A Single Deoxynucleoside Kinase Variant from ... - ACS Publications

Letter pubs.acs.org/synthbio

A Single Deoxynucleoside Kinase Variant from Drosophila melanogaster Synthesizes Monophosphates of Nucleosides That Are Components of an Expanded Genetic System Mariko F. Matsuura,†,§ Christian B. Winiger,† Ryan W. Shaw,†,∥ Myong-Jung Kim,†,∥ Myong-Sang Kim,†,∥ Ashley B. Daugherty,⊥,∇ Fei Chen,†,# Patricia Moussatche,†,∥ Jennifer D. Moses,† Stefan Lutz,⊥ and Steven A. Benner*,†,∥ †

The Foundation for Applied Molecular Evolution (FfAME), 13709 Progress Blvd., Box 17, Alachua, Florida 32615, United States Department of Chemistry, University of Florida, Gainesville, Florida 32611, United States ∥ Firebird Biomolecular Sciences, LLC, 13709 Progress Blvd., Box 17, Alachua, Florida 32615, United States ⊥ Department of Chemistry, Emory University, 1515 Dickey Drive, Atlanta, Georgia 30322, United States §

S Supporting Information *

ABSTRACT: Deoxynucleoside kinase from D. melanogaster (DmdNK) has broad specificity; although it catalyzes the phosphorylation of natural pyrimidine more efficiently than natural purine nucleosides, it accepts all four 2′-deoxynucleosides and many analogues, using ATP as a phosphate donor to give the corresponding deoxynucleoside monophosphates. Here, we show that replacing a single amino acid (glutamine 81 by glutamate) in DmdNK creates a variant that also catalyzes the phosphorylation of nucleosides that form part of an artificially expanded genetic information system (AEGIS). By shuffling hydrogen bonding groups on the nucleobases, AEGIS adds potentially as many as four additional nucleobase pairs to the genetic “alphabet”. Specifically, we show that DmdNK Q81E creates the monophosphates from the AEGIS nucleosides dP, dZ, dX, and dK (respectively 2-amino-8-(1′-β-D-2′deoxyribofuranosyl)-imidazo[1,2-a]-1,3,5-triazin-4(8H)-one, dP; 6-amino-3-(1′-β-D-2′-deoxyribofuranosyl)-5-nitro-1H-pyridin2-one, dZ; 8-(1′β-D-2′-deoxy-ribofuranosyl)imidazo[1,2-a]-1,3,5-triazine-2(8H)-4(3H)-dione, dX; and 2,4-diamino-5-(1′-β-D-2′deoxyribofuranosyl)-pyrimidine, dK). Using a coupled enzyme assay, in vitro kinetic parameters were obtained for three of these nucleosides (dP, dX, and dK; the UV absorbance of dZ made it impossible to get its precise kinetic parameters). Thus, DmdNK Q81E appears to be a suitable enzyme to catalyze the first step in the biosynthesis of AEGIS 2′-deoxynucleoside triphosphates in vitro and, perhaps, in vivo, in a cell able to manage plasmids containing AEGIS DNA. KEYWORDS: enzyme engineering, artificially expanded genetic information system, nucleoside/nucleotide analogues, nucleotide biosynthesis, artificial metabolism that used a polymerase active site to enforce an “edge on” pair based on geometric complementarity. Lacking the directionality of internucleotide hydrogen bonding, the Romesberg pair intercalates when not constrained by a polymerase active site. Undoubtedly because of their considerably different molecular structure, the Romesberg group reported that it could not find ways to biosynthesize the triphosphates needed to replicate that single nucleotide pair. As a result, Romesberg placed a nucleoside triphosphate transporter into the cells, and fed triphosphates of their analogues to the cells in an environment rich in inorganic phosphate. The abundance of inorganic phosphate suppressed phosphatases sufficient to prevent the cell from destroying the triphosphates before they could be transported and used.

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ne of the more ambitious long-term goals that synthetic biology seeks is to construct living cells that have different biopolymers (DNA, RNA, and proteins) at the core of their molecular biology. In pursuit of this challenge, we developed a set of alternative nucleobase pairs that create a 12letter artificially expanded genetic information system (AEGIS)1−5 (Figure 1). Rather than removing hydrogen bonds and relying upon hydrophobic interactions to allow specificity of base pairing (an approach pioneered by the Kool, Hirao, and Romesberg groups6−8), AEGIS pairs are joined by alternative patterns of hydrogen bond donor and acceptor groups. Evidence for the interest in this challenge among scientists and within the public at large, was obtained in 2014 by the Romesberg group at The Scripps Research Institute. The group reported that a strain of E. coli could maintain within a plasmid a single exemplar of a nonstandard base pair for a day,9 a pair © XXXX American Chemical Society

Received: August 17, 2016

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DOI: 10.1021/acssynbio.6b00228 ACS Synth. Biol. XXXX, XXX, XXX−XXX

Letter

ACS Synthetic Biology

Figure 1. Structure of AEGIS nucleobases showing the hydrogen-bonding pattern, which differs from that of standard nucleotides. P, X, and K are highlighted in pink.

Figure 2. Planned biosynthetic pathway that will allow the replication of plasmids containing two AEGIS pairs, Z:P and K:X, a pathway that requires the kinase developed in this study to carry out the second step (indicated in red). When the pathway is complete, an E. coli cell will be fed Z, P, K, X nucleosides (dZ, dP, dK, dX), which will be transported into the cell, where they will be phosphorylated by kinases developed in this work to give the corresponding nucleoside monophosphates. These will then be carried forward to their corresponding diphosphates and triphosphates (dZTP, dPTP, dKTP, dXTP), the last being the substrates for polymerases that incorporate them into plasmid DNA containing ZP and/or KX base pairs. B = Z, P, K, and X.

Two of our unnatural nucleobase pairs have become especially well-developed. The first uses the hydrogen bonding complementarity of the small pyrimidine analogue 6-amino-3(1′-β-D-2′-deoxyribofuranosyl)-5-nitro-1H-pyridin-2-one (trivially called dZ), which presents a hydrogen bond donor− donor−acceptor pattern from the major to the minor groove and 2-amino-8-(1′-β-D-2′-deoxyribofuranosyl)-imidazo[1,2-a]1,3,5-triazin-4(8H)-one (trivially called dP), which presents a hydrogen bond acceptor-acceptor−donor pattern. DNA polymerases, RNA polymerases, and reverse transcriptases have all been developed to copy and transcribe DNA and RNA containing Z and P.11−13 Further, restriction enzymes, sequencing technology, and other tools taken for granted in classical molecular biology have been developed to exploit Z and P.11 Indeed, the Z:P system has been used to support in

Growing cells on media that contain nucleoside triphosphates is not a long-term solution to meet this “grand challenge” goal. Rather, a semi-synthetic cell must create unnatural nucleoside triphosphates from a less expensive and more stable “food”, just as the triphosphates of natural nucleosides are biosynthesized in many living cells in salvage pathways.10 To do this, the cell must have enzymes that phosphorylate unphosphorylated nucleosides taken up from the growth medium (or, more ambitiously, biosynthesized de novo within the cell), phosphorylate the resulting nucleoside monophosphates, phosphorylate the resulting nucleoside diphosphates to give triphosphates, the substrates for DNA polymerases. B


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ACS Synthetic Biology vitro evolution to create by GACTZP Darwinism aptamers33,34 and catalysts13 “on demand”. The second nucleobase pair uses the hydrogen bonding complementarity of the small pyrimidine analogue 2,4-diamino5-(1′-β-D-2′-deoxyribofuranosyl)-pyrimidine (trivially called dK, which presents a hydrogen bond donor−acceptor−donor pattern) and 8-(1′-β-D-2′-deoxyribofuranosyl)-imidazo[1,2-a]1,3,5-triazine-2(8H)-4(3H)-dione (dX, which presents a hydrogen bond acceptor−donor−acceptor pattern). This system is less well developed, although various polymerases and certain analytical tools have also been developed for this and certain of its analogues.14−16 These results make the Z:P pair and the K:X pair perhaps the best suited for use within living cells, if intracellular metabolism can be created that delivers their triphosphates by intracellular biosynthesis (Figure 2). As the first step in this metabolism, the development and characterization of a nucleoside kinase variant is reported that makes the monophosphates dP, dZ, dX, and dK from their corresponding nucleosides. To identify an enzyme that might phosphorylate these AEGIS nucleotides, we examined a deoxynucleoside kinase (EC 2.7.1.145) from D. melanogaster that, in its native form, accepts all four natural deoxynucleosides.17 It also accepts many nucleoside analogues.18 Further, the structure of this enzyme is available19 to support the generation of variants that might perform better with our nucleoside analogues. Unfortunately, this kinase in its native form did not accept the 2′-deoxyribo-AEGIS nucleosides well (dP: KM 122 ± 27 μM, kcat 15.4 ± 1.1 s−1, kcat/KM 1.26 × 105 M−1 s−1; dZ: KM > 790 μM, kcat > 0.27 s−1; dK: KM ∼ 500 μM see Figure S1). Therefore, we turned to literature reports that the substrate specificity of DmdNK might be altered by amino acid substitution.20 These variants have been used to accept nucleoside analogues that have therapeutic value.21 For example, Liu and co-workers showed that DmdNK variants with four to eight amino acid replacements have improved abilities to accept 3-deoxythymidine (ddT) and fluorescently tagged ddT, over natural dT, dC, dA, and dG).22,23 Other site-directed mutagenesis studies24 showed that a single replacement of glutamine at position 81 can change the specificity of the enzyme as well. For example, wild-type DmdNK has a preference for pyrimidine nucleosides. In contrast, the Q81N variant prefers purine (dG). However, Q81N variant did not accept AEGIS nucleosides well (dP: KM 434.6 ± 34.1 μM, kcat 12.54 ± 0.39 s−1, kcat/KM 29 × 103 M−1 s−1; dZ: KM > 285 μM, kcat > 0.04 s−1). Thus, following a screen, we examined more closely the Q81E variant to accept AEGIS nucleosides.

TLC Assay. TLC mobilities were assigned using authentic deoxyribonucleoside monophosphates. Kinase activity was assessed by following the appearance of 32P at the appropriate spot, using gamma-32P labeled ATP as a substrate. As seen in Figure 3, DmdNK Q81E phosphorylates all deoxynucleosides, with monophosphate formation detected

Figure 3. Image of a PEI-cellulose TLC plate showing the deoxyribonucleoside monophosphate products from samples without substrates (negative control), and samples with each substrate (GACTZPKX nucleosides) and DmdNK Q81E respectively. The plate is visualized by phosphorimager detecting P-32 transferred to the deoxyribonucleoside substrates from γ-labeled ATP. Here, with 30 min incubation and addition of sufficient enzymes, kinetic data cannot be extracted. However, this end point assay allows us to confidently identify the products themselves, something that is not to be taken for granted using the enzyme-coupled assay.

using a PEI-cellulose plate and 1 M acetic acid (pH adjusted to 3.5 with NH4OH) as a solvent. Monophosphate formation was observed for all substrates including Z, P, K, and X. Enzyme-Coupled Assay. DmdNK Q81E activity examined using an enzyme-coupled assay18 allowed us to estimate Michaelis−Menten kinetic parameters for three of the four AEGIS nucleosides (Figure 4, Table 1, Figure S2). In this assay,

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RESULTS AND DISCUSSION Expression and Purification of the Kinase Variant. To obtain the enzyme variant so that we can study its ability to phosphorylate the unnatural nucleoside substrates, expression of the transformed bacteria was induced as described in the Methods. SDS-PAGE applied to cells following expression and purification showed production of the desired enzyme with the expected size. Although the C-terminus of this enzyme is important for its catalytic activity and specificity,18 N-terminal truncation is known to not affect the activity of human thymidine kinase 225 (phylogenetically close to DmdNK35). Therefore, the activity of the DmdNK variant with the (His)6tag at the N-terminus was analyzed without removing the tag.

Figure 4. Rates of phosphorylation of each nucleoside substrate by the Q81E variant of DmdNK. The graph shows an absorbance change in samples containing DmdNK Q81E and its substrates at 340 nm, a change arising by the oxidation of NADH by lactate dehydrogenase acting on the pyruvate generated in the coupled enzyme assay by the phosphorylation of the substrate. The x-axis shows reaction time in minutes; the y-axis shows an absorbance at 340 nm. The reaction was initiated by incubating nucleoside substrate (500 μM, 1 mM for dG and dK due to their low kcat), and DmdNK (33 ng) in a reaction buffer (100 μL, 50 mM Tris-HCl (pH 7.5), 100 mM KCl, 2.5 mM MgCl2, 0.18 mM NADH, 0.21 mM PEP, 1 mM ATP, 1 mM DTT, 0.1 mg/mL BSA, 30 U pyruvate kinase and 33 U lactate dehydrogenase). In order of overall reaction rate (≈ kcat), dK ≈ dG < dT ≈ dC < dP ≈ dA ≈ dX. C


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ACS Synthetic Biology Table 1. Kinetic Parameters of DmdNK Q81E for Substrates kcat (s−1) dG dA dC dT dP dX dK

1.9 ± 0.6 5.7 ± 0.2 4.5 ± 1.3 4.5 ± 0.6 4.9 ± 0.2 18.7 ± 2.4 1.1 ± 0.06

kcat/KM (M−1 s−1)

KM (M) −3

pyrimidines19,27 (Figure 5A and B). In one orientation, “donor−acceptor” pattern of the amide interacts with the

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1.4 × 10 ± 7.2 × 10 3.9 × 10−6 ± 5.5 × 10−7 1.0 × 10−5 ± 5.5 × 10−6 5.8 × 10−6 ± 1.8 × 10−7 9.9 × 10−6 ± 1.3 × 10−6 1.7 × 10−4 ± 5.6 × 10−5 1.16 × 10−5 ± 2.7 × 10−6

1.4 1.5 4.4 7.8 5.0 1.1 9.5

× × × × × × ×

103 106 105 105 105 105 104

Experiments with dK and dX run by CBW, all others by MFM.

the phosphorylation of the nucleoside by ATP leads to the generation of ADP, which serves as a substrate for pyruvate kinase, which transfers a phosphate group from phosphoenolpyruvate to generate pyruvate. The resulting pyruvate is then reduced by lactate dehydrogenase, consuming NADH to cause a loss of absorbency at 340 nm. This assay proved to not provide precise values when dZ was the substrate, as its nitroamino heterocycle absorbs strongly at 340 nm, interfering with the absorption measurements required to measure the rate of oxidation of NADH. To assess whether products inhibited the reaction, the respective products were added up to 1600 μM; no inhibition was observed (Figure S3). Thus, for the most active substrates (where the reaction was followed up to ∼100% conversion), these results exclude the possibility that kinetic parameters were perturbed by product inhibition. These experiments found that the DmdNK Q81E variant accepts all of the four AEGIS nucleosides and, for the three mentioned above, with useful kinetic parameters. For example, for dP as a substrate, the Michaelis constant (KM) was ∼10 μM, the turnover rate (kcat) was 4.9 s−1, and the kcat/KM was 5.0 × 105 M−1 s−1. This is similar to the values obtained with the natural nucleosides. Thus, for kcat, the order of increasing activity is dK ≈ dG < dT/dC/dP/dA < dX (Table 1). These results showed that the Q81E mutation allowed the DmdNK to accept dZ, dP, dK, and dX as substrates. Kinetic parameters were similar for the AEGIS and standard nucleosides, with natural dG having the worst kcat/KM value. Excluding dG and dK, KM values range from 3.9 to 10 μM (dX: 170 μM), kcat values range from ∼4.5 to 18.7 s−1, and specificity constants (kcat/KM) range from 1.1 to 15 × 105 M−1 s−1. Therefore, this Q81E replacement effectively broadens the substrate specificity excluding dG and dK. dP and dX proved to be fairly good substrates compared with other unnatural nucleosides.26 Parallel experiments with the native DmdNK showed that dP, dZ, dK were not good substrates compared with natural nucleosides (dP: KM: 122 ± 27 μM, kcat: 15.4 ± 1.1 s−1, kcat/ KM: 1.26 × 105 M−1 s−1; dZ: KM: 790 μM, kcat: 0.27 s−1; dK: KM: ∼ 500 μM). Further, the kinase variant showed an increased activity for dA and dP compared to the wild-type enzyme, but not for dG. This result was structurally quite surprising. Although differing in the atoms at the purine ring positions 5 and 7, and having a different interstrand hydrogen bonding pattern, P otherwise resembles G. Perhaps naively, we might expect that a variant that accepts P better would also accept G better. Crystal structures are available for the kinase complexed to both dT (PDB ID: 1OT327) and dC (PDB ID: 2VP519), but not to dA or dG. Here, the glutamate interacts with the hydrogen bonding units on the Watson−Crick “edge” of these

Figure 5. Crystal structure of DmdNK wildtype complexed with dT (A; PDB ID: 1OT3), dC (B; PDB ID: 2VP5), and dGTP (C; PDB ID: 2VP2). Note the glutamine at position 81 can rotate to make two productive hydrogen bonding contacts with both of the pyrimidines. With the active site containing G, only a single hydrogen bond is made. Any interaction between the glutamine CO oxygen and nitrogen-7 of the purine is repulsive. As both dX and dP lack nitrogen7, we might speculate that they would be better substrates than G. Possible hydrogen bond acceptors and donors are colored in red and blue, respectively.

“acceptor−donor” pattern of dT; rotating the amide by 180° allows the “acceptor−donor” hydrogen bonding pattern of the glutamine amide to form two hydrogen bonds to the “donor− acceptor” of dC. For purine, the crystal structure that contains an inhibitor dGTP is available19 (PDB ID: 2VP2). It shows that the “donor” of the “donor−acceptor” pattern of the amide interacts with the carbonyl group of guanine. (Figure 5C) D


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available from Firebird Biomolecular Sciences LLC (firebirdbio.com). TLC Assays. Phosphoryl transfers were performed in a buffer (20 μL) containing 16.7 nM [γ-32P] ATP (Perkin Elmer, Waltham, MA, USA), 0.5 mM deoxynucleoside substrates (GACTZPKX), buffer (50 mM Tris-HCl (pH 7.6), 5 mM MgCl2, 100 mM KCl, 0.5 mg/mL BSA, 1 mM DTT), and 3.3 ng of DmdNK. The samples were incubated for 2 h at 37 °C then mixed rapidly with cold formic acid (11 M, 2 μL) to terminate the reaction. Inorganic phosphates were removed by adding a solution (2 μL) containing sodium tungstate (400 mM) tetraethylammonium chloride (500 mM), and procaineHCl (500 mM) in the ratio 5:4:1.31 The reaction mixture (2 μL) was spotted on polyethylenimine (PEI) cellulose F thin layer chromatography sheets (EMD Millipore, Billerica, MA, USA) and the nucleotides were separated in a buffer containing acetic acid (1 M, pH adjusted to 3.5 with NH4OH).32 The chromatograms were autoradiographed using phosphor imaging screens (BioRad, Hercules, CA, USA) and the TLC images were captured by a Personal Molecular Imager (PMI) System (BioRad). Each compound was confirmed by comparing spots with standards by UV shadowing. Enzyme-Coupled Assay. The activity of the enzyme was measured in a three-step assay described previously.18 This assay couples the phosphorylation of the nucleoside: production of ADP; the conversion of the ADP to ATP using pyruvate kinases and phosphoenolpyruvate; and reduction of the product pyruvate by lactate dehydrogenase in the presence of NADH to give NAD+. Loss of absorbance at 340 nm corresponding to oxidation of NADH was followed by spectroscopy. In detail, a mixture (250 μL) containing 50 mM Tris-HCl (pH 7.5), 100 mM KCl, 2.5 mM MgCl2, 0.18 mM NADH, 0.21 mM PEP, 1 mM ATP, 1 mM DTT, 0.1 mg/mL BSA, 30 U pyruvate kinase (Roche, Basel, Switzerland) and 33 U lactate dehydrogenase (Roche) was placed in a quartz cuvette. DmdNK Q81E (125−500 ng) was added, and the reaction was initiated by adding substrate (0.125−400 μM). The cuvette was placed in a DU 640 spectrophotometer (Beckman Coulter, Brea, CA, USA) and absorbance at 340 nm was monitored for 45 min at room temperature. Mixtures containing DmdNK Q81E or nucleoside substrate were used as negative controls. dG is not soluble in its solvent (DMSO) when its concentration exceeded 250 mM (solution that was added to the reaction mixture), a challenge because the KM for dG and dX proved to be higher than other substrates. Therefore, the assay for dG and dX was performed in a range of 50−1600 μM and 25−600 μM as a final concentration of the reaction mixture, respectively. This assay was also performed by using a spectrophotometer (SPECTROstar Omega, BMG LABTECH, Ortenberg, Germany) and 96 well plate (Nunc flat-bottom 96 well plate, Thermo Fisher Scientific, Waltham, MA, USA) to obtain Figure 4, Figure S1, and Figure S3. The components of the reaction mixture were the same as above, however, the volume of the reaction was 100 μL. Also, 33 or 160 ng of DmdNK was used, and the final concentrations of nucleoside/ tide were in a range of 125−2000 μM. Kinetic Parameters. Assays were run in triplicate (six runs for dK) and kinetic parameters were determined by fitting data to the Michaelis−Menten equation, using nonlinear regression analysis in SciDAVis (for natural nucleosides and dP; scidavis. sourceforge.net/index.html) and KaleidaGraph (for dK and dX; synergy.com/wordpress_650164087/kaleidagraph/).

According to the crystal structures, we hypothesize that Q81 interacts with position 3 and 4 of the pyrimidine nucleobases. Thus, the Q81E variant may be able to accept dZ because (i) glutamic acid has the same length of carbon chain and (ii) its “acceptor-acceptor” hydrogen-bonding pattern matches the “donor−donor” pattern of Z base. However, that hydrogen bonding pattern does not explain why the Q81E variant accepts dK, dP, and dX. If we assume that K, P, and X bases bind to Q81E in the same orientation as T/C or G (Figure 5A,B,C), hydrogen bonds would not be formed (or only one hydrogen bond would be formed for K), as glutamic acid does not provide hydrogen (“acceptor−acceptor”) although P and X will interact Q81E with its carbonyl group (acceptor). These problems would be solved if K, P, and X exist as their minor imino and enol forms, or if glutamic acid is not charged (“donor−acceptor”), which seems unlikely at pH 7. Nevertheless, the uncharged side chain would still not interact well with dZ although it would interact well with dK. Of course, it is also possible that dGTP-DmdNK complex does not resemble dG-DmdNK complex, and the hydrogen-bond interaction(s) with the purine occurs at different position(s) of purine bases. These considerations notwithstanding, this engineered kinase provides the enzymology needed for the first step for constructing a biosynthetic pathway to make the triphosphates of four AEGIS 2′-deoxynucleosides. We showed elsewhere that the endogenous Escherichia coli nucleoside diphosphate kinase (NDPK) catalyzes the third phosphorylation step to synthesize dZTP, dPTP, dKTP, and dXTP.28 Therefore, for the construction of an artificial metabolism in vitro, we only lack nucleoside monophosphate kinases (NMPK) able to phosphorylate dZMP, dPMP, dKMP and dXMP to give dZDP, dPDP, dKDP and dXDP to complete the pathway.

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METHODS All chemicals used in this study are from Sigma Aldrich (St. Louis, MO, USA) unless otherwise stated. Construction, Expression, and Purification of the Kinase Variant. A gene encoding the nucleoside kinase from Drosophila melanogaster with the Q81E amino acid replacement was previously constructed by the Lutz laboratory.22,23 The gene was ligated with a tag that encoded six consecutive histidines at the N-teminus, and placed behind a tetracycline promoter. Plasmid carrying the gene was transformed into Escherichia coli by electroporation, and expression was initiated with anhydrotetracyclin. The cells were incubated at 37 °C for 4 h, recovered by centrifugation, and lysed by sonication (20 s, 10 W, three times with 1 min rest intervals). The crude supernatant was recovered by centrifugation and applied to a cobalt column (HisPur Cobalt Resin, Thermo Fisher Scientific, Waltham, MA, USA). The column was washed with wash buffer (50 mM Tris-HCl, pH 7.5, 300 mM NaCl, 0.03% Igepal CA600, 10 mM imidazole), and the kinase was eluted with elution buffer (50 mM Tris-HCl, pH 7.5, 300 mM NaCl, 0.03% Igepal CA-600, 1 mM DTT, 200 mM imidazole). The size and purity of the isolated enzyme was confirmed by SDS-PAGE. Concentration of the enzyme was determined by the Bradford assay. Preparation of Unnatural Nucleosides. The AEGIS deoxyribonucleosides and their monophosphates were synthesized using the previously described methods28−30 with some modifications, and the routes shown in the schemes in Supporting Information (Scheme S1−S3). These are all E


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ASSOCIATED CONTENT

oxyribofuranosyl)-imidazo[1,2-a]-1,3,5-triazin-2(8H)-one; AEGIS, Artificially Expanded Genetic Information System.

S Supporting Information *

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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssynbio.6b00228. Figure S1: Enzyme-coupled assay: DmdNK WT + dK; Figure S2: Nonlinear regression analysis; Figure S3: Production inhibition assay; Scheme S1−S3: Synthesis of AEGIS nucleosides P, X, and K (PDF)

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REFERENCES

(1) Yang, Z., Hutter, D., Sheng, P., Sismour, A. M., and Benner, S. A. (2006) Artificially expanded genetic information system: a new base pair with an alternative hydrogen bonding pattern. Nucleic Acids Res. 34, 6095−6101. (2) Yang, Z., Sismour, A. M., Sheng, P., Puskar, N. L., and Benner, S. A. (2007) Enzymatic incorporation of a third nucleobase pair. Nucleic Acids Res. 35, 4238−4249. (3) Yang, Z., Chen, F., Chamberlin, S. G., and Benner, S. A. (2010) Expanded genetic alphabets in the polymerase chain reaction. Angew. Chem., Int. Ed. 49, 177−180. (4) Switzer, C. Y., Moroney, S. E., and Benner, S. A. (1993) Enzymatic recognition of the base pair between isocytidine and isoguanosine. Biochemistry 32, 10489−10496. (5) Benner, S. A. (2004) Understanding nucleic acids using synthetic chemistry. Acc. Chem. Res. 37, 784−797. (6) Kool, E. T. (2002) Replacing the nucleobases in DNA with designer molecules. Acc. Chem. Res. 35, 936−943. (7) Hirao, I., Harada, Y., Kimoto, M., Mitsui, T., Fujiwara, T., and Yokoyama, S. (2004) A two-unnatural-base-pair system toward the expansion of the genetic code. J. Am. Chem. Soc. 126, 13298−13305. (8) Henry, A. A., and Romesberg, F. E. (2003) Beyond A, C, G and T: augmenting nature’s alphabet. Curr. Opin. Chem. Biol. 7, 727−733. (9) Malyshev, D. A., Dhami, K., Lavergne, T., Chen, T., Dai, N., Foster, J. M., Corrêa, I. R., and Romesberg, F. E. (2014) A semisynthetic organism with an expanded genetic alphabet. Nature 509, 385−388. (10) Henderson, J. F., Robb, A., and Paterson, P. (2014) Nucleotide Metabolism: An Introduction, Academic Press. (11) Yang, Z., Chen, F. J., Alvarado, B. J., and Benner, S. A. (2011) Amplification, mutation, and sequencing of a six-letter synthetic genetic system. J. Am. Chem. Soc. 133, 15105−15112. (12) Leal, N. A., Kim, H. J., Hoshika, S., Kim, M. J., Carrigan, M. A., and Benner, S. A. (2014) Transcription, reverse transcription, and analysis of RNA containing artificial genetic components. ACS Synth. Biol. 17, 407−13. (13) Laos, R., Shaw, R. W., Leal, N. A., Gaucher, E., and Benner, S. A. (2013) Directed evolution of polymerases to accept nucleotides with nonstandard hydrogen bond patterns. Biochemistry 52, 5288−5294. (14) Sismour, A. M., Lutz, S., Park, J. H., Lutz, M. J., Boyer, P. L., Hughes, S. H., and Benner, S. A. (2004) PCR amplification of DNA containing non-standard base pairs by variants of reverse transcriptase from human immunodeficiency virus-1. Nucleic Acids Res. 32, 728− 735. (15) Laos, R., Thomson, J. M., and Benner, S. A. (2014) DNA polymerases engineered by directed evolution to incorporate nonstandard nucleotides. Front. Microbiol. 5, 00565. (16) Winiger, C. B., Kim, M. J., Hoshika, S., Shaw, R. W., Moses, J. D., Matsuura, M. F., Gerloff, D. L., and Benner, S. A. (2016) Polymerase interactions with wobble mismatches in synthetic genetic systems and their evolutionary implications. Biochemistry 55, 3847− 3850. (17) Munch-Petersen, B., Piskur, J., and Søndergaard, L. (1998) Four deoxynucleoside kinase activities from Drosophila melanogaster are contained within a single monomeric enzyme, a new multifunctional deoxynucleoside kinase. J. Biol. Chem. 273, 3926−3931. (18) Munch-Petersen, B., Knecht, W., Lenz, C., Søndergaard, L., and Pišk ur, J. (2000) Functional expression of a multisubstrate deoxyribonucleoside kinase from Drosophila melanogaster and its Cterminal deletion mutants. J. Biol. Chem. 275, 6673−6679. (19) Mikkelsen, E. N., Munch-Petersen, B., and Eklund, H. (2008) Structural studies of nucleoside analog and feedback inhibitor binding to Drosophila melanogaster multisubstrate deoxyribonucleoside kinase. FEBS J. 275, 2151−2160. (20) Knecht, W., Sandrini, PB. M., Johansson, K., Eklund, H., Munch-Petersen, B., and Piškur, J. (2002) A few amino acid

AUTHOR INFORMATION

Corresponding Author

*Tel.: 386-418-8085. E-mail: manuscript@ffame.org. ORCID

Mariko F. Matsuura: 0000-0002-2072-8141 Steven A. Benner: 0000-0002-3318-9917 Present Addresses #

CAS Key Laboratory of Genome Sciences and Information, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, China. ∇ Nephron Pharmaceuticals Corporation. West Columbia, South Carolina 29172, United States. Author Contributions

M.F.M., C.B.W., and R.W.S. designed the assays, M.F.M. and C.B.W. performed the assays. M.-J.K. and M.-S.K. synthesized AEGIS nucleosides and nucleotides. R.W.S. and S.A.B. supervised the project. A.B.D., F.C. and P.M. performed the preliminary kinase screening, and S.L. supervised the work at Emory. M.F.M. and S.A.B. wrote the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS MFM is indebted to Prof. Nicole Horenstein for advice concerning the kinetic experiments. CBW acknowledges a grant from the Swiss National Science Foundation. Grant No. P2BEP3_158965. This project/publication was made possible through the support under grants from the National Science Foundation (Grant No. MCB01412869), the Templeton World Charity Foundation, Inc. (0092/AB57), NASA (award Nos NNX14AK37G and NNX15AF46G), and the Defense Advanced Research Projects Agency Microsystems Technology Office (DSO) under contract CLIO: N66001-12-C-4019, ARPA Order No. 8657\00. The views and conclusions contained in this document are those of the authors and should not be interpreted as representing the official policies, either expressly or implied, of the U.S. Government, nor the Department of Defense, nor NASA, nor the NSF, nor the TWCF.

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ABBREVIATIONS DmdNK, nucleoside kinase from Drosophila melanogaster; Z, 6amino-3-(1′-β-D-2′-deoxyribofuranosyl)-5-nitro-1H-pyridin-2one; P, 2-amino-8-(1′-β-D-2′-deoxyribofuranosyl)-imidazo[1,2a]-1,3,5-triazin-4(8H)-one; K, 2,4-diamino-5-(1′-β-D-2′-deoxyribofuranosyl)-pyrimidine; X, 8-(1′-β-D-2′-deoxyribofuranosyl)imidazo[1,2-a]-1,3,5-triazine-2(8H)-4(3H)-dione; S, 2-amino1-(1′-β-D-2′-deoxyribofuranosyl)-5-methyl-pyrimidin-4(1H)one; B, 6-amino-1,9-dihydro-9-(1′-β-D-2′-deoxyribofuranosyl)3H-7-deazapurin-2-one; V, 2-amino-3-(1′-β-D-2′-deoxyribofuranosyl)-5-nitro-1H-pyridin-6-one; J, 4-amino-8-(1′-β-D-2′-deF


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