Preparation of 3′-O-allyl ether NTPs (A, U, G, C)
A mixture of 3′-O-allyl ether adenosine nucleoside (molecular weight: 307.3 g mol−1, 1.04 g, 3.38 mmol) and proton sponge (molecular weight: 214.31 g mol−1, 2.03 g, 9.47 mmol) was prepared as described in the general procedure, dissolved in trimethoxy phosphate (25.0 ml) and cooled to −5 °C. This was followed by slow addition of phosphoryl oxychloride (molecular weight: 153.32 g mol−1, density: 1.64 g cm−3, 0.35 ml, 3.7 mmol). After 3 min, another portion of phosphoryl oxychloride (molecular weight: 153.32 g mol−1, density: 1.64 g cm−3, 0.1 ml, 1.1 mmol) was added. After stirring for 10 min, a prechilled mixture of tributyl ammonium pyrophosphate (molecular weight: 548.68 g mol−1, 7.0 g, 12.8 mmol), acetonitrile (55 ml) and tributyl amine (12 ml) was quickly added to the reaction. This was stirred for 2 h and slowly warmed to room temperature. The reaction was quenched by the addition of water (~150 ml) and worked up, isolated and purified according to the general procedure (yield 33%, formula weight for the tetra-triethylammonium salt: 951.5 g mol−1, 1.65 g, 1.11 mmol). 1H NMR (400 MHz, D2O) δ 8.43 (s, 1H), 8.15 (s, 1H), 6.04 (d, J = 6 Hz, 1H), 5.96 (m, J = 17, 11 Hz, 1H), 5.37 (dd, J = 17, 2 Hz, 1H), 5.23 (dd, J = 11, 2 Hz, 1H), 4.80 (t, J = 6 Hz, 1H), 4.42 (t, J = 3 Hz, 1H), 4.30 (dd, J = 6, 3 Hz, 1H), 4.19 (d, J = 6 Hz, 2H), 4.15 (t, J = 3 Hz, 2H), 3.09 (q, 16 H), 1.18 (t, 24H). 31P NMR (400 MHz, D2O) δ −10.6 (d, J = 20, 1H), −11.5 (d, J = 20, 1H), −23.1 (t, J = 20). ESI-MS: calculated for [C13H21N5O13P3]+ = 548.0343; found: 548.0347.
A mixture of 3′-O-allyl ether uridine nucleoside (molecular weight: 284.10 g mol−1, 0.57 mg, 2.00 mmol) and proton sponge (molecular weight: 214.31 g mol−1, 1.00 g, 4.67 mmol) was prepared as described in the general procedure, dissolved in trimethoxy phosphate (9.0 ml) and cooled to −5 °C. This was followed by slow addition of phosphoryl oxychloride (molecular weight: 153.32 g mol−1, density: 1.64 g cm−3, 0.2 ml, 2.1 mmol). After 3 min, another portion of phosphoryl oxychloride (molecular weight: 153.32 g mol−1, density: 1.64 g cm−3, 0.1 ml, 1.1 mmol) was added. After stirring for 10 min, a prechilled mixture of tributyl ammonium pyrophosphate (molecular weight: 548.68 g mol−1, 3.7 g, 6.7 mmol), acetonitrile (32 ml) and tributyl amine (6 ml) was quickly added to the reaction. This was stirred for 2 h and slowly warmed to room temperature. The reaction was quenched by the addition of water (~200 ml) and worked up, isolated and purified according to the general procedure (yield 29%, formula weight for the tetra-triethylammonium salt: 928.5 g mol−1, 0.54 g, 0.58 mmol). 1H NMR (400 MHz, D2O) δ 7.88 (d, J = 8 Hz, 1H), 5.93 (m, 3 H), 5.32 (dd, J = 17, 2 Hz, 1H), 5.22 (dd, J = 11, 2 Hz, 1H), 4.40 (t, J = 6 Hz, 1H), 4.32 (m, J = 3 Hz, 1H), 4.15 (m, J = 3 Hz, 3H), 3.12 (q, 19 H), 1.20 (t, 29 H). 31P NMR (400 MHz, D2O) δ −10.8 (d, J = 20, 1H), −11.6 (d, J = 20, 1H), −23.3 (t, J = 20, 1H). ESI-MS: calculated for [C12H18N2O15P3]− = 522.9926; found: 522.9928.
A mixture of 3′-O-allyl ether guanine nucleoside (molecular weight: 323.12 g mol−1, 1.1 g, 3.40 mmol) and proton sponge (molecular weight: 214.31 g mol−1, 2.21 g, 10.3 mmol) was prepared as described in the general procedure, dissolved in trimethoxy phosphate (25.0 ml) and cooled to −5 °C. This was followed by slow addition of phosphoryl oxychloride (molecular weight: 153.32 g mol−1, density: 1.64 g cm−3, 0.35 ml, 3.7 mmol). After 3 min, another portion of phosphoryl oxychloride (molecular weight: 153.32 g mol−1, density: 1.64 g cm−3, 0.1 ml, 1.1 mmol) was added. After stirring for 10 min, a prechilled mixture of tributyl ammonium pyrophosphate (molecular weight: 548.68 g mol−1, 7.0 g, 12.8 mmol), acetonitrile (55 ml) and tributyl amine (12 ml) and was quickly added to the reaction. This was stirred for 2 h and slowly warmed to room temperature. The reaction was quenched by the addition of water (~200 ml) and worked up, isolated and purified according to the general procedure (yield 40%, formula weight for the tetra-triethylammonium salt: 967.5 g mol−1, 1.33 g, 1.37 mmol). 1H NMR (400 MHz, D2O) δ 8.03 (s, 1H), 5.96 (ddt, J = 6 Hz, 1H), 5.84 (d, J = 7 Hz, 1H), 5.35 (dd, J = 17 Hz, 1H), 5.23 (dd, J = 11 Hz, 1H), 4.84 (dd, J = 7 Hz, 1H), 4.39 (m, J = 3 Hz, 1H), 4.29 (dd, J = 5, 3 Hz, 1H), 4.19 (d, J = 6 Hz, 2H), 4.15 (m, J = 6 Hz, 2H), 3.11 (q, 19H), 1.19 (t, 29H). 31P NMR (400 MHz, D2O) δ −10.6 (d, J = 20 Hz, 1H), −11.5 (d, J = 20 Hz, 1H), −23.1 (t, J = 20 Hz, 1H). ESI-MS: calculated for [C13H19N5O14P3]− = 562.0147; found: 562.0150.
The 3′-O-allyl ether cytidine nucleoside (molecular weight: 283.3 g mol−1, 0.22 g, 0.78 mmol) was dissolved in a mixture of dimethylformamide (DMF) and 1,1-dimethoxytrimethylamine (~6:1, 2.1 ml) and stirred for 36 h. The crude product was concentrated in vacuo. To the resultant solid was added dry proton sponge (molecular weight: 214.3 g mol−1, 10.45 g, 2.1 mmol), and the mixture was dried on a lyophilizer overnight in the reaction flask. Under a blanket of argon, the mixture was dissolved in trimethoxy phosphate (5.0 ml). This solution was cooled to −5 °C, and phosphoryl oxychloride was added (0.07 ml, 0.75 mmol); after 3 min, another portion of phosphoryl oxychloride (0.03 ml, 0.32 mmol) was added. This solution was stirred in a cold bath for about 20 min. After this time, prechilled tributyl ammonium pyrophosphate (1.4 g, 2.6 mmol) and tributyl amine (2.4 ml) in acetonitrile (11 ml) were added in one portion. This was stirred for 2 h and slowly warmed to room temperature. The reaction was quenched by the addition of water and worked up, isolated and purified according to the general procedure (yield 39%, formula weight for the tetra-triethylammonium salt: 927.5 g mol−1, 283.0 mg, 0.31 mmol). 1H NMR (400 MHz, D2O) δ 7.90 (d, J = 8 Hz, 1H), 6.09 (d, J = 8 Hz, 1H), 5.93 (m, J = 6, 5 Hz, 2H), 5.32 (dd, J = 17, 2 Hz, 1H), 5.22 (dd, J = 10, 2 Hz, 1H), 4.36 (t, J = 5 Hz, 1H), 4.30 (dt, J = 3 Hz, 1H), 4.22 (ddd, J = 3 Hz, 1H), 4.14 (m, 2H), 3.13 (q, 11H), 1.21 (t, 18H). 31P NMR (400 MHz, D2O) δ −10.42 (d, J = 20 Hz, 1H), −11.47 (d, J = 20 Hz, 1H), −23.12 (t, J = 20 Hz, 1H). ESI-MS: calculated for [C12H19N3O14P3]− = 522.0085; found: 522.0089.
PUP expression and purification
The DNA sequences for the wild-type CID1 S. pombe PUP (SEQ1) and mutant variants (H336R (SEQ2) and H336R-N171A-T172S (SEQ3)) were codon optimized for expression in E. coli, ordered as gBlocks (IDT) fragments and inserted into the pET-28-a-(+) expression vector (EMD Millipore 69864-3) using 2X Gibson Assembly Master Mix (NEB E2611) per the manufacturer’s instructions. High-efficiency T7 Express chemically competent E. coli cells (NEB C2566) were transformed with the fully assembled plasmid per the manufacturer’s instructions, and positive transformants were selected for on LB-kanamycin plates. Several bacterial colonies were picked, and sent for Sanger sequencing (Azenta) using the T7 forward and T7-Term primers. Those with correct sequences were grown in liquid LB-kanamycin media (Fisher 10-855-021) overnight at 37 °C, diluted the next morning (1:400) in fresh liquid LB supplemented with 50 µg ml−1 kanamycin (Sigma K1377) and induced with high-grade isopropyl β-d-1-thiogalactopyranoside (Sigma I5502) at an optical density at 600 nm of 0.6. The induced liquid cultures were incubated overnight at 15 °C, with shaking at 250 rpm. Cultures were then pelleted at 3,500g for 10 min and His-Tag purified using HisTalon Metal Affinity Resin per the manufacturer’s instructions (Takara 635503, 635623 and 635651). The eluted enzyme samples were concentrated and buffer-exchanged into 1× PUP storage buffer (10 mM Tris-HCl (Thermo AM9855G), 250 mM NaCl (Thermo AM9760G), 1 mM DTT (Sigma D9779), 0.1 mM EDTA (Thermo AM9260G), pH 7.5 at 25 °C) using Amicon 30K MWCO 15 ml filter columns (Sigma UFC9030), flash frozen using liquid nitrogen and stored at −80 °C until needed.
Endonuclease V expression and purification
Wild-type E. coli endonuclease V (SEQ4) and endonuclease V fused to a maltose binding protein at the amino terminus (SEQ 5) were expressed and purified as described for PUP, with the exception of the 1× Endo V storage buffer being composed of 10 mM Tris-HCl, 250 mM NaCl, 0.1 mM EDTA and 1 mM DTT, pH 8.0, at 25 °C. Expressed enzyme was flash frozen using liquid nitrogen and stored at −80 °C until needed.
Standard liquid bulk phase reactions
Controlled enzymatic extension reactions with PUP
A standard master mix for controlled oligonucleotide enzymatic extension in the bulk liquid phase was composed of 1× extension buffer (50 mM NaCl, 10 mM Tris-HCl, 8 mM MgCl2 (Thermo AM9530G), 2 mM MnCl2 (RPI M20100) and 1 mM DTT at pH 7.9), 0.1 mg ml−1 purified enzyme, 1 mM 3′-O-allyl ether RT-NTP and 2.5 pmol µl−1 initiator oligo. All extension reactions were carried out at 37 °C for 30 min unless otherwise specified. Following incubation, 2 µl proteinase K (NEB P8107) was added to the samples, followed by gentle mixing and incubation for 5 min at 37 °C. Extension products were then isolated and purified using Oligonucleotide Clean and Concentrator spin-columns (Zymo D4060) following the manufacturer’s instructions and eluted in MilliQ water. All standard liquid bulk phase extension reactions used an internally Cy5-labeled, 19-nt RNA initiator oligo comprised of the sequence 5-AmMC12/-rU-rU-rU-/iCy5/-rU-rU-rU-rU-rU-rU-rU-rU-rU-rU-rU-rU-rU-rU-rU-rU (IDT) and PUP mutant variant H336R unless otherwise specified.
Allyl ether deblocking reactions
A standard allyl ether deblocking reaction consisted of degassed 10 mM Tris-HCl (pH 6.7), 1.15 nmol µl−1 sodium tetrachloropalladate(II) (Na2PdCl4) (Sigma 205818), 8.80 nmol µl−1 triphenylphosphine-3,3′,3′′-trisulfonic acid trisodium salt (P(PhSO3Na)3) (Sigma 744034) and 2.5 pmol µl−1 blocked RNA oligonucleotide in MilliQ water. All deblocking reactions were carried out at 62 °C for 12 min. Deblocked oligonucleotide was then purified using Oligonucleotide Clean and Concentrator spin-columns (Zymo) and eluted in MilliQ water.
Azido methyl ether deblocking reactions
A standard azido methyl ether deblocking reaction was composed of degassed 10 mM Tris-HCl, 0.25 M Tris(2-carboxyethyl)phosphine hydrochloride (Sigma C4706) and 2.5 pmol µl−1 blocked RNA oligonucleotide in MilliQ water. Deblocking reactions were carried out at room temperature (~20 °C) for approximately 5 min. Deblocked oligonucleotide was then purified using Oligonucleotide Clean and Concentrator spin-columns (Zymo) and eluted in MilliQ water.
Endonuclease V-mediated oligonucleotide cleavage reactions
A standard endonuclease V-mediated cleavage reaction in the liquid bulk phase was carried out by incubating 2.5 pmol µl−1 initiator oligonucleotide containing a deoxy- or riboinosine base in 1× cleavage buffer (50 mM potassium acetate (Thermo J60832.AK), 20 mM Tris-acetate (Bioworld 42020180), 10 mM magnesium acetate (Thermo J60041.AE), 1 mM DTT, pH 7.9 at 25 °C) and 0.05 mg ml−1 endonuclease V at 37 °C for 30 min. For commercially sourced endonuclease V (NEB M0305), 20 U enzyme was added to reactions. Cleaved oligonucleotide was purified using Oligonucleotide Clean and Concentrator spin-columns (Zymo) and eluted into MilliQ water for downstream analysis. Wild-type endonuclease V prepared in-house was used for all standard cleavage reactions unless otherwise noted.
Phosphatase-mediated oligonucleotide dephosphorylation reactions
A standard dephosphorylation reaction in the liquid bulk phase was carried out by incubating an 5′-phosphate modified oligonucleotide with 100 U Antarctic phosphatase (NEB M0289) in 1× NEB 4 (50 mM bis-Tris-propane-HCl, 1 mM MgCl2 and 0.1 mM ZnCl2, pH 6 at 25 °C) at a concentration of 2.5 pmol µl−1 for 30 min at 37 °C. Dephosphorylated oligonucleotide was purified using Oligonucleotide Clean and Concentrator spin-columns (Zymo) and eluted in MilliQ water.
Preparation of CPG solid support derivatized with initiator oligonucleotide
The initiator-oligonucleotide-labeled solid support was prepared by covalently attaching a 5′-amine-modified oligonucleotide to the surface of LCAA-CPG using a bis-N-hydroxysuccinimide ester linker. Then, 2 g of dry LCAA-CPG with a pore size of 1,000 Å (ChemGenes N-5100-10) was added to a 20 ml scintillation vial and washed three times with 15 ml anhydrous DMF (Sigma 227056). The vial containing LCAA-CPG was rotated for 15 min during each DMF wash, and liquid waste was discarded. A 100 mg ml−1 solution of bis-PEG5-NHS ester linker (BroadPharm BP-20429) was prepared in anhydrous DMF. After the final wash of the LCAA-CPG, 5 ml of the 100 mg ml−1 bis-PEG5-NHS ester linker solution was added. Additional anhydrous DMF was added (~4–5 ml) to bring the solution to volume, and then the vial was incubated at room temperature for 2 h with rotation. After incubation, the bis-PEG5-NHS solution was discarded, and the LCAA-CPG was washed three times with anhydrous DMF. To link the initiator oligonucleotide to the now-derivatized LCAA-CPG, 500 µl of a 1 mM solution containing the 5′-amine-modified oligonucleotide (sequence: 5′-NH2-C12-rU-rC-rU-rA-rC-rC-rA-rU-rA-rU-rA-rU-dI-rA-rA-rC-rA-rA-rG-rC-rA-rC-rA-rCr-U-rA-rA-rA-rU-rU) (IDT), where dI is deoxyinosine) was prepared in MilliQ water and directly added to the LCAA-CPG in the vial, along with an additional 10 ml of fresh anhydrous DMF. This solution was incubated at room temperature for at least 4 h with rotation. After incubation, the initiator-oligonucleotide-labeled CPG solid support was washed three times with anhydrous DMF and then with a 0.1 M solution of succinimide anhydride (Sigma 239690) to cap any remaining primary amine sites on the surface of the LCAA-CPG. The solid support was then transferred to a 20 ml solid phase extraction (SPE) column with filter and washed in excess with a 10 mM Tris-HCl solution using a vacuum manifold. The resultant labeled CPG solid support was then stored at 4 °C until needed for enzymatic RNA oligonucleotide synthesis.
Standard solid phase reactions
Controlled enzymatic extension reactions on CPG solid support
A standard solid phase enzymatic extension reaction was conducted by incubating 150 mg of initiator-oligonucleotide-labeled CPG solid support with 1× extension buffer (50 mM NaCl, 10 mM Tris-HCl, 8 mM MgCl2, 2 mM MnCl2, 1 mM DTT, at pH 7.9), 0.1 mg ml−1 enzyme and 1 mM allyl ether RT-NTP terminator in a total volume of 1.5 ml. Reactions were carried out in a ‘stir format’, in which the CPG solid support and extension reaction master mix were combined in a capped 3 ml SPE column containing a small flea-sized magnetic stir bar and placed on a custom-made heat block/magnetic stir plate set to 37 °C and 1,500 rpm, respectively, for 30 min (Supplementary Fig. 18). Following incubation, the SPE column was uncapped and placed on a vacuum manifold, where the extension master mix was discarded. The solid support was then washed two times with 3 ml of DNA wash buffer (Zymo D4003) and five times with 3 ml of 10 mM Tris-HCl (pH 6.7). During each wash, the CPG solid support was gently agitated with a 1 ml pipette to ensure complete washing. The SPE column was then removed from the vacuum manifold, capped and placed on ice or stored at 4 °C until needed.
Allyl ether deblocking reactions on CPG solid support
To remove the 3′-O-allyl ether blocking group from the growing oligonucleotide on the surface of the CPG solid support, 1 ml of deblocking solution (degassed, 10 mM Tris-HCl (pH 6.7), 1.15 nmol µl−1 Na2PdCl4 and 8.80 nmol µl−1 P(PhSO3Na)3) was prepared and added directly to the SPE column. The SPE column was then placed on the combination heat block/magnetic stir plate and incubated at 62 °C for 12 min without stirring. After incubation, the SPE column was placed on a vacuum manifold, where the deblocking solution was immediately discarded. The solid support was then washed once with 3 ml of 3% ammonium hydroxide (Sigma 05002), two times with 3 ml of DNA wash buffer and five times with 3 ml of a 10 mM Tris-HCl solution (pH 6.7). During each wash, the CPG solid support was gently agitated with a 1 ml pipette to ensure complete washing. The SPE column was then removed from the vacuum manifold, capped and placed on ice until the subsequent enzymatic extension step or cleavage from the surface.
Enzymatic cleavage from the CPG solid support
Once enzymatic RNA synthesis had been completed, the oligonucleotide product was cleaved and collected by incubating 150 mg of CPG solid support with 1× cleavage buffer (50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 1 mM DTT, pH 7.9 at 25 °C) and 0.05 mg ml−1 endonuclease V at 37 °C for 30 min in a total volume of 0.750 ml. Cleavage reactions were carried out as before in a ‘stir format’ (Supplementary Fig. 18), in which the same SPE column containing the CPG solid support and magnetic flea was incubated at 37 °C and spun at 1,500 rpm for 30 min. After incubation, the cleaved oligonucleotide was collected by placing the uncapped SPE column in a 15 ml empty falcon tube and centrifuging for 1 min at 1,000g. The RNA oligonucleotide product was then stored at −20 °C until needed for analysis or downstream applications.
Conjugation of GalNAc ligand to propargyl functional handles using click chemistry
The following stock solutions were prepared before the click chemistry protocol was performed: 5 mM ascorbic acid (Sigma A92902) in MilliQ water, 10 mM copper (II)-TBTA (Tris(benzyltriazolylmethyl)amine) in 55% dimethyl sulfoxide (DMSO); prepared by dissolving 25 mg copper (II) sulfate pentahydrate (Sigma 209198) in 10 ml MilliQ water and mixing with a solution of 58 mg of TBTA ligand (Sigma 678937) in 11 ml of anhydrous DMSO) and 2 M triethylammonium acetate buffer, pH 7.0 (prepared by mixing 2.78 ml triethylamine (TEA, Chem-Impex 00319) with 1.14 ml of glacial acetic acid (Fisher A38-500), bringing the volume to 10 ml and adjusting the pH to 7.0). A stock solution of α-GalNAc-PEG3-azide ligand (Sigma SMB00392) was prepared at a final concentration of 10 mM in 100% anhydrous DMSO. Click chemistry reactions took place in a 1.5 ml high-performance LC (HPLC) glass vial with the following standard components: 200 mM triethylammonium acetate buffer, 0.5 mM ascorbic acid, 0.5 mM copper (II)-TBTA complex, 30 µM α-GalNAc-PEG3-azide and 20 µM 3′-O-propargyl-ether-modified RNA oligonucleotide (previously dissolved in MilliQ water) in a total volume of 100 µl. A low flow of high-purity argon was bubbled through the click reaction for 30 s, and then the HPLC vial was sealed tightly. Reactions were carried out overnight for 12 h at room temperature, and the α-GalNAc-PEG3-labeled RNA oligonucleotides were purified using Oligonucleotide Clean and Concentrator spin-columns (Zymo) and eluted in MilliQ water for downstream analysis.
Analysis of RNA oligonucleotide product mass, purity and concentration
Enzymatic RNA oligonucleotide synthesis product profiles were analyzed by a combination of high-resolution gel electrophoresis, MALDI-TOF mass spectrometry and LC/MS. A NanoDrop spectrophotometer (Thermo) was used to determine the concentrations of all oligonucleotide products based on absorbance at 260 nm. In instances where an oligonucleotide initiator, intermediate or final product featured an internal Cy5 dye, the absorbance at 649 nm was used to directly assess its crude purity in the presence of impurities that absorbed at 260 nm (which were generally buffer components and additives, such as guanidinium chloride, used for isolation of oligonucleotide from bulk liquid phase reactions).
High-resolution gel electrophoresis
For high-resolution gel electrophoresis, 15% TBE-urea denaturing gels (Thermo EC68855) were loaded with approximately 10–100 pmol of oligonucleotide material and run for 90 min at 185 V per the manufacturer’s instructions. If necessary, gels were then incubated with 1X GelStar nucleic acid stain (Lonza 50535) for 10 min on an orbital shaker. Gels were imaged with a Azure Sapphire Biomolecular Imager using the appropriate laser and filter settings (SYBR: 497 nm | 520 nm; Cy5: 651 nm | 670 nm).
MALDI-TOF mass spectrometry
Oligonucleotide masses were analyzed using MALDI-TOF by mixing 0.5 µl prepared MALDI matrix (50 mg ml−1 3-hydroxypicolinic acid (Sigma 56197) and 10 mg ml−1 ammonium citrate (Sigma 247561) in a solution of 50/50 MS-grade acetonitrile (Sigma 900667) and MilliQ water) with 0.75 µl purified oligonucleotide directly on a 384-spot polished steel target plate. Samples were dried under vacuum for 5 min before analysis on a Bruker autoflex MALDI-TOF using flexControl software (v.3.4). Peak acquisition was performed in positive polarity mode using an in-source decay with reflector engaged method. Analysis of acquired data was performed using Bruker flexAnalysis software (v.3.4), with all peaks transformed and smoothed using the built-in baseline subtraction feature.
LC/MS analysis
The final mass and purity of oligonucleotide intermediates and final products were assessed with an Agilent 1200 series LC system with diode array detection and XBridge Oligonucleotide BEH C18 column (130 Å, 2.5 µm, 4.6 mm × 50 mm) (Waters 186003953) using a reversed phase method (mobile phase A: 5:95 methanol/water, 400 mM 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) (Chem-Impex 00080), 15 mM TEA (Chem-Impex 00319); mobile phase B: 50:50 methanol/water, 400 mM HFIP, 15 mM TEA; method: 56% isocratic over 60 min). Mass spectra were obtained by running an Agilent 6400 series single-quadrupole MS module in scanning negative mode. Deconvolution was performed using the Agilent Bioanalysis software package.
Preparation of nucleoside triphosphate building blocks from nucleoside intermediates
Procurement and preparation of reaction components
All natural (2′-OH) and 2′-modified (-F, -OMe) nucleoside intermediates were purchased from ChemGenes Corporation with the 3′-O-allyl ether blocking group preinstalled as a custom order. Installation of the α-PS during triphosphorylation was outsourced as a custom order to Jena Bioscience using the nucleoside intermediates purchased from ChemGenes. The α-PS-modified 3′-O-azido-methyl ether RT-NTPs were accessed similarly, with ChemGenes providing nucleosides and Jena Bioscience performing triphosphorylation. Propargyl-modified nucleotides including 3′-O-propargyl-A, U, G, C, as well as N6-propargyl-ATP and 2-ethynyl-ATP, were purchased from Jena Bioscience (catalog numbers NU-945, NU-946, NU-947, NU-948, CLK-NU-001 and CLK-NU-004). Our method for the synthesis of nucleoside triphosphates from their nucleoside intermediates followed that previously reported in the literature59. Cytidine nucleosides were base transformed to their N4-DMF-C protected versions before triphosphorylation (Fig. 3b). Triphosphorylation reactions were carried out in dried glassware, under an argon atmosphere, using anhydrous acetonitrile (Sigma 271004) and tributyl amine (Sigma 90781). In addition, for triphosphorylation reactions, the nucleoside and proton sponge were premixed in their reaction flasks and vacuum dried overnight. Nucleosides that would not dissolve readily were gently heated until they were almost completely dispersed in the solution.
Standard triphosphorylation conditions
Several specific triphosphorylation reactions are discussed in detail in the ‘Preparation of 3′-O-allyl ether NTPs (A, U, G, C)’ section. All triphosphorylation reactions followed this general procedure: a mixture of nucleoside (3.38 mmol, 1 eq.) and proton sponge (9.47 mmol, 2.8 eq.) was prepared as described in the general procedure above. This mixture was dissolved in trimethoxy phosphate (25.0 ml) and cooled to −5 °C, followed by slow addition of phosphoryl oxychloride (3.7 mmol, 1.1 eq.). After 3 min, another portion of phosphoryl oxychloride (1.1 mmol, 0.3 eq.) was added. After stirring for 10 min, a prechilled mixture of tributyl ammonium pyrophosphate (12.8 mmol, 3.8 eq.), acetonitrile (55 ml) and tributyl amine (12 ml) was quickly added to the reaction. This was stirred for 2 h and then warmed to room temperature. The reaction was quenched by the addition of water (~150 ml) and worked up, isolated and purified according to the general procedure defined in the section entitled ‘Isolation and purification of prepared nucleoside triphosphates’.
Isolation and purification of prepared nucleoside triphosphates
Isolation and purification of nucleoside triphosphates was optimized and generally carried out as follows. Crude, quenched reaction mixture was washed with dichloromethane. The aqueous layer containing triphosphorylated product was washed with hexane and concentrated in vacuo. The isolated material was purified via ion-exchange chromatography (DEAE Sepharose resin; mobile phase A: MilliQ water; mobile phase B: 1 M triethyl ammonium bicarbonate buffer, pH 8 ± 0.5). The fractions containing primarily triphosphate (assayed via LC-MS) were combined and concentrated in vacuo. The sample was then further purified via preparatory HPLC (1260 Infinity Preparative LC System and Phenomenex Jupiter C18 reverse-phase column; 10 µm particle size, 300 Å pore size, 250 mm length, 21.2 mm diameter). Generally, a single method provided excellent purities for the various NTP products (mobile phase A: 0.1% ammonium acetate in acetonitrile; mobile phase B: 0.1% ammonium acetate in 1:667 water/acetonitrile; general method: 5% isocratic over 20 min, then to 90% over 30 min). Pure fractions were combined, frozen and lyophilized. The counter-ions on the triphosphate were exchanged by diluting the lyophilized sample in triethyl ammonium bicarbonate (1 M) and concentrating on the lyophilizer. Excess triethyl ammonium bicarbonate was removed from the sample by additional dilution with water and freezing, followed by lyophilization until the sample reached constant mass. Note that for this protocol, yields and stock solutions of the nucleoside triphosphates were prepared with the presumption that all final products would exist as tetra-triethylammonium salts. However, after additional, scrupulous lyophilization of analytical samples for the NMR analysis, we generally saw two to three triethylammoniums present in the final product.
Evaluation of prepared nucleoside triphosphates
Analytical HPLC was performed on an Agilent 1260 series LC system with diode array detection using a reversed phase method (mobile phase A: water, 400 mM HFIP, 15 mM TEA; mobile phase B: methanol, 400 mM HFIP, 15 mM TEA, unless otherwise specified). The best triphosphate resolution was obtained using a Waters XBridge Oligonucleotide BEH C18 column (130 Å, 2.5 µm, 4.6 mm × 50 mm). High-resolution mass spectra were obtained via ESI-MS-HiRes on a Thermo q-Exactive Plus spectrometer. 1H NMR (400 MHz on a Varian Mercury instrument) and 31P NMR (400 MHz on a Varian Mercury instrument) spectra were measured. Chemical shifts are reported relative to the central line of residual solvent.
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
