A Synthetic Biology Pathway to Nucleoside Triphosphates for Expanded Genetic Alphabets

Abstract

One horizon in synthetic biology seeks alternative forms of DNA that store, transcribe, and support evolution of biological information. Here, hydrogen bond donor and acceptor groups are rearranged within a Watson Crick geometry to get 12 nucleotides that form 6 independently replicating pairs. Such artificially expanded genetic information systems (AEGIS) support Darwinian evolution in vitro. To move AEGIS into living cells, metabolic pathways are next required to make AEGIS triphosphates economically from their nucleosides, eliminating the need to feed these expensive compounds in growth media. We report that “polyphosphate kinases” can be recruited for such pathways, working with natural diphosphate kinases and engineered nucleoside kinases. This pathway in vitro makes AEGIS triphosphates, including third generation triphosphates having improved ability to survive in living bacterial cells. In α-³²P labeled forms, produced here for the first time, they were used to study DNA polymerases, finding cases where third generation AEGIS triphosphates perform better with natural enzymes than natural triphosphates.

Graphical Abstract

Introduction

One horizon in synthetic biology¹ seeks non-standard forms of DNA and RNA that store, replicate, and transcribe information, reminiscent of these processes in natural DNA and RNA, but on different scaffolds. Alternative forms of DNA may store information more stably and at higher density, to be retrieved long after magnetically stored information has decayed.^2,3 Different nucleic acids are better than standard DNA/RNA to support “laboratory in vivo evolution” (LIVE), where nucleic acid libraries are challenged to evolve receptors, ligands, and catalysts under scientist-generated selective pressures.⁴

One strategy to approach this horizon retains Watson-Crick complementarity, both size and hydrogen bonding, in the synthetic genetic system. Fig. 1 shows a “second generation” form of a 12 letter genetic alphabet that has been developed in the Benner laboratory that does this.⁵ These Artificially Expanded Genetic Information Systems (AEGIS) support laboratory in vitro evolution,^{6 ,7,8, 9} surmounting limitations that have prevented LIVE in various forms from achieving its original promise.¹⁰

Figure 1.

Second generation components of an Artificially Expanded Genetic Information System (Aegis), now commercially available, ¹¹ were obtained by rearranging hydrogen bonding donor and acceptor groups on base pairs while retaining Watson-Crick size complementarity (large two ring systems pair with small one ring systems), and Watson-Crick hydrogen bonding complementarity (hydrogen bond donor groups pair with hydrogen bond acceptor groups). Polymerases have been developed to replicate Aegis,¹² allowing it to support laboratory in vitro evolution (LIVE);^5–8 Aegis-containing aptamers have potential in cancer therapy.¹³

In other forays into this genre of synthetic biology, Kool,¹⁴ Hirao,¹⁵ and Romesberg^16,17 have explored the versatility of size complementarity (Fig. 2). Kool has even created an eight-letter genetic system that supports duplex formation in vitro (Fig. 2).¹⁸ Romesberg has reported “semisynthetic” E. coli that replicates, transcribes, and translates DNA and RNA that contained size-complementary hydrophobic base pairs not joined by any hydrogen bonds.^{19, 20} Unfortunately, Romesberg was required to feed expensive triphosphates to E. coli growth medium, after placing in the bacterium a gene for a plastid triphosphate transporter^{21, 22}. This is likely to expensive for any but research use.

Figure 2.

Structures of various non-standard nucleobases used in various efforts to support synthetic biology that alters the biopolymers at the center of life.

(Top panel) Three examples of hydrophobic pairs that approximate the geometry of standard Watson-Crick pairs but lack inter-nucleotide hydrogen bonding.

(Middle panel) Eight-letter genetic system that retains inter-nucleotide hydrogen bonding complementarity, but with geometrically larger pairs.

(Bottom panel) AEGIS nucleobases examined here. Those with superscript “2” are second-generation AEGIS components that implement the indicated hydrogen bonding pattern (red H-bond acceptors; blue = hydrogen bond donors). Those with superscript “3” are third-generation AEGIS species with properties better suited for intracellular performance.

This exemplifies a general problem with all synthetic biology: Elements of natural biology are almost obligatorily recruited to work with unnatural synthetic parts. Thus, enzymes that “phosphorylate up” standard nucleotides inside of cells (Fig. 3) would deliver triphosphates less expensively. This, however, requires that the natural phosphorylating kinases directly, or in engineered form, accept non-standard nucleoside and nucleotide substrates.

Figure 3.

Enzymatic pathways to make nucleoside triphosphates (right, magenta) from nucleosides (left, blue). The standard pathway concept (center) adds single phosphate groups stepwise, one at a time, to convert a nucleoside to its nucleoside monophosphate, the nucleoside monophosphate to its nucleoside diphosphate, and the nucleoside diphosphate to its nucleoside triphosphate, with ATP providing the phosphate moiety at each step. It has proven difficult to find a natural nucleoside monophosphate kinase, or to engineer one, to accept triphosphates that have non-standard nucleobases (Figs. 1 and 2). Accordingly, polyphosphate kinases (green) were sought to convert monophosphates to di- and triphosphates using polyphosphate as a phosphate donor.

The more “alien” the nucleoside, the less likely that such recruitments might be possible. However, recruiting natural enzymes to phosphorylate even the structurally similar AEGIS nucleosides has been challenging. Many unnatural nucleosides are taken up from the growth medium by the NupC, NupG, and/or XapB (for xanthosine analog) transporter,²³ and AEGIS nucleotides appear to be no different. Further, the base substrate specificities of the endogenous E. coli nucleoside diphosphate kinases are sufficiently broad that they convert most second-generation AEGIS nucleoside diphosphates to their triphosphates.²⁴

However, mutation of the nucleoside kinase from Drosophila melanogaster was required to create an enzyme that converts various AEGIS nucleosides in Fig. 1 to their monophosphates.²⁵ It proved even more difficult to engineer nucleoside monophosphate kinases to convert AEGIS monophosphates to their diphosphates. Rational design by ROSETTA and semi-random screening failed to generate enzymes that converted AEGIS monophosphates to diphosphates.

Accordingly, we sought to engineer a different path to generate AEGIS nucleoside triphosphates from AEGIS nucleosides. This began by surveying enzymes that use polyphosphate, rather than ATP, as a phosphate donor, and act on nucleoside monophosphates. We asked whether such “polyphosphate kinases”²⁶ (named for the reverse reaction that they catalyze, (P_i)_n + ADP ➔ (P_i)_n+1 + AMP) might be more adaptable to accept AEGIS nucleotide monophosphates.

We report here experiments that show that polyphosphate kinases act on a series of second generation AEGIS nucleoside monophosphates. This, together with other natural and engineered kinases, allowed us to engineer an in vitro metabolic path that delivered AEGIS nucleoside triphosphates from AEGIS nucleosides, ATP, and polyphosphate (Fig. 3). This pathway was then used to solve an important problem in practical synthetic biology: making AEGIS triphosphates whose alpha phosphorus atoms are ³²P radiolabeled.

We then applied this pathway to “third generation” AEGIS structures engineered to manage various challenges in developing AEGIS for intracellular use. These included variations of X carrying an ethynyl (acetylene) group (Fig. 2). This third generation heterocycle implementing the large acceptor-donor-acceptor had been found, in DNA, to resist intracellular endonuclease V degradation, while still being transported by the NupG or XapB (Li et al. in preparation). This was extended to third generation version of P, also with an ethynyl group.

A third generation form of the Waston-Crick complement of X, a pyrimidine analog with a nitro group implementing a donor-acceptor-donor hydrogen bonding pattern, was designed (Fig. 2). This group is analogous to the nitro group found on AEGIS Z (Fig. 1) (Kim et al., in preparation). Interestingly, we were able to find cases where these third generation “alien” DNA components perform better with natural enzymes than natural DNA components.

Results and Discussion

Q81E variant of D. melanogaster nucleoside kinase accepts third generation AEGIS nucleosides.

In separate literature, we found that that a single amino acid replacement in a nucleoside kinase from D. melanogaster (Q81E) allowed this kinase to accept many first and second generation AEGIS nucleosides.²⁵ With new variants in development, we asked whether this mutant kinase would also accept third generation species. Data shown in Figure S1 shows that they do. Table 1 summarizes kinetic parameters of this kinase with various AEGIS nucleosides.

Table 1.

Summary of DmdNKE activities with selected AEGIS nucleosides

^*All values are means ± standard deviation from three repeats.

Interestingly, adding an ethynyl group to the puADA AEGIS component (converting X² to X³) improved its k_cat/K_M performance with the nucleoside kinase. A similar, also modest, improvement was seen when attaching an ethynyl unit to the puAAD AEGIS component. In contrast, the nitropyrimidone version of K, K³, was a 100 fold poorer substrate than second generation K² (Fig. 1).

Polyphosphate kinases phosphorylate AEGIS nucleoside monophosphates

We then asked whether polyphosphate kinases might convert a spectrum of AEGIS nucleoside monophosphates prepared by the Q81E mutant of nucleoside kinase to their diphosphates or triphosphates. Polyphosphate kinases come in various versions, based on (i) an evolutionary analysis of their natural history and (ii) the products that they produce from the substrates.²⁷ Standard nomenclature divides these into classes: Class I enzymes generate triphosphates from the diphosphates; Class II enzymes generate diphosphates from monophosphates; Class III enzymes generate mixtures of diphosphates and triphosphates from monophosphates.

Several kinases Class III were comparatively examined (Fig. S2), including those obtain from clones provided by the Matsuura lab.²⁸ This comparison found that the Chu PPK2 (Class III, from C. hutchinsonii) catalyzes the best conversion of most AEGIS ribo and deoxyribo nucleoside monophosphates to mixtures of diphosphates and triphosphates (Fig. S3). These AEGIS components included rZ²MP, rK²MP, rX²MP, dZ²MP, dK²MP, dX²MP, dP²MP, dK³MP, dX³MP, and dP³MP (Fig. 2). Similarly broad activity was seen with the Class III PPK2 from Matsuura lab and the Class III PPK2 from A. aurescens (Fig. S4).

Polyphosphate kinases support an in vitro path to get α-³²P-labeled AEGIS triphosphates

Table 2 summarizes data showing various product yields of triphosphate formed. Since polyphosphate is a heterogeneous substrate, and is also a product, of this enzyme, and since mixtures of products are formed in this easily reversible reaction, standard kinetic studies are unrevealing. However, the data were sufficient to select the preferred enzyme for most AEGIS monophosphates (Chu PPK2), and to use this enzyme to solve a long-standing problem in this branch of synthetic biology: How to make alpha radiolabeled AEGIS triphosphates.

Table 2.

Summary of PPK2 activities on AEGIS nucleoside monophosphates^*

	Substrate	DtCs PPK2	ChuPPK2	AaurPPK2
2nd generation ribonucleotide	rP2MP	di-: 49% tri: 3%	21% 70%	32% 61%
	rX2MP	di-: 53% tri: 10%	24% 71%	21% 51%
	rZ2MP	di: 32% tri: 43%	26% 59%	37% 55%
	rK2MP	di: 66% tri: 23%	19% 77%	19% 76%
2nd generation deoxy-ribonucleotide	dP2MP	di: 5% tri: 2%	8% 3%	3% 2%
	dX2MP	di: 21% tri: 3%	18% 32%	3% 6%
	dZ2MP	di: 17% tri: 26%	22% 58%	27% 24%
	dK2MP	di: 26% tri: 42%	19% 72%	13% 44%
3rd generation deoxy-ribonucleotide	dP3MP	di: 27% tri: 4%	19% 27%	19% 47%
	dX3MP	di: 16% tri: none	21% 27%	7% 5%
	dK3MP	di: 25% tri: 45%	19% 72%	13% 44%

^*By UV absorbance of the respective HPLC peaks. Addition of unreacted monophophate makes the sum 100%.

The pathway in Fig. 3 was implemented where the first step used γ-³²P-labeled ATP and the D. melanogaster nucleoside kinase Q81E mutant to make the AEGIS nucleoside monophosphate with a ³²P-label. Here, it was expedient to remove leftover excess γ-³²P-labeled ATP by treating with sodium periodate (NaIO₄). This reagent cleaves the ribose ring, but not the deoxyribose ring. Further with heat treatment at 85 °C for 10 min, both ATP and ADP were efficiently converted to adenosine dialdehyde (Fig. 4A); the single step caused the elimination of the phosphate groups. The products did not interfere with further phosphorylation reactions. Thus, nucleosides were smoothly converted to triphosphates, in one “pot”, by sequentially adding ATP and the nucleoside kinase, periodate, heating, and then adding polyphosphate (commercial hexametaphosphate) and polyphosphate kinase (Fig. 4B).

Figure 4.

Two alternative pathways to make AEGIS nucleoside triphosphates

A. Deoxynucleoside dP³ was converted into monophosphate by DmdNKE with ATP as the phosphate donor. Excess ATP and ADP were destroyed by NaIO₄ and heating. Blue arrows: substrates; red arrows: products. AdOx: adenosine dialdehyde. B. dP³MP was converted into dP³DP and dP³TP by incubation with ChuPPK2 with PolyP₆. Nucleotide products were confirmed by their UV spectra and retention time compared to authentic standards.

α-³²P-labeled AEGIS triphosphates made by this pathway were substrates for DNA polymerases

After the kinases were inactivated by heat, the α-³²P-labeled AEGIS triphosphates could be directly used without purification in primer extension reactions catalyzed by DNA polymerases. The following templates containing with 2K¹ and 5Z² (respectively 45 and 30 nucleotides long) were prepared by solid phase synthesis from AEGIS phosphoramidites (Firebird Biomolecular Sciences LLC): KK template: 5’-CAT GTC TGA TCC TGC ACT GCT GGK¹ K¹GG CCT TGA CTC TCG TAC CTG-3’; 5Z template: 5′-AGA GZ²Z² Z²CZ² Z²CC ACC ACA CGC TGC TCC GAC-3’.

Gel electrophoresis showed that α-³²P-labeled AEGIS nucleoside triphosphates gave full-length radiolabeled oligonucleotides with various DNA polymerases (Fig. 5A). Further after purification through ion-exchange HPLC, α-³²P labeled AEGIS triphosphates were confirmed by TLC analysis (Fig. 5B) and applied in primer extension assay (Fig. 5C).

Figure 5.

Analyses of α-³²P labeled triphosphates by polymerase incorporation and TLC.

A. Primer extension with unpurified reaction mixtures that contain α-³²P labeled dX³TP or dP³TP. Templates contain two K¹s or five Z²s and reactions were carried out with DNA polymerases Klenow fragment at 37 °C or KODexo- at 72 °C for 5min. Samples were resolved on 18% urea denature PAGE.

B. TLC analysis of HPLC purified α-³²P labeled AEGIS triphosphates. 10 nmol of unlabeled authentic dZ²TP, dK²TP, dP³TP and dX³TP alone, or mixed with 50-80 pmol of HPLC purified α-³²P-labeled (~1000 CPM) triphosphates was spotted on PEI cellulose plates and developed with 0.85M KH₂PO₄ pH 3.5 as the mobile phase. Both fluorescent images under UV exposure and phosphor images were recorded.

C. Primer extension assay with HPLC purified α-³²P labeled AEGIS triphosphates. Primer extension assay was carried out with Klenow large fragment at 37°C for 90s and samples were resolved on 18% PAGE with 7 M urea after mixed with 1 vol of 90% formamide and 20 mM EDTA. In left panel, lane 3, 4, 7, 8, 13 and 15 are products from 5’ ³²P -labeled primer and unlabeled AEGIS triphosphates, served as controls; lane 5, 6, 9,10, 14 and 16 are products from unlabeled primer and α-³²P labeled AEGIS triphosphates (marked with asterisk). P: 5’ ³²P -labeled primer only.

Competition assays experiments to measure the fidelity of incorporation

The α-³²P-labeled AEGIS triphosphates allowed us to do competition studies to compare second and third generation AEGIS deoxyribonucleoside triphosphates in polymerase extension reactions. With α-³²P-labeled AEGIS triphosphates, it is possible to compete hot and cold unlabeled material in symmetric pairs of reactions to measure infidelity when both matching and mismatching triphosphates are present. Fig. 6 shows an example of such experiments.

Figure 6.

Competition experiments used various α-³²P-labeled AEGIS triphosphates to examine the preference of the Klenow fragment of E. coli DNA polymerase I for second (Fig. 1) versus third generation (Fig. 2) heterocycles implementing the puADA or pyDAD hydrogen bonding pattern.

Primer extension assays were carried out with fixed amounts of α-³²P-labeled triphosphates and increasing amounts of unlabeled competitors. The full length ³²P-labeled products were resolved on denaturing PAGE and quantified by phosphor imaging and densitometry. The relative amounts of products were plotted against ratio of radiolabeled vs unlabeled triphosphates, where the intensity of full-length product without unlabeled competitors was taken as 100%. A. dX²TP vs dX³TP; B. dK²TP vs dK³TP.

Results in Fig. 6 show that the dX³TP third generation implementation of the puADA hydrogen bonding pattern is a better substrate for the Klenow fragment of DNA polymerase 1 than second generation dX²TP. This is unexpected, as the structure of dX²TP is more “natural” than dX³TP. Likewise, the dK³TP third generation implementation of the pyDAD hydrogen bonding pattern is a better substrate for this polymerase 1 than second generation dK²TP. This is also unexpected, as the structure of dK²TP seems more “natural” than dK³TP.

These results make available for the first time AEGIS nucleoside triphosphates where the α-phosphate is ³²P labeled. This in vitro pathway is a useful addition to the tools available to manipulate AEGIS, and it is made possible by the broad substrate specificity of the polyphosphate kinase. In particular, the labeled species are useful for analysis of the extent to which AEGIS nucleotides are incorporated into DNA, for example, inside of cells. This is applicable to the Romesberg’s strategy, where the triphosphate is imported from the growth medium by a triphosphate transporter to import it into the cell. Separately, data not reported here show that these second and third generation AEGIS triphosphates are also taken up into cells by the triphosphate importer originating from plastids and heterologously expressed in E. coli.

More importantly, this path offers a way of discarding the triphosphate importer architecture entirely when creating a semi-synthetic organism. Since polyphosphate is abundant as a phosphate storage molecule, it should be possible to simply feed nucleosides to such semi-synthetic organisms, have it enter the cell via nucleoside transporters, and be converted to triphosphates. These would then be available to replicate AEGIS DNA in plasmids and, eventually, chromosomes.

Methods

Chemicals, enzymes, and other reagents

If not mentioned elsewhere, chemicals were purchased from Sigma and enzymes for DNA manipulation such as restriction enzymes, DNA polymerases for PCR and T4 polynucleotide kinase for 5’ labeling of primers were purchased from New England Biolabs (NEB). AEGIS nucleosides, nucleotides, and oligos were purchased from Firebird Biomolecular Sciences (Alachua, FL). Primers and oligos with natural nucleotides (ATGC) were purchased from Integrated DNA technologies (IDT) (Coralville, Iowa). Plasmids and E.coli strains used in this studies are listed in Table S3.

Expression and purification of recombinant His-tagged PPK2s

Amino acid sequences of ChuPPK2 from Cytophaga hutchinsonii ATCC 33406 (ABG57400.1) and AaurPPK2 from Aenarthrobacter aurescens TC1(ABM08865.1)²⁶ were retrieved from NCBI and converted to their DNA coding sequences using E. coli optimized codons with the online program provided by IDT (Table S4). DNA molecules encoding the kinase proteins were synthesized as gBlocks by IDT and cloned into a pASK-IBA43plus vector (IBA Lifesciences, Goettingen, Germany) using In-Fusion HD cloning reagent (TaKaRa) following the manufactural instruction. The expressed proteins carried a 6xHis-tag fused to their C-termini respectively.

Both enzymes were expressed in Novablue (DE3) (Novagen) strain with 100 ng/mL of anhydrotetracycline (α-TC) induction in overnight cultures growing in LB media at 30 °C. Cells were lysed by sonication, and the supernatant following centrifugation was applied to TALON^® metal affinity resin (Clontech). Proteins were eluted in elution buffer (50mM Na-phosphate buffer pH 8.0, 300mM NaCl, 150mM imidazole, 0.1% triton X-100) and then buffer-exchanged into 2x enzyme storage buffer (40 mM Tris-HCl pH 7.5, 200 mM KCl, 2 mM DTT, 0.1% Tween-20) using Pierce^™ Protein Concentrators PES, 10K MWCO (ThermoFisher) to the concentration of imidazole was less than 1uM and the purity was checked by Coomassie blue stained SDS-PAGE. Different batches of final products were combined and quantified with QuickStart Bradford reagent (Bio-Rad) then stored in storage buffer (20 mM Tris-HCl pH 7.5, 100 mM KCl, 1 mM DTT, 0.05% Tween-20, 40% glycerol) at −20 °C.

For expression and purification of His-tagged DtCsPPK2 (Delftia tsuruhatensis), DtCsPPK2 coding region from a construct pET28-DtCsPPK2 kindly provided by Matsuura group²⁸ was PCR amplified and cloned into a home-constructed pRS vector derived from pASK-IBA43+. Protein expression and purification were followed the procedure described above.

Phylogenetic studies

The analysis involved a group of amino acid sequences of Class III PPK2 retrieved from NCBI based on previously listed in literature²⁷ with addition of recently characterized PPK2s.^26,28 Evolutionary analyses were conducted in MEGA11.²⁹ Multiple sequence alignment of listed PPK2s was generated with Clustal Omega and used to construct the phylogenetic tree with the UPGMA method. The bootstrap consensus tree inferred from 500 replicates is taken to represent the evolutionary history of the taxa analyzed. Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (500 replicates) are shown next to the branches. The evolutionary distances were computed using the JTT matrix-based method and are in the units of the number of amino acid substitutions per site. All ambiguous positions were removed for each sequence pair (pairwise deletion option). There was a total of 423 positions in the final dataset. The full list of PPK2s is included in table S2.

PPK2 kinase assay

In 20 μL reactions, 2 mM of NMP or dNMP was mixed with 1x PPK2 buffer (50 mM MOPs pH 7.0, 10 mM MnCl₂)¹⁸, 1 mM PolyP₆ (Sodium hexametaphosphate, Sigma), and 1 μM PPK2 on ice. After incubated at 37 °C for 90 min, the mixtures were diluted to 100 μL by adding 80 μL of sterilized water and enzymes were removed by centrifuge through Microcon^® 10KD-cut-off spin column (MilliporeSigma) at 14000x g for 20 min. The pass-through product (~90 μL) was transferred to a new tube and further centrifuged to move particulates at 17000x g for 10 min. 80 μL of aliquots of each sample was transferred to HPLC sample vials and 10 μL of samples was injected and resolved by ion exchange HPLC (Water 2695 separation module equipped with Water 2996 photodiode array detector and DNAPac^™ PA-100 BioLC 4 x 250 mm analytical column (ThermoFisher)), using water (A)/1M NH₄HCO₃ (B) as the mobile phase with the gradient (0-20min 0-20% B, 20-22min 20-50% B, 22-25min 50% B, 25-27min 0% B). The amounts of product (nucleoside diphosphate, triphosphate, etc.) were calculated from the overall integrated area of the resolved peaks, assuming that the extinction coefficient did not change as the level of phosphorylation changed.

Kinetics of DmdNKE kinase on AEGIS nucleosides

DmdNKE kinase activities on AEGIS nucleosides were examined using the pyruvate kinase and lactate dehydrogenase coupled-enzyme assay.³⁰ The kinase reaction was carried out in solution contained 50 mM Tris-HCl (pH 7.5), 100 mM KCl and 2.5 mM MgCl₂, 0.18 mM NADH, 0.21 mM phosphoenolpyruvate, 1 mM ATP, 1 mM 1,4-dithio-DL-threitol, 30 U/mL pyruvate kinase, 33 unit/mL lactate dehydrogenase and 250 nM of DmdNKE. Substrate nucleoside ranges in mM: 0, 0.05, 0.1, 0.2, 0.5, 1, 1.5, 2, 2.5, 3. Buffer components were prepped in 5x stock solution that filter sterilized with a 0.22 um PVDF filter. Substrate component stocks were stored at −20 °C, except for NADH which was stored at −80 °C. All components were prepared in a Master Mix excluding the nucleoside substrate. Reaction mixtures were then loaded into the 96 well plate with a multichannel pipette in succession. Reactions were performed in a 96 well plate (Axygen), in 30 μL volumes and read in the Omega reader at 340 nm to monitor the decrease in absorbance. For kinetic calculation, the fluorescent signal obtained was converted to velocity (V) and plotted against substrate concentrations [S]. The data were plotted as the Lineweaver -Burk Plot ($\frac{1}{V}=\frac{Km}{V\mathit{max}[S]}+\frac{1}{V\mathit{max}}$) to obtain Km and Vmax. Mean values from three replicates were used to calculate Kcat/Km.

DmdNKE kinase assay with AEGIS nucleosides were also analyzed with ion-exchange HPLC. In brief, in total 20 μL reaction, 1 mM of ATP was mixed with 0.8 mM of AEGIS nucleosides in 1x DmdNKE buffer, and 1 μM DmdNKE, incubated for 1 hr at 37 °C, followed by centrifuge through Microcon^® 10KD-cut-off spin column (MilliporeSigma) to remove kinases. The products were diluted with deionized water to 100 μL and 10 μL was injected into ThermoFisher UHPLC (Dionex ultimate 3000 system equipped with diode array detector and DNAPac^™ PA-100 BioLC 4 x 250 mm analytical column) and resolved with H2O (A): 1M NH4HCO3(B) gradient (0 – 1 min 0 - 0.5%B, 1 - 3 min 0.5-10% B, 3 - 10 min 10-30% B, 10 - 25 min 30-70% B, 25 - 30 min 70-80% B, 30 – 33 min 80-90% B, 33 – 36 min 0% B).

Synthesis of α-³²P labeled AEGIS nucleoside triphosphates in a coupled kinase pathway

The coupled kinase reactions were carried out in 30 μL volume. AEGIS nucleosides (0.8 mM final concentration), including dX², dX³, dP³, dK², dK³ and dZ², were individually mixed with 1x DmdNKE buffer 2 (50 mM Hepes-KOH, pH 7.5, 20 mM KCl, 1 mM DTT), 1 mM ATP, 3 μL of γ-³²P-ATP (6000 Ci/mmol,10mCi/mL, Perkin Elmer), and 1 μM of glycerol-free DmdNKE. The reaction mixture was incubated at 37 °C for 2 hr, followed by addition of 10 mM NaIO₄ and incubation at 25 °C for 10 min. After the excess of NaIO₄ was neutralized by addition of 20 mM pinacol at 25 °C for another 10 min, the reaction mixture was incubated at 85 °C for 10 min then cooled on ice. The second step of coupled reaction was continued by adding 1x PPK2 buffer, 0.5 mM of polyP₆, and 1 μM ChuPPK2, followed by incubation at 37 °C for 90 min. The final volume of the reaction mixture was 60 μL. The reactions were stopped by heating at 85 °C for 10min and the mixtures were centrifuged at 17000x g for 10 min. ~55 μL of supernatant was recovered, diluted to 80 μL by adding sterilized water, and manually injected into HPLC (Water 600) equipped with DNAPac^™ PA-100 BioLC 4 x 250 mm analytical column (ThermoFisher), 100 μL injection-loop and Dual λ UV detector (Water 2487). The fractions of deoxynucleoside triphosphates were collected, frozen in liquid nitrogen, and dried in speedVac for overnight. The dried samples were dissolved in 20 μL of sterilized water and the concentrations of purified nucleoside triphosphates were quantified using Nanodrop2000 (ThermoFisher) with their maximum UV absorbance, respectively. The total radioactivity of each purified nucleoside triphosphates was measured with scintillation counter (Tri-Carb 2800TR, Perkin Elem) using the Cherenkov method.

To monitor the yields of products in each step of reaction, the same set of reactions were carried out in parallel except no radio-labeled ATP was added. Aliquots (5 μL) of the mixtures was taken at the end of each kinase reaction and diluted to 50 μL with deionized water, followed by injecting 10 μL for HPLC analysis using ThermoFisher UHPLC equipped with the same column using the same setting of mobile phase gradient.

Primer extension assay

For regular primer extension assay with 5’-³²P labeled primers, reactions were carried out in 10 μL volume. 10 pmol of DNA templates was mixed with 7 pmol of the primer (supplemental table 1), 0.1 pmol of 5’-³²P labeled primer, 100 μM natural dNTP (ATGC), 100 μM of AEGIS deoxy nucleoside triphosphates and 1x KOD buffer (TOYOBO, Japan) or 1x Klenow buffer (NEB) on ice. The mixture was heated at 94 °C for 1 min and cooled to 25 °C at 0.1C/s to anneal primers and templates. The primer extension was initiated by adding 0.02 U of KODexo-DNA polymerases (TOYOBO, Japan) into prewarmed primer-template mixture at 72 °C or 0.1U of Klenow at 37 °C. After 1 min incubation, the reaction was stopped by addition of one volume of sample buffer (95% formamide, 20 mM EDTA, 0.05% bromophenol blue, and 0.05% Xylene cyanol FF). All samples were resolved on 18 or 20% 7M urea PAGE in 1xTBE buffer. Autoradiography was carried out by exposure of dried gels to the phosphor screen which was then scanned with Personal Molecular Imager (Bio-Rad).

For primer extension with α-³²P labeled AEGIS deoxynucleoside triphosphates, the assay was followed the procedure above except non-radiolabeled primers were used and the unlabeled AEGIS nucleoside triphosphates was replaced with 100 μM α-³²P labeled AEGIS deoxynucleoside triphosphates (0.1-0.2 μCi) purified from coupled kinase reactions.

For competition experiments, α-³²P labeled AEGIS deoxynucleoside triphosphates at fixed concentration 50 uM was mixed with variable concentrations of unlabeled competitor at 0, 50, 100, and 200 uM, and 50 uM of dNTPs (ATGC) in the assay. Primer extension reactions were carried out as mentioned above. The signals of full-length products were quantified from scanned phosphor images by densiometric analysis of bands with Quantity One software (Bio-Rad).