Enzymatic synthesis of key RNA therapeutic building blocks using simple phosphate donors

Domain: chemistry
Published: 2025-12-17
Editorial outcome: major_revision

Abstract
The rapid emergence of RNA therapeutics has highlighted the need for more efficient, scalable and sustainable methods for their manufacture. Biocatalytic approaches hold particular promise, but rely on a secure, sustainable and low-cost supply of nucleoside triphosphate (NTP) building blocks, including those containing chemical modifications. Here we report the development of a biocatalytic approach and engineered enzymes to convert widely available nucleosides into NTPs featuring pharmaceutically relevant modifications using inexpensive phosphate donors. Importantly our strategy obviates the need for ATP as a phosphate donor that complicates NTP isolation using existing methods. To showcase the utility of our approach, we employ an engineered acid phosphatase, polyphosphate kinase and acetate kinase to produce 2′-O-methoxyethyl-ATP (2′-MOE-ATP) and 2′-fluoro-ATP, key building blocks of commercial therapeutics. Finally, we show that crude NTPs from our process can be used directly in enzymatic oligonucleotides synthesis, obviating the need for costly NTP isolation or purification steps.

Introduction
Recent years have seen rapid expansion in the field of RNA therapeutics1,2. For example, mRNA vaccines were developed in response to the COVID-19 pandemic, and mRNA therapeutics are now emerging for the treatment of cancers and genetic disorders3–7. An increasing number of antisense oligonucleotides (ASOs) and small-interfering RNAs (siRNAs) have also been approved and many more are in advanced stages of clinical trials8,9. To address the increasing demand for high volumes of RNA products, more efficient and scalable synthetic methods are urgently needed10,11. In this regard, several complementary biocatalytic strategies have been developed using terminal deoxyribonucleotidyl transferases (TdTs)12–15, RNA-16,17, DNA-18, or Poly(U)- polymerases19. A common feature of these approaches is the requirement for nucleoside triphosphates (NTPs) as monomeric building blocks. However, NTPs are notoriously challenging to chemically synthesize due to poor solubility in organic solvent and the presence of multiple reactive groups on the ribose sugar and nucleobases. The challenges of efficient NTP synthesis are amplified by the common requirement for chemical modifications to the ribose sugar, nucleobase and/or phosphate backbone, which confer beneficial pharmacokinetic and pharmacodynamic properties2,9,20. Traditional P(III)-based approaches such as the Yoshikawa and Ludwig-Eckstein methods work well for some NTPs, but are generally hampered by low yields, the need for moisture- and air-sensitive reagents, the requirement for protecting groups and complex purifications21,22. Alternative approaches involving the use of derivatized P(III) phosphoramidites23–25, P(V) organic pyrophosphates26 or triphosphate transfer reagents27 offer some advantages and facilitate isolation/purification of NTP products. However, these methods rely on specialized reagents that are produced through multi-step chemical synthesis. As a result, production of modified NTPs in a scalable, economical and sustainable fashion remains a major challenge.

Beyond enzymatic synthesis of RNA therapeutics, biocatalysis could offer a more efficient route to the requisite NTP building blocks. In nature, nucleosides can be sequentially phosphorylated through the action of kinases that transfer a γ-phosphate from ATP to nucleoside, nucleoside monophosphate (NMP), or nucleoside diphosphate (NDP) substrates. This approach has formed the basis of biocatalytic strategies to produce NTPs (Fig. 1A)28–34; however these kinases have not been shown to operate on many substrates featuring common 2′-ribose modifications or 3′- protecting groups that are required for enzymatic synthesis of oligonucleotide therapeutics. Moreover, the use of ATP as a phosphate donor raises several challenges. ATP is expensive, necessitating the use of ancillary enzymes for cofactor recycling that must operate in conjunction with the three kinases required for NTP synthesis. Furthermore, when producing NTPs that are close structural analogues of ATP (e.g., 2′-flouro ATP), the presence of ATP and ADP by-products complicates downstream purification. Given that ATP is commonly an excellent substrate for downstream enzymes used during oligonucleotide synthesis16–19, even low levels of ATP impurities can result in heterogeneous product mixtures. An elegant solution to avoid the requirement for ATP involves the development of engineered kinases that can use catalytic concentrations of target NTP products as a phosphate donor in conjunction with an appropriate recycling system29. This approach was recently used to produce 2′-F-(Sp)-thioATP for production of a cyclic dinucleotide analogue29, however, it unknown whether cofactor specificity can be more extensively reprogrammed towards alternative bases and/or ribose modifications.

To address existing limitations of NTP synthesis, here we establish a versatile ATP-free biocatalytic approach (Fig. 1B) that is suitable for preparative scale synthesis and enables production of a wide variety of therapeutically relevant NTP building blocks. We began by selecting an engineered non-specific acid phosphatase from Morganella morganii containing G92D and I171T mutations (PhoC_1) that had previously been shown to reduce competing enzyme catalyzed hydrolysis of 5′-phosphorylated nucleoside products35. This variant has been used to prepare 5′-inosine monophosphate on a 100 g scale, making it an attractive candidate for further development as an industrial biocatalyst for modified NTP production35. However, despite its potential there is little known about the ability of this enzyme to operate on chemically modified nucleosides as required for our target applications. Indeed, initial activity assays performed on a small panel of commercial nucleosides reveal that enzyme activity is severely compromised on substrates containing modifications that are commonly found in therapeutic sequences (see below). To overcome these limitations, we elected to broaden the substrate tolerance of PhoC_1 through enzyme engineering. We selected 2′-O-methoxyethyl adenosine (2′-MOE-A, 1a) as a target substrate for evolutionary optimization using pyrophosphate as a phosphate donor (Fig. 2A). 2′-MOE modified nucleotides are found in approved oligonucleotide therapeutics, including mipomersen and inotersen, yet the corresponding 2′-MOE-NTP building blocks are not widely available from commercial suppliers2,9,10. The evolutionary strategy consisted of two rounds of saturation mutagenesis targeting residues in the active site, second coordination sphere and flexible loop regions. In each round 20 positions were individually randomized using NNK degenerate codons (theoretical library size of 640 genetic variants per round). Individual variants were arrayed as clarified cell lysates in 96-well microtitre plates and evaluated using a high-throughput UPLC assay monitoring the conversion of 1a to 1b. The most promising (ca. 1%) clones were then evaluated as purified proteins for improved activity (Supplementary Fig. 1). Beneficial mutations identified during each round were combined by overlap extension PCR. The most active variant (PhoC_4) to emerge following evaluation of more than 3500 clones contains three mutations (A90E, N151A and D154L) and displays a substantial eightfold improvement in the conversion of 1a to 1b under the assay conditions used during evolution (96% and 12% conversion with PhoC_4 and PhoC_1, respectively) (Fig. 2B). This improvement arises from a tenfold increase in the rate of 1a phosphorylation, alongside a more modest 1.4-fold increase in the rate of competing 2′-MOE-AMP (1b) hydrolysis, showing how the mutations installed during evolution have led to a more favourable partitioning of PhoC activities (Fig. 2C, D). Kinetic analysis shows PhoC_4 displays a ninefold improvement in catalytic efficiency (kcat/KM) compared with the parent enzyme PhoC_1. These improvements are attributed to a 3.5-fold reduction in KM and a threefold improvement in kcat (PhoC_1 kcat = 0.3 s−1; PhoC_4 kcat = 0.8 s−1, Supplementary Fig. 2).

Results
To understand the role of mutations installed during evolution, PhoC_1 and PhoC_4 were both crystallized and diffracted to 2.2 and 2.0 Å, respectively. Crystals formed in space group P3 12 with three molecules present in the asymmetric unit. Expansion of the crystal symmetry, supported by PISA analysis43,44, indicates a stable hexameric quaternary structure (trimer of dimers) similar to that observed in the acid phosphatase from Escherichia blattae (pdb codes 1D2T & 1EOI)45. Individual subunits from PhoC_1 and PhoC_4 can be superimposed with an RMSD of 0.15 Å (Supplementary Fig. 3). A comparative analysis of PhoC_4 with 1D2T and 1EOI suggests that the three mutations installed during PhoC_4 evolution (A90E, N151A and D154L) all reside in dynamic regions of the structure. N151A and D154L, reside on a poorly resolved loop region (residues 151–159) similar to that observed in 1D2T (residues 133–143). In 1EOI, which contains molybdate as a transition state analogue, this loop undergoes a structural rearrangement into a helical architecture to create a more occluded binding pocket. Based upon sequence and structural alignment, A90E in PhoC_4 is equivalent to residue S72 in 1EOI, which undergoes a 6 Å reorientation upon molybdate binding. In the absence of a substrate or product-bound PhoC_4 complex, docking studies were performed on a PhoC_4 model (PhoC_4*) generated using molecular dynamics to produce a conformation similar to that observed in 1EOI45. The observed docking pose of 2′-MOE-AMP and key interactions are presented in Fig. 2E. In this model, Arg201 and His168 adopt alternative conformers to those observed in the apo-structures to facilitate phosphate binding through polar interactions. Replacement of His168 by alanine in PhoC_4 led to a 48-fold reduction in activity, while mutation of Arg201 to alanine completely abolishes activity. Although further studies are needed to elucidate the specific role of the polar to hydrophobic N151A and D154L mutations, this analysis suggests that the flexible loop region 151–159 plays an important role in substrate recognition and/or the partitioning of phosphoryl transfer vs hydrolysis activity, marking this region as an attractive target for more extensive engineering to develop more proficient biocatalysts.

With an improved PhoC variant in hand, we next evaluated its activity towards a diverse panel of canonical and modified nucleosides (1a–32a, Fig. 3). Compared with the PhoC_1 starting variant, PhoC_4 displays improved efficiency towards all substrates tested. In addition to 2′-MOE-A (1a), PhoC_4 displays high levels of activity towards other 2′-MOE nucleosides (20–22a) as well as substrates with other therapeutically important 2′-substiuents including 2′-fluoro- (12a–14a), 2′-methoxy- (16a–18a) and challenging locked nucleic acids (23a–24a & 26a). Interestingly C1 and base modifications are also well tolerated. PhoC_4 was also efficient at converting protected 3′-substituted nucleosides (27a, 29a–30a) that are important building blocks used in non-templated approaches to enzymatic oligonucleotide synthesis12–15,19. With a view to future process intensification and scale-up, we next explored the activity of PhoC_4 at elevated nucleoside concentrations (25–100 mM, Supplementary Table 1). Importantly even at low catalyst loadings (20 µM) and using only twofold excess of pyrophosphate, high reactions conversions are maintained across a range of modified substrates. These studies reveal that PhoC_4 can readily tolerate 10% DMSO (Supplementary Fig. 4) as a cosolvent and in favourable cases can achieve >5000 turnovers.

Next, our attention turned to the elaboration of PhoC-produced NMPs into the corresponding NTPs using a combination of PPKs and AcK. To this end, we produced a panel of 9 PPK homologs39,40 and evaluated their activity towards a subset of NMPs, including those containing each of the four nucleobases, 3′-protecting groups and the most common 2′-modifications found in oligonucleotide therapeutics. These NMP substrates were prepared via PhoC_4 mediated biotransformations under intensified conditions followed by filtration to remove PhoC_4, precipitation of unreacted pyrophosphate using MgCl2 and pH adjustment to the optimal operating window of PPK. From these assays, we were able to identify PPK homologs with activity towards all substrates tested to produce mixtures of NDP and NTP products (Fig. 4A and Supplementary Table 2). As an alternative to this two-step biotransformation PhoC_4 and PPK can be used in a single enzymatic cascade if pyrophosphate, which inhibits PPK activity, is replaced by phenyl phosphate as a donor (Supplementary Fig. 5). To further improve conversion to the target NTPs, we included a wild-type acetate kinase (AcK) from Thermotoga maritima that phosphorylates NDPs using acetyl phosphate as a donor29. With all substrates tested, addition of AcK resulted in substantially improved NTP conversion demonstrating the substrate promiscuity of the wild-type enzyme. Overall, using our three-enzyme system we successfully produced nine structurally diverse NTPs, featuring different nucleobases, 2′- and 3′-modifications, from the parent nucleoside in conversion ranging from 40 to 82% (Fig. 4B).

To demonstrate the practical utility of our biocatalytic approach, we next performed preparative scale transformations using 2′-MOE-ATP (1d) as a synthetic target. Under optimized conditions, the parent nucleoside (163 mg) was converted to 2′-MOE-ATP in 76% conversion (2′-MOE-ADP, 8%; 2′-MOE-AMP, 6%; 2′-MOE-A, 10%) using <0.06 mol% loading of each of the PhoC_4, PPK and AcK biocatalysts. Importantly, the synthesis required no costly intermediate purification steps. The reaction conversions are almost identical to those achieved in small-scale biotransformations, showing that our approach can be readily scaled from 100 µL to 10 mL without compromising efficiency. The 2′-MOE-ATP product was isolated following DEAE Sepharose ion exchange purification to provide 1d in >99% purity and 65% isolated yield (Fig. 5A and Supplementary Fig. 6). Finally, we evaluated the performance of our enzymatically synthesized 2′-MOE-ATP (1d) as a substrate for biocatalytic oligonucleotide synthesis. Here, we employed our recently reported method that uses an engineered DNA polymerase and endonuclease V that work in synergy to amplify a catalytic self-priming template to furnish modified oligonucleotide sequences in a single operation (Fig. 5B)18. Using an engineered polymerase Tgo2M46 and endonuclease V (EndoV) from Thermatoga neopolitana18, enzymatically synthesized 2′-MOE-ATP was converted to a poly-2′-MOE-A 5mer oligonucleotide sequence. Importantly, analogous reactions using chemically synthesized 2′-MOE-ATP purified by silica chromatography gave near identical results, affording the target sequence in >90% purity alongside truncated sequences as minor impurities (Fig. 5C). Thus, these results confirm the suitability of our enzymatically synthesized NTPs as building blocks for modified oligonucleotide production.

To further showcase the utility of our approach, we next elected to prepare 2′-F-ATP and investigate its incorporation into oligonucleotides. Given its close structural similarity to ATP, isolation of high-purity 2′-F-ATP from established ATP-dependent biocatalytic processes can be especially challenging. In this case, we prepared 2′-F-ATP from the parent nucleoside (713 mg) in 77% conversion (Supplementary Fig. 7). Seeking further improvements in efficiency, we recognized the potential to use enzymatically produced 2′-F-ATP in crude form directly in biocatalytic oligonucleotide synthesis, thus obviating the need for NTP purification steps. To our delight, crude 2′-F-ATP could be used directly in our polymerase and endonuclease cascade to furnish a poly-2′-F-A 8mer in 70% conversion. Efforts to produce poly-2′-F-A sequences using 2′-F-ATP in the presence of low concentrations (from 1 to 10 mol%) of ATP contaminants gave complex product distributions arising from competing ATP and 2′-F-ATP incorporation (Supplementary Fig. 8).

We have established a sustainable and potentially scalable biocatalytic approach to NTP synthesis using inexpensive and widely available phosphate donors. Importantly, our method obviates the need for ATP, which poses numerous challenges for existing biocatalytic routes to NTP production. Our method is versatile and can be applied to the synthesis of a wide range of modified products containing 3′-protecting groups and 2′-modifications commonly found in therapeutic products. Key to the success of our platform was the development of an engineered acid phosphatase with broad substrate specificity. Moving forward, there are clear opportunities to make further improvements to our process. The development of highly evolved PhoC, PPK and AcK variants that are optimized toward target substrates of interest and minimize off-pathway processes (e.g., NMP hydrolysis by PhoC) will lead to enhanced productivity and reduced enzyme loadings. Here, there is also the potential to develop enzymes with similar pH-optima to avoid the need for pH adjustments during the process. For large scale NTP manufacture, product isolation and purification will preferably be carried out by precipitation and/or crystallization to avoid the need for ion exchange resins29. Alternatively, as shown within this study, crude NTPs from our process can be used directly in enzymatic oligonucleotide synthesis, avoiding the need for NTP purification. It is unlikely that such an approach could be adopted with ATP-dependent methods, as even low concentrations of ATP contaminants severely compromise the purity of downstream oligonucleotide products. Our approach also offers several advantages over established chemical methods for NTP synthesis that rely on the use of toxic and difficult to remove solvents such as trimethylphosphate, air/moisture-sensitive and counterion-specific reagents, and the preparation and isolation of reactive intermediates [P(V) or P(III) species]. In light of these advantages, we anticipate that our biocatalytic platform will be widely adopted for the preparation of canonical and modified NTPs needed to underpin the biocatalytic production of important RNA therapeutics12–19.

Methods
Materials
Reagents and chemicals were obtained from commercial suppliers and were used without further purification unless otherwise stated. Lysozyme, kanamycin sulphate, tetrapotassium pyrophosphate, sodium hexametaphosphate, lithium potassium acetyl phosphate, 2c and 2d were purchased from Sigma-Aldrich, polymyxin B sulfate, 12a, and 15a from Alfa Aesar, LB agar, LB medium, and Isopropyl β-D-1-thiogalactopyranoside (IPTG) from Formedium. Phenyl phosphate, 2b, 13a, 16a, 17a, and 19a were purchased from Thermo Scientific, 2a, 4a–8a, and 18a from Fluorochem, 3a and 9a from Tokyo Chemical Industry, 1a, 14a, 20a–22a from Biosynth, 10a and 11a from MedChemExpress, 23a–26a from Sapala Organics. Compounds 29a–32a were provided by Nuclera Ltd (Cambridge). Nucleosides 27a and 28a, standards of 1b and 1d were chemically synthesized in house (see supplementary information). E. coli DH5α, E. coli BL21(DE3), Q5 DNA polymerase, T4 DNA ligase and restriction enzyme BsaI were purchased from New England BioLabs and E. coli C43(DE3) from Cambridge Bioscience. Oligonucleotides were synthesized by Integrated DNA Technologies. PPK4-PPK9 were obtained from Prozomix. Diafiltration devices (Vivaspin) were purchased from Sartorius AG. Sequencing was performed by Source BioScience (Nottingham).
Expression plasmids: Genes encoding PhoC_1, PPK1, PPK2, PPK3, and AcK were ordered from Integrated DNA Technologies (IDT) as gBlocks with codon optimization for recombinant expression in E. coli. The genes were cloned into pET-28a using Golden Gate Assembly (Nucleotide sequences of ORFs are shown below).

Protein expression and purification
For recombinant expression of PPKs and AcK, the respective plasmids were transformed into chemically competent E. coli BL21(DE3) cells. For expression of PhoC and variants, chemically competent E. coli C43(DE3) were transformed with the relevant pET28a_PhoC constructs. Following transformation and plating on LB agar containing 50 µg/mL kanamycin, 5 mL starter cultures of LB medium (50 µg/mL kanamycin) were inoculated with single colonies of freshly transformed cells and incubated for 18 h at 37 °C and 180 rpm. Starter cultures were used to inoculate 50 mL of LB medium supplemented with 50 µg/mL kanamycin. Cultures were grown at 37 °C, 180 rpm to an optical density of ~0.6 at 600 nm (OD600). Protein expression was induced with the addition of IPTG to a final concentration of 1 mM. Induced cultures were incubated for 20 h (PPKs and AcK) or 4 h (PhoC and variants) at 30 °C, 180 rpm. Cells were subsequently harvested by centrifugation (3220 × g for 10 min). Pelleted cells were resuspended in lysis buffer (PhoC and variants: 1 mg/mL lysozyme, 0.5 mg/mL polymyxin B, 20 mM sodium acetate, 300 mM NaCl, pH 7.5; PPKs & AcK: 1 mg/mL lysozyme, 0.5 mg/mL polymyxin B, 50 mM Tris-HCl, 300 mM NaCl, pH 7.5) and lysed by sonication. Cell lysates were cleared by centrifugation (27,216 × g for 30 min). Supernatants were loaded onto Ni-NTA agarose (Qiagen) gravity-flow columns for affinity purification and washed with wash buffer (PhoC and variants: 20 mM sodium acetate, 300 mM NaCl, pH 7.5, 10 mM imidazole; PPKs & AcK: 50 mM Tris-HCl, 300 mM NaCl, 10 mM imidazole, pH 7.5). Proteins were eluted using 500 mM imidazole, 300 mM NaCl, 20 mM sodium acetate, pH 7.5 for PhoC and variants, or 50 mM Tris-HCl, pH 7.5 for PPKs & AcK. Proteins were desalted on 10DG gel filtration columns (Bio-Rad) equilibrated with buffer (PhoC and variants: 20 mM sodium acetate, pH 5.0; PPKs & AcK: 50 mM Tris-HCl, pH 7.5) and analysed by SDS–PAGE. Proteins were aliquoted, flash-frozen in liquid nitrogen and stored at −80 °C. Protein concentrations were determined by measuring the absorbance at 280 nm on a NanoDrop spectrometer (Nanodrop 1000.3.8.1). Extinction coefficients were calculated based on the respective amino acid sequence using Expasy ProtParam (https://web.expasy.org/protparam/).

PhoC library generation
Selected amino acid positions were individually randomized using degenerate NNK primers (see supplementary Table S3). DNA libraries were generated by overlap extension polymerase chain reaction (PCR). Assembled and gel purified genes were subsequently inserted into a customised pET28a vector using Golden Gate Assembly in a 5:1 ratio, respectively, using T4 DNA ligase (20 U/µL) and BsaI (1 U/µL). Golden gate reactions were directly transformed into E. coli DH5α cells. The resulting colonies on LB-agar (50 µg/ml kanamycin) plates were pooled together, and plasmids were extracted by Miniprep (QIAGEN) to yield plasmid DNA for each library.

PhoC library screening
For protein expression and screening, all transfer and aliquoting steps were performed using Hamilton liquid-handling robots running Alvaro Cuevas 2010 software. Chemically competent E. coli C43(DE3) cells were transformed with the appropriate library plasmids and plated on LB agar (50 µg/ml kanamycin) plates. Freshly transformed colonies were used to inoculate 180 μL of LB (50 µg/mL kanamycin) in Corning Costar 96-well microtitre round-bottom plates. Each plate contained six freshly transformed clones of the parent template PhoC_1 and two catalytically inactive clones (PhoC_1_H168A/H207A) in E. coli C43(DE3). Plates were sealed with gas permeable membranes (Wolflabs) and incubated overnight at 30 °C, 80% humidity in a shaking incubator at 850 rpm. 20 µL of overnight culture was used to inoculate 480 μL of LB medium supplemented with 50 µg/mL kanamycin in 2 mL 96 deep well plates. The cultures were incubated at 30 °C, 80% humidity with shaking at 850 rpm until an OD600 of ~0.6 was reached. Protein expression was induced by the addition of IPTG to a final concentration of 1 mM. Induced plates were incubated for 4 h at 30 °C, 80% humidity, with shaking at 850 rpm. Cells were harvested by centrifugation at 3220 × g for 10 min. The supernatant was discarded and the cell pellets were resuspended in 200 μL of lysis buffer (20 mM sodium acetate, 1 mg/mL lysozyme, and 0.5 mg/mL polymixin B) and incubated for 2 h at 30 °C, 80% humidity with shaking at 850 rpm. Cell debris was removed by centrifugation at 3220 × g for 10 min, and 50 μL of cell free extract was transferred to fresh 96-well microtitre plates. 50 μL Reaction mixture was added to give a final concentration of 200 mM potassium pyrophosphate, 1 mM 2′-MOE-adenosine (1a), and 20 mM sodium acetate (pH 4). Plates were incubated at 30 °C, 850 rpm, and 80% humidity and after 16 h, the reaction was terminated by the addition of 100 µL methanol (final concentration 50% v/v). After 10 min centrifugation at 3220 × g, 50 μL of the reaction mixture was transferred to fresh 96-well microtitre plates for UPLC analysis. Plasmids from variants with improved activity were isolated and sequenced. The most active clones from each round were rescreened as purified proteins. Following each round of evolution, beneficial mutations were combined by overlap extension PCR and evaluated as purified proteins. The best variant was selected as the parent template for the subsequent round of directed evolution.

Chromatographic analysis
LC-MS analysis was performed using an Agilent 1290 Infinity II LC/MSD running in negative ESI mode at 4.0 kV capillary, acquiring up to 2000 m/z. Signals were recorded at 260 nm and analysed using Agilent OpenLab software (3.6). Nucleosides and NMPs were analysed using an InfinityLab Poroshell 120 EC-C18 column (2.1 × 100 mm, 1.9 µm). Compounds were eluted over 10 min using a gradient of 5–70% eluent B at 0.6 mL/min. Eluent A: 10 mM ammonium acetate, pH 3.6; Eluent B: Acetonitrile. Oligonucleotides were analysed on an AdvanceBio Oligonucleotides 2.7 µm column, 50 × 2.1 mm (Agilent) at 60 °C. Oligonucleotide sequences were separated over 14 min using a gradient of 2–50% eluent B at 0.3 mL/min. Eluent A: 400 mM hexafluoro isopropanol and 20 mM triethylamine in water; Eluent B: Methanol.
All HPLC analysis was performed on an Agilent 1290 Infinity II LC system, signals were recorded at 260 nm. Peak areas were integrated using Agilent OpenLab software (3.6) and assigned by comparison to commercial or chemically synthesized standards. PhoC libraries were evaluated using an InfinityLab Poroshell 120 EC-C18 column (3.0 × 30 mm, 2.7 µm). Eluent A: 60 mM ammonium acetate, pH 3.6; Eluent B: Acetonitrile. Separation of 2′-MOE-adenosine monophosphate (1b) from 2′-MOE-adenosine (1a) was achieved using a gradient of 5–12% B at 1 mL/min for 1 min. PPK and AcK reactions were analysed using a ZORBAX Eclipse Plus C18 column (2.1 × 50 mm, 1.8 µm) at 40 °C. Eluent A: 50 mM hydroxylamine adjusted to pH 7 with acetic acid; Eluent B: 65:35 (v/v) mixture of isopropanol: acetonitrile. Nucleosides and nucleotides (NMP, NDP, NTP) were eluted over 4 min using a gradient of 5–90% eluent B at 0.6 mL/min.

Steady state kinetic assays
Kinetic assays were performed using pyrophosphate (200 mM), 2′-MOE-A (1a, 0–256 mM) and enzyme (5 µM) in 20 mM sodium acetate buffer pH 4 at 30 °C. Samples taken at different time points were quenched with the addition of 1 volume methanol. Initial rates were measured using HPLC and product concentration was determined by comparison to commercial standards. Assays were performed in triplicate, and the averaged initial rates were fitted to the Michaelis–Menten equation using GraphPad Prism 9 software (9.1.2) (Supplementary Fig. 2).

Substrate profiling of PhoC_1 and PhoC_4
100 µL reactions containing 1 mM nucleoside (1–32a), 200 mM pyrophosphate, 20 µM purified PhoC_1 or PhoC_4, 20 mM sodium acetate, pH 4 were assembled and incubated at 30 °C, 850 rpm. Reactions containing no enzyme served as a negative control. Reactions were quenched with the addition of 100 µL methanol and precipitated protein was removed by centrifugation. Cleared reactions were analysed by LC-MS analysis. In total 96 samples were evaluated, using a single replicate.

Evaluation of PhoC_4 R201A and H168A variants
Arg201 and His168 were individually mutated to alanine in PhoC_4. DNA genes were generated by overlap extension PCR. Assembled and gel purified genes were subsequently inserted into a customised pET 28a vector using Golden Gate Assembly in a 5:1 ratio, using T4 DNA ligase (20 U/µL), and BsaI (1 U/µL). Golden gate reactions were directly transformed into E. coli DH5α cells. Plasmids were extracted by Miniprep (QIAGEN) and mutations were confirmed by sequencing. PhoC_4 R201A and PhoC_4 H168A were expressed and purified according to the general protocol. To evaluate their activity, reactions containing 1 mM 2′-MOE-A, 200 mM pyrophosphate, 20 µM purified PhoC_4, PhoC_4 R201A or PhoC_4 H168A, 20 mM sodium acetate, pH 4 were assembled and incubated at 30 °C, 850 rpm. Reactions were quenched with the addition of 100 µL methanol and precipitated protein was removed by centrifugation. Cleared reactions were analysed by LC-MS analysis and the conversion to product was determined by comparison to chemically synthesized 2′-MOE-AMP (see supplementary information).

Substrate profiling of a PPK panel
1 mL reactions containing 25–100 mM nucleoside (Supplementary Table 1), 200 mM potassium pyrophosphate, 20 µM purified PhoC_4, 20 mM sodium acetate, pH 4 were assembled and incubated at 30 °C, 850 rpm for 4–16 h. Next, PhoC was removed by diafiltration using a 10 kDa filter to terminate the reaction. The filtrate was collected and pH adjusted to 7 by addition of 400 µL 200 mM Tris-HCl, pH 9 and 200 µL 5 M NaOH. Residual pyrophosphate was precipitated by addition of 400 µL 750 mM MgCl2. After centrifugation, 50 µL of the buffer adjusted NMP containing solution was mixed with 50 µL reaction mixture. The final reaction contained 20 mg/mL sodium hexametaphosphate, 50 mM MgCl2, 1 mg/mL lyophilized PPK cell free extract (Prozomix), 50 mM Tris-HCl, pH 7 and was incubated at 30 °C, 850 rpm for 16 h. Reactions were terminated by diafiltration (10 kDa filter) and analysed by HPLC.

One-pot PhoC_4 and PPK cascade
A 100 µL reaction containing 10 mM adenosine (2a), 200 mM potassium pyrophosphate or phenyl phosphate, 20 mg/mL sodium hexametaphosphate, 50 mM MgCl2, 20 µM purified PhoC_4, PPK5 (Prozomix) as lyophilized cell-free extracts (1 mg/mL), 20 mM sodium acetate and 50 mM Tris-HCl pH 5 were assembled and incubated at 30 °C and 850 rpm for 16 h. Reactions were terminated by diafiltration (10 kDa filter) and analysed by HPLC.

Analytical scale NTP synthesis
1 mL reactions containing 25–100 mM nucleoside, 200 mM potassium pyrophosphate, 20 µM purified PhoC_4 and 20 mM sodium acetate pH 4 were assembled and incubated at 30 °C, 850 rpm for 4–16 h (Supplementary Table 1). Next, PhoC_4 was removed by diafiltration using a 10 kDa filter to terminate the reaction. The filtrate was collected and pH adjusted to 7 by addition of 400 µL 200 mM Tris-HCl pH 9 and 200 µL 5 M NaOH. Residual pyrophosphate was precipitated by addition of 400 µL 750 mM MgCl2. After centrifugation, 50 µL of the buffer-adjusted NMP solution was mixed with 50 µL reaction mixture. The final reaction contained 20 mg/mL sodium hexametaphosphate, 50 mM MgCl2, 1 mg/mL lyophilized PPK cell free extract (Prozomix) and 50 mM Tris-HCl pH 7, and was incubated at 30 °C, 850 rpm for 16 h. The reaction was terminated by diafiltration (10 kDa filter) and 50 µL of filtrate was added to 50 µL AcK reaction mixture. The final reaction contained 50 mM lithium potassium acetyl phosphate, 5 mM MgCl2, 25 µM purified AcK, and 50 mM Tris-HCl pH 7, and was incubated at 30 °C, 850 rpm for 16 h. Reactions were terminated by diafiltration (10 kDa filter) and analysed by HPLC.

Preparative scale 2′-MOE-ATP (1d) synthesis
All incubation steps were performed using an EasyMax 102 Advanced Thermostat reactor (Mettler Toledo) equipped with a magnetic stir bar and incubated at 30 °C, 100 rpm. A 10 mL reaction was prepared containing 50 mM 2′-MOE-adenosine (1a), 200 mM potassium pyrophosphate, 20 µM purified PhoC_4 and 20 mM sodium acetate, pH 4. Following 12 h incubation PhoC_4 was removed by diafiltration using a 10 kDa filter to terminate the reaction. The filtrate containing 2′-MOE-AMP (1b) was collected and pH adjusted to 7 by adding 4 mL 200 mM Tris-HCl, pH 9 and 2 mL 5 M NaOH. Residual pyrophosphate was removed by precipitation with 4 mL 750 mM MgCl2. Following centrifugation, the 2′-MOE-AMP (1b) containing supernatant was added to a 20 mL reaction mixture containing 20 mg/mL sodium hexametaphosphate, 50 mM MgCl2, 30 µM purified PPK2 and 50 mM Tris-HCl pH 7. Following a further 16 h incubation, the reaction was terminated by diafiltration (10 kDa filter), and the filtrate was added to a 25 mL final reaction mixture containing 50 mM lithium potassium acetyl phosphate, 5 mM MgCl2, 25 µM purified AcK, and 50 mM Tris-HCl, pH 7. After 16 h AcK was removed by diafiltration (10 kDa filter).

Purification and isolation of 2′-MOE-ATP (1d)
2′-MOE-ATP (1d) was purified from the crude reaction mixture by DEAE sepharose ion exchange chromatography on an ÄKTA pureTM (running Cytiva Unicorn software 3.6), equipped with a Cytiva GEXK26 column packed with DEAE sepharose Fast Flow resin. Signals were recorded at 260 nm. Eluent A: water; Eluent B: 1 M ammonium bicarbonate. Separation of 1d from 1a, 1b and 1c was achieved using a gradient of 15–100% B. The purified product was isolated by freeze drying overnight and stored at −80 °C (Supplementary Fig. 4). 1H NMR (400 MHz, D2O) δ 8.51 (s, 1H), 8.25 (s, 1H), 6.19 (d, J = 6.2 Hz, 1H), 4.68 (dd, J = 5.2, 3.1 Hz, 1H), 4.59 (t, J = 5.7 Hz, 1H), 4.45–4.35 (m, 1H), 4.31–4.13 (m, 2H), 3.83 (ddd, J = 11.8, 5.8, 3.2 Hz, 1H), 3.70 (ddd, J = 11.9, 5.4, 3.2 Hz, 1H), 3.57–3.42 (m, 2H), 3.14 (s, 3H) ppm; 31P{1H} NMR (162 MHz, D2O) δ −7.68 (br s), −11.20 (d, J = 19.4 Hz), −22.30 (t, J = 19.4 Hz).

Poly-2′-MOE-A 5mer biosynthesis
A solution containing 4 µM DNA template (5′- (Bio)TTTTTCACGTGCACCATTGGTGCACGIG) and 1 mM 1d in buffer (50 mM Tris-AcOH, pH 8, 20 mM MgSO4, 100 mM potassium glutamate, 50 mM KOAc, 0.01 mg/ml Acetylated BSA, 30 mM DTT) was prepared according to previous reports18. The reaction was initiated with the addition of 5 µM DNA polymerase Tgo2M46 and 0.3 µM TnEndoV18 and incubated at 70 °C for 12 h. Reactions were quenched with the addition of one volume 20 mM EDTA. Samples were heated at 98 °C for 2 h and precipitated proteins were removed by centrifugation (14,000 × g) for 15 min. The supernatant was analysed by LC-MS.

Preparative scale 2′-F-ATP (12d) synthesis
All incubation steps were performed using an EasyMax 102 Advanced Thermostat reactor (Mettler Toledo) equipped with a magnetic stir bar and incubated at 30 °C, 100 rpm. A 50 mL reaction was prepared containing 50 mM 2′-F-adenosine (12a), 200 mM potassium pyrophosphate, 20 µM purified PhoC_4 and 20 mM sodium acetate, pH 4. Following 12 h incubation, PhoC_4 was removed by diafiltration using a 10 kDa filter to terminate the reaction. The filtrate containing 2′-F-AMP (12b) was collected and pH adjusted to 7 by adding 20 mL 200 mM Tris-HCl, pH 9 and 10 mL 5 M NaOH. Residual pyrophosphate was removed by precipitation with 20 mL 750 mM MgCl2. Following centrifugation, the 2′-F-AMP (12b) containing supernatant was added to a 150 mL reaction mixture containing 20 mg/mL sodium hexametaphosphate, 50 mM MgCl2, 30 µM purified PPK3 and 50 mM Tris-HCl pH 7. Following a further 16 h incubation, the reaction was terminated by diafiltration (10 kDa filter), and the filtrate was added to a 200 mL final reaction mixture containing 50 mM lithium potassium acetyl phosphate, 5 mM MgCl2, 25 µM purified AcK, and 50 mM Tris-HCl, pH 7. After 16 h AcK was removed by diafiltration (10 kDa filter) and the filtrate containing 2′-F-ATP (12d) was collected. The 2′-F-ATP concentration was measured using HPLC analysis by comparison to a commercial standard.

Poly-2′-F-A 8mer biosynthesis
A solution containing 4 µM DNA template (5′- (Bio)TTTTTTTTCACGTGCACCATTGGTGCACGIG), 10% formamide, 10 mM (NH4)2SO4, 10 mM KCl, 2 mM MgSO4, 0.1% Triton X-100, 2 mM NiCl2 and 20 mM Tris-HCl pH 8.8 was prepared according to previous reports18. Enzymatically synthesized 2′-F-ATP was added to give a final concentration of 1 mM. The reaction was initiated with the addition of 1 µM DNA polymerase TfPol*, 2 µM TnEndoV, and 4.5 U/mL pyrophosphatase, and incubated at 70 °C for 12 h. Reactions were quenched with the addition of one volume of chloroform. After centrifugation, the supernatant was analysed by LC-MS.

PhoC crystallization and modelling
Single crystals of PhoC_1 and PhoC_4 were prepared by mixing 200 nl of 10 mg ml−1 protein in crystallization buffer with equal volumes of precipitant. All trials were conducted by sitting drop vapour diffusion and incubated at 4 °C. Crystals of PhoC_1 appeared from a precipitant of 0.1 M Phosphate/Citrate pH 5.5 and 30% PEG Smear Low (12.5% v/v PEG 400, 12.5% v/v PEG 500 MME, 12.5% v/v PEG 600 and 12.5% PEG 1000) (BCS screen A2). PhoC_4 crystals were grown from a precipitant solution containing 0.1 M Carboxylic acids (0.2 M Sodium formate, 0.2 M Ammonium acetate, 0.2 M Sodium citrate tribasic dihydrate, 0.2 M Potassium sodium tartrate tetrahydrate and 0.2 M Sodium oxamate), 0.1 M Buffer system 3 pH 8.5 (1.0 M Tris base, Bicine pH 8.5), 37.5% v/v Precipitant Mix 4 (25% v/v MPD, 25% PEG 1000, 25% PEG 3350) (Morpheus G12). Crystals were flash cooled in liquid nitrogen prior to data collection. Data were collected from single crystals of PhoC_1 and PhoC_4 at Diamond Light Source (MX31850) and subsequently scaled and reduced with Xia247. Preliminary phasing was performed by molecular replacement using an AlphaFold 2 derived search model. Iterative cycles of rebuilding and refinement were performed in COOT48 and Phenix.refine49, respectively. Structure validation with MolProbity50 and PDBREDO51,52 was integrated into the iterative rebuild and refinement process. The resolution cutoff of the data was determined by paired refinement as implemented in PDBREDO. Complete data collection and refinement statistics can be found in Table S4. Coordinates and structure factors were deposited in the Protein Data Bank (PDB) under accession codes 9QCI (PhoC_1) and 9 QCJ (PhoC_4). Representative electron density maps for PhoC_1 and PhoC_4 are shown in Supplementary Fig. S9. Docking studies were performed in ICM-Pro (Molsoft) utilising a model derived from the crystal structure of PhoC_4. In the absence of a substrate or product bound complex of PhoC_4, it was necessary to generate a model incorporating features observed in 1EOI (the crystal structure of Escherichia blattae acid phosphatase complexed with the transition state analogue molybdate)45. The conformation of 1EOI was imposed onto the coordinates of PhoC_4 using molecular mechanics. All subsequent docking studies were directed towards this derived model of PhoC_4*.

Statistics and reproducibility
No statistical method was used to predetermine sample sizes. No data were excluded from the analyses. The experiments were not randomized and the investigators were not blinded to allocation during experiments and outcome assessment.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This manuscript describes an ATP-free biocatalytic approach that can be utilized to convert nucleosides of diverse chemistry into their corresponding nucleoside triphosphates (NTPs). To start the pipeline, the authors engineered a non-specific acid phosphatase (PhoC) to synthesize nucleoside monophosphates (NMPs) using inorganic pyrophosphatase as the phosphate donor. The most promising variant (PhoC_4), obtained by combing beneficial mutations identified by screening variants in microtiter plates, achieves 96% conversion of 2’-MOE-A into 2’-MOE-AMP. Structure determination studies and molecular modeling of PhoC_4 suggest that the flexible loop plays an important role in substrate recognition. Importantly, PhoC_4 exhibits improved activity across a range of nucleosides relative to the starting variant (PhoC_1). An optimized enzymatic cascade was then established utilizing PPK and AcK to convert NMPs into NTPs, with the successful demonstration of nine structurally diverse examples from their starting nucleoside. The enzymatic pathway was scaled, under optimized conditions to generate >150 mg of 2’-MOE-ATP, which was used to produce a poly-2’-MOE-oligonucleotide pentamer. Overall, the manuscript is interesting, but will require significant revision prior to publication.\n\nComments:\n1. The main concern is that certain areas of the manuscript are underdeveloped, lacking appropriate context, discussion, and referencing. Other areas contain jargony terms that should be avoided.\n2. Important details in the results and discussion section are lacking. Some examples include: (i) undefined statements such as “high conversion” on line 75; (ii) theoretical size of the library; (iii) number of clones evaluated; and (iv) a clear description of the model in Figure 2E as a chimera from prior structures.\n3. Nonstandard terms should be removed from the text. This includes “holistic biocatalytic approach”, “telescoped fashion”, “vide infra”, and “pleasingly”.\n4. The manuscript lacks adequate citations. Some examples include: (i) the MOE chemical modification (line 95); (ii) the synthesis of 100 g of 5’ IMP (line 86); and the entire methods paragraph on the crystal structure which fails to cite any of the programs used for structure refinement.\n5. The source of compound 1a is missing. Was this synthesized or purchased? If it was synthesized, the details are needed.\n6. The synthesis of a pentamer of 2’-MOE-modified polyA is not a suitable demonstration of an oligonucleotide therapeutic. The language around this example should be tempered. Discussion about the length versus Tm values of the extended product is warranted.\n7. The x-ray crystal structures of PhoC_1 and PhoC_4 warrant a more detailed structural comparison.\n8. The functional role of H168 and R183 should be empirically determined.\n9. The structural consequences of adaptive PhoC mutations should be discussed and shown in a figure, in particular E90, which is not located in the flexible loop.\n10. Molybdate is mentioned in the text, but not in the structure.\n11. In certain places, figure callouts are unclear. For example, the substrates from Figures 2 and 3 are being called out as 1a, 1b, etc. It is not clear which figure they are referring to.\n12. The comparison between the enzymatically produced poly-2’-MOE-A 5-mer and the authentic standard in Figure 5c should be more clearly described in the text.\n13. The manuscript would benefit from a summary of the strengths and weaknesses of the current strategy relative to other NTP synthesis approaches. Yields, time, purification, solvent, etc. would be useful information to share.", "reviewer_2": "The manuscript by Meng et al. reports the development of a fully enzymatic, ATP-free cascade for the production of nucleoside triphosphates (NTPs), including chemically modified variants used in therapeutic oligonucleotides. This work elegantly builds on the seminal work in Science (DOI: 10.1126/science.add589), also by the Lovelock group, to address a key bottleneck in the RNA therapeutics field, namely, the scalable and selective synthesis of modified NTPs, which are often challenging to prepare chemically. The work describes a three-enzyme system comprising an engineered acid phosphatase (PhoC), polyphosphate kinase (PPK), and acetate kinase (AcK), which together enable phosphorylation of a wide array of canonical and modified nucleosides using inexpensive phosphate donors and without the need for ATP. This approach significantly simplifies purification by eliminating ATP and ADP impurities that would otherwise complicate downstream processes.\n\nThe authors demonstrate a well-executed protein engineering campaign on PhoC to enhance its activity on therapeutically relevant substrates such as 2′-MOE-adenosine. Directed evolution led to a triple mutant (PhoC_4) with an 8-fold improvement in conversion and an expanded substrate scope to several therapeutically relevant compounds. Structural characterization of PhoC_4 and docking of 2′-MOE-AMP provide insight into the mutational effects and highlight a loop region as likely contributing to substrate engagement and activity partitioning. This sets the stage for future engineering efforts and adds mechanistic depth to the study. Following this, a panel of PPKs and a promiscuous AcK were used to advance monophosphates to the triphosphate level across a range of substrates, achieving conversions up to 82%. The enzymatic synthesis of 2′-MOE-ATP on preparative scale and its subsequent use in oligonucleotide assembly via a polymerase–endonuclease cascade further demonstrates the practical value of the method.\n\nThe manuscript is clearly written, methodically executed, and of broad relevance. Overall, this is an excellent contribution to the field and represents a clear advance in enabling the biocatalytic synthesis of NTP building blocks. The work will be of interest to researchers in biocatalysis, nucleic acid chemistry, and RNA therapeutic development. As such I recommend publication after minor revisions below:\n- While the engineering of PhoC_4 is thoroughly presented in terms of conversion data and structural interpretation, a kinetic comparison between PhoC_1 and PhoC_4 (e.g., kcat, Km values) would provide more quantitative context to the improvements and help evaluate trade-offs between rate and specificity.\n- The preparative scale synthesis is a great addition to the manuscript. It may also be helpful to include a brief comment on how the system behaves at larger scales - any limitations observed with respect to enzyme stability, precipitation issues, solubility, for example.", "reviewer_3": "Meng et al report on the phosphorylation of nucleosides by enzymatic cascade reactions. The resulting nucleoside triphosphates are substrates of engineered RNA polymerases in the synthesis of oligonucleotides. The authors use transphosphorylation catalyzed by an engineered phosphatase to produce the nucleoside monophosphates (NMP). The NMPs are phosphorylated sequentially to yield the nucleoside triphosphates (NTP). The nucleoside diphosphates (NDP) are formed as intermediates. The phosphorylations shown can be done chemically or through enzymatic cascade reactions, typically using NTP dependent kinases. The authors use enzymatic reactions independent of NTP and claim that NTP-free cascade transformations represent a major advance. This reviewer did not agree. The study is sound but the results do not constitute a significant improvement to existing methods of synthesis. The enzymatic reactions are all well-known and the developments are rather incremental. Most importantly, complicated mixtures of nucleoside, NMP, NDP and NTP are produced (Figure 4) that require extensive purification before the NTP product can be used in further transformations. The argument used against the use of established NTP kinases, that the intermediate donor (e.g., ATP) must be removed as part of the product work-up, appears to be biased. The requirement for work-up of mixtures from reactions as shown in Figure 4 certainly exceeds the purification needed for a classical NTP-kinase cascade reaction.\n\nFigure 1 is biased in the description of the different methods. While the reviewer agrees on some limitations of the chemical methods, these reactions aren’t as low yielding and complex as the authors would like to suggest. The distinction made between reactions in B and C are not clear and appear to reflect the subjective preferences of the authors. The effort in product purification required in each of these reactions depends on the specific transformation used and the extent to which the particular transformation was optimized. In an optimized reaction at elevated substrate concentration (100 mM; see the Extended Data Table 1 as an example), the presence of 1 – 5 mol% of NTP substrate for the kinases appears to be hardly of concern for the product isolation. Moreover, the substrates in Figure 3 are structurally quite different from ATP, so it would seem that they aren’t really so difficult to separate from ATP/ADP. HPLC purification may be used in certain cases but just a normal chromatographic separation will probably be fine in synthesis. And purification will be required in cases B and C (see line 201 and following).\n\nAn advantage of the kinase reaction (Figure 1B) is ignored. The activities of the individual enzymes can be adjusted to avoid accumulation of intermediates. Figure 4 shows the formation of product mixtures. The composition of the mixture varies with the substrate used. This renders the synthetic method unattractive in the opinion of this reviewer.\n\nThe cascade phosphorylation uses three different donor substrates for phosphate: pyrophosphate, polyphosphate and acetyl phosphate. A complex mixture of side products is generated which cause significant burden on the product isolation. Even the control of the reaction is complicated. Pyrophosphate and polyphosphate form complexes with metal ions required for enzyme activity. Polyphosphate is usually not well defined structurally as obtained commercially and the kinases differ in their ability to use the polyphosphate to reasonable completeness. The reviewer raises these points to emphasize that scheme C is not in fact a simpler reaction in practice than scheme B.\n\nIt has been known that kinases show promiscuity in the phosphorylation of NMP and NDP. For example, pyruvate kinase can work with different NMP and NDP substrates. Here, the authors show promiscuous reactivities of polyphosphate kinases and acetate kinase. It appears that they add acetate kinase because the polyphosphate kinase is insufficiently active to convert NDP into NTP. The characterization of polyphosphate kinases (Extended Data Table 2) is not sufficient. They show conversions after 18 h of incubation at large enzyme loading. Presumably the polyphosphate kinases are by less active than kinases typically used in these phosphorylation reactions.\n\nEngineering of the phosphatase PhoC has yielded enzymes with higher activity for the nucleoside substrates. However, while interesting the development is incremental to earlier works of Asano and others. The role of the protein structures in the overall context of the study was not clear. It was not clear how the mutations have affected the enzyme activity. Moreover, the authors appear to gloss over critical issues of the reaction of the phosphatase in diversity-oriented synthesis. The outcome of the reaction with different substrates is extremely difficult to predict/control. Extended Data Table 1 illustrates this point. In reactions of NTP kinases, adding more enzyme is usually sufficient to overcome limitations of enzyme reactivity. In the transphosphorylation, the complex interplay of transfer and hydrolysis reactions requires optimization in a substrate-specific manner.\n\nOverall, the reviewer felt that the study was better suited for a different journal within NPG (e.g., Communications Chemistry) or elsewhere (e.g., Green Chemistry)."}, "author_response": "Response to Reviewers comments\nWe would like to thank the reviewers for their constructive feedback on our previous submission.\nWe have now uploaded a revised version of the paper to the Nature Communications website\nthat addresses the issues raised by the reviewers and editors. The specific changes made are\noutlined in the reviewer response below and are also highlighted in red in the marked-up versions\nof the manuscript and supporting information.\nReviewer #1:\nReviewer: This manuscript describes an ATP-free biocatalytic approach that can be utilized to\nconvert nucleosides of diverse chemistry into their corresponding nucleoside triphosphates\n(NTPs). To start the pipeline, the authors engineered a non-specific acid phosphatase (PhoC) to\nsynthesize nucleoside monophosphates (NMPs) using inorganic pyrophosphatase as the\nphosphate donor. The most promising variant (PhoC_4), obtained by combing beneficial\nmutations identified by screening variants in microtiter plates, achieves 96% conversion of 2'-\nMOE-A into 2'-MOE-AMP. Structure determination studies and molecular modeling of PhoC_4\nsuggest that the flexible loop plays an important role in substrate recognition. Importantly,\nPhoC_4 exhibits improved activity across a range of nucleosides relative to the starting variant\n(PhoC_1). An optimized enzymatic cascade was then established utilizing PPK and AcK to\nconvert NMPs into NTPs, with the successful demonstration of nine structurally diverse\nexamples from their starting nucleoside. The enzymatic pathway was scaled, under optimized\nconditions to generate >150 mg of 2'-MOE-ATP, which was used to produce a poly-2'-MOE-\noligonucleotide pentamer. Overall, the manuscript is interesting, but will require significant\nrevision prior to publication. The main concern is that certain areas of the manuscript are\nunderdeveloped, lacking appropriate context, discussion, and referencing. Other areas contain\njargony terms that should be avoided.\nResponse: We thank the reviewer for their positive and constructive feedback, and for their\nrecognition of the significance of our work.\nReviewer: Important details in the results and discussion section are lacking. Some examples\ninclude: (i) undefined statements such as “high conversion” on line 75; (ii) theoretical size of\nthe library; (iii) number of clones evaluated; and (iv) a clear description of the model in Figure 2E\nas a chimera from prior structures.\nResponse: We have now revised the manuscript text to address the reviewer comments.\nSpecifically, we have removed the term ‘high conversions’ on line 75 and added details of the\ntheoretical library size in the main text and Supplementary Fig. 1. The number of clones evaluated\nis already indicated in the text ‘following evaluation of more than 3,500 clones’ and\nSupplementary Fig. 1. The PhoC_4 model shown in Figure 2E was generated using molecular\ndynamics simulations. Here the loop (residues 151-159) closed to adopt a conformation similar\nto that observed in 1EOI. Additional details have been added to the main text.\nReviewer: Nonstandard terms should be removed from the text. This includes “holistic\nbiocatalytic approach”, “telescoped fashion”, “vide infra”, and “pleasingly”.\nResponse: We have decided to keep one instance of the term ‘telescoped’ as it is a commonly\nused term, however we have added additional text to clarify its meaning. “Ideally these enzymes\nwould also be able to operate in a telescoped process, where consecutive biotransformations\nare performed without isolation or purification of intermediates.” All other terms have been\nremoved in line with the reviewer’s suggestion.\nReviewer: The manuscript lacks adequate citations. Some examples include: (i) the MOE\nchemical modification (line 95); (ii) the synthesis of 100 g of 5'- IMP (line 86); and the entire\nmethods paragraph on the crystal structure which fails to cite any of the programs used for\nstructure refinement.\nResponse: We apologize for this oversight and are grateful to the reviewer for highlighting this.\nAdditional references have been included in the revised manuscript.\nReviewer: The source of compound 1a is missing. Was this synthesized or purchased? If it was\nsynthesized, the details are needed.\nResponse: Compound 1a was purchased from Biosynth, this information was provided within\nthe ‘materials and methods’ section in the original manuscript.\nReviewer: The synthesis of a pentamer of 2'-MOE-modified polyA is not a suitable demonstration\nof an oligonucleotide therapeutic. The language around this example should be tempered.\nDiscussion about the length versus Tm values of the extended product is warranted.\nResponse: In line with the reviewer’s suggestions, we have removed the claim ‘showcasing a\ncomplete holistic biocatalytic approach to therapeutic RNA synthesis’ from the abstract. We\nhave reviewed our discussion on the synthesis of the 2'-MOE-modified polyA sequence and\nconfirm that we have not described this sequence as an oligonucleotide therapeutic.\nRegarding the discussion on the length vs Tm values, we are not clear on what the reviewer is\nrequesting. We originally produced a 2'-MOE-modified pentamer as this is the typical length of\nthe 2’-MOE modified flanking region of gapmers. However, in response to a comment from\nreviewer 3 we have now also produced a longer 8-mer sequence (in this case 2’F-modified polyA)\nwhich has a higher Tm value (see response to reviewer 3 for further details). We note that in our\nprevious work, we have produced sequences up to 21 nucleotides in length using our biocatalytic\ncascade (Science 380, 1150–1154, 2023).\nReviewer: 7. The x-ray crystal structures of PhoC_1 and PhoC_4 warrant a more detailed\nstructural comparison.\nResponse: We have now included additional discussion as requested.\nReviewer: The functional role of H168 and R183 should be empirically determined.\nResponse: We apologize there was a numbering error in our initial submission, these residues\nshould be H168 and R201. We have now shown that mutation of R201to alanine abolishes activity\nin PhoC_4, while a H168A substitution resulted in a 48-fold reduction in activity – this data is\nincluded in the revised draft.\nReviewer: The structural consequences of adaptive PhoC mutations should be discussed and\nshown in a figure, in particular E90, which is not located in the flexible loop.\nResponse: A comparative analysis of PhoC_4 with 1D2T and 1EOI suggests that the three\nmutations installed during PhoC_4 evolution (A90E, N151A and D154L) all reside in dynamic\nregions of the structure. N151A and D154L, reside on a poorly resolved loop region (residues 151-\n159) similar to that observed in 1D2T (residues 133-143). In 1EOI, which contains molybdate as\na transition state analogue, this loop undergoes a structural rearrangement into a helical\narchitecture to create a more occluded binding pocket. Based upon sequence and structural\nalignment, A90E in PhoC_4 is equivalent to residue S72 in 1EOI, which undergoes a 6Å\nreorientation upon molybdate binding. We have now included a supplementary figure and\nadditional discussion in the main manuscript.\nReviewer: Molybdate is mentioned in the text, but not in the structure.\nResponse: Molybdate was present as a transition state analogue in the previously reported\nstructure of E. blattae acid phosphatase (pdb 1EOI) but is not present in our PhoC structures\nreported in this manuscript.\nReviewer: In certain places, figure callouts are unclear. For example, the substrates from Figures\n2 and 3 are being called out as 1a, 1b, etc. It is not clear which figure they are referring to.\nResponse: We thank the reviewer for the comment – we have reviewed the manuscript text and\nensured the figure callouts are clear and appropriate.\nReviewer: The comparison between the enzymatically produced poly-2'-MOE-A 5-mer and the\nauthentic standard in Figure 5c should be more clearly described in the text.\nResponse: We have now included additional text as requested.\nReviewer: The manuscript would benefit from a summary of the strengths and weaknesses of\nthe current strategy relative to other NTP synthesis approaches. Yields, time, purification,\nsolvent, etc. would be useful information to share.\nResponse: We thank the reviewer for the suggestion. We have now expanded the conclusions to\nbetter emphasize some of the advantages of our approach, as well as highlighting some current\nlimitations and opportunities for future improvements. Notably, since the original submission we\nhave now shown that crude NTPs from our process can be used directly in enzymatic\noligonucleotide synthesis, thus avoiding the need for NTP purification (Supplementary Fig. 7). It\nis unlikely that such an approach could be adopted with ATP-dependent methods, as even low\nconcentrations of ATP contaminants severely compromise the purity of downstream\noligonucleotide products (Supplementary Fig. 8).\nReviewer #2:\nReviewer: The manuscript by Meng et al. reports the development of a fully enzymatic, ATP-free\ncascade for the production of nucleoside triphosphates (NTPs), including chemically modified\nvariants used in therapeutic oligonucleotides. This work elegantly builds on the seminal work in\nScience (DOI: 10.1126/science.add589), also by the Lovelock group, to address a key bottleneck\nin the RNA therapeutics field, namely, the scalable and selective synthesis of modified NTPs, which are often\nchallenging to prepare chemically. The work describes a three-enzyme system comprising an engineered acid phosphatase (PhoC), polyphosphate kinase (PPK), and acetate kinase (AcK), which together enable phosphorylation of a wide array of canonical and modified nucleosides using inexpensive phosphate donors and without the need for ATP. This approach significantly simplifies purification by eliminating ATP and ADP impurities that would otherwise complicate downstream processes.\n\nThe authors demonstrate a well-executed protein engineering campaign on PhoC to enhance its\nactivity on therapeutically relevant substrates such as 2'-MOE-adenosine. Directed evolution led\nto a triple mutant (PhoC_4) with an 8-fold improvement in conversion and an expanded substrate\nscope to several therapeutically relevant compounds. Structural characterization of PhoC_4 and\ndocking of 2'-MOE-AMP provide insight into the mutational effects and highlight a loop region as\nlikely contributing to substrate engagement and activity partitioning. This sets the stage for future\nengineering efforts and adds mechanistic depth to the study. Following this, a panel of PPKs and\na promiscuous AcK were used to advance monophosphates to the triphosphate level across a\nrange of substrates, achieving conversions up to 82%. The enzymatic synthesis of 2'-MOE-ATP\non preparative scale and its subsequent use in oligonucleotide assembly via a polymerase–\nendonuclease cascade further demonstrates the practical value of the method.\n\nThe manuscript is clearly written, methodically executed, and of broad relevance. Overall, this is\nan excellent contribution to the field and represents a clear advance in enabling the biocatalytic\nsynthesis of NTP building blocks. The work will be of interest to researchers in biocatalysis,\nnucleic acid chemistry, and RNA therapeutic development. As such I recommend publication\nafter minor revisions below:\nResponse: We thank the reviewer for their positive comments.\nReviewer: While the engineering of PhoC_4 is thoroughly presented in terms of conversion data\nand structural interpretation, a kinetic comparison between PhoC_1 and PhoC_4 (e.g., kcat, Km\nvalues) would provide more quantitative context to the improvements and help evaluate trade-\noffs between rate and specificity.\nResponse: We are grateful for the reviewer’s suggestion and have now carried out the kinetic\ncomparison of PhoC_1 and PhoC_4. This data shows that directed evolution has led to a 10-fold\nincrease in catalytic efficiency, due to a 3-fold improvement in k and a 3.5-fold reduction in K\ncat M.\nThis information is now included in the revised draft.\nReviewer: The preparative scale synthesis is a great addition to the manuscript. It may also be\nhelpful to include a brief comment on how the system behaves at larger scales - any limitations\nobserved with respect to enzyme stability, precipitation issues, solubility, for example.\nResponse: We have now included additional text in line with the reviewer’s suggestion.\nReviewer #3\nReviewer: Meng et al report on the phosphorylation of nucleosides by enzymatic cascade\nreactions. The resulting nucleoside triphosphates are substrates of engineered RNA\npolymerases in the synthesis of oligonucleotides. The authors use transphosphorylation\ncatalyzed by an engineered phosphatase to produce the nucleoside monophosphates (NMP).\nThe NMPs are phosphorylated sequentially to yield the nucleoside triphosphates (NTP).\nThe nucleoside diphosphates (NDP) are formed as intermediates. The phosphorylations shown can\nbe done chemically or through enzymatic cascade reactions, typically using NTP dependent\nkinases. The authors use enzymatic reactions independent of NTP and claim that NTP-free\ncascade transformations represent a major advance. This reviewer did not agree. The study is\nsound but the results do not constitute a significant improvement to existing methods of\nsynthesis. The enzymatic reactions are all well-known and the developments are rather\nincremental.\nResponse: We respectfully disagree on several points raised by the reviewer. Scalable\nproduction of NTPs containing clinically important modifications remains a major challenge, and\nin fact the work contained within this manuscript is supported by diverse industry partners\nincluding leading suppliers of NTP building blocks and pharmaceutical end-users.\n1. Relating to the advantages of ATP-independent synthesis. Although powerful in some\ninstances, ATP-dependent methods have several drawbacks, and chemical synthesis remains\nthe dominant method of modified NTP-production. Even when using ancillary enzymes for ATP\nrecycling (which leads to additional costs), relatively high loadings of ATP are typically required.\nFurthermore, the typical stoichiometric phosphate donors used are far more expensive than\npyrophosphate used for our PhoC biotransformations. Perhaps more importantly, when\nproducing NTPs that are close structural analogues of ATP (e.g. 2'-F-ATP), the presence of ATP\nand ADP by-products complicates downstream purification. In response to the reviewer’s\ncomments, in our revised draft we have now shown that crude NTPs produced using our\nbiocatalytic process can be used directly in enzymatic oligonucleotide synthesis, avoiding the\nneed for NTP isolation/purification (Supplementary Fig. 7). It is unlikely that such an approach\ncould be adopted with ATP-dependent methods, as even low concentrations of ATP\ncontaminants severely compromise the purity of downstream oligonucleotide products\n(Supplementary Fig. 8). Finally, we were unable to find any previous reports showing that ATP-\ndependent kinases are active towards 2'-OMe, 2'-MOE, LNA or 3'-protecting groups required for\nenzymatic synthesis of oligonucleotide therapeutics. In the revised manuscript, we have\nincluded an updated figure 1 and included additional text to better emphasize this important\npoint. We note that we have evaluated a panel of ATP-dependent nucleoside kinases towards 2'-\nribose modified substrates and detected no activity towards 2'-MOE or LNA nucleosides (data\nnot shown).\n2. Relating to the incremental nature of the study. Again, we respectfully disagree with the\nreviewer’s commentary. While acid phosphatases have previously been shown to accept inosine\nand adenosine, to date there are no reports showing activity towards ribose modified substrates.\nIndeed, initial PhoC_1 activity towards the target modified substrates was very low making it\nunsuitable for direct use in NTP synthesis. Key to the success of our platform was the directed\nevolution of PhoC_4 which displays substantially improved catalytic efficiency towards 2'-MOE-\nA, the target substrate used for evolutionary optimization. Importantly, PhoC_4 also displays\nimproved efficiency towards a diverse panel of clinically relevant substrates. The high degree of\nsubstrate promiscuity displayed by PhoC_4 is extremely valuable, and provides a common\nstarting basis for engineering more efficient and specialized enzymes moving forward. We note\nthat that nucleoside kinases do not appear to display this broad substrate tolerance towards\nribose modifications.\nWe were also unable to find reports showing PPK or AcK activity towards diverse 2'- or 3'-modified\nsubstrates. Although PPKs have been used previously to produce nucleobase modified NTPs\nfrom the corresponding NMP, their applications have been limited by low conversions to the\ntarget NTP. This is the first report combining a PPK with an AcK to address equilibrium challenges\nand achieve high NTP conversions. Finally, we have now shown that the combination of an\nengineered PhoC, PPK and AcK can be used to produce important NTP building blocks needed to\nunderpin biocatalytic production of RNA therapeutics.\nReviewer: Most importantly, complicated mixtures of nucleoside, NMP, NDP and NTP are\nproduced (Figure 4) that require extensive purification before the NTP product can be used in\nfurther transformations. The argument used against the use of established NTP kinases, that the\nintermediate donor (e.g., ATP) must be removed as part of the product work-up, appears to be\nbiased. The requirement for work-up of mixtures from reactions as shown in Figure 4 certainly\nexceeds the purification needed for a classical NTP-kinase cascade reaction. The effort in\nproduct purification required in each of these reactions depends on the specific transformation\nused and the extent to which the particular transformation was optimized. In an optimized\nreaction at elevated substrate concentration (100 mM; see the Extended Data Table 1 as an\nexample), the presence of 1 – 5 mol% of NTP substrate for the kinases appears to be hardly of\nconcern for the product isolation. Moreover, the substrates in Figure 3 are structurally quite\ndifferent from ATP, so it would seem that they aren’t really so difficult to separate from ATP/ADP.\nHPLC purification may be used in certain cases but just a normal chromatographic separation\nwill probably be fine in synthesis. And purification will be required in cases B and C (see line 201\nand following).\nResponse: We strongly disagree with reviewer that the NTPs produced in this study require\nextensive purification. As shown in figure 5A we were able to isolate 139 mg 2'-MOE-ATP from our\nbiotransformation using standard purification on DEAE Sepharose resin. Here, the NTP product\nwas removed from minor NMP and NDP impurities, which are also generated in ATP-dependent\nkinase reactions. Furthermore, using ATP-dependent methods leads to additional ATP and ADP\nimpurities which present additional purification challenges, especially when producing NTPs\nthat are close structural analogues of ATP (e.g. 2'-F-ATP).\nTo further address the reviewer’s concerns, we have now shown that enzymatically produced 2’-\nF-ATP in crude form can be used directly in biocatalytic oligonucleotide synthesis, thus obviating\nthe need for NTP purification altogether (Supplementary Fig. 8). To our delight, crude 2′-F-ATP\ncould be used directly in our polymerase and endonuclease cascade to furnish a poly-2′-F-A\n8mer in 70% conversion. In contrast, efforts to produce poly-2′-F-A using 2’-F-ATP in the\npresence of low concentrations (from 1-10 mol%) of ATP contaminants gave complex product\ndistributions arising from competing ATP and 2′-F-ATP incorporation (Supplementary Fig. 8).\nReviewer: Figure 1 is biased in the description of the different methods. While the reviewer\nagrees on some limitations of the chemical methods, these reactions aren’t as low yielding and\ncomplex as the authors would like to suggest. The distinction made between reactions in B and\nC are not clear and appear to reflect the subjective preferences of the authors.\nResponse: We have now revised figure 1 in light of the reviewer’s concerns.\nReviewer: An advantage of the kinase reaction (Figure 1B) is ignored. The activities of the\nindividual enzymes can be adjusted to avoid accumulation of intermediates. Figure 4 shows the\nformation of product mixtures. The composition of the mixture varies with the substrate used.\nThis renders the synthetic method unattractive in the opinion of this reviewer.\nResponse: While we appreciate the concentrations of individual enzymes can be adjusted in\nkinase reactions, the same is true of any enzyme cascade including ours developed within this\nstudy. Although we agree that there is some substrate-dependent variation in composition, this\nis to be expected – some of these substrates feature sterically demanding substituents and are\nchallenging biocatalytic conversions. Moreover, the reactions have not been intensively\noptimized in each case to further drive higher conversions. Similarly, the enzymes have not been\nspecifically engineered towards each substrate. Moving forward there are clear opportunities for\nefficiency gains using a combination of reaction and enzyme engineering. Finally, we note that a\ndirect comparison to ATP-dependent kinases is simply not possible, as nucleoside kinases have\nnot been shown to accept the ribose modified substrates used within this study.\nTo reflect the points above, we have now included additional text in the manuscript conclusions\nhighlighting some current limitations of our approach and opportunities for future\nimprovements.\nReviewer: The cascade phosphorylation uses three different donor substrates for phosphate:\npyrophosphate, polyphosphate and acetyl phosphate. A complex mixture of side products is\ngenerated which cause significant burden on the product isolation. Even the control of the\nreaction is complicated. Pyrophosphate and polyphosphate form complexes with metal ions\nrequired for enzyme activity. Polyphosphate is usually not well defined structurally as obtained\ncommercially and the kinases differ in their ability to use the polyphosphate to reasonable\ncompleteness. The reviewer raises these points to emphasize that scheme C is not in fact a\nsimpler reaction in practice than scheme B.\nResponse: The side products produced in the reaction (monophosphate, polyphosphate and\nacetic acid) did not present any challenges during product isolation (Figure 5A). Furthermore, the\npyrophosphate and magnesium complex can be easily removed following the first step by\nfiltration. Moving forward there are opportunities to engineer PhoC to operate efficiently using\nonly 1 equivalent of pyrophosphate which would avoid the need for a filtration step. As described\nin the text, we have also shown that PhoC_4 and PPK can be used in a single enzymatic cascade\nif pyrophosphate is replaced by phenylphosphate, which does not coordinate to the metal ions.\nReviewer: It has been known that kinases show promiscuity in the phosphorylation of NMP and\nNDP. For example, pyruvate kinase can work with different NMP and NDP substrates. Here, the\nauthors show promiscuous reactivities of polyphosphate kinases and acetate kinase. It appears\nthat they add acetate kinase because the polyphosphate kinase is insufficiently active to convert\nNDP into NTP. The characterization of polyphosphate kinases (Supplementary Table 2) is not\nsufficient. They show conversions after 18 h of incubation at large enzyme loading. Presumably\nthe polyphosphate kinases are by less active than kinases typically used in these\nphosphorylation reactions.\nResponse: As mentioned previously, we were unable to find any previous reports showing that\nATP-dependent kinases are active towards 2'-OMe, 2'-MOE, LNA or 3'-protecting groups.\nFurthermore, we screened a panel of ATP-dependent nucleoside kinases towards 2'-ribose\nmodified substrates and detected no activity towards 2'-MOE or LNA nucleosides.\nAs described in the text, polyphosphate kinases converted NMPs under thermodynamic control\nto mixtures of NDPs and NTPs. The acetate kinase (AcK) is used to phosphorylate any remaining\nNDP to improve yields of the target NTPs. The PPKs were initially evaluated using lyophilized cell-\nfree extracts (Supplementary Table 2) so the exact enzyme loading is not known. However, for\npreparative scale reactions (2'-MOE-ATP and 2'-F-ATP) we used only 30 µM purified PPK (0.06\nmol%). This enzyme loading is comparable to that used in ATP-dependent kinase reactions (e.g.\nAngew. Chem. Int. Ed. 64, e202506330 (2025)).\nReviewer: Engineering of the phosphatase PhoC has yielded enzymes with higher activity for the\nnucleoside substrates. However, while interesting the development is incremental to earlier\nworks of Asano and others.\nResponse: We strongly disagree with the reviewer. Previous studies involving PhoC have\nfocussed on the production of inosine monophosphate (a flavour enhancer), AMP and\nphosphorylated aliphatic alcohols. As described previously in our response, we have been\nunable to find any reports showing activity towards nucleosides containing 2'-modifications and\n3'-protecting groups required to produce RNA therapeutics. Moreover, we have shown that the\nstarting PhoC variant had very poor activity towards our target substrates. Following our study,\nwe now have access to an engineered PhoC variant that displays good activity towards a broad\nrange of substrates featuring different bases, 1’- and 2’-modifications and 3’-protecting groups.\nReviewer: The role of the protein structures in the overall context of the study was not clear.\nIt was not clear how the mutations have affected the enzyme activity.\nResponse: The loop region harbouring the N151A and D154L mutations is poorly resolved in the\nPhoC_1 and PhoC_4 structures, precluding any detailed structural comparison. However,\nmolecular dynamics simulations suggest that the loop spanning residues 151-159 may reorder\nduring substrate binding to adopt a more closed conformation, and this loop may play an\nimportant role in substrate recognition and/or the partitioning of phosphoryl transfer vs\nhydrolysis activity. Thus, this structural characterization suggests targeting this loop region (151-\n159) for more extensive engineering should lead to the develop of more proficient biocatalysts.\nIn response to the reviewer’s comment, we have added more detail to the structural\ncharacterization section, including information relating to the role of A90E.\nReviewer: Moreover, the authors appear to gloss over critical issues of the reaction of the\nphosphatase in diversity-oriented synthesis.\nResponse: We are not clear what the reviewer is suggesting. The focus of this study is the\ndevelopment of a low cost, sustainable method for NTP manufacturing. The connection to\ndiversity-oriented synthesis is not immediately obvious.\nReviewer: The outcome of the reaction with different substrates is extremely difficult to\npredict/control. Extended Data Table 1 illustrates this point. In reactions of NTP kinases,\nadding more enzyme is usually sufficient to overcome limitations of enzyme reactivity. In the\ntransphosphorylation, the complex interplay of transfer and hydrolysis reactions requires\noptimization in a substrate-specific manner. Overall, the reviewer felt that the study was better\nsuited for a different journal within NPG (e.g., Communications Chemistry) or elsewhere (e.g.,\nGreen Chemistry).\nResponse: Predicting the outcome of any enzymatic transformation can be challenging.\nExtended Data Table 1 shows that high conversions can be achieved across a range of substrates\nat elevated substrate loadings. The different substrate and DMSO concentrations used in each\nreaction are a result of nucleoside solubility and are not related to the enzyme efficiency. We\nnote that when exploring the substrate scope of enzymes, it is perfectly common to make\nadjustments to reaction conditions (e.g. time) to improve conversions. Finally, we note that\nnucleoside kinases have not been shown to operate on these substrates."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have addressed all of my concerns, so I can now recommend acceptance of this manuscript. Very nice work!", "reviewer_2": "The revisions the authors have made have satisfied my previous comments and, along with the additional revisions requested by the other reviewers, have significantly strengthened the manuscript. I support publication.", "reviewer_3": "Authors have responded to the comments of my original review. They disagree on several points raised. The reviewer retains the criticism that the overall cascade reaction is not convincing for production. Important elements of the proposed cascade reaction have been demonstrated in earlier studies.\n\nAuthors justify their efforts in engineering PhoC by the lack of broadly specific nucleoside kinases. In particular, they mention nucleosides modified at the 2’- and 3’-O position. The authors will know the field better than the reviewer, but only a very quick literature search revealed paper (Catalysts 2022, 12, 1401) describing broadly specific nucleoside kinases tolerating -O-Me substitution at both positions. The engineered PhoC may be a valuable addition to the toolbox of enzymes for phosphorylation, but as already mentioned in the first review it was not clear that this enzyme was a breakthrough.\n\nInformation gained from structural analysis of the engineered phosphatase was rather low. Based on simple docking analysis in the experimental protein structure a flexible loop was suggested to play a role in substrate recognition and reaction selectivity.\n\nAuthors claim that NTP synthesis free of ATP represents a major advance. The point was not clear. The NTP solution from enzymatic production (≥ 50 mmol/L) will contain ATP in low amount, probably not more than 5 mol%. It was not evident which applications in RNA synthesis would be compromised by this level of contamination. However, if a specific application of NTP did not tolerate any ATP, it would always be possible to run the kinase reaction on the nucleoside in the presence of catalytic amounts of the synthetic NTP. This would avoid ATP altogether. The small amount of NTP required to start the reaction might be synthesized chemically. The phosphatase cascade reaction is therefore not the only option to exclude ATP.\n\nFigure 1A does not describe the state of the art correctly. Various papers show phosphorylation of NMP and NDP substrates by PPK. The PPK reactions do not require extra ATP as it is shown in the figure. For example, paper by Li et al. (ACS Synth. Biol. 2023, 12, 1772−1781) discusses the use of different PPKs for NMP conversion into NTP in the absence of ATP. Various PPKs were evaluated in this study. The conversion of NDP into NTP by other kinases often used for nucleotide donor regeneration (pyruvate kinase, AcK) is known.\n\nAuthors claim that the enzymatic cascade reaction is efficient for synthesis and might even be useful for production as the title implies. The reviewer disagrees. The syntheses of 2’-MOE-ATP and 2’-F-ATP involve complicated multistep procedures. Mixture of the phosphatase reaction must be filtered to remove enzyme and the pH be readjusted by titration and addition of a different buffer. Pyrophosphate must be precipitated by adding 750 mmol/L (!) of MgCl2 and mixture be centrifuged. The reaction is then performed in the presence of PPK2 using hexametaphosphate (20 mg/mL; about 33 mmol/L assuming mass of 611 Da). Dilution up to this point was around 4-fold. After the PPK reaction the mixture is again filtered and further diluted. Reaction of AcK involves 50 mmol/L acetyl phosphate. The composition of the product solution is not stated but it will contain a lot of monomeric and oligomeric phosphate, Mg2+ and acetate/acetyl phosphate. Additionally, the product is a mixture of NTP (sometimes below 50% of total product; see Figure 4), NMP, NDP and nucleoside. The reviewer expressed concern about the purification required. Authors respond that a single “standard ion exchange chromatography” was sufficient to isolate the product. The purification was only superficially described. The point was not about whether the compounds can be fractionated on an ion exchange resin in principle. This is well documented in the literature. The point was about how practical is this purification (e.g., dilution of the target NTP, contamination of NTP by salt needed for elution and later removal of the salt etc.) considering the authors’ claim that a new method of NTP synthesis, applicable for production, is presented.\n\nConsidering mixture of products formed (NTP, NDP, NMP, nucleoside) the reviewer asked about control of the product formation by adjusting the enzyme activities. A true one-pot transformation based on coupled reactions would allow for such control. Authors respond that their reactions could also be optimized by adjusting the individual enzyme activities. But this is not true. Their enzymatic reactions are performed uncoupled one from another. Only one enzyme is present at a time. Advantage of reaction coupling in cascade transformations is lost. The reaction of the phosphatase is not compatible with the reactions of PPK and AcK for several reasons, foremost the different pH requirements.\n\nThe kinase reactions are supposed to operate under thermodynamic control. Variation in composition of the product mixture was therefore not clear. Is this dependent on the ability of the enzymes to use certain NMP and NDP substrates?\n\nThe additional experiments showing use of the NTPs in oligonucleotide synthesis are appreciated. However, the evidence is demonstration of robustness of their method of DNA synthesis rather than demonstration of the efficiency of their NTP production.\n\nOther points\nPhenyl phosphate appears to be unattractive as donor for phosphorylation in production."}, "author_response": "Response to Reviewer 3\nReviewer: Authors have responded to the comments of my original review. They\ndisagree on several points raised. The reviewer retains the criticism that the overall\ncascade reaction is not convincing for production. Important elements of the proposed\ncascade reaction have been demonstrated in earlier studies. Authors justify their\nefforts in engineering PhoC by the lack of broadly specific nucleoside kinases. In\nparticular, they mention nucleosides modified at the 2’- and 3’-O position. The authors\nwill know the field better than the reviewer, but only a very quick literature search\nrevealed paper (Catalysts 2022, 12, 1401) describing broadly specific nucleoside\nkinases tolerating -O-Me substitution at both positions. The engineered PhoC may be\na valuable addition to the toolbox of enzymes for phosphorylation, but as already\nmentioned in the first review it was not clear that this enzyme was a breakthrough.\nResponse: The manuscript highlighted by the reviewer describes a nucleoside kinase\nwith low level activity (24% conversion) towards 2'-OMe-uridine but it does not show\nconversion to the corresponding NTP product. As stated in our previous response we\nwere unable to find examples of the use of ATP-dependent kinases in the synthesis of\nNTPs containing 2'-MOE and LNA modifications or 3'-protecting groups required for\nenzymatic synthesis of oligonucleotide therapeutics. In fact, in the paper highlighted\nby the reviewer, the authors evaluate 3’-acetyl protecting groups but detected no initial\nactivity. This reference has been added to the revised manuscript.\nReviewer: Information gained from structural analysis of the engineered phosphatase\nwas rather low. Based on simple docking analysis in the experimental protein structure\na flexible loop was suggested to play a role in substrate recognition and reaction\nselectivity.\nResponse: We respectfully disagree with the reviewer. Our structural analysis\nhighlighted key regions in the protein to be targeted in future engineering campaigns\nand will be of interest to researchers in the field.\nReviewer: Authors claim that NTP synthesis free of ATP represents a major advance.\nThe point was not clear. The NTP solution from enzymatic production (≥ 50 mmol/L)\nwill contain ATP in low amount, probably not more than 5 mol%. It was not evident\nwhich applications in RNA synthesis would be compromised by this level of\ncontamination.\nResponse: The NTP solutions generated using our approach will not contain any ATP.\nTo highlight the negative impacts of low-level ATP contamination on oligonucleotide\nsynthesis, in our previous revision we performed polymerase catalyzed reactions\nusing NTPs spiked with 1-10% ATP. Using NTPs containing only 1% ATP impurity led\nto a substantial (~3-fold) reduction in oligonucleotide yield and formation of impurities\narising from misincorporation of ATP (Figure S8). This misincorporation of ATP will also\nlikely be observed for enzymatic oligonucleotide synthesis using terminal\ndeoxyribonucleotidyl transferases (TdTs) and poly(U)polymerases (PUPs).\nReviewer: However, if a specific application of NTP did not tolerate any ATP, it would\nalways be possible to run the kinase reaction on the nucleoside in the presence of\ncatalytic amounts of the synthetic NTP. This would avoid ATP altogether. The small\namount of NTP required to start the reaction might be synthesized chemically. The\nphosphatase cascade reaction is therefore not the only option to exclude ATP.\nResponse: We agree with the reviewer that this is another potential approach to NTP\nsynthesis. Indeed, this has been shown by the Merck biocatalysis group who used 2′F-\n(Sp)-thioATP (a close structural analogue of ATP) as their phosphate donor during\nsynthesis of a cyclic dinucleotide. However, despite its close structural similarity to\nATP, kinase activity towards the non-canonical donor was only modest and\nengineering was required to reprogramme cofactor selectivity. It remains to be seen\nwhether ATP-dependent kinases can accept a wider range of modified NTPs as the\nphosphate donor. For example, as highlighted above we were unable to find examples\nof ATP-dependent kinases with activity towards 2'-MOE, LNA or 3'-protecting groups.\nReviewer: Figure 1A does not describe the state of the art correctly. Various papers\nshow phosphorylation of NMP and NDP substrates by PPK. The PPK reactions do not\nrequire extra ATP as it is shown in the figure. For example, paper by Li et al. (ACS\nSynth. Biol. 2023, 12, 1772−1781) discusses the use of different PPKs for NMP\nconversion into NTP in the absence of ATP. Various PPKs were evaluated in this study.\nThe conversion of NDP into NTP by other kinases often used for nucleotide donor\nregeneration (pyruvate kinase, AcK) is known.\nResponse: Figure 1A describes an ATP-dependent enzyme cascade and does not\ndescribe the use of PPKs. PPKs are described in Figure 1B: ATP-independent\nbiocatalytic approach. We acknowledge that PPKs have previously been evaluated for\nactivity towards nucleobase modified NTPs but were unable to find any examples\nPPKs with activity towards 2’ and 3’-modifications. Additional text has been added for\nclarification.\nReviewer: Authors claim that the enzymatic cascade reaction is efficient for synthesis\nand might even be useful for production as the title implies. The reviewer disagrees.\nThe syntheses of 2’-MOE-ATP and 2’-F-ATP involve complicated multistep\nprocedures. Mixture of the phosphatase reaction must be filtered to remove enzyme\nand the pH be readjusted by titration and addition of a different buffer. Pyrophosphate\nmust be precipitated by adding 750 mmol/L (!) of MgCl2 and mixture be centrifuged.\nThe reaction is then performed in the presence of PPK2 using hexametaphosphate\n(20 mg/mL; about 33 mmol/L assuming mass of 611 Da). Dilution up to this point was\naround 4-fold. After the PPK reaction the mixture is again filtered and further diluted.\nReaction of AcK involves 50 mmol/L acetyl phosphate. The composition of the product\nsolution is not stated but it will contain a lot of monomeric and oligomeric phosphate,\nMg2+ and acetate/acetyl phosphate. Additionally, the product is a mixture of NTP\n(sometimes below 50% of total product; see Figure 4), NMP, NDP and nucleoside. The\nreviewer expressed concern about the purification required. Authors respond that a\nsingle “standard ion exchange chromatography” was sufficient to isolate the product.\nThe purification was only superficially described. The point was not about whether the\ncompounds can be fractionated on an ion exchange resin in principle. This is well\ndocumented in the literature. The point was about how practical is this purification\n(e.g., dilution of the target NTP, contamination of NTP by salt needed for elution and\nlater removal of the salt etc.) considering the authors’ claim that a new method of NTP\nsynthesis, applicable for production, is presented.\nResponse: The process described in the manuscript has not been extensively\noptimized and we agree further process optimization would be required to enable\nscalable NTP production. These points are clearly discussed in the manuscript\nconclusions. For example, we suggest that optimization of individual enzymes towards\ntarget substrates of interest would minimize off-pathway processes (e.g. NMP\nhydrolysis by PhoC) and lead to enhanced productivity. To avoid the need for pH\nadjustments and dilutions there is the potential to develop enzymes with similar pH-\noptima. Alternatively, the NMP intermediate could be precipitated following the first\nstep, avoiding the requirement for additional MgCl2 and dilution. We have already\nbegun process development for some selected NTP products and are working towards\nisolating the NMP intermediates and NTP products using precipitation and\nrecrystallization. We have also shown that enzymatically produced NTPs in crude form\ncan be used directly in biocatalytic oligonucleotide synthesis, obviating the need for\npurification altogether (Supplementary Fig. 8).\nReviewer: Considering mixture of products formed (NTP, NDP, NMP, nucleoside) the\nreviewer asked about control of the product formation by adjusting the enzyme\nactivities. A true one-pot transformation based on coupled reactions would allow for such\ncontrol. Authors respond that their reactions could also be optimized by adjusting the\nindividual enzyme activities. But this is not true. Their enzymatic reactions are\nperformed uncoupled one from another. Only one enzyme is present at a time.\nAdvantage of reaction coupling in cascade transformations is lost. The reaction of the\nphosphatase is not compatible with the reactions of PPK and AcK for several reasons,\nforemost the different pH requirements.\nResponse: Although the reactions were performed sequentially in the preparative\nscale synthesis, we have also shown that PhoC_4 and PPK can be used in a single\nenzymatic cascade if pyrophosphate is replaced by phenyl phosphate as a donor\n(Supplementary Fig. 5). Both enzymes are active at pH 5.\nReviewer: The kinase reactions are supposed to operate under thermodynamic\ncontrol. Variation in composition of the product mixture was therefore not clear. Is this\ndependent on the ability of the enzymes to use certain NMP and NDP substrates?\nResponse: For many substrates, the variation in the NDP to NTP ratio is small (Fig\n4A). However, for some challenging substrates (e.g. 3’-aminoxy modified AMP) the\nNTP yield was slightly lower suggesting the reactions have not yet reached\nequilibrium.\nReviewer: The additional experiments showing use of the NTPs in oligonucleotide\nsynthesis are appreciated. However, the evidence is demonstration of robustness of\ntheir method of DNA synthesis rather than demonstration of the efficiency of their NTP\nproduction.\nResponse: The additional oligonucleotide synthesis reactions were designed to\nshowcase the advantages of ATP-free synthesis. We highlight how even low\nconcentrations of ATP contaminants severely compromise the purity of downstream\noligonucleotide products (Supplementary Fig. 8). In contrast, crude NTPs produced\nusing our biocatalytic process can be used directly in enzymatic oligonucleotide\nsynthesis, avoiding the need for NTP isolation/purification (Supplementary Fig. 7).\nReviewer: Other points: Phenyl phosphate appears to be unattractive as donor for\nphosphorylation in production.\nResponse: It is not clear why the reviewer believes that phenyl phosphate is\nunattractive, but we respectfully disagree."}]

Decision letter