CN)4PF6 and TBTA were added to the solution and the reaction stirred at room temperature for 2 days. After evaporation of the solvent, the crude was precipitated with Pet. Ether and centrifuged (repeated three times) in order to remove the excess of capping azide. The obtained solid was used in the next step without further purification.
Scheme S8 shows the CuAAC capping with phenyl propargyl ether of the duplexes from the five different experiments carried out in the previous section.
Phenyl propargyl ether was added to a solution of the corresponding duplex mixtures in dry and degassed THF. Cu(CH3CN)4PF6 and TBTA were added to the previous solution and the reaction stirred overnight at room temperature. After evaporation of the solvent, the crude was precipitated with Pet. Ether and centrifuged (repeated three times) in order to remove the excess of capping alkyne. The obtained solid was used in the next step without further purification.
Scheme S9 shows the cleavage step of the capped duplexes from the five different experiments carried out in the previous section. Basic hydrolysis of the ester bonds regenerates the template 1 along with products 8-15, whose proportion is related to the mutation rate, and the traceless biphenyl linker.
The crude reaction mixture from previous step was dissolved in THF:H2O 3:1 and 1 M LiOH soln. was added. The solution was left to react overnight at 5 o C. Then, the crude was diluted with H2O and acidified with 0.1 M HCl soln. to pH 2-3. The aqueous phase was extracted with EtOAc (3x) and the combined organic phase was washed with 0.01M EDTA soln. (2x), H2O (1x), brine (1x), dried over MgSO4 and evaporated to dryness.

Determination of sequence population after covalent template-directed mutation
The populations of the obtained products 8-15 were calculated by deconvoluting the UPLC peaks using the open-source curve-fitting software Fityk (version 1.3.1). S8 The UPLC traces were exported in CSV format from Waters MassLynx™ software (version 4.2) using Microsoft® Excel®. The peaks corresponding to the templated products 8-15 were fitted to a Gaussian function using the PRAXIS fitting method provided in Fityk. PAP-APP and PAA-APA pairs were fitted to single Gaussian functions as their peaks are not resolved enough. Fig. S24 shows the fitting model, individual functions and residuals obtained in experiments 2-4.

S72
Tables S2-4 show the areas of the individual curves and the population of each sequence extracted from the fitted UPLC curves for experiments 2-4, which are the values used in the y axis in Fig. 8 in the text (for experiments 1 and 5, the single templated product obtained is considered as 100%). Table S2. Areas of the fitted curves shown in green in Fig. S24 for experiment 2. Table S3. Areas of the fitted curves shown in green in Fig. S24 for experiment 3. Table S4. Areas of the fitted curves shown in green in Fig. S24