WO2021247851A2 - Modified template-independent enzymes for polydeoxynucleotide synthesis - Google Patents
Modified template-independent enzymes for polydeoxynucleotide synthesis Download PDFInfo
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- WO2021247851A2 WO2021247851A2 PCT/US2021/035685 US2021035685W WO2021247851A2 WO 2021247851 A2 WO2021247851 A2 WO 2021247851A2 US 2021035685 W US2021035685 W US 2021035685W WO 2021247851 A2 WO2021247851 A2 WO 2021247851A2
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- 239000000126 substance Substances 0.000 description 1
- YBBRCQOCSYXUOC-UHFFFAOYSA-N sulfuryl dichloride Chemical compound ClS(Cl)(=O)=O YBBRCQOCSYXUOC-UHFFFAOYSA-N 0.000 description 1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
- C12N9/1241—Nucleotidyltransferases (2.7.7)
- C12N9/1264—DNA nucleotidylexotransferase (2.7.7.31), i.e. terminal nucleotidyl transferase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
- C12P19/28—N-glycosides
- C12P19/30—Nucleotides
- C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
- C12Y207/07—Nucleotidyltransferases (2.7.7)
- C12Y207/07031—DNA nucleotidylexotransferase (2.7.7.31), i.e. terminal deoxynucleotidyl transferase
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1034—Isolating an individual clone by screening libraries
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
Definitions
- the invention relates to modified enzymes for de novo synthesis of polynucleotides with a desired sequence, and without the use of a template.
- the invention provides the capability to make libraries of polynucleotides of varying sequence and varying length for research, genetic engineering, and gene therapy.
- 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.
- 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.
- TdT terminal deoxynucleotidyl transferase
- 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.
- 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.
- SAP Shrimp Alkaline Phosphatase
- 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.
- 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- chip fluorescence detection of DNA hybridization by surface-initiated enzymatic polymerization,” Anal. Chem., 2011; 83:5153-5159 (2011).
- TdT terminal deoxynucleotidyl transferase
- dATP deoxyadenosine triphosphate
- fluorescent strand initiator 5’-Cy5-dA10 fluorescent strand initiator 5’-Cy5-dA10
- TdT modified terminal deoxynucleotidyl transferase
- 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 +
- 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 triphosphate (3'-0-M0M-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- T A AT A AT A AT A AT A AT A ATTTTTT-P04-3 ’ .
- FIG. 14 shows CGE traces showing the removal of 3’-P04 by treatment with Vietnamese pot Alkaline Phosphatase.
- FIG. 15 shows CGE traces comparing: A) 5 ’ -FAM-T A AT A AT A AT A ATTTTT after treatment with murine WT TdT and no dNTP; B) 5’-FAM-
- FIG. 16 shows CGE traces comparing A) 5 ’ -FAM-TAAT AAT AATAAT AATTTTT -3 ’ after treatment with murine WT TdT and no dNTP for 60 minutes at 37oC; B) 5’-FAM- T AAT AAT AAT AATTTTT after treatment with murine TdT E180K + M192K + L381K + R454K + N474R and 500 uM 3’-P04-dTTP for 60 minutes at 37oC; C) 5’-FAM- TAATAATAATAATAATTTTT-3’ after treatment with murine WT TdT and 500 uM 3’-P04- dTTP for 60 minutes at 37oC, followed by treatment with 0.2 units of Micromp Alkaline Phosphatase for 15 minutes at 37oC; D) Homopolymer dT extension ladder created by treatment of 5 ’-FAM-T AAT AATAAT AAT AATTTTT-3’ with murine WT
- the invention facilitates the synthesis of polynucleotides, such as DNA, by providing modified enzymes that can be used with nucleic acid analogs.
- modified enzymes that can be used with nucleic acid analogs.
- TdT template-independent terminal deoxynucleotidyl transferase
- 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 ’-O-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.
- 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.
- 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.
- TdT terminal deoxynucleotidyl transferases
- 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.
- 3'-0-blocked nucleotides being incorporated into single- stranded oligonucleotide by an enzyme in the absence of a template.
- substitution of the 3'-hydroxyl group results in complete inactivity of available transferase enzymes.
- TdT terminal deoxynucleotidyl transferase
- dATP deoxyadenosine triphosphate
- fluorescent strand initiator 5’-Cy5-dA10 fluorescent strand initiator 5’-Cy5-dA10 at different time points.
- 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 Delame et ah, EMBQ 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.
- TdT can use substrates having modifications and/or substitutions at the deoxyribose sugar ring as well as the purine/pyrimidine nucleobases.
- 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.
- 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.
- TdT Native TdT is a very efficient enzyme. It has been demonstrated that TdT can polymerize extremely long homopoly deoxynucleotides of 1000 to 10,000 nucleotides in length (see Hoard et al., J of Biol Chem, 1969244(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.
- TdT 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.
- Figure 3 shows a time course of a solution phase synthesis of 1-1.5 kb homopolymers.
- 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.
- FIG. 3 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 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'-
- 3'-0-blocked dNTP analogs include, but are not limited to, the 3'-0-allyl, 3'-0-azidomethyl, 3'-0-NH 2 , 3'-0-CH 2 N 3, 3'-0- 0NHC(0)H, 3'-0-CH 2 SSCH 3, and 3'-0-CH 2 CN blocking groups.
- 3'- O-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.
- FIG. 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 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.
- Arg336 is near the reaction center in the active site, Arg 336 is highly conserved, and early studies found 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.
- Gly452 and Ser453 exist in a cis-peptide bond conformation (see Delarue et al., EMBQ 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.
- the instability created by modifying position 336 may be overcome by using proline residues to stabilize cis-peptide bond conformation.
- the entire TGSR motif (positions 451, 452, 435, 454) may also have to be modified to compensate for this change.
- 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.
- 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.
- substitutions at Arg454 to accommodate the steric bulk of a 3'- O-blocking group may require additional modifications to the al4 region to compensate for substitutions of glycine or alanine at Arg454.
- 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.
- residues such as Gly332, Gly333, Gly452, Thr451, Trp450, Ser453, and Q455 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, Lysl99, Serl96, Metl92, Glul80 or Leul61 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.
- insertion of residues into the modified TdT 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 Glul80 including E180F, E180R, E180D, or E180K.
- Contemplated modifications to Met 192 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.
- combinations of two or more modified residues may be used such as, for example, E180D+W450H, E180K+R454A, M192K+E180K, E180K+R454I
- TdTs include the GGFRR and TGSR motifs.
- 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.
- 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.
- 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.
- 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).
- affinity column Hitrap, Amersham Pharmacia Biotech, Uppsala, Sweden.
- 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).
- 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.
- Table 5 Amino Acid Sequence of a A151 and His-tagged Bovine TdT.
- modified enzymes of the invention may include an N-terminus truncation relative to their respective native TdT enzyme.
- 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
- SEQ ID No. 11 Murine del- 131 with His-tag and linker
- 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.
- 3'-0-modified dNTPs and NTPs may be used with the disclosed proteins for de novo synthesis.
- the preferred removable 3'-0-blocking group is a 3'- O-amino, a 3'-0-allyl or a 3'-0-azidomethyl.
- the removable 3'-0- blocking moiety is selected from the group consisting of O-phenoxyacetyl; O-methoxyacetyl; O- acetyl; 0-(p-toluene )-sulfonate; O-phosphate; O-nitrate; 0-[4-methoxy ]-tetrahydrothiopyranyl; O-tetrahydrothiopyranyl; 0-[5-methyl]-tetrahydrofuranyl; O- [2-methyl, 4-methoxy]- tetrahydropyranyl; 0-[5-methyl]-tetrahydropyranyl; and O-tetrahydrothiofuranyl (see US 8,133,669).
- 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).
- 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 N ⁇ -benzoyl-5'-0-(tert- butyldimethylsilyl)-2'-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 NaHC03 solution was added and the aqueous layer was extracted with CH2C12. The combined organic extract was washed with saturated NaHC03 solution and dried over Na2S04.
- N 6 -Benzoyl-3'-0- (methylthiomethyl)-5'-0-(tert-butyldimethylsilyl)-2'-deoxyadenosine shown as compound 1 in FIG. 7
- 400 mg of N 6 -Benzoyl-3'-0-(methylthiomethyl)- 5'-0-(tert-butyldimethylsilyl)-2'-deoxyadenosine was dissolved in dry CH2CI2 (7ml) under nitrogen to create a solution (0.76 mmol).
- N 6 -Benzoyl-3'-0-(azidomethyl)-2'-deoxyadenosine (123 mg; 0.3 mmol) and a proton sponge (75.8 mg; 0.35 mmol) were then dried in a vacuum desiccator over P2O5 overnight before dissolving in trimethyl phosphate (600m1).
- Next freshly distilled POCI3 (40m1; 0.35 mmol) was added dropwise at 0°C and the mixture was stirred at 0°C for 2 h.
- 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 NaHCCF solution (100 ml) was added, and the aqueous layer was extracted with ethyl acetate (3x100 ml). The combined organic extract was washed with a saturated solution of NaHCCF and dried over NaiSCF.
- 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 N 4 -benzoyl-5'-0-(tert- butyldimethylsilyl)-2'-deoxycytidine [CNH Technologies, Woburn, MA] was added to 14.7 ml of DMSO to produce a 7.65 mmol solution. To this solution, acetic acid (6.7 ml) and acetic anhydride (21.6ml) were added, and the reaction mixture was stirred at room temperature for 48 h. A saturated NaHCCE solution (100 ml) was then added and the aqueous layer was extracted with CH 2 Cl 2 (3xl00 ml).
- the reaction mixture was dispersed in distilled water (50 ml) and extracted with CH 2 Cl 2 (3x50 ml). The combined organic extract was dried over Na 2 S0 4 and concentrated under reduced pressure. The residue was dissolved in MeOH (5 ml) and reacted with NH 4 F (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 CH 2 Cl 2 (3x50 ml). The combined organic extract was dried over Na 2 S0 4 and concentrated under reduced pressure. The crude product was purified by flash column chromatography (hexane/ethyl acetate) to produce
- N 4 -Benzoyl-3'-0-(azidomethyl)-2'-deoxycytidine (FIG. 9, compound 8) as a white powder (200 mg; 50% yield).
- N 4 -Benzoyl-3'-0-(azidomethyl)-2'-deoxycytidine and a proton sponge (0.35 mmol) were dried in a vacuum desiccator over P2O5 overnight before dissolving in trimethyl phosphate (600m1).
- freshly distilled POCI3 40m1; 0.35 mmol was added dropwise at 0°C and the mixture was stirred at 0°C for 2 h.
- 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.
- N - Isobutyryl-3'-0-(methylthiomethyl)-5'-0-(tert-butyldimethylsilyl)-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).
- SIGMA N,N-diisopropylethylamine
- N 2 -Isobutyryl-0 6 - (diphenylcarbamoyl)-3'-0-(methylthiomethyl)-5'-0-(tert-butyldimethylsilyl)-2'-deoxyguanosine was then dissolved in dry CH2CI2 (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 CH 2 Cl 2 (3x50 ml). The combined organic extract was dried over
- 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.
- 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. Qrg. Chem..
- 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.
- a 3'-0-N02 group is removed from a oligonucleotide by a 40% w/v solution of ammonium sulfide for ⁇ 5 minutes at R.T.
- a 3'-0-CH2CN group is removed from an oligonucleotide by treatment with 0.5M KOH at 70°C.
- the removal of the 3'-0-blocking group does not include chemical cleavage but uses a cleaving enzyme such as alkaline phosphatase.
- an enzymatic reaction is used for removal of the 3'-blocking group.
- dvsAP Shrimp Alkaline Phosphatase
- SAP has one of the fastest enzymatic rates reported in the literature and has a wide range of substrate utilization.
- 5’-0-Benzoyl-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 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.
- TEAB triethylammonium bicarbonate buffer
- 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%).
- 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.
- 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.
- the bead slurry was then washed 3 x 50mM Tris-HCl, pH 8, 500mM NaCl (500 uL), followed by washing 4 x 50mM Tris-HCl, pH 8, 500mM NaCl, 50 mM Imidazole (200 uL).
- the protein was then recovered by treating with 50mM Tris-HCl, pH 8, 500mM NaCl, 300 mM Imidazole (50 uL), then 50mM Tris-HCl, pH 8, 500mM NaCl, 300 mM Imidazole (130 uL), and finally 50mM Tris-HCl, pH 8, 500mM NaCl, 1M Imidazole (50 uL).
- TdT activity screening was performed via a dNTP polymerase extension reaction using different 3’-0-blocked dNTP analogs and a biotinylated oligonucleotide:
- 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),
- 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 30pl 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
- 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’-P04-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.
- 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.
- AvNAPSA Neighboring Atoms Per Sidechain Atom
- 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.
- FIGS. 13-16 illustrate the superior nucleotide incorporation of modified TdT over the wild type with respect to 3’-P04-dNTPs .
- FIG. 13 Panel A is the CGE analysis of a chemically synthesized oligonucleotide (IDT) (21-mer; 5 ’ -FAM-TAAT AAT AATAAT AATTTTTT-P0 4 -3 ’ ), while Panel B shows that the addition of one nucleotide bearing a 3’-P0 4 group, causes faster electrophoretic mobility than a comparable 20-mer (IDT) (5’-FAM-
- IDTT chemically synthesized oligonucleotide
- FIG. 14 is the CGE analysis demonstrating that Shrimp Akaline Phosphatase (SAP) (NEB #P0757) quantitatively removes a 3’-P0 4 group in 1 minute or less at a concentration of 1.23 x 10 3 U/ul per pmol of oligonucleotide.
- SAP Shrimp Akaline Phosphatase
- 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’-P0 4 -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’-P0 4 -dTTP is shown in panel C of FIG. 15 as demonstrated by the lack of reactivity of the oligonucleotide starting material (Panel A).
- 16 is a CGE analysis of the partial incorporation of a 3’-P0 4 -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.
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