Compositions, methods, and cells for making 2-aminoadenine (DZ) DNA

Abstract

The present disclosure relates, according to some embodiments, to compositions, cells, methods and systems for producing dZ-containing DNA in vivo.

Description

[0001]PATENT APPLICATION NEB-491-PCT COMPOSITIONS, METHODS, AND CELLS FOR MAKING 2-AMINOADENINE (DZ) DNA SEQUENCE LISTING STATEMENT This disclosure includes a Sequence Listing submitted electronically in .xml format under the file name “NEB-491” created on September 28, 2023, and having a size of 31.7 KB. This Sequence Listing is incorporated herein in its entirety by this reference. INTRODUCTION In the biological arms race between bacterial hosts and phages, phages deploy DNA base modifications to counteract the host-encoded restriction systems. DNA base modifications can occur on four regular bases to become: modified cytosines (e.g. phage Xp12 with 5- methylcytosine (5mC), phage T4gt with 5-hydroxymethylcytosine (5hmC), and wild-type T4 with glucosylated (glc)-5hmC), modified guanine (e.g. phage 9g, deoxyarchaeosine (dG+)), modified thymidine phi W-14 with α-putrescinylthymidine (putT), phage Mu with carbamoylmethyl (glycinamide) group at the N6-position of adenine (abbreviated as 6- NcmdA), and cyanophage S-2L with 2-aminoadenine (base (d)Z) that can form three hydrogen bonds with thymidine. Phage genomes containing these hypermodified dZ bases are more resistant to Type II restrictions. Although a limited number of REases have been tested on dZ- modified phage DNA, these results may be obscured by the possibility of other modification systems encoded by the hosts when phage DNA replications occur. Bacterial hosts can harbor multiple R-M systems or orphan methylases (MTases), especially in some cyanobacteria where R-M systems and orphan M systems can number as high as 40-50. SUMMARY The present disclosure relates to cells, systems, apparatus, compositions, and / or methods for making and using DNA comprising dZ. In some embodiments, a microbial cell may comprise or may be modified to comprise one or more enzymes for 2-aminoadenine biosynthesis. For example, a microbial cell (e.g., an E. coli cell) may comprise at least one, at least two, or all three of (a) a phage 2-adenylosuccinate synthetase (PurZ), (b) a phage dATP triphosphohydrolase (MazZ), and (c) a phage (d)GTP-specific diphosphohydrolase (DatZ). A microbial cell (e.g., an E. coli cell) may comprise a DNA polymerase or a reverse transcriptase, for example, capable of incorporating dZ into DNA. A microbial cell may comprise at least one, at least two, at least three or all four of (a) a phage 2-adenylosuccinate synthetase (PurZ), (b) a phage dATP triphosphohydrolase (MazZ), (c) a phage (d)GTP-specific PATENT APPLICATION NEB-491-PCT diphosphohydrolase (DatZ), and (d) a DNA polymerase or a reverse transcriptase. In some embodiments, a microbial cell (e.g., an E. coli cell) may comprise a plasmid. A microbial cell (e.g., an E. coli cell) may comprise, in some embodiments, an expressible purZ gene encoding the phage adenylosuccinate synthetase, an expressible mazZ gene encoding the phage dATP triphosphohydrolase; and / or an expressible datZ gene encoding the phage (d)GTP-specific diphosphohydrolase, one or more of which may be present on a plasmid in the microbial cell. In some embodiments, a microbial cell (e.g., an E. coli cell) may comprise an expressible phage dpoZ gene encoding a DNA polymerase. In some embodiments, a microbial cell (e.g., an E. coli cell) may comprise a polynucleotide template encoding a heterologous (e.g., with respect to the genome of the host cell) sequence of interest. For example, a microbial cell (e.g., an E. coli cell) may comprise a nucleic acid template to be copied into dZ DNA, which copy comprises a sequence encoding an RNA and / or protein of interest. In some embodiments, a microbial cell (e.g., an E. coli cell) may comprise one or more molecules of DNA having a dZ content of 0% - 60%. (e.g., 0% - 60% of all bases in the DNA molecule are dZ or 0% to 60% of all dA positions are dZ, in each case, where 0% means at least one base is dZ). In some embodiments, one or more of the molecules comprising dZ is a plasmid DNA molecule. In some embodiments, a microbial cell (e.g., an E. coli cell) may comprise at least one transcription product of at least one of the dZ- containing DNA molecules. The present disclosure further relates to DNA molecule (e.g., comprised in a microbial cell) that are resistant and / or sensitive to one or more type II restriction endonucleases. For example, a DNA molecule (e.g., present in a microbial cell or cell lysate) may comprise a recognition site for a type II restriction enzyme that is effective to cut such DNA if it is free of dZ but not effective to cut such DNA if it comprises dZ. For example, a DNA molecule (e.g., present in a microbial cell or cell lysate) may comprise a recognition site for a type II restriction enzyme that is effective to cut such DNA without regard to whether the DNA comprises dZ. In some embodiments, a dZ DNA molecule may comprise one or more recognition sites for one or more type II restriction endonucleases that will not cut such dZ DNA and one or more recognition sites for one or more type II restriction endonucleases that will cut such dZ DNA molecule. For example, a dZ-containing DNA molecule may comprise (a) a recognition site for a type II restriction endonuclease selected from the group consisting of AatII, AccI, AfeI, ApaLI, AseI, AvaII, BamHI-HF, BbsI, BciVI, BfaI, BfuAI, BpmI, BsaWI, BsgI, BsiHKAI, BspDI, BspEI, BspHI, BstZ17I, BtsIMUTI, ClaI, DpnII, DraI, EarI, EcoNI, EcoP15I, FatI, PATENT APPLICATION NEB-491-PCT HindIII-HF, HinfI, MboI, MscI, NdeI, NheI-HF, NruI-HF, PciI, PflMI, PpuMI, PstI-HF, SphI- HF, SspI-HF, StyI-HF, TfiI, TspRI, and XmnI or (b) a recognition site for a type II restriction endonuclease selected from the group consisting of AflIII, AhdI, AluI, AlwI, AlwNI, ApeKI, ApoI, AvaI, BanI, BbvI, BccI, BceAI, BcoDI, BmrI, BsaHI, BsaI-V2, BseYI, BsiEI, BsmAI, BsmFI, BsoBI, Bsp1286I, BspCNI, BsrBI, BsrFI-V2, BsrI, BstAPI, BstYI, BtgI, BtsCI, CviAII, DrdI, EaeI, EciI, EcoO109I, FokI, HaeII, HgaI, HphI, Hpy166II, Hpy188I, Hpy188III, HpyAV, HpyCH4III, HpyCH4IV, HpyCH4V, MboII, MlyI, MnlI, MseI, MslI, MspA1I, NlaIII, PflFI, PleI, PshAI, PspGI, RsaI, SalI-HF, SfaNI, SfcI, SmlI, TaqI, TseI, Tsp45I, and Tth111I, or (c) both (i) a recognition site for a type II restriction endonuclease selected from the group consisting of AatII, AccI, AfeI, ApaLI, AseI, AvaII, BamHI-HF, BbsI, BciVI, BfaI, BfuAI, BpmI, BsaWI, BsgI, BsiHKAI, BspDI, BspEI, BspHI, BstZ17I, BtsIMUTI, ClaI, DpnII, DraI, EarI, EcoNI, EcoP15I, FatI, HindIII-HF, HinfI, MboI, MscI, NdeI, NheI-HF, NruI-HF, PciI, PflMI, PpuMI, PstI-HF, SphI-HF, SspI-HF, StyI-HF, TfiI, TspRI, and XmnI , and (ii) a recognition site for a type II restriction endonuclease selected from the group consisting of AflIII, AhdI, AluI, AlwI, AlwNI, ApeKI, ApoI, AvaI, BanI, BbvI, BccI, BceAI, BcoDI, BmrI, BsaHI, BsaI-V2, BseYI, BsiEI, BsmAI, BsmFI, BsoBI, Bsp1286I, BspCNI, BsrBI, BsrFI-V2, BsrI, BstAPI, BstYI, BtgI, BtsCI, CviAII, DrdI, EaeI, EciI, EcoO109I, FokI, HaeII, HgaI, HphI, Hpy166II, Hpy188I, Hpy188III, HpyAV, HpyCH4III, HpyCH4IV, HpyCH4V, MboII, MlyI, MnlI, MseI, MslI, MspA1I, NlaIII, PflFI, PleI, PshAI, PspGI, RsaI, SalI-HF, SfaNI, SfcI, SmlI, TaqI, TseI, Tsp45I, and Tth111I. The present disclosure further relates to lysates of any of the microbial cells disclosed herein. Such lysates may have any desired degree of processing including, for example, crude lysates and fractionated lysates (e.g., supernatant). The present disclosure also relates, according to some embodiments, to methods for making dZ- containing DNA. For example, a method may comprise contacting any of the disclosed microbial cells (e.g., E. coli cells) or any of the disclose cell lysates (e.g., E. coli cell lysates) with a polynucleotide template exogenous to the E. coli cell or exogenous to the lysate, wherein the cell or the cell lysate comprises one or more dNTPs, at least one of which is dZTP, and one or more DNA polymerases and / or one or more reverse transcriptases to produce a dZ-containing DNA. In some embodiments, a method may comprise contacting a dZ-containing DNA and an exonuclease or a type II restriction endonuclease (e.g., any of the restriction endonucleases listed in Table 2, for example, enzymes marked as “R”, marked as “MR” marked as “PR” or marked as or “S”). PATENT APPLICATION NEB-491-PCT BRIEF DESCRIPTION OF THE FIGURES FIGURE 1A shows an example dZTP synthetic scheme. FIGURE 1B shows an example molecular mass determination by ESI-HRMS of synthesized dZTP. HRMS [ESI-MS] calc. for C10H16N6O12P3-, [M-H]-: m / z 505.0045, found m / z 505.0040. FIGURE 2 shows an example LCMS base composition analysis after nucleoside digestion mix treatment of dZ-containing PCR DNA and regular PCR DNA. Two samples dZ-containing PCR DNA (marked “dZ) and two samples of regular PCR DNA (marked “dA”) were analyzed, which showed nearly identical results. FIGURE 3A and FIGURE 3B show results of example restriction digests of modified (dZ-containing DNA) and unmodified DNA (without dZ). dZ-containing PCR DNA (~0.3 ^g) was digested by REases (15 U to 20 U) in 30 ^l reaction in the recommended restriction buffer and temperature. Substrates and cleavage products were analyzed in 1% agarose gel by electrophoresis. REases used in digestions: 1) ApoI (RAATTY), 2) BstYI (RGATCY), 3) HindIII (AAGCTT), 4) BmtI (GCTAGC), 5) AatII (GACGTC), 6) BamHI (GGATCC), 7) BsaI-HF v2 (GGTCTC), 8) ClaI (ATCGAT), 9) EcoRI (GAATTC), 10) EcoRV (GATATC), 11) NheI (GCTAGC), 12) NruI (TCGCGA), 13) PstI-HF (CTGCAG), 14) PvuI-HF (CTGCAG), 15) SalI (GTCGAC), 16) ScaI-HF (AGTACT), 17) SphI-HF (GCATGC), 18) SspI (AATATT), 19) uncut PCR DNA, 20) 1 kb plus (2 log) DNA ladder (0.1 – 10 kb, NEB). In dZ-modified PCR DNA, most of adenine bases have been substituted by dZ (98%). FIGURE 3A shows results of example restriction digests of Z-modified PCR DNA (3 kb). FIGURE 3B shows results of example restriction digests of dA PCR DNA (3 kb). FIGURE 4A shows an example dZ base composition analysis of 4-gene plasmids (purZ, mazZ, datZ, and phage DNA polymerase gene dpoZ) extracted from E. coli following IPTG induction at 25oC overnight. Mini-prep plasmid DNA (1 ^g) of 12 samples along with a control was treated by nucleoside digestion mix and RNase A overnight at 37oC and subjected to LCMS base composition analysis. The minor peaks are RNA degradation products (nucleosides). FIGURE 4B shows an example bar graph of dZ level in 12 samples derived from IPTG-induced plasmids expressing four genes (purZ, mazZ, datZ, dpoZ). Same results are shown in Table 2 as numbers. The control plasmid is the empty vector pET21b which does not contain any dZ bases. PATENT APPLICATION NEB-491-PCT FIGURE 5A and FIGURE 5B show example RecBCD and phage T5 exonuclease digests of dZ-modified and unmodified PCR DNA in a time course (1 to 30 min). FIGURE 5A shows exonuclease digestion of dZ-modified PCR DNA (3 kb). RecBCD (5 U) and ATP (1 mM) was incubated with 0.35 ^g of PCR DNA at the indicated time and the reaction was terminated by addition of gel loading buffer with 0.1% SDS. Phage T5 exonuclease (2 U) was incubated with 0.35 ^g of PCR DNA at the indicated time in 1x CutSmart buffer. FIGURE 5B shows exonuclease digestion of unmodified PCR DNA (3 kb) under the same condition. Same enzyme units and amount of DNA were used in the digests. MluCI (AATT) and HpaII (CCGG) were used as controls. dZ modification affects MluCI digestion (mostly resistant) and did not impact HpaII digestion. FIGURE 6 shows an example LCMS base composition analysis of IPTG-induced or non-induced plasmids containing 3, 4, and 6 genes for dZ production in E. coli cells. The percentage of dZ incorporation is shown in Table 3 as calculated from this original peak data. Samples 1-6 (+IPTG), 4 genes, pET21b-purZ-mazZ-datZ-dpoZ. Samples 7-9 (+IPTG), 3 genes, pET21b-purZ-mazZ-datZ. Samples 10-12, 6 genes, pET21b-purZ-mazZ-datZ and pACYCT7-DNA helicase-SSB-dpoZ. Sample 13, pBR322 (negative control). Samples 14-16 (no IPTG), 3 genes, pET21b-purZ-mazZ-datZ. FIGURE 7 shows dZ levels in example expression plasmids with 3-6 genes involved in dZ biosynthesis. dZ cluster = purZ-mazZ-datZ.6-genes plasmids = pET21b-purZ-mazZ- datZ and pACYCT7-DNA helicase-SSB-dpoZ. The original LCMS base composition peak data is shown in Suppl. Fig. S1. FIGURE 8 shows dZ levels in example expression plasmids with 3-6 genes involved in dZ biosynthesis, including two novel DpoZ homolog DNA polymerase discovered from a phage metagenome study performed in-house at NEB. dZ cluster = purZ-mazZ-datZ; dZ + SpDpoZ = purZ-mazZ-datZ + Salmonella DpoZ; dZ + VpDpoZ = purZ-mazZ-datZ + Vibrio phage phiVC8 DpoZ; dZ + phiDpoZ1 = purZ-mazZ-datZ + WY DNA polymerase 1; dZ+ phiDpoZ2 = purZ-mazZ-datZ + WY DNA polymerase 2; dZ + SpDpoZ-helicase-ssb = pET21b- purZ-mazZ-datZ + pACYCT7-DNA helicase-SSB-Salmonella DpoZ. N=8 from 2 biological experiment replicates. Statistical analysis performed with t-test. ns: p value not significant; *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001. BRIEF DESCRIPTION OF THE SEQUENCES Some embodiments of this disclosure relate to the following provided sequences of example polynucleotides and / or example polypeptides. PATENT APPLICATION NEB-491-PCT SEQ ID NO:1 is an example amino acid sequence of a Salmonella phage PMBT28 adenylosuccinate synthetase. SEQ ID NO:2 is an example amino acid sequence of a Salmonella phage PMBT28 nucleotide pyrophosphohydrolase. SEQ ID NO:3 is an example amino acid sequence of a Salmonella phage PMBT28 hydrolase. SEQ ID NO:4 is an example nucleotide acid sequence of a Salmonella phage PMBT28 comprising 3 genes encoding PurZ (nt 28-970), MazZ (nt 971-1810), DatZ (nt 1810-2639)(nt 1-27 and nt 2513-2639 = vector sequence for cloning / assembly into pET21b vector; nt 1-27, nt 941-970, and nt 1781-1810 = vector sequence for cloning / assembly into pET21b vector). SEQ ID NO:5 is an example nucleotide acid sequence of a Salmonella phage PMBT28 comprising a T7 promoter and 4 genes (5’-T7 promoter- purZ-mazZ-datZ-dpoZ-3’), the last gene encoding Salmonella DNA polymerase (start ATG at nt 41-43 and stop TGA at 1916-1918; nt 1- 28 and 1919-1945 = required for assembly into pET21b vector). SEQ ID NO:6 is an example nucleotide acid sequence of a Salmonella phage PMBT28 encoding an ATP-dependent helicase (helicase; nt 56-1533)-dpoZ (DNA pol; nt 1534-3414)-ssDNA binding protein (SSB; nt 3488-3829) (respective start ATG at nt 56-58, 1534-1536, and 3488-3490 and respective stop TGA at nt 1534-1536 and 3409-3414). SEQ ID NO:7 is an example nucleotide acid sequence of a co-transformation construct encoding an ATP-dependent helicase (28-1,000) dpoZ (DNA pol, 1,001-1,970) SSB (1,971-2,940) (NT 1-27, 971-1000, 1941-1970, 2911-2940, and 3802-3831 = sequences for cloning / assembly into vector and among the gene blocks; 25-30 and 3802-3807 = restriction sites for cloning and assembly). SEQ ID NO:8 is an amino acid sequence of an example of DNA pol genes enriched from meta-phage genomes. SEQ ID NO:9 is an example nucleotide acid sequence of WY DNA polymerase 1 (coding sequence nt 38-1,891) (nt 1-1000 = gene block 1; nt 971-1000 = overlapping sequence between gene blocks). SEQ ID NO:10 is an example amino acid sequence of WY DNA polymerase 2. SEQ ID NO:11 is an example nucleotide sequence encoding WY DNA polymerase 2 (coding sequence nt 41-1,852) (nt 1-1000 = gene block 1 sequence; nt 971-1000 = for assembly of blocks 1 and 2). PATENT APPLICATION NEB-491-PCT SEQ ID NO:12 is an example amino acid sequence of a Vibrio phage phiVC8 DNA polymerase I – 3’-5’ exonuclease. SEQ ID NO:13 is an example nucleotide acid sequence encoding Vibrio phage phiVC8 DNA polymerase I – 3’-5’ exonuclease (nt 1-1000 = gene block 1; nt 1001-1940 = gene block; nt 41-43 = ATG start codon and, following a 6xHis tag, nt 1964-1966 TGA stop codon). DETAILED DESCRIPTION The present disclosure relates, in some embodiments, modified DNA molecules with improved resistance to cleavage by one or more restriction enzymes and compositions, kits, methods, cells, and systems for making and using such DNA molecules. It may be advantageous to make DNA that has a higher melting temperature and / or that is more resistant to cleavage by nucleases (e.g., Type I restriction endonucleases, Type II restriction endonucleases, Type III restriction endonucleases, Type IV restriction endonucleases, and / or exonucleases). For example, a therapeutic DNA comprising dZ DNA may be easier to formulate, transport, and / or deliver to a subject and / or may have a longer half-life in a subject’s cells and / or tissues. The modified DNA base 2,6 diaminopurine (2-aminoadenine, (d)Z base) was originally found in phages to counteract host encoded restriction systems. Only a limited number of restriction endonucleases (REases) have been tested on dZ-modified DNA. Cleavage activity results are reported here for 147 restriction enzymes on dZ-modified PCR DNA. Among the enzymes tested, 53.1% are resistant or partially resistant, and 46.9% are sensitive when the restriction sites contain 1 to 6 modified dZ bases. Sites with 4-6 dZ substitutions are most likely resistant to Type II restriction. dZ-modified DNA can also “slow down” phage T5 exonuclease degradation, but it has no difference in RecBCD digestion. When two genes for dZ biosynthesis and one gene for dATP hydrolysis from Salmonella phage PMBT28 (purZ (adenylosuccinate synthetase), datZ (dATP triphosphohydrolase), and mazZ ((d)GTP-specific diphosphohydrolase) were cloned into E. coli plasmid, dZ incorporation level reached 19-20% dZ / (dZ+dA). dZ level can be further increased to 28.9-44.3% with co-expression of a DNA polymerase gene from the same phage. High level of dZ incorporation in recombinant plasmid is possible by co-expression of purZ, mazZ, datZ and a phage DNA polymerase gene dpoZ. This work has a general interest for molecular biologists working on dZ DNA modification and restriction systems. It provides a foundation for future research on screening dZ-dependent Type IV restriction systems. The results presented herein may have implication in gene therapy PATENT APPLICATION NEB-491-PCT utilizing dZ-modified DNA, provided that human RNA polymerase variants can efficiently perform transcription from a dZ-modified template. In this work, PCR DNA substrates amplified in vitro that lack all other types of base modification except dZ were prepared using pure dZTP / dCTP / dGTP / dTTP. dZTP was prepared by chemical synthesis and used in PCR amplification to produce dZ-containing DNA for restriction digests. In addition, exonuclease digests of the dZ-modified DNA were examined to see any slow-down in degradation. A few exonucleases are less efficient in digestion of dZ-modified substrate which may have implication in DNA stability in practical applications such as gene therapy, gene targeting, and DNA vaccine. It has been shown herein that by expression of three phage genes in E. coli (e.g., purZ, datZ, mazZ), dZ-containing plasmid DNA can be generated with, for example, up to 20% dZ replacing adenine. The dZ level in dZ-containing plasmids was enhanced so the plasmids could be used more effectively to screen for dZ-dependent restriction endonucleases (REases). dZ- containing plasmids may be useful in certain cost-sensitive therapeutic applications, production of such modified DNA in E. coli. To increase dZ level in E. coli plasmid, three genes from Salmonella phage PMBT28 (purZ (2-adenylosuccinate synthetase), datZ (dATP triphosphohydrolase, dATPase), and mazZ ((d)GTP-specific diphosphohydrolase) were cloned into a T7 expression vector pET21b. Since the codon usage of Salmonella is more compatible with E. coli expression systems, the dZ level may be improved by expression under IPTG induction. In addition, the DNA polymerase gene from Salmonella phage PMBT28 was cloned in the same plasmid containing dZ modification genes, or co-expressed in a compatible plasmid to further increase dZ incorporation. Co-expression of six phage genes in E. coli was also attempted to enhance dZ base incorporation. When modified DNA enters E. coli cells, it may encounter modification-dependent restriction systems such as McrBC (RglBC), Mrr, and McrA, GmrSD, SauUSI, DpnI and winged helix fusion endonucleases or other EVE-HNH domain containing endonucleases. Therefore, in this work the transformation efficiency of dZ-modified pUC19 DNA into commonly used E. coli lab strains, particularly Mrr+ strains, that are involved in restriction of modified adenine in DNA was examined. The present disclosure provides, in some embodiments, dZ DNA-producing cells. A dZ DNA-producing cell may include, for example, an E. coli cell comprising at least one, at least two, at least three or all four of PurZ, MazZ, DatZ, and Salmonella DNA polymerase. A dZ DNA-producing cell (e.g., a dZ DNA-producing E. coli cell) may comprise one or more PATENT APPLICATION NEB-491-PCT dZ-containing DNA molecules having a dZ content of 0% - 60% (where, in context, 0% refers to trace amounts of dZ (e.g., ≥0.001, ≥0.01, ≥0.1, or ≥1 dZ per 106bases)). A dZ DNA- producing cell (e.g., a dZ DNA-producing E. coli cell) may comprise (may further comprise) at least one transcription product of at least one of the dZ-containing DNA molecules. According to some embodiments and examples disclosed herein, dZ-modified PCR DNA may be resistant or partially resistant to one or more restriction endonucleases (e.g., 53% of REases examined) and may be useful for fighting against host-encoded restriction systems. Another feature of dZ-containing phages is phage-encoded DNA polymerase (PolI family) or primase-polymerase fusion (Pri-pol family) to efficiently replicate phage DNA from a nucleotide pool with dZTP. Co-expression of PurZ, MazZ, and DatZ with a Salmonella DNA polymerase in E. coli, achieved 29% to 40% dZ incorporation in plasmids under IPTG induction. While DNA comprising dZ is not digested by T5 exonuclease as rapidly as DNA without dZ DNA, DNA comprising dZ can be degraded equally well by ATP-dependent exonuclease RecBCD as regular DNA. In some embodiments, cells comprising a 3-gene containing plasmid (pET21b-purZ- mazZ-datZ) may incorporate dZ bases in plasmid DNA at a rate of ~20%, which is higher than the three gene cluster cloned from cyanophage S-2L (7%-14%). Without being limited to any mechanism of action, this may relate to Salmonella phage genes being more efficiently expressed in E. coli (since the original Salmonella phage DNA sequences were used in gene block synthesis). Mrr is a Type IV REase in E. coli that restricts 6mA- and 5mC-modified plasmid and phage DNA. The other two Type IV REases involved in restriction of modified adenines are winged-helix domain fusion endonucleases (e.g. DpnI and HhiV4I) and possibly Yth domain containing fusion endonucleases. DpnI and HhivV4I endonucleases do not digest dZ- containing PCR DNA (unpublished result. Submitted and under review in Frontiers in Microbiology). But when the dZ-containing pUC19 PCR DNA was transferred into Mrr+E. coli host, there is a small reduction (~50-fold) in transformation efficiency. Without limiting any embodiment to any particular mechanism of action, it is not clear whether this reduction is due to the transcription-coupled nucleotide-excision repair (TC-NER) in the host or the result of true restriction by Mrr endonuclease. Further experiments are needed to answer this question. Regardless of restriction or DNA repair-based nicking, it is recommended to use Mrr- deficient cells for transformation of dZ-modified plasmid DNA. General Considerations PATENT APPLICATION NEB-491-PCT Aspects of the present disclosure can be understood in light of the provided descriptions, figures, sequences, embodiments, section headings, and examples, none of which should be construed as limiting the entire scope of the present disclosure in any way. Accordingly, the innovations set forth herein should be construed in view of the full breadth and spirit of the disclosure. Each of the individual embodiments described and illustrated herein has discrete components and features which can be readily separated from or combined with the components and / or features of any of the other several embodiments without departing from the scope or spirit of the present teachings. Lists of example species within a particular genus may vary in length at different places throughout the disclosure. Species lists shortened for convenience shall not be construed to exclude example species listed elsewhere in the specification. Any recited method can be carried out in the order of events recited or in any other order which is logically possible. Unless otherwise expressly stated to be required herein, each component, feature, and method step disclosed herein is optional and the disclosure contemplates embodiments in which each optional element may be expressly excluded. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements or use of a “negative” limitation. It is further intended to serve as antecedent basis for use of such elective terminology as “optionally” and the like in connection with the recitation of one or more claim elements. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Still, certain terms are defined herein with respect to embodiments of the disclosure and for the sake of clarity and ease of reference. Sources of commonly understood terms and symbols may include: standard treatises and texts such as Kornberg and Baker, DNA Replication, Second Edition (W.H. Freeman, New York, 1992); Lehninger, Biochemistry, Second Edition (Worth Publishers, New York, 1975); Strachan and Read, Human Molecular Genetics, Second Edition (Wiley-Liss, New York, 1999); Eckstein, editor, Oligonucleotides and Analogs: A Practical Approach (Oxford University Press, New York, 1991); Gait, editor, Oligonucleotide Synthesis: A Practical Approach (IRL Press, Oxford, 1984); Singleton, et al., Dictionary of Microbiology and Molecular biology, 2d ed., John Wiley and Sons, New York (1994), and Hale & Markham, the Harper Collins Dictionary of Biology, Harper Perennial, N.Y. (1991) and the like. PATENT APPLICATION NEB-491-PCT As used herein and in the appended claims, the singular forms “a” and “an” include plural referents unless the context clearly dictates otherwise. For example, the term “a protein” refers to one or more proteins, i.e., a single protein and multiple proteins. Numeric ranges are inclusive of the numbers defining the range. All numbers should be understood to encompass the midpoint of the integer above and below the integer i.e., the number 2 encompasses 1.5-2.5. The number 2.5 encompasses 2.45-2.55 etc. When sample numerical values are provided, each alone may represent an intermediate value in a range of values and together may represent the extremes of a range unless specified. Percent ranges with only one end point (e.g., ≥ 90% or ≤ 10%) optionally include a second endpoint at the maximum or minimum percentage (e.g., ≥ 90% includes a range of 90%-100% and ≤ 10% includes a range of 0%-10%). Ranges (including percent ranges) with only one end point (e.g., ≥ 90 or ≤ 10) optionally include a second endpoint 10% higher or 10% lower than the provided endpoint (e.g., ≥ 90 includes a range of 90-99 and ≤ 10 includes a range of 1-10). Concentration percentages are w / v percentages unless otherwise indicated. In the context of the present disclosure, “buffer” and “buffering agent” refer to a chemical entity or composition that itself resists and, when present in a solution, allows such solution to resist changes in pH when such solution is contacted with a chemical entity or composition having a higher or lower pH (e.g., an acid or alkali). Examples of suitable non- naturally occurring buffering agents that may be used in disclosed compositions, kits, and methods include HEPES, MES, MOPS, TAPS, tricine, and Tris. Additional examples of suitable buffering agents that may be used in disclosed compositions, kits, and methods include ACES, ADA, BES, Bicine, CAPS, carbonic acid / bicarbonic acid, CHES, citric acid, DIPSO, EPPS, histidine, MOPSO, phosphoric acid, PIPES, POPSO, TAPS, TAPSO, and triethanolamine. In the context of the present disclosure, “container” refers to a human-made container. A container may comprise one or more walls (e.g., defining an interior volume) and optionally one or more openings. Containers comprising one or more openings may further comprise one or more closures (e.g., a removable closures) for some or all such openings. A closure optionally may comprise an aperture or a septum, for example, to provide fluid communication with a volume of the container and a connected or inserted tube or syringe. Examples of containers include boxes, cartons, bottles, tubes (e.g., test tubes, microcentrifuge tubes), plates (e.g., 96-well, 384-well plates), vials, pipette tips, and ampules. Containers and / or closures may comprise any desired material including paper, plastics, glass, silicone, composites, PATENT APPLICATION NEB-491-PCT metals, alloys, or combinations thereof. Containers and / or closures may comprise materials that are compostable, recyclable, and / or sustainable. In the context of the present disclosure, “dZ” refers to the nucleoside comprising the nucleobase 2,6 diaminopurine (IUPAC: 7H-purine-2,6-diamine). A dZ base may be included in a nucleotide (e.g., dZ triphosphate or dZTP). In the context of the present disclosure, “DNA comprising dZ DNA”, “dZ-containing DNA”, and “dZ-modified DNA” refer to any DNA molecule comprising one or more 2,6 diaminopurine (dZ) bases. The dZ content of a DNA molecule may be expressed as an absolute percentage calculated by dividing the number of dZ bases in the sequence by the sum of the number of dA bases and the number of dZ bases in the sequence (dZ / (dA+dZ). A DNA comprising dZ DNA may have a dZ content in a range of 0.1%, 1%, 5%, 10%, 15%, 20%, or 25% to 10%, 20%, 40%, 60%, 75%, 90%, or 100%. For example, a DNA comprising dZ DNA may have a dZ content in a range of 0.1% - 100%, 0.1% - 10%, 5% - 20%, 10% - 40%, 10% - 60%, or 20% - 60%. A DNA molecule may be synthesized under conditions that permit or favor incorporation of dZ bases resulting in a dZ-modified DNA or a DNA molecule may be contacted with one or more reagents that convert dA to dZ to produce dZ-modified DNA. In the context of the present disclosure, a “dZ DNA-producing cell” refers to a non- naturally occurring bacterial cell or a non-naturally occurring yeast cell comprising one or more expressible dZ biosynthesis genes, wherein at least one of the expressible dZ biosynthesis genes is an artificial gene, arises from another organism, or is modified from its native form. An artificial gene may arise from any desired source including, for example, an ancestral reconstruction or a consensus sequence shared by two or more genes that encode the same enzyme. An artificial gene may be assembled (e.g., by Golden Gate assembly) or chemically synthesized. One or more expressible dZ biosynthesis genes may come from another organism. For example, an E. coli cell may be transformed with one or more expressible dZ biosynthesis genes from Salmonella. One or more expressible dZ biosynthesis genes may be modified from its native form. For example, a Salmonella cell may comprise one or more mutations (e.g., deletions) in one of its native expressible dZ biosynthesis gene and comprise a phage or plasmid having an intact copy of that expressible dZ biosynthesis gene (e.g., under the control of a different promoter and / or fused to another coding sequence). In some embodiments, a dZ DNA-producing E. coli cell may comprise at least one, at least two, at least three or all four of PurZ, MazZ, DatZ, and Salmonella DNA polymerase. For example, an E. coli cell may comprise at least one, at least two, at least three or all four of an PATENT APPLICATION NEB-491-PCT expressible purZ gene, an expressible mazZ gene, an expressible datZ gene, and an expressible dpoZ gene. A dZ DNA-producing E. coli cell may comprise one or more DNA molecules having a dZ content of, for example, 0.1% - 100%, 0.1% - 10%, 5% - 20%, 10% - 40%, 10% - 60%, or 20% - 60%. In some embodiments, a dZ DNA-producing E. coli cell may comprise one or more transcription products of a dZ-containing DNA molecule. A dZ-containing DNA may be transcribed by one or more native or engineered RNA polymerases, for example, T3 RNA polymerase, T7 RNA polymerase, SP6 polymerase, H6 RNA polymerase, among others and variants thereof including thermostable variants (e.g., International PCT Publication No. WO2017123748 and U.S. Patent Nos.10,519,431 and 11,259,184). In the context of the present disclosure, “lysate” refers to the products of cellular lysis. Lysates may comprise cellular materials, including, for example, organelles, proteins, lipids, carbohydrates, polynucleotides, amino acids, nucleic acids, salts, and metabolites. Lysates may comprise few or no intact cells, but may comprise membrane fragments (e.g., as micelles, liposomes, or other forms). Lysates of any cells of interest may be prepared, for example, bacterial, yeast, plant, insect, and / or mammalian cells. A lysate may be prepared by any available means for disrupting a cell envelope and, optionally, one or more organelle membranes. Example means include any available chemical or physical means for lysis, examples of which include mechanical grinding (with or without beads), sonication, freeze- thaw, shearing, osmotic, detergent, and enzymatic lysis. In the context of the present disclosure, “modified nucleotide” refers to nucleotides having a modification on the sugars (e.g., 2′‐fluororibose, ribose, 2′‐deoxyribose, arabinose, and hexose); and / or in the phosphate groups (e.g., phosphorothioates and 5′‐N‐ phosphoramidite linkages); and / or in the nucleotide base (e.g., as described in US 8,383,340; WO 2013 / 151666; US 9,428,535 B2; US 2016 / 0032316). Examples of modified nucleotides include dZTP, pseudouridine and N1-methyl-pseudouridine. In the context of the present disclosure, “non-naturally occurring” refers to a molecule (e.g., a polynucleotide, polypeptide, carbohydrate, or lipid) or composition that does not exist in nature. Such a molecule or composition may differ from naturally occurring molecules or compositions in one or more respects. For example, a polymer (e.g., a polynucleotide, polypeptide, or carbohydrate) may differ in the kind and arrangement of the component parts (e.g., nucleotide sequence, amino acid sequence, or sugar molecules). A polymer may differ from a naturally occurring polymer with respect to the molecule(s) to which it is linked. For example, a “non-naturally occurring” polypeptide (e.g., protein) may differ from naturally PATENT APPLICATION NEB-491-PCT occurring polypeptides in its secondary, tertiary, or quaternary structure, by having (or lacking) a chemical bond (e.g., a covalent bond including a peptide bond, a phosphate bond, a disulfide bond, an ester bond, and ether bond, and others) to a lipid, a carbohydrate, a second polypeptide (e.g., a fusion protein), or any other molecule. Similarly, a “non-naturally occurring” polynucleotide or nucleic acid may comprise (or lack) one or more other modifications (e.g., an added label or other moiety) to the 5’- end, the 3’ end, and / or between the 5’- and 3’-ends (e.g., methylation) of the nucleic acid. A “non-naturally occurring” molecule or composition may differ from naturally occurring compositions in one or more of the following respects: (a) having components that are not combined in nature, (b) having components in ratios and / or concentrations not found in nature, (c) lacking one or more components otherwise found in naturally occurring molecules or compositions (e.g., a cell-free composition, a chromosome- free composition, a histone-free composition, a polymerase-free composition, a cell membrane- free composition), (d) having a form not found in nature (e.g., dried, freeze dried, lyophilized, crystalline, aqueous, immobilized), and (e) having one or more additional components beyond those found in nature (e.g., a buffering agent, a detergent, a dye, a solvent or a preservative). All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. Reagents referenced in this disclosure may be made using available materials and techniques, obtained from the indicated source, and / or obtained from New England Biolabs, Inc. (Ipswich, MA). Compositions and Kits The present disclosure provides, in some embodiments, compositions, cells, lysates, systems, and kits for producing DNA comprising dZ DNA and / or transcription products thereof. For example, compositions, cells, lysates, systems, and kits may comprise (non- naturally occurring combinations of) one or more dZ biosynthesis enzymes, one or more DNA polymerases capable of incorporating dZ in a DNA, and optionally one or more RNA polymerases capable of transcribing a DNA comprising dZ. In some embodiments, each of a composition, cell, lysate, system, and kit may comprise an adenylosuccinate synthetase, an ATP triphosphohydrolase, a (d)GTP-specific diphosphohydrolase, and a polymerase (e.g., a DNA polymerase and / or an RNA polymerase). Examples of DNA polymerases include those disclosed herein. RNA polymerases include RNA polymerases classified into families I, II, III, IV, and V. Examples of polymerases further include Q5 polymerases, Taq polymerases, PATENT APPLICATION NEB-491-PCT Bst polymerases, Bsu polymerases, phi29 polymerases, T4 DNA polymerase, T7 DNA polymerase, DNA pol I, Therminator™, Klenow, Vent® polymerases, Deep Vent® polymerases, terminal transferase, 9°N polymerase, wild-type or variant polymerase D (e.g., as described in WO2021163561), T3 RNA polymerase, wild type or a variant T7 RNA polymerases (e.g., as described in US10519431), SP6 polymerases, wild-type or variant H6 RNA polymerases (e.g., as described in US63582625 filed 2023-09-14), E. coli RNA polymerase, signa factor 70 core enzyme, polyA polymerases, and polyU polymerases. A cell may comprise one or more expressible polynucleotides encoding one or more of an adenylosuccinate synthetase, an ATP triphosphohydrolase, a (d)GTP-specific diphosphohydrolase, and a polymerase (e.g., a DNA polymerase and / or an RNA polymerase). Compositions, cells, lysates, systems, and kits for producing DNA comprising dZ DNA and / or transcription products thereof may comprise, in some embodiments, dNTPs, rNTPs, primers, other enzymes (e.g., other polymerases, enzymes other than polymerases, or both), buffering agents, or combinations thereof. Enzymes may be included in a storage buffer. Any suitable storage buffer may be used, for example, buffers comprising one or more of a cryoprotectant (e.g., a polyol such as glycerol, an antifreeze protein), a salt, a detergent, a reducing agent, a sugar, a chelator, and an antimicrobial agent and having a pH tolerated by the enzyme to be stored, for example, between pH 6 and 9. A composition, system, or kit may include a reaction buffer which may be in concentrated form, and the buffer may contain additives (e.g. glycerol), salt (e.g. NaCl, KCl), reducing agent, EDTA or detergents, among others. Detergents include nonionic detergents (e.g., t- octylphenoxypolyethoxyethanol), anionic detergents (e.g., alkylbenzene sulfonates), cationic detergents (e.g., alkylbenzene quaternary ammonium), and zwitterionic detergents. A composition, system, or kit comprising dNTPs may include one, two, three of all four of dATP, dTTP, dGTP and dCTP. A composition, system, or kit comprising rNTPs may include one, two, three of all four of rATP, rUTP, rGTP and rCTP. A composition, system, or kit may further comprise one or more modified nucleotides. A composition, system, or kit may optionally comprise one or more primers (random primers, bump primers, exonuclease- resistant primers, chemically-modified primers, custom sequence primers, or combinations thereof). Systems and kits may be configured to have one or more enzymes in a separate container or all enzymes together in a single container. A composition, system, or kit may be a non-natural collection of components configured, for example, for convenient storage, shipping, delivery, and / or use. One or more components of a system or kit may be included PATENT APPLICATION NEB-491-PCT in one container for a single step reaction, or one or more components may be contained in one container, but separated from other components for sequential use or parallel use. The contents of a system or kit may be formulated for use in a desired method or process. Example compositions may comprise a cell or a lysate of a cell, in each case, where the cell comprises one or more dZ biosynthesis enzymes, one or more DNA polymerases capable of incorporating dZ in a DNA, optionally one or more RNA polymerases capable of transcribing a DNA comprising dZ, and optionally any of the other components disclosed herein (e.g., one or more buffers, one or more dNTPs (including modified dNTPs), one or more NTPs (including modified NTPs), one or more detergents, one or more salts, one or more reducing agents, one or more sugars, and / or one or more additives). A composition comprising a cell or lysate may further include a polynucleotide of interest, for example, to serve as a template for the synthesis of the dZ DNA. A kit is provided that contains: (i) one or more of an adenylosuccinate synthetase, an ATP triphosphohydrolase, a (d)GTP-specific diphosphohydrolase, and a polymerase (e.g., a DNA polymerase and / or an RNA polymerase); and (ii) a buffer. One or more of the enzymes may have a lyophilized form or may be included in a buffer (e.g., a storage buffer or a reaction buffer in concentrated form). A kit may contain one or more of the enzymes in a mastermix suitable for making, receiving, amplifying, and / or transcribing a DNA comprising dZ. One or more of the enzymes may be a purified so as to contain substantially no DNA or RNA and no nucleases. The reaction buffer in (ii) and / or storage buffers containing enzymes in (i) may include non-ionic, ionic e.g. anionic or zwitterionic surfactants and crowding agents. A kit may include one or more of the enzymes and the reaction buffer in a single tube or in different tubes. A subject kit may further include instructions for using the components of the kit to practice a desired method. The instructions may be recorded on a suitable recording medium. For example, instructions may be printed on a substrate, such as paper or plastic, etc. As such, the instructions may be present in the kits as a package insert, in the labeling of the container of the kit or components thereof (i.e., associated with the packaging or subpackaging) etc. Instructions may be present as an electronic storage data file residing on a suitable computer readable storage medium (e.g. a CD-ROM, a flash drive). Instructions may be provided remotely using, for example, cloud or internet resources with a link or other access instructions provided in or with a kit. In some embodiments, the present disclosure relates to therapeutic compositions comprising dZ DNA. A therapeutic composition may comprise a DNA (e.g., a therapeutic PATENT APPLICATION NEB-491-PCT DNA) comprising dZ in a range of 0.1%, 1%, 5%, 10%, 15%, 20%, or 25% to 10%, 20%, 40%, 60%, 75%, 90%, or 100%. A therapeutic composition may comprise (may further comprise) one or more pharmaceutically acceptable additives, binders, buffers, coatings, colors, controlled release agents, delivery agents (e.g., liposomes, propellants), diluents, disintegrants, dyes, excipients, fillers, lipids, lubricants, salts, sorbents, stabilizers, and / or other agents to produce a therapeutic DNA dosage form. A therapeutic DNA may be combined with (e.g., in a single dosage form) or delivered concurrently or in sequence with one or more other active pharmaceutical agents. A therapeutic DNA and / or its encoded transcription / translation product(s) may function in a subject as an active pharmaceutical agent, according to some embodiments. A therapeutic DNA may be administered by any suitable route of administration, including transdermal, oral, enteral, parenteral, ocular, ottic, transmucosal, sublingual, and pulmonary (e.g., by nebulization and / or inhalation) routes, and combinations thereof. Methods of Making and Methods of Use The present disclosure relates, in some embodiments, to methods of making dZ and dZ-containing DNA. For example, a method may comprise drying 2-amino-2′- deoxyadenosine in-vacuo overnight over phosphorus pentoxide to produce dried 2-amino-2′- deoxyadenosine, drying trimethyl phosphate over molecular sieves to produce dried trimethyl phosphate, contacting dried 2-amino-2′-deoxyadenosine, dried trimethyl phosphate, and phosphorus oxychloride (e.g., at ≤ 4 °C, ≤ 2 °C, ≤ 0 °C) to produce a reaction mixture, contacting the reaction mixture, tributylammonium pyrophosphate, and tributylamine in DMF to produce products, and optionally contacting the products and triethylammonium bicarbonate, wherein the products comprise dZTP. In some embodiments, dZTP may be made enzymatically with PurZ, MazZ, and DatZ. Microbial cells (e.g., E. coli cells) and cell lysates comprising PurZ, MazZ, and DatZ that are operable individually and collectively to produce dZTP may produce sufficient amounts of dZTP for incorporation into DNA synthesized, for example, by a polymerase or reverse transcriptase. The present disclosure further relates to making dZ-containing DNA. A method may comprise, for example, contacting a polynucleotide template, one or more dNTPs, at least one of which is dZTP, and one or more DNA polymerases and / or one or more reverse transcriptases to produce a dZ-containing DNA. A method may include transforming a dZ DNA-producing cell (e.g., a dZ DNA-producing E. coli cell) with a polynucleotide template and culturing (e.g., maintaining, incubating, growing) the cells, wherein the cells optionally PATENT APPLICATION NEB-491-PCT comprise at least one, at least two, at least three or at least four of PurZ, MazZ, DatZ, a DNA polymerase (e.g., a Salmonella DNA polymerase), and a reverse transcriptase (e.g., a Salmonella reverse transcriptase). In some embodiments, a method may comprise contacting a polynucleotide template, a cell lysate comprising one or more dNTPs, at least one of which is dZTP, and one or more DNA polymerases and / or one or more reverse transcriptases to produce a dZ-containing DNA. A method may comprise, in some embodiments, contacting a polynucleotide template, a cell lysate comprising one or more dNTPs, at least one of which is dZTP, and one or more DNA polymerases and / or one or more reverse transcriptases to produce a dZ-containing DNA. In the context of the present disclosure, a cell lysate may comprise all of the components needed to produce a desired dZ-containing DNA or it may lack one or more of the components need to produce a desired dZ-containing DNA. In some embodiments, it may be desirable to regulate production of a desired dZ-containing DNA. For example, a method of producing a dZ-containing DNA of interest may comprise contacting (a) a cell or cell lysate having all but lacking one or two of the following: (i) PurZ, (ii) MazZ, (iii) DatZ, (iv) a means to copy a polynucleotide template (e.g., a DNA polymerase (e.g., a Salmonella DNA polymerase), and a reverse transcriptase (e.g., a Salmonella reverse transcriptase)), and (v) a polynucleotide template for the dZ-containing DNA of interest, and (b) the one or two lacking components. A cell or cell lysate may be provided, for example, having PurZ, MazZ, and DatZ operable individually and collectively to produce dZTP, but lacking both the means to copy a polynucleotide template (e.g., a polymerase or reverse transcriptase) and the polynucleotide template for the dZ-containing DNA of interest. A cell or cell lysate comprising such operable PurZ, MazZ, and DatZ may be contacted with the lacking means to copy a polynucleotide template (e.g., a polymerase or reverse transcriptase) and the polynucleotide template for the dZ-containing DNA of interest to produce the dZ-containing DNA of interest. Similarly, a cell or cell lysate may be provided, for example, having PurZ, MazZ, and DatZ operable individually and collectively to produce dZTP, and having the means to copy a polynucleotide template (e.g., a polymerase or reverse transcriptase) , but lacking the polynucleotide template for the dZ-containing DNA of interest. A cell or cell lysate comprising such operable PurZ, MazZ, and DatZ and means for copying a polynucleotide may be contacted with the lacking polynucleotide template for the dZ-containing DNA of interest to produce the dZ-containing DNA of interest. EXAMPLES PATENT APPLICATION NEB-491-PCT Some specific example embodiments may be illustrated by one or more of the examples provided herein. EXAMPLE 1: dZTP chemical synthesis Unless otherwise noted all reagents were purchased from Sigma-Aldrich (St. Louis, MO) and used without further purification.2-amino-2′deoxyadenosine was purchased from Toronto Research Chemicals (North York, ON, Canada). Analytical reverse-phase HPLC was performed on an Agilent 1200 series LC / MS system equipped with a G1315D diode array UV-Vis detector and a 6120 quadrupole LC / MS detector in both positive (+ESI) and negative mode (-ESI). LC was performed on a Phenomenex (Torrance, CA) Clarity Oligo-XT C18 column (4.6 x 100 mm, 2.6µm, 100 Å) with a gradient mobile phase consisting of 100 mM 1,1,1,3,3,3-hexafluoro-2-propanol and 4 mM hexylamine in water, and methanol. Relative abundance of product to reactants was determined by UV. Preparative-scale HPLC was done on an Agilent 1290 Infinity II, with a Phenomenex Gemini NX C-18 column (30 x 100 mm, 10 µm, 110 Å) using a gradient mobile phase of 200 mM triethylammonium bicarbonate in water and acetonitrile. Preparative-scale anion exchange was done on an AKTA Pure 150 FPLC from Cytiva (Marlborough, MA), with a 50 x 250 mm column containing Ceramic HyperDF DEAE resin from Pall (Port Washington, New York), with a gradient mobile phase of water and 1.0 M triethylammonium bicarbonate in water, pH 7.4. Nuclear Magnetic Resonance (NMR) and Hi-Resolution MS (HRMS) was done by Novatia LLC (Newtown, PA). Weighed 1.1 g (4.1 mmol, 1.0 eq.) of 2-amino-2′-deoxyadenosine into a round bottom flask, dried in-vacuo overnight over phosphorus pentoxide. In a separate flask, 30 mL of trimethyl phosphate was dried over molecular sieves for two days. Dissolved dried 2-amino- 2′deoxyadenosine in 30 mL of trimethyl phosphate, then cooled solution on ice to 0 °C, under Ar(g). Added 0.76 mL (8.2 mmol, 2.0 eq.) of phosphorus oxychloride to the solution over the period of 1 minute with vigorous stirring. Continued stirring the reaction at 0 °C until high conversion was observed via LCMS. Added an ice-cold solution of tributylammonium pyrophosphate (7.52g, 16.5 mmol, 4.0 eq.) and tributylamine (3.9 mL, 16.5 mmol, 4.0eq.) in dry DMF with vigorous stirring at 0 °C. Monitored the reaction by LCMS and quenched with 500 mL 0.2 M triethylammonium bicarbonate when appreciable product conversion was observed. The crude reaction product was diluted to 1 L with dH2O, and then purified by DEAE. Isolated product fraction was evaporated to dryness, co-evaporated with methanol PATENT APPLICATION NEB-491-PCT two times, then polished by reverse phase HPLC. Product fractions were combined, evaporated to dryness, then co-evaporated three times with 20 mL methanol to yield 1.2 mmol of the final product as the triethylammonium salt (28% yield) as a clear oil, and the purity of dZTP was determined by LCMS to be 98% FIGURE 1A shows the reaction scheme for dZTP synthesis and FIGURE 1B shows mass confirmation of the synthetic product by LCMS. EXAMPLE 2: Restriction and modification enzymes, plasmids and E. coli strains Restriction endonucleases (REases), high fidelity Phusion and Q5 DNA polymerases are provided by New England Biolabs (NEB). Restriction digests were carried out in the recommended restriction buffers at the appropriate temperatures: buffer 1.1 (10 mM Bis-Tris- Propane-HCl, 10 mM MgCl2, 100 µg / ml BSA or recombinant albumin, pH 7.0 at 25 °C); 2.1 (50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, 100 µg / ml BSA or recombinant albumin, pH 7.9 at 25 °C); 3.1 (100 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2,100 µg / ml BSA or recombinant albumin, pH 7.9 at 25 °C) or CutSmart buffer (50 mM Potassium Acetate, 20 mM Tris-acetate, 10 mM Magnesium Acetate, 100 µg / ml BSA or recombinant albumin, pH 7.9 at 25 °C). Plasmids pBR322, pUC19, 2 log DNA ladder (1 kb plus), phage ^ ^DNA were also from NEB. The T7 expression vector pET21b with C-terminal 6xHis tag was originally purchased from Novagen (NdeI-XhoI). E. coli competent cells of the following strains were produced in- house at NEB: NEB Turbo (Mrr+, E. coli K strain), C2925 (Mrr+, Dam-, Dcm-, McrA-, McrB- ), NEB 10 ^ (RecA-), C2683 (Mrr-, McrBC-, K strain), T7 shuffle (C3026, K strain), Nico ( ^DE3) (C2529, B strain), NEB Express (C2523, B strain). Synthetic gene blocks were purchased from IDT. Plasmid mini-prep kit and PCR DNA clean-up kits were provided by NEB. EXAMPLE 3: LC-MS LC-MS analysis was performed using an Agilent 1290 Infinity II UHPLC equipped with G7117A Diode Array Detector and 6135XT MS Detector, on a Waters XSelect HSS T3 XP column (2.1 × 100 mm, 2.5 µm) with the gradient mobile phase consisting of methanol and 10 mM ammonium acetate buffer (pH 4.5). The relative abundance of each nucleoside was determined by the integration of each peak at 260 nm or their respective UV absorption maxima. EXAMPLE 4: Restriction digestion of dZ-modified PCR DNA PATENT APPLICATION NEB-491-PCT Two sets of primers and Q5 DNA polymerase were use to amplify dZ-modified PCR DNA (3 and 4 kb) from pBR322 and used the DNA for restriction analysis. LCMS base composition analysis indicated that 98.56 % of dZ in the DNA substrate, which is consistent with the purity of dZTP at ~98%. Results are shown in FIGURE 2. REases with 1-6 dZ / dA within the recognition sequences were selected for restriction digest. FIGURE 3A and FIGURE 3B show 18 examples of digests of dZ or dA PCR DNA. Four types of digestion were detected: fully resistant (R), mostly resistant (MR, i.e. very partial digestion), partially resistant (PR, less than 50% of the DNA is resistant), sensitive (S, complete or nearly complete digestions). Among the 18 digests, four enzymes (ApoI, BstYI, BsaI-HF v2, and SalI) can fully or nearly digest the dZ-PCR DNA. The modified substrate is resistant or partially resistant to 15 restriction enzymes (FIGURE 3A), comparing restriction patterns on dZ (top) and dA (bottom) substrates. As expected, the same REases can digest regular (dA) PCR DNA (FIGURE 3B). A total of 147 REases were examined in digests of dZ-containing PCR DNA and regular PCR DNA and found the modified substrate is resistant or partially resistant to ~53.1% of REases. 46.9% of REases are able to completely digest the modified DNA. Table 1 summarizes the restriction results. Table 2 lists individual restriction results. Compared to other hypermodified phage DNA such as glc-5hmC modified T4 genome that is resistant to >90 REases with CG recognition sequences, the dZ-modified DNA displays moderate level of resistance to Type II restrictions. Similar results are expected for dZ-DNA resistance to Type I restriction in vivo. Only one ATP-dependent Type III restriction enzyme EcoP15I (CAGCAG N25 / 27) was tested on the dZ substrate in a test tube, which did not cut dZ-containing PCR DNA. One Type IV restriction system (Mrr) was tested in vivo by plasmid transformation because its restriction activity had been shown on 6mA- and 5mC-modified DNA (see below). TABLE 1. Type II restriction of dZ-containing PCR DNA. Type II restriction level Number of REases Percentage among 147 RE PATENT APPLICATION NEB-491-PCT TABLE 2. Listing of restriction enzyme digestions tested on dZ-PCR DNA. Cleavage on dZ- REase Recognition Site modified DNA PATENT APPLICATION NEB-491-PCT BclI TGATCA BccI CCATCN4 S PATENT APPLICATION NEB-491-PCT BsoBI CYCGRG S Bsp1286I GDGCHC S PATENT APPLICATION NEB-491-PCT DdeI CTNAG PR DpnI G6mATC PATENT APPLICATION NEB-491-PCT Hpy188I TCNGA S Hpy188III TCNNGA S PATENT APPLICATION NEB-491-PCT NsiI-HF ATGCAT NspI RCATGY PR PATENT APPLICATION NEB-491-PCT SfoI GGCGCC SgrAI CRCCGGYG , , , resistant; S, sensitive to restriction. 53.1% tested REases are resistant or partially resistant. Some REases tested have similar recognition sequences, but they have different outcome on restriction of dZ-modified DNA. For example, EarI (CTCTTC N1 / N4) is resistant to dZ DNA, but SapI and BspQI (GCTCTTCN1 / N4) are partially resistant. EcoO109I (RGGNCCY) is sensitive to dZ DNA, but PpuMI (RGGWCCY) is resistant. A single dZ in GGWCC can block AvaII digestion, but GCWGC is sensitive to ApeKI restriction in dZ- modified PCR DNA. It is noted that a limited number of Type IIS REases (enzyme cleavage downstream / outside of recognition sequences) are resistant to dZ modifications such as BbsI, PATENT APPLICATION NEB-491-PCT BciVI, BfuAI, BpmI, BsgI, BsmI, and EcoP15I. This property may be utilized in recombinant DNA techniques (e.g. restriction sites in the primer with dZ modification could not cut, but internal sites in PCR DNA in the absence of modifications can be cut, a strategy similar to methylated or phosphorothioated sites in the primers). EXAMPLE 5: Incorporation of dZ base in E. coli The dZ biosynthesis pathway can be expressed in E. coli to generate partial dZ-modified plasmid and genomic DNA. The level of dZ incorporation can reach over 20% in plasmid DNA carrying the dZ biosynthesis genes under IPTG induction. The goal was to improve dZ level by co-expression of three genes from Salmonella phage for dZ biosynthesis and a 4thgene encoding phage DNA polymerase. In a preliminary experiment, the dZ level in expression plasmids was examined and the three-gene plasmid (pET21b-purZ-mazZ-datZ) and four-gene plasmid (pET21b-purZ-mazZ-datZ-dpoZ) were found to generate 19.2% and 29.8% of dZ (dZ divided by dZ+dA), respectively (IPTG induction overnight at 18oC). This dZ incorporation level is slightly higher than the reported result from expression of three genes in E. coli from cyanophage S-2L in the range of 7-16%. To further increase the dZ level, the T7 expression strain carrying four genes was grown to mid-log phase and performed IPTG-induction at 25oC overnight. TABLE 3 shows that dZ level reached as high as 44.3% in sample #9. Among the 12 samples (plasmids) tested, 11 samples gave rise to a dZ level in the range of 29% to 40%. The original base composition data of LCMS analysis is shown in FIGURE 4A and bar representation of dZ level is shown in FIGURE 4B. Expression of four Salmonella phage genes (purZ, mazZ, datZ, and phage DNA polymerase gene dpoZ) can increase dZ incorporation in E. coli plasmids up to 44%. In theory, the host genomic DNA should also contain a high level of dZ, but it is difficult to accurately determine the percentage of dZ due to contaminating plasmid in genomic DNA preparation using the commercially available kit. The use of CsCl2 gradient separation of plasmid DNA from genomic DNA may be necessary to obtain clean data. TABLE 3. dZ / (dZ+dA) level in 4-gene plasmids (samples 1-12) by LCMS analysis. dC dG dT dZ dA dZ / (dZ+dA) PATENT APPLICATION NEB-491-PCT 1.04 1.00 0.94 0.39 0.54 0.42 1.02 1.00 0.96 0.13 0.80 0.14 Sample 4 l 13 samples. Expression of six phage genes together in the same host was attempted by expression of phage DNA helicase-SSB-dpoZ in a compatible plasmid pACYCT7. When both plasmids were co-transformed into a T7 expression stain, the host carries two plasmid C2566 [pET21b- purZ-mazZ-datZ, pACYCT7-helicase-SSB-dpoZ]. After IPTG induction at 25oC overnight, the dZ level reached 35.4% to 55.6% in 11 samples, which is higher than the 4-gene plasmid on average. While successful, the T7 expression host carrying six phage genes grew slowly and reached lower cell density after overnight culture. To see whether the dZ level is reproducible, the experiment was repeated by IPTG induction of two expression plasmids in the same host. TABLE 4 showed that the plasmids extracted from C2566 [pET21b-purZ-mazZ-datZ, pACYCT7-helicase-SSB-dpoZ] cells gave rise to the best dZ incorporation at 50% (average of three samples / plasmids). The 4-genes plasmid yielded dZ level at nearly 40% (average of six samples / plasmids). Surprisingly, the non-induced cells with 3-genes plasmid generated higher dZ level than IPTG-induced plasmid (27.5% vs 9.0%). The reason for this unexpected result is not clearly understood. TABLE 4. dZ level in three expression plasmids.1). PATENT APPLICATION NEB-491-PCT dC dG dT dZ dA dZ / (dZ+dA) dZaverage1.036 1.000 0.931 0.319 0.569 35.91%39.8% 2). 4-genes plasmid: pET21b-purZ-mazZ-datZ-dpoZ, + IPTG (samples 1-6). 3). 6-genes plasmids: pET21b-purZ-mazZ-datZ and pACYCT7-DNA helicase-SSB-dpoZ, +IPTG (samples 10-12). EXAMPLE 6: Transfer of dZ-modified plasmid DNA into E. coli lab strains by transformation To examine the transformation efficiency of dZ-modified plasmid into commonly used E. coli lab strains, two sets of primers were used to amplify pUC19 by inverse PCR. The PCR DNA have overlapping sequences at the priming sites and annealed to form circular DNA which can be transferred into E. coli competent cells without ligation. The dZ-modified pUC19 and regular pUC19 inverse PCR DNA were transferred into E. coli lab strains by transformation using 20 ng DNA: NEB Turbo (Mrr+, E. coli K strain), C2925 (Mrr+, Dam-, Dcm-, McrA-, McrB-), NEB 10 ^ (Mrr-, RecA-), C2683 (Mrr-, McrBC-, K strain), T7 shuffle (C3026, K strain), Nico ( ^DE3) (C2529, B strain), NEB Express (C2523, B strain). Less than 2-fold differences were observed in transformation efficiency when the dZ-modified pUC19 PCR DNA was transferred into the above strains compared to unmodified inverse PCR DNA, with the exception of two strains, NEB Turbo and C2925. In NEB Turbo and C2925 strains, there was a reduction of 46 to 63-fold in transformation efficiency using dZ-modified DNA (average of two experiments). Mrr endonuclease which restricts modified adenine (N6mA, 6mA) is implicated in the reduced PCR DNA transformation although other host genetic PATENT APPLICATION NEB-491-PCT background may also be involved. Further study is needed to pinpoint the molecular basis of this reduced transformation. Typical restriction systems restrict foreign DNA or phage DNA at 103to 106-fold. Thus, a ~50-fold reduced transformation into Mrr+strain is not a significant large restriction. For cloning and working with dZ-modified DNA, it is recommended to use Mrr-deficient E. coli strains: NEB 10 ^, C2683 (Mrr-, McrBC-), T7 shuffle (C3026), Nico ( ^DE3) (C2529), NEB Express (C2523), C2566 (T7 Express). The dZ-modified pUC19 DNA or modified pET21b-purZ-mazZ-datZ-dnpZ) can be used to transform WT E. coli strains and screen for modification-dependent restriction systems specifically towards dZ DNA if such dZ- dependent REases exist in WT host strains. In the biological arms race between host and phages, coliphages such as T4 utilize DNA base hypermodifications to counteract host- encoded Type II restriction systems. Subsequently, E. coli hosts evolve modification- dependent REases such as GmrSD and EcoO157SI to restrict modified T4 genomes with glc- 5hmC (glucosylated-5hmC), or McrBC and McrA to restrict modified cytosines in T4gt. EXAMPLE 7: Exonuclease digestion of dZ-modified PCR DNA In light of the resistance to Type II restriction endonucleases displayed by dZ-modified DNA shown here, susceptibility to exonuclease digestion was tested. PCR DNA substrates comprising dZ or dA were digested by exonucleases RecBCD (supplemented with ATP), E. coli exonuclease III (exo III), phage T5 exonuclease (T5 exo), phage T7 exonuclease (T7 exo), E. coli exonuclease VIII (exo VIII, truncated), phage ^ exonuclease ( ^ exo), and Bal31 nuclease. There appeared to be very minor differences in exonuclease digestions of dZ- modified PCR DNA in 20 min digestion compared to digestion of regular PCR substrate (data not shown). Bal31 exonuclease digestions were inconclusive due to the complete degradation of both dZ-modified and unmodified PCR DNA (1 U incubated with 0.35 ^g DNA, data not shown). As expected, MluCI (AATT) cleaved the unmodified PCR DNA, while most of the dZ-modified PCR DNA was not digested. To further confirm the digestion of dZ-modified DNA by RecBCD and T5 exonucleases, a timecourse of exonuclease digests was performed with samples collected after between 1 and 30 mins. Results are shown in FIGURE 5. There was very little difference in RecBCD digestion of dZ-modified and unmodified PCR DNA in 1-30 min. A minor difference was detected in T5 exonuclease digestion of dZ-modified DNA. After 30 min digestion, the dZ end products are slightly larger (0.5-1.5 kb) than the end products of dA PCR DNA (0.5-0.7 kb), suggesting that dZ-modified DNA slowed down T5 exonuclease degradation. The slowed-down digestion of dZ-modified DNA by certain PATENT APPLICATION NEB-491-PCT exonucleases may be a desired feature for gene therapy and DNA vaccine applications to prolong the half-life of target genes, provided that human RNA polymerase variant is compatible to utilize dZ-modified DNA for transcription. EXAMPLE 8: Cloning and expression of dZ cluster (purZ-mazZ-datZ) from Salmonella phage PMBT28 into E. coli T7 expression strain C2566 Grow 6 ml cells C2566 [pET21b-purZ-mazZ-datZ](SEQ ID NO:4 encoding SEQ ID NOS:1-3) in LB+Amp to late log phase, add IPTG (0.5 mM) and incubate at 25oC overnight. Extract plasmid DNA by a mini-prep kit from NEB. Digest 1 µg of plasmid DNA in nucleoside digest mix overnight at 37oC (1 µg DNA, 2 µL 10x buffer, 1 ml nucleoside digest enzyme mix, 1µL RNase A (10 mg / ml), add TE to 20 µL). Analyzed base composition by LC-MS. EXAMPLE 9: Co-expression of dZ cluster from Salmonella phage PMBT28 with DNA pol gene dpoZ from the same phage in pET21b in E. coli T7 expression strain C2566 XhoI digestion of pET21b-purZ-mazZ-datZ plasmid, insert Salmonella DNA pol gene (two gene blocks) into the XhoI site. ATG start codon, TGA stop codon are shown in red text. The gene order is T7 promoter- purZ-mazZ-datZ-dpoZ. Grow 6 ml cells C2566 [pET21b-purZ-mazZ-datZ-dpoZ] (SEQ ID NO:5) in LB+Amp to late log phase, add IPTG (0.5 mM) and incubate at 25oC overnight. Extract plasmid DNA by a mini-prep kit from NEB. Digest 1 µg of plasmid DNA in nucleoside digest mix overnight at 37oC (1 µg DNA, 2 µL 10x buffer, 1 µL nucleoside digest enzyme mix, 1 µL RNase A (10 mg / ml), add TE to 20 µL). Analyzed base composition by LC-MS. EXAMPLE 10: Co-expression of dZ cluster (purZ-mazZ-datZ) from Salmonella phage PMBT28 and DNA helicase-dpoZ-SSB in a compatible plasmid pACYCT7 in E. coli T7 expression strain C2566: C2566 [pET21b- purZ-mazZ-datZ, pACYCT7-DNA helicase-dpoZ- SSB] Grow 6 ml cells C2566 DNA helicase- dpoZ-SSB](SEQ ID NO:6) in LB+Amp+Cm to late log phase, add IPTG (0.5 mM) and incubate at 25oC overnight. Extract plasmid DNA by a mini-prep kit from NEB. Digest 1 µg of plasmid DNA in nucleoside digest mix overnight at 37oC (1 µg DNA, 2 µL 10x buffer, 1 µL nucleoside digest enzyme mix, 1 µL RNase A (10 mg / ml), add TE to 20 µL). Analyzed base composition by LC-MS. PATENT APPLICATION NEB-491-PCT Phage PMBT28 ATP-dependent helicase (helicase)-dpoZ (DNA pol)-ssDNA binding protein (SSB) Helicase=nt 26816 DNA pol=nt 28294 DNA binding protein nt30248-30589. ATG start codons are highlighted for the three genes. (numbering is the same from phage genome) 3 synthetic genes = 3774 bp in total length (to be cloned into NdeI-BamHI cut pACYC-T7) Co-expression of helicase, DNA pol, ssDNA binding protein-SSB with dZ cluster to increase dZTP incorporation. Note: underlined sequences (SEQ ID NO:7) are overlapping sequence with the vector or among gene blocks for cloning / assembly. Experiment strategy: co-transformation of two plasmids into the same host C2566 and select for AmpR and CmR. C2566 [pET21b- purZ-mazZ-datZ, pACYCT7-DNA helicase- dpoZ-SSB]. Grow 6 ml cells in LB+ Amp to late log phase, add IPTG (0.5 mM) and incubate at 25oC overnight. Extract plasmid DNA by a mini-prep kit from NEB. Digest 1 µg of plasmid DNA in nucleoside digest mix overnight at 37oC (1 µg DNA, 2 µL 10x buffer, 1 µL nucleoside digest enzyme mix, 1 µL RNase A (10 mg / ml), add TE to 20 µL). Analyzed base composition by LC-MS. EXAMPLE 11: Co-expression of uncultured phage metagenomic phage DNA polymerase with Salmonella dZ cluster The uncultured phage DNA pol (WY1) (SEQ ID NO:8) was selected from a phage metagenome library after restriction enzyme challenge (dZ modified DNA is resistant to the restriction enzymes and survived in the library). Assemble / clone two gblocks of WY1 DNA pol gene into pET21b-purZ-mazZ-datZ, XhoI cut expression plasmid) using NEB HiFi assembly cloning mix enzymes (2X master mix). Selection of successful plasmid transformation by AmpR. DNA pol gene insert was confirmed by XhoI digestion. C2566 [pET21b- purZ-mazZ-datZ-dpoZ (WY1] (SEQ ID NO:9). Grow 6 ml cells in LB+ Amp to late log phase, add IPTG (0.5 mM) and incubate at 25oC overnight. Extract plasmid DNA by a mini-prep kit from NEB. Digest 1 µg of plasmid DNA in nucleoside digest mix overnight at 37oC (1 µg DNA, 2 µL 10x buffer, 1 µL nucleoside digest enzyme mix, 1 µL RNase A (10 mg / ml), add TE to 20 µL). Analyzed base composition by LC-MS. PATENT APPLICATION NEB-491-PCT EXAMPLE 12: Co-expression of uncultured phage metagenomic phage DNA polymerase with Salmonella dZ cluster The uncultured phage DNA pol (WY2) (SEQ ID NO:10) was selected from a phage metagenome library after restriction enzyme challenge (dZ modified DNA is resistant to the restriction enzymes and survived in the library). Assemble / clone two gblocks of WY2 DNA pol gene into pET21b-purZ-mazZ-datZ, XhoI cut expression plasmid) using NEB HiFi assembly cloning mix enzymes (2X master mix). Selection of AmpR and inserts. DNA pol gene WY2 insert was confirmed by XhoI digestion. C2566 [pET21b- purZ-mazZ-datZ-dpoZ (WY2] (SEQ ID NO:11). Grow 6 ml cells in LB+ Amp to late log phase, add IPTG (0.5 mM) and incubate at 25oC overnight. Extract plasmid DNA by a mini-prep kit from NEB. Digest 1 µg of plasmid DNA in nucleoside digest mix overnight at 37oC (1 µg DNA, 2 µL 10x buffer, 1 µL nucleoside digest enzyme mix, 1 µL RNase A (10 mg / ml), add TE to 20 µL). Analyzed base composition by LC-MS. Assemble / clone two gblocks into pET21b-PurZ-MazZ-DatZ, XhoI cut expression plasmid)using NEB HiFi assembly cloning mix enzymes (2X master mix). Selection of AmpR and inserts, grow 6 ml cells to late log phase, induce cells at 25°C with IPTG (0.5 mM). Purify plasmid DNA, perform nucleoside digestion overnight at 37°C, Analyze dZ level by MS-LS. EXAMPLE 13: Co-expression of Vibrio phage DNA polymerase with dZ cluster Assemble / clone two gblocks of Vibrio DNA pol gene into pET21b-purZ-mazZ-datZ, XhoI cut expression plasmid) using NEB HiFi assembly cloning mix enzymes (2X master mix). Selection of AmpR and inserts. C2566 [pET21b- purZ-mazZ-datZ-dpoZ(Vibrio)] (SEQ ID NO:13). Grow 6 ml cells in LB+ Amp to late log phase, add IPTG (0.5 mM) and incubate at 25oC overnight. Extract plasmid DNA by a mini-prep kit from NEB. Digest 1 µg of plasmid DNA in nucleoside digest mix overnight at 37oC (1 µg DNA, 2 µL 10x buffer, 1 µL nucleoside digest enzyme mix, 1 µL RNase A (10 mg / ml), add TE to 20 µL). Analyzed base composition by LC-MS.

Claims

PATENT APPLICATION NEB-491-PCT CLAIMS What is claimed is:

1. An E. coli cell comprising at least one, at least two, at least three or all four of: (a) a phage 2-adenylosuccinate synthetase (PurZ), (b) a phage dATP triphosphohydrolase (MazZ), (c) a phage (d)GTP-specific diphosphohydrolase (DatZ), and (d) a DNA polymerase or a reverse transcriptase.

2. An E. coli cell according to Claim 1, wherein the cell comprises all three of (a), (b), and (c).

3. An E. coli cell according to Claim 1 or Claim 2, wherein the E. coli cell comprises the DNA polymerase or the reverse transcriptase.

4. An E. coli cell according to any preceding claim, wherein the DNA polymerase is capable of incorporating dZ into DNA or the reverse transcriptase is capable of incorporating dZ into DNA.

5. An E. coli cell according to any preceding claim, wherein the E. coli cell comprises a plasmid.

6. An E. coli cell according to any preceding claim, wherein the E. coli cell comprises: an expressible purZ gene encoding the phage adenylosuccinate synthetase, an expressible mazZ gene encoding the phage dATP triphosphohydrolase; and / or an expressible datZ gene encoding the phage (d)GTP-specific diphosphohydrolase.

7. An E. coli cell according to any preceding claim, wherein the E. coli cell comprises: an expressible phage dpoZ gene encoding the DNA polymerase.

8. An E. coli cell according to any preceding claim, wherein the cell further comprises a polynucleotide template encoding a heterologous sequence of interest.

9. An E. coli cell according to any preceding claim, wherein the cell comprises one or more dZ-containing DNA molecules having a dZ content of 0% - 60%.PATENT APPLICATION NEB-491-PCT 10. An E. coli cell according to any preceding claim, wherein at least one of the dZ- containing DNA molecules is a plasmid DNA.

11. An E. coli cell according to any preceding claim, wherein at least one of the dZ- containing DNA molecules comprises a recognition site for a type II restriction endonuclease selected from the group consisting of AatII, AccI, AfeI, ApaLI, AseI, AvaII, BamHI-HF, BbsI, BciVI, BfaI, BfuAI, BpmI, BsaWI, BsgI, BsiHKAI, BspDI, BspEI, BspHI, BstZ17I, BtsIMUTI, ClaI, DpnII, DraI, EarI, EcoNI, EcoP15I, FatI, HindIII-HF, HinfI, MboI, MscI, NdeI, NheI-HF, NruI-HF, PciI, PflMI, PpuMI, PstI-HF, SphI-HF, SspI-HF, StyI-HF, TfiI, TspRI, and XmnI.

12. An E. coli cell according to any preceding claim, wherein at least one of the dZ- containing DNA molecules comprises a recognition site for a type II restriction endonuclease selected from the group consisting of AflIII, AhdI, AluI, AlwI, AlwNI, ApeKI, ApoI, AvaI, BanI, BbvI, BccI, BceAI, BcoDI, BmrI, BsaHI, BsaI-V2, BseYI, BsiEI, BsmAI, BsmFI, BsoBI, Bsp1286I, BspCNI, BsrBI, BsrFI-V2, BsrI, BstAPI, BstYI, BtgI, BtsCI, CviAII, DrdI, EaeI, EciI, EcoO109I, FokI, HaeII, HgaI, HphI, Hpy166II, Hpy188I, Hpy188III, HpyAV, HpyCH4III, HpyCH4IV, HpyCH4V, MboII, MlyI, MnlI, MseI, MslI, MspA1I, NlaIII, PflFI, PleI, PshAI, PspGI, RsaI, SalI-HF, SfaNI, SfcI, SmlI, TaqI, TseI, Tsp45I, and Tth111I.

13. An E. coli cell according to any preceding claim, wherein at least one of the dZ- containing DNA molecules comprises: (a) a recognition site for a type II restriction endonuclease selected from the group consisting of AatII, AccI, AfeI, ApaLI, AseI, AvaII, BamHI-HF, BbsI, BciVI, BfaI, BfuAI, BpmI, BsaWI, BsgI, BsiHKAI, BspDI, BspEI, BspHI, BstZ17I, BtsIMUTI, ClaI, DpnII, DraI, EarI, EcoNI, EcoP15I, FatI, HindIII-HF, HinfI, MboI, MscI, NdeI, NheI-HF, NruI-HF, PciI, PflMI, PpuMI, PstI-HF, SphI-HF, SspI-HF, StyI- HF, TfiI, TspRI, and XmnI; and (b) a recognition site for a type II restriction endonuclease selected from the group consisting of AflIII, AhdI, AluI, AlwI, AlwNI, ApeKI, ApoI, AvaI, BanI, BbvI, BccI, BceAI, BcoDI, BmrI, BsaHI, BsaI-V2, BseYI, BsiEI, BsmAI, BsmFI, BsoBI, Bsp1286I, BspCNI, BsrBI, BsrFI-V2, BsrI, BstAPI, BstYI, BtgI, BtsCI, CviAII, DrdI, EaeI, EciI, EcoO109I, FokI, HaeII, HgaI, HphI, Hpy166II, Hpy188I, Hpy188III, HpyAV, HpyCH4III, HpyCH4IV, HpyCH4V, MboII, MlyI, MnlI, MseI, MslI,PATENT APPLICATION NEB-491-PCT MspA1I, NlaIII, PflFI, PleI, PshAI, PspGI, RsaI, SalI-HF, SfaNI, SfcI, SmlI, TaqI, TseI, Tsp45I, and Tth111I.

14. An E. coli cell according to any preceding claim, wherein the cell further comprises at least one transcription product of at least one of the dZ-containing DNA molecules.

15. A lysate of an E. coli cell according to any preceding claim.

16. A method of synthesizing a DNA comprising dZ, the method comprising contacting: (a) an E. coli cell according to any of Claims 1-14 or a lysate according to Claim 15; and (b) a polynucleotide template exogenous to the E. coli cell or exogenous to the lysate, wherein the E. coli cell or the lysate comprises one or more dNTPs, at least one of which is dZTP, and one or more DNA polymerases or one or more reverse transcriptases to produce a dZ-containing DNA.

Images