Engineered template-independent enzymes for polydeoxynucleotide synthesis

Abstract

The present invention includes methods for identifying polymerases, such as modified terminal nucleotidyl transferases (TdT), that are capable of attaching nucleotides containing removable 3'-O-blocking moieties to nucleic acid initiators without the use of a template. The present invention further includes the identified polymerases and methods of using the polymerases for de novo synthesis of predetermined oligonucleotide sequences. The methods of the present invention include nucleic acid synthesis using 3'-O-blocked nucleotide analogs and shrimp alkaline phosphatase (SAP) for the controlled addition of selected nucleotides.

Description

[Technical Field]

[0001] Related Applications This application claims priority to and the benefit of U.S. patent application Ser. No. 16 / 891,449, filed Jun. 3, 2020, the contents of which are incorporated herein by reference.

[0002] FIELD OF THE INVENTION The present invention relates to engineered enzymes for the de novo synthesis of polynucleotides having desired sequences without the use of a template. As such, the present invention provides the ability to generate libraries of polynucleotides of varying sequence and length for research, genetic engineering, and gene therapy. [Background technology]

[0003] background Most de novo nucleic acid sequences are synthesized using solid-phase phosphoramidite technology, which was developed more than 30 years ago. This technology involves sequential deprotection and synthesis of sequences built from phosphoramidite reagents corresponding to natural (or unnatural) nucleic acid bases. However, phosphoramidite nucleic acid synthesis is length-limited in that nucleic acids longer than 200 base pairs (bp) experience high rates of cleavage and side reactions. Moreover, phosphoramidite synthesis produces toxic by-products, and disposal of this waste limits the availability of nucleic acid synthesizers and increases the cost of contract oligonucleotide production (annual demand for oligonucleotide synthesis is estimated to result in over 300,000 gallons of hazardous chemical waste, including acetonitrile, trichloroacetic acid, toluene, tetrahydrofuran, and pyridine. See LeProust et al., Nucleic Acids Res., vol. 38(8), pp. 2522-2540, (2010), which is incorporated herein by reference in its entirety). Thus, there is a need for more efficient and cost-effective methods for oligonucleotide synthesis. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] LeProust et al., Nucleic Acids Res. (2010)38(8)2522~2540 Summary of the Invention [Means for solving the problem]

[0005] overview The present invention discloses modified terminal deoxynucleotidyl transferase (TdT) enzymes that can be used for the de novo synthesis of oligonucleotides in the absence of a template. Also disclosed is a method for creating template-independent polymerases by combining computer-guided and saturation mutagenesis, followed by screening to identify functional mutants. Native TdT enzymes are inefficient or completely unable to incorporate different blocked nucleotide analogs used in template-independent synthesis schemes. The present invention provides various TdT modifications that expand the enzyme's functionality, particularly for blocked nucleotide analogs with 3'-O-blocking groups. In particular, the modified TdT of the present invention can be used to incorporate 3'-O-phosphate blocked nucleotide analogs, which wild-type TdT may not be able to do.

[0006] The methods of the present invention involve nucleic acid synthesis using 3'-O-blocked nucleotide analogs and shrimp alkaline phosphatase (SAP) for the controlled addition of selected nucleotides.

[0007] Using the enzymes and methods of the present invention, it will be possible to synthesize de novo polynucleotides faster and more cheaply. As such, the present invention dramatically reduces the overall cost of synthesizing custom nucleic acids. In particular, the present methods can be used to create template-free transferases that can synthesize custom oligos in a stepwise manner using modified 3' hydroxyl-blocked nucleotides. Due to the terminal group, synthesis pauses with each addition of a new base, at which point the terminal group is cleaved, leaving a polynucleotide that is essentially identical to a naturally occurring nucleotide (i.e., recognized by the enzyme as a substrate for further nucleotide incorporation).

[0008] The methods and enzymes of the present invention represent an important step forward in synthetic biology, as the enzymes will enable aqueous-phase, template-free oligonucleotide synthesis. Such methods represent an improvement over the prior art in that they will significantly reduce the chemical waste produced during oligonucleotide synthesis, while enabling the production of longer polynucleotides. Furthermore, because the methods replace chemical processes with biological processes, the cost and complexity of automated synthesis systems will be reduced. In certain embodiments, a simple five-reagent delivery system could be used to build oligonucleotides in a stepwise fashion, allowing unused reagents to be recycled. In certain embodiments, for example, the following items are provided: (Item 1) 1. A modified terminal deoxynucleotidyl transferase (TdT) comprising a mutation selected from the group consisting of E33K, E180L, E180K, M192E, M192K, M192W, W303H, L381K, L381Q, L381R, L381V, W450H, R454I, R454T, R454K, E457K, R461V, R461Q, R461V, N474R and N474K, wherein the modified TdT is capable of adding a nucleotide analog comprising a removable blocking moiety on the 3'-oxygen of the analog to the 3'-OH of a nucleic acid initiator in the absence of a nucleic acid template. (Item 2) 2. The modified TdT according to item 1, comprising the mutation E457K. (Item 3) 2. The modified TdT according to item 1, comprising the mutations E180K, M192W, L381R and W450H. (Item 4) 2. The modified TdT according to item 1, comprising the mutations L381Q and W450H. (Item 5) 2. The modified TdT according to item 1, comprising the mutations E180L, M193E, L381K, R461Q and N457K. (Item 6) 2. The modified TdT according to item 1, comprising the mutations E180K, L381Q, W450H and R461V. (Item 7) 2. The modified TdT according to item 1, comprising the mutations L381Q and W450H. (Item 8) 2. The modified TdT according to item 1, comprising the mutations E180L, M192E, L381K, R461Q and N457K. (Item 9) 2. The modified TdT according to item 1, comprising the mutations E180K, M192E, L381K, R454T and N47K. (Item 10) 2. The modified TdT according to item 1, comprising the mutations E180K, M192K, L381K, R454T and N457R. (Item 11) 2. The modified TdT according to item 1, comprising the mutations E180K, M192K, L381K, R454K and N457K. (Item 12) 2. The modified TdT according to item 1, comprising the mutations M192E, L381V, R454I and R461V. (Item 13) 2. The modified TdT according to item 1, comprising the mutations E180K and L381R. (Item 14) 2. The modified TdT according to item 1, comprising the mutations E180K, M192K, L381K, R454K and N474R. (Item 15) 2. The modified TdT of item 1, wherein the nucleotide analog containing the removable 3'-O-blocking moiety can be added to the 3'-OH of the nucleic acid initiator at an increased rate compared to native TdT. (Item 16) 2. The modified TdT according to item 1, comprising an N-terminal truncation compared to native TdT. (Item 17) 16. The modified TdT of item 15, comprising an N-terminal t-131 mouse TdT and a protein tag sequence attached to the N-terminus. (Item 18) 16. The modified TdT of item 15, comprising an N-terminal t-147 mouse TdT and a protein tag sequence attached to the N-terminus. (Item 19) 2. The modified TdT according to item 1, which is capable of adding adenine, cytosine, guanine and thymine deoxyribonucleotides modified with a removable 3'-O-blocking moiety. (Item 20) 19. The modified TdT of item 18, wherein the nucleotide is a 2'-deoxyribonucleotide. (Item 21) 2. The modified TdT according to item 1, which is capable of adding adenine, cytosine, guanine and uracil ribonucleotides modified with a removable 3'-O-blocking moiety. (Item 22) The removable 3'-O-blocking moiety is CH 2 N 3 , N.H. 2 , ONHC(O)H, allyl, CH 2 SSCH 3 , phenoxyacetyl, methoxyacetyl, acetyl, (p-toluene)sulfonate, phosphate, nitrate, [4-methoxy]-tetrahydrothiopyranyl, tetrahydrothiopyranyl, [5-methyl]-tetrahydrofuranyl, [2-methyl,4-methoxy]-tetrahydropyranyl, [5-methyl]-tetrahydropyranyl; and O-tetrahydrothiofuranyl. (Item 23) 2. The modified TdT of item 1, wherein the modified TdT is capable of incorporating a 3'-O-blocked nucleotide 5'-triphosphate, and the removable blocking moiety comprises a group selected from an ester, an ether, a carbonitrile, a phosphate, a carbonate, a carbamate, a hydroxylamine, a borate, a nitrate, a sugar, a phosphoramide, a phosphoramidate, a phenylsulfenate, a sulfate, a sulfone, and an amino acid. (Item 24) 2. The modified TdT according to item 1, which can incorporate modified nucleotides at a reaction temperature of about 30°C to about 80°C. (Item 25) 2. The modified TdT according to item 1, which is capable of incorporating modified nucleotides at a concentration of 1000 μM or less. (Item 26) 2. The modified TdT according to item 1, which is capable of incorporating modified nucleotides at a concentration of 100 μM or less. (Item 27) 15. The modified TdT according to item 14, wherein a nucleotide analog containing a removable 3'-O-phosphate can be added to the 3'-OH of a nucleic acid initiator. [Brief explanation of the drawings]

[0009] [Figure 1] Figure 1 shows agarose gels of a solution-phase polymerization reaction consisting of terminal deoxynucleotidyl transferase (TdT), deoxyadenosine triphosphate (dATP), and the fluorescent chain initiator 5'-Cy5-dA10 at different time points from Tjong et al. "Amplified on-chip fluorescence detection of DNA hybridization by surface-initiated enzymatic polymerization," Anal. Chem., 2011; 83:5153-5159 (2011).

[0010] [Figure 2] FIG. 2 illustrates an exemplary modified terminal deoxynucleotidyl transferase (TdT)-mediated polynucleotide synthesis cycle using a support-bound initiator and a 3′-O-blocked nucleotide triphosphate, including (A) incorporation of a nucleotide analog containing a cleavable 3′-O-blocking group (designated by R), and (B) removal of the 3′-O-blocking group (thereby allowing the next 3′-O-blocked nucleotide analog to be incorporated) (where N=A, G, C, or T).

[0011] [Figure 3]Figure 3 shows a polyacrylamide gel analysis of solution-phase reaction time courses for commercially available TdT and nucleic acid initiators with 3'-O-azidomethyl-dCTP or 3'-O-azidomethyl-dATP. Lane 1 - 100 bp ladder size standard; Lane 2 - oligonucleotide standard; Lane 3 - 15-minute reaction time for 3'-O-azidomethyl-dCTP + TdT; Lane 4 - 1 hour; Lane 5 - 2 hours; Lane 6 - 4 hours; Lane 7 - 24 hours; Lane 8 - 15-minute reaction time for 3'-O-azidomethyl-dATP + TdT; Lane 9 - 1 hour; Lane 10 - 2 hours; Lane 10 - 4 hours; Lane 11 - 24 hours; Lane 12 - 15-minute reaction time for dATP + TdT; Lane 13 - 1 hour; Lane 14 - 4 hours; Lane 15 - 24 hours.

[0012] [Figure 4] Figure 4 shows a computer-generated image of the active site of TdT using the PDB crystal structure 4I29, showing the computationally docked catalytically productive position and the 3'-O-dATP analog (blue, red, and orange frames), each complexed with two active site metal ions (large green spheres). Residues in close proximity to the incoming dNTP and residues that are targets for mutagenesis and screening are indicated.

[0013] [Figure 5] FIG. 5 shows a table of TdT variants selected for increased incorporation of selected 3′-O-blocked dNTP analogs described herein.

[0014] [Figure 6] FIG. 6 shows exemplary 3′-O-azidomethyl deoxynucleotides that can be used to synthesize custom DNA oligomers using the modified TdT described herein.

[0015] [Figure 7]FIG. 7 shows a synthetic scheme for producing 3′-O-azidomethyldeoxyadenosine triphosphate (3′-O-azidomethyl-dATP).

[0016] [Figure 8] FIG. 8 shows a synthetic scheme for producing 3′-O-azidomethyldeoxythymidine triphosphate (3′-O-azidomethyl-dTTP).

[0017] [Figure 9] FIG. 9 shows a synthetic scheme for producing 3′-O-azidomethyldeoxycytidine triphosphate (3′-O-azidomethyl-dCTP).

[0018] [Figure 10] FIG. 10 shows a synthetic scheme for producing 3′-O-azidomethyldeoxyguanosine triphosphate (3′-O-azidomethyl-dGTP).

[0019] [Figure 11] FIG. 11 shows a synthetic scheme for producing 3′-O-methoxymethyldeoxythymidine triphosphate (3′-O-MOM-dTTP).

[0020] [Figure 12] FIG. 12 shows a synthetic scheme for producing 3′-O-thiomethyldeoxycytidine triphosphate (3′-O-MTM-dCTP).

[0021] [Figure 13] FIG. 13 shows CGE (capillary gel electrophoresis) traces showing the migration of A) a chemically synthesized, certified standard of 5′-FAM-TAATAATAATAATAATTTTT compared to B) a chemically synthesized, certified standard of 5′-FAM-TAATAATAATAATAATTTTT-PO4-3′.

[0022] [Figure 14]Figure 14 shows CGE traces demonstrating the removal of 3'-PO4 by treatment with shrimp alkaline phosphatase. A) Chemically synthesized 5'-TAATAATAATAATAATTTTT-PO4-3' before treatment with shrimp alkaline phosphatase, and B) 4.1 x 10-4 U / ul, C) 1.23 x 10-3 U / ul, D) 3.7 x 10-3 U / ul, E) 1.1 x 10-2, F) 3.33 x 10-2 U / ul, G) 1.0 x 10-1 U / ul of shrimp alkaline phosphatase after treatment for 1 minute at 37°C.

[0023] [Figure 15] Figure 15 shows CGE traces comparing A) 5'-FAM-TAATAATAATAATAATTTTT after treatment with mouse WT TdT and no dNTPs; B) 5'-FAM-TAATAATAATAATAATTTTT after treatment with mouse WT TdT and 500uM 3'-PO4-dTTP for 60 minutes at 37°C; and C) 5'-FAM-TAATAATAATAATAATTTTT after treatment with mouse WT TdT and 500uM 3'-PO4-dTTP for 60 minutes at 37°C followed by treatment with 0.2 units of shrimp alkaline phosphatase for 15 minutes at 37°C.

[0024] [Figure 16]Figure 16 shows A) 5'-FAM-TAATAATAATAATAATTTTT-3' after treatment with mouse WT TdT and no dNTPs for 60 minutes at 37°C; B) 5'-FAM-TAATAATAATAATAATTTTT after treatment with mouse TdT E180K+M192K+L381K+R454K+N474R and 500uM 3'-PO4-dTTP for 60 minutes at 37°C; C) 5'-FAM-TAATAATAATAATAATTTTT-3' after treatment with mouse WT TdT and 500uM 3'-PO4-dTTP for 60 minutes at 37°C followed by treatment with 0.2 units of shrimp alkaline phosphatase for 15 minutes at 37°C; D) 5'-FAM-TAATAATAATAATAATTTTT-3' after treatment with mouse WT TdT and 500uM 3'-PO4-dTTP for 60 minutes at 37°C. 1 shows CGE traces comparing homopolymeric dT extension ladders created by treatment of 5'-FAM-TAATAATAATAATAATTTTT-3' with TdT and dTTP. DETAILED DESCRIPTION OF THE INVENTION

[0025] Description of the Invention The present invention facilitates the synthesis of polynucleotides, such as DNA, by providing modified enzymes that can be used with nucleic acid analogs. Using the disclosed methods, modified template-independent terminal deoxynucleotidyl transferases (TdTs) are obtained that enable the enzymatic synthesis of de novo oligodeoxynucleotides, thereby enabling their use in routine assembly for gene synthesis. The enzymes of the present invention are useful in aqueous-based, enzyme-mediated methods for synthesizing polynucleotides of predetermined sequence on a solid support.

[0026] The modified enzymes of the present invention allow 3'-O-blocked dNTP analogs to be used in a step-by-step manner to extend starting nucleic acids to user-defined sequences (see Figure 2). Furthermore, after each nucleotide extension step, the reactants can be recovered from the solid support and recycled back to the original reagent reservoir. Upon completion of this step, the 3'-O-blocking group would be removed, allowing the cycle to begin anew. At the end of n cycles of extension-recovery-unblocking-wash, the full-length single-stranded polydeoxynucleotide would be cleaved from the solid support and isolated for subsequent use. While a variety of 3'-O-blocked deoxynucleotides can be used, the selection of a specific 3'-O-blocking group is dictated by 1) the smallest possible bulk to maximize substrate utilization by TdT, and 2) removal of the blocking group in the shortest time period under the mildest, preferably aqueous, conditions.

[0027] The cost savings of this approach will be achieved by utilizing higher yields of final oligonucleotide product at lower starting scales (i.e., less than 1 nanomole) than currently used as the existing industry standard. Future adaptation of this enzymatic approach to array-based formats will enable even greater and more dramatic reductions in the cost of synthesizing long oligonucleotides achievable through highly parallel synthesis. Furthermore, our proposed enzymatic synthesis process uses only aqueous-based chemistry, such as buffers and salts, thus significantly reducing the environmental burden of organic waste generated by existing phosphoramidite methods.

[0028] The methods of the present invention can be used to modify terminal deoxynucleotidyl transferase (TdT), but similar methods can be used to modify other enzymes. TdT is potentially a successful starting enzyme because it is capable of 3'-extension activity using a single-stranded starting primer in template-independent polymerization. However, prior to the invention described herein, there had been no reports of 3'-O-blocked nucleotides being enzymatically incorporated into single-stranded oligonucleotides in the absence of a template. Indeed, as reported by Chang and Bollum, replacement of the 3'-hydroxyl group results in complete inactivation of the available transferase enzyme. See Chang and Bollum, "Molecular Biology of Terminal Transferase," CRC Critical Reviews in Biochemistry, vol. 21 (1), pp. 27-52 (1986), which is incorporated herein by reference in its entirety. Nevertheless, when TdT is used with natural dNTPs (i.e., not 3'-O-blocked) and without a template, oligonucleotide elongation continues without termination. Such uncontrolled incorporation is evidenced by the time-dependent gel electrophoresis images shown in FIG. 1, which shows an agarose gel of a solution-phase polymerization reaction composed of terminal deoxynucleotidyl transferase (TdT), deoxyadenosine triphosphate (dATP), and the fluorescent chain initiator 5'-Cy5-dA10 at different time points (Tjong et al. "Amplified on-chip fluorescence detection of DNA hybridization by surface-initiated enzymatic polymerization," Anal. Chem., 2004, 10, 111-115, which is incorporated herein by reference in its entirety). (Adapted with permission from 2011; 83:5153-5159 (2011)). Moreover, TdT can extend primers in a nearly quantitative manner, resulting in the addition of thousands of nucleotides, while TdT can accept a wide variety of modified and substituted dNTPs as efficient substrates.Moreover, a substantial library of mechanistic and structural information on TdT already exists, see Delarue et al., EMBO J. 2002;21(3):427-39; Gouge et al., J Mol Biol. 2013 Nov 15;425(22):4334-52 and Romain et al., Nucleic Acids Res. 2009;37(14):4642-56, both of which are incorporated by reference in their entirety.

[0029] It is known that TdT can use substrates with modifications and / or substitutions in deoxyribose sugar ring and purine / pyrimidine nucleobase.For example, TdT accepts bulky modifications at C5 of pyrimidine and C7 of purine.See Sorensen et al., "Enzymatic Ligation of Large Biomolecules to DNA," ACS Nano 2013, 7(9):8098-104; Figeys et al., Anal. Chem. 1994, 66(23):4382-3; Li et al., Cytometry, 1995, 20(2):172-80, all of which are incorporated herein by reference in their entirety.In some instances, TdT can also accept non-nucleotide triphosphates. See Barone et al., Nucleotides and Nucleic Acids 2001, 20(4-7):1141-5 and Alexandrova et al., Bioconjug Chem., 2007, 18(3):886-93, both of which are incorporated herein by reference in their entireties. However, there is little evidence in the prior art that TdT can accept 3'-O-blocked nucleotides. See, for example, Knapp et al., Chem. Eur. J., 2011, 17:2903, both of which are incorporated herein by reference in their entireties. Although the lack of activity of TdT was not the focus of Knapp et al., the authors reported that when they tested their own 3'-OH modified analogs with TdT, they did not observe incorporation of this relatively small 3'-OH modification into oligonucleotides.

[0030] Native TdT is a highly efficient enzyme. It has been demonstrated that TdT can polymerize extremely long homopolydeoxynucleotides ranging from 1,000 to 10,000 nucleotides in length (see Hoard et al., J of Biol Chem, 1969 244(19):5363-73; Bollum, The Enzymes, Volume 10, New York: Academic Press; 1974. pp. 141-71; Tjong et al., Anal Chem, 2011, 83:5153-59, all of which are incorporated herein by reference in their entirety). Random sequence oligomers consisting of all four nucleotides have also been polymerized by TdT, but there have been no reports of ordered polynucleotides synthesized in the absence of a template. See Damiani, et al., Nucleic Acids Res, 1982, 10(20):6401-10, all of which are incorporated herein by reference in their entirety. Support-bound synthesis of polynucleotides by TdT is further supported by reports of homopolymer synthesis of a 150-bp initiator covalently attached to a self-assembled monolayer on a gold surface. See Chow et al., J Am Chem Soc 2005; 127:14122-3 and Chow and Chilikoti, Langmuir 2007, 23:11712-7, both of which are incorporated herein by reference in their entireties. The authors also observed a TdT preference of dATP>dTTP>>dGTP≒dCTP for homopolymer incorporation. In a more recent report, Tjong et al. demonstrated TdT-mediated synthesis of long (>1 Kb) homopolymeric ssDNA from an initiator primer immobilized on a glass surface.

[0031] The distribution behavior of TdT is reinforced by Figure 3, which shows the time course of the solution-phase synthesis of a 1-1.5 kb homopolymer. After each addition of an unmodified (native) dNTP, the enzyme dissociates, thereby allowing random extension of any strand in the population. The product length distribution in such a system should follow a Poisson distribution, as reported by Bollum and coworkers in 1974. When TdT is used with a terminating nucleotide species, i.e., a species with a blocked 3'-O-position, the reaction should proceed to completion, resulting in no distribution of product lengths but essentially pure products of single nucleotide addition.

[0032] Nevertheless, as described above, nucleotide synthesis with 3'-O-blocked dNTPs does not proceed with commercially available TdT protein. This fact is reinforced by Figure 3, which shows a gel shift assay used to monitor the solution-phase incorporation kinetics of 3'-O-azidomethyl dATP and 3'-O-azidomethyl dCTP using commercially available recombinant TdT. The data in Figure 3 clearly show that neither 3'-O-modified dNTP analog is a substrate for TdT, i.e., there is no polynucleotide elongation compared to reactions containing dATP as a positive control (lanes 12 to 15). Thus, Figure 3 provides further evidence that commercially available TdT cannot synthesize oligomers by incorporating dNTPs with modified 3'-OH groups.

[0033] With appropriate modification, a variety of different 3'-O-blocked dNTP analogs will be suitable for the controlled addition of nucleotides by TdT. Modified 3'-O-blocked dNTP analogs include, but are not limited to, 3'-O-allyl, 3'-O-azidomethyl, 3'-O-NH, 3'-O-CHN, 3'-O-ONHC(O)H, 3'-O-CHSSCH, and 3'-O-CHCN blocking groups. Overall, the selection of a 3'-O-blocking group will be dictated by 1) the smallest possible bulk to maximize substrate utilization by TdT, which may affect kinetic incorporation, and 2) the blocking group with the mildest removal conditions, preferably aqueous, in the shortest time period. Suitable 3'-O-blocking groups for use in accordance with the present invention are described in WO2003 / 048387; WO2004 / 018497; WO1996 / 023807; WO2008 / 037568; Hutter D, et al. Nucleosides Nucleotides Nucleic Acids, 2010, 29(11): 879-95; and Knapp et al., Chem. Eur. J., 2011, 17:2903, all of which are incorporated by reference in their entirety.

[0034] A computer model of the active site of mouse TdT was created to understand the structural basis for TdT's lack of utilization of 3'-O-blocked dNTPs. Furthermore, the computer model allowed various modified dNTPs to be "fitted" into the active site. Figure 4 shows the docking of -dATP (shown in blue, red, magenta, and orange) with mouse TdT (see SEQ ID NO: 9 below) using the PDB crystal structure 4I29 and AutoDock 4.2 (Molecular Graphics Laboratory, Scripps Research Institute, La Jolla, CA).

[0035] The phosphate moiety of dATP (orange) complexes with the catalytic metal ion (green), while the alpha phosphate is positioned for attack by the 3'-OH of the bound oligonucleotide. The model shown in Figure 4 indicates the selection of amino acid residues that may interfere with the formation of a catalytically productive complex in the presence of a 3'-O-blocked dNTP. Other residues that can interact with the nearest residue, such as Glu180 or Met192, are also targets for modification. The amino acid numbering and positions are provided with reference to mouse TdT of SEQ ID NO: 9, but the referenced amino acid modifications are applicable to any TdT with a similar sequence containing a GGFRR or TGSR motif.

[0036] The binding mode predicted by AutoDock suggests that modifications to the 3'-OH would alter the electrostatic interaction between two residues, Arg336 and Arg454. Although Arg336 is close to the reaction center in the active site, it is highly conserved, and initial studies have found that replacing Arg336 with Gly or Ala reduced dNTP activity by 10-fold (Yang B et al. J. Mol. Biol. 1994; 269(16):11859-68). Therefore, one motif for modification is the GGFRR motif, which includes Arg336 in the structural model above.

[0037] Furthermore, Gly452 and Ser453 exist in a cis-peptide-binding conformation (see Delarue et al., EMBO J., 2002; 21(3):427-39, incorporated herein by reference in its entirety), and the guanidinium group of Arg336 is thought to help stabilize this conformation. The stability provided by Arg336 may help explain why substitution at this position has a negative impact on the reactivity of modified TdT proteins. In some instances, the instability created by modifying position 336 can be overcome by using a proline residue to stabilize the cis-peptide-binding conformation. However, if Arg336 is substituted, for example, with alanine or glycine, the entire TGSR motif (positions 451, 452, 435, and 454) may also need to be modified to compensate for this change. For example, the TGSR motif can be modified into a TPSR or TGPR. Therefore, the TGSR motif containing Gly452 in the above structural model was targeted for modification.

[0038] On the other hand, sequence analysis of the TdT family demonstrates a wide range of amino acids that can be accommodated at position 454. This analysis suggests structural flexibility at position 454 and surrounding residues. In another embodiment, substitutions at Arg454 to accommodate the steric bulk of the 3'-O-blocking group may require additional modifications to the α14 region to compensate for the substitution of glycine or alanine at Arg454. In other embodiments, substitutions to other residues in the α11 region may be required to compensate for the substitution to Arg336, either instead of or in addition to modifications of the TGSR motif.

[0039] While modifications to Arg336 and Arg454 can alter the binding interactions of 3'-O-modified dNTPs, it may be necessary to explore substitutions that result in improved steric interactions of 3'-O-modified dNTPs with TdT. To test computationally predicted enzyme variants that exhibit increased substrate utilization of 3'-O-blocked dNTPs, synthetic genes specifying specific amino acid substitutions were generated in appropriate plasmid vectors and introduced into cells. After expression and isolation, protein variants were screened for activity by polymerase incorporation assays using selected 3'-O-blocked dNTP analogs. Figure 5 shows the results of screening various synthetically generated mouse TdT variants. In some embodiments, single amino acid changes are important, while in other embodiments, combinations of one and two amino acids also produce increased incorporation of 3'-O-blocked dNTPs. Interactions with residues such as Gly332, Gly333, Gly452, Thr451, Trp450, Ser453, and Q455 of mouse TdT are important. Each of these residues is within 0.6 nm of the 3'-OH of a typical dNTP. These residues are also potential targets for substitution to allow for the extra steric bulk of a 3'-blocking group such as 3'-O-azidomethyl or 3'-O-aminoxy. In addition to or in combination with Arg336 and Arg454, residues within 1.2 nm of the 3'-OH, such as Glu457, Ala510, Asp509, Arg508, Lys199, Ser196, Met192, Glu180, or Leu161, can also potentially interfere with substrate utilization of 3'-O-blocked dNTPs and are therefore targets for substitution. Additional residues of interest include Arg461 and Asn474.

[0040] Although the TGSR and GGFRR motifs are emphasized here, modifications to adjacent amino acids such as Thr331, Gly337, Lys338, Gly341, or His342 are also contemplated (alone or in combination) to provide increased incorporation of the 3'-O-blocked dNTPs discussed herein. Various in silico modeled TdT modifications capable of increased incorporation are discussed in Example 2 below.

[0041] In addition to amino acid substitutions at positions 500-510, it may be necessary to delete residues to eliminate interference with the 3'-O-blocking group. Because these amino acids are located near the C-terminus of the protein and are in a relatively unstructured region, they can be deleted alone or together, either instead of or in combination with the modifications described above. In certain embodiments, inserting residues into modified TdT, such as inserting residues in the GGFRR or TGSR motif or adjacent regions, can increase the rate of incorporation of 3'-O-blocked dNTPs by the modified TdT. TdT modifications can include inserting a tyrosine residue between (or substituting for) the Phe334 and Arg335 residues of the GGFRR motif.

[0042] Modified TdTs of the present invention include those depicted in Figure 5. Modified TdTs can include one or more modifications to Glu180, including E180L, E180R, E180D, or E180K. Contemplated modifications to Met192 include, for example, M192E, M192W, M192K, or M192R. Contemplated modifications to Gln455 include, for example, Q455I. Contemplated modifications to Trp450 include, for example, W450H. Contemplated modifications to ARG454 include, for example, R454I, R454K, R454A, or R454T. Contemplated modifications to Arg461 include, for example, R461V, and modifications to Asn474 can include N474R. In various embodiments, combinations of two or more altered residues can be used, such as, for example, E180D+W450H, E180K+R454A, M192K+E180K, E180K+R454I, E180D+M192E, E180D+M192E+R454T, or E180K+W450H.

[0043] As shown below, most TdTs contain GGFRR and TGSR motifs. In the following sequences, the GGFRR and TGSR motifs are shown in bold and underlined for ease of reference. Native calf thymus TdT is a candidate for modification of its primary structure to achieve a suitable template-independent polymerase. However, various other proteins, including human and mouse TdT, can be explored to identify suitable candidates for use with 3'-O-blocked dNTP analogs. The amino acid sequence corresponding to native calf TdT is listed in Table 1 as SEQ ID NO: 1, while the nucleic acid sequence is listed in Table 2 as SEQ ID NO: 2. In some embodiments, the resulting protein adapted for sequence-specific de novo polynucleotide synthesis using 3'-O-modified dNTPs and NTPs will be at least 85% identical to SEQ ID NO: 1, i.e., at least 90% identical, i.e., at least 93% identical, i.e., at least 95% identical, i.e., at least 97% identical, i.e., at least 98% identical, i.e., at least 99% identical. Furthermore, it may be possible to truncate portions of the amino acid sequence of bovine TdT and still maintain catalytic activity. Table 1. Amino acid sequence of bovine TdT SEQ ID NO: 1: (520 aa) [Table 1] Table 2. Nucleic acid sequence of bovine TdT SEQ ID NO: 2: (1923 nt) [Table 2-1] [Table 2-2]

[0044] Furthermore, to facilitate easier isolation of recombinant proteins, it is common to add an N-terminal His tag sequence to recombinant proteins, which is used in combination with an affinity column (Hitrap, Amersham Pharmacia Biotech, Uppsala, Sweden) (see Boule JB et al., Molecular Biotechnology, 1998;10:199-208, incorporated herein by reference in its entirety). Alternatively, an N-terminal truncated form of the enzyme with an added His tag sequence will function in the present invention (see, e.g., US Pat. No. 7,494,797, incorporated herein by reference in its entirety). The His-tagged bovine TdT amino acid sequence is shown in Tables 3, 5, and 7 below, while the His-tagged bovine TdT nucleic acid sequence is shown in Tables 4, 6, and 8 below. The His tag can be engineered at other positions as desired. In some embodiments, the resulting protein adapted for sequence-specific de novo polynucleotide synthesis using 3'-O-modified dNTPs and NTPs will be at least 85% identical, i.e., at least 90% identical, i.e., at least 93% identical, i.e., at least 95% identical, i.e., at least 97% identical, i.e., at least 98% identical, i.e., at least 99% identical to SEQ ID NO: 3, 5 or 7. Table 3. Amino acid sequences of Δ138- and His-tagged bovine TdT SEQ ID NO: 3: (392 aa) [Table 3] Table 4. Nucleotide sequences of Δ138- and His-tagged bovine TdT SEQ ID NO: 4: (1187nt) [Table 4-1] [Table 4-2] Table 5: Amino acid sequences of Δ151 and His-tagged bovine TdT SEQ ID NO: 5: (379aa) [Table 5] Table 6. Nucleotide sequences of Δ151 and His-tagged bovine TdT SEQ ID NO: 6: (1148nt) [Table 6-1] [Table 6-2] Table 7. Amino acid sequences of Δ160 and His-tagged bovine TdT SEQ ID NO: 7: (370 aa) [Table 7] Table 8. Nucleotide sequences of Δ160 and His-tagged bovine TdT SEQ ID NO: 8: (1121nt) [Table 8] Table 9. Amino acid sequence of mouse TdT SEQ ID NO: 9: (510 aa) [Table 9] In certain embodiments, the modified enzymes of the present invention can comprise an N-terminal truncation compared to the respective native TdT enzyme. For example, in a preferred embodiment, the native enzyme can be the mouse TdT provided in SEQ ID NO: 9 above. The modified TdT can be truncated at the equivalent positions of 147 or 131 of native mouse TdT, as shown in SEQ ID NOs: 10 and 11, respectively. The modified TdT can comprise a protein tag sequence, such as a His tag, and an additional linker at its N-terminus, as illustrated in SEQ ID NOs: 10 and 11. The His tag portion is underlined in each of the sequences, and the linker is presented in bold. SEQ ID NO: 10: Mouse del-147 with His tag and linker [ka] SEQ ID NO: 11: Mouse del-131 with His tag and linker [ka]

[0045] Additional TdT modifications that can increase the efficiency of incorporation of 3'-O-blocked or other nucleotide analogs are listed in Table 10 below. While the modifications are described with reference to murine TdT listed in SEQ ID NO:9, the present invention contemplates such modifications applied to the equivalent amino acids in any TdT, including the truncated enzymes disclosed in SEQ ID NOs:10 and 11 above, with or without His tags and linkers. In various embodiments, contemplated modifications include deletion of amino acids S420 to E424. Various combinations of amino acid substitutions of the present invention are listed in rows 1-175 of Table 10. Table 10 [Table 10-1] [Table 10-2] [Table 10-3] [Table 10-4] [Table 10-5] [Table 10-6]

[0046] Various 3'-O-modified dNTPs and NTPs can be used with the disclosed proteins for de novo synthesis. In some embodiments, the preferred removable 3'-O-blocking group is 3'-O-amino, 3'-O-allyl or 3'-O-azidomethyl. In other embodiments, the removable 3'-O-blocking moiety is selected from the group consisting of O-phenoxyacetyl; O-methoxyacetyl; O-acetyl; O-(p-toluene)-sulfonate; O-phosphate; O-nitrate; O-[4-methoxy]-tetrahydrothiopyranyl; O-tetrahydrothiopyranyl; O-[5-methyl]-tetrahydrofuranyl; O-[2-methyl,4-methoxy]-tetrahydropyranyl; O-[5-methyl]-tetrahydropyranyl; and O-tetrahydrothiofuranyl (see US 8,133,669). In other embodiments, the removable blocking moiety is selected from the group consisting of esters, ethers, carbonitriles, phosphates, carbonates, carbamates, hydroxylamines, borates, nitrates, sugars, phosphoramides, phosphoramidates, phenylsulfenates, sulfates, sulfones, and amino acids (see Metzker ML et al. Nuc Acids Res. 1994;22(20):4259-67; U.S. Pat. Nos. 5,763,594, 6,232,465, 7,414,116; and 7,279,563, all of which are incorporated by reference in their entireties).

[0047] Synthesis of Exemplary 3'-O-Blocked dNTP Analogs Figure 6 shows four exemplary 3'-O-blocked dNTP analogs: 3'-O-azidomethyl-dATP, 3'-O-azidomethyl-dCTP, 3'-O-azidomethyl-dGTP, and 3'-O-azidomethyl-dTTP. The synthesis of each 3'-O-azidomethyl analog is described below and detailed in Figures 7-12. 3'-O-blocked dNTP analogs can also be purchased from specialty suppliers such as Azco Biotech, Oceanside, CA. It should be understood that the corresponding 3'-O-blocked ribonucleotides can be formed by similar synthetic methods to enable the creation of custom RNA oligos.

[0048] 3'-O-azidomethyl-dATP: See Figure 7. N in DMSO (12 ml), acetic acid (5.5 ml), and acetic anhydride (17.6 ml) 6 A solution of 2'-benzoyl-5'-O-(tert-butyldimethylsilyl)-2'-deoxyadenosine (3.0 g; 6.38 mmol) [CNH Technologies, Woburn, MA] was prepared. The mixture was stirred at room temperature for 48 hours. Approximately 100 ml of saturated NaHCO3 solution was added, and the aqueous layer was extracted with CHCl2. The combined organic extracts were washed with saturated NaHCO3 solution and dried over NaSO4. The residue was purified by flash column chromatography (hexane / ethyl acetate, 1:1 to 1:4) to give N 6 400 mg of N-benzoyl-3'-O-(methylthiomethyl)-5'-O-(tert-butyldimethylsilyl)-2'-deoxyadenosine (shown in Figure 7 as Compound 1) was recovered as a white powder (2.4 g; 71% yield). 63'-Benzoyl-3'-O-(methylthiomethyl)-5'-O-(tert-butyldimethylsilyl)-2'-deoxyadenosine was dissolved in dry CHCl (7 ml) under nitrogen to create a solution (0.76 mmol). Next, cyclohexene (400 μl) and SOCl (155 μl; 1.91 mmol, redistilled) were added. The reaction mixture was stirred at 0°C for 2 hours. The solvent was then removed under reduced pressure and then under high vacuum for 10 minutes. The resulting residue was dissolved in dry DMF (5 ml) and reacted with NaN (400 mg; 6.6 mmol) at room temperature for 3 hours. The reaction mixture was dispersed in distilled water (50 ml) and extracted with CHCl. ​​The combined organic layers were dried over NaSO and concentrated under reduced pressure. The residue was dissolved in MeOH (5 ml) and stirred with NHF (300 mg; 8.1 mmol) at room temperature for 24 hours. The solvent was then removed under reduced pressure. The reaction mixture was concentrated under reduced pressure and partitioned between water and CH2Cl2. The organic layer was separated and dried over Na2SO4. After concentration, the crude product was purified by flash column chromatography (ethyl acetate / methanol) to give N 6 3'-benzoyl-3'-O-(azidomethyl)-2'-deoxyadenosine (compound 2; Figure 7) was produced as a white powder (150 mg; 48% yield). 6123 mg (0.3 mmol) of benzoyl-3'-O-(azidomethyl)-2'-deoxyadenosine and 75.8 mg (0.35 mmol) of proton sponge were dried overnight over PO in a vacuum desiccator and then dissolved in trimethyl phosphate (600 μL). Freshly distilled POCl (40 μL; 0.35 mmol) was then added dropwise at 0°C, and the mixture was stirred at 0°C for 2 hours. A mixture of tributylammonium pyrophosphate (552 mg) and tributylamine (0.55 mL; 2.31 mmol) in anhydrous DMF (2.33 mL) was then added at room temperature and stirred for 30 minutes. Next, 15 mL of triethylammonium bicarbonate solution (TEAB) (0.1 M; pH 8.0) was added, and the mixture was stirred at room temperature for 1 hour. 15 mL of concentrated NHOH was then added, and the mixture was stirred at room temperature overnight. The resulting mixture was concentrated under vacuum, and the residue was diluted with 5 mL of water. The crude mixture was then purified by anion exchange chromatography on DEAE-Sephadex A-25 at 4 °C using a gradient of TEAB (pH 8.0; 0.1-1.0 M). The crude product was purified by reverse-phase HPLC to yield the nucleotide analog, 3'-O-azidomethyl-dATP (Figure 7, compound 3), to be used for subsequent synthesis.

[0049] 3'-O-Azidomethyl-dTTP: Acetic acid (4.8 ml) and acetic anhydride (15.4 ml) were added to a stirred solution of 5'-O-(tert-butyldimethylsilyl)thymidine (2.0 g; 5.6 mmol) [CNH Technologies, Woburn, MA] in DMSO. The reaction mixture was stirred at room temperature for 48 hours. Saturated NaHCO3 solution (100 ml) was added, and the aqueous layer was extracted with ethyl acetate (3 × 100 ml). The combined organic extracts were washed with a saturated solution of NaHCO3 and dried over Na2SO4. After concentration, the crude product was purified by flash column chromatography (hexane / ethyl acetate) to produce 3'-O-(methylthiomethyl)-5'-O-(tert-butyldimethylsilyl)thymidine (Figure 8; compound 4) as a white powder (1.75 g; 75% yield). Approximately 1 gram of 3'-O-(methylthiomethyl)-5'-O-(tert-butyldimethylsilyl)thymidine was dissolved in dry CHCl (10 ml) under nitrogen. To this mixture, cyclohexene (1.33 ml) and SOCl (284 μl; 3.5 mmol, redistilled) were added. The resulting mixture was then stirred at 0°C for 1.5 hours. The solvent was then removed under reduced pressure and then under high vacuum for 10 minutes. The residue was dissolved in dry DMF (5 ml) and reacted with NaN (926 mg; 15.4 mmol) at room temperature for 3 hours. The reaction mixture was then dispersed in distilled water (50 ml) and extracted with CHCl (3 × 50 ml). The combined organic extracts were dried over NaSO and concentrated under reduced pressure. The residue was dissolved in MeOH (5 ml) and reacted with NHF (600 mg; 16.2 mmol) at room temperature for 24 hours. The reaction mixture was concentrated under reduced pressure and partitioned between water and CHCl. ​​The organic layer was then separated and dried over NaSO. After concentration, the residue was purified by flash column chromatography (hexane / ethyl acetate) to produce 3'-O-(azidomethyl)thymidine (Figure 8, compound 5) as a white powder (550 mg; 71% yield). Next, 3'-O-(azidomethyl)thymidine and proton sponge (0.35 mmol) were dried over PO in a vacuum desiccator overnight and then dissolved in trimethyl phosphate (600 μL).Freshly distilled POCl3 (40 μL; 0.35 mmol) was then added dropwise at 0 °C, and the mixture was stirred at 0 °C for 2 h. A mixture of tributylammonium pyrophosphate (552 mg) and tributylamine (0.55 mL; 2.31 mmol) in anhydrous DMF (2.33 mL) was then added at room temperature and stirred for 30 min. Next, triethylammonium bicarbonate solution (TEAB) (0.1 M; pH 8.0; 15 mL) was added, and the mixture was stirred at room temperature for 1 h. Concentrated NH4OH (15 mL) was then added, and the mixture was stirred overnight at room temperature. The resulting mixture was concentrated under vacuum, and the residue was diluted with 5 mL of water. The crude mixture was then purified by anion exchange chromatography on DEAE-Sephadex A-25 at 4 °C using a gradient of TEAB (pH 8.0; 0.1–1.0 M). The crude product was purified by reverse-phase HPLC to yield the nucleotide analog, 3'-O-azidomethyl-dTTP (Figure 8, compound 6), to be used for subsequent synthesis.

[0050] 3'-O-azidomethyl-dCTP: 3.5 grams N 4 5'-Benzoyl-5'-O-(tert-butyldimethylsilyl)-2'-deoxycytidine [CNH Technologies, Woburn, MA] was added to 14.7 ml of DMSO to produce a 7.65 mmol solution. To this solution, acetic acid (6.7 ml) and acetic anhydride (21.6 ml) were added, and the reaction mixture was stirred at room temperature for 48 hours. Saturated NaHCO3 solution (100 ml) was then added, and the aqueous layer was extracted with CHCl2 (3 x 100 ml). The combined organic extracts were washed with a saturated solution of NaHCO3 and then dried over NaSO4. After concentration, the crude product was purified by flash column chromatography (ethyl acetate / hexanes) to give the N 4 3'-benzoyl-3'-O-(methylthiomethyl)-5'-O-(tert-butyldimethylsilyl)-2'-deoxycytidine (Figure 9; Compound 7) was produced as a white powder (2.9 g; 73% yield). 45'-O-benzoyl-3'-O-(methylthiomethyl)-5'-O-(tert-butyldimethylsilyl)-2'-deoxycytidine (558 mg; 1.04 mmol) was dissolved in a 1000 ml solution of 2-benzoyl-3'-O-(methylthiomethyl)-5'-O-(tert-butyldimethylsilyl)-2'-deoxycytidine, followed by the addition of cyclohexene (560 μL) and SO2Cl2 (220 μL; 2.7 mmol). The reaction mixture was stirred at 0°C for 1 hour. The volatiles were then removed under reduced pressure. The remaining residue was dissolved in dry DMF (5 mL) and reacted with NaN3 (400 mg; 6.6 mmol) at room temperature for 2 hours. The reaction mixture was dispersed in distilled water (50 mL) and extracted with CHCl2 (3 × 50 mL). The combined organic extracts were dried over Na2SO4 and concentrated under reduced pressure. The residue was dissolved in MeOH (5 mL) and reacted with NHF (600 mg; 16.2 mmol) at room temperature for 24 hours. The solvent was removed under reduced pressure. The resulting residue was suspended in water (50 ml) and extracted with CH2Cl2 (3 x 50 ml). The combined organic extracts were dried over Na2SO4 and concentrated under reduced pressure. The crude product was purified by flash column chromatography (hexane / ethyl acetate) to give N 4 N-benzoyl-3'-O-(azidomethyl)-2'-deoxycytidine (Figure 9, compound 8) was produced as a white powder (200 mg; 50% yield). 42'-Benzoyl-3'-O-(azidomethyl)-2'-deoxycytidine and proton sponge (0.35 mmol) were dried overnight over PO in a vacuum desiccator and then dissolved in trimethyl phosphate (600 μL). Freshly distilled POCl (40 μL; 0.35 mmol) was then added dropwise at 0°C, and the mixture was stirred at 0°C for 2 hours. A mixture of tributylammonium pyrophosphate (552 mg) and tributylamine (0.55 mL; 2.31 mmol) in anhydrous DMF (2.33 mL) was then added at room temperature and stirred for 30 minutes. Next, triethylammonium bicarbonate solution (TEAB) (0.1 M; pH 8.0; 15 mL) was added, and the mixture was stirred at room temperature for 1 hour. Concentrated NHOH (15 mL) was then added and stirred overnight at room temperature. The resulting mixture was concentrated under vacuum, and the residue was diluted with 5 mL of water. The crude mixture was then purified by anion exchange chromatography on DEAE-Sephadex A-25 using a gradient of TEAB (pH 8.0; 0.1-1.0 M) at 4° C. The crude product was purified by reverse-phase HPLC to yield the nucleotide analog, 3′-O-azidomethyl-dCTP (Figure 9, compound 9), to be used for subsequent synthesis.

[0051] 3'-O-azidomethyl-dGTP: N in dry DMSO (21 ml) 2 To a stirred solution of 5'-isobutyryl-5'-O-(tert-butyldimethylsilyl)-2'-deoxyguanosine (5 g; 11.0 mmol) [CNH Technologies, Woburn, MA], acetic acid (10 ml) and acetic anhydride (32 ml) were added. The reaction mixture was stirred at room temperature for 48 hours. Saturated NaHCO3 solution (100 ml) was added, and the aqueous layer was extracted with ethyl acetate (3 x 100 ml). The combined organic extracts were washed with saturated NaHCO3 solution and dried over Na2SO4. After concentration, the crude product was purified by flash column chromatography (CHCl / MeOH) to give N 21 gram of N-isobutyryl-3'-O-(methylthiomethyl)-5'-O-(tert-butyldimethylsilyl)-2'-deoxyguanosine (Figure 10, compound 10) was produced as a white powder (3.9 g; 69% yield). 2 N,N-isobutyryl-3'-O-(methylthiomethyl)-5'-O-(tert-butyldimethylsilyl)-2'-deoxyguanosine was then added to dry pyridine (22 ml; 2.0 mmol) along with diphenylcarbamoyl chloride (677 mg; 2.92 mmol) and DIEA (N,N-diisopropylethylamine; SIGMA) (1.02 ml; 5.9 mmol). The reaction mixture was stirred under nitrogen atmosphere at room temperature for 3 hours. The solvent was removed under high vacuum. The crude product was purified by flash column chromatography (ethyl acetate / hexane) to give N,N-isobutyryl-3'-O-(methylthiomethyl)-5'-O-(tert-butyldimethylsilyl)-2'-deoxyguanosine, which appeared as a yellowish powder. 2 -Isobutyryl-O 6 1-(diphenylcarbamoyl)-3'-O-(methylthiomethyl)-5'-O-(tert-butyldimethylsilyl)-2'-deoxyguanosine (Figure 10, compound 11) was produced (1.09 g; 80% yield). 2 -Isobutyryl-O 6-(Diphenylcarbamoyl)-3'-O-(methylthiomethyl)-5'-O-(tert-butyldimethylsilyl)-2'-deoxyguanosine was dissolved in dry CHCl (1.1 mmol) and stirred under a nitrogen atmosphere at 0°C for 1.5 hours. The solvent was removed under reduced pressure and then under high vacuum for 10 minutes. The resulting residue was dissolved in dry DMF (5 mL) and reacted with NaN (600 mg; 10 mmol) at room temperature for 3 hours. The reaction mixture was then dispersed in distilled water (50 mL) and extracted with CHCl (3 × 50 mL). The combined organic extracts were dried over NaSO and concentrated under reduced pressure. The resulting residue was dissolved in MeOH (5 mL) and reacted with NHF (500 mg; 13.5 mmol) at room temperature for 24 hours. The solvent was removed under reduced pressure. The residue was suspended in water (50 mL) and extracted with CHCl (3 × 50 mL). The combined organic extracts were dried over Na2SO4 and concentrated under reduced pressure. The crude product was purified by flash column chromatography (hexane / ethyl acetate) to give N 2 -Isobutyryl-O 6 1-(diphenylcarbamoyl)-3'-O-azidomethyl-2'-deoxyguanosine (Figure 10, compound 12) was produced as a white powder (230 mg; 36% yield). 2 -Isobutyryl-O 6-(Diphenylcarbamoyl)-3'-O-azidomethyl-2'-deoxyguanosine and proton sponge (0.35 mmol) were dried overnight over PO in a vacuum desiccator and then dissolved in trimethyl phosphate (600 μL). Freshly distilled POCl (40 μL; 0.35 mmol) was then added dropwise at 0°C, and the mixture was stirred at 0°C for 2 hours. A mixture of tributylammonium pyrophosphate (552 mg) and tributylamine (0.55 mL; 2.31 mmol) in anhydrous DMF (2.33 mL) was then added at room temperature and stirred for 30 minutes. Next, triethylammonium bicarbonate solution (TEAB) (0.1 M; pH 8.0; 15 mL) was added, and the mixture was stirred at room temperature for 1 hour. Concentrated NHOH (15 mL) was then added and stirred overnight at room temperature. The resulting mixture was concentrated in vacuo, and the residue was diluted with 5 mL of water. The crude mixture was then purified by anion exchange chromatography on DEAE-Sephadex A-25 using a gradient of TEAB (pH 8.0; 0.1-1.0 M) at 4° C. The crude product was purified by reverse-phase HPLC to yield the nucleotide analog, 3′-O-azidomethyl-dGTP (Figure 10, compound 13), to be used for subsequent synthesis.

[0052] As described with respect to Figure 2, once a 3'-O-blocked dNTP or rNTP has been added, it will be necessary to remove the blocking group so that an additional dNTP or rNTP can be added. In some embodiments, the 3'-O-blocking group can be removed by palladium catalysis in neutral aqueous solution at elevated temperatures, hydrochloric acid to pH 2, a reducing agent such as mercaptoethanol, or the addition of tris-(2-carboxyethyl)phosphine. See, e.g., U.S. Pat. No. 6,664,079; Meng, et al. J. Org. Chem., 2006, 71(81):3248-52; Bi et al., J. Amer. Chem. Soc. 2006; 2542-2543; U.S. Pat. No. 7,279,563; and U.S. Pat. No. 7,414,116, all of which are incorporated by reference in their entireties. In other embodiments, 3'-substituents can be removed by UV irradiation (see, e.g., WO 92 / 10587, incorporated herein by reference in its entirety). Most 3'-O-blocking groups are removed by oxidative, reductive, or hydrolytic chemical reactions. In some embodiments, 3'-O-NO groups are removed from oligonucleotides with a 40% w / v solution of ammonium sulfide at room temperature for <5 minutes. In some embodiments, 3'-O-CHCN groups are removed from oligonucleotides by treatment with 0.5 M KOH at 70°C. In some embodiments, removal of 3'-O-blocking groups does not involve chemical cleavage, but uses a cleavage enzyme such as alkaline phosphatase.

[0053] In a preferred embodiment, an enzymatic reaction is used to remove the 3'-blocking group. Shrimp alkaline phosphatase (SAP) can be used in certain embodiments. SAP has one of the fastest enzymatic rates reported in the literature and has a wide range of substrate utilization.

[0054] 3'-O-Methoxymethyl-dTTP:5'-O-benzoylthymidine (173 mg, 0.5 mmol, 1 equiv.) was dissolved in 10 mL of dichloromethane at ambient temperature under argon. Diisopropylethylamine (128 mg, 1 mmol, 2 equiv.) was added, followed by methoxymethyl bromide (124 mg, 1 mmol, 2 equiv.). The mixture was stirred at ambient temperature for 18 hours. The mixture was diluted with 10 mL of dichloromethane and washed sequentially with 20 mL of 5% aqueous HCl and brine. The organic layer was dried over sodium sulfate and evaporated. 5'-O-Benzoyl-3'-O-methoxymethylthymidine (50 mg, 0.13 mmol) was dissolved in 5 mL of concentrated ammonium hydroxide at ambient temperature. The mixture was stirred overnight at ambient temperature. The mixture was diluted and extracted three times with 10 mL portions of dichloromethane. The combined extracts were washed with brine. The organic layer was dried over sodium sulfate and evaporated. 3'-O-Methoxymethylthymidine (23 mg, 0.08 mmol) was coevaporated with pyridine (1.5 mL × 3) and dried under high vacuum overnight. The nucleoside was dissolved in a mixture of 1.5 mL of trimethyl phosphate and 0.6 mL of dry pyridine under Ar. The mixture was cooled in an ice bath. A first aliquot of 10 μL of POCl3 was added dropwise. After 5 min, a second aliquot of 10 μL was added. The mixture was stirred for an additional 30 min. A solution of TBA phosphate salt in dry DMF (1.25 mL) was cooled in an ice bath in a vial under Ar. This was added dropwise to the reaction mixture over 10 s. Immediately, a pre-weighed amount of solid proton sponge (21 mg, 1.25 equiv.) was added as a solid in one portion. After this addition, the mixture was stirred for 25 min and quenched with 5 mL of cold TEAB buffer. The mixture was stirred in an ice bath for 10 min and then transferred to a small RB flask for FPLC separation. Final separation was achieved by reverse-phase HPLC using a water / acetonitrile gradient containing 0.1 mM formic acid.

[0055] 3'-O-Methylthiomethyl-dCTP: To a suspension of deoxycytidine (1 g, 4.4 mmol) in 25 mL of methanol was added N,N-dimethylformamide dimethyl acetal (1.75 mL, 13.2 mmol). The mixture was stirred overnight at ambient temperature. The reaction mixture was evaporated, and the residue was purified by flash chromatography using a DCM / methanol gradient as eluent. N6-Formamidino-5'-O-benzoyldeoxy-3'-O-methylthiomethyldeoxycytidine (250 mg, 0.41 mmol) was dissolved in 10 mL of methanol and 10 mL of concentrated aqueous ammonium hydroxide. The mixture was stirred at ambient temperature for 18 h and then evaporated under reduced pressure. The residue was purified by column chromatography (DCM / methanol 98:2 to 90:10) to give 170 mg (93%) of the desired nucleoside as a slightly yellow solid. 3'-O-Methylthiomethyldexoxycytidine (25.0 mg, 0.09 mmol) in a 25 mL vial was coevaporated with anhydrous pyridine (3 × 1 mL) and dried over the weekend. Trimethyl phosphate (0.7 mL) was added to dissolve the nucleoside and cooled to 0 °C in an ice bath. Phosphoryl chloride (28 μL, 0.3 mmol) was added slowly (12 μL, 8 μL after 5 min, 8 μL after 30 min), and the reaction was stirred for 2 h at 0 °C. Di(tetrabutylammonium) hydrogen pyrophosphate was dissolved in anhydrous DMF (1 mL), and this mixture was cooled to 0 °C and added to the reaction mixture. Proton sponge (9.2 mg, 0.04 mmol) was added, and the reaction was stirred for 2 h at 0 °C. 1 M triethylammonium bicarbonate buffer (TEAB) (2 mL) was added to the reaction mixture, and the mixture was stirred for 1 h. The mixture was then transferred to a round-bottom flask, 50 mL x 3 of miliQ water was added, and the mixture was concentrated to dryness. The residue was dissolved in miliQ water (11 mL) and loaded onto an AKTA FPLC at room temperature. The fractions containing the triphosphates (F48-F52) were evaporated under reduced pressure at 40 °C, and the residue was then lyophilized. The triphosphates were dried to give the desired triphosphates (12 mg, 16.5%). [Example]

[0056] Example 1 Protein modification The murine (mur) TdT variant was derived from a 380-aa synthetic gene. This backbone is a truncated version of WT murine TdT, representing the catalytic core of the ET sequence. The chemically synthesized TdT construct was cloned into the pRSET A bacterial expression vector (ThermoFisher Scientific GeneArt Gene Synthesis), which features an N-terminal 6x histidine tag and an enterokinase cleavage site. The synthetic TdT plasmid was maintained in DH5 alpha cells (Biopioneer) plated on LB agar plates containing 100 μg / ml carbenicillin. For expression, the pRSETA-murine TdT plasmid was transformed into BL21(DE3)pLysS cells (ThermoFisher) by incubating the plasmid and cells on ice for 20 minutes, followed by a 30-second heat shock at 42°C, followed by the addition of SOC medium and incubation at 37°C for 30–60 minutes with shaking. After addition of SOC medium to the cells, the entire volume (typically 60 ul) was plated onto LB agar plates containing 100 ug / mL carbenicillin and 34 ug / mL chloramphenicol.

[0057] Cells from 10 mL cultures (24-well plates, Corning) were harvested by centrifugation (3000 × g, 15 min) and then lysed in B-PER lysis buffer (Thermo-Fisher) containing lysozyme, protease inhibitors, and 100 mM NaCl. The pellet was soaked in TBS buffer for 1 × 60 min, and the supernatant was collected for purification. The supernatant was bound to 50 μL Ni-NTA bead (GE Life Sciences) slurry in a 24-well plate for 30 min. The bead slurry was then washed 3 × with 50 mM Tris-HCl, pH 8, 500 mM NaCl (500 μL), followed by 4 × with 50 mM Tris-HCl, pH 8, 500 mM NaCl, 50 mM imidazole (200 μL). Protein was then recovered by treatment with 50 mM Tris-HCl, pH 8, 500 mM NaCl, 300 mM imidazole (50 uL), then 50 mM Tris-HCl, pH 8, 500 mM NaCl, 300 mM imidazole (130 uL), and finally 50 mM Tris-HCl, pH 8, 500 mM NaCl, 1 M imidazole (50 uL).

[0058] A 2.5 μl sample was collected and the collected fractions were analyzed by running an 8% NuPage gel (Thermo-Fisher) at 200 V for 50 minutes under denaturing conditions. The gel was stained with Coomassie blue. The eluted protein was buffer exchanged using a 7.5 MWCO desalting column (Thermo-Fisher) and stored at -80°C (storage buffer = 20 mM Tris-HCl, pH 6.8, 50 mM NaOAc; 0.01% Triton X-100 and 10% glycerol).

[0059] Activity Screening: TdT activity screening was performed by dNTP polymerase extension reactions using different 3'-O-blocked dNTP analogs and biotinylated oligonucleotides: 5BiosG / TAATAATAATAATAATAATAATAATAATAATAATAATAATAATTTTTT (ChemGenes Corporation) SEQ ID NO: 12

[0060] Reactions were typically set up in 96-well plates. Reactions were performed by creating a master mix with the following final concentrations of components: 0.2U PPase (Thermo-Fisher), 10 pmol of oligonucleotide, 75 uM dNTP (see below), and 1x TdT reaction buffer (5x from Thermo-Fisher) to a final volume of 10 ul. Reactions were initiated by adding a defined volume (typically 2 ul) of TdT variant to different wells and incubating the reaction mix at 37°C for 5 minutes. At the 60 minute time point, the reaction was terminated by removing a 10 ul aliquot and adding it to 5 ul of 250 mM EDTA. Tested dNTPs: 3'-O-azidomethyl-dTTP See above 3'-O-azidomethyl-dATP See above 3'-O-azidomethyl-dGTP See above 3'-O-MOM-dTTP See above 3'-O-MTM-dCTP See above 3'-aminooxy-dTTP Firebird BioMolecular Sciences LLC 3'-Aminooxy-dATP Firebird BioMolecular Sciences LLC 3'-Aminooxy-dGTP Firebird BioMolecular Sciences LLC 3'-O-methyl-dATP TriLink BioTechnologies LLC 3'-O-methyl-dGTP TriLink BioTechnologies LLC 3'-O-methyl-dCTP TriLink BioTechnologies LLC

[0061] The biotinylated oligos in the quenched reaction mix were bound to streptavidin beads (0.77 μm, Spherotech). The beads were then transferred to a filter plate (Pall Corporation) and washed several times with water. The oligonucleotides were cleaved from the solid support by incubating the plate with cleavage buffer (10% diisopropylamine in methanol) at 50°C for 30 minutes, followed by elution in water. The eluted samples were dried and dissolved in 30 μl of water containing oligonucleotide sizing standards (two oligonucleotides (ChemGenes Corporation) approximately 15–20 bases smaller or larger than the starting 42-mer oligonucleotide). The oligonucleotides were then analyzed for extension efficiency by capillary gel electrophoresis (Oligo Pro II, Advanced Analytical Technologies Inc.).

[0062] Example 2 In silico modeling Several amino acid modifications to the GGFRR and TGSR motifs and adjacent amino acids discussed above were modeled in silico to determine modifications capable of increasing the incorporation of the 3'-O-blocked dNTP analogs described above. Single, double, and triple amino acid substitutions, as well as amino acid insertions, were modeled. Table 11 below shows the modifications found to induce increased incorporation. The amino acid positions are presented with reference to mouse TdT, but are applicable to any TdT conserved sequence. The rows of Table 11 represent base modifications to one or more amino acids in or adjacent to the GGFRR motif. The columns include additional combinations of modifications to other amino acids, such as those in and adjacent to the TGSR motif. Table 11. [Table 11-1] [Table 11-2] [Table 11-3] [Table 11-4]

[0063] Example 3 Incorporation of dNTPs with phosphate blocking groups DNA and nucleotides, including DNA, are highly negatively charged due to the phosphate groups in the nucleotides. See Lipfert J, Doniach S, Das R, Herschlag D. Understanding Nucleic Acid-Ion Interactions, Annu Rev Biochem. 2014; 83: 813-841, incorporated herein by reference. 3'-PO4-dNTPs have an even greater negative charge compared to natural nucleotides due to the additional phosphate group at the 3' position. The increased negative charge can affect the ability of TdT to incorporate modified nucleotides. In certain embodiments, the engineered TdT enzyme of the present invention can be modified for efficient incorporation of 3'-phosphate-dNTPs by neutralizing the negative charge with a positive charge in the modified TdT.

[0064] The Average Number of Neighboring Atoms Per Sidechain Atom (AvNAPSA) algorithm in Rosetta protein software suite 3 was used to identify mutations that would increase the positive charge in and around the enzyme active site of TdT. Sequence positions in the active site of TdT were targeted by increasing a key parameter of the AvNAPSA algorithm, termed the surface_atom_cutoff. The surface charge of the protein was manipulated by mutating solvent-exposed polar residues to charged residues, with the amount of solvent exposure determined by the number of neighboring non-self atoms. See Miklos AE, et al., Structure-Based Design of Supercharged, Highly Thermoresistant Antibodies, Chemistry & Biology, Volume 19, Issue 4, 20 April 2012, Pages 449-455; Kaufmann KW, et al., Practically useful: what the Rosetta protein modeling suite can do for you, Biochemistry. 2010 Apr 13; 49(14):2987-98, the contents of each of which are incorporated herein by reference. Increasing the surface_atom_cutoff term allows AvNAPSA to consider sequence positions with a larger number of neighboring atoms, such as positions within an enzyme active site. A summary of positions in TdT identified using AvNAPSA as potentially useful for more efficient incorporation of 3'-phosphate-dNTPs is shown in Table 12. Table 12: TdT modifications for incorporation of phosphate-blocked dNTPs [Table 12]

[0065] Figures 13-16 illustrate the superior nucleotide incorporation of modified TdT over wild-type with respect to 3'-PO4-dNTP. Figure 13, panel A, is a CGE analysis of a chemically synthesized oligonucleotide (IDT) (21-mer; 5'-FAM-TAATAATAATAATAATTTTTT-PO4-3'), while panel B shows that the addition of a single nucleotide with a 3'-PO4 group results in faster electrophoretic mobility than the comparable 20-mer (IDT) (5'-FAM-TAATAATAATAATAATTTTT). Figure 14 shows that shrimp alkaline phosphatase (SAP) (NEB#P0757) induces a 1.23 x 10 per pmol of oligonucleotide incorporation. -3 This CGE analysis demonstrates quantitative removal of 3'-PO4 groups in 1 minute or less at concentrations of 0 U / ul (Panel A) to 1.0 x 10 U / ul. -Figure 15, panel B, shows a titration series of increasing amounts of SAP to 1 U / ul (panel G). Figure 15, panel B, is a CGE analysis of the mouse WT TdT reaction mixture demonstrating that the polymerase did not mediate extension, even in the presence of 500 μM 3'-PO4-dTTP (MyChem LLC), as evidenced by no change from the starting oligonucleotide shown in panel A. Further evidence of the lack of substrate utilization of 3'-PO4-dTTP is shown in panel C of Figure 15, as evidenced by the lack of reactivity of the oligonucleotide starting material (panel A). Figure 16 is a CGE analysis of the partial incorporation of 3'-PO4-dTTP by the variant TdT enzyme (E180K+M192K+L381K+R454K+N474R), as shown in panel B, demonstrating the appearance of a new oligonucleotide species with faster electrophoretic mobility (circled new peak), as expected based on the results shown in Figure 13. Further evidence of 3'-PO4 incorporation by variant TdT is demonstrated by post-extension removal of 3'-PO4 by treatment with SAP and the appearance of a new oligonucleotide species (Panel C - new peak circled) with a slower electrophoretic migration rate than the oligonucleotide starting material, as expected from the poly-dT size ladder shown in Panel D, and the disappearance of the species formed in Panel B, as indicated by the arrow in Panel C. In another embodiment, increased incorporation of 3'-PO4-dTTP is demonstrated by the variant enzyme (E180K+M192K+R454K+R461V+N474R).

[0066] Incorporation by Reference References and citations are made throughout this disclosure to other documents, such as patents, patent applications, patent publications, journals, books, articles, web content, etc. All such documents are hereby incorporated by reference herein in their entirety for all purposes.

[0067] equivalent Various modifications of the present invention and many further embodiments thereof, in addition to those shown and described herein, will become apparent to those skilled in the art from the entire contents of this document, including the scientific and patent references cited herein. The subject matter herein contains important information, exemplification and guidance that can be adapted to the practice of this invention in its various embodiments and equivalents thereof.

Claims

1. (i) an E33K mutation with reference to the sequence of SEQ ID NO: 10 without the His tag and linker, (ii) an E180K mutation with reference to the sequence of SEQ ID NO: 9, (iii) an E180K mutation and one further mutation selected from M192K, W450H, or R454I with reference to the sequence of SEQ ID NO: 9, or (iv) a modified terminal deoxynucleotidyl transferase (TdT) comprising the mutations E180K, M192K, L381K, R454K, and N474R with reference to the sequence of SEQ ID NO: 9, wherein the modified TdT is capable of adding a nucleotide analogue, which comprises a removable blocking moiety on the 3'-oxygen of the analogue, to the 3'-OH of a nucleic acid initiator in the absence of a nucleic acid template.

2. The modified TdT of claim 1, comprising the mutations E180K, M192K, L381K, R454K and N474R, with reference to the sequence of SEQ ID NO:

9.

3. 2. The modified TdT of claim 1, wherein the modified TdT can add the nucleotide analog containing the removable 3'-O-blocking moiety to the 3'-OH of the nucleic acid initiator at an increased rate compared to native TdT.

4. The modified TdT of claim 1, which comprises an N-terminal truncation compared to native TdT.

5. The modified TdT of claim 4, wherein the N-terminal truncation is a 131 amino acid truncation or a 147 amino acid truncation.

6. 5. The modified TdT of claim 4, wherein the N-terminal truncation is a 131 amino acid truncation and the modified TdT comprises a protein tag sequence attached to the N-terminus.

7. 5. The modified TdT of claim 4, wherein the N-terminal truncation is a 147 amino acid truncation and the modified TdT comprises a protein tag sequence attached to the N-terminus.

8. 2. The modified TdT of claim 1, wherein the modified TdT is capable of adding nucleotide analogs including adenine, cytosine, guanine, and thymine deoxyribonucleotides modified with a removable 3'-O-blocking moiety.

9. 9. The modified TdT of claim 8, wherein the deoxyribonucleotides are 2'-deoxyribonucleotides.

10. 2. The modified TdT of claim 1, wherein the modified TdT is capable of adding adenine, cytosine, guanine, and uracil ribonucleotides modified with a removable 3'-O-blocking moiety.

11. The removable 3'-O-blocking moiety is CH ２ N ３ , N.H. ２ , ONHC(O)H, allyl, CH ２ SSCH ３ , phenoxyacetyl, methoxyacetyl, acetyl, (p-toluene)sulfonate, phosphate, nitrate, [4-methoxy]-tetrahydrothiopyranyl, tetrahydrothiopyranyl, [5-methyl]-tetrahydrofuranyl, [2-methyl,4-methoxy]-tetrahydropyranyl, [5-methyl]-tetrahydropyranyl; and O-tetrahydrothiofuranyl.

12. 2. The modified TdT of claim 1, wherein the modified TdT is capable of incorporating a 3'-O-blocked nucleotide 5'-triphosphate and the removable blocking moiety comprises a group selected from an ester, an ether, a carbonitrile, a phosphate, a carbonate, a carbamate, a hydroxylamine, a borate, a nitrate, a sugar, a phosphoramide, a phosphoramidate, a phenylsulfenate, a sulfate, a sulfone, and an amino acid.

13. The modified TdT of claim 1, wherein the modified TdT is capable of incorporating the nucleotide analog at a reaction temperature of 30°C to 80°C.

14. 2. The modified TdT of claim 1, wherein the modified TdT is capable of incorporating the nucleotide analog at a concentration of 1000 μM or less.

15. 2. The modified TdT of claim 1, wherein the modified TdT is capable of incorporating the nucleotide analog at a concentration of 100 μM or less.

16. 3. The modified TdT of claim 2, wherein the modified TdT is capable of adding a nucleotide analog containing a removable 3'-O-phosphate to the 3'-OH of a nucleic acid initiator.

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