CA3185851A1 - Modified template-independent enzymes for polydeoxynucleotide synthesis - Google Patents
Modified template-independent enzymes for polydeoxynucleotide synthesisInfo
- Publication number
- CA3185851A1 CA3185851A1 CA3185851A CA3185851A CA3185851A1 CA 3185851 A1 CA3185851 A1 CA 3185851A1 CA 3185851 A CA3185851 A CA 3185851A CA 3185851 A CA3185851 A CA 3185851A CA 3185851 A1 CA3185851 A1 CA 3185851A1
- Authority
- CA
- Canada
- Prior art keywords
- tdt
- modified
- modified tdt
- mutations
- lys
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
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- 238000003786 synthesis reaction Methods 0.000 title abstract description 31
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- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 44
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Abstract
The invention includes methods for identifying polymerases, such as modified terminal nucleotidyl transferases (TdT), that are capable of binding nucleotides comprising removable 3'- O-blocking moieties to a nucleic acid initiator, without the use of a template. The invention further includes the identified polymerases, and methods of using the polymerases for de novo synthesis of predetermined oligonucleotide sequences.
Description
MODIFIED TEMPLATE-INDEPENDENT ENZYMES FOR
POLYDEOXYNUCLEOTIDE SYNTHESIS
RELATED APPLICATIONS
This Application claims priority to and the benefit of U.S. Application Ser.
No. 16/891,449, filed June 3, 2020, the content of which is incorporated by reference herein.
FIELD OF THE INVENTION
The invention relates to modified enzymes for de novo synthesis of polynucleotides with a desired sequence, and without the use of a template. As such, the invention provides the capability to make libraries of polynucleotides of varying sequence and varying length for research, genetic engineering, and gene therapy.
BACKGROUND
Most de novo nucleic acid sequences are synthesized using solid phase phosphoramidite-techniques developed more than 30 years ago. The technique involves the sequential de-protection and synthesis of sequences built from phosphoramidite reagents corresponding to natural (or non-natural) nucleic acid bases. Phosphoramidite nucleic acid synthesis is length-limited, however, in that nucleic acids greater than 200 base pairs (bp) in length experience high rates of breakage and side reactions. Additionally, phosphoramidite synthesis produces toxic by-products, and the disposal of this waste limits the availability of nucleic acid synthesizers, and increases the costs of contract oligo production. (It is estimated that the annual demand for oligonucleotide synthesis is responsible for greater than 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), p.2522-2540, (2010), incorporated by reference herein in its entirety). Thus, there is a need for more efficient and cost-effective methods for oligonucleotide synthesis.
SUMMARY
The invention discloses modified terminal deoxynucleotidyl transferase (TdT) enzymes that can be used for de novo synthesis of oligonucleotides in the absence of a template.
Methods for creating a template-independent polymerase through a combination of computational guidance and saturation mutagenesis, with a subsequent screen to identify functional mutants, are also disclosed. Native TdT enzymes are either inefficient or completely unable to incorporate the different blocked nucleotide analogs used in template-independent synthesis schemes. The present invention provides various TdT modifications that expand the enzyme's functionality with respect to blocked nucleotide analogs, especially those with 3'-0 blocking groups. In particular, modified TdTs of the invention can be used to incorporate 3'-0-Phosphate-blocked nucleotide analogs where wild type TdTs may be unable to do so.
Methods of the invention include nucleic acid synthesis using 3'-0-blocked nucleotide analogs and Shrimp Alkaline Phosphatase (SAP) for controlled addition of selected nucleotides.
Using enzymes and methods of the invention, it will be possible to synthesize de novo polynucleotides faster and more cheaply. As such, the invention dramatically reduces the overall cost of synthesizing custom nucleic acids. In particular, the methods can be used to create template-independent transferases that can synthesize custom oligos in a stepwise fashion using modified 3' hydroxyl-blocked nucleotides. Because of the terminating group, synthesis pauses with the addition of each new base, whereupon the terminating group is cleaved, leaving a polynucleotide that is essentially identical to a naturally occurring nucleotide (i.e., is recognized by the enzyme as a substrate for further nucleotide incorporation).
The methods and enzymes of the invention represent an important step forward in synthetic biology because the enzymes will allow for aqueous phase, template-independent oligonucleotide synthesis. Such methods represent an improvement over the prior art in that they will greatly reduce the chemical waste produced during oligonucleotide synthesis while allowing for the production of longer polynucleotides. Furthermore, because the methods replace a chemical process with a biological one, costs will be reduced, and the complexity of automated synthetic systems will also be reduced. In an embodiment, a simple five-reagent delivery system can be used to build oligonucleotides in a stepwise fashion and will enable recycling of unused reagents.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows an agarose gel of a solution phase polymerization reaction composed of terminal deoxynucleotidyl transferase (TdT), deoxyadenosine triphosphate (dATP) and fluorescent strand initiator 5'-Cy5-dA10 at different time points from Tjong et al. "Amplified on-
POLYDEOXYNUCLEOTIDE SYNTHESIS
RELATED APPLICATIONS
This Application claims priority to and the benefit of U.S. Application Ser.
No. 16/891,449, filed June 3, 2020, the content of which is incorporated by reference herein.
FIELD OF THE INVENTION
The invention relates to modified enzymes for de novo synthesis of polynucleotides with a desired sequence, and without the use of a template. As such, the invention provides the capability to make libraries of polynucleotides of varying sequence and varying length for research, genetic engineering, and gene therapy.
BACKGROUND
Most de novo nucleic acid sequences are synthesized using solid phase phosphoramidite-techniques developed more than 30 years ago. The technique involves the sequential de-protection and synthesis of sequences built from phosphoramidite reagents corresponding to natural (or non-natural) nucleic acid bases. Phosphoramidite nucleic acid synthesis is length-limited, however, in that nucleic acids greater than 200 base pairs (bp) in length experience high rates of breakage and side reactions. Additionally, phosphoramidite synthesis produces toxic by-products, and the disposal of this waste limits the availability of nucleic acid synthesizers, and increases the costs of contract oligo production. (It is estimated that the annual demand for oligonucleotide synthesis is responsible for greater than 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), p.2522-2540, (2010), incorporated by reference herein in its entirety). Thus, there is a need for more efficient and cost-effective methods for oligonucleotide synthesis.
SUMMARY
The invention discloses modified terminal deoxynucleotidyl transferase (TdT) enzymes that can be used for de novo synthesis of oligonucleotides in the absence of a template.
Methods for creating a template-independent polymerase through a combination of computational guidance and saturation mutagenesis, with a subsequent screen to identify functional mutants, are also disclosed. Native TdT enzymes are either inefficient or completely unable to incorporate the different blocked nucleotide analogs used in template-independent synthesis schemes. The present invention provides various TdT modifications that expand the enzyme's functionality with respect to blocked nucleotide analogs, especially those with 3'-0 blocking groups. In particular, modified TdTs of the invention can be used to incorporate 3'-0-Phosphate-blocked nucleotide analogs where wild type TdTs may be unable to do so.
Methods of the invention include nucleic acid synthesis using 3'-0-blocked nucleotide analogs and Shrimp Alkaline Phosphatase (SAP) for controlled addition of selected nucleotides.
Using enzymes and methods of the invention, it will be possible to synthesize de novo polynucleotides faster and more cheaply. As such, the invention dramatically reduces the overall cost of synthesizing custom nucleic acids. In particular, the methods can be used to create template-independent transferases that can synthesize custom oligos in a stepwise fashion using modified 3' hydroxyl-blocked nucleotides. Because of the terminating group, synthesis pauses with the addition of each new base, whereupon the terminating group is cleaved, leaving a polynucleotide that is essentially identical to a naturally occurring nucleotide (i.e., is recognized by the enzyme as a substrate for further nucleotide incorporation).
The methods and enzymes of the invention represent an important step forward in synthetic biology because the enzymes will allow for aqueous phase, template-independent oligonucleotide synthesis. Such methods represent an improvement over the prior art in that they will greatly reduce the chemical waste produced during oligonucleotide synthesis while allowing for the production of longer polynucleotides. Furthermore, because the methods replace a chemical process with a biological one, costs will be reduced, and the complexity of automated synthetic systems will also be reduced. In an embodiment, a simple five-reagent delivery system can be used to build oligonucleotides in a stepwise fashion and will enable recycling of unused reagents.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows an agarose gel of a solution phase polymerization reaction composed of terminal deoxynucleotidyl transferase (TdT), deoxyadenosine triphosphate (dATP) and fluorescent strand initiator 5'-Cy5-dA10 at different time points from Tjong et al. "Amplified on-
2 chip fluorescence detection of DNA hybridization by surface-initiated enzymatic polymerization," Anal. Chem., 2011; 83:5153-5159 (2011).
FIG. 2 illustrates an exemplary modified terminal deoxynucleotidyl transferase (TdT) mediated polynucleotide synthesis cycle using a support bound initiator and 3'-0-blocked nucleotide triphosphate including (A) incorporation of a nucleotide analog comprising a cleavable 3'-0-blocking group (indicated by R), and (B) removal of the 3'-0-blocking group thus enabling the next 3'-0-blocked nucleotide analog to be incorporated, wherein N=A, G, C, or T.
FIG. 3 shows the polyacrylamide gel analysis of a solution phase reaction time course of commercially-available TDT and a nucleic acid initiator with 3'-0-azidomethyl-dCTP or 3'-0-azidomethyl-dATP. Lane 1 ¨ 100 bp ladder size standard, Lane 2 ¨
oligonucleotide standard, Lane 3 ¨ 3'-0-azidomethyl-dCTP + TdT 15' reaction time, Lane 4 ¨ 1 hour, Lane 5 ¨ 2 hours, Lane 6 ¨ 4 hours, Lane 7 ¨ 24 hours, Lane 8 ¨ 3'-0-azidomethyl-dATP + TdT 15' reaction time, Lane 9 ¨ 1 hour, Lane 10 ¨ 2 hours, Lane 10 ¨ 4 hours, Lane 11 ¨ 24 hours, Lane 12 ¨ dATP +
TdT 15' reaction time, Lane 13 ¨ 1 hour, Lane 14 ¨ 4 hours, Lane 15 ¨ 24 hours.
FIG. 4 shows a computer-generated image of the active site of TdT using the PDB crystal structure 4129, showing the computationally docked catalytically productive position a 3'-0--dATP analog (blue, red, orange frame), each complexed to the two active-site metal ions (large greenspheres). Shown are the residues, that are in close proximity to the incoming dNTP and the targets of mutagenesis and screening.
FIG. 5 shows a table of TdT variants that were selected for increased incorporation of selected 3'-0-blocked dNTP analogs as described herein.
FIG. 6 shows exemplary 3'-0-azidomethyl deoxynucleotides that can be used to synthesize custom DNA oligomers using modified TdTs, as described herein.
FIG. 7 shows a synthetic scheme for producing 3'-0-azidomethyl deoxyadenosine triphosphate (3'-0-azidomethyl-dATP).
FIG. 8 shows a synthetic scheme for producing 3'-0-azidomethyl deoxythymidine triphosphate (3'-0-azidomethyl-dTTP).
FIG. 9 shows a synthetic scheme for producing 3'-0-azidomethyl deoxycytidine triphosphate (3'-0-azidomethyl-dCTP).
FIG. 10 shows a synthetic scheme for producing 3'-0-azidomethyl deoxyguanosine triphosphate (3'-0-azidomethyl-dGTP).
FIG. 11 shows a synthetic scheme for producing 3'-0-methoxymethyl deoxythymidine
FIG. 2 illustrates an exemplary modified terminal deoxynucleotidyl transferase (TdT) mediated polynucleotide synthesis cycle using a support bound initiator and 3'-0-blocked nucleotide triphosphate including (A) incorporation of a nucleotide analog comprising a cleavable 3'-0-blocking group (indicated by R), and (B) removal of the 3'-0-blocking group thus enabling the next 3'-0-blocked nucleotide analog to be incorporated, wherein N=A, G, C, or T.
FIG. 3 shows the polyacrylamide gel analysis of a solution phase reaction time course of commercially-available TDT and a nucleic acid initiator with 3'-0-azidomethyl-dCTP or 3'-0-azidomethyl-dATP. Lane 1 ¨ 100 bp ladder size standard, Lane 2 ¨
oligonucleotide standard, Lane 3 ¨ 3'-0-azidomethyl-dCTP + TdT 15' reaction time, Lane 4 ¨ 1 hour, Lane 5 ¨ 2 hours, Lane 6 ¨ 4 hours, Lane 7 ¨ 24 hours, Lane 8 ¨ 3'-0-azidomethyl-dATP + TdT 15' reaction time, Lane 9 ¨ 1 hour, Lane 10 ¨ 2 hours, Lane 10 ¨ 4 hours, Lane 11 ¨ 24 hours, Lane 12 ¨ dATP +
TdT 15' reaction time, Lane 13 ¨ 1 hour, Lane 14 ¨ 4 hours, Lane 15 ¨ 24 hours.
FIG. 4 shows a computer-generated image of the active site of TdT using the PDB crystal structure 4129, showing the computationally docked catalytically productive position a 3'-0--dATP analog (blue, red, orange frame), each complexed to the two active-site metal ions (large greenspheres). Shown are the residues, that are in close proximity to the incoming dNTP and the targets of mutagenesis and screening.
FIG. 5 shows a table of TdT variants that were selected for increased incorporation of selected 3'-0-blocked dNTP analogs as described herein.
FIG. 6 shows exemplary 3'-0-azidomethyl deoxynucleotides that can be used to synthesize custom DNA oligomers using modified TdTs, as described herein.
FIG. 7 shows a synthetic scheme for producing 3'-0-azidomethyl deoxyadenosine triphosphate (3'-0-azidomethyl-dATP).
FIG. 8 shows a synthetic scheme for producing 3'-0-azidomethyl deoxythymidine triphosphate (3'-0-azidomethyl-dTTP).
FIG. 9 shows a synthetic scheme for producing 3'-0-azidomethyl deoxycytidine triphosphate (3'-0-azidomethyl-dCTP).
FIG. 10 shows a synthetic scheme for producing 3'-0-azidomethyl deoxyguanosine triphosphate (3'-0-azidomethyl-dGTP).
FIG. 11 shows a synthetic scheme for producing 3'-0-methoxymethyl deoxythymidine
3 triphosphate (3'-0-MOM-dTTP).
FIG. 12 shows a synthetic scheme for producing 3'-0-thiomethyl deoxycytidine triphosphate (3'-0-MTM-dCTP).
FIG. 13 shows CGE (Capillary Gel Electrophoresis) traces showing migration of chemically synthesized authentic standard of A) 5'-FAM-TAATAATAATAATAATTTTT
compared to chemically synthesized authentic standard of B) 5'-FAM-TAATAATAATAATAATTTTTT-PO4-3'.
FIG. 14 shows CGE traces showing the removal of 3'-PO4 by treatment with Shrimp Alkaline Phosphatase. A) Chemically synthesized 5'-TAATAATAATAATAATTTTT-PO4-3' prior to treatment with Shrimp Alkaline Phosphatase and chemically synthesized 5'-TAATAATAATAATAATTTTT-PO4-3' after treatment with B) 4.1 x 10-4 U/ul, C) 1.23 x 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 for 1 minute at 37 C.
FIG. 15 shows CGE traces comparing: A) 5'-FAM-TAATAATAATAATAATTTTT
after treatment with murine WT TdT and no dNTP; B) 5'-FAM-TAATAATAATAATAATTTTT after treatment with murine WT TdT and 500 uM 3'-PO4-dTTP for 60 minutes at 37oC; C) 5'-FAM-TAATAATAATAATAATTTTT after treatment with murine WT TdT and 500 uM 3'-PO4-dTTP for 60 minutes at 37oC, followed by treatment with 0.2 units of Shrimp Alkaline Phosphatase for 15 minutes at 37oC.
FIG. 16 shows CGE traces comparing A) 5'-FAM-TAATAATAATAATAATTTTT-3' after treatment with murine WT TdT and no dNTP for 60 minutes at 37oC; B) 5'-FAM-TAATAATAATAATAATTTTT after treatment with murine TdT E180K + M192K + L381K +
R454K + N474R and 500 uM 3'-PO4-dTTP for 60 minutes at 37oC; C) 5'-FAM-TAATAATAATAATAATTTTT-3' after treatment with murine WT TdT and 500 uM 3'-PO4-dTTP for 60 minutes at 37oC, followed by treatment with 0.2 units of Shrimp Alkaline Phosphatase for 15 minutes at 37oC; D) Homopolymer dT extension ladder created by treatment of 5'-FAM-TAATAATAATAATAATTTTT-3' with murine WT TdT and dTTP.
DESCRIPTION OF THE INVENTION
The 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, a modified template-independent terminal deoxynucleotidyl transferase (TdT) is obtained that
FIG. 12 shows a synthetic scheme for producing 3'-0-thiomethyl deoxycytidine triphosphate (3'-0-MTM-dCTP).
FIG. 13 shows CGE (Capillary Gel Electrophoresis) traces showing migration of chemically synthesized authentic standard of A) 5'-FAM-TAATAATAATAATAATTTTT
compared to chemically synthesized authentic standard of B) 5'-FAM-TAATAATAATAATAATTTTTT-PO4-3'.
FIG. 14 shows CGE traces showing the removal of 3'-PO4 by treatment with Shrimp Alkaline Phosphatase. A) Chemically synthesized 5'-TAATAATAATAATAATTTTT-PO4-3' prior to treatment with Shrimp Alkaline Phosphatase and chemically synthesized 5'-TAATAATAATAATAATTTTT-PO4-3' after treatment with B) 4.1 x 10-4 U/ul, C) 1.23 x 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 for 1 minute at 37 C.
FIG. 15 shows CGE traces comparing: A) 5'-FAM-TAATAATAATAATAATTTTT
after treatment with murine WT TdT and no dNTP; B) 5'-FAM-TAATAATAATAATAATTTTT after treatment with murine WT TdT and 500 uM 3'-PO4-dTTP for 60 minutes at 37oC; C) 5'-FAM-TAATAATAATAATAATTTTT after treatment with murine WT TdT and 500 uM 3'-PO4-dTTP for 60 minutes at 37oC, followed by treatment with 0.2 units of Shrimp Alkaline Phosphatase for 15 minutes at 37oC.
FIG. 16 shows CGE traces comparing A) 5'-FAM-TAATAATAATAATAATTTTT-3' after treatment with murine WT TdT and no dNTP for 60 minutes at 37oC; B) 5'-FAM-TAATAATAATAATAATTTTT after treatment with murine TdT E180K + M192K + L381K +
R454K + N474R and 500 uM 3'-PO4-dTTP for 60 minutes at 37oC; C) 5'-FAM-TAATAATAATAATAATTTTT-3' after treatment with murine WT TdT and 500 uM 3'-PO4-dTTP for 60 minutes at 37oC, followed by treatment with 0.2 units of Shrimp Alkaline Phosphatase for 15 minutes at 37oC; D) Homopolymer dT extension ladder created by treatment of 5'-FAM-TAATAATAATAATAATTTTT-3' with murine WT TdT and dTTP.
DESCRIPTION OF THE INVENTION
The 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, a modified template-independent terminal deoxynucleotidyl transferase (TdT) is obtained that
4 allows the enzymatically mediated synthesis of de novo oligodeoxynucleotides, thereby enabling their use in routine assembly for gene synthesis. The enzymes of the invention lend themselves to aqueous-based, enzyme-mediated methods of synthesizing polynucleotides of a predetermined sequence on a solid support.
The modified enzymes of the invention will allow 3'-0-blocked dNTP analogs to be used in a step-by-step method to extend an initiating nucleic acid into a user defined sequence (see Figure 2). Furthermore, after each nucleotide extension step, the reactants can be recovered and recycled from the solid support back to the original reagent reservoir. Once that step is complete, the 3'-0-blocking group will be removed, allowing the cycle to start anew. At the conclusion of n cycles of extension-recover-deblock-wash, the full length, single strand polydeoxynucleotide will be cleaved from the solid support and isolated for subsequent use. A
variety of 3'-0-blocked deoxynucleotides, may be used, but the choice of specific 3'-0-blocking groups is dictated by: 1) the smallest possible bulk to maximize substrate utilization by TdT and 2) removal of the blocking group with the mildest and preferably aqueous conditions in the shortest period of time.
Cost savings by this approach will be achieved by exploiting the higher yield of final oligonucleotide product at a lower starting scale than currently being used as the existing industry standard (i.e., less than 1 nanomole). Future adaptation of this enzymatic approach to array based formats will allow even further and more dramatic reductions in the cost of synthesis of long oligonucleotides achievable by highly parallel synthesis. Furthermore, the enzymatic synthesis process that we propose uses only aqueous based chemistries like buffers and salts, thus greatly reducing the environmental burden of the organic waste generated by the existing phosphoramidite method.
The methods of the invention may be used to modify terminal deoxynucleotidyl transferases (TdT), however other enzymes could be modified with similar methods. TdT is likely to be a successful starting enzyme because it is capable of 3'-extension activity using single strand initiating primers in a template-independent polymerization.
However, prior to the invention described herein, there have been no reports of 3'-0-blocked nucleotides being incorporated into single-stranded oligonucleotide by an enzyme in the absence of a template. In fact, as Chang and Bollum reported, substitution of the 3'-hydroxyl group results in complete inactivity of available transferase enzymes. See Chang and Bollum, "Molecular Biology of Terminal Transferase, CRC Critical Reviews in Biochemistry, vol. 21(1), p. 27-52 (1986), incorporated herein by reference in its entirety. Nonetheless, when TdT is used with natural dNTPs (i.e., not 3'-0-blocked), and without a template, oligonucleotide extension continues without stopping. Such uncontrolled incorporation is evidenced by the time-dependent gel electrophoresis images shown in FIG. 1. FIG. 1 shows an agarose gel of a solution phase polymerization reaction composed of terminal deoxynucleotidyl transferase (TdT), deoxyadenosine triphosphate (dATP) and fluorescent strand initiator 5'-Cy5-dA10 at different time points. (Adapted with permission from Tjong et al. "Amplified on-chip fluorescence detection of DNA hybridization by surface-initiated enzymatic polymerization,"
Anal. Chem., 2011; 83:5153-5159 (2011), incorporated by reference herein in its entirety.) Additionally, TdT
can extend primers in a near quantitative manner resulting in the addition of thousands of nucleotides, while TdT is likely to accept a wide variety of modified and substituted dNTPs as efficient substrates. Furthermore, a substantial library of mechanistic and structural information regarding 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 entireties.
It is known that TdT can use substrates having modifications and/or substitutions at the deoxyribose sugar ring as well as the purine/pyrimidine nucleobases. For example, TdT accepts bulky modifications at the C5 of pyrimidines and the C7 of purines. 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 by reference in their entireties. In some instances, TdT can even accept non-nucleotide triphosphates. See Barone et al., Nucleotides and Nucleic Acids 2001, 20(4-7):1141-
The modified enzymes of the invention will allow 3'-0-blocked dNTP analogs to be used in a step-by-step method to extend an initiating nucleic acid into a user defined sequence (see Figure 2). Furthermore, after each nucleotide extension step, the reactants can be recovered and recycled from the solid support back to the original reagent reservoir. Once that step is complete, the 3'-0-blocking group will be removed, allowing the cycle to start anew. At the conclusion of n cycles of extension-recover-deblock-wash, the full length, single strand polydeoxynucleotide will be cleaved from the solid support and isolated for subsequent use. A
variety of 3'-0-blocked deoxynucleotides, may be used, but the choice of specific 3'-0-blocking groups is dictated by: 1) the smallest possible bulk to maximize substrate utilization by TdT and 2) removal of the blocking group with the mildest and preferably aqueous conditions in the shortest period of time.
Cost savings by this approach will be achieved by exploiting the higher yield of final oligonucleotide product at a lower starting scale than currently being used as the existing industry standard (i.e., less than 1 nanomole). Future adaptation of this enzymatic approach to array based formats will allow even further and more dramatic reductions in the cost of synthesis of long oligonucleotides achievable by highly parallel synthesis. Furthermore, the enzymatic synthesis process that we propose uses only aqueous based chemistries like buffers and salts, thus greatly reducing the environmental burden of the organic waste generated by the existing phosphoramidite method.
The methods of the invention may be used to modify terminal deoxynucleotidyl transferases (TdT), however other enzymes could be modified with similar methods. TdT is likely to be a successful starting enzyme because it is capable of 3'-extension activity using single strand initiating primers in a template-independent polymerization.
However, prior to the invention described herein, there have been no reports of 3'-0-blocked nucleotides being incorporated into single-stranded oligonucleotide by an enzyme in the absence of a template. In fact, as Chang and Bollum reported, substitution of the 3'-hydroxyl group results in complete inactivity of available transferase enzymes. See Chang and Bollum, "Molecular Biology of Terminal Transferase, CRC Critical Reviews in Biochemistry, vol. 21(1), p. 27-52 (1986), incorporated herein by reference in its entirety. Nonetheless, when TdT is used with natural dNTPs (i.e., not 3'-0-blocked), and without a template, oligonucleotide extension continues without stopping. Such uncontrolled incorporation is evidenced by the time-dependent gel electrophoresis images shown in FIG. 1. FIG. 1 shows an agarose gel of a solution phase polymerization reaction composed of terminal deoxynucleotidyl transferase (TdT), deoxyadenosine triphosphate (dATP) and fluorescent strand initiator 5'-Cy5-dA10 at different time points. (Adapted with permission from Tjong et al. "Amplified on-chip fluorescence detection of DNA hybridization by surface-initiated enzymatic polymerization,"
Anal. Chem., 2011; 83:5153-5159 (2011), incorporated by reference herein in its entirety.) Additionally, TdT
can extend primers in a near quantitative manner resulting in the addition of thousands of nucleotides, while TdT is likely to accept a wide variety of modified and substituted dNTPs as efficient substrates. Furthermore, a substantial library of mechanistic and structural information regarding 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 entireties.
It is known that TdT can use substrates having modifications and/or substitutions at the deoxyribose sugar ring as well as the purine/pyrimidine nucleobases. For example, TdT accepts bulky modifications at the C5 of pyrimidines and the C7 of purines. 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 by reference in their entireties. In some instances, TdT can even 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 by reference in their entireties. However, there is little evidence in the prior art that TdT can accept 3'-0-blocked nucleotides. See, for example, Knapp et al., Chem. Eur.
J., 2011, 17:2903, incorporated herein by reference in its entirety. While the lack of activity of TdT was not a focus
J., 2011, 17:2903, incorporated herein by reference in its entirety. While the lack of activity of TdT was not a focus
6 of Knapp et al., the authors reported that they tested their 3'-OH modified analog with TdT, and saw no incorporation of this relatively small 3'-OH modification into an oligonucleotide.
Native TdT is a very efficient enzyme. It has been demonstrated that TdT can polymerize extremely long homopolydeoxynucleotides of 1000 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. p. 141-71; Tjong et al., Anal Chem, 2011, 83:5153-59, all of which are incorporated by reference in their entireties). Random sequence oligomers consisting of all four nucleotides have also been polymerized by TdT, however there are no reports of ordered polynucleotides being synthesized in the absence of a template. See Damiani, et al., Nucleic Acids Res, 1982, 10(20):6401-10, incorporated by reference herein in its entirety.
Support-bound synthesis of polynucleotides by TdT is additionally supported by reports of homopolymer synthesis of 150 bps initiators covalently attached to self-assembled monolayers on gold surfaces. 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 by reference in their entireties.
These authors also observed preference by TdT of dATP>dTTP>>dGTPdCTP for incorporation of homopolymers. In a more recent report, Tjong et al.
demonstrated the TdT
mediated synthesis of long (>1 Kb) homopolymer ssDNA from initiator primers immobilized on glass surfaces.
The distributive behavior of TdT is reinforced by Figure 3, which shows a time course of a solution phase synthesis of 1-1.5 kb homopolymers. After each addition of an unmodified (natural) dNTP, the enzyme dissociates, thus allowing the random extension of any strand in the population. The distribution of product lengths in such a system should follow a Poisson distribution, as reported by Bollum and co-workers in 1974. If TdT were used with a terminating nucleotide species, i.e., one with the 3'-0-position blocked, the reaction should proceed to completion, resulting not in a distribution of product lengths, but essentially a pure product of a single nucleotide addition.
Nonetheless, as described above, nucleotide synthesis with 3'-0-blocked dNTPs does not proceed with commercially-available TdT proteins. This fact is reinforced by FIG. 3, which shows a gel shift assay used to monitor the solution phase incorporation kinetics of 3'-0-azidomethyl dATP and 3'-0-azidomethyl dCTP using a commercially-available, recombinant TdT. The data in FIG. 3 clearly show that neither 3'-0-modified dNTP analog is a substrate for
Native TdT is a very efficient enzyme. It has been demonstrated that TdT can polymerize extremely long homopolydeoxynucleotides of 1000 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. p. 141-71; Tjong et al., Anal Chem, 2011, 83:5153-59, all of which are incorporated by reference in their entireties). Random sequence oligomers consisting of all four nucleotides have also been polymerized by TdT, however there are no reports of ordered polynucleotides being synthesized in the absence of a template. See Damiani, et al., Nucleic Acids Res, 1982, 10(20):6401-10, incorporated by reference herein in its entirety.
Support-bound synthesis of polynucleotides by TdT is additionally supported by reports of homopolymer synthesis of 150 bps initiators covalently attached to self-assembled monolayers on gold surfaces. 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 by reference in their entireties.
These authors also observed preference by TdT of dATP>dTTP>>dGTPdCTP for incorporation of homopolymers. In a more recent report, Tjong et al.
demonstrated the TdT
mediated synthesis of long (>1 Kb) homopolymer ssDNA from initiator primers immobilized on glass surfaces.
The distributive behavior of TdT is reinforced by Figure 3, which shows a time course of a solution phase synthesis of 1-1.5 kb homopolymers. After each addition of an unmodified (natural) dNTP, the enzyme dissociates, thus allowing the random extension of any strand in the population. The distribution of product lengths in such a system should follow a Poisson distribution, as reported by Bollum and co-workers in 1974. If TdT were used with a terminating nucleotide species, i.e., one with the 3'-0-position blocked, the reaction should proceed to completion, resulting not in a distribution of product lengths, but essentially a pure product of a single nucleotide addition.
Nonetheless, as described above, nucleotide synthesis with 3'-0-blocked dNTPs does not proceed with commercially-available TdT proteins. This fact is reinforced by FIG. 3, which shows a gel shift assay used to monitor the solution phase incorporation kinetics of 3'-0-azidomethyl dATP and 3'-0-azidomethyl dCTP using a commercially-available, recombinant TdT. The data in FIG. 3 clearly show that neither 3'-0-modified dNTP analog is a substrate for
7 TdT, i.e., there is no polynucleotide extension when compared to reactions containing dATP as a positive control (lanes 12 thru 15). FIG. 3, thus, adds further evidence that commercially-available TdTs are not able to synthesize oligomers by incorporating dNTPs with modified 3'-OHs.
With suitable modifications, a variety of different 3'-0-blocked dNTP analogs will be suitable for the controlled addition of nucleotides by TdT. Modified 3'-0-blocked dNTP analogs include, but are not limited to, the 3'-0-allyl, 3'-0-azidomethyl, 3'-0-NH2, 3'-0-CH2N3,3'-0-ONHC(0)H, 3'-0-CH2SSCH3, and 3'-0-CH2CN blocking groups. Overall, the choice of the 3'-0-blocking group will be dictated by: 1) the smallest possible bulk to maximize substrate utilization by TdT, which is likely to affect kinetic uptake, and 2) the blocking group with the mildest removal conditions, preferably aqueous, and in the shortest period of time. 3'-0-blocking groups that are the suitable for use with this invention are described in WO
2003/048387; WO 2004/018497; WO 1996/023807; WO 2008/037568; 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 entireties.
A computational model of the active site of murine TdT was created to understand the structural basis for the lack of utilization of 3'-0-blocked dNTPs by TdT.
Additionally, the computer model made it possible to "fit" various modified dNTPs into the active site. Figure 4 shows the docking of a -dATP (shown in blue, red, magenta, orange) with murine TdT (see SEQ ID NO. 9, below) using the PDB crystal structure 4129 and AutoDock 4.2 (Molecular Graphics Laboratory, Scripps Research Institute, La Jolla, CA).
The phosphate portions of the dATPs (orange) are in complex with the catalytic metal ions (green) while the alpha phosphate is positioned to be attacked by the 3'-OH of the bound oligonucleotide . The model shown in FIG. 4 indicates the choice of amino acid residues likely to interfere with the formation of a catalytically productive complex when a 3'-0-blocked dNTP is present. Other residues that may interact with the closest residues, like Glu 180 or Met 192, are also targets of modification. Amino acid numbering and positions are provided with reference to the murine TdT of SEQ ID NO. 9 but the referenced amino acid modifications are applicable to any TdT having similar sequence including the GGFRR or TGSR
motifs.
AutoDock's predicted binding mode suggests that modification to the 3'-OH will change the electrostatic interactions between two residues, Arg336 and Arg454.
Although Arg336 is near the reaction center in the active site, Arg 336 is highly conserved, and early studies found
With suitable modifications, a variety of different 3'-0-blocked dNTP analogs will be suitable for the controlled addition of nucleotides by TdT. Modified 3'-0-blocked dNTP analogs include, but are not limited to, the 3'-0-allyl, 3'-0-azidomethyl, 3'-0-NH2, 3'-0-CH2N3,3'-0-ONHC(0)H, 3'-0-CH2SSCH3, and 3'-0-CH2CN blocking groups. Overall, the choice of the 3'-0-blocking group will be dictated by: 1) the smallest possible bulk to maximize substrate utilization by TdT, which is likely to affect kinetic uptake, and 2) the blocking group with the mildest removal conditions, preferably aqueous, and in the shortest period of time. 3'-0-blocking groups that are the suitable for use with this invention are described in WO
2003/048387; WO 2004/018497; WO 1996/023807; WO 2008/037568; 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 entireties.
A computational model of the active site of murine TdT was created to understand the structural basis for the lack of utilization of 3'-0-blocked dNTPs by TdT.
Additionally, the computer model made it possible to "fit" various modified dNTPs into the active site. Figure 4 shows the docking of a -dATP (shown in blue, red, magenta, orange) with murine TdT (see SEQ ID NO. 9, below) using the PDB crystal structure 4129 and AutoDock 4.2 (Molecular Graphics Laboratory, Scripps Research Institute, La Jolla, CA).
The phosphate portions of the dATPs (orange) are in complex with the catalytic metal ions (green) while the alpha phosphate is positioned to be attacked by the 3'-OH of the bound oligonucleotide . The model shown in FIG. 4 indicates the choice of amino acid residues likely to interfere with the formation of a catalytically productive complex when a 3'-0-blocked dNTP is present. Other residues that may interact with the closest residues, like Glu 180 or Met 192, are also targets of modification. Amino acid numbering and positions are provided with reference to the murine TdT of SEQ ID NO. 9 but the referenced amino acid modifications are applicable to any TdT having similar sequence including the GGFRR or TGSR
motifs.
AutoDock's predicted binding mode suggests that modification to the 3'-OH will change the electrostatic interactions between two residues, Arg336 and Arg454.
Although Arg336 is near the reaction center in the active site, Arg 336 is highly conserved, and early studies found
8 that replacement of Arg336 with Gly or Ala reduced dNTP activity by 10-fold (Yang B et al. J.
Mol. Biol. 1994; 269(16):11859-68). Accordingly, one motif for modification is the GGFRR
motif including Arg 336 in the above structural model.
Additionally, it is thought that Gly452 and Ser453 exist in a cis-peptide bond conformation (see Delarue et al., EMBO J., 2002; 21(3):427- 39, incorporated herein by reference in its entirety) and that the guanidinium group of Arg336 assists in the stabilization of this conformation. The stability provided by Arg336 may help explain why substitutions at this position have a negative impact on the reactivity of modified TdT proteins. In some instances, the instability created by modifying position 336 may be overcome by using proline residues to stabilize cis-peptide bond conformation. However, if Arg336 is substituted, e.g., with alanine or glycine, the entire TGSR motif (positions 451, 452, 435, 454) may also have to be modified to compensate for this change. For example, the TGSR motif may be modified to TPSR or TGPR. Accordingly, the TGSR motif, including Gly452 in the above structural model was targeted for modification.
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 a 3'- 0-blocking group may require additional modifications to the al4 region to compensate for substitutions of glycine or alanine at Arg454. In other embodiments, substitutions to other residues in the all region may be required to compensate for substitution to Arg336 either instead of, or in addition to, modification of the TGSR motif.
While modification to Arg336 and Arg454 may change the binding interactions of 3'-0-modified dNTPs, it may also be necessary to explore substitutions that would result in improved steric interactions of 3'-0-modified dNTPs with TdT. In order to test computationally predicted enzyme variants that show increased substrate utilization of 3'-0-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 a polymerase incorporation assay with selected 3'-0-blocked dNTP
analogs. FIG. 5 shows the results of the screening of various synthetically generated murine TdT variants. In some embodiments, single amino acid changes are important while in other, combinations of one & two amino acids also produce increased incorporation of 3'-0-blocked dNTPs.
Interactions with residues such as Gly332, Gly333, Gly452, Thr451, Trp450, Ser453, and Q455
Mol. Biol. 1994; 269(16):11859-68). Accordingly, one motif for modification is the GGFRR
motif including Arg 336 in the above structural model.
Additionally, it is thought that Gly452 and Ser453 exist in a cis-peptide bond conformation (see Delarue et al., EMBO J., 2002; 21(3):427- 39, incorporated herein by reference in its entirety) and that the guanidinium group of Arg336 assists in the stabilization of this conformation. The stability provided by Arg336 may help explain why substitutions at this position have a negative impact on the reactivity of modified TdT proteins. In some instances, the instability created by modifying position 336 may be overcome by using proline residues to stabilize cis-peptide bond conformation. However, if Arg336 is substituted, e.g., with alanine or glycine, the entire TGSR motif (positions 451, 452, 435, 454) may also have to be modified to compensate for this change. For example, the TGSR motif may be modified to TPSR or TGPR. Accordingly, the TGSR motif, including Gly452 in the above structural model was targeted for modification.
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 a 3'- 0-blocking group may require additional modifications to the al4 region to compensate for substitutions of glycine or alanine at Arg454. In other embodiments, substitutions to other residues in the all region may be required to compensate for substitution to Arg336 either instead of, or in addition to, modification of the TGSR motif.
While modification to Arg336 and Arg454 may change the binding interactions of 3'-0-modified dNTPs, it may also be necessary to explore substitutions that would result in improved steric interactions of 3'-0-modified dNTPs with TdT. In order to test computationally predicted enzyme variants that show increased substrate utilization of 3'-0-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 a polymerase incorporation assay with selected 3'-0-blocked dNTP
analogs. FIG. 5 shows the results of the screening of various synthetically generated murine TdT variants. In some embodiments, single amino acid changes are important while in other, combinations of one & two amino acids also produce increased incorporation of 3'-0-blocked dNTPs.
Interactions with residues such as Gly332, Gly333, Gly452, Thr451, Trp450, Ser453, and Q455
9 of murine TdT are important. Each of these residues is within 0.6nm of the 3'-OH of a typical dNTP. These residues are also potential targets for substitution to allow the extra steric bulk of a 3'-blocking group like 3'-0-azidomethyl or 3'-0-aminoxy. Residues that are within 1.2 nm of the 3'-OH such as Glu457, Ala510, Asp509, Arg508, Lys199, Ser196, Met192, Glu180 or Leu161 may also potentially interfere with the substrate utilization of a 3'-0-blocked dNTP and are thus targets for substitution in addition to or in combination with Arg336 and Arg454.
Additional residues of interest include Arg461 and Asn474.
While the TGSR and GGFRR motifs are highlighted here, modifications to the flanking amino acids such as Thr331, Gly337, Lys338, Gly341, or His342 are also contemplated for providing (alone or in combination) increased incorporation of 3'-0-blocked dNTPs as discussed herein. Various in silico modeled TdT modifications capable of increased incorporation are discussed in Example 2 below.
In addition to amino acid substitutions at positions 500- 510 it may be necessary to delete residues to remove interference with a 3'-0-blocking group. Since these amino acids are located near the C-terminus of the protein, and exist in a relatively unstructured region, they may be deleted singly or altogether, either instead of or in combination with the modifications described above. In certain embodiments, insertion of residues into the modified TdT.
For example, insertions of residues in the GGFRR or TGSR motifs or flanking regions can allow an increased rate of incorporation of 3'-0-blocked dNTP by the modified TdT. TdT
modifications can include insertion of a Tyrosine residue between the Phe334 and Arg335 residues (or substitutions thereof) of the GGFRR motif.
Modified TdT's of the invention include those described in FIG. 5. Modified TdT's may include one or more of a modification 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 may include N474R. In various embodiments combinations of two or more modified residues may be used such as, for example, E180D+W450H, E180K+R454A, M192K+E180K, E180K+R454I, E180D+M192E, E180D+M192E+R454T, or E180K+W450H.
As shown below, most TdTs include the GGFRR and TGSR motifs. In the following sequences, the GGFRR and TGSR motifs have been bolded and underlined for easy reference.
Native calf thymus TdT is a candidate for alteration of the primary structure to achieve a suitable template-independent polymerase. However, a variety of other proteins may be explored to identify a candidate suitable for the use with 3'-0-blocked dNTP analogs, including human and murine TdT. 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 with 3'-0-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, with SEQ ID NO. 1.
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) MAQQRQHQRL PMDPLCTASS GPRKKRPRQV GASMASPPHD IKFQNLVLFI
LEKKMGTTRR NFLMELARRK GFRVENELSD SVTHIVAENN SGSEVLEWLQ
VQNIRASSQL ELLDVSWLIE SMGAGKPVEI TGKHQLVVRT DYSATPNPGF
QKTPPLAVKK ISQYACQRKT TLNNYNHIFT DAFEILAENS EFKENEVSYV
TFMRAASVLK SLPFTIISMK DTEGIPCLGD KVKCIIEEII EDGESSEVKA VLNDERYQSF
KLFTSVFGVG LKTSEKWFRM GFRSLSKIMS DKTLKFTKMQ KAGFLYYEDL
VSCVTRAEAE AVGVLVKEAV WAFLPDAFVT MTGGFRRGKK IGHDVDFLIT
SPGSAEDEEQ LLPKVINLWE KKGLLLYYDL VESTFEKFKL PSRQVDTLDH
FQKCFLILKL HHQRVDSSKS NQQEGKTWKA IRVDLVMCPY ENRAFALLGW
TGSRQFERDI RRYATHERKM MLDNHALYDK TKRVFLKAES EEEIFAHLGL
DYIEPWERNA
Table 2. Nucleic Acid Sequence of Bovine TdT
SEQ ID NO. 2: (1923 nt) ctcttctgga gataccactt gatggcacag cagaggcagc atcagcgtct tcccatggat ccgctgtgca cagcctcctc aggccctcgg aagaagagac ccaggcaggt gggtgcctca atggcctccc ctcctcatga catcaagttt caaaatttgg tcctcttcat tttggagaag aaaatgggaa ccacccgcag aaacttcctc atggagctgg ctcgaaggaa aggtttcagg gttgaaaatg agctcagtga ttctgtcacc cacattgtag cagaaaacaa ctctggttca gaggttctcg agtggcttca ggtacagaac ataagagcca gctcgcagct agaactcctt gatgtctcct ggctgatcga aagtatggga gcaggaaaac cagtggagat tacaggaaaa caccagcttg ttgtgagaac agactattca gctaccccaa acccaggctt ccagaagact ccaccacttg ctgtaaaaaa gatctcccag tacgcgtgtc aaagaaaaac cactttgaac aactataacc acatattcac ggatgccttt gagatactgg ctgaaaattc tgagtttaaa gaaaatgaag tctcttatgt gacatttatg agagcagctt ctgtacttaa atctctgcca ttcacaatca tcagtatgaa ggatacagaa ggaattccct gcctggggga caaggtgaag tgtatcatag aggaaattat tgaagatgga gaaagttctg aagttaaagc tgtgttaaat gatgaacgat atcagtcctt caaactcttt acttctgttt ttggagtggg actgaagaca tctgagaaat ggttcaggat ggggttcaga tctctgagta aaataatgtc agacaaaacc ctgaaattca caaaaatgca gaaagcagga tttctctatt atgaagacct tgtcagctgc gtgaccaggg ccgaagcaga ggcggttggc gtgctggtta aagaggctgt gtgggcattt ctgccggatg cctttgtcac catgacagga ggattccgca ggggtaagaa gattgggcat gatgtagatt ttttaattac cagcccagga tcagcagagg atgaagagca acttttgcct aaagtgataa acttatggga aaaaaaggga ttacttttat attatgacct tgtggagtca acatttgaaa agttcaagtt gccaagcagg caggtggata ctttagatca ttttcaaaaa tgctttctga ttttaaaatt gcaccatcag agagtagaca gtagcaagtc caaccagcag gaaggaaaga cctggaaggc catccgtgtg gacctggtta tgtgccccta cgagaaccgt gcctttgccc tgctaggctg gactggctcc cggcagtttg agagagacat ccggcgctat gccacacacg agcggaagat gatgctggat aaccacgctt tatatgacaa gaccaagagg gtatttctca aagcggaaag tgaagaagaa atctttgcac atctgggatt ggactacatt gaaccatggg aaagaaatgc ttaggagaaa gctgtcaact tttttctttt ctgttctttt tttcaggtta gacaaattat gcttcatatt ataatgaaag atgccttagt caagtttggg attctttaca ttttaccaag atgtagattg cttctagaaa taagtagttt tggaaacgtg atcaggcacc ccctgggtta tgctctggca agccatttgc aggactgatg tgtagaactc gcaatgcatt ttccatagaa acagtgttgg aattggtggc tcatttccag ggaagttcat caaagcccac tttgcccaca gtgtagctga aatactgtat acttgccaat aaaaatagga aac Additionally, to make isolation of recombinant proteins easier, it is common to append an N-terminal His tag sequence to the recombinant protein (see Boule J-B et al., Molecular Biotechnology, 1998;10:199-208, incorporated by reference herein in its entirety), which is used in combination with an affinity column (Hitrap, Amersham Pharmacia Biotech, Uppsala, Sweden). Alternatively, N-terminal truncated forms of the enzyme with appended His-tag sequence will work with the current invention (see, e.g., US 7,494,797, incorporated by reference herein in its entirety). His-tagged Bovine TdT amino acid sequences are shown below in Tables 3, 5, and 7, while His-tagged Bovine TdT nucleic acid sequences are shown below in Tables 4, 6, and 8. His tags may be engineered at other positions as required. In some embodiments, the resulting protein, adapted for sequence-specific de novo polynucleotide synthesis with 3'-0-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, with SEQ ID NOS. 3, 5, or 7.
Table 3. Amino Acid Sequence of a A138 and His-tagged Bovine TdT.
SEQ ID No. 3: (392 aa) Met Arg Gly Ser His His His His His His Arg Thr Asp Tyr Ser Ala Thr Pro Asn Pro Gly Phe Gln Lys Thr Pro Pro Leu Ala Val Lys Lys Be Ser Gln Tyr Ala Cys Gln Arg Lys Thr Thr Leu Asn Asn Tyr Asn His Be Asp Ala Phe Glu Be Leu Ala Glu Asn Ser Glu Phe Lys Glu Asn Glu Val Ser Tyr Val Thr Phe Met Arg Ala Ala Ser Val Leu Lys Ser Leu Pro Phe Thr Ile Be Ser Met Lys Asp Thr Phe Thr Glu Gly Ile Pro Cys Leu Gly Asp Lys Val Lys Cys Be Be Glu Glu Ile Ile Glu Asp Gly Glu Ser Ser Glu Val Lys Ala Val Leu Asn Asp Glu Arg Tyr Gln Ser Phe Lys Leu Ser Val Phe Gly Val Gly Leu Lys Thr Ser Glu Lys Trp Phe Arg Met Gly Phe Thr Phe Arg Ser Leu Ser Lys Ile Met Ser Asp Lys Thr Leu Lys Lys Met Gln Lys Ala Gly Phe Leu Tyr Tyr Glu Asp Leu Val Ser Cys Val Thr Arg Ala Glu Ala Glu Ala Val Gly Val Leu Val Lys Glu Ala Val Trp Ala Phe Leu Pro Asp Ala Phe Val Thr Met Thr Gly Gly Phe Arg Arg Gly Lys Lys Ile Gly His Asp Val Asp Phe Leu Ile Thr Ser Pro Gly Ser Ala Glu Asp Glu Glu Gln Leu Leu Pro Lys Val Be Asn Leu Trp Glu Lys Lys Gly Leu Leu Leu Tyr Tyr Asp Leu Val Glu Ser Thr Phe Glu Lys Phe Lys Phe Thr Leu Pro Ser Arg Gln Val Asp Thr Leu Asp His Phe Gln Lys Cys Phe Leu Ile Leu Lys Leu His His Gln Arg Val Asp Ser Ser Lys Ser Asn Gln Gln Glu Gly Lys Thr Trp Lys Ala Ile Arg Val Asp Leu Val Met Cys Pro Tyr Glu Asn Arg Ala Phe Ala Leu Leu Gly Trp Thr Gly Ser Arg Gln Phe Glu Arg Asp Ile Arg Arg Tyr Ala Thr His Glu Arg Lys Met Met Leu Asp Asn His Ala Leu Tyr Asp Lys Thr Lys Arg Val Phe Leu Lys Ala Glu Ser Glu Glu Glu Ile Phe Ala His Leu Gly Leu Asp Tyr Be Glu Pro Trp Glu Arg Asn Ala Table 4. Nucleotide Sequence of a A138 and His-tagged Bovine TdT.
SEQ ID No. 4: (1187 nt) atgagaggat cgcatcacca tcaccatcac agaacagact attcagctac cccaaaccca ggcttccaga agactccacc acttgctgta aaaaagatct cccagtacgc gtgtcaaaga aaaaccactt tgaacaacta taaccacata ttcacggatg cctttgagat actggctgaa aattctgagt ttaaagaaaa tgaagtctct tatgtgacat ttatgagagc agcttctgta cttaaatctc tgccattcac aatcatcagt atgaaggata cagaaggaat tccctgcctg ggggacaagg tgaagtgtat catagaggaa attattgaag atggagaaag ttctgaagtt aaagctgtgt taaatgatga acgatatcag tccttcaaac tctttacttc tgtttttgga gtgggactga agacatctga gaaatggttc aggatggggt tcagatctct gagtaaaata atgtcagaca aaaccctgaa attcacaaaa atgcagaaag caggatttct ctattatgaa gaccttgtca gctgcgtgac cagggccgaa gcagaggcgg ttggcgtgct ggttaaagag gctgtgtggg catttctgcc ggatgccttt gtcaccatga caggaggatt ccgcaggggt aagaagattg ggcatgatgt agatttttta attaccagcc caggatcagc agaggatgaa gagcaacttt tgcctaaagt gataaactta tgggaaaaaa agggattact tttatattat gaccttgtgg agtcaacatt tgaaaagttc aagttgccaa gcaggcaggt ggatacttta gatcattttc aaaaatgctt tctgatttta aaattgcacc atcagagagt agacagtagc aagtccaacc agcaggaagg aaagacctgg aaggccatcc gtgtggacct ggttatgtgc ccctacgaga accgtgcctt tgccctgcta ggctggactg gctcccggca gtttgagaga gacatccggc gctatgccac acacgagcgg aagatgatgc tggataacca cgctttatat gacaagacca agagggtatt tctcaaagcg gaaagtgaag aagaaatctt tgcacatctg ggattggact acattgaacc atgggaaaga aatgcttaag cttgcgc Table 5: Amino Acid Sequence of a A151 and His-tagged Bovine TdT.
SEQ ID No. 5: (379 aa) Met Arg Gly Ser His His His His His His Lys Thr Pro Pro Leu Ala Val Lys Lys Be Ser Gln Tyr Ala Cys Gln Arg Lys Thr Thr Leu Asn Asn Tyr Asn His Be Asp Ala Phe Glu Be Leu Ala Glu Asn Ser Glu Phe Lys Glu Asn Glu Val Ser Tyr Val Thr Phe Met Arg Ala Ala Ser Val Leu Lys Ser Leu Pro Phe Thr Be Ile Ser Met Lys Asp Thr Phe Thr Glu Gly Be Pro Cys Leu Gly Asp Lys Val Lys Cys Be Be Glu Glu Be Ile Glu Asp Gly Glu Ser Ser Glu Val Lys Ala Val Leu Asn Asp Glu Arg Tyr Gln Ser Phe Lys Leu Ser Val Phe Gly Val Gly Leu Lys Thr Ser Glu Lys Trp Phe Arg Met Gly Phe Thr Phe Arg Ser Leu Ser Lys Be Met Ser Asp Lys Thr Leu Lys Lys Met Gln Lys Ala Gly Phe Leu Tyr Tyr Glu Asp Leu Val Ser Cys Val Thr Arg Ala Glu Ala Glu Ala Val Gly Val Leu Val Lys Glu Ala Val Trp Ala Phe Leu Pro Asp Ala Phe Val Thr Met Thr Gly Gly Phe Arg Arg Gly Lys Lys Ile Gly His Asp Val Asp Phe Leu Be Thr Ser Pro Gly Ser Ala Glu Asp Glu Glu Gln Leu Leu Pro Lys Val Be Asn Leu Trp Glu Lys Lys Gly Leu Leu Leu Tyr Tyr Asp Leu Val Glu Ser Thr Phe Glu Lys Phe Lys Phe Thr Leu Pro Ser Arg Gln Val Asp Thr Leu Asp His Phe Gln Lys Cys Phe Leu Ile Leu Lys Leu His His Gln Arg Val Asp Ser Ser Lys Ser Asn Gln Gln Glu Gly Lys Thr Trp Lys Ala Be Arg Val Asp Leu Val Met Cys Pro Tyr Glu Asn Arg Ala Phe Ala Leu Leu Gly Trp Thr Gly Ser Arg Gln Phe Glu Arg Asp Ile Arg Arg Tyr Ala Thr His Glu Arg Lys Met Met Leu Asp Asn His Ala Leu Tyr Asp Lys Thr Lys Arg Val Phe Leu Lys Ala Glu Ser Glu Glu Glu Be Phe Ala His Leu Gly Leu Asp Tyr Be Glu Pro Trp Glu Arg Asn Ala Table 6. Nucleotide Sequence of a A151 and His-tagged Bovine TdT.
SEQ ID No. 6: (1148 nt) atgagaggat cgcatcacca tcaccatcac aagactccac cacttgctgt aaaaaagatc tcccagtacg cgtgtcaaag aaaaaccact ttgaacaact ataaccacat attcacggat gcctttgaga tactggctga aaattctgag tttaaagaaa atgaagtctc ttatgtgaca tttatgagag cagcttctgt acttaaatct ctgccattca caatcatcag tatgaaggat acagaaggaa ttccctgcct gggggacaag gtgaagtgta tcatagagga aattattgaa gatggagaaa gttctgaagt taaagctgtg ttaaatgatg aacgatatca gtccttcaaa ctctttactt ctgtttttgg agtgggactg aagacatctg agaaatggtt caggatgggg ttcagatctc tgagtaaaat aatgtcagac aaaaccctga aattcacaaa aatgcagaaa gcaggatttc tctattatga agaccttgtc agctgcgtga ccagggccga agcagaggcg gttggcgtgc tggttaaaga ggctgtgtgg gcatttctgc cggatgcctt tgtcaccatg acaggaggat tccgcagggg taagaagatt gggcatgatg tagatttttt aattaccagc ccaggatcag cagaggatga agagcaactt ttgcctaaag tgataaactt atgggaaaaa aagggattac ttttatatta tgaccttgtg gagtcaacat ttgaaaagtt caagttgcca agcaggcagg tggatacttt agatcatttt caaaaatgct ttctgatttt aaaattgcac catcagagag tagacagtag caagtccaac cagcaggaag gaaagacctg gaaggccatc cgtgtggacc tggttatgtg cccctacgag aaccgtgcct ttgccctgct aggctggact ggctcccggc agtttgagag agacatccgg cgctatgcca cacacgagcg gaagatgatg ctggataacc acgctttata tgacaagacc aagagggtat ttctcaaagc ggaaagtgaa gaagaaatct ttgcacatct gggattggac tacattgaac catgggaaag aaatgcttaa gcttgcgc Table 7. Amino Acid Sequence of a A160 and His-tagged Bovine TdT.
SEQ ID No. 7: (370 aa) Met Arg Gly Ser His His His His His His Be Ser Gln Tyr Ala Cys Gln Arg Lys Thr Thr Leu Asn Asn Tyr Asn His Be Asp Ala Phe Glu Ile Leu Ala Glu Asn Ser Glu Phe Lys Glu Asn Glu Val Ser Tyr Val Thr Phe Met Arg Ala Ala Ser Val Leu Lys Ser Leu Pro Phe Thr Be Be Ser Met Lys Asp Thr Phe Thr Glu Gly Be Pro Cys Leu Gly Asp Lys Val Lys Cys Be Ile Glu Glu Ile Ile Glu Asp Gly Glu Ser Ser Glu Val Lys Ala Val Leu Asn Asp Glu Arg Tyr Gln Ser Phe Lys Leu Ser Val Phe Gly Val Gly Leu Lys Thr Ser Glu Lys Trp Phe Arg Met Gly Phe Thr Phe Arg Ser Leu Ser Lys Ile Met Ser Asp Lys Thr Leu Lys Lys Met Gln Lys Ala Gly Phe Leu Tyr Tyr Glu Asp Leu Val Ser Cys Val Thr Arg Ala Glu Ala Glu Ala Val Gly Val Leu Val Lys Glu Ala Val Trp Ala Phe Leu Pro Asp Ala Phe Val Thr Met Thr Gly Gly Phe Arg Arg Gly Lys Lys Ile Gly His Asp Val Asp Phe Leu Ile Thr Ser Pro Gly Ser Ala Glu Asp Glu Glu Gln Leu Leu Pro Lys Val Be Asn Leu Trp Glu Lys Lys Gly Leu Leu Leu Tyr Tyr Asp Leu Val Glu Ser Thr Phe Glu Lys Phe Lys Phe Thr Leu Pro Ser Arg Gln Val Asp Thr Leu Asp His Phe Gln Lys Cys Phe Leu Be Leu Lys Leu His His Gln Arg Val Asp Ser Ser Lys Ser Asn Gln Gln Glu Gly Lys Thr Trp Lys Ala Ile Arg Val Asp Leu Val Met Cys Pro Tyr Glu Asn Arg Ala Phe Ala Leu Leu Gly Trp Thr Gly Ser Arg Gln Phe Glu Arg Asp Ile Arg Arg Tyr Ala Thr His Glu Arg Lys Met Met Leu Asp Asn His Ala Leu Tyr Asp Lys Thr Lys Arg Val Phe Leu Lys Ala Glu Ser Glu Glu Glu Ile Phe Ala His Leu Gly Leu Asp Tyr Ile Glu Pro Trp Glu Arg Asn Ala Table 8. Nucleotide Sequence of a A160 and His-tagged Bovine TdT.
SEQ ID No. 8: (1121 nt) atgagaggat cgcatcacca tcaccatcac atctcccagt acgcgtgtca aagaaaaacc actttgaaca actataacca catattcacg gatgcctttg agatactggc tgaaaattct gagtttaaag aaaatgaagt ctcttatgtg acatttatga gagcagcttc tgtacttaaa tctctgccat tcacaatcat cagtatgaag gatacagaag gaattccctg cctgggggac aaggtgaagt gtatcataga ggaaattatt gaagatggag aaagttctga agttaaagct gtgttaaatg atgaacgata tcagtccttc aaactcttta cttctgtttt tggagtggga ctgaagacat ctgagaaatg gttcaggatg gggttcagat ctctgagtaa aataatgtca gacaaaaccc tgaaattcac aaaaatgcag aaagcaggat ttctctatta tgaagacctt gtcagctgcg tgaccagggc cgaagcagag gcggttggcg tgctggttaa agaggctgtg tgggcatttc tgccggatgc ctttgtcacc atgacaggag gattccgcag gggtaagaag attgggcatg atgtagattt tttaattacc agcccaggat cagcagagga tgaagagcaa cttttgccta aagtgataaa cttatgggaa aaaaagggat tacttttata ttatgacctt gtggagtcaa catttgaaaa gttcaagttg ccaagcaggc aggtggatac tttagatcat tttcaaaaat gctttctgat tttaaaattg caccatcaga gagtagacag tagcaagtcc aaccagcagg aaggaaagac ctggaaggcc atccgtgtgg acctggttat gtgcccctac gagaaccgtg cctttgccct gctaggctgg actggctccc ggcagtttga gagagacatc cggcgctatg ccacacacga gcggaagatg atgctggata accacgcttt atatgacaag accaagaggg tatttctcaa agcggaaagt gaagaagaaa tctttgcaca tctgggattg gactacattg aaccatggga aagaaatgct taagcttgcg c Table 9. Amino Acid Sequence of murine TdT
SEQ lD NO. 9: (510 aa) MDPLQAVHLG PRKKRPRQLG TPVASTPYDI RFRDLVLFIL EKKMGTTRRA
FLMELARRKG FRVENELSDS VTHIVAENNS GSDVLEWLQL QNIKASSELE
LLDISWLIEC MGAGKPVEMM GRHQLVVNRN SSPSPVPGSQ NVPAPAVKKI
SQYACQRRTT LNNYNQLFTD ALDILAENDE LRENEGSCLA FMRASSVLKS
LPFPITSMKD TEGIPCLGDK VKSIIEGIIE DGESSEAKAV LNDERYKSFK LFTSVFGVGL
KTAEKWFRMG FRTLSKIQSD KSLRFTQMQK AGFLYYEDLV SCVNRPEAEA
VSMLVKEAVV TFLPDALVTM TGGFRRGKMT GHDVDFLITS PEATEDEEQQ
LLHKVTDFWK QQGLLLYCDI LESTFEKFKQ PSRKVDALDH FQKCFLILKL
DHGRVHSEKS GQQEGKGWKA IRVDLVMCPY DRRAFALLGW TGSRQFERDL
RRYATHERKM MLDNHALYDR TKRVFLEAES EEEIFAHLGL DYIEPWERNA
In certain embodiments, modified enzymes of the invention may include an N-terminus truncation relative to their respective native TdT enzyme. For example, in preferred embodiments, the native enzyme may be murine TdT as provided in SEQ ID NO. 9 above. The modified TdT
may be truncated at the equivalent of position 147 or 131 of the native murine TdT as shown in SEQ ID Nos. 10 and 11 respectively. Modified TdTs may include a protein tag sequence such as a His tag and additional linkers at their N-terminus as illustrated in SEQ ID
Nos. 10 and 11. The His-tag portion if underlined in each of the sequences and the linker is provided in bold.
SEQ ID No. 10: Murine del-147 with His-tag and linker MRGSHHHHHHGMAS MT GGQQMGRDLYDD DDKDRWGS ELE KKIS QYACQRRTTLNNY
NQLFTDALDILAENDELRENEGSCLAFMRAS S VLKSLPFPITSMKDTEGIPCLGDKVKSIIEG
IIED GE S SEAKAVLNDERYKSFKLFTS VFGVGLKTAEKWFRMGFRTLS KIQS D KS LRFT QM
QKAGFLYYEDLVSCVNRPEAEAVSMLVKEAVVTFLPDALVTMTGGFRRGKMTGHDVDF
LIT S PEATED EE QQLLHKVTDFWKQQGLLLYC DILES TFEKFKQPSRKVDALDHFQKCFLIL
KLDHGRVHSEKS GQQEGKGWKAIRVDLVMC PYDRRAFALLGWT GS RQFERD LRRYATH
ERKMMLDNHALYDRTKRVFLEAESEEEIFAHLGLDYIEPWERNA
SEQ ID No. 11: Murine del-131 with His-tag and linker MRGSHHHHHHGMAS MT GGQQMGRENLYF Q GS PS PVPGS QNVPAPAVKKIS QYACQRRT
TLNNYNQLFTDALDILAENDELRENEGSCLAFMRAS S VLKS LPFPIT S MKD TEGIPCLGD KV
KS IIEGIIED GE S SEAKAVLNDERYKSFKLFTS VFGVGLKTAEKWFRMGFRTLS KIQS D KS L
RFTQMQKAGFLYYEDLVSCVNRPEAEAVSMLVKEAVVTFLPDALVTMTGGFRRGKMTG
HDVDFLITSPEATEDEEQQLLHKVTDFWKQQGLLLYCDILES TFEKFKQPS RKVDALDHFQ
KCFLILKLDHGRVHS E KS GQQEGKGWKAIRVDLVMCPYDRRAFALLGWTGS RQFERDLR
RYATHERKMMLDNHALYDRTKRVFLEAES EEEIFAHLGLDYIEPWERNA
Additional TdT modifications that may increase incorporation efficiency of 3'-0-blocked or other nucleotide analogs are listed in Table 10 below. While the modifications are described with referenced to the murine TdT listed in SEQ ID NO. 9, such the 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 the His-tags and linkers. In various embodiments, contemplated modifications include deletion of the S420 through E424 amino acids.
Various combinations of amino acid substitutions of the invention are listed in each row 1-175 of Table 10.
Table 10 7 [177K E180K R454A
8 [177K E180K
9 [177K
E180D M192[ L381 K R454T R461Q
16 E180D M192[ L381Q R454K N474A
17 E180D M192[ R454K
24 E180K M192[ L381 K R454T N474K
E180K M192[ L381A W450H R454T R461V
26 E180K M192[ L381 K W450H R4541 R461Q
27 E180K M192[ L381V N474A
28 E180K M192[ L381W R4541 R461V
29 E180K M192[ R454I
E180K M192[ R454T
98 M192[ L381R R454T R461V N474A
99 M192[ L381V R4541 R461V
100 M192[ L381V R4541 R461V N474K
101 M192[ L381V W450 H R454 K
102 M192[ L381V W450 H R454 K R461V N474A
103 M192[ R454A
111 Q455[
A variety of 3'-0-modified dNTPs and NTPs may be used with the disclosed proteins for de novo synthesis. In some embodiments, the preferred removable 3'-0-blocking group is a 3'-0-amino, a 3'-0-ally1 or a 3'-0-azidomethyl. In other embodiments, the removable 3'-0-blocking moiety is selected from the group consisting of 0-phenoxyacetyl; 0-methoxyacetyl; 0-acetyl; 0-(p-toluene )-sulfonate; 0-phosphate; 0-nitrate; 0-[4-methoxy [-tetrahydrothiopyranyl;
0-tetrahydrothiopyranyl; 0-[5-methyl[-tetrahydrofuranyl; 0-[2-methy1,4-methoxy]-tetrahydropyranyl; 045-methyThtetrahydropyranyl; and 0-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, hydroxylamine, borates, nitrates, sugars, phosphoramide, phosphoramidates, phenylsulfenates, sulfates, sulfones and amino acids (see Metzker ML et al. Nuc Acids Res. 1994;22(20):4259-67, U.S.P.N.
5,763,594, 6,232,465, 7,414,116; and 7,279,563, all of which are incorporated by reference in their entireties).
Synthesis of exemplary 3'-0-blocked dNTP analogs FIG. 6 shows four exemplary 3'-0-blocked dNTP analogs, namely 3'-0-azidomethyl-dATP, 3'-0-azidomethyl-dCTP, 3'-0-azidomethyl-dGTP, and 3'-0-azidomethyl-dTTP.
The synthesis of each 3'-0-azidomethyl analog is described below and detailed in FIGS. 7-12. The 3'-0-blocked dNTP analogs can also be purchased from specialty suppliers, such as Azco Biotech, Oceanside, CA. It is to be understood that corresponding 3'-0-blocked ribonucleotides can be formed with similar synthetic methods to enable the creation of custom RNA oligos.
3'-0-azidomethyl-dATP: With reference to FIG. 7, a solution of N6-benzoy1-5'-0-(tert-butyldimethylsily1)-T-deoxyadenosine (3.0 g; 6.38 mmol) [CNH Technologies, Woburn, MA] in DMSO (12 ml), acetic acid (5.5 ml) and acetic anhydride (17.6 ml) was prepared. The mixture was stirred at room temperature for 48 h. Approximately 100 ml of a saturated NaHCO3 solution was added and the aqueous layer was extracted with CH2C12. The combined organic extract was washed with saturated NaHCO3 solution and dried over Na2SO4. The residue was purified by flash column chromatography (hexane/ethyl acetate, 1:1 to 1:4) to recover N6-Benzoy1-3'-0-(methylthiomethyl)-5'-0-(tert-butyldimethylsily1)-2'-deoxyadenosine (shown as compound 1 in FIG. 7) as a white powder (2.4 g; 71% yield). 400 mg of N6-Benzoy1-3'-0-(methylthiomethyl)-5'-0-(tert-butyldimethylsily1)-2'-deoxyadenosine was dissolved in dry CH2C12 (7m1) under nitrogen to create a solution (0.76 mmol). Cyclohexene (400 1), and S02C12(155 1; 1.91 mmol, redistilled) were then added. The reaction mixture was stirred at 0 C for 2 h.
The solvent was then removed under reduced pressure and then under a high-vacuum pump for 10 min. The resulting residue was dissolved in dry DMF (5 ml) and reacted with NaN3 (400 mg; 6.6 mmol) at room temperature for 3 h. The reaction mixture was dispersed in distilled water (50 ml) and extracted with CH2C12. The combined organic layer was dried over Na2SO4 and concentrated under reduced pressure. The residue was dissolved in Me0H (5 ml) and stirred with NH4F (300 mg; 8.1 mmol) at room temperature for 24 h. The solvent was then removed under reduced pressure. The reaction mixture was concentrated under reduced pressure and partitioned between water and CH2C12. The organic layer was separated and dried over Na2SO4. After concentration, the crude product was purified by flash column chromatography (ethyl acetate/methanol) to produce N6-Benzoy1-3'-0-(azidomethyl)-2'-deoxyadenosine (compound 2; FIG. 7) as a white powder (150 mg; 48% yield). N6-B enzoy1-3*-0-(azidomethyl)-T-deoxyadenosine (123 mg; 0.3 mmol) and a proton sponge (75.8 mg; 0.35 mmol) were then dried in a vacuum desiccator over P205 overnight before dissolving in trimethyl phosphate (6000). Next freshly distilled P0C13 (400; 0.35 mmol) was added dropwise at 0 C and the mixture was stirred at 0 C
for 2 h.
Subsequently, a mixture of tributylammonium pyrophosphate (552 mg) and tributylamine (0.55 ml; 2.31 mmol) in anhydrous DMF (2.33 ml) was added at room temperature and stirred for 30 min. Triethyl ammonium bicarbonate solution (TEAB) (0.1 M; pH 8.0; 15 ml) was then added, and the mixture was stirred for 1 hour at room temperature. Subsequently, concentrated NH4OH
(15 ml) was 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 with 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 with reverse-phase HPLC to produce 3'-0-azidomethyl-dATP (FIG. 7, compound 3), a nucleotide analog to be used for later synthesis.
3'-0-azidomethyl-dTTP: Acetic acid (4.8 ml) and acetic anhydride (15.4 ml) were added to a stirred solution of 5'-0-(tertbutyldimethylsilyl)thymidine (2.0 g;
5.6 mmol) [CNH
Technologies, Woburn, MA] in DMSO. The reaction mixture was stirred at room temperature for 48 h. A saturated NaHCO3 solution (100 ml) was added, and the aqueous layer was extracted with ethyl acetate (3x100 m1). The combined organic extract was 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'-0-(Methylthiomethyl)-5'-0-(tert-butyldimethylsilyl)thymidine (FIG. 8; Compound 4) as a white powder (1.75 g; 75%
yield). Approximately 1 gram of 3'-0-(Methylthiomethyl)-5'-0-(tert-butyldimethylsilyl)thymidine was then dissolved in dry CH2C12 (10 ml) under nitrogen. To this mixture cyclohexene (1.33 ml) and S02C12 (2840; 3.5 mmol, redistilled) were added. The resulting mixture was then stirred at 0 C for 1.5 h. The solvent was then removed under reduced pressure and then under high vacuum for 10 min. The residue was dissolved in dry DMF (5 ml) and reacted with NaN3 (926 mg; 15.4 mmol) at room temperature for 3 h. That reaction mixture was next dispersed in distilled water (50 ml) and extracted with CH2C12 (3x50 m1). The combined organic extract was dried over Na2SO4 and concentrated under reduced pressure. The residue was dissolved in Me0H (5 ml) and reacted with NH4F (600 mg; 16.2 mmol) at room temperature for 24 h. The reaction mixture was concentrated under reduced pressure and partitioned between water and CH2C12. The organic layer was then separated and dried over Na2SO4. After concentration, the residue was purified by flash column chromatography (hexane/ethyl acetate) to produce 3'-0-(azidomethyl)thymidine (FIG. 8, Compound 5) as a white powder (550 mg; 71% yield). Next, the 3'-0-(azidomethyl)thymidine and a proton sponge (0.35 mmol) were dried in a vacuum desiccator over P205 overnight before dissolving in trimethyl phosphate (6000). Next, freshly distilled P0C13 (400; 0.35 mmol) was added dropwise at 0 C
and the mixture was stirred at 0 C for 2 h. Subsequently, a mixture of tributylammonium pyrophosphate (552 mg) and tributylamine (0.55 ml; 2.31 mmol) in anhydrous DMF
(2.33 ml) was added at room temperature and stirred for 30 min. Triethyl ammonium bicarbonate solution (TEAB) (0.1 M; pH 8.0; 15 ml) was then added, and the mixture was stirred for 1 hour at room temperature. Subsequently, concentrated NH4OH (15 ml) was 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 with 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 with reverse-phase HPLC to produce 3'-0-azidomethyl-dTTP (FIG. 8, compound 6), a nucleotide analog to be used for later synthesis.
3'-0-azidomethyl-dCTP: Three and a half grams of N4-benzoy1-5'-0-(tert-butyldimethylsily1)-T-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 (21.6m1) were added, and the reaction mixture was stirred at room temperature for 48 h. A saturated NaHCO3 solution (100 ml) was then added and the aqueous layer was extracted with CH2C12(3x100 m1). The combined organic extract was washed with a saturated solution of NaHCO3 and then dried over Na2SO4. After concentration, the crude product was purified by flash column chromatography (ethyl acetate/hexane) to produce N4-Benzoy1-3'-0-(methylthiomethyl)-5'-0-(tert-butyldimethylsily1)-T-deoxycytidine (FIG. 9;
compound 7) as a white powder (2.9 g; 73% yield). In 8 ml of CH2C12 N4-Benzoy1-3'-0-(methylthiomethyl)-51-0-(tert-butyldimethylsily1)-T-deoxycytidine (558 mg; 1.04 mmol) was dissolved and then cyclohexene (5600) and S02C12(220 ill; 2.7 mmol) were added. The reaction mixture was stirred at 0 C for 1 h. The volatiles were then removed with reduced pressure.
The remaining residue was dissolved in dry DMF (5 ml) and reacted with NaN3(400 mg; 6.6mmo1) at room temperature for 2 h. The reaction mixture was dispersed in distilled water (50 ml) and extracted with CH2C12(3x50 m1). The combined organic extract was dried over Na2SO4 and concentrated under reduced pressure. The residue was dissolved in Me0H (5 ml) and reacted with NH4F (600 mg; 16.2 mmol) at room temperature for 24 h. The solvent was removed under reduced pressure.
The resulting residue was suspended in water (50 ml) and extracted with CH2C12(3x50 m1). The combined organic extract was dried over Na2SO4 and concentrated under reduced pressure. The crude product was purified by flash column chromatography (hexane/ethyl acetate) to produce N4-Benzoy1-3'-0-(azidomethyl)-2'-deoxycytidine (FIG. 9, compound 8) as a white powder (200 mg; 50% yield). Next, the N4-Benzoy1-3'-0-(azidomethyl)-2'-deoxycytidine and a proton sponge (0.35 mmol) were dried in a vacuum desiccator over P205 overnight before dissolving in trimethyl phosphate (6000). Then freshly distilled P0C13 (400; 0.35 mmol) was added dropwise at 0 C and the mixture was stirred at 0 C for 2 h. Subsequently, a mixture of tributylammonium pyrophosphate (552 mg) and tributylamine (0.55 ml; 2.31 mmol) in anhydrous DMF (2.33 ml) was added at room temperature and stirred for 30 min.
Triethyl ammonium bicarbonate solution (TEAB) (0.1 M; pH 8.0; 15 ml) was then added, and the mixture was stirred for 1 hour at room temperature. Subsequently, concentrated NH4OH (15 ml) was 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 with 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 with reverse-phase HPLC to produce 3'-0-azidomethyl-dCTP (FIG. 9, compound 9), a nucleotide analog to be used for later synthesis.
3'-0-azidomethyl-dGTP: To a stirred solution of N2-isobutyry1-5'-0-(tert-butyldimethylsily1)-T-deoxyguanosine (5 g; 11.0 mmol) [CNH Technologies, Woburn, MA] in dry DMSO (21 ml), acetic acid (10 ml) and acetic anhydride (32 ml) were added.
The reaction mixture was stirred at room temperature for 48 h. A saturated NaHCO3 solution (100 ml) was added and the aqueous layer was extracted with ethyl acetate (3x100 m1). The combined organic extract was washed with a saturated NaHCO3 solution and dried over Na2SO4.
After concentration, the crude product was purified by flash column chromatography (CH2C12/Me0H) to produce N2-Isobutyry1-3*-0-(methylthiomethyl)-5'-0-(tert-butyldimethylsily1)-T-deoxyguanosine (FIG. 10, compound 10) as a white powder (3.9 g; 69% yield).
One gram of N2-Isobutyry1-3*-0-(methylthiomethyl)-5'-0-(tert-butyldimethylsily1)-2'-deoxyguanosine was subsequently 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 h. The solvent was removed under high vacuum. The crude product was purified by flash column chromatography (ethyl acetate/hexane) to produce N2-Isobutyry1-06-(diphenylcarbamoy1)-3'-0-(methylthiomethyl)-5'-0-(tert-butyldimethylsily1)-T-deoxyguanosine (FIG. 10, compound 11), which appeared as a yellowish powder (1.09 g; 80% yield). N2-Isobutyry1-06-(diphenylcarbamoy1)-3*-0-(methylthiomethyl)-5'-0-(tert-butyldimethylsily1)-T-deoxyguanosine was then dissolved in dry CH2C12 (1.1 mmol) and stirred under nitrogen atmosphere at 0 C for 1.5 h. The solvent was removed under reduced pressure and then under high vacuum for 10 min.
The resulting residue was dissolved in dry DMF (5 ml) and reacted with NaN3 (600 mg; 10 mmol) at room temperature for 3 h. The reaction mixture was then dispersed in distilled water (50 ml) and extracted with CH2C12(3x50 m1). The combined organic extract was dried over Na2SO4 and concentrated under reduced pressure. The resultant residue was dissolved in Me0H
(5 ml) and reacted with NH4F (500 mg; 13.5 mmol) at room temperature for 24 h.
The solvent was removed under reduced pressure. The residue was suspended in water (50 ml) and extracted with CH2C12(3x50 m1). The combined organic extract was dried over Na2SO4 and concentrated under reduced pressure. The crude product was purified by flash column chromatography (hexane/ethyl acetate) to produce N2-Isobutyry1-06-(diphenylcarbamoy1)-3*-0-azidomethyl-T-deoxyguanosine (FIG. 10, compound 12) as a white powder (230 mg; 36% yield).
Finally, the N2-Isobutyry1-06-(diphenylcarbamoy1)-3'-0-azidomethyl-T-deoxyguanosine and a proton sponge (0.35 mmol) were dried in a vacuum desiccator over P205 overnight before dissolving in trimethyl phosphate (6000). Then freshly distilled POC13 (400; 0.35 mmol) was added dropwise at 0 C and the mixture was stirred at 0 C for 2 h. Subsequently, a mixture of tributylammonium pyrophosphate (552 mg) and tributylamine (0.55 ml; 2.31 mmol) in anhydrous DMF (2.33 ml) was added at room temperature and stirred for 30 min.
Triethyl ammonium bicarbonate solution (TEAB) (0.1 M; pH 8.0; 15 ml) was then added, and the mixture was stirred for 1 hour at room temperature. Subsequently, concentrated NH4OH (15 ml) was 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 with 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 with reverse-phase HPLC to produce 3'-0-azidomethyl-dGTP (FIG. 10, compound 13), a nucleotide analog to be used for later synthesis.
As described with respect to FIG. 2, once a 3'-0-blocked dNTP or 3'-0-blocked rNTP is added, it will be necessary to remove the blocking group so that additional dNTPs or rNTPs can be added. In some embodiments, the 3'-0-blocking group can be removed with a palladium catalyst in neutral aqueous solution at elevated temperature hydrochloric acid to pH 2, a reducing agent such as mercaptoethanol, or by the addition of tris-(2-carboxyethyl) phosphine.
See, e.g., U.S.P.N. 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.P.N. 7,279,563, and U.S.P.N. 7,414,116, all of which are incorporated herein by reference in their entireties. In other embodiments, the 3'-substitution group may be removed by UV irradiation (see, e.g., WO 92/10587, incorporated by reference herein in its entirety). Most 3'-0-blocking groups are removed by oxidative, reductive or hydrolytic chemical reactions. In some embodiments, a 3'-0-NO2 group is removed from a oligonucleotide by a 40% w/v solution of ammonium sulfide for <5 minutes at R.T. In some embodiments, a 3'-0-CH2CN group is removed from an oligonucleotide by treatment with 0.5M
KOH at 70 C. In some embodiments, the removal of the 3'-0-blocking group does not include chemical cleavage but uses a cleaving enzyme such as alkaline phosphatase.
In preferred embodiments an enzymatic reaction is used for removal of the 3'-blocking group. Shrimp Alkaline Phosphatase (SAP) may 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.
3'-0-Methoxymethyl-dTTP: 5'-0-Benzoylthymidine (173 mg, 0.5 mmol, 1 equiv) was dissolved in 10 mL of dichloromethane under argon at ambient T. Di-isopropylethylamine (128 mg, 1 mmol, 2 equiv) was added followed by methoxymethyl bromide (124 mg, 1 mmol, 2 equiv). The mixture was stirred at ambient T for 18h. The mixture was diluted with 10 mL
dichloromethane and this was washed successively with 20 mL of 5% aq HC1, and brine. The organic layer was dried with sodium sulfate and evaporated. 5'-0-Benzoy1-3'-0-methoxymethylthymidine (50 mg, 0.13 mmol) was dissolved in 5 mL of concentrated ammonium hydroxide at ambient temperature. The mixture was stirred at ambient T overnight.
The mixture was diluted extracted 3 times with 10 mL portions of dichloromethane. The combined extracts were washed with brine. The organic layer was dried with sodium sulfate and evaporated. 3'-0-Methoxymethylthymidine (23 mg, 0.08 mmol) was co-evaporated with pyridine (1.5 mL x 3) and dried overnight under high vacuum. The nucleoside was dissolved in a mixture of 1.5 mL of trimethylphosphate and 0.6 mL dry pyridine under Ar.
The mixture was cooled in an ice bath. a first aliquot of 10 uL of P0C13 was added dropwise. Five minutes later, a second aliquot of 10 uL was added. The mixture was stirred an additional 30 min. A
solution of the TBA phosphate salt in dry DMF (1.25 mL) was cooled in an ice bath in a vial under Ar. This was added to the rxn mixture dropwise over 10 sec. Immediately the pre-weighed solid proton sponge (21 mg, 1.25 equiv) was added as a solid in one portion. The mixture was stirred for 25 min after this addition and was quenched with 5 mL
of cold TEAB
buffer. The mixture was stirred in the ice bath for 10 min and then transferred to a small RB
flask for FPLC separation. Final separation was accomplished by reverse phase HPLC using a water/acetonitrile gradient containing 0.1 mM formic acid.
3'-0-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 eluant. N6-Formamidino-5'-0-benzoyldeoxy-3'-0-methylthiomethyldeoxycytidine (250 mg, 0.41 mmol) was dissolved in 10 mL of methanol and 10 mL conc 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 afford 170 mg (93%) of the desired nucleoside as a slightly yellow solid. 3'-0-Methylthiomethyl dexoxycytidine (25.0 mg, 0.09 mmol) in a 25 mL vial was co-evaporated with anhydrous pyridine (3 x lmL) and dried over the weekend. Trimethyl phosphate (0.7 mL) was added to dissolve the nucleoside and cooled in an ice bath to 0 C.
Phosphoryl chloride (28 [IL, 0.3 mmol) was added slowly (12 [IL, 5 min later 8 [IL, 30 min later 8 [IL) and the reaction was stirred for 2 h at 0 C. The di(tetrabutylammonium) hydrogen pyrophosphate was dissolved in anhydrous DMF (1 mL), 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 at 0 C for 2 h. To the reaction mixture was added 1 M triethylammonium bicarbonate buffer (TEAB) (2 mL) and the mixture was stirred for 1 h. The mixture was then transferred to round-bottom flask, 50 mL x 3 of miliQ water was added and 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 triphosphate (F48-F52) were evaporated under reduced pressure at 40 C, and the residue was then lyophilized. The triphosphate was dried to afford the desired triphosphate (12 mg, 16.5%).
EXAMPLES
Example 1: Protein Modifications.
Murine (mur) TdT variants originated from 380 aa synthetic gene. This backbone is a truncated version of WT murine TdT and represents a catalytic core of the ET
sequence.
Chemically synthesized TdT constructs were cloned into a pRSET A bacterial expression vector, featuring an N-terminal 6x-histidine tag and enterokinase cleavage site (ThermoFisher Scientific GeneArt Gene Synthesis). Synthetic TdT plasmids were maintained in DH5alpha cells (Biopioneer) plated on LB agar plates containing 100 ug/ml carbenicillin. For expression, the pRSETA-murine TdT plasmids were transformed into BL21 (DE3) pLysS cells (Thermo-Fisher) by incubating plasmids and cells on ice for 20 min., followed by a 30 sec.
heat shock at 42 C, followed by addition of SOC media and incubation with shaking at 37 C for 30-60 min. After addition of SOC media to cells, the entire volume (typically 60 ul) were plated on LB agar plates containing 100 ug/mL carbenicillin plus 34 ug/mL chloramphenicol.
Cells from 10 mL cultures (24-well plates, Corning) were harvested by centrifugation (3000xg, 15 min), then lysed in B-PER lysis buffer (Thermo-Fisher) containing lysozyme, protease inhibitors, and 100 mM NaCl. Pellets were soaked 1 x 60 min. in TBS buffer and supernatants collected for purification. The supernatant was bound onto 50 uL Ni-NTA bead (GE Life Sciences) slurry in 24-well plates for 30 min. The bead slurry was then washed 3 x 50mM
Tris-HC1, pH 8, 500mM NaCl (500 uL), followed by washing 4 x 50mM Tris-HC1, pH 8, 500mM NaCl, 50 mM
Imidazole (200 uL). The protein was then recovered by treating with 50mM Tris-HC1, pH 8, 500mM NaCl, 300 mM Imidazole (50 uL), then 50mM Tris-HC1, pH 8, 500mM NaCl, 300 mM
Imidazole (130 uL), and finally 50mM Tris-HC1, pH 8, 500mM NaCl, 1M Imidazole (50 uL).
Recovered fractions were analyzed by taking 2.5 ul sample and running on 8%
NuPage gel (Thermo-Fisher) , 200 V for 50 min, denaturing conditions. Gel stained with Coomassie Blue. The eluted protein was buffer exchanged using a 7.5 MWCO desalting column (Thermo-Fisher) and sored at -80 C (Storage Buffer = 20mM Tris-HC1, pH 6.8, 50 mM Na0Ac; 0.01%
Triton X-100 and 10% Glycerol).
Activity Screens:
TdT activity screening was performed via a dNTP polymerase extension reaction using different 3'-0-blocked dNTP analogs and a biotinylated oligonucleotide:
5BiosG/TAATAATAATAATAATAATAATAATAATAATAATAATTTTTT (ChemGenes Corporation) SEQ ID NO. 12 Reactions were typically set up in a 96 well plate. Reactions were performed by making a master mix with final concentrations of the following components: 0.2U PPase (Thermo-Fisher), pmol of oligonucleotide, 75 uM dNTP (see below), 1X TdT reaction buffer (5X
from Thermo-Fisher) to a final volume of 10 ul. Reactions were initiated by adding a defined volume (typically 2u1) of TdT variants in different wells and incubating the reaction mix at 37 C for 5 min and 60 min time points. Reactions were terminated by removal of a 10 ul aliquot and adding to 5 ul of 250 mM EDTA.
dNTPs tested:
3'-0-azidomethyl-dTTP see description above 3'-0-azidomethyl-dATP see description above 3'-0-azidomethyl-dGTP see description above 3'-0-MOM-dTTP see description above 3'-0-MTM-dCTP see description above 3'-aminoxy-dTTP Firebird BioMolecular Sciences LLC
3'-aminoxy-dATP Firebird BioMolecular Sciences LLC
3'-aminoxy-dGTP Firebird BioMolecular Sciences LLC
3'-0-methyl-dATP TriLink BioTechnologies LLC
3'-0-methyl-dGTP TriLink BioTechnologies LLC
3'-0-methyl-dCTP TriLink BioTechnologies LLC
Biotinylated oligos in the quenched reaction mix were bound to Streptavidin beads (0.77 um, Spherotech). The beads were then transferred to filter plates (Pall Corporation) and washed several times with water. The oligonucleotides were cleaved from the solid support by incubating the plate with cleavage buffer (10% Diisopropyl-amine in methanol) at 50 C for 30 min followed by elution in water. The eluted samples were dried and dissolved in 300 of water containing oligonucleotide sizing standards (two oligonucleotides (ChemGenes Corporation) that are approximately 15-20 bases smaller or larger than the starting 42-mer oligonucleotide).
Oligonucleotides were then analyzed for extension efficiency by Capillary Gel Electrophoresis (Oligo Pro II, Advanced Analytical Technologies Inc.).
Example 2: In silico modeling Several amino acid modifications to the GGFRR and TGSR motifs and flanking amino acids discussed above were modeled in silico to determine modifications capable of increased incorporation of 3'-0-blocked dNTP analogs as described above. Single, double, and triple amino acid substitutions as well amino acid insertions were modeled. Table 11 below shows modifications found to elicit increased incorporation. Amino acid positions are provided with reference to murine TdT but are applicable to conserved sequences of any TdT.
Rows in Table 11 describe a base modification to one or more amino acids in or flanking the GGFRR motif.
Columns include additional combinations of modifications to other amino acids such as those in and flanking the TGSR motif.
Table 11.
Residue Single Combination Combinati Combination Combination Combination Position Site with E180K on with with E180K with R461V with N474R
Mutation R454T and R454T
T331 T331M, T331M + T331M + T331M + T331M + T331M +
T3315 E180K, R454T, E180K + R461V, N474R, T331A, T3315+ T3315+ R454T, T3315+ T3315+
T331V, E180K, R454T, T3315+ R461V, N474R, T331G, T331A + T331A + E180K + T331A + T331A +
T331I, E180K, R454T, R454T, R461V, N474R, T331N, T331V + T331V + T331A + T331V + T331V +
T331C, E180K, R454T, E180K + R461V, N474R, T331L T331G + T331G + R454T, T331G + T331G +
E180K, R454T, T331V + R461V, N474R, T3311+ T3311+ E180K + T3311+ T3311+
E180K, R454T, R454T, R461V, N474R, T331N + T331N + T331G + T331N + T331N +
E180K, R454T, E180K + R461V, N474R, T331C + T331C + R454T, T331C + T331C +
E180K, R454T, T3311+ R461V, N474R, T331L + T331L + E180K + T331L + T331L +
E180K R454T R454T, R461V N474R
T331N +
E180K +
R454T, T331C +
E180K +
R454T, T331L +
E180K +
G332 G332A G332A + G332A + G332A +
G332A +
G332A + E180K
R454T [180K+ R461V N474R
G333 G333S +
E180K +
R454T, G333S + G333S + G333S +
G333A +
G333S + E180K, R454T, R461V, N474R, E180K +
G333A + G333A + G333A +
G333A +
G333S, R454T, E180K, R454T, R461V, N474R, G333A, G333D +
G333D + G333D + G333D +
G333D +
G333D, E180K +
E180K, R454T, R461V, N474R, G333P, R454T, G333P + G333P + G333P +
G333P +
G333E G333P +
E180K, R454T, R461V, N474R, E180K +
G333E + E180K G333[+ G333[+
G333[+
R454T, G333E +
E180K +
G333 and G333S +
G333S + G333S +
G333S +
G333S + G333S + F334Y F334Y +
F334 F334Y + F334Y +
F334Y +
F334Y +[180K [180K+
F334 F334H +
E180K +
F334H + F334H + F334H +
R454T, R454T, R461V, N464R, F334H, F334H + E180K' F334Y + F334Y +
F334Y +
F334Y +
F334Y, F334Y + E180K' [180K+
R454T, R461V, N474R, F334N F334N + E180K R454T, F334N + F334N + F334N +
F334N +
E180K +
F334 and Y
F334S +
insertion F334S + F334S + F334S +
F334S + F334S +
334 335insY
334 335in 334 335insY + 334 335ins - 334 335insY
334 335insY
_ _ _ _ _ between + E180K +
sY E180K Y + R454T + R461V +
F334 and R335 R335L +
R335L + [180K+ R335L +
R335L +
R454T, R454T, R461V, N474R, R335L + E180K, R335S + R335S R335S +
R335S +
R335 L, R335S +E180K, R454T, +[180K+ R461V, N474R, R3355' R335K + E180K, R335K + R454T, R335K +
R335K +
R335K, R335W + R454T, R335K + R461V, N474R, R335W, E180K, R335W + E180K + R335W +
R335W +
R335T + E180K R454T, R454T, R461V, N474R, R335T + R335W + R335T +
R335T +
R454T [180K+ R461V
R454T, R335T +
E180K +
R336 R336K +
E180K +
R454T, R336K + R336S + R336K +
R336K +
R454T, E180K + R461V, N474R, R336S + R454T, R336S +
R336S +
R336K + E180K' R336K' R454T, R336I + R461V, N474R, R336S + E180K' R336I + E180K + R336I +
R336I +
R336S' R336I +E180K, R336I, R454T, R454T, R461V, N474R, R336N +
R336N, R336N + R336N + R336N + R336N +
E180K, R336V' R454T, [180K+ R461V, N474R, R336V + E180K' R336Q R336V+ R454T, R336V + R336V
+
R336Q + E180K
R454T, R336V + R461V, N474R, R336Q + [180K+ R336Q +
R336Q +
R454T R454T, R461V
R336Q +
E180K +
G337 R336K +
E180K +
R454T, R336S +
G337K + G337K + G337K +
E180K +
R454T, R461V, N474R, R454T, G337K + G337[+ G337[+
G337[+
R336I +
E180K, R454T, R461V, N474R, E180K +
G337K, G337E + E180K, G337A G337A G337A +
R454T, G337E, G337A +[180K, +R454T, +R461V, N474R, R336N +
G337A, G337D + G337D + G337D + G337D +
E180K +
G337D, E180K, R454T, R461V, N474R, R454T, G337H, G337H +E180K, G337H G337H G337H +
R336I +
G337S G337H + +R454T, +R461V, N474R, E180K +
E180K, G337H + G337H + G337H +
R454T, G337S + E180K R454T, R461V, N474R, R336V +
G337S + G337S + G337S +
E180K +
R454T, R336Q +
E180K +
K338 K338R +
K338R + [180K+ K338R +
K338R +
K338R, K338R + E180K, R454T, R454T, R461V, N474R, K338A K338A + E180K K338A + K338A + K338A
+ K338A +
R454T [180K+ R461V
G341 G341C, G341C + G341C + G341C + G341C +
G341C +
G341S, E180K, R454T, [180K+ R461V, N474R, G341V, G341S + E180K, G341S + R454T, G341S +
G341S +
G341I G341V + R454T, G341S + R461V, N464R, E180K, G341V + E180K + G341V +
G341V +
G341I + E180K R454T, R454T, R461V, N474R, G341I + G341V + G341I +
G341I +
R454T E180K + R461V
R454T, G341I +
E180K +
H342 H342G +
E180K +
H342G + R454T, H342G +
H342G +
H342G +
R454T, H342K + R461V, N474R, E180K, H342G, H342K + E180K + H342K + H342K +
H342K +
H342K, R454T, R454T, R461V, N474R
E180K, H342R, H342R + H342R + H342R + H342R +
H342R +
H342D R454T, [180K+ R461V, N474R, E180K, H342D + R454T, H342D +
H342D +
H342D + E180K
R454T H342D + R461V
E180K +
Example 3: Incorporation of dNTPs with phosphate blocking groups DNA and the nucleotides that comprise DNA are highly negatively charged due to the phosphate groups within 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 relative to natural nucleotides due to the additional phosphate group at the 3'- position. The increased negative charge may affect the ability of the TdT to incorporate the modified nucleotides. In certain embodiments, engineered TdT enzymes of the invention may be modified for efficient incorporation of 3'-phosphate-dNTPs by neutralizing the negative charges with positive charges on the modified TdT.
The Average number of Neighboring Atoms Per Sidechain Atom (AvNAPSA) algorithm within the Rosetta protein software suite3 was used to identify mutations that will increase the positive charge in and around the enzymatic active site of TdT. By increasing a key parameter of the AvNAPSA algorithm, termed surface atom cutoff, sequence positions in the active site of TdT
were targeted. The surface charge of proteins 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 content of each of which is incorporate herein by reference. Increasing the surface atom cutoff term allows AvNAPSA to consider sequence positions with a higher number of neighboring atoms, such as positions within an enzyme active site. A summary of positions identified in TdT using AvNAPSA as being potentially useful for more efficient incorporation of 3'-phosphate-dNTP is shown in Table 12.
Table 12: TdT modifications for incorporation of phosphate-blocked dNTPs FIGS. 13-16 illustrate the superior nucleotide incorporation of modified TdT
over the wild type with respect to 3'-PO4-dNTPs . FIG. 13, Panel A is the CGE analysis of a chemically synthesized oligonucleotide (IDT) (21-mer; 5'-FAM-TAATAATAATAATAATTTTTT-PO4-3'), while Panel B shows that the addition of one nucleotide bearing a 3'-PO4 group, causes faster electrophoretic mobility than a comparable 20-mer (IDT) (5'-FAM-TAATAATAATAATAATTTTT). FIG. 14 is the CGE analysis demonstrating that Shrimp Akaline Phosphatase (SAP) (NEB #P0757) quantitatively removes a 3'-PO4 group in 1 minute or less at a concentration of 1.23 x 10-3 U/ul per pmol of oligonucleotide. The figure shows a titration series of increasing amounts of SAP from 0 U/ul (Panel A) to 1.0 x 10-1 U/ul (Panel G).
FIG. 15, Panel B is the CGE analysis of a murine WT TdT reaction mixture that demonstrates no polymerase mediated extension even in the presence of 500 uM 3'-PO4-dTTP (MyChem LLC) as evidenced by no change to the starting material oligonucleotide shown in Panel A. Further evidence of the lack of substrate utilization of 3'-PO4-dTTP is shown in panel C of FIG. 15 as demonstrated by the lack of reactivity of the oligonucleotide starting material (Panel A). FIG. 16 is a CGE analysis of the partial incorporation of a 3'-PO4-dTTP by a variant TdT enzyme (E180K + M192K
+ L381K +
R454K + N474R) as shown in panel B that demonstrates the appearance of a new oligonucleotide species (new peak circled) with a faster electrophoretic mobility as would be expected based on the results shown in FIG. 13. Further evidence of the incorporation of a 3'-PO4 by the variant TdT is demonstrated by the post-extension removal of the 3'-PO4 by treatment with SAP
and the appearance of a new oligonucleotide species (Panel C - new peak circled) with an electrophoretic migration rate slower than the oligonucleotide starting material as would be 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 a variant enzyme (E180K+M192K+R454K+R461V+N474R) Incorporation by Reference References and citations to other documents, such as patents, patent applications, patent publications, journals, books, papers, web contents, have been made throughout this disclosure.
All such documents are hereby incorporated herein by reference in their entirety for all purposes.
Equivalents Various modifications of the 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 full contents of this document, including references to the scientific and patent literature 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.
Additional residues of interest include Arg461 and Asn474.
While the TGSR and GGFRR motifs are highlighted here, modifications to the flanking amino acids such as Thr331, Gly337, Lys338, Gly341, or His342 are also contemplated for providing (alone or in combination) increased incorporation of 3'-0-blocked dNTPs as discussed herein. Various in silico modeled TdT modifications capable of increased incorporation are discussed in Example 2 below.
In addition to amino acid substitutions at positions 500- 510 it may be necessary to delete residues to remove interference with a 3'-0-blocking group. Since these amino acids are located near the C-terminus of the protein, and exist in a relatively unstructured region, they may be deleted singly or altogether, either instead of or in combination with the modifications described above. In certain embodiments, insertion of residues into the modified TdT.
For example, insertions of residues in the GGFRR or TGSR motifs or flanking regions can allow an increased rate of incorporation of 3'-0-blocked dNTP by the modified TdT. TdT
modifications can include insertion of a Tyrosine residue between the Phe334 and Arg335 residues (or substitutions thereof) of the GGFRR motif.
Modified TdT's of the invention include those described in FIG. 5. Modified TdT's may include one or more of a modification 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 may include N474R. In various embodiments combinations of two or more modified residues may be used such as, for example, E180D+W450H, E180K+R454A, M192K+E180K, E180K+R454I, E180D+M192E, E180D+M192E+R454T, or E180K+W450H.
As shown below, most TdTs include the GGFRR and TGSR motifs. In the following sequences, the GGFRR and TGSR motifs have been bolded and underlined for easy reference.
Native calf thymus TdT is a candidate for alteration of the primary structure to achieve a suitable template-independent polymerase. However, a variety of other proteins may be explored to identify a candidate suitable for the use with 3'-0-blocked dNTP analogs, including human and murine TdT. 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 with 3'-0-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, with SEQ ID NO. 1.
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) MAQQRQHQRL PMDPLCTASS GPRKKRPRQV GASMASPPHD IKFQNLVLFI
LEKKMGTTRR NFLMELARRK GFRVENELSD SVTHIVAENN SGSEVLEWLQ
VQNIRASSQL ELLDVSWLIE SMGAGKPVEI TGKHQLVVRT DYSATPNPGF
QKTPPLAVKK ISQYACQRKT TLNNYNHIFT DAFEILAENS EFKENEVSYV
TFMRAASVLK SLPFTIISMK DTEGIPCLGD KVKCIIEEII EDGESSEVKA VLNDERYQSF
KLFTSVFGVG LKTSEKWFRM GFRSLSKIMS DKTLKFTKMQ KAGFLYYEDL
VSCVTRAEAE AVGVLVKEAV WAFLPDAFVT MTGGFRRGKK IGHDVDFLIT
SPGSAEDEEQ LLPKVINLWE KKGLLLYYDL VESTFEKFKL PSRQVDTLDH
FQKCFLILKL HHQRVDSSKS NQQEGKTWKA IRVDLVMCPY ENRAFALLGW
TGSRQFERDI RRYATHERKM MLDNHALYDK TKRVFLKAES EEEIFAHLGL
DYIEPWERNA
Table 2. Nucleic Acid Sequence of Bovine TdT
SEQ ID NO. 2: (1923 nt) ctcttctgga gataccactt gatggcacag cagaggcagc atcagcgtct tcccatggat ccgctgtgca cagcctcctc aggccctcgg aagaagagac ccaggcaggt gggtgcctca atggcctccc ctcctcatga catcaagttt caaaatttgg tcctcttcat tttggagaag aaaatgggaa ccacccgcag aaacttcctc atggagctgg ctcgaaggaa aggtttcagg gttgaaaatg agctcagtga ttctgtcacc cacattgtag cagaaaacaa ctctggttca gaggttctcg agtggcttca ggtacagaac ataagagcca gctcgcagct agaactcctt gatgtctcct ggctgatcga aagtatggga gcaggaaaac cagtggagat tacaggaaaa caccagcttg ttgtgagaac agactattca gctaccccaa acccaggctt ccagaagact ccaccacttg ctgtaaaaaa gatctcccag tacgcgtgtc aaagaaaaac cactttgaac aactataacc acatattcac ggatgccttt gagatactgg ctgaaaattc tgagtttaaa gaaaatgaag tctcttatgt gacatttatg agagcagctt ctgtacttaa atctctgcca ttcacaatca tcagtatgaa ggatacagaa ggaattccct gcctggggga caaggtgaag tgtatcatag aggaaattat tgaagatgga gaaagttctg aagttaaagc tgtgttaaat gatgaacgat atcagtcctt caaactcttt acttctgttt ttggagtggg actgaagaca tctgagaaat ggttcaggat ggggttcaga tctctgagta aaataatgtc agacaaaacc ctgaaattca caaaaatgca gaaagcagga tttctctatt atgaagacct tgtcagctgc gtgaccaggg ccgaagcaga ggcggttggc gtgctggtta aagaggctgt gtgggcattt ctgccggatg cctttgtcac catgacagga ggattccgca ggggtaagaa gattgggcat gatgtagatt ttttaattac cagcccagga tcagcagagg atgaagagca acttttgcct aaagtgataa acttatggga aaaaaaggga ttacttttat attatgacct tgtggagtca acatttgaaa agttcaagtt gccaagcagg caggtggata ctttagatca ttttcaaaaa tgctttctga ttttaaaatt gcaccatcag agagtagaca gtagcaagtc caaccagcag gaaggaaaga cctggaaggc catccgtgtg gacctggtta tgtgccccta cgagaaccgt gcctttgccc tgctaggctg gactggctcc cggcagtttg agagagacat ccggcgctat gccacacacg agcggaagat gatgctggat aaccacgctt tatatgacaa gaccaagagg gtatttctca aagcggaaag tgaagaagaa atctttgcac atctgggatt ggactacatt gaaccatggg aaagaaatgc ttaggagaaa gctgtcaact tttttctttt ctgttctttt tttcaggtta gacaaattat gcttcatatt ataatgaaag atgccttagt caagtttggg attctttaca ttttaccaag atgtagattg cttctagaaa taagtagttt tggaaacgtg atcaggcacc ccctgggtta tgctctggca agccatttgc aggactgatg tgtagaactc gcaatgcatt ttccatagaa acagtgttgg aattggtggc tcatttccag ggaagttcat caaagcccac tttgcccaca gtgtagctga aatactgtat acttgccaat aaaaatagga aac Additionally, to make isolation of recombinant proteins easier, it is common to append an N-terminal His tag sequence to the recombinant protein (see Boule J-B et al., Molecular Biotechnology, 1998;10:199-208, incorporated by reference herein in its entirety), which is used in combination with an affinity column (Hitrap, Amersham Pharmacia Biotech, Uppsala, Sweden). Alternatively, N-terminal truncated forms of the enzyme with appended His-tag sequence will work with the current invention (see, e.g., US 7,494,797, incorporated by reference herein in its entirety). His-tagged Bovine TdT amino acid sequences are shown below in Tables 3, 5, and 7, while His-tagged Bovine TdT nucleic acid sequences are shown below in Tables 4, 6, and 8. His tags may be engineered at other positions as required. In some embodiments, the resulting protein, adapted for sequence-specific de novo polynucleotide synthesis with 3'-0-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, with SEQ ID NOS. 3, 5, or 7.
Table 3. Amino Acid Sequence of a A138 and His-tagged Bovine TdT.
SEQ ID No. 3: (392 aa) Met Arg Gly Ser His His His His His His Arg Thr Asp Tyr Ser Ala Thr Pro Asn Pro Gly Phe Gln Lys Thr Pro Pro Leu Ala Val Lys Lys Be Ser Gln Tyr Ala Cys Gln Arg Lys Thr Thr Leu Asn Asn Tyr Asn His Be Asp Ala Phe Glu Be Leu Ala Glu Asn Ser Glu Phe Lys Glu Asn Glu Val Ser Tyr Val Thr Phe Met Arg Ala Ala Ser Val Leu Lys Ser Leu Pro Phe Thr Ile Be Ser Met Lys Asp Thr Phe Thr Glu Gly Ile Pro Cys Leu Gly Asp Lys Val Lys Cys Be Be Glu Glu Ile Ile Glu Asp Gly Glu Ser Ser Glu Val Lys Ala Val Leu Asn Asp Glu Arg Tyr Gln Ser Phe Lys Leu Ser Val Phe Gly Val Gly Leu Lys Thr Ser Glu Lys Trp Phe Arg Met Gly Phe Thr Phe Arg Ser Leu Ser Lys Ile Met Ser Asp Lys Thr Leu Lys Lys Met Gln Lys Ala Gly Phe Leu Tyr Tyr Glu Asp Leu Val Ser Cys Val Thr Arg Ala Glu Ala Glu Ala Val Gly Val Leu Val Lys Glu Ala Val Trp Ala Phe Leu Pro Asp Ala Phe Val Thr Met Thr Gly Gly Phe Arg Arg Gly Lys Lys Ile Gly His Asp Val Asp Phe Leu Ile Thr Ser Pro Gly Ser Ala Glu Asp Glu Glu Gln Leu Leu Pro Lys Val Be Asn Leu Trp Glu Lys Lys Gly Leu Leu Leu Tyr Tyr Asp Leu Val Glu Ser Thr Phe Glu Lys Phe Lys Phe Thr Leu Pro Ser Arg Gln Val Asp Thr Leu Asp His Phe Gln Lys Cys Phe Leu Ile Leu Lys Leu His His Gln Arg Val Asp Ser Ser Lys Ser Asn Gln Gln Glu Gly Lys Thr Trp Lys Ala Ile Arg Val Asp Leu Val Met Cys Pro Tyr Glu Asn Arg Ala Phe Ala Leu Leu Gly Trp Thr Gly Ser Arg Gln Phe Glu Arg Asp Ile Arg Arg Tyr Ala Thr His Glu Arg Lys Met Met Leu Asp Asn His Ala Leu Tyr Asp Lys Thr Lys Arg Val Phe Leu Lys Ala Glu Ser Glu Glu Glu Ile Phe Ala His Leu Gly Leu Asp Tyr Be Glu Pro Trp Glu Arg Asn Ala Table 4. Nucleotide Sequence of a A138 and His-tagged Bovine TdT.
SEQ ID No. 4: (1187 nt) atgagaggat cgcatcacca tcaccatcac agaacagact attcagctac cccaaaccca ggcttccaga agactccacc acttgctgta aaaaagatct cccagtacgc gtgtcaaaga aaaaccactt tgaacaacta taaccacata ttcacggatg cctttgagat actggctgaa aattctgagt ttaaagaaaa tgaagtctct tatgtgacat ttatgagagc agcttctgta cttaaatctc tgccattcac aatcatcagt atgaaggata cagaaggaat tccctgcctg ggggacaagg tgaagtgtat catagaggaa attattgaag atggagaaag ttctgaagtt aaagctgtgt taaatgatga acgatatcag tccttcaaac tctttacttc tgtttttgga gtgggactga agacatctga gaaatggttc aggatggggt tcagatctct gagtaaaata atgtcagaca aaaccctgaa attcacaaaa atgcagaaag caggatttct ctattatgaa gaccttgtca gctgcgtgac cagggccgaa gcagaggcgg ttggcgtgct ggttaaagag gctgtgtggg catttctgcc ggatgccttt gtcaccatga caggaggatt ccgcaggggt aagaagattg ggcatgatgt agatttttta attaccagcc caggatcagc agaggatgaa gagcaacttt tgcctaaagt gataaactta tgggaaaaaa agggattact tttatattat gaccttgtgg agtcaacatt tgaaaagttc aagttgccaa gcaggcaggt ggatacttta gatcattttc aaaaatgctt tctgatttta aaattgcacc atcagagagt agacagtagc aagtccaacc agcaggaagg aaagacctgg aaggccatcc gtgtggacct ggttatgtgc ccctacgaga accgtgcctt tgccctgcta ggctggactg gctcccggca gtttgagaga gacatccggc gctatgccac acacgagcgg aagatgatgc tggataacca cgctttatat gacaagacca agagggtatt tctcaaagcg gaaagtgaag aagaaatctt tgcacatctg ggattggact acattgaacc atgggaaaga aatgcttaag cttgcgc Table 5: Amino Acid Sequence of a A151 and His-tagged Bovine TdT.
SEQ ID No. 5: (379 aa) Met Arg Gly Ser His His His His His His Lys Thr Pro Pro Leu Ala Val Lys Lys Be Ser Gln Tyr Ala Cys Gln Arg Lys Thr Thr Leu Asn Asn Tyr Asn His Be Asp Ala Phe Glu Be Leu Ala Glu Asn Ser Glu Phe Lys Glu Asn Glu Val Ser Tyr Val Thr Phe Met Arg Ala Ala Ser Val Leu Lys Ser Leu Pro Phe Thr Be Ile Ser Met Lys Asp Thr Phe Thr Glu Gly Be Pro Cys Leu Gly Asp Lys Val Lys Cys Be Be Glu Glu Be Ile Glu Asp Gly Glu Ser Ser Glu Val Lys Ala Val Leu Asn Asp Glu Arg Tyr Gln Ser Phe Lys Leu Ser Val Phe Gly Val Gly Leu Lys Thr Ser Glu Lys Trp Phe Arg Met Gly Phe Thr Phe Arg Ser Leu Ser Lys Be Met Ser Asp Lys Thr Leu Lys Lys Met Gln Lys Ala Gly Phe Leu Tyr Tyr Glu Asp Leu Val Ser Cys Val Thr Arg Ala Glu Ala Glu Ala Val Gly Val Leu Val Lys Glu Ala Val Trp Ala Phe Leu Pro Asp Ala Phe Val Thr Met Thr Gly Gly Phe Arg Arg Gly Lys Lys Ile Gly His Asp Val Asp Phe Leu Be Thr Ser Pro Gly Ser Ala Glu Asp Glu Glu Gln Leu Leu Pro Lys Val Be Asn Leu Trp Glu Lys Lys Gly Leu Leu Leu Tyr Tyr Asp Leu Val Glu Ser Thr Phe Glu Lys Phe Lys Phe Thr Leu Pro Ser Arg Gln Val Asp Thr Leu Asp His Phe Gln Lys Cys Phe Leu Ile Leu Lys Leu His His Gln Arg Val Asp Ser Ser Lys Ser Asn Gln Gln Glu Gly Lys Thr Trp Lys Ala Be Arg Val Asp Leu Val Met Cys Pro Tyr Glu Asn Arg Ala Phe Ala Leu Leu Gly Trp Thr Gly Ser Arg Gln Phe Glu Arg Asp Ile Arg Arg Tyr Ala Thr His Glu Arg Lys Met Met Leu Asp Asn His Ala Leu Tyr Asp Lys Thr Lys Arg Val Phe Leu Lys Ala Glu Ser Glu Glu Glu Be Phe Ala His Leu Gly Leu Asp Tyr Be Glu Pro Trp Glu Arg Asn Ala Table 6. Nucleotide Sequence of a A151 and His-tagged Bovine TdT.
SEQ ID No. 6: (1148 nt) atgagaggat cgcatcacca tcaccatcac aagactccac cacttgctgt aaaaaagatc tcccagtacg cgtgtcaaag aaaaaccact ttgaacaact ataaccacat attcacggat gcctttgaga tactggctga aaattctgag tttaaagaaa atgaagtctc ttatgtgaca tttatgagag cagcttctgt acttaaatct ctgccattca caatcatcag tatgaaggat acagaaggaa ttccctgcct gggggacaag gtgaagtgta tcatagagga aattattgaa gatggagaaa gttctgaagt taaagctgtg ttaaatgatg aacgatatca gtccttcaaa ctctttactt ctgtttttgg agtgggactg aagacatctg agaaatggtt caggatgggg ttcagatctc tgagtaaaat aatgtcagac aaaaccctga aattcacaaa aatgcagaaa gcaggatttc tctattatga agaccttgtc agctgcgtga ccagggccga agcagaggcg gttggcgtgc tggttaaaga ggctgtgtgg gcatttctgc cggatgcctt tgtcaccatg acaggaggat tccgcagggg taagaagatt gggcatgatg tagatttttt aattaccagc ccaggatcag cagaggatga agagcaactt ttgcctaaag tgataaactt atgggaaaaa aagggattac ttttatatta tgaccttgtg gagtcaacat ttgaaaagtt caagttgcca agcaggcagg tggatacttt agatcatttt caaaaatgct ttctgatttt aaaattgcac catcagagag tagacagtag caagtccaac cagcaggaag gaaagacctg gaaggccatc cgtgtggacc tggttatgtg cccctacgag aaccgtgcct ttgccctgct aggctggact ggctcccggc agtttgagag agacatccgg cgctatgcca cacacgagcg gaagatgatg ctggataacc acgctttata tgacaagacc aagagggtat ttctcaaagc ggaaagtgaa gaagaaatct ttgcacatct gggattggac tacattgaac catgggaaag aaatgcttaa gcttgcgc Table 7. Amino Acid Sequence of a A160 and His-tagged Bovine TdT.
SEQ ID No. 7: (370 aa) Met Arg Gly Ser His His His His His His Be Ser Gln Tyr Ala Cys Gln Arg Lys Thr Thr Leu Asn Asn Tyr Asn His Be Asp Ala Phe Glu Ile Leu Ala Glu Asn Ser Glu Phe Lys Glu Asn Glu Val Ser Tyr Val Thr Phe Met Arg Ala Ala Ser Val Leu Lys Ser Leu Pro Phe Thr Be Be Ser Met Lys Asp Thr Phe Thr Glu Gly Be Pro Cys Leu Gly Asp Lys Val Lys Cys Be Ile Glu Glu Ile Ile Glu Asp Gly Glu Ser Ser Glu Val Lys Ala Val Leu Asn Asp Glu Arg Tyr Gln Ser Phe Lys Leu Ser Val Phe Gly Val Gly Leu Lys Thr Ser Glu Lys Trp Phe Arg Met Gly Phe Thr Phe Arg Ser Leu Ser Lys Ile Met Ser Asp Lys Thr Leu Lys Lys Met Gln Lys Ala Gly Phe Leu Tyr Tyr Glu Asp Leu Val Ser Cys Val Thr Arg Ala Glu Ala Glu Ala Val Gly Val Leu Val Lys Glu Ala Val Trp Ala Phe Leu Pro Asp Ala Phe Val Thr Met Thr Gly Gly Phe Arg Arg Gly Lys Lys Ile Gly His Asp Val Asp Phe Leu Ile Thr Ser Pro Gly Ser Ala Glu Asp Glu Glu Gln Leu Leu Pro Lys Val Be Asn Leu Trp Glu Lys Lys Gly Leu Leu Leu Tyr Tyr Asp Leu Val Glu Ser Thr Phe Glu Lys Phe Lys Phe Thr Leu Pro Ser Arg Gln Val Asp Thr Leu Asp His Phe Gln Lys Cys Phe Leu Be Leu Lys Leu His His Gln Arg Val Asp Ser Ser Lys Ser Asn Gln Gln Glu Gly Lys Thr Trp Lys Ala Ile Arg Val Asp Leu Val Met Cys Pro Tyr Glu Asn Arg Ala Phe Ala Leu Leu Gly Trp Thr Gly Ser Arg Gln Phe Glu Arg Asp Ile Arg Arg Tyr Ala Thr His Glu Arg Lys Met Met Leu Asp Asn His Ala Leu Tyr Asp Lys Thr Lys Arg Val Phe Leu Lys Ala Glu Ser Glu Glu Glu Ile Phe Ala His Leu Gly Leu Asp Tyr Ile Glu Pro Trp Glu Arg Asn Ala Table 8. Nucleotide Sequence of a A160 and His-tagged Bovine TdT.
SEQ ID No. 8: (1121 nt) atgagaggat cgcatcacca tcaccatcac atctcccagt acgcgtgtca aagaaaaacc actttgaaca actataacca catattcacg gatgcctttg agatactggc tgaaaattct gagtttaaag aaaatgaagt ctcttatgtg acatttatga gagcagcttc tgtacttaaa tctctgccat tcacaatcat cagtatgaag gatacagaag gaattccctg cctgggggac aaggtgaagt gtatcataga ggaaattatt gaagatggag aaagttctga agttaaagct gtgttaaatg atgaacgata tcagtccttc aaactcttta cttctgtttt tggagtggga ctgaagacat ctgagaaatg gttcaggatg gggttcagat ctctgagtaa aataatgtca gacaaaaccc tgaaattcac aaaaatgcag aaagcaggat ttctctatta tgaagacctt gtcagctgcg tgaccagggc cgaagcagag gcggttggcg tgctggttaa agaggctgtg tgggcatttc tgccggatgc ctttgtcacc atgacaggag gattccgcag gggtaagaag attgggcatg atgtagattt tttaattacc agcccaggat cagcagagga tgaagagcaa cttttgccta aagtgataaa cttatgggaa aaaaagggat tacttttata ttatgacctt gtggagtcaa catttgaaaa gttcaagttg ccaagcaggc aggtggatac tttagatcat tttcaaaaat gctttctgat tttaaaattg caccatcaga gagtagacag tagcaagtcc aaccagcagg aaggaaagac ctggaaggcc atccgtgtgg acctggttat gtgcccctac gagaaccgtg cctttgccct gctaggctgg actggctccc ggcagtttga gagagacatc cggcgctatg ccacacacga gcggaagatg atgctggata accacgcttt atatgacaag accaagaggg tatttctcaa agcggaaagt gaagaagaaa tctttgcaca tctgggattg gactacattg aaccatggga aagaaatgct taagcttgcg c Table 9. Amino Acid Sequence of murine TdT
SEQ lD NO. 9: (510 aa) MDPLQAVHLG PRKKRPRQLG TPVASTPYDI RFRDLVLFIL EKKMGTTRRA
FLMELARRKG FRVENELSDS VTHIVAENNS GSDVLEWLQL QNIKASSELE
LLDISWLIEC MGAGKPVEMM GRHQLVVNRN SSPSPVPGSQ NVPAPAVKKI
SQYACQRRTT LNNYNQLFTD ALDILAENDE LRENEGSCLA FMRASSVLKS
LPFPITSMKD TEGIPCLGDK VKSIIEGIIE DGESSEAKAV LNDERYKSFK LFTSVFGVGL
KTAEKWFRMG FRTLSKIQSD KSLRFTQMQK AGFLYYEDLV SCVNRPEAEA
VSMLVKEAVV TFLPDALVTM TGGFRRGKMT GHDVDFLITS PEATEDEEQQ
LLHKVTDFWK QQGLLLYCDI LESTFEKFKQ PSRKVDALDH FQKCFLILKL
DHGRVHSEKS GQQEGKGWKA IRVDLVMCPY DRRAFALLGW TGSRQFERDL
RRYATHERKM MLDNHALYDR TKRVFLEAES EEEIFAHLGL DYIEPWERNA
In certain embodiments, modified enzymes of the invention may include an N-terminus truncation relative to their respective native TdT enzyme. For example, in preferred embodiments, the native enzyme may be murine TdT as provided in SEQ ID NO. 9 above. The modified TdT
may be truncated at the equivalent of position 147 or 131 of the native murine TdT as shown in SEQ ID Nos. 10 and 11 respectively. Modified TdTs may include a protein tag sequence such as a His tag and additional linkers at their N-terminus as illustrated in SEQ ID
Nos. 10 and 11. The His-tag portion if underlined in each of the sequences and the linker is provided in bold.
SEQ ID No. 10: Murine del-147 with His-tag and linker MRGSHHHHHHGMAS MT GGQQMGRDLYDD DDKDRWGS ELE KKIS QYACQRRTTLNNY
NQLFTDALDILAENDELRENEGSCLAFMRAS S VLKSLPFPITSMKDTEGIPCLGDKVKSIIEG
IIED GE S SEAKAVLNDERYKSFKLFTS VFGVGLKTAEKWFRMGFRTLS KIQS D KS LRFT QM
QKAGFLYYEDLVSCVNRPEAEAVSMLVKEAVVTFLPDALVTMTGGFRRGKMTGHDVDF
LIT S PEATED EE QQLLHKVTDFWKQQGLLLYC DILES TFEKFKQPSRKVDALDHFQKCFLIL
KLDHGRVHSEKS GQQEGKGWKAIRVDLVMC PYDRRAFALLGWT GS RQFERD LRRYATH
ERKMMLDNHALYDRTKRVFLEAESEEEIFAHLGLDYIEPWERNA
SEQ ID No. 11: Murine del-131 with His-tag and linker MRGSHHHHHHGMAS MT GGQQMGRENLYF Q GS PS PVPGS QNVPAPAVKKIS QYACQRRT
TLNNYNQLFTDALDILAENDELRENEGSCLAFMRAS S VLKS LPFPIT S MKD TEGIPCLGD KV
KS IIEGIIED GE S SEAKAVLNDERYKSFKLFTS VFGVGLKTAEKWFRMGFRTLS KIQS D KS L
RFTQMQKAGFLYYEDLVSCVNRPEAEAVSMLVKEAVVTFLPDALVTMTGGFRRGKMTG
HDVDFLITSPEATEDEEQQLLHKVTDFWKQQGLLLYCDILES TFEKFKQPS RKVDALDHFQ
KCFLILKLDHGRVHS E KS GQQEGKGWKAIRVDLVMCPYDRRAFALLGWTGS RQFERDLR
RYATHERKMMLDNHALYDRTKRVFLEAES EEEIFAHLGLDYIEPWERNA
Additional TdT modifications that may increase incorporation efficiency of 3'-0-blocked or other nucleotide analogs are listed in Table 10 below. While the modifications are described with referenced to the murine TdT listed in SEQ ID NO. 9, such the 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 the His-tags and linkers. In various embodiments, contemplated modifications include deletion of the S420 through E424 amino acids.
Various combinations of amino acid substitutions of the invention are listed in each row 1-175 of Table 10.
Table 10 7 [177K E180K R454A
8 [177K E180K
9 [177K
E180D M192[ L381 K R454T R461Q
16 E180D M192[ L381Q R454K N474A
17 E180D M192[ R454K
24 E180K M192[ L381 K R454T N474K
E180K M192[ L381A W450H R454T R461V
26 E180K M192[ L381 K W450H R4541 R461Q
27 E180K M192[ L381V N474A
28 E180K M192[ L381W R4541 R461V
29 E180K M192[ R454I
E180K M192[ R454T
98 M192[ L381R R454T R461V N474A
99 M192[ L381V R4541 R461V
100 M192[ L381V R4541 R461V N474K
101 M192[ L381V W450 H R454 K
102 M192[ L381V W450 H R454 K R461V N474A
103 M192[ R454A
111 Q455[
A variety of 3'-0-modified dNTPs and NTPs may be used with the disclosed proteins for de novo synthesis. In some embodiments, the preferred removable 3'-0-blocking group is a 3'-0-amino, a 3'-0-ally1 or a 3'-0-azidomethyl. In other embodiments, the removable 3'-0-blocking moiety is selected from the group consisting of 0-phenoxyacetyl; 0-methoxyacetyl; 0-acetyl; 0-(p-toluene )-sulfonate; 0-phosphate; 0-nitrate; 0-[4-methoxy [-tetrahydrothiopyranyl;
0-tetrahydrothiopyranyl; 0-[5-methyl[-tetrahydrofuranyl; 0-[2-methy1,4-methoxy]-tetrahydropyranyl; 045-methyThtetrahydropyranyl; and 0-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, hydroxylamine, borates, nitrates, sugars, phosphoramide, phosphoramidates, phenylsulfenates, sulfates, sulfones and amino acids (see Metzker ML et al. Nuc Acids Res. 1994;22(20):4259-67, U.S.P.N.
5,763,594, 6,232,465, 7,414,116; and 7,279,563, all of which are incorporated by reference in their entireties).
Synthesis of exemplary 3'-0-blocked dNTP analogs FIG. 6 shows four exemplary 3'-0-blocked dNTP analogs, namely 3'-0-azidomethyl-dATP, 3'-0-azidomethyl-dCTP, 3'-0-azidomethyl-dGTP, and 3'-0-azidomethyl-dTTP.
The synthesis of each 3'-0-azidomethyl analog is described below and detailed in FIGS. 7-12. The 3'-0-blocked dNTP analogs can also be purchased from specialty suppliers, such as Azco Biotech, Oceanside, CA. It is to be understood that corresponding 3'-0-blocked ribonucleotides can be formed with similar synthetic methods to enable the creation of custom RNA oligos.
3'-0-azidomethyl-dATP: With reference to FIG. 7, a solution of N6-benzoy1-5'-0-(tert-butyldimethylsily1)-T-deoxyadenosine (3.0 g; 6.38 mmol) [CNH Technologies, Woburn, MA] in DMSO (12 ml), acetic acid (5.5 ml) and acetic anhydride (17.6 ml) was prepared. The mixture was stirred at room temperature for 48 h. Approximately 100 ml of a saturated NaHCO3 solution was added and the aqueous layer was extracted with CH2C12. The combined organic extract was washed with saturated NaHCO3 solution and dried over Na2SO4. The residue was purified by flash column chromatography (hexane/ethyl acetate, 1:1 to 1:4) to recover N6-Benzoy1-3'-0-(methylthiomethyl)-5'-0-(tert-butyldimethylsily1)-2'-deoxyadenosine (shown as compound 1 in FIG. 7) as a white powder (2.4 g; 71% yield). 400 mg of N6-Benzoy1-3'-0-(methylthiomethyl)-5'-0-(tert-butyldimethylsily1)-2'-deoxyadenosine was dissolved in dry CH2C12 (7m1) under nitrogen to create a solution (0.76 mmol). Cyclohexene (400 1), and S02C12(155 1; 1.91 mmol, redistilled) were then added. The reaction mixture was stirred at 0 C for 2 h.
The solvent was then removed under reduced pressure and then under a high-vacuum pump for 10 min. The resulting residue was dissolved in dry DMF (5 ml) and reacted with NaN3 (400 mg; 6.6 mmol) at room temperature for 3 h. The reaction mixture was dispersed in distilled water (50 ml) and extracted with CH2C12. The combined organic layer was dried over Na2SO4 and concentrated under reduced pressure. The residue was dissolved in Me0H (5 ml) and stirred with NH4F (300 mg; 8.1 mmol) at room temperature for 24 h. The solvent was then removed under reduced pressure. The reaction mixture was concentrated under reduced pressure and partitioned between water and CH2C12. The organic layer was separated and dried over Na2SO4. After concentration, the crude product was purified by flash column chromatography (ethyl acetate/methanol) to produce N6-Benzoy1-3'-0-(azidomethyl)-2'-deoxyadenosine (compound 2; FIG. 7) as a white powder (150 mg; 48% yield). N6-B enzoy1-3*-0-(azidomethyl)-T-deoxyadenosine (123 mg; 0.3 mmol) and a proton sponge (75.8 mg; 0.35 mmol) were then dried in a vacuum desiccator over P205 overnight before dissolving in trimethyl phosphate (6000). Next freshly distilled P0C13 (400; 0.35 mmol) was added dropwise at 0 C and the mixture was stirred at 0 C
for 2 h.
Subsequently, a mixture of tributylammonium pyrophosphate (552 mg) and tributylamine (0.55 ml; 2.31 mmol) in anhydrous DMF (2.33 ml) was added at room temperature and stirred for 30 min. Triethyl ammonium bicarbonate solution (TEAB) (0.1 M; pH 8.0; 15 ml) was then added, and the mixture was stirred for 1 hour at room temperature. Subsequently, concentrated NH4OH
(15 ml) was 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 with 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 with reverse-phase HPLC to produce 3'-0-azidomethyl-dATP (FIG. 7, compound 3), a nucleotide analog to be used for later synthesis.
3'-0-azidomethyl-dTTP: Acetic acid (4.8 ml) and acetic anhydride (15.4 ml) were added to a stirred solution of 5'-0-(tertbutyldimethylsilyl)thymidine (2.0 g;
5.6 mmol) [CNH
Technologies, Woburn, MA] in DMSO. The reaction mixture was stirred at room temperature for 48 h. A saturated NaHCO3 solution (100 ml) was added, and the aqueous layer was extracted with ethyl acetate (3x100 m1). The combined organic extract was 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'-0-(Methylthiomethyl)-5'-0-(tert-butyldimethylsilyl)thymidine (FIG. 8; Compound 4) as a white powder (1.75 g; 75%
yield). Approximately 1 gram of 3'-0-(Methylthiomethyl)-5'-0-(tert-butyldimethylsilyl)thymidine was then dissolved in dry CH2C12 (10 ml) under nitrogen. To this mixture cyclohexene (1.33 ml) and S02C12 (2840; 3.5 mmol, redistilled) were added. The resulting mixture was then stirred at 0 C for 1.5 h. The solvent was then removed under reduced pressure and then under high vacuum for 10 min. The residue was dissolved in dry DMF (5 ml) and reacted with NaN3 (926 mg; 15.4 mmol) at room temperature for 3 h. That reaction mixture was next dispersed in distilled water (50 ml) and extracted with CH2C12 (3x50 m1). The combined organic extract was dried over Na2SO4 and concentrated under reduced pressure. The residue was dissolved in Me0H (5 ml) and reacted with NH4F (600 mg; 16.2 mmol) at room temperature for 24 h. The reaction mixture was concentrated under reduced pressure and partitioned between water and CH2C12. The organic layer was then separated and dried over Na2SO4. After concentration, the residue was purified by flash column chromatography (hexane/ethyl acetate) to produce 3'-0-(azidomethyl)thymidine (FIG. 8, Compound 5) as a white powder (550 mg; 71% yield). Next, the 3'-0-(azidomethyl)thymidine and a proton sponge (0.35 mmol) were dried in a vacuum desiccator over P205 overnight before dissolving in trimethyl phosphate (6000). Next, freshly distilled P0C13 (400; 0.35 mmol) was added dropwise at 0 C
and the mixture was stirred at 0 C for 2 h. Subsequently, a mixture of tributylammonium pyrophosphate (552 mg) and tributylamine (0.55 ml; 2.31 mmol) in anhydrous DMF
(2.33 ml) was added at room temperature and stirred for 30 min. Triethyl ammonium bicarbonate solution (TEAB) (0.1 M; pH 8.0; 15 ml) was then added, and the mixture was stirred for 1 hour at room temperature. Subsequently, concentrated NH4OH (15 ml) was 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 with 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 with reverse-phase HPLC to produce 3'-0-azidomethyl-dTTP (FIG. 8, compound 6), a nucleotide analog to be used for later synthesis.
3'-0-azidomethyl-dCTP: Three and a half grams of N4-benzoy1-5'-0-(tert-butyldimethylsily1)-T-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 (21.6m1) were added, and the reaction mixture was stirred at room temperature for 48 h. A saturated NaHCO3 solution (100 ml) was then added and the aqueous layer was extracted with CH2C12(3x100 m1). The combined organic extract was washed with a saturated solution of NaHCO3 and then dried over Na2SO4. After concentration, the crude product was purified by flash column chromatography (ethyl acetate/hexane) to produce N4-Benzoy1-3'-0-(methylthiomethyl)-5'-0-(tert-butyldimethylsily1)-T-deoxycytidine (FIG. 9;
compound 7) as a white powder (2.9 g; 73% yield). In 8 ml of CH2C12 N4-Benzoy1-3'-0-(methylthiomethyl)-51-0-(tert-butyldimethylsily1)-T-deoxycytidine (558 mg; 1.04 mmol) was dissolved and then cyclohexene (5600) and S02C12(220 ill; 2.7 mmol) were added. The reaction mixture was stirred at 0 C for 1 h. The volatiles were then removed with reduced pressure.
The remaining residue was dissolved in dry DMF (5 ml) and reacted with NaN3(400 mg; 6.6mmo1) at room temperature for 2 h. The reaction mixture was dispersed in distilled water (50 ml) and extracted with CH2C12(3x50 m1). The combined organic extract was dried over Na2SO4 and concentrated under reduced pressure. The residue was dissolved in Me0H (5 ml) and reacted with NH4F (600 mg; 16.2 mmol) at room temperature for 24 h. The solvent was removed under reduced pressure.
The resulting residue was suspended in water (50 ml) and extracted with CH2C12(3x50 m1). The combined organic extract was dried over Na2SO4 and concentrated under reduced pressure. The crude product was purified by flash column chromatography (hexane/ethyl acetate) to produce N4-Benzoy1-3'-0-(azidomethyl)-2'-deoxycytidine (FIG. 9, compound 8) as a white powder (200 mg; 50% yield). Next, the N4-Benzoy1-3'-0-(azidomethyl)-2'-deoxycytidine and a proton sponge (0.35 mmol) were dried in a vacuum desiccator over P205 overnight before dissolving in trimethyl phosphate (6000). Then freshly distilled P0C13 (400; 0.35 mmol) was added dropwise at 0 C and the mixture was stirred at 0 C for 2 h. Subsequently, a mixture of tributylammonium pyrophosphate (552 mg) and tributylamine (0.55 ml; 2.31 mmol) in anhydrous DMF (2.33 ml) was added at room temperature and stirred for 30 min.
Triethyl ammonium bicarbonate solution (TEAB) (0.1 M; pH 8.0; 15 ml) was then added, and the mixture was stirred for 1 hour at room temperature. Subsequently, concentrated NH4OH (15 ml) was 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 with 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 with reverse-phase HPLC to produce 3'-0-azidomethyl-dCTP (FIG. 9, compound 9), a nucleotide analog to be used for later synthesis.
3'-0-azidomethyl-dGTP: To a stirred solution of N2-isobutyry1-5'-0-(tert-butyldimethylsily1)-T-deoxyguanosine (5 g; 11.0 mmol) [CNH Technologies, Woburn, MA] in dry DMSO (21 ml), acetic acid (10 ml) and acetic anhydride (32 ml) were added.
The reaction mixture was stirred at room temperature for 48 h. A saturated NaHCO3 solution (100 ml) was added and the aqueous layer was extracted with ethyl acetate (3x100 m1). The combined organic extract was washed with a saturated NaHCO3 solution and dried over Na2SO4.
After concentration, the crude product was purified by flash column chromatography (CH2C12/Me0H) to produce N2-Isobutyry1-3*-0-(methylthiomethyl)-5'-0-(tert-butyldimethylsily1)-T-deoxyguanosine (FIG. 10, compound 10) as a white powder (3.9 g; 69% yield).
One gram of N2-Isobutyry1-3*-0-(methylthiomethyl)-5'-0-(tert-butyldimethylsily1)-2'-deoxyguanosine was subsequently 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 h. The solvent was removed under high vacuum. The crude product was purified by flash column chromatography (ethyl acetate/hexane) to produce N2-Isobutyry1-06-(diphenylcarbamoy1)-3'-0-(methylthiomethyl)-5'-0-(tert-butyldimethylsily1)-T-deoxyguanosine (FIG. 10, compound 11), which appeared as a yellowish powder (1.09 g; 80% yield). N2-Isobutyry1-06-(diphenylcarbamoy1)-3*-0-(methylthiomethyl)-5'-0-(tert-butyldimethylsily1)-T-deoxyguanosine was then dissolved in dry CH2C12 (1.1 mmol) and stirred under nitrogen atmosphere at 0 C for 1.5 h. The solvent was removed under reduced pressure and then under high vacuum for 10 min.
The resulting residue was dissolved in dry DMF (5 ml) and reacted with NaN3 (600 mg; 10 mmol) at room temperature for 3 h. The reaction mixture was then dispersed in distilled water (50 ml) and extracted with CH2C12(3x50 m1). The combined organic extract was dried over Na2SO4 and concentrated under reduced pressure. The resultant residue was dissolved in Me0H
(5 ml) and reacted with NH4F (500 mg; 13.5 mmol) at room temperature for 24 h.
The solvent was removed under reduced pressure. The residue was suspended in water (50 ml) and extracted with CH2C12(3x50 m1). The combined organic extract was dried over Na2SO4 and concentrated under reduced pressure. The crude product was purified by flash column chromatography (hexane/ethyl acetate) to produce N2-Isobutyry1-06-(diphenylcarbamoy1)-3*-0-azidomethyl-T-deoxyguanosine (FIG. 10, compound 12) as a white powder (230 mg; 36% yield).
Finally, the N2-Isobutyry1-06-(diphenylcarbamoy1)-3'-0-azidomethyl-T-deoxyguanosine and a proton sponge (0.35 mmol) were dried in a vacuum desiccator over P205 overnight before dissolving in trimethyl phosphate (6000). Then freshly distilled POC13 (400; 0.35 mmol) was added dropwise at 0 C and the mixture was stirred at 0 C for 2 h. Subsequently, a mixture of tributylammonium pyrophosphate (552 mg) and tributylamine (0.55 ml; 2.31 mmol) in anhydrous DMF (2.33 ml) was added at room temperature and stirred for 30 min.
Triethyl ammonium bicarbonate solution (TEAB) (0.1 M; pH 8.0; 15 ml) was then added, and the mixture was stirred for 1 hour at room temperature. Subsequently, concentrated NH4OH (15 ml) was 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 with 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 with reverse-phase HPLC to produce 3'-0-azidomethyl-dGTP (FIG. 10, compound 13), a nucleotide analog to be used for later synthesis.
As described with respect to FIG. 2, once a 3'-0-blocked dNTP or 3'-0-blocked rNTP is added, it will be necessary to remove the blocking group so that additional dNTPs or rNTPs can be added. In some embodiments, the 3'-0-blocking group can be removed with a palladium catalyst in neutral aqueous solution at elevated temperature hydrochloric acid to pH 2, a reducing agent such as mercaptoethanol, or by the addition of tris-(2-carboxyethyl) phosphine.
See, e.g., U.S.P.N. 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.P.N. 7,279,563, and U.S.P.N. 7,414,116, all of which are incorporated herein by reference in their entireties. In other embodiments, the 3'-substitution group may be removed by UV irradiation (see, e.g., WO 92/10587, incorporated by reference herein in its entirety). Most 3'-0-blocking groups are removed by oxidative, reductive or hydrolytic chemical reactions. In some embodiments, a 3'-0-NO2 group is removed from a oligonucleotide by a 40% w/v solution of ammonium sulfide for <5 minutes at R.T. In some embodiments, a 3'-0-CH2CN group is removed from an oligonucleotide by treatment with 0.5M
KOH at 70 C. In some embodiments, the removal of the 3'-0-blocking group does not include chemical cleavage but uses a cleaving enzyme such as alkaline phosphatase.
In preferred embodiments an enzymatic reaction is used for removal of the 3'-blocking group. Shrimp Alkaline Phosphatase (SAP) may 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.
3'-0-Methoxymethyl-dTTP: 5'-0-Benzoylthymidine (173 mg, 0.5 mmol, 1 equiv) was dissolved in 10 mL of dichloromethane under argon at ambient T. Di-isopropylethylamine (128 mg, 1 mmol, 2 equiv) was added followed by methoxymethyl bromide (124 mg, 1 mmol, 2 equiv). The mixture was stirred at ambient T for 18h. The mixture was diluted with 10 mL
dichloromethane and this was washed successively with 20 mL of 5% aq HC1, and brine. The organic layer was dried with sodium sulfate and evaporated. 5'-0-Benzoy1-3'-0-methoxymethylthymidine (50 mg, 0.13 mmol) was dissolved in 5 mL of concentrated ammonium hydroxide at ambient temperature. The mixture was stirred at ambient T overnight.
The mixture was diluted extracted 3 times with 10 mL portions of dichloromethane. The combined extracts were washed with brine. The organic layer was dried with sodium sulfate and evaporated. 3'-0-Methoxymethylthymidine (23 mg, 0.08 mmol) was co-evaporated with pyridine (1.5 mL x 3) and dried overnight under high vacuum. The nucleoside was dissolved in a mixture of 1.5 mL of trimethylphosphate and 0.6 mL dry pyridine under Ar.
The mixture was cooled in an ice bath. a first aliquot of 10 uL of P0C13 was added dropwise. Five minutes later, a second aliquot of 10 uL was added. The mixture was stirred an additional 30 min. A
solution of the TBA phosphate salt in dry DMF (1.25 mL) was cooled in an ice bath in a vial under Ar. This was added to the rxn mixture dropwise over 10 sec. Immediately the pre-weighed solid proton sponge (21 mg, 1.25 equiv) was added as a solid in one portion. The mixture was stirred for 25 min after this addition and was quenched with 5 mL
of cold TEAB
buffer. The mixture was stirred in the ice bath for 10 min and then transferred to a small RB
flask for FPLC separation. Final separation was accomplished by reverse phase HPLC using a water/acetonitrile gradient containing 0.1 mM formic acid.
3'-0-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 eluant. N6-Formamidino-5'-0-benzoyldeoxy-3'-0-methylthiomethyldeoxycytidine (250 mg, 0.41 mmol) was dissolved in 10 mL of methanol and 10 mL conc 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 afford 170 mg (93%) of the desired nucleoside as a slightly yellow solid. 3'-0-Methylthiomethyl dexoxycytidine (25.0 mg, 0.09 mmol) in a 25 mL vial was co-evaporated with anhydrous pyridine (3 x lmL) and dried over the weekend. Trimethyl phosphate (0.7 mL) was added to dissolve the nucleoside and cooled in an ice bath to 0 C.
Phosphoryl chloride (28 [IL, 0.3 mmol) was added slowly (12 [IL, 5 min later 8 [IL, 30 min later 8 [IL) and the reaction was stirred for 2 h at 0 C. The di(tetrabutylammonium) hydrogen pyrophosphate was dissolved in anhydrous DMF (1 mL), 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 at 0 C for 2 h. To the reaction mixture was added 1 M triethylammonium bicarbonate buffer (TEAB) (2 mL) and the mixture was stirred for 1 h. The mixture was then transferred to round-bottom flask, 50 mL x 3 of miliQ water was added and 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 triphosphate (F48-F52) were evaporated under reduced pressure at 40 C, and the residue was then lyophilized. The triphosphate was dried to afford the desired triphosphate (12 mg, 16.5%).
EXAMPLES
Example 1: Protein Modifications.
Murine (mur) TdT variants originated from 380 aa synthetic gene. This backbone is a truncated version of WT murine TdT and represents a catalytic core of the ET
sequence.
Chemically synthesized TdT constructs were cloned into a pRSET A bacterial expression vector, featuring an N-terminal 6x-histidine tag and enterokinase cleavage site (ThermoFisher Scientific GeneArt Gene Synthesis). Synthetic TdT plasmids were maintained in DH5alpha cells (Biopioneer) plated on LB agar plates containing 100 ug/ml carbenicillin. For expression, the pRSETA-murine TdT plasmids were transformed into BL21 (DE3) pLysS cells (Thermo-Fisher) by incubating plasmids and cells on ice for 20 min., followed by a 30 sec.
heat shock at 42 C, followed by addition of SOC media and incubation with shaking at 37 C for 30-60 min. After addition of SOC media to cells, the entire volume (typically 60 ul) were plated on LB agar plates containing 100 ug/mL carbenicillin plus 34 ug/mL chloramphenicol.
Cells from 10 mL cultures (24-well plates, Corning) were harvested by centrifugation (3000xg, 15 min), then lysed in B-PER lysis buffer (Thermo-Fisher) containing lysozyme, protease inhibitors, and 100 mM NaCl. Pellets were soaked 1 x 60 min. in TBS buffer and supernatants collected for purification. The supernatant was bound onto 50 uL Ni-NTA bead (GE Life Sciences) slurry in 24-well plates for 30 min. The bead slurry was then washed 3 x 50mM
Tris-HC1, pH 8, 500mM NaCl (500 uL), followed by washing 4 x 50mM Tris-HC1, pH 8, 500mM NaCl, 50 mM
Imidazole (200 uL). The protein was then recovered by treating with 50mM Tris-HC1, pH 8, 500mM NaCl, 300 mM Imidazole (50 uL), then 50mM Tris-HC1, pH 8, 500mM NaCl, 300 mM
Imidazole (130 uL), and finally 50mM Tris-HC1, pH 8, 500mM NaCl, 1M Imidazole (50 uL).
Recovered fractions were analyzed by taking 2.5 ul sample and running on 8%
NuPage gel (Thermo-Fisher) , 200 V for 50 min, denaturing conditions. Gel stained with Coomassie Blue. The eluted protein was buffer exchanged using a 7.5 MWCO desalting column (Thermo-Fisher) and sored at -80 C (Storage Buffer = 20mM Tris-HC1, pH 6.8, 50 mM Na0Ac; 0.01%
Triton X-100 and 10% Glycerol).
Activity Screens:
TdT activity screening was performed via a dNTP polymerase extension reaction using different 3'-0-blocked dNTP analogs and a biotinylated oligonucleotide:
5BiosG/TAATAATAATAATAATAATAATAATAATAATAATAATTTTTT (ChemGenes Corporation) SEQ ID NO. 12 Reactions were typically set up in a 96 well plate. Reactions were performed by making a master mix with final concentrations of the following components: 0.2U PPase (Thermo-Fisher), pmol of oligonucleotide, 75 uM dNTP (see below), 1X TdT reaction buffer (5X
from Thermo-Fisher) to a final volume of 10 ul. Reactions were initiated by adding a defined volume (typically 2u1) of TdT variants in different wells and incubating the reaction mix at 37 C for 5 min and 60 min time points. Reactions were terminated by removal of a 10 ul aliquot and adding to 5 ul of 250 mM EDTA.
dNTPs tested:
3'-0-azidomethyl-dTTP see description above 3'-0-azidomethyl-dATP see description above 3'-0-azidomethyl-dGTP see description above 3'-0-MOM-dTTP see description above 3'-0-MTM-dCTP see description above 3'-aminoxy-dTTP Firebird BioMolecular Sciences LLC
3'-aminoxy-dATP Firebird BioMolecular Sciences LLC
3'-aminoxy-dGTP Firebird BioMolecular Sciences LLC
3'-0-methyl-dATP TriLink BioTechnologies LLC
3'-0-methyl-dGTP TriLink BioTechnologies LLC
3'-0-methyl-dCTP TriLink BioTechnologies LLC
Biotinylated oligos in the quenched reaction mix were bound to Streptavidin beads (0.77 um, Spherotech). The beads were then transferred to filter plates (Pall Corporation) and washed several times with water. The oligonucleotides were cleaved from the solid support by incubating the plate with cleavage buffer (10% Diisopropyl-amine in methanol) at 50 C for 30 min followed by elution in water. The eluted samples were dried and dissolved in 300 of water containing oligonucleotide sizing standards (two oligonucleotides (ChemGenes Corporation) that are approximately 15-20 bases smaller or larger than the starting 42-mer oligonucleotide).
Oligonucleotides were then analyzed for extension efficiency by Capillary Gel Electrophoresis (Oligo Pro II, Advanced Analytical Technologies Inc.).
Example 2: In silico modeling Several amino acid modifications to the GGFRR and TGSR motifs and flanking amino acids discussed above were modeled in silico to determine modifications capable of increased incorporation of 3'-0-blocked dNTP analogs as described above. Single, double, and triple amino acid substitutions as well amino acid insertions were modeled. Table 11 below shows modifications found to elicit increased incorporation. Amino acid positions are provided with reference to murine TdT but are applicable to conserved sequences of any TdT.
Rows in Table 11 describe a base modification to one or more amino acids in or flanking the GGFRR motif.
Columns include additional combinations of modifications to other amino acids such as those in and flanking the TGSR motif.
Table 11.
Residue Single Combination Combinati Combination Combination Combination Position Site with E180K on with with E180K with R461V with N474R
Mutation R454T and R454T
T331 T331M, T331M + T331M + T331M + T331M + T331M +
T3315 E180K, R454T, E180K + R461V, N474R, T331A, T3315+ T3315+ R454T, T3315+ T3315+
T331V, E180K, R454T, T3315+ R461V, N474R, T331G, T331A + T331A + E180K + T331A + T331A +
T331I, E180K, R454T, R454T, R461V, N474R, T331N, T331V + T331V + T331A + T331V + T331V +
T331C, E180K, R454T, E180K + R461V, N474R, T331L T331G + T331G + R454T, T331G + T331G +
E180K, R454T, T331V + R461V, N474R, T3311+ T3311+ E180K + T3311+ T3311+
E180K, R454T, R454T, R461V, N474R, T331N + T331N + T331G + T331N + T331N +
E180K, R454T, E180K + R461V, N474R, T331C + T331C + R454T, T331C + T331C +
E180K, R454T, T3311+ R461V, N474R, T331L + T331L + E180K + T331L + T331L +
E180K R454T R454T, R461V N474R
T331N +
E180K +
R454T, T331C +
E180K +
R454T, T331L +
E180K +
G332 G332A G332A + G332A + G332A +
G332A +
G332A + E180K
R454T [180K+ R461V N474R
G333 G333S +
E180K +
R454T, G333S + G333S + G333S +
G333A +
G333S + E180K, R454T, R461V, N474R, E180K +
G333A + G333A + G333A +
G333A +
G333S, R454T, E180K, R454T, R461V, N474R, G333A, G333D +
G333D + G333D + G333D +
G333D +
G333D, E180K +
E180K, R454T, R461V, N474R, G333P, R454T, G333P + G333P + G333P +
G333P +
G333E G333P +
E180K, R454T, R461V, N474R, E180K +
G333E + E180K G333[+ G333[+
G333[+
R454T, G333E +
E180K +
G333 and G333S +
G333S + G333S +
G333S +
G333S + G333S + F334Y F334Y +
F334 F334Y + F334Y +
F334Y +
F334Y +[180K [180K+
F334 F334H +
E180K +
F334H + F334H + F334H +
R454T, R454T, R461V, N464R, F334H, F334H + E180K' F334Y + F334Y +
F334Y +
F334Y +
F334Y, F334Y + E180K' [180K+
R454T, R461V, N474R, F334N F334N + E180K R454T, F334N + F334N + F334N +
F334N +
E180K +
F334 and Y
F334S +
insertion F334S + F334S + F334S +
F334S + F334S +
334 335insY
334 335in 334 335insY + 334 335ins - 334 335insY
334 335insY
_ _ _ _ _ between + E180K +
sY E180K Y + R454T + R461V +
F334 and R335 R335L +
R335L + [180K+ R335L +
R335L +
R454T, R454T, R461V, N474R, R335L + E180K, R335S + R335S R335S +
R335S +
R335 L, R335S +E180K, R454T, +[180K+ R461V, N474R, R3355' R335K + E180K, R335K + R454T, R335K +
R335K +
R335K, R335W + R454T, R335K + R461V, N474R, R335W, E180K, R335W + E180K + R335W +
R335W +
R335T + E180K R454T, R454T, R461V, N474R, R335T + R335W + R335T +
R335T +
R454T [180K+ R461V
R454T, R335T +
E180K +
R336 R336K +
E180K +
R454T, R336K + R336S + R336K +
R336K +
R454T, E180K + R461V, N474R, R336S + R454T, R336S +
R336S +
R336K + E180K' R336K' R454T, R336I + R461V, N474R, R336S + E180K' R336I + E180K + R336I +
R336I +
R336S' R336I +E180K, R336I, R454T, R454T, R461V, N474R, R336N +
R336N, R336N + R336N + R336N + R336N +
E180K, R336V' R454T, [180K+ R461V, N474R, R336V + E180K' R336Q R336V+ R454T, R336V + R336V
+
R336Q + E180K
R454T, R336V + R461V, N474R, R336Q + [180K+ R336Q +
R336Q +
R454T R454T, R461V
R336Q +
E180K +
G337 R336K +
E180K +
R454T, R336S +
G337K + G337K + G337K +
E180K +
R454T, R461V, N474R, R454T, G337K + G337[+ G337[+
G337[+
R336I +
E180K, R454T, R461V, N474R, E180K +
G337K, G337E + E180K, G337A G337A G337A +
R454T, G337E, G337A +[180K, +R454T, +R461V, N474R, R336N +
G337A, G337D + G337D + G337D + G337D +
E180K +
G337D, E180K, R454T, R461V, N474R, R454T, G337H, G337H +E180K, G337H G337H G337H +
R336I +
G337S G337H + +R454T, +R461V, N474R, E180K +
E180K, G337H + G337H + G337H +
R454T, G337S + E180K R454T, R461V, N474R, R336V +
G337S + G337S + G337S +
E180K +
R454T, R336Q +
E180K +
K338 K338R +
K338R + [180K+ K338R +
K338R +
K338R, K338R + E180K, R454T, R454T, R461V, N474R, K338A K338A + E180K K338A + K338A + K338A
+ K338A +
R454T [180K+ R461V
G341 G341C, G341C + G341C + G341C + G341C +
G341C +
G341S, E180K, R454T, [180K+ R461V, N474R, G341V, G341S + E180K, G341S + R454T, G341S +
G341S +
G341I G341V + R454T, G341S + R461V, N464R, E180K, G341V + E180K + G341V +
G341V +
G341I + E180K R454T, R454T, R461V, N474R, G341I + G341V + G341I +
G341I +
R454T E180K + R461V
R454T, G341I +
E180K +
H342 H342G +
E180K +
H342G + R454T, H342G +
H342G +
H342G +
R454T, H342K + R461V, N474R, E180K, H342G, H342K + E180K + H342K + H342K +
H342K +
H342K, R454T, R454T, R461V, N474R
E180K, H342R, H342R + H342R + H342R + H342R +
H342R +
H342D R454T, [180K+ R461V, N474R, E180K, H342D + R454T, H342D +
H342D +
H342D + E180K
R454T H342D + R461V
E180K +
Example 3: Incorporation of dNTPs with phosphate blocking groups DNA and the nucleotides that comprise DNA are highly negatively charged due to the phosphate groups within 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 relative to natural nucleotides due to the additional phosphate group at the 3'- position. The increased negative charge may affect the ability of the TdT to incorporate the modified nucleotides. In certain embodiments, engineered TdT enzymes of the invention may be modified for efficient incorporation of 3'-phosphate-dNTPs by neutralizing the negative charges with positive charges on the modified TdT.
The Average number of Neighboring Atoms Per Sidechain Atom (AvNAPSA) algorithm within the Rosetta protein software suite3 was used to identify mutations that will increase the positive charge in and around the enzymatic active site of TdT. By increasing a key parameter of the AvNAPSA algorithm, termed surface atom cutoff, sequence positions in the active site of TdT
were targeted. The surface charge of proteins 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 content of each of which is incorporate herein by reference. Increasing the surface atom cutoff term allows AvNAPSA to consider sequence positions with a higher number of neighboring atoms, such as positions within an enzyme active site. A summary of positions identified in TdT using AvNAPSA as being potentially useful for more efficient incorporation of 3'-phosphate-dNTP is shown in Table 12.
Table 12: TdT modifications for incorporation of phosphate-blocked dNTPs FIGS. 13-16 illustrate the superior nucleotide incorporation of modified TdT
over the wild type with respect to 3'-PO4-dNTPs . FIG. 13, Panel A is the CGE analysis of a chemically synthesized oligonucleotide (IDT) (21-mer; 5'-FAM-TAATAATAATAATAATTTTTT-PO4-3'), while Panel B shows that the addition of one nucleotide bearing a 3'-PO4 group, causes faster electrophoretic mobility than a comparable 20-mer (IDT) (5'-FAM-TAATAATAATAATAATTTTT). FIG. 14 is the CGE analysis demonstrating that Shrimp Akaline Phosphatase (SAP) (NEB #P0757) quantitatively removes a 3'-PO4 group in 1 minute or less at a concentration of 1.23 x 10-3 U/ul per pmol of oligonucleotide. The figure shows a titration series of increasing amounts of SAP from 0 U/ul (Panel A) to 1.0 x 10-1 U/ul (Panel G).
FIG. 15, Panel B is the CGE analysis of a murine WT TdT reaction mixture that demonstrates no polymerase mediated extension even in the presence of 500 uM 3'-PO4-dTTP (MyChem LLC) as evidenced by no change to the starting material oligonucleotide shown in Panel A. Further evidence of the lack of substrate utilization of 3'-PO4-dTTP is shown in panel C of FIG. 15 as demonstrated by the lack of reactivity of the oligonucleotide starting material (Panel A). FIG. 16 is a CGE analysis of the partial incorporation of a 3'-PO4-dTTP by a variant TdT enzyme (E180K + M192K
+ L381K +
R454K + N474R) as shown in panel B that demonstrates the appearance of a new oligonucleotide species (new peak circled) with a faster electrophoretic mobility as would be expected based on the results shown in FIG. 13. Further evidence of the incorporation of a 3'-PO4 by the variant TdT is demonstrated by the post-extension removal of the 3'-PO4 by treatment with SAP
and the appearance of a new oligonucleotide species (Panel C - new peak circled) with an electrophoretic migration rate slower than the oligonucleotide starting material as would be 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 a variant enzyme (E180K+M192K+R454K+R461V+N474R) Incorporation by Reference References and citations to other documents, such as patents, patent applications, patent publications, journals, books, papers, web contents, have been made throughout this disclosure.
All such documents are hereby incorporated herein by reference in their entirety for all purposes.
Equivalents Various modifications of the 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 full contents of this document, including references to the scientific and patent literature 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 (27)
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, said modified TdT capable of adding a nucleotide analog comprising a removable blocking moiety at a 3'-Oxygen of the analog to a 3'-OH
of a nucleic acid initiator in the absence of a nucleic acid template.
of a nucleic acid initiator in the absence of a nucleic acid template.
2. The modified TdT of claim 1, comprising a mutation E457K.
3. The modified TdT of claim 1, comprising the mutations E180K, M192W, L381R, and W450H.
4. The modified TdT of claim 1, comprising the mutations L381Q and W450H.
5. The modified TdT of claim 1, comprising the mutations E180L, M193E, L381K, R461Q, and N457K.
6. The modified TdT of claim 1, comprising the mutations E180K, L381Q, W450H
and R461V.
and R461V.
7. The modified TdT of claim 1, comprising the mutations L381Q and W450H.
8. The modified TdT of claim 1, comprising the mutations E180L, M192E, L381K, R461Q, and N457K.
9. The modified TdT of claim 1, comprising the mutations E180K, M192E, L381K, R454T, and N47K.
10. The modified TdT of claim 1, comprising the mutations E180K, M192K, L381K, R454T, and N457R.
11. The modified TdT of claim 1, comprising the mutations E180K, M192K, L381K, R454K, and N457K.
12. The modified TdT of claim 1, comprising the mutations M192E, L381V, R4541, and R461V.
13. The modified TdT of claim 1, comprising the mutations E180K and L381R.
14. The modified TdT of claim 1, comprising the mutations E180K, M192K, L381K, R454K, and N474R.
15. The modified TdT of claim 1, wherein the modified TdT is capable of adding the nucleotide analog comprising the removable 3'-0-blocking moiety to the 3'-OH of the nucleic acid initiator at an increased rate compared to native TdT.
16. The modified TdT of claim 1, comprising an N-terminus truncation relative to native TdT
17. The modified TdT of claim 15, wherein the modified TdT comprises an N-terminus t-131 murine TdT and a protein tag sequence attached to the N-terminus.
18. The modified TdT of claim 15, wherein the modified TdT comprises an N-terminus t-147 murine TdT and a protein tag sequence attached to the N-terminus.
19. The modified TdT of claim 1, wherein the modified TdT is capable of adding adenine, cytosine, guanine, and thymine deoxyribonucleotides modified with a removable 3'-O-blocking moiety.
20. The modified TdT of claim 18, wherein said nucleotides are 2'-deoxyribonucleotides.
21. 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'-0-blocking moiety.
22. The modified TdT of claim 1, wherein said removable 3'-0-blocking moiety comprises a 3'-0-blocking group selected from the group consisting of CH2N3, NH2, ONHC(0)H, allyl, CH2SSCH3, phenoxyacetyl, methoxyacetyl, acetyl, (p-toluene)sulfonate, phosphate, nitrate, [4-methoxy] -tetrahydrothiopyranyl, tetrahydrothiopyranyl, [5-methyThtetrahydrofuranyl, [2-methy1,4-methoxy] -tetrahydropyranyl, [5-methyThtetrahydropyranyl; and 0-tetrahydrothiofuranyl.
23. The modified TdT of claim 1, wherein the modified TdT is capable of incorporating a 3'-0-blocked nucleotide 5'-triphosphate, and said removable blocking moiety comprises a group selected from esters, ethers, carbonitriles, phosphates, carbonates, carbamates, hydroxylamine, borates, nitrates, sugars, phosphoramide, phosphoramidates, phenylsulfenates, sulfates, sulfones and amino acids.
24. The modified TdT of claim 1, wherein the modified TdT is capable of incorporating modified nucleotides at a reaction temperature from about 30 C to about 80 C.
25. The modified TdT of claim 1, wherein the modified TdT is capable of incorporating modified nucleotides at a concentration of 1000 p.A4 or less.
26. The modified TdT of claim 1, wherein the modified TdT is capable of incorporating modified nucleotides at a concentration of 100 p.A4 or less.
27. The modified TdT of claim 14, said modified TdT capable of adding a nucleotide analog comprising a removable 3'-0-phosphate to the 3'-OH of a nucleic acid initiator.
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US16/891,449 US11390858B2 (en) | 2014-10-20 | 2020-06-03 | Modified template-independent enzymes for polydeoxynucleotide synthesis |
US16/891,449 | 2020-06-03 | ||
PCT/US2021/035685 WO2021247851A2 (en) | 2020-06-03 | 2021-06-03 | Modified template-independent enzymes for polydeoxynucleotide synthesis |
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US10059929B2 (en) * | 2014-10-20 | 2018-08-28 | Molecular Assemblies, Inc. | Modified template-independent enzymes for polydeoxynucleotide synthesis |
GB201502152D0 (en) * | 2015-02-10 | 2015-03-25 | Nuclera Nucleics Ltd | Novel use |
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