Enzymes and methods

Abstract

Described is a terminal deoxynucleotidyl transferase (TdT) comprising an amino acid sequence that has at least 75% sequence identity to SEQ ID NO: 3. The TdT may comprise mutations to increase thermos

Description

FIELD OF THE INVENTION The present invention relates to engineered template-independent polymerases, such as terminal deoxynucleotidyl transferases (TdTs). The invention also relates to methods of producing oligonucleotides using enzymes, in particular TdTs. BACKGROUND TO THE INVENTION There is an increasing demand for artificial synthesis of polynucleotides. Using readily available techniques of molecular biology, it is possible to replicate and amplify polynucleotides from natural sources. Such techniques additionally enable engineering of natural polynucleotide sequences, for example through substitution, insertion or deletion of one or more nucleotides, and hence provide access to polynucleotides that are not naturally available. However, such approaches are often time-consuming and labour-intensive. In addition, a reliance on natural sequences as a starting point may limit the scope of sequences that are practically achievable. Furthermore, difficulty in accessing natural polynucleotides themselves may give rise to additional obstacles. De novo synthesis of polynucleotides offers a route to synthesise theoretically any nucleic acid sequence and may therefore overcome some of the issues associated with traditional molecular biology-based approaches. In vitro synthesis of relatively short oligonucleotides, for example via solid-phase synthesis using the phosphoramidite method, is well known. Indeed, traditional molecular biology often relies on synthesised oligonucleotide primers for use in polymerase chain reaction (PCR) and site-directed mutagenesis methods. Polynucleotides may be synthesised by connecting a number of separately synthesised oligonucleotides. Typically, under this approach a group of oligonucleotides may be synthesised, for example using automated solid-phase synthesis, purified, and then the individual oligonucleotides may be subsequently connected together by ligation of the short synthesised oligonucleotides into longer polynucleotides. Alternatively, short oligonucleotides are synthesised with some complementary sequences, hybridised and ligated to make longer partially or fully double-stranded polynucleotides. However, oligonucleotide synthesis via traditional synthetic chemistry-based approaches may be associated with issues, such as inaccuracy and low yield. Inaccuracies in oligonucleotide sequences may arise, as random errors accumulate with the length of the oligonucleotides 1 being synthesised; errors may be introduced into the synthesised oligonucleotides due to unintended side reactions, or incomplete or missed reactions. For example, a coupling failure may result when a growing oligonucleotide chain does not react with the next building block but retains a reactive 5’-OH group, which may then participate in the next coupling round, resulting in an oligonucleotide with a missing nucleotide (a deletion error). Typical oligonucleotide synthesis techniques do not give rise to 100% yields for each coupling step. Even with a yield of 99.5% per coupling round, the yields multiply over the length of a nucleic acid sequence and may lead to significant difficulties in the provision of longer polynucleotides, such as full length genes and genomes, resulting in very low overall yields, decreased purity, and a waste of starting materials and intermediates. In addition, harsh solvent conditions and moisture sensitive reagents may be required, which may increase environmental impact, lead to unintended modifications to products and further inefficiencies. Such issues have prompted an interest in using template-independent polymerases, such as terminal deoxynucleotidyl transferases (TdTs) in methods of polynucleotide synthesis. These enzymes can extend nucleic acid initiators or growing oligonucleotide chains without the requirement of a template nucleic acid strand, and are active in mild aqueous conditions. The enzymes may be engineered to tolerate protecting groups added to nucleotide building blocks that prevent uncontrolled multiple extensions. Highly parallel synthesis of many oligonucleotides can be achieved by spatial localisation of reaction conditions to selectively remove protecting groups. The use of heat is advantageous, as protecting groups can be engineered to be highly thermally labile, which increases the accuracy of synthesis by comparison to other approaches. There remains a significant need for methods of accurately and efficiently producing oligonucleotides. In particular, there is a need for engineered template-independent polymerases that may exhibit improved properties, such as thermostability, soluble expression and enzymatic activity, and that may tolerate suitable protected substrates. SUMMARY OF THE INVENTION The inventors have provided engineered terminal deoxynucleotidyl transferases (TdTs) with improved thermostability, solubility and activity, and that are capable of adding protected nucleotides comprising a 3’-O-protecting group to a 3’-OH of a nucleic acid initiator or a growing oligonucleotide chain in the absence of a nucleic acid template (TdTs may use nucleoside triphosphates as substrates). The engineered TdTs may be used in methods of producing oligonucleotides. In addition, the inventors have developed a method of producing oligonucleotides using template-independent polymerases, such as TdTs, wherein protected nucleotide substrates comprise a first 3’ protecting group that may be small in size, and which may for example be stable under typical oligonucleotide synthesis conditions. Particularly, the first 3’ protecting group may be activated once the nucleotide has been added to a nucleic acid initiator (e.g. a growing oligonucleotide) to form a second protecting group that may be more readily removable, for example may be thermo-labile. This enables smaller protecting groups to be used during the step of enzyme-catalysed addition, which results in a reduced need for engineering of the template-independent polymerase in order for the active site to accommodate the nucleotide that comprises the first 3’ protecting group. In one aspect, the invention provides a terminal deoxynucleotidyl transferase (TdT) comprising an amino acid sequence that has at least 75% sequence identity to SEQ ID NO: 3. Preferably, the TdT is capable of adding a protected nucleotide comprising a 3’-O-protecting group to a 3’-OH of a nucleic acid initiator in the absence of a nucleic acid template. In preferred embodiments, the nucleotide is a deoxyribonucleotide. Suitably, the TdT comprises or consists of an amino acid sequence that has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 3. Particularly, the TdT comprises the amino acid sequence SEQ ID NO: 3. In some embodiments, the TdT consists of the amino acid sequence SEQ ID NO: 3. In some embodiments, the TdT comprises one or more mutation with respect to SEQ ID NO 3 at an amino acid position selected from the group consisting of 315, 52 and 312, wherein the amino acids are numbered with reference to SEQ ID NO: 3. In some embodiments, the TdT comprises one or more mutation with respect to SEQ ID NO 3 at an amino acid position selected from the group consisting of 315 and 52, wherein the amino acids are numbered with reference to SEQ ID NO: 3. In some embodiments, the TdT comprises a mutation with respect to SEQ ID NO 3 at amino acid position 315, wherein the amino acids are numbered with reference to SEQ ID NO: 3. In some embodiments, the TdT comprises a mutation with respect to SEQ ID NO 3 at amino acid position 52, wherein the amino acids are numbered with reference to SEQ ID NO: 3. In some embodiments, the TdT comprises a mutation with respect to SEQ ID NO 3 at amino acid position 312, wherein the amino acids are numbered with reference to SEQ ID NO: 3. In some embodiments, the TdT comprises one or more mutation with respect to SEQ ID NO 3, wherein the one or more mutation is selected from the group consisting of E315K, R52Q, E315R, E315Q, E315M, E315G, E315F, R312T, R312L, R312I, R312A and R312E, wherein the amino acids are numbered with reference to SEQ ID NO: 3. In some embodiments, the TdT comprises one or more mutation with respect to SEQ ID NO 3, wherein the one or more mutation is selected from the group consisting of E315K and E315G, wherein the amino acids are numbered with reference to SEQ ID NO: 3. In some embodiments, the TdT comprises the mutation E315G, wherein the amino acids are numbered with reference to SEQ ID NO: 3. In preferred embodiments, the TdT comprises the mutation E315K, wherein the amino acids are numbered with reference to SEQ ID NO: 3. In some embodiments, the TdT comprises one or more mutation with respect to SEQ ID NO 3, wherein the one or more mutation is selected from the group consisting of R312T, R312L, and R312I, wherein the amino acids are numbered with reference to SEQ ID NO: 3. In some embodiments, the TdT comprises the mutation R312I, wherein the amino acids are numbered with reference to SEQ ID NO: 3. In some embodiments, the TdT comprises the mutation R52Q, wherein the amino acids are numbered with reference to SEQ ID NO: 3. In preferred embodiments, the TdT comprises the mutations R52Q and E315K, wherein the amino acids are numbered with reference to SEQ ID NO: 3. In some embodiments, the TdT exhibits an improved property as compared to SEQ ID NO: 2. In some embodiments, the TdT exhibits an improved property as compared to SEQ ID NO: 3. In some embodiments, the improved property is improved thermostability and / or improved enzymatic activity. In some embodiments, the improved property is improved thermostability. In some embodiments, the improved property is improved enzymatic activity. In some embodiments, the improved property is improved soluble expression. In some embodiments, the TdT exhibits an increase in melting temperature (Tm) of at least 5°C, 10°C, 15°C, 20°C or25°C compared to SEQ ID NO: 2. In some embodiments, the TdT exhibits an increase in melting temperature (Tm) of at least 20°C compared to SEQ ID NO: 2. In some embodiments, the TdT exhibits an increase in enzymatic activity of at least 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold or 15-fold compared to SEQ ID NO: 2. In some embodiments, the TdT exhibits an increase in enzymatic activity of at least 1.1-fold compared to SEQ ID NO: 2. In some embodiments, the TdT exhibits an increase in enzymatic activity of at least 1.5-fold compared to SEQ ID NO: 2. In some embodiments, the TdT exhibits an increase in soluble expression of at least 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold or 5-fold compared to SEQ ID NO: 2. In some embodiments, the TdT exhibits an increase in melting temperature (Tm) of at least 1CC, 2°C, 3°C, 4°C, 5°C, 10°C, 15°C, 20°C or 25°C compared to SEQ ID NO: 3. In some embodiments, the TdT exhibits an increase in enzymatic activity of at least 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold or 10-fold compared to SEQ ID NO: 3. In some embodiments, the TdT exhibits an increase in enzymatic activity of at least 1.1-fold compared to SEQ ID NO: 3. In some embodiments, the TdT exhibits an increase in enzymatic activity of at least 1.5-fold compared to SEQ ID NO: 3. In some embodiments, the TdT comprises or consists of an amino acid sequence that has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 4 preferably, the TdT comprises a lysine at amino acid position 315, wherein the amino acids are numbered with reference to SEQ ID NO: 3. In some embodiments, the TdT comprises or consists of an amino acid sequence that has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 5. Preferably, the TdT comprises a glutamine at amino acid position 52 and a lysine at amino acid position 315, wherein the amino acids are numbered with reference to SEQ ID NO: 3. In some embodiments, the TdT comprises the amino acid sequence SEQ ID NO: 4. In some embodiments, the TdT consists of the amino acid sequence SEQ ID NO: 4. In preferred embodiments, the TdT comprises the amino acid sequence SEQ ID NO: 5. In preferred embodiments, the TdT consists of the amino acid sequence SEQ ID NO: 5. In some embodiments, the TdT comprises the amino acid sequence SEQ ID NO: 6. In some embodiments, the TdT consists of the amino acid sequence SEQ ID NO: 6. In another aspect, the invention provides a polynucleotide comprising a nucleic acid sequence encoding the terminal deoxynucleotidyl transferase (TdT) of the invention. In some embodiments, the polynucleotide comprises a nucleic acid sequence that has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 7. In some embodiments, the polynucleotide comprises the nucleic acid sequence SEQ ID NO: 7. In some embodiments, the polynucleotide consists of the nucleic acid sequence SEQ ID NO: 7. In some embodiments, the polynucleotide comprises a nucleic acid sequence that has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 8. Preferably, the polynucleotide encodes a TdT comprising a lysine at amino acid position 315, wherein the amino acids are numbered with reference to SEQ ID NO: 3. In some embodiments, the polynucleotide comprises the nucleic acid sequence SEQ ID NO: 8. In some embodiments, the polynucleotide consists of the nucleic acid sequence SEQ ID NO: 8. In some embodiments, the polynucleotide comprises a nucleic acid sequence that has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 9. Preferably, the polynucleotide encodes a TdT comprising a glutamine at amino acid position 52 and a lysine at amino acid position 315, wherein the amino acids are numbered with reference to SEQ ID NO: 3. In some embodiments, the polynucleotide comprises the nucleic acid sequence SEQ ID NO: 9. In some embodiments, the polynucleotide consists of the nucleic acid sequence SEQ ID NO: 9. In some embodiments, the polynucleotide comprises a nucleic acid sequence that has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 10. Preferably, the polynucleotide encodes a TdT comprising an isoleucine at amino acid position 312, wherein the amino acids are numbered with reference to SEQ ID NO: 3. In some embodiments, the polynucleotide comprises the nucleic acid sequence SEQ ID NO: 10. In some embodiments, the polynucleotide consists of the nucleic acid sequence SEQ ID NO: 10 In another aspect, the invention provides a vector comprising the polynucleotide of the invention. In another aspect, the invention provides a cell comprising the polynucleotide or vector of the invention. In another aspect, the invention provides a kit comprising the terminal deoxynucleotidyl transferase (TdT) of the invention and one or more protected nucleotide (preferably a nucleoside triphosphate) comprising a 3’-O-protecting group. In another aspect, the invention provides use of the terminal deoxynucleotidyl transferase (TdT) of the invention for producing an oligonucleotide. In another aspect, the invention provides a method of producing an oligonucleotide comprising a step of contacting the terminal deoxynucleotidyl transferase (TdT) of the invention with a nucleic acid initiator and a protected nucleotide comprising a 3’-O-protecting group (particularly a 3’-O-protected nucleoside triphosphate). According to any embodiment of the method, suitable 3’-O-protecting groups can include: azidomethyl (i.e. -CH2N3), amino (i.e. -NH2), allyl, CH2SSCH3, phenoxyacetyl [i.e. -C(=O)-CH2-O-Ph], methoxyacetyl [i.e. -C(=O)-CH2OMe], acetyl, p-toluenesulfonate, phosphate, nitrate, [4-methoxy]-tetrahydrothiopyranyl, tetrahydrothiopyranyl, [5-methyl]-tetrahydrofuranyl, [2-methyl,4-methoxy]-tetrahydropyranyl, [5-methyl]-tetrahydropyranyl, tetrahydrothiofuranyl and 2-nitrobenzyl. According to any embodiment of the method, suitable 3’-O-protecting groups can include: azidomethyl (i.e. -CH2N3), amino (i.e. -NH2), allyl, CH2SSCH3, phenoxyacetyl [i.e. -C(=O)-CH2-O-Ph], methoxyacetyl [i.e. -C(=O)-CH2OMe], acetyl, p-toluenesulfonate, [4-methoxy]-tetrahydrothiopyranyl, tetrahydrothiopyranyl, [5-methyl]-tetrahydrofuranyl, [2-methyl,4-methoxy]-tetrahydropyranyl, [5-methyl]-tetrahydropyranyl, tetrahydrothiofuranyl and 2-nitrobenzyl. Particularly, the 3’-O-protecting group is azidomethyl, amino or allyl. More particularly, the 3’-O-protecting group is a 2-nitrobenzyl group. In particularly preferred methods of the invention, the 3’-O-protecting group is an azidomethyl group. In some embodiments, the 3’-O-protecting group is -CH2ONH2. According to any embodiment, the method may further comprise a step of deprotecting the protected nucleotide that has been conjugated to the nucleic acid initiator by removal of the 3’-O-protecting group. Preferably, the deprotecting step is a thermal deprotection step. In another aspect, the invention provides a method of producing an oligonucleotide comprising a step of (a) contacting the terminal deoxynucleotidyl transferase (TdT) of the invention with a nucleic acid initiator and a protected nucleotide comprising a 3’-O-protecting group (particularly a 3’-O-protected nucleoside triphosphate). According to any embodiment, the method may further comprise a step of (b) deprotecting the protected nucleotide that has been conjugated to the nucleic acid initiator by removal of the 3’-O-protecting group. Preferably, the deprotecting step is a thermal deprotection step. In some embodiments, the method further comprises repeating steps (a)-(b), wherein the product of step (b) of cycle n of steps (a)-(b) is the nucleic acid initiator of cycle n+1 of steps (a)-(b). In another aspect, the invention provides a method of producing the terminal deoxynucleotidyl transferase (TdT) of the invention comprising culturing the cell of the invention under conditions for expressing the TdT. The method may further comprising purifying the TdT. In some embodiments, the TdT comprises a protein tag. In some embodiments, the TdT comprises a His tag (e.g. a 6xHis tag). In some embodiments, the TdT comprises a thioredoxin tag. In some embodiments, the TdT comprises a His tag (e.g. a 6xHis tag) and a thioredoxin tag. In another aspect, the invention provides a method of producing an oligonucleotide, wherein the method comprises the steps: (a) contacting a nucleic acid initiator with a terminal deoxynucleotidyl transferase (TdT) and a protected nucleotide (particularly a nucleoside triphosphate) comprising a first 3’-O-protecting group; (b) activating the first 3’-O-protecting group to form a second 3’-O-protecting group; and (c) cleaving the second 3’-O-protecting group to form a 3’-OH group. In preferred embodiments, the second 3’-O-protecting group is a thermo-labile 3’-O-protecting group. Particularly, the first 3’-O-protecting group is a thermo-stable 3’-O-protecting group. In any embodiment of the above-described method for producing an oligonucleotide, the first 3’-O-protecting group is -CH2ONH2. Preferably, the second 3’-O-protecting group is -CH2ONC(CH3)COOH, or a salt thereof. This second 3’-O-protecting group may be formed in accordance with step (b) of the above method, by reaction of a first 3’-O-protecting group which is -CH2ONH2 with, for example, pyruvic acid (optionally in the form of a salt, such as sodium pyruvate, potassium pyruvate, or ammonium pyruvate), optionally in the presence of an acid such as acetic acid. In any embodiment of the above-described method for producing an oligonucleotide, the second 3’-O-protecting group is preferably thermo-labile and the cleavage step (c) is preferably conducted by heating, optionally in the presence of an acid or base. In some embodiments, the TdT comprises or consists of an amino acid sequence that has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 2. In some embodiments, the TdT comprises the amino acid sequence SEQ ID NO: 2. In some embodiments, the TdT consists of the amino acid sequence SEQ ID NO: 2. In some embodiments, the TdT is the TdT as described in any aspect or embodiment of the invention. In some embodiments, the method further comprises repeating steps (a)-(c), wherein the product of step (c) of cycle n of steps (a)-(c) is the nucleic acid initiator of cycle n+1 of steps (a)-(c). In some embodiments, the method further comprises removing or inactivating the TdT after step (a) and before step (b). In some embodiments, the method further comprises removing the TdT after step (a) and before step (b). In some embodiments, the method further comprises inactivating the TdT after step (a) and before step (b). DESCRIPTION OF THE DRAWINGS FIGURE 1 Schematic of a method for producing an oligonucleotide. The schematic shows one cycle of: enzymatic incorporation of a protected nucleotide on a nucleic acid initiator; activation of a protecting group; and removal of an activated protecting group. Repeating cycles of incorporation enables a desired sequence to be synthesised in a controlled manner. Using a thermally-addressable chip, different sites on the chip may be deprotected or maintained in protected form to enable sites to be chosen to allow or prevent addition of a further nucleotide in each cycle. FIGURE 2 SDS-PAGE analysis of expression and purification of an engineered TdT and a mouse TdT. (A) Production of 6xHis-TrxA-EVO33 (the EVO33 is SEQ ID NO: 3). (B) Production of 6xHis-TrxA-EVO2 (mouse TdT catalytic domain; the EVO2 is SEQ ID NO: 2). SF, soluble fraction. FT, flow-through after passage of the soluble fraction through the purification column. FIGURE 3 Comparison of EVO2 and EVO33. Comparison of soluble expression levels (%), relative activity and thermostability (Tm, °C) between EVO2 and EVO33. FIGURE 4 Example of a capillary electrophoresis spectrum. FIGURE 5 Fold change of enzymatic activity relative to the parent (EVO33) for EVO33 variant libraries tested (L7 (E315), L8 (R312) and L9 (R196)) when assayed with 3’-O-AzMe-dTTP. The thresholds for improved, neutral, deleterious and strongly deleterious fold changes of the variants are also shown. FIGURE 6 Fold changes of enzymatic activity relative to the parent EVO33 and unique residue hits from EVO33 variant libraries. (A) L7 (E315) and (B) L8 (R312), with 3’-O-AzMe-dTTP and 3-0-MHA-dTTP. FIGURE 7 Comparison of EVO2 (control), EVO33 (parent) and mutants thereof. Comparison of soluble expression levels (%), thermostability (Tm, °C) and relative activity between EVO2, EVO33 and mutants thereof with 3’-O-MHA-dTTP. DETAILED DESCRIPTION OF THE INVENTION The terms “comprising”, “comprises” and “comprised of” as used herein are synonymous with “including” or “includes”; or “containing” or “contains”, and are inclusive or open-ended and do not exclude additional, non-recited members, elements or steps. The terms “comprising”, “comprises” and “comprised of” also include the term “consisting of’. The term “thermo-stable” as used herein, particularly with reference to a 3’-O-protecting group, refers to a protecting group which is stable (i.e. remains intact on the 3’-position) during the enzymatic conjugation of the nucleotide and a nucleic acid initiator. The term “thermo-labile” as used herein, particularly with reference to an activated 3’-O-protecting group, refers to a protecting group which can be cleaved (i.e. removed) by heating, optionally in the presence of an agent, particularly an acid or base, or water. As used herein, AzMe refers to azidomethyl. As used herein, MHA (methylhydroxyamino) refers to -CH2ONH2. TERMINAL DEOXYNUCLEOTIDYL TRANSFERASE (TdT) Terminal deoxynucleotidyl transferase (TdT), also known as DNA nucleotidylexotransferase (DNTT) or terminal transferase, is a template-independent polymerase that catalyses the addition of nucleotides at the 3’-hydroxyl terminus of a nucleic acid. TdT does not require a template. TdTs may be naturally expressed in immature, pre-B, pre-T lymphoid cells and acute lymphoblastic leukaemia / lymphoma cells. In a natural role, TdT adds nucleotides to the V, D and J exons of the TCR and BCR genes during antibody gene recombination. An example natural TdT amino acid sequence is the mouse TdT: MDPLQAVHLGPRKKRPRQLGTPVASTPYDIRFRDLVLFILEKKMGTTRRAFLMELARRKGFR VENELSDSVTHIVAENNSGSDVLEWLQLQNIKASSELELLDISWLIECMGAGKPVEMMGRHQ LVVNRNSSPSPVPGSQNVPAPAVKKISQYACQRRTTLNNYNQLFTDALDILAENDELRENEG SCLAFMRASSVLKSLPFPITSMKDTEGIPCLGDKVKSIIEGIIEDGESSEAKAVLNDERYKS FKLFTSVFGVGLKTAEKWFRMGFRTLSKIQSDKSLRFTQMQKAGFLYYEDLVSCVNRPEAEA VSMLVKEAWTFLPDALVTMTGGFRRGKMTGHDVDFLITSPEATEDEEQQLLHKVTDFWKQQ GLLLYCDILESTFEKFKQPSRKVDALDHFQKCFLILKLDHGRVHSEKSGQQEGKGWKAIRVD LVMCPYDRRAFALLGWTGSRQFERDLRRYATHERKMMLDNHALYDRTKRVFLEAESEEEIFA HLGLDYIEPWERNA (SEQ ID NO: 1) Full length TdT may comprise a BRCT domain, for example towards the N-terminus of the protein. The BRCT domain may not be required for artificial synthesis of polynucleotides. Truncated TdT proteins, for example truncated to delete part of the sequence at the N-terminus (preferably to delete a BRCT domain), preferably retaining the natural C-terminus, may retain activity in catalysing the addition of nucleotides at the 3’-hydroxyl terminus of a nucleic acid. An example truncated mouse TdT amino acid sequence is: VPAPAVKKISQYACQRRTTLNNYNQLFTDALDILAENDELRENEGSCLAFMRASSVLKSLPF PITSMKDTEGIPCLGDKVKSIIEGIIEDGESSEAKAVLNDERYKSFKLFTSVFGVGLKTAEK WFRMGFRTLSKIQSDKSLRFTQMQKAGFLYYEDLVSCVNRPEAEAVSMLVKEAVVTFLPDAL VTMTGGFRRGKMTGHDVDFLITSPEATEDEEQQLLHKVTDFWKQQGLLLYCDILESTFEKFK QPSRKVDALDHFQKCFLILKLDHGRVHSEKSGQQEGKGWKAIRVDLVMCPYDRRAFALLGWT GSRQFERDLRRYATHERKMMLDNHALYDRTKRVFLEAESEEEIFAHLGLDYIEPWERNA (SEQ ID NO: 2) The present invention may use template-independent polymerases, such as TdT, for the synthesis of oligonucleotides. In particular, engineered TdTs may be used that accept 3’-O-protected nucleotides as substrates and allow the enzymatic synthesis of oligonucleotides, for example as disclosed herein, for example during gene synthesis. In preferred embodiments, the oligonucleotide is a deoxyribooligonucleotide. In one aspect, the invention provides a terminal deoxynucleotidyl transferase (TdT) comprising an amino acid sequence that has at least 75% sequence identity to SEQ ID NO: 3. MGPASAVARISQYACQRRTTLNNHNKIFTDAFEILAENAEFNENEGRCLAFRRAASVLKSLP YAVRSMEDLEGLPCLGDQTKAVIEEILEDGKSSKVESILNDERYQALKLFTSVFGVGLKTAE KWYRMGLRTLEEVQADPTIKLTRMQKAGFLYYEDISSAVSKAEADAVGQIVEETVHKFLPDA IVTLTGGFRRGKEIGHDVDFLITSPEEGKEEGLLPKVINRLKKQGLLLYHDIQESTFDKSKL PSRRFDAMDHFEKCFLILKLHRSQVEAGQSSQSESRGWKAVRVDLVVAPIDQYAFALLGWTG SRQFERDLRRFARHEKKMLLDNHALYDKTKKVFLPAKTEEEIFAHLGLEYIEPWERNA (SEQ ID NO: 3) In some embodiments, the TdT comprises or consists of an amino acid sequence that has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 3. Preferably, the TdT is capable of adding a protected nucleotide comprising a 3’-O-protecting group to a 3’-OH of a nucleic acid initiator in the absence of a nucleic acid template. Preferably, the TdT is capable of adding protected adenine, cytosine, guanine and thymine nucleotides comprising a 3’-O-protecting group to a 3’-OH of a nucleic acid initiator in the absence of a nucleic acid template. Preferably, the nucleotide is a deoxyribonucleotide. The capability of a candidate TdT for adding a protected nucleotide comprising a 3’-O-protecting group to a 3’-OH of a nucleic acid initiator may be determined using any of a number of suitable assays known in the art, which include assays disclosed herein. For example, suitable assays for measuring the incorporation of nucleotides into a substrate by a candidate TdT may utilise detection and characterisation of products by agarose gel electrophoresis, capillary electrophoresis (CE) or liquid chromatography-mass spectrometry (LCMS) approaches. These approaches may also be readily implemented to compare various properties of candidate TdTs, such as catalytic activity. In some embodiments, the TdT exhibits an improved property as compared to SEQ ID NO: 2. In some embodiments, the improved property is improved thermostability and / or improved enzymatic activity and / or improved soluble expression. In some embodiments, the improved property is improved thermostability. In some embodiments, the improved property is improved enzymatic activity. In some embodiments, the improved property is improved soluble expression. Thermostability, enzymatic activity and soluble expression may be determined using any of a number of suitable assays known in the art, which include assays disclosed herein. Example assays suitable for characterising thermostability include analysing the melting temperature of a protein. The skilled person is readily able to determine the melting temperature of a protein using methods of the art. Example methods for determining melting temperature are also disclosed herein. Example assays suitable for characterising enzymatic activity include those described above and in the Examples of the disclosure. Example assays suitable for characterising soluble expression levels include those described herein (e.g. use of SDS-PAGE analysis). In some embodiments, the TdT exhibits an increase in melting temperature (Tm) of at least 5°C, 10°C, 15°C, 20°C or25°C compared to SEQ ID NO: 2. In some embodiments, the TdT exhibits an increase in melting temperature (Tm) of at least 20°C compared to SEQ ID NO: 2. In some embodiments, the TdT exhibits a melting temperature at least 5°C, for example at least 10°C, at least 15°C, at least 20°C, at least 21°C, at least 22°C, at least 23°C, at least 24°C or at least 25 C, greater than SEQ ID NO: 2. In some embodiments, the TdT exhibits a melting temperature at least 20°C greater than SEQ ID NO: 2. Preferably, the melting temperature of the TdT and the protein of SEQ ID NO: 2 are determined using the same method. In some embodiments, the TdT exhibits an increase in enzymatic activity of at least 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold or 15-fold compared to SEQ ID NO: 2. In some embodiments, the TdT exhibits an increase in enzymatic activity of at least 1.1-fold compared to SEQ ID NO: 2. In some embodiments, the TdT exhibits an increase in enzymatic activity of at least 1.5-fold compared to SEQ ID NO: 2. In some embodiments, the TdT exhibits an increase in soluble expression of at least 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold or 5-fold compared to SEQ ID NO: 2. In some embodiments, the TdT exhibits an increase in soluble expression of at least 1.1-fold compared to SEQ ID NO: 2. In some embodiments, the TdT exhibits an increase in soluble expression of at least 1.5-fold compared to SEQ ID NO: 2. In some embodiments, the TdT exhibits an increase in soluble expression of at least 2-fold compared to SEQ ID NO: 2. In some embodiments, the TdT comprises the amino acid sequence SEQ ID NO: 3. In some embodiments, the TdT consists of the amino acid sequence SEQ ID NO: 3. In some embodiments, the TdT comprises one or more mutation with respect to SEQ ID NO 3 at an amino acid position selected from the group consisting of 315, 52 and 312, wherein the amino acids are numbered with reference to SEQ ID NO: 3. In some embodiments, the TdT comprises one or more mutation with respect to SEQ ID NO 3 at an amino acid position selected from the group consisting of 315 and 52, wherein the amino acids are numbered with reference to SEQ ID NO: 3. In some embodiments, the TdT comprises a mutation with respect to SEQ ID NO 3 at amino acid position 315, wherein the amino acids are numbered with reference to SEQ ID NO: 3. In some embodiments, the TdT comprises a mutation with respect to SEQ ID NO 3 at amino acid position 52, wherein the amino acids are numbered with reference to SEQ ID NO: 3. In some embodiments, the TdT comprises a mutation with respect to SEQ ID NO 3 at amino acid position 312, wherein the amino acids are numbered with reference to SEQ ID NO: 3. In some embodiments, the TdT comprises mutations with respect to SEQ ID NO 3 at amino acid positions 315 and 52, wherein the amino acids are numbered with reference to SEQ ID NO: 3. In some embodiments, the TdT comprises mutations with respect to SEQ ID NO 3 at amino acid positions 315 and 312, wherein the amino acids are numbered with reference to SEQ ID NO: 3. In some embodiments, the TdT comprises mutations with respect to SEQ ID NO 3 at amino acid positions 52 and 312, wherein the amino acids are numbered with reference to SEQ ID NO: 3. In some embodiments, the TdT comprises mutations with respect to SEQ ID NO 3 at amino acid positions 315, 52 and 312, wherein the amino acids are numbered with reference to SEQ ID NO: 3. The term “mutation” may refer to a substitution of the amino acid that is at a certain position of a protein sequence with another amino acid. For example, a mutation with respect to SEQ ID NO: 3 at position 315, wherein the amino acids are numbered with reference to SEQ ID NO: 3, may refer to substitution of the amino acid that is present at position 315 in SEQ ID NO: 3 with a different amino acid. The terms “corresponding to”, “reference to” and “relative to” when used in the context of the numbering of a given amino acid or polynucleotide sequence may refer to the numbering of the residues of a specified reference sequence when the given amino acid or polynucleotide sequence is compared to the reference sequence. In other words, the residue number or residue position of a given polymer is designated with respect to the reference sequence rather than by the actual numerical position of the residue within the given amino acid or polynucleotide sequence. For example, a given amino acid sequence, such as that of a TdT, can be aligned to a reference sequence by introducing gaps to optimise residue matches between the two sequences. In these cases, although the gaps are present, the numbering of the residue in the given amino acid or polynucleotide sequence is made with respect to the reference sequence to which it has been aligned. In some embodiments, the TdT exhibits an improved property as compared to SEQ ID NO: 3. In some embodiments, the improved property is improved thermostability and / or improved enzymatic activity. In some embodiments, the improved property is improved thermostability. In some embodiments, the improved property is improved enzymatic activity. In some embodiments, the improved property is improved soluble expression. In some embodiments, the TdT exhibits an increase in soluble expression of at least 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold or 5-fold compared to SEQ ID NO: 3. In some embodiments, the TdT exhibits an increase in melting temperature (Tm) of at least 1°C, 2°C, 3°C, 4°C, 5°C, 10°C, 15°C, 20°C or 25°C compared to SEQ ID NO: 3. In some embodiments, the TdT exhibits an increase in enzymatic activity of at least 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold or 10-fold compared to SEQ ID NO: 3. In some embodiments, the TdT exhibits an increase in enzymatic activity of at least 1.1-fold compared to SEQ ID NO: 3. In some embodiments, the TdT exhibits an increase in enzymatic activity of at least 1.5-fold compared to SEQ ID NO: 3. In some embodiments, the TdT comprises the mutation E315K, wherein the amino acids are numbered with reference to SEQ ID NO: 3. In some embodiments, the TdT comprises the mutation R52Q, wherein the amino acids are numbered with reference to SEQ ID NO: 3. In preferred embodiments, the TdT comprises the mutations R52Q and E315K, wherein the amino acids are numbered with reference to SEQ ID NO: 3. In some embodiments, the TdT comprises or consists of an amino acid sequence that has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 4. Preferably, the TdT comprises a lysine at amino acid position 315, wherein the amino acids are numbered with reference to SEQ ID NO: 3. MGPASAVARISQYACQRRTTLNNHNKIFTDAFEILAENAEFNENEGRCLAFRRAASVLKSLP YAVRSMEDLEGLPCLGDQTKAVIEEILEDGKSSKVESILNDERYQALKLFTSVFGVGLKTAE KWYRMGLRTLEEVQADPTIKLTRMQKAGFLYYEDISSAVSKAEADAVGQIVEETVHKFLPDA IVTLTGGFRRGKEIGHDVDFLITSPEEGKEEGLLPKVINRLKKQGLLLYHDIQESTFDKSKL PSRRFDAMDHFEKCFLILKLHRSQVEAGQSSQSESRGWKAVRVDLVVAPIDQYAFALLGWTG SRQFKRDLRRFARHEKKMLLDNHALYDKTKKVFLPAKTEEEIFAHLGLEYIEPWERNA (SEQ ID NO: 4) In some embodiments, the TdT comprises the amino acid sequence SEQ ID NO: 4. In some embodiments, the TdT consists of the amino acid sequence SEQ ID NO: 4. In some embodiments, the TdT comprises or consists of an amino acid sequence that has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 5. Preferably, the TdT comprises a glutamine at amino acid position 52 and a lysine at amino acid position 315, wherein the amino acids are numbered with reference to SEQ ID NO: 3. MGPASAVARISQYACQRRTTLNNHNKIFTDAFEILAENAEFNENEGRCLAFQRAASVLKSLP YAVRSMEDLEGLPCLGDQTKAVIEEILEDGKSSKVESILNDERYQALKLFTSVFGVGLKTAE KWYRMGLRTLEEVQADPTIKLTRMQKAGFLYYEDISSAVSKAEADAVGQIVEETVHKFLPDA IVTLTGGFRRGKEIGHDVDFLITSPEEGKEEGLLPKVINRLKKQGLLLYHDIQESTFDKSKL PSRRFDAMDHFEKCFLILKLHRSQVEAGQSSQSESRGWKAVRVDLVVAPIDQYAFALLGWTG SRQFKRDLRRFARHEKKMLLDNHALYDKTKKVFLPAKTEEEIFAHLGLEYIEPWERNA (SEQ ID NO: 5) In preferred embodiments, the TdT comprises the amino acid sequence SEQ ID NO: 5. In preferred embodiments, the TdT consists of the amino acid sequence SEQ ID NO: 5. In some embodiments, the TdT comprises or consists of an amino acid sequence that has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 6. Preferably, the TdT comprises an isoleucine at amino acid position 312, wherein the amino acids are numbered with reference to SEQ ID NO: 3. MGPASAVARISQYACQRRTTLNNHNKIFTDAFEILAENAEFNENEGRCLAFRRAASVLKSLP YAVRSMEDLEGLPCLGDQTKAVIEEILEDGKSSKVESILNDERYQALKLFTSVFGVGLKTAE KWYRMGLRTLEEVQADPTIKLTRMQKAGFLYYEDISSAVSKAEADAVGQIVEETVHKFLPDA IVTLTGGFRRGKEIGHDVDFLITSPEEGKEEGLLPKVINRLKKQGLLLYHDIQESTFDKSKL PSRRFDAMDHFEKCFLILKLHRSQVEAGQSSQSESRGWKAVRVDLVVAPIDQYAFALLGWTG SIQFKRDLRRFARHEKKMLLDNHALYDKTKKVFLPAKTEEEIFAHLGLEYIEPWERNA (SEQ ID NO: 6) In some embodiments, the TdT comprises the amino acid sequence SEQ ID NO: 6. In some embodiments, the TdT consists of the amino acid sequence SEQ ID NO: 6. POLYNUCLEOTIDES In one aspect, the invention provides a polynucleotide comprising a nucleic acid sequence encoding the terminal deoxynucleotidyl transferase (TdT) of the invention. In some embodiments, the polynucleotide comprises a nucleic acid sequence that has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 7. ATGGGTCCTGCAAGCGCAGTTGCACGTATTAGCCAGTATGCATGTCAGCGTCGTACCACACT GAATAACCATAACAAAATCTTTACCGACGCCTTTGAAATTCTGGCAGAAAATGCCGAATTTA ACGAAAATGAAGGTCGTTGTCTGGCATTTCGTCGTGCAGCAAGCGTTCTGAAAAGCCTGCCG TATGCAGTTCGTAGCATGGAAGATCTGGAAGGTCTGCCGTGTCTGGGTGATCAGACCAAAGC AGTTATTGAAGAAATCCTGGAAGATGGCAAAAGCAGCAAAGTTGAAAGCATTCTGAATGATG AACGTTACCAGGCACTGAAACTGTTTACCAGCGTTTTTGGTGTTGGTCTGAAAACCGCAGAA AAATGGTATCGTATGGGTCTGCGTACCCTGGAAGAAGTTCAGGCAGATCCGACCATTAAACT GACCCGCATGCAGAAAGCAGGTTTTCTGTATTATGAAGATATTAGCAGCGCAGTGAGCAAAG CCGAAGCAGATGCAGTTGGTCAGATTGTTGAAGAAACCGTGCATAAATTTCTGCCGGATGCA ATTGTTACCCTGACCGGTGGTTTTCGTCGCGGTAAAGAAATTGGTCATGATGTGGATTTTCT GATCACAAGTCCGGAAGAAGGTAAAGAAGAAGGCCTGCTGCCGAAAGTTATTAATCGTCTGA AAAAACAGGGTCTGCTGCTGTATCACGATATTCAAGAAAGCACCTTCGACAAAAGCAAACTG CCGAGCCGTCGTTTTGATGCAATGGATCATTTTGAGAAGTGCTTTCTGATCCTGAAACTGCA TCGTAGCCAGGTTGAAGCAGGTCAGAGCAGCCAGAGCGAAAGCCGTGGTTGGAAAGCAGTTC GTGTTGATCTGGTTGTTGCACCGATTGATCAGTATGCCTTTGCACTGTTAGGTTGGACCGGT AGCCGTCAGTTTGAACGTGATCTGCGTCGCTTTGCACGTCATGAGAAAAAAATGCTGCTGGA TAATCATGCCCTGTACGACAAAACCAAAAAAGTTTTTCTGCCTGCCAAAACCGAAGAAGAAA TTTTTGCCCATCTGGGCCTTGAGTATATTGAACCGTGGGAACGTAATGCATAA (SEQ ID NO: 7) In some embodiments, the polynucleotide comprises the nucleic acid sequence SEQ ID NO: 7. In some embodiments, the polynucleotide consists of the nucleic acid sequence SEQ ID NO: 7. In some embodiments, the polynucleotide comprises a nucleic acid sequence that has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 8. Preferably, the polynucleotide encodes a TdT comprising a lysine at amino acid position 315, wherein the amino acids are numbered with reference to SEQ ID NO: 3. ATGGGTCCTGCAAGCGCAGTTGCACGTATTAGCCAGTATGCATGTCAGCGTCGTACCACACT GAATAACCATAACAAAATCTTTACCGACGCCTTTGAAATTCTGGCAGAAAATGCCGAATTTA TATGCAGTTCGTAGCATGGAAGATCTGGAAGGTCTGCCGTGTCTGGGTGATCAGACCAAAGC AGTTATTGAAGAAATCCTGGAAGATGGCAAAAGCAGCAAAGTTGAAAGCATTCTGAATGATG AACGTTACCAGGCACTGAAACTGTTTACCAGCGTTTTTGGTGTTGGTCTGAAAACCGCAGAA AAATGGTATCGTATGGGTCTGCGTACCCTGGAAGAAGTTCAGGCAGATCCGACCATTAAACT GACCCGCATGCAGAAAGCAGGTTTTCTGTATTATGAAGATATTAGCAGCGCAGTGAGCAAAG CCGAAGCAGATGCAGTTGGTCAGATTGTTGAAGAAACCGTGCATAAATTTCTGCCGGATGCA ATTGTTACCCTGACCGGTGGTTTTCGTCGCGGTAAAGAAATTGGTCATGATGTGGATTTTCT GATCACAAGTCCGGAAGAAGGTAAAGAAGAAGGCCTGCTGCCGAAAGTTATTAATCGTCTGA AAAAACAGGGTCTGCTGCTGTATCACGATATTCAAGAAAGCACCTTCGACAAAAGCAAACTG CCGAGCCGTCGTTTTGATGCAATGGATCATTTTGAGAAGTGCTTTCTGATCCTGAAACTGCA TCGTAGCCAGGTTGAAGCAGGTCAGAGCAGCCAGAGCGAAAGCCGTGGTTGGAAAGCAGTTC GTGTTGATCTGGTTGTTGCACCGATTGATCAGTATGCCTTTGCACTGTTAGGTTGGACCGGT AGCCGTCAGTTTAAGCGTGATCTGCGTCGCTTTGCACGTCATGAGAAAAAAATGCTGCTGGA TAATCATGCCCTGTACGACAAAACCAAAAAAGTTTTTCTGCCTGCCAAAACCGAAGAAGAAA TTTTTGCCCATCTGGGCCTTGAGTATATTGAACCGTGGGAACGTAATGCATAA (SEQ ID NO: 8) In some embodiments, the polynucleotide comprises the nucleic acid sequence SEQ ID NO: 8. In some embodiments, the polynucleotide consists of the nucleic acid sequence SEQ ID NO: 8. In some embodiments, the polynucleotide comprises a nucleic acid sequence that has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 9. Preferably, the polynucleotide encodes a TdT comprising a glutamine at amino acid position 52 and a lysine at amino acid position 315, wherein the amino acids are numbered with reference to SEQ ID NO: 3. ATGGGTCCTGCAAGCGCAGTTGCACGTATTAGCCAGTATGCATGTCAGCGTCGTACCACACT GAATAACCATAACAAAATCTTTACCGACGCCTTTGAAATTCTGGCAGAAAATGCCGAATTTA ACGAAAATGAAGGTCGTTGTCTGGCATTTCAGCGTGCAGCAAGCGTTCTGAAAAGCCTGCCG TATGCAGTTCGTAGCATGGAAGATCTGGAAGGTCTGCCGTGTCTGGGTGATCAGACCAAAGC AGTTATTGAAGAAATCCTGGAAGATGGCAAAAGCAGCAAAGTTGAAAGCATTCTGAATGATG AACGTTACCAGGCACTGAAACTGTTTACCAGCGTTTTTGGTGTTGGTCTGAAAACCGCAGAA AAATGGTATCGTATGGGTCTGCGTACCCTGGAAGAAGTTCAGGCAGATCCGACCATTAAACT GACCCGCATGCAGAAAGCAGGTTTTCTGTATTATGAAGATATTAGCAGCGCAGTGAGCAAAG CCGAAGCAGATGCAGTTGGTCAGATTGTTGAAGAAACCGTGCATAAATTTCTGCCGGATGCA ATTGTTACCCTGACCGGTGGTTTTCGTCGCGGTAAAGAAATTGGTCATGATGTGGATTTTCT GATCACAAGTCCGGAAGAAGGTAAAGAAGAAGGCCTGCTGCCGAAAGTTATTAATCGTCTGA AAAAACAGGGTCTGCTGCTGTATCACGATATTCAAGAAAGCACCTTCGACAAAAGCAAACTG CCGAGCCGTCGTTTTGATGCAATGGATCATTTTGAGAAGTGCTTTCTGATCCTGAAACTGCA TCGTAGCCAGGTTGAAGCAGGTCAGAGCAGCCAGAGCGAAAGCCGTGGTTGGAAAGCAGTTC GTGTTGATCTGGTTGTTGCACCGATTGATCAGTATGCCTTTGCACTGTTAGGTTGGACCGGT AGCCGTCAGTTTAAGCGTGATCTGCGTCGCTTTGCACGTCATGAGAAAAAAATGCTGCTGGA TAATCATGCCCTGTACGACAAAACCAAAAAAGTTTTTCTGCCTGCCAAAACCGAAGAAGAAA TTTTTGCCCATCTGGGCCTTGAGTATATTGAACCGTGGGAACGTAATGCATAA (SEQ ID NO: 9) In some embodiments, the polynucleotide comprises the nucleic acid sequence SEQ ID NO: 9. In some embodiments, the polynucleotide consists of the nucleic acid sequence SEQ ID NO: 9. In some embodiments, the polynucleotide comprises a nucleic acid sequence that has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 10. Preferably, the polynucleotide encodes a TdT comprising an isoleucine at amino acid position 312, wherein the amino acids are numbered with reference to SEQ ID NO: 3. ATGGGTCCTGCAAGCGCAGTTGCACGTATTAGCCAGTATGCATGTCAGCGTCGTACCACACT GAATAACCATAACAAAATCTTTACCGACGCCTTTGAAATTCTGGCAGAAAATGCCGAATTTA ACGAAAATGAAGGTCGTTGTCTGGCATTTCAGCGTGCAGCAAGCGTTCTGAAAAGCCTGCCG TATGCAGTTCGTAGCATGGAAGATCTGGAAGGTCTGCCGTGTCTGGGTGATCAGACCAAAGC AGTTATTGAAGAAATCCTGGAAGATGGCAAAAGCAGCAAAGTTGAAAGCATTCTGAATGATG AACGTTACCAGGCACTGAAACTGTTTACCAGCGTTTTTGGTGTTGGTCTGAAAACCGCAGAA AAATGGTATCGTATGGGTCTGCGTACCCTGGAAGAAGTTCAGGCAGATCCGACCATTAAACT GACCCGCATGCAGAAAGCAGGTTTTCTGTATTATGAAGATATTAGCAGCGCAGTGAGCAAAG CCGAAGCAGATGCAGTTGGTCAGATTGTTGAAGAAACCGTGCATAAATTTCTGCCGGATGCA ATTGTTACCCTGACCGGTGGTTTTCGTCGCGGTAAAGAAATTGGTCATGATGTGGATTTTCT GATCACAAGTCCGGAAGAAGGTAAAGAAGAAGGCCTGCTGCCGAAAGTTATTAATCGTCTGA AAAAACAGGGTCTGCTGCTGTATCACGATATTCAAGAAAGCACCTTCGACAAAAGCAAACTG CCGAGCCGTCGTTTTGATGCAATGGATCATTTTGAGAAGTGCTTTCTGATCCTGAAACTGCA TCGTAGCCAGGTTGAAGCAGGTCAGAGCAGCCAGAGCGAAAGCCGTGGTTGGAAAGCAGTTC GTGTTGATCTGGTTGTTGCACCGATTGATCAGTATGCCTTTGCACTGTTAGGTTGGACCGGT AGCATTCAGTTTAAGCGTGATCTGCGTCGCTTTGCACGTCATGAGAAAAAAATGCTGCTGGA TAATCATGCCCTGTACGACAAAACCAAAAAAGTTTTTCTGCCTGCCAAAACCGAAGAAGAAA TTTTTGCCCATCTGGGCCTTGAGTATATTGAACCGTGGGAACGTAATGCATAA (SEQ ID NO: 10) In some embodiments, the polynucleotide comprises the nucleic acid sequence SEQ ID NO: 10. In some embodiments, the polynucleotide consists of the nucleic acid sequence SEQ ID NO: 10. Polynucleotides of the invention that encode a template-independent polymerase, such as a TdT, may comprise DNA or RNA, preferably DNA. They may be single-stranded or doublestranded. Preferably the polynucleotides are isolated polynucleotides. It will be understood by a skilled person that numerous different polynucleotides can encode the same polypeptide as a result of the degeneracy of the genetic code. In addition, it is to be understood that skilled persons may, using routine techniques, make nucleotide substitutions that do not affect the polypeptide sequence encoded by the polynucleotides of the invention to reflect the codon usage of any particular host organism in which the polypeptides of the invention are to be expressed. The polynucleotides that encode a template-independent polymerase, such as a TdT, may be modified by any method available in the art. Such modifications may be carried out in order to enhance the in vivo activity or lifespan of the polynucleotides. Polynucleotides that encode a template-independent polymerase, such as a TdT, such as DNA polynucleotides, may be produced recombinantly, synthetically or by any means available to the skilled person. They may also be cloned by standard techniques. The polynucleotide that encodes a template-independent polymerase, such as a TdT, may comprise a promoter and / or enhancer operably linked to the nucleotide sequence encoding the template-independent polymerase, such as the TdT, of the invention. The term “operably linked”, as used herein, may mean that two components are linked together in a manner, which enables both to carry out their function substantially unhindered. For example, the promoter and / or enhancer may facilitate and / or enhance expression of the templateindependent polymerase, such as the TdT. VECTORS A vector is a tool that allows or facilitates the transfer of an entity from one environment to another. In accordance with the invention, and by way of example, some vectors used in recombinant nucleic acid techniques allow entities, such as a segment of nucleic acid (e.g. a heterologous DNA segment, such as a heterologous cDNA segment), to be transferred into a target cell. The vector may serve the purpose of maintaining the heterologous nucleic acid (DNA or RNA) within the cell, facilitating the replication of the vector comprising a segment of nucleic acid or facilitating the expression of the protein encoded by a segment of nucleic acid. Vectors may be non-viral or viral. Examples of vectors used in recombinant nucleic acid techniques include, but are not limited to, plasmids, mRNA molecules (e.g. in vitro transcribed mRNAs), chromosomes, artificial chromosomes and viruses. The vector may also be, for example, a naked nucleic acid (e.g. DNA). In its simplest form, the vector may itself be a nucleotide of interest. Vectors may be introduced into cells using a variety of techniques known in the art, such as transfection, transformation and transduction. Non-viral delivery systems include but are not limited to DNA transfection methods. Here, transfection includes a process using a non-viral vector to deliver a gene to a target cell. Typical transfection methods include electroporation, DNA biolistics, lipid-mediated transfection, compacted DNA-mediated transfection, liposomes, immunoliposomes, lipofectin, cationic agent-mediated transfection, cationic facial amphiphiles (CFAs) and combinations thereof. VARIANTS, DERIVATIVES, ANALOGUES, HOMOLOGUESAND FRAGMENTS In addition to the specific proteins and polynucleotides mentioned herein, the invention also encompasses variants, derivatives, analogues, homologues and fragments thereof. The term “polynucleotide” in the following section may relate to a polynucleotide that encodes a template-independent polymerase, such as a TdT. In the context of the invention, a “variant’ of any given sequence is a sequence in which the specific sequence of residues (whether amino acid or nucleic acid residues) has been modified in such a manner that the polypeptide or polynucleotide in question retains at least one of its endogenous functions (e.g. TdT activity or encoding a polypeptide with TdT activity). A variant sequence can be obtained by addition, deletion, substitution, modification, replacement and / or variation of at least one residue present in the naturally occurring polypeptide or polynucleotide. The term “derivative” as used herein in relation to proteins or polypeptides of the invention includes any substitution of, variation of, modification of, replacement of, deletion of and / or addition of one (or more) amino acid residue from or to the sequence, providing that the resultant protein or polypeptide retains at least one of its endogenous functions (e.g. TdT activity). The term “analogue” as used herein in relation to polypeptides or polynucleotides includes any mimetic, that is, a chemical compound that possesses at least one of the endogenous functions of the polypeptides or polynucleotides which it mimics (e.g. TdT activity or encoding a polypeptide with TdT activity). Typically, amino acid substitutions may be made, for example from 1, 2 or 3, to 10 or 20 substitutions, provided that the modified sequence retains the required activity or ability. Amino acid substitutions may include the use of non-naturally occurring analogues. Proteins used in the invention may also have deletions, insertions or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent protein. Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and / or the amphipathic nature of the residues as long as the endogenous function is retained. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include asparagine, glutamine, serine, threonine and tyrosine. Conservative substitutions may be made, for example according to the table below. Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other: ALIPHATIC Non-polar GAP I LV Polar - uncharged CSTM NQ Polar - charged D E KR H AROMATIC F WY The term “homologue” as used herein means an entity having a certain homology with the wild type amino acid sequence or the wild type nucleotide sequence. The term “homology” can be equated with “identity”. In the present context, a homologous sequence is taken to include an amino acid sequence which may be at least 50%, 55%, 65%, 75%, 85% or 90% identical, preferably at least 95%, 96% or 97% or 98% or 99% identical to the subject sequence. Typically, the homologues will comprise the same active sites etc. as the subject amino acid sequence. Although homology can also be considered in terms of similarity (i.e. amino acid residues having similar chemical properties / functions), in the context of the present invention it is preferred to express homology in terms of sequence identity. In the present context, a homologous sequence is taken to include a nucleotide sequence which may be at least 50%, 55%, 65%, 75%, 85% or 90% identical, preferably at least 95%, 96% or 97% or 98% or 99% identical to the subject sequence. Although homology can also be considered in terms of similarity, in the context of the present invention it is preferred to express homology in terms of sequence identity. Preferably, reference to a sequence which has a percent identity to any one of the SEQ ID NOs detailed herein refers to a sequence which has the stated percent identity over the entire length of the SEQ ID NO referred to. Homology comparisons can be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate percent homology or identity between two or more sequences. Percent homology may be calculated over contiguous sequences, i.e. one sequence is aligned with the other sequence and each amino acid or nucleotide in one sequence is directly compared with the corresponding amino acid or nucleotide in the other sequence, one residue at a time. This is called an “ungapped” alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues. Although this is a very simple and consistent method, it fails to take into consideration that, for example, in an otherwise identical pair of sequences, one insertion or deletion in the amino acid or nucleotide sequence may cause the following residues or codons to be put out of alignment, thus potentially resulting in a large reduction in percent homology when a global alignment is performed. Consequently, most sequence comparison methods are designed to produce optimal alignments that take into consideration possible insertions and deletions without penalising unduly the overall homology score. This is achieved by inserting “gaps” in the sequence alignment to try to maximise local homology. However, these more complex methods assign “gap penalties” to each gap that occurs in the alignment so that, for the same number of identical amino acids or nucleotides, a sequence alignment with as few gaps as possible, reflecting higher relatedness between the two compared sequences, will achieve a higher score than one with many gaps. “Affine gap costs” are typically used that charge a relatively high cost for the existence of a gap and a smaller penalty for each subsequent residue in the gap. This is the most commonly used gap scoring system. High gap penalties will of course produce optimised alignments with fewer gaps. Most alignment programs allow the gap penalties to be modified. However, it is preferred to use the default values when using such software for sequence comparisons. For example when using the GCG Wisconsin Bestfit package the default gap penalty for amino acid sequences is -12 for a gap and -4 for each extension. Calculation of maximum percent homology therefore firstly requires the production of an optimal alignment, taking into consideration gap penalties. A suitable computer program for carrying out such an alignment is the GCG Wisconsin Bestfit package (University of Wisconsin, USA; Devereux et al. (1984) Nucleic Acids Research 12: 387). Examples of other software that can perform sequence comparisons include, but are not limited to, the BLAST package, FASTA (Atschul et al. (1990) J. Mol. Biol. 403-410) and the GENEWORKS suite of comparison tools. Both BLAST and FASTA are available for offline and online searching. However, for some applications, it is preferred to use the GCG Bestfit program. Another tool, BLAST 2 Sequences, is also available for comparing protein and nucleotide sequences (FEMS Microbiol. Lett. (1999) 174(2):247-50; FEMS Microbiol. Lett. (1999) 177(1):187-8). Although the final percent homology can be measured in terms of identity, the alignment process itself is typically not based on an all-or-nothing pair comparison. Instead, a scaled similarity score matrix is generally used that assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance. An example of such a matrix commonly used is the BLOSUM62 matrix (the default matrix for the BLAST suite of programs). GCG Wisconsin programs generally use either the public default values or a custom symbol comparison table if supplied (see the user manual for further details). For some applications, it is preferred to use the public default values for the GCG package, or in the case of other software, the default matrix, such as BLOSUM62. Once the software has produced an optimal alignment, it is possible to calculate percent homology, preferably percent sequence identity. The software typically does this as part of the sequence comparison and generates a numerical result. “Fragments” are also variants and the term typically refers to a selected region of the polypeptide or polynucleotide that is of interest either functionally or, for example, in an assay. “Fragment” thus refers to an amino acid or nucleic acid sequence that is a portion of a full-length polypeptide or polynucleotide. Such variants may be prepared using standard recombinant DNA techniques such as site-directed mutagenesis. Where insertions are to be made, synthetic DNA encoding the insertion together with 5’ and 3’ flanking regions corresponding to the naturally-occurring sequence either side of the insertion site may be made. The flanking regions will contain convenient restriction sites corresponding to sites in the naturally-occurring sequence so that the sequence may be cut with the appropriate enzyme(s) and the synthetic DNA ligated into the cut. The DNA is then expressed in accordance with the invention to make the encoded protein. These methods are only illustrative of the numerous standard techniques known in the art for manipulation of DNA sequences and other known techniques may also be used. METHOD OF PRODUCING AN OLIGONUCLEOTIDE Template-independent polymerases, such as TdTs, may be used for the synthesis of oligonucleotides, such as DNA. As disclosed herein, engineered TdTs may be used that accept 3’-O-protected nucleotides, such as 3’-O-protected nucleoside triphosphates, as substrates and allow the enzymatic synthesis of oligonucleotides, for example during gene synthesis. By way of example, template-independent polymerases, such as TdTs, may enable a 3’-O-protected nucleotide, preferably a 3’-O-protected nucleoside triphosphate, to be used in a step-by-step method to extend an initiating nucleic acid into a defined sequence. For example, in a first extension step a 3’-O-protected nucleotide may be added to a nucleic acid initiator by a TdT (for example a TdT of the invention). The 3’-O-protecting group may prevent the addition of more than one nucleotide to the initiator during the first step (e.g. once the nucleotide comprising a 3’-O-protecting group has been added to a nucleic acid initiator, the extended nucleic acid may not comprise a 3’ hydroxyl (3’-OH) to which a further nucleotide may be added). Reagents, such as the TdT and unreacted protected nucleotide (e.g. unreacted protected nucleoside triphosphate), and / or waste products may then be removed, for example by washing. Subsequently, the 3’ protecting group may be cleaved from the nucleotide that has just been conjugated to the nucleotide initiator (in one or more step) to form a 3’-OH group. The product of the first step, which comprises the 3’-OH group, may then be used as an initiator for a second extension step. Repeating cycles of extension steps may provide a product oligonucleotide with a defined sequence. The sequence may be controlled through the selection of the protected nucleotide (particularly a 3’-O-protected nucleoside triphosphate) that is used for each extension step. For example, because the use of 3’-O-protected nucleotide, particularly a 3’-O-protected nucleoside triphosphate allows the addition of a single nucleotide to an initiator during each step, it may be possible to prevent addition of multiples of the same nucleotide during each extension step, and the consequent ability to select which type of nucleotide is added in each step allows a desired sequence to be built in a step-by-step method. In one aspect, the invention provides use of the terminal deoxynucleotidyl transferase (TdT) of the invention for producing an oligonucleotide. In another aspect, the invention provides a method of producing an oligonucleotide comprising a step of contacting the terminal deoxynucleotidyl transferase (TdT) of the invention with a nucleic acid initiator and a 3’-O-protected nucleotide (preferably a 3’-O-protected nucleoside triphosphate). According to any embodiment of this method, the method may further comprise removing the TdT and / or unreacted protected nucleotide. The removal may be, for example, by washing. According to any embodiment of this method, the method may further comprise inactivating the TdT. According to any embodiment, the method may further comprise cleaving the 3’-O-protecting group (e.g. from the nucleic acid initiator-protected nucleotide conjugate) to form a 3’-OH group. Suitably, the method comprises repeating cycles each comprising the step of contacting and the step of deprotecting or cleaving. Preferably, the cycles are repeated until a target oligonucleotide is produced. Preferably, the method comprises the steps: (a) contacting the nucleic acid initiator with the TdT and the 3’-O-protected nucleotide (preferably a 3’-O-protected nucleoside triphosphate) comprising a 3’-O-protecting group (e.g. to form a nucleic acid initiator-protected nucleotide conjugate); (b) removing or inactivating the TdT and / or unreacted protected nucleotide (preferably a 3’-O-protected nucleoside triphosphate) after step (a); and (c) cleaving the 3’-O-protecting group (e.g. from the nucleic acid initiator-protected nucleotide conjugate) to form a 3’-OH group. Optionally, the method further comprises repeating steps (a)-(c). In this way, the product of step (c) of cycle n of steps (a)-(c) is the nucleic acid initiator of cycle n+1 of steps (a)-(c). The term “nucleic acid initiator” may refer to a site to which a protected nucleotide may be added by a template-independent polymerase, such as a TdT. The nucleic acid initiator may be, for example, a nucleotide or an oligonucleotide with a free 3’-end (e.g. a 3’-OH group) to which the protected nucleotide (particularly a 3’-O-protected nucleotide) can be attached. According to any embodiment of this process, the nucleic acid initiator is a deoxyribooligonucleotide. Particularly, the nucleic acid initiator is an oligonucleotide from 5 to 50 nucleotides in length, such as from 5 to 30 nucleotides in length, from 5 to 20 nucleotides in length, or from 5 to 15 nucleotides in length. More particularly, the nucleic acid initiator comprises a single-stranded oligonucleotide. The nucleic acid initiator may be bound to a solid support, for example an array, which may aid washing and purification. At the conclusion of the required number of extension steps a product oligonucleotide may be optionally deprotected (e.g. where the base moieties are protected) and cleaved from the solid support and isolated for subsequent use. Increased efficiencies may be achieved with such methods as they are amenable to highly parallel synthesis. For example, highly parallel synthesis of many oligonucleotides can be achieved by spatial localisation of reaction conditions to selectively remove protecting groups. According to any embodiment of this process, the nucleic acid initiator is attached to a solid support, for example an array or a chip (such as a thermally-addressable chip). A solid support may allow removal of the TdT and unreacted protected nucleotide (particularly unreacted protected nucleoside triphosphate) without concomitant removal of a product oligonucleotide. Particularly, the nucleic acid initiator is attached to a solid support by a reversible interacting moiety, such as a cleavable linker, an antibody-epitope complex, a biotin-biotin binding protein (e.g. avidin or streptavidin) complex, or glutathione-GST complex. The nucleic acid initiator may, for example, be attached to a solid support by a photo-labile, pH-labile, electrochemically-labile or thermally cleavable linker. More particularly, the nucleic acid initiator is attached to a solid support by a chemically-cleavable linker, such as a disulfide, allyl or azide-masked hemiaminal ether linker. In some embodiments, the method further comprises releasing a product oligonucleotide from the solid support by cleaving the linker, for example by addition of tris(2-carboxyethyl)phosphine (TCEP) or dithiothreitol (DTT) for a disulfide linker; palladium complexes for an allyl linker or TCEP for an azide-masked hemiaminal ether linker. The term “3’-O-protecting group” refers to a protecting group at the 3’ position of a nucleotide, which may be removed to form a 3’-hydroxyl (3’-OH). For example, if the 3’-O-protecting group is a group X, this may form -3’-O-X. According to any embodiment of this process, the 3’-O-protecting group is a thermo-labile 3’-O-protecting group, a photolabile 3’-O-protecting group, a pH-labile protecting group or an electrochemically-labile protecting group. In preferred embodiments, the 3’-O-protecting group is a thermo-labile 3’-O-protecting group. According to any embodiment of the above-described process, the 3’-O-protecting group may be selected from: CH2N3, -NH2, O-allyl, -CH2SSCH3, phenoxyacetyl, methoxyacetyl, acetyl, p-toluenesulfonate, phosphate, nitrate, [4-methoxy]-tetrahydrothiopyranyl, tetrahydrothiopyranyl, [5-methyl]-tetrahydrofuranyl, [2-methyl,4-methoxy]-tetrahydropyranyl, [5-methyl]-tetrahydropyranyl, tetrahydrothiofuranyl and 2-nitrobenzyl. According to any embodiment of the above-described process, the 3’-O-protecting group may be selected from: CH2N3, -NH2, O-allyl, -CH2SSCH3, phenoxyacetyl, methoxyacetyl, acetyl, p-toluenesulfonate, [4-methoxy]-tetrahydrothiopyranyl, tetrahydrothiopyranyl, [5-methyl]-tetrahydrofuranyl, [2-methyl,4-methoxy]-tetrahydropyranyl, [5-methyl]-tetrahydropyranyl, tetrahydrothiofuranyl and 2-nitrobenzyl. Particularly, the 3’-O-protecting group is azidomethyl, amino or allyl. In some embodiments, the 3’-O-protecting group is a 2-nitrobenzyl group. In some embodiments, the 3’-O-protecting group is an azidomethyl group. In some embodiments, the 3’-O-protecting group is -CH2ONH2. In some embodiments, the method further comprises a step of deprotecting the protected nucleotide after the protected nucleotide has been conjugated to the nucleic acid initiator. In some embodiments, the method further comprises a step of cleaving the 3’-O-protecting group to form a 3’-OH group The term “deprotection” may refer to the removal of a protecting group, for example a 3’ protecting group may be deprotected to form a 3’-hydroxyl (3’-OH). The term “conjugated” may refer to the joining (e.g. through formation of a covalent bond) of two entities, for example conjugation of a nucleotide and a nucleic acid initiator may result in the nucleotide being covalently bonded to a 3’ position of the nucleic acid initiator. In preferred embodiments, the step of deprotecting or cleaving is a thermal deprotection step. The term “thermal deprotection” may refer to the removal of a protecting group by the application of conditions that include heat. The invention also relates to a method of producing oligonucleotides using a templateindependent polymerase, such as a TdT, wherein protected nucleotide substrates may be used that comprise a first 3’ protecting group that may be small in size, but which may for example not be removable under reaction conditions that do not damage a product oligonucleotide. The first 3’ protecting group may be sized to reduce or prevent steric hindrance with the template-independent polymerase, such as the TdT. The first 3’ protecting group may be activated once the protected nucleotide has been added to a nucleic acid initiator (e.g. a growing oligonucleotide) to form a second protecting group. The second protecting group may be removable, for example may be thermo-labile. The term “activating” may refer to the modification of a first entity (e.g. a first protecting group) to form a second entity (e.g. a second protecting group), wherein the second entity has different properties (e.g. reduced stability under certain conditions, such as high temperature) to the first entity. In one aspect, the invention provides a method of producing an oligonucleotide, wherein the method comprises the steps: (a) contacting a nucleic acid initiator with a template-independent polymerase and a protected nucleotide (preferably a nucleoside triphosphate)comprising a first 3’-O-protecting group; (b) activating the first 3’-O-protecting group to form a second 3’-O-protecting group; and (c) cleaving the second 3’-O-protecting group to form a 3’-OH group. Particularly, the template-independent polymerase is a terminal deoxynucleotidyl transferase (TdT). In one aspect, the invention provides a method of producing an oligonucleotide, wherein the method comprises the steps: (a) contacting a nucleic acid initiator with a terminal deoxynucleotidyl transferase (TdT) and a protected nucleotide, particularly a protected nucleoside triphosphate, comprising a first 3’-O-protecting group; (b) activating the first 3’-O-protecting group to form a second 3’-O-protecting group; and (c) cleaving the second 3’-O-protecting group to form a 3’-OH group. In the methods of the invention, preferably the method steps are carried out in the order (a) -> (b) —> (c). Preferably, the first 3’-O-protecting group is activated to form the second 3’-O-protecting group after the protected nucleotide has been added to the nucleic acid initiator. According to this process, in step (a), the TdT effects the conjugation of the protected nucleotide (i.e. the incoming protected nucleotide) onto the nucleic acid initiator (for example a nucleotide or oligonucleotide), to produce a nucleic acid initiator that is increased in length by one nucleotide. Particularly, the first 3’-O-protecting group is stable under the conditions of step (a). The conjugated nucleotide, still containing the first 3’-O-protecting group, is then activated in step (b), e.g. the first 3’-O-protecting group is derivatised to form a second 3’-O-protecting group, which is labile to cleavage (e.g under thermal, pH, LIV, electrochemical conditions). In step (c), the second 3’-O-protecting group is cleaved under suitable conditions to release the 3’-OH group, thereby making it available for another cycle of conjugation, activation and cleavage, in order to grow the oligonucleotide. In preferred embodiments, the second 3’-O-protecting group is a thermo-labile 3’-O-protecting group. Preferably, the first 3’-O-protecting group is a thermo-stable 3’-O-protecting group. Preferably, the first 3’ protecting group does not sterically interfere with the templateindependent polymerase (e.g. the TdT). Particularly the first 3’-O-protecting group remains intact during the enzymatic conjugation of the nucleotide and a nucleic acid initiator. As discussed above, in step (b), the first 3’-O-protecting group may be activated to form the second 3’-O-protecting group. In particular, the first 3’-O-protecting group is derivatised, for example by reaction with a suitable reagent to form the second 3’-O-protecting group. The term “thermo-labile” as used herein, particularly with reference to the second 3’-O-protecting group, refers to a protecting group which can be cleaved (i.e. removed) by heating, optionally in the presence of an acid or base, or water, and preferably an acid or base, more preferably an acid. In preferred embodiments, the step of cleaving is a thermal deprotection step. In some embodiments, the method further comprises repeating steps (a)-(c), wherein the product of step (c) of cycle n of steps (a)-(c) is the nucleic acid initiator of cycle n+1 of steps (a)-(c). In some embodiments, the method further comprises removing the TdT after step (a) and before step (b). In some embodiments, the method further comprises removing the TdT and / or 31 unreacted protected nucleotide, preferably unreacted protected nucleoside triphosphate, after step (a) and before step (b). In some embodiments, the method further comprises inactivating the TdT after step (a) and before step (b). In some embodiments, the method further comprises inactivating the TdT and / or removing unreacted protected nucleotide, preferably unreacted protected nucleoside triphosphate, after step (a) and before step (b). In some embodiments, the second 3’-O-protecting group is a thermo-labile 3’-O-protecting group, a photolabile 3’-O-protecting group, a pH-labile protecting group or an electrochemically-labile protecting group. In preferred embodiments, the second 3’-O-protecting group is a thermo-labile 3’-O-protecting group. In some embodiments, the first 3’-O-protecting group is a thermo-stable 3’-O-protecting group. In some embodiments, the first 3’-O-protecting group is -CH2ONH2. In some embodiments, the second 3’-O-protecting group is -CH2ONC(CH3)COOH, or a salt thereof. Preferably, the TdT is capable of adding the protected nucleotide comprising the first 3’-O-protecting group to a 3’-OH of a nucleic acid initiator in the absence of a nucleic acid template. In some embodiments, the TdT comprises or consists of an amino acid sequence that has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 2. In some embodiments, the TdT comprises the amino acid sequence SEQ ID NO: 2. In some embodiments, the TdT consists of the amino acid sequence SEQ ID NO: 2. In some embodiments, the TdT is the TdT of the invention. KIT In another aspect, the invention provides a kit comprising the terminal deoxynucleotidyl transferase (TdT) of the invention. In some embodiments, the kit comprises one or more protected nucleotides, preferably one or more protected nucleoside triphosphates, comprising a 3’-O-protecting group. Particularly, the kit comprises protected adenine, cytosine, guanine and / or thymine nucleotides each comprising a 3’-O-protecting group, more particularly adenine, cytosine, guanine and / or thymine nucleoside triphosphates each comprising a 3’-O-protecting group. The kit may include instructions for use. The skilled person will understand that they can combine all features of the invention disclosed herein without departing from the scope of the invention as disclosed. Preferred features and embodiments of the invention will now be described by way of nonlimiting examples. The practice of the present invention will employ, unless otherwise indicated, conventional techniques of chemistry, biochemistry, molecular biology, microbiology and immunology, which are within the capabilities of a person of ordinary skill in the art. Such techniques are explained in the literature. See, for example, Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press; Ausubel, F.M. et al. (1995 and periodic supplements) Current Protocols in Molecular Biology, Ch. 9, 13 and 16, John Wiley &Sons; Roe, B., Crabtree, J. and Kahn, A. (1996) DNA Isolation and Sequencing: Essential Techniques, John Wiley &Sons; Polak, J.M. and McGee, J.O’D. (1990) In Situ Hybridization: Principlesand Practice, Oxford University Press; Gait, M.J. (1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press; and Lilley, D.M. and Dahlberg, J.E. (1992) Methods in Enzymology: DNA Structures Part A: Synthesis and Physical Analysis of DNA, Academic Press. Each of these general texts is herein incorporated by reference. EXAMPLES METHODS Expression and purification of TdT candidates Plasmids for the expression of the desired TdT candidates were transformed into E. coli BL21 (DE3). Resulting strains were used to inoculate 1 L culture media (LB, 100 pg / mL ampicillin) after a pre-culture step. IPTG was added when the OD600 reached around 0.6. For His-tag affinity purification, cell pellets were collected after expression culture by centrifugation, washed in 30 mL PBS (in a 50 mL tube), centrifuged again and frozen (-20°C). On the next day, cell pellets were thawed completely with 10 mL lysis buffer (Tris 20 mM pH8, 500 mM NaCI, Bugbuster 1X, Benzonase 1 pL / mL) and incubated under mild agitation at room temperature for 30 min. Cell debris was removed by centrifugation (1h, 4°C) to collect the clarified lysate (soluble fraction). A HisTrap HP protein purification column (1 mL) was washed with 5 volumes of His-binding buffer (Tris 20 mM pH8, 500 mM NaCI, 5 mM imidazole) before application of the soluble fraction. The column was then washed with >5 volumes of wash buffer (Tris 20 mM pH8, 500 mM NaCI, 30 mM imidazole) before elution of the protein of interest using elution buffer (Tris 20 mM pH8, 500 mM NaCI, 250 mM imidazole). Methods for detecting TdT activity Incorporation assays with analysis using agarose gels For multiple nucleotide incorporation experiments, oligonucleotide 5’-FAM-dT26 at 2 pM, was incubated (e.g. with 10 U TdT) in a 10 pL reaction volume consisting of TdT buffer (50 mM Potassium Acetate pH 7.9, 20 mM Tris-acetate, 10 mM Magnesium Acetate), 1 mM dNTP, 250 pM CoCh. Reactions were performed at 25°C. Reaction products were purified by silica columns (Oligo Clean and Concentrator, Zymo), using a 15 pL volume of water for elution. A 5% agarose gel was prepared with 1x TBE buffer (LC6675, Invitrogen). Capillary electrophoresis (CE) The standard reagent concentrations in the assays were 20 mM Tris-acetate, pH 7.9, 50 mM potassium acetate, 250 pM nucleotide, 1.5 mM cobalt chloride, 50 nM oligonucleotide and 10 U / ml yeast inorganic pyrophosphatase (YiPP). The TdT was added either from diluted cell lysate or purified enzyme. Assays were performed at either 20 °C or 37 °C. Reactions (10 pL) were terminated with 190 pl 26.3 mM EDTA. Samples were prepared using 3 pL of assay in 17 pL Hi-Di formamide (4311320, ThermoFisher Scientific) containing GeneScan 120 size standard (4324287, ThermoFisher Scientific). Samples were then analysed by capillary electrophoresis at the University of Cambridge Department of Biochemistry sequencing facility or SourceBioscience. Alternatively, reactions were performed in 50 pl volumes at 20°C by mixing a 50 nM oligonucleotide initiator (60-mer), 25 pM dTTP (ratio nucleotide / oligo = 500), 0.5 pM purified enzyme in the presence of 50 mM Potassium Acetate, 20 mM Tris-acetate, 10 mM Magnesium Acetate, pH 7.9) and 250 pM C0CI2. Samples were taken after 20 and 60 min of reaction and analysed by CE. TdT activities were calculated using Excel by inferring from the CE profiles the quantity of incorporated dTTP and thus the rate of incorporation. LCMS analysis of oligonucleotides Standard conditions for LCMS assays were 100 mM HEPES, pH 8.0, 50 mM potassium acetate, 1 mM nucleotide, 1.5 mM cobalt chloride, 100 pM unlabelled oligonucleotide, 10 U / ml 34 YiPP and 50 pM purified enzyme at 37 °C. Samples were either heat-treated (80 °C, 10 mins) or run through the micron 10kDa filters (MRCPRT010, Merck Life Science UK Limited) at 14000xg for >30 mins. Samples were analysed on an Agilent LCMS using an Acquity UPLC Oligonucleotide BEH C18 column (186003950, Waters). Thermal denaturation assays Heat-induced unfolding of TdT variants was measured in triplicate over a range between 25 and 95 °C in a BioRad CFX Connect, using purified protein (5 and 10 pM final concentration) and SYPRO™ Orange Protein Gel Stain (5Xand 10X final concentrations). Protein unfolding was monitored by measuring the change in fluorescence caused by binding of the dye (Aexcitation = 488 nm; Aemission, 500-750 nm) and the midpoint of denaturation (Tm) was determined as the maximum of the first derivative for each temperature-fluorescence curve and averaged. NNK saturation mutagenesis by Golden Gate cloning Primers were designed to provide primer pairs with template-complementary 3’-ends and nonannealing 5’-ends that contained Bsal recognition sites. Only the forward primer carried an NNK codon to saturate the target site. A whole-plasmid PCR was carried out using Q5 DNA polymerase master mix. The PCR product was Dpnl-treated, column purified and then used in a single-fragment GoldenGate reaction. Following the GoldenGate reaction, DNA was again column purified before transformation. This was used to transform E. cloni electrocompetent cells. A few E. cloni colonies were subjected to sequencing to verify correct saturation mutagenesis. The rest of the E. cloni colonies were scraped, pooled and miniprepped. The miniprepped library was also directly sequenced to verify correct NNK saturation. Screening of saturation mutagenesis libraries Glycerol stocks of each library in 96-well format were used to inoculate deep-well microplates containing 300 pL LB and 100 pg / mL ampicillin (Amp). Library culture plates were incubated at 30 °C, 230 rpm for 16-18 hrs. From the overnight culture, 50 pL was used to inoculate an expression culture containing 850 pL LB and 100 pg / mL Amp per well. When the culture reached an OD600 of >0.5, it was induced with IPTG (10356553, Fisher Scientific) in LB and 100 pg / mL Amp. The final IPTG concentration in the culture was 0.5 mM. Protein expression was performed at 20 °C for 20 hrs at 230 rpm. Prior to pelleting the cells (4000 rpm, 15 mins, 4 °C), the OD600 value was measured (used for normalising the incorporation ratio) using a Molecular Device SpectraMax iD3 Multi-Mode Microplate Reader. After supernatant removal, the cell pellets were frozen at-80°C and then thawed. 200 pL 1x lysis buffer [1:1 vol: vol mixture of BugBuster (70921, Merck Millipore) and 1x TdT Buffer (50 mM Potassium Acetate, 20 mM Tris-acetate, pH 7.9)] was added to each well and the plate vortexed before incubating the plate at 25°C for 30 mins. Cell debris was removed by centrifugation (4000 rpm, 30 mins, 4°C) and the lysate transferred to a fresh plate. At this point, the lysate was left undiluted or diluted up to 30-fold in 1x TdT buffer. A 10 pL assay consisted of 5 pL of undiluted or diluted lysate, 1x TdT buffer, 1.5 mM cobalt chloride, 50 nM 5’-FAM-dT60, 10 U / mL yeast inorganic pyrophosphatase (M2403S, NEB) and substrate. The substrate was either 50 pM ddTTP, 250 pM 3’-O-AzMe-dTTP or 250 pM 3’-O-MHA-dTTP. For ddTTP assays, 2 U / mL yeast inorganic pyrophosphatase was optionally added or used. Reactions were incubated at 20°C for 20 mins for ddTTP assays and 60 mins for 3’-O-AzMe-dTTP and 3’-O-MHA-dTTP assays. Reactions were terminated with 190 pL26.3 mM EDTAand frozen at-20°C. ForCE, 3 pL of each sample was placed into a MicroAmp Optical 96-well reaction plate (N8010560, Thermo Fisher Scientific) with 17 pL Hi-Di Formamide (4311320, Thermo Fisher Scientific) containing GeneScan 120 Size Standard (4324287, Thermo Fisher Scientific). EXAMPLE 1 Terminal deoxynucleotidyl transferase (TdT) engineering Candidate engineered enzyme sequences were generated following analysis of a number of terminal deoxynucleotidyl transferase (TdT) and DNA polymerase mu (Pol p) sequences. Sequences were tested for expression, thermostability and TdT activity in the absence of a template. Mouse TdT (SEQ ID NO: 2) was used as a control for comparing activity and additional characteristics, such as soluble expression and thermostability. EXAMPLE 2 Expression and purification of TdTs Genes corresponding to desired candidate TdTs were synthesised and cloned into a bacterial expression vector under the control of a T7 promoter. The candidate TdT-coding sequences were cloned to enable expression of the candidate TdT fused to a His tag-Thioredoxin tag fusion at the N-terminus of the candidate TdT. A thrombin cleavage site was positioned between the thioredoxin tag and the candidate TdT to allow for removal of the tags. Fusion constructs comprised from N- to C-terminus: (1) a 6xHis Tag, (2) a Thioredoxin tag (TrxA, 109 residues), (3) a thrombin cleavage site, and (4) the catalytic domain of the candidate TdT. Candidate TdTs were expressed in E. coli BL21(DE3) and optionally purified using Ni-NTA affinity chromatography. A number of TdTs were obtained with good yield and purity, including mouse TdT (EVO2; SEQ ID NO: 2), and engineered TdTs EVO33 (SEQ ID NO: 3) and mutants thereof (e.g. SEQ ID NOs: 4 and 5) (Figure 2A shows expression analysis of a protein comprising the EVO33 sequence SEQ ID NO: 3; Figure 2B shows expression analysis of a protein comprising the EVO2 sequence SEQ ID NO: 2). EXAMPLE 3 Characterisation of candidate TdTs Candidate TdTs were compared against mouse TdT (EVO2) as a control. EVO33 was found to have improved soluble expression, improved activity in terminal transferase assays, and improved thermostability (Figure 3). Terminal transferase assays were performed to calculate the rate of incorporation at 20°C for the purified enzymes. EVO33 demonstrated an activity about 1.5-fold greater than that of the EVO2 control. Thermostability was also analysed and EVO33 demonstrated considerably higher thermostability compared to the EVO2 control (Tm values up to about 61 °C for EVO33, which is about 21°C greater than the EVO2 control). EXAMPLE 4 Further TdT engineering The EVO33 TdT was used for further engineering towards improved catalytic and stability properties. Saturation mutagenesis was performed on residues in close proximity to the 3’ hydroxyl of the deoxyribose moiety or the base of the incoming nucleotide, based on an analysis of the active site of murine TdT (PDB ID 4I2J), in particular R312, E315 and R52 (based on a numbering convention with reference to SEQ ID NO: 3). To introduce NNK saturation individually to chosen sites, a Golden-Gate style approach was used. A functional assay was carried out to screen libraries for activity against 3’-O-protected nucleotides. This consisted of (i) subjecting the naive libraries to a primary screen using commercially available 3’-O-protected nucleotides (ddTTP and 3’-O-AzMe-dTTP (3’-O-azidomethyl-dTTP)) to identify active variants before (ii) confirming the properties of those potential hits in triplicate in a secondary screen against a target substrate, 3’-O-MHA-dTTP (3’-O-CH2ONH2-dTTP). Libraries were subjected to a medium-throughput functional screen assaying TdT activity in cell lysate using capillary electrophoresis (CE) as a read-out. Primary screen of TdT libraries Upon cell growth and protein expression, E. coli cell pellets in each well of the library plates were lysed, cell debris removed and then incubated with an oligonucleotide, the 3’-O-protected nucleotide and cobalt ions at 20 °C. All reactions were terminated with the addition of EDTA to strip the enzymes of the cobalt ions before analysis of the oligonucleotide products by capillary electrophoresis (CE) (Figure 4A). The areas of the N and N+1 peaks enable the change between the variant and parent enzymes to be calculated using the incorporation ratio (Figure 4B) followed by normalisation with the optical density value at 600 nm (OD600) of the E. coli culture prior to cell lysis. This analysis utilised a 5’-FAM-labelled poly-deoxythymidine oligonucleotide (5’-FAM-dT60) so peaks representing 60 nt (N) and 61 nt (N+1) were the peaks of interest. TdTs typically show higher activity with ddNTP than with large 3’-O-protecting groups. To identify the optimal conditions for testing each of the EVO2 and EVO33 libraries, they were initially assayed with ddTTP to determine the conditions for library activity and to identify any variants with improved activity compared to the parent. Any variants that showed increased activity were Sanger sequenced. EVO33 libraries had high activity with ddTTP and the lysates had to be diluted 30-fold before assaying to produce an incorporation ratio of <75% conversion to allow sufficient comparison between parent and variant TdTs. No activity was observed with the EVO2 libraries when using undiluted lysate. Each library was then assayed with 3’-O-AzMe-dTTP. As the protecting group is much larger than the natural 3’-hydroxyl, it was expected that all enzymes would be slower to incorporate 3’-O-AzMe-dTTP than ddTTP. When using undiluted lysate with EVO2 libraries, no activity was observed suggesting that these TdT enzymes were too inefficient with the larger protecting group for an N+1 peak to be observed on the CE spectra or that the TdTs may be slower than the exonucleases in the E. coli cell lysate preventing activity of these enzymes being observed. The EVO33 libraries showed activity with 3’-O-AzMe-dTTP requiring a 4-fold lysate dilution rather than undiluted lysate to prevent too high incorporation ratios so that the parent and variant enzymes could be better compared (Figure 5). Unique hits were identified for L7 (E315) and L8 (R312) libraries with improved activity with 3’-O-AzMe-dTTP. Secondary screen of unique TdT variants The variants selected for the second round of screening for L7 (E315) were Ala, Arg, Cys, Gin, Gly, His, Lys, Met, Pro, Ser, Trp, Phe and Tyr. For L8 (R312), the variants were Ala, Glu, lie, Leu and Thr. All the individual hit variants identified from the 3’-O-AzMe-dTTP assays for L7 and L8 were grown in triplicate and assayed with both 3’-O-AzMe-dTTP and 3’-O-MHA-dTTP separately. This was performed to identify whether the same unique hit variants used with 3’-O-AzMe-dTTP would result in also being unique hits with 3’-O-MHA-dTTP. Based on the primary screen with 3’-O-AzMe-dTTP, 29 variants were selected that represented 12 unique residue hits for L7 (Figure 6A). Three further hits were also included that also encoded glutamate at the E315 position. While most of the tested variants clustered around the parent, variants containing lysine and glycine were >1.5-fold better than the parent with 3’-O-MHA-dTTP where only lysine was >1.5-fold better than the parent with both substrates. For L8,11 variants representing five unique residue hits were screened (Figure 6B), all hits showed a >1.5-fold increase relative to the parent for both nucleotides, but more so for 3’-O-MHA-dTTP. From this analysis, lysine and glycine could be placed in the E315 position and / or threonine, leucine and isoleucine in the R312 position of EVO33. Table 1. Relative fold changes of each variant from L7 (E315) and L8 (R312) with 3’-O-AzMe-dTTP and 3’-O-MHA-dTTP. The error shown is standard error. Library Variant Fold change relative to parent Library Variant Fold change relative to parent 3’-O-AzMe-dTTP 3’-O-MHA-dTTP 3’-O-AzMe-dTTP 3’-O-MHA-dTTP L7 E315K 1.92±0.17 2.31±0.11 L8 R312A 1.57±0.05 1.61±0.05 E315W 0.75±0.15 0.81±0.18 R312E 1.80±0.17 1.72±0.33 E315Q 0.69±0.04 1.43±0.17 R312T 1.82±0.22 2.95±0.30 E315M 0.81±0.02 1.07±0.11 R312L 1.80±0.26 3.32±0.41 E315G 0.65±0.06 1.65±0.12 R312I 1.65±0.15 4.31±0.55 E315R 1.13±0.05 1,27±0.06 E315H 0.68±0.07 0.93±0.18 Parent (EVO33) 1.00±0.12 1.00±0.12 E315A 0.89±0.07 0.89±0.07 E315C 0.91±0.04 0.91±0.04 E315P 0.63±0.04 0.63±0.04 E315S 0.81±0.06 0.81 ±0.06 E315Y 0.91±0.40 0.91 ±0.04 E315F 1.20±0.07 0.88±0.09 Further rational design of engineered TdTs was then carried out by incorporation of an R52Q mutation into EVO33 and the E315K mutation thereof. The EVO33 R52Q / E315K TdT was demonstrated to have further improved activity in DNA synthesis (Figure 7), with over 10-fold improvement in the rate of DNA synthesis compared to an EVO2 control experiment. EXAMPLE 5 Protecting group activation To aid accommodation of a 3’ protecting group by TdTs, a small and stable protecting group which can be activated, for example to form a thermally sensitive protecting group, may be used. The activated protecting group may then be selectively removed, for example heat may be used to trigger a selective deprotection, allowing the addition of a further nucleotide by TdT. We used MHA (-CH2ONH2) as a small, stable group to protect the 3’ position of a nucleotide (e.g. a thymidine analogue). We then demonstrated conversion of the -MHA protecting group to an activated protecting group, as shown in the schematic below. To a stirred solution of 3’-O-MHA-dT (15 pL) in water (2.5 mg / mL) was added a solution (15 pL) of sodium pyruvate (50 mg / mL) and AcOH (0.5 pL / mL) and the reaction was followed by LCMS. The reaction was complete after 5 minutes after which time the solvent was removed under reduced pressure to give the crude material. This compound was dissolved in aqueous 1 M NaOH (50 pL). The solution was kept at room temperature for 6 h to cleave the ethyl ester. 1 M aq. AcOH was added to neutralise excess NaOH. The reaction mixture was then evaporated to dryness under reduced pressure to give the sodium salt of the product. This was purified by HPLC for use in kinetics experiments. We then tested the kinetics of deprotection of the activated protecting group under different conditions: Using TBACI (0.5 M) in water Using TBACI (0.5 M) in DMSO / Water (9:1) Temp (°C) Half life ti / 2 (sec) Temp (°C) Half life ti / 2 (sec) 70 10555 50 2802 80 3106 60 771 90 908 70 233 Ea -127 kJ / mol Ea -114 kJ / mol Optimal reaction time -13400 sec Optimal reaction time -329 sec Minimum error rate -0.05% Minimum error rate -0.11% The reaction kinetics show its suitability as a thermally-sensitive protecting group. Particularly good results were obtained in the DMSO-water system. We also showed activation of the -MHA protecting group in a single step using pyruvate 5 acetate, including on a chip. All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the disclosed enzymes, polynucleotides, cells, kits, uses and methods of the invention will be apparent to the skilled person without departing from the scope and spirit of the invention. Although the invention has been disclosed in 10 connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the disclosed modes for carrying out the invention, which are obvious to the skilled person are intended to be within the scope of the following claims.

Claims

1. A terminal deoxynucleotidyl transferase (TdT) comprising an amino acid sequence that has at least 75% sequence identity to SEQ ID NO: 3.

2. The TdT of claim 1, wherein the TdT comprises one or more mutation with respect to SEQ ID NO: 3 at an amino acid position selected from the group consisting of 315, 52 and 312, wherein the amino acids are numbered with reference to SEQ ID NO: 3.

3. The TdT of claim 1 or 2, wherein the TdT comprises one or more mutation with respect to SEQ ID NO: 3, wherein the one or more mutation is selected from the group consisting of E315K, R52Q, E315R, E315Q, E315M, E315G, E315F, R312T, R312L, R312I, R312A and R312E, wherein the amino acids are numbered with reference to SEQ ID NO: 3.

4. The TdT of any preceding claim, wherein the TdT comprises the mutation E315K, wherein the amino acids are numbered with reference to SEQ ID NO: 3.

5. The TdT of any preceding claim, wherein the TdT comprises the mutations R52Q and E315K, wherein the amino acids are numbered with reference to SEQ ID NO: 3.

6. The TdT of any preceding claim, wherein the TdT comprises the mutation R312I, wherein the amino acids are numbered with reference to SEQ ID NO: 3.

7. The TdT of any preceding claim, wherein the TdT exhibits an improved property as compared to SEQ ID NO: 2 or 3.

8. The TdT of claim 7, wherein the improved property is improved thermostability, improved enzymatic activity and / or improved soluble expression.

9. The TdT of any preceding claim, wherein the TdT exhibits at least 1.1-fold the thermostability, enzymatic activity or soluble expression of SEQ ID NO: 2 or 3.

10. The TdT of any preceding claim, wherein the TdT comprises the amino acid sequence SEQ ID NO: 3, 4, 5 or 6.

11. A polynucleotide comprising a nucleic acid sequence encoding the terminal deoxynucleotidyl transferase (TdT) of any preceding claim.

12. A vector comprising the polynucleotide of claim 11.

13. A cell comprising the polynucleotide or vector of claim 11 or 12.

14. A kit comprising the terminal deoxynucleotidyl transferase (TdT) of any one of claims 1-10 and one or more protected nucleoside triphosphate comprising a 3’-O-protecting group.

15. Use of the terminal deoxynucleotidyl transferase (TdT) of any one of claims 1-10 for producing an oligonucleotide.

16. A method of producing an oligonucleotide comprising a step of contacting the terminal deoxynucleotidyl transferase (TdT) of any one of claims 1-10 with a nucleic acid initiator and a protected nucleoside triphosphate comprising a 3’-O-protecting group.

17. The method of claim 16 further comprising a step of deprotecting the protected nucleotide after the protected nucleotide has been conjugated to the nucleic acid initiator.

18. The method of claim 17, wherein the step of deprotecting is a thermal deprotection step.

19. A method of producing the terminal deoxynucleotidyl transferase (TdT) of any one of claims 1-10 comprising culturing the cell of claim 13 under conditions for expressing the TdT.

20. A method of producing an oligonucleotide, wherein the method comprises the steps:(a) contacting a nucleic acid initiator with a terminal deoxynucleotidyl transferase (TdT) and a protected nucleotide (preferably a protected nucleoside triphosphate) comprising a first 3’-O-protecting group;(b) activating the first 3’-O-protecting group to form a second 3’-O-protecting group; and(c) cleaving the second 3’-O-protecting group to form a 3’-OH group.

21. The method of claim 20, wherein the second 3’-O-protecting group is a thermo-labile 3’-O-protecting group.

22. The method of claim 20 or 21, wherein the first 3’-O-protecting group is -CH2ONH2.

23. The method of any one of claims 20-22, wherein the second 3’-O-protecting group isa -CH2ONC(CH3)COOH, or a salt thereof.

24. The method of any one of claims 20-23, wherein the TdT is:(a) a TdT comprising an amino acid sequence that has at least 75% sequence identity to SEQ ID NO: 2; or(b) theTdT of any one of claims 1-10.

25. The method of any one of claims 20-24 further comprising repeating steps (a)-(c), wherein the product of step (c) of cycle n of steps (a)-(c) is the nucleic acid initiator of cycle n+1 of steps (a)-(c).5 26. The method of any one of claims 20-25 further comprising removing or inactivating theTdT after step (a) and before step (b).Application No: GB2313749.0Examiner: Dr Andrew GuyClaims searched: 1-19Date of search: 22 February 2024Patents Act 1977: Search Report under Section 17Documents considered to be relevant:Category Relevant to claims Identity of document and passage or figure of particular relevance X 1-19 WO 2020 / 099451 Al (DNA SCRIPT) See SEQ ID NO 28 X 1-19 WO 2021 / 213903 Al (DNA SCRIPT) See SEQ ID NO 18 X 1-19 WO 2022 / 063835 Al (DNA SCRIPT) See SEQ ID NO 22 v A 1-19 WO 2020 / 161480 Al (NUCLERA LTD) See at least SEQ ID NOs 547, 570, 675 A - ACS Synthetic Biology, vol. 9, 2020, Chua et al, "Evolving a thermostable terminal deoxynucleotidyl transferase" pp. 1725-1735 See abstract A - Genes, vol. 11, 2020, Barthel et al, "Enhancing terminal deoxynucleotidyl transferase activity on substrates..." art. 102 See abstractCategories:X Document indicating lack of novelty or inventive step A Document indicating technological background and / or state of the art. Y Document indicating lack of inventive step if P Document published on or after the declared priority date but combined with one or more other documents of same category. before the filing date of this invention. & Member of the same patent family E Patent document published on or after, but with priority date earlier than, the filing date of this application.Field of Search:International Classification:Subclass Subgroup Valid From C12N 0009 / 12 01 / 01 / 2006 C12P 0019 / 34 01 / 01 / 2006

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