Amplification, mutation, and sequencing of a six-letter synthetic genetic system

Abstract

The next goals in the development of a synthetic biology that uses artificial genetic systems will require chemistry-biology combinations that allow the amplification of DNA containing any number of sequential and nonsequential nonstandard nucleotides. This amplification must ensure that the nonstandard nucleotides are not unidirectionally lost during PCR amplification (unidirectional loss would cause the artificial system to revert to an all-natural genetic system). Further, technology is needed to sequence artificial genetic DNA molecules. The work reported here meets all three of these goals for a six-letter artificially expanded genetic information system (AEGIS) that comprises four standard nucleotides (G, A, C, and T) and two additional nonstandard nucleotides (Z and P). We report polymerases and PCR conditions that amplify a wide range of GACTZP DNA sequences having multiple consecutive unnatural synthetic genetic components with low (0.2% per theoretical cycle) levels of mutation. We demonstrate that residual mutation processes both introduce and remove unnatural nucleotides, allowing the artificial genetic system to evolve as such, rather than revert to a wholly natural system. We then show that mechanisms for these residual mutation processes can be exploited in a strategy to sequence "six-letter" GACTZP DNA. These are all not yet reported for any other synthetic genetic system.

Figure 1. The expanded GACTZP genetic system

Left column: Matched C:G, Z:P, and T:A pairs all fit the Watson-Crick geometry (a small pyrimidine analog with one ring complements in size a large purine analog with two rings and are all joined by three hydrogen bonds). Electron density presented to the minor groove is represented as shaded green lobes. Right column: Mismatched C:P, Z:G, and T:P pairs. Note clashes between electrons (gray lobes) or hydrogens, which can be mitigated by protonation/deprotonation, respectively.

Figure 2. Agarose gel (3%) resolving amplicons from “six-letter” GACTZP PCR with standard templates and synthetic templates containing multiple consecutive dPs

Lane 1: Control without template; Lane 2: Amplification of standard template, without dZTP and dPTP; Lane 3: Amplification of standard template, with dZTP and dPTP; Lane 4 and 6: Amplification of synthetic template, without dZTP and dPTP; Lane 5 and 7: Amplification of synthetic template, with dZTP and dPTP. dNTPs (0.1 mM for each), dZTP (0.05 mM) and dPTP (0.6 mM). M: 25 bp marker. See Methods and Materials for PCR conditions.

Figure 3. Mutation interconverting Z:P, C:G and T:A Pairs

a. Measuring the “forward” mutation converting C:G pairs into Z:P pairs using digestion with the Bsp120I restriction endonuclease. Standard oligonucleotide (Bsp-G, Table 1) containing the Bsp120I recognition sequence (5′-GGGCCC-3′) were 1000 fold amplified using Taq DNA polymerase at pH = 8.8 or 8.0 with standard dNTPs (0.2 mM of each) with or without dZTP and dPTP. Then, PCR amplicon were digested by endonuclease (Bsp120I). Lane 1: In the absence of both dZTP and dPTP; Lane 2: With dPTP (0.2 mM); Lane 3: With dZTP (0.2 mM); Lane 4: With both dZTP and dPTP (0.2 mM for each), Not digested: indicates the fraction of PCR product resisted endonuclease digestion; Digested: indicates the fraction of PCR product was digested. See Methods and Materials for PCR conditions. b. Measure of the “reverse” mutation of Z:P to give C:G and T:A using restriction endonuclease. Two complementary synthetic templates (Bsp-Z and Bsp-P, Table 1) containing 5′-GGGCCZ-3′ and 3′-CCCGGP-5′, were 1000 fold amplified using Taq with standard dNTPs (200 μM), dZTP and dPTP (with various concentration, lane 1 (20 μM), lane 2 (10 μM), lane 3 (5 μM)), then, PCR amplicon were digested by endonuclease (Bsp120I). See Methods and Materials for details. c. Measuring the mutation of Z into C and T (left) and P into A and G (right) using restriction endonuclease digestion. Single-stranded synthetic oligonucleotide containing either 5′-GGGCCZ-3′ (left, lane 1, Bsp-Z) or 3′-CCCGGP-5′ (right, lane 2, Bsp-P), was 1000 fold amplified using Taq with only four standard dNTPs (0.2 mM) in 1x ThermoPol reaction buffer (pH 8.8 at room temperature). Then, PCR amplicon was digested by endonuclease (Bsp120I). See Methods and Materials for details. d. Observed pathways of mutation between non-standard nucleotides and standard nucleotides. Conversion of C:G pair to Z:P pair (forward mutation) involves mis-incorporation of dZTP opposite template-G to form Z:G mismatch at high pH (8.8) (Fig. 3a); Conversion of Z:P pair to C:G pair (reverse mutation) involves two pathways: 1) the most likely pathway, mis-incorporation of dGTP opposite template-Z to form G:Z mismatch at high pH (8.8) (Fig. 3b); 2) mis-incorporation of dCTP opposite template-P to form C:P mismatch (Fig. 3c, lane 2); Conversion of Z:P pair to T:A pair (reverse mutation) involves two pathways: 1) the most likely pathway, mis-incorporation of dTTP opposite template-P to form T:P mismatch; 2) misincorporation of dATP opposite template-Z to form A:Z mismatch (Fig. 3c). See Figure 1 for corresponding matched and mismatched base pairs.

Figure 3. Mutation interconverting Z:P, C:G and T:A Pairs

a. Measuring the “forward” mutation converting C:G pairs into Z:P pairs using digestion with the Bsp120I restriction endonuclease. Standard oligonucleotide (Bsp-G, Table 1) containing the Bsp120I recognition sequence (5′-GGGCCC-3′) were 1000 fold amplified using Taq DNA polymerase at pH = 8.8 or 8.0 with standard dNTPs (0.2 mM of each) with or without dZTP and dPTP. Then, PCR amplicon were digested by endonuclease (Bsp120I). Lane 1: In the absence of both dZTP and dPTP; Lane 2: With dPTP (0.2 mM); Lane 3: With dZTP (0.2 mM); Lane 4: With both dZTP and dPTP (0.2 mM for each), Not digested: indicates the fraction of PCR product resisted endonuclease digestion; Digested: indicates the fraction of PCR product was digested. See Methods and Materials for PCR conditions. b. Measure of the “reverse” mutation of Z:P to give C:G and T:A using restriction endonuclease. Two complementary synthetic templates (Bsp-Z and Bsp-P, Table 1) containing 5′-GGGCCZ-3′ and 3′-CCCGGP-5′, were 1000 fold amplified using Taq with standard dNTPs (200 μM), dZTP and dPTP (with various concentration, lane 1 (20 μM), lane 2 (10 μM), lane 3 (5 μM)), then, PCR amplicon were digested by endonuclease (Bsp120I). See Methods and Materials for details. c. Measuring the mutation of Z into C and T (left) and P into A and G (right) using restriction endonuclease digestion. Single-stranded synthetic oligonucleotide containing either 5′-GGGCCZ-3′ (left, lane 1, Bsp-Z) or 3′-CCCGGP-5′ (right, lane 2, Bsp-P), was 1000 fold amplified using Taq with only four standard dNTPs (0.2 mM) in 1x ThermoPol reaction buffer (pH 8.8 at room temperature). Then, PCR amplicon was digested by endonuclease (Bsp120I). See Methods and Materials for details. d. Observed pathways of mutation between non-standard nucleotides and standard nucleotides. Conversion of C:G pair to Z:P pair (forward mutation) involves mis-incorporation of dZTP opposite template-G to form Z:G mismatch at high pH (8.8) (Fig. 3a); Conversion of Z:P pair to C:G pair (reverse mutation) involves two pathways: 1) the most likely pathway, mis-incorporation of dGTP opposite template-Z to form G:Z mismatch at high pH (8.8) (Fig. 3b); 2) mis-incorporation of dCTP opposite template-P to form C:P mismatch (Fig. 3c, lane 2); Conversion of Z:P pair to T:A pair (reverse mutation) involves two pathways: 1) the most likely pathway, mis-incorporation of dTTP opposite template-P to form T:P mismatch; 2) misincorporation of dATP opposite template-Z to form A:Z mismatch (Fig. 3c). See Figure 1 for corresponding matched and mismatched base pairs.

Figure 3. Mutation interconverting Z:P, C:G and T:A Pairs

a. Measuring the “forward” mutation converting C:G pairs into Z:P pairs using digestion with the Bsp120I restriction endonuclease. Standard oligonucleotide (Bsp-G, Table 1) containing the Bsp120I recognition sequence (5′-GGGCCC-3′) were 1000 fold amplified using Taq DNA polymerase at pH = 8.8 or 8.0 with standard dNTPs (0.2 mM of each) with or without dZTP and dPTP. Then, PCR amplicon were digested by endonuclease (Bsp120I). Lane 1: In the absence of both dZTP and dPTP; Lane 2: With dPTP (0.2 mM); Lane 3: With dZTP (0.2 mM); Lane 4: With both dZTP and dPTP (0.2 mM for each), Not digested: indicates the fraction of PCR product resisted endonuclease digestion; Digested: indicates the fraction of PCR product was digested. See Methods and Materials for PCR conditions. b. Measure of the “reverse” mutation of Z:P to give C:G and T:A using restriction endonuclease. Two complementary synthetic templates (Bsp-Z and Bsp-P, Table 1) containing 5′-GGGCCZ-3′ and 3′-CCCGGP-5′, were 1000 fold amplified using Taq with standard dNTPs (200 μM), dZTP and dPTP (with various concentration, lane 1 (20 μM), lane 2 (10 μM), lane 3 (5 μM)), then, PCR amplicon were digested by endonuclease (Bsp120I). See Methods and Materials for details. c. Measuring the mutation of Z into C and T (left) and P into A and G (right) using restriction endonuclease digestion. Single-stranded synthetic oligonucleotide containing either 5′-GGGCCZ-3′ (left, lane 1, Bsp-Z) or 3′-CCCGGP-5′ (right, lane 2, Bsp-P), was 1000 fold amplified using Taq with only four standard dNTPs (0.2 mM) in 1x ThermoPol reaction buffer (pH 8.8 at room temperature). Then, PCR amplicon was digested by endonuclease (Bsp120I). See Methods and Materials for details. d. Observed pathways of mutation between non-standard nucleotides and standard nucleotides. Conversion of C:G pair to Z:P pair (forward mutation) involves mis-incorporation of dZTP opposite template-G to form Z:G mismatch at high pH (8.8) (Fig. 3a); Conversion of Z:P pair to C:G pair (reverse mutation) involves two pathways: 1) the most likely pathway, mis-incorporation of dGTP opposite template-Z to form G:Z mismatch at high pH (8.8) (Fig. 3b); 2) mis-incorporation of dCTP opposite template-P to form C:P mismatch (Fig. 3c, lane 2); Conversion of Z:P pair to T:A pair (reverse mutation) involves two pathways: 1) the most likely pathway, mis-incorporation of dTTP opposite template-P to form T:P mismatch; 2) misincorporation of dATP opposite template-Z to form A:Z mismatch (Fig. 3c). See Figure 1 for corresponding matched and mismatched base pairs.

Figure 3. Mutation interconverting Z:P, C:G and T:A Pairs

a. Measuring the “forward” mutation converting C:G pairs into Z:P pairs using digestion with the Bsp120I restriction endonuclease. Standard oligonucleotide (Bsp-G, Table 1) containing the Bsp120I recognition sequence (5′-GGGCCC-3′) were 1000 fold amplified using Taq DNA polymerase at pH = 8.8 or 8.0 with standard dNTPs (0.2 mM of each) with or without dZTP and dPTP. Then, PCR amplicon were digested by endonuclease (Bsp120I). Lane 1: In the absence of both dZTP and dPTP; Lane 2: With dPTP (0.2 mM); Lane 3: With dZTP (0.2 mM); Lane 4: With both dZTP and dPTP (0.2 mM for each), Not digested: indicates the fraction of PCR product resisted endonuclease digestion; Digested: indicates the fraction of PCR product was digested. See Methods and Materials for PCR conditions. b. Measure of the “reverse” mutation of Z:P to give C:G and T:A using restriction endonuclease. Two complementary synthetic templates (Bsp-Z and Bsp-P, Table 1) containing 5′-GGGCCZ-3′ and 3′-CCCGGP-5′, were 1000 fold amplified using Taq with standard dNTPs (200 μM), dZTP and dPTP (with various concentration, lane 1 (20 μM), lane 2 (10 μM), lane 3 (5 μM)), then, PCR amplicon were digested by endonuclease (Bsp120I). See Methods and Materials for details. c. Measuring the mutation of Z into C and T (left) and P into A and G (right) using restriction endonuclease digestion. Single-stranded synthetic oligonucleotide containing either 5′-GGGCCZ-3′ (left, lane 1, Bsp-Z) or 3′-CCCGGP-5′ (right, lane 2, Bsp-P), was 1000 fold amplified using Taq with only four standard dNTPs (0.2 mM) in 1x ThermoPol reaction buffer (pH 8.8 at room temperature). Then, PCR amplicon was digested by endonuclease (Bsp120I). See Methods and Materials for details. d. Observed pathways of mutation between non-standard nucleotides and standard nucleotides. Conversion of C:G pair to Z:P pair (forward mutation) involves mis-incorporation of dZTP opposite template-G to form Z:G mismatch at high pH (8.8) (Fig. 3a); Conversion of Z:P pair to C:G pair (reverse mutation) involves two pathways: 1) the most likely pathway, mis-incorporation of dGTP opposite template-Z to form G:Z mismatch at high pH (8.8) (Fig. 3b); 2) mis-incorporation of dCTP opposite template-P to form C:P mismatch (Fig. 3c, lane 2); Conversion of Z:P pair to T:A pair (reverse mutation) involves two pathways: 1) the most likely pathway, mis-incorporation of dTTP opposite template-P to form T:P mismatch; 2) misincorporation of dATP opposite template-Z to form A:Z mismatch (Fig. 3c). See Figure 1 for corresponding matched and mismatched base pairs.

Figure 4. Measuring the retention and mutation of Z:P pair in optimized six-letter PCR

Standard template (Bsp-G, Table 1) and synthetic template (Bsp-P, Table 1) were amplified using Taq DNA polymerase under 1x Thermopol buffer (pH 8.0), followed by endonuclease digestion(Bsp120I). dA,T,G/TPs = 0.1 mM, dCTP = 0.4 mM, dZTP = 0.05 mM, and dPTP = 0.6 mM. Lane 1 and 2: Standard template was amplified 10⁴ fold using Taq, without (lane 1) and with (lane 2) dZTP and dPTP; Lane 3, 4, and 5: Synthetic template, 10³ (lane 3), 10⁴ (lane 4), and 10⁵ (lane 5) fold amplification, with both dZTP and dPTP; Not digested: indicates the fraction of PCR product retained Z:P pair, therefore, resisted endonuclease digestion; Digested: indicates the fraction of PCR product was digested. See Methods and Materials for details.

Figure 5. Strategy for sequencing GACTZP DNA

(a) The positions of Z and P in an amplicon are inferred by a process that converts Z:P pairs into a mixture of T:A pairs and C:G pairs, followed by standard Sanger sequencing. Comparison of the resulting sequences shows only T:A or C:G pairs at sites where T:A or C:G pairs were present in the initial amplicon, but mixtures of T:A and C:G pairs at sites where Z:P pairs were present in the initial amplicon. (b) Manipulation of the concentrations of dPTP without dZTP allows stepwise conversion of Z:P pairs into C:G pairs or into T:A pairs.