Patent Description:
Nucleic acid synthesis is vital to modern biotechnology. The rapid pace of development in the biotechnology arena has been made possible by the scientific community's ability to artificially synthesise DNA, RNA and proteins.

Artificial DNA synthesis - a E1 billion and growing market - allows biotechnology and pharmaceutical companies to develop a range of peptide therapeutics, such as insulin for the treatment of diabetes. It allows researchers to characterise cellular proteins to develop new small molecule therapies for the treatment of diseases our aging population faces today, such as heart disease and cancer. It even paves the way forward to creating life, as the Venter Institute demonstrated in <NUM> when they placed an artificially synthesised genome into a bacterial cell.

However, current DNA synthesis technology does not meet the demands of the biotechnology industry. While the benefits of DNA synthesis are numerous, an oft-mentioned problem prevents the further growth of the artificial DNA synthesis industry, and thus the biotechnology field. Despite being a mature technology, it is practically impossible to synthesise a DNA strand greater than <NUM> nucleotides in length, and most DNA synthesis companies only offer up to <NUM> nucleotides. In comparison, an average protein-coding gene is of the order of <NUM> - <NUM> nucleotides, and an average eukaryotic genome numbers in the billions of nucleotides. Thus, all major gene synthesis companies today rely on variations of a "synthesise and stitch' technique, where overlapping <NUM>-<NUM>-mer fragments are synthesised and stitched together by PCR (see <NPL>). Current methods offered by the gene synthesis industry generally allow up to <NUM> kb in length for routine production.

The reason DNA cannot be synthesised beyond <NUM>-<NUM> nucleotides at a time is due to the current methodology for generating DNA, which uses synthetic chemistry (i.e., phosphoramidite technology) to couple a nucleotide one at a time to make DNA. As the efficiency of each nucleotide-coupling step is <NUM> - <NUM>% efficient, it is mathematically impossible to synthesise DNA longer than <NUM> nucleotides in acceptable yields. The Venter Institute illustrated this laborious process by spending <NUM> years and <NUM> million USD to synthesise the relatively small genome of a bacterium (see <NPL>).

Known methods of DNA sequencing use template-dependent DNA polymerases to add <NUM>'-reversibly terminated nucleotides to a growing double-stranded substrate (see,<NPL>). In the 'sequencing-by-synthesis' process, each added nucleotide contains a dye, allowing the user to identify the exact sequence of the template strand. Albeit on double-stranded DNA, this technology is able to produce strands of between <NUM>-<NUM> bps long. However, this technology is not suitable for de novo nucleic acid synthesis because of the requirement for an existing nucleic acid strand to act as a template.

Various attempts have been made to use a terminal deoxynucleotidyl transferase for controlled de novo single-stranded DNA synthesis (see <NPL>, <CIT> and <CIT>). Uncontrolled de novo single-stranded DNA synthesis, as opposed to controlled, takes advantage of TdT's deoxynucleotide triphosphate (dNTP) <NUM>' tailing properties on single-stranded DNA to create, for example, homopolymeric adaptor sequences for next-generation sequencing library preparation (see <NPL> and <CIT>). A reversible deoxynucleotide triphosphate termination technology needs to be employed to prevent uncontrolled addition of dNTPs to the <NUM>'-end of a growing DNA strand. The development of a controlled single-stranded DNA synthesis process through TdT would be invaluable to in situ DNA synthesis for gene assembly or hybridization microarrays as it removes the need for an anhydrous environment and allows the use of various polymers incompatible with organic solvents (see <NPL> and <CIT>).

However, TdT has not been shown to efficiently add nucleotide triphosphates containing <NUM>'-O reversibly terminating moieties for building up a nascent single-stranded DNA chain necessary for a de novo synthesis cycle. A <NUM>'-O reversible terminating moiety would prevent a terminal transferase like TdT from catalysing the nucleotide transferase reaction between the <NUM>'-end of a growing DNA strand and the <NUM>'-triphosphate of an incoming nucleotide triphosphate. Data is presented herein which demonstrates that the widely commercially available recombinant TdT sourced from calf thymus is unable to add <NUM>'-O-terminated nucleotide triphosphates in a quantitative fashion (see <FIG>). In previous reports, the TdT specifically mentioned is recombinant TdT from calf thymus (see <NPL>, <CIT> and <CIT>) or uses a different reversible terminating mechanism not located on the <NUM>' end of the deoxyribose moiety (see <CIT>).

Most DNA and RNA polymerases contain highly selective sugar steric gates to tightly discriminate between deoxyribose and ribose nucleotide triphosphate substrates (see<NPL>). The result of this sugar steric gate is the enormous challenge of finding and/or engineering polymerases to accept sugar variants for biotechnology reasons, such as sequencing-by-synthesis (see <NPL> and <CIT>). The challenge of finding a polymerase that accepts a <NUM>'-O reversibly terminating nucleotide is so large, various efforts have been made to create reversible terminating nucleotides where the polymerase termination mechanism is located on the nitrogenous base of the terminating nucleotide (see <NPL> and <CIT>).

There is therefore a need to identify terminal deoxynucleotidyl transferases that readily incorporate <NUM>'-O reversibly terminated nucleotides and modified said terminal deoxynucleotidyl transferases to incorporate <NUM>'-O reversibly terminated nucleotides in a fashion useful for biotechnology and single-stranded DNA synthesis processes in order to provide an improved method of nucleic acid synthesis that is able to overcome the problems associated with currently available methods.

There is disclosed herein the use of a terminal deoxynucleotidyl transferase (TdT) enzyme comprising an amino acid sequence selected from either: (a) any one of SEQ ID NOS: <NUM> to <NUM> and <NUM> or a functional equivalent or fragment thereof having at least <NUM>% sequence homology to said amino acid sequence; or (b) a modified derivative of SEQ ID NO: <NUM>; in a method of nucleic acid synthesis.

According to a first aspect of the invention, there is provided a method of nucleic acid synthesis, which comprises the steps of:.

According to a further aspect of the invention, there is provided the use of a kit in a method of nucleic acid synthesis, wherein said kit comprises a TdT as defined in the first aspect of the invention, one or more nucleoside triphosphates blocked by either a <NUM>'-O-azidomethyl, <NUM>'-aminoxy or <NUM>'-O-allyl group, and an initiator sequence which is an oligonucleotide with a free <NUM>'-end and is between <NUM> and <NUM> nucleotides long, optionally in combination with one or more components selected from: inorganic pyrophosphatase, such as purified, recombinant inorganic pyrophosphatase from Saccharomyces cerevisiae, and a cleaving agent; further optionally together with instructions for use of the kit in accordance with any of the methods defined herein.

There is also disclosed the use of a <NUM>'-blocked nucleotide triphosphate in a method of template independent nucleic acid synthesis, wherein the <NUM>'-blocked nucleotide triphosphate is selected from a compound of formula (I), (II), (III) or (IV):
<CHM>
<CHM>
wherein.

Also disclosed herein is the use of inorganic pyrophosphatase in a method of nucleic acid synthesis.

According to a first aspect of the disclosure, there is provided the use of a terminal deoxynucleotidyl transferase (TdT) enzyme comprising an amino acid sequence selected from either: (a) any one of SEQ ID NOS: <NUM> to <NUM> and <NUM> or a functional equivalent or fragment thereof having at least <NUM>% sequence homology to said amino acid sequence; or (b) a modified derivative of SEQ ID NO: <NUM>; in a method of nucleic acid synthesis.

According to one particular aspect of the disclosure which may be mentioned, there is provided the use of a terminal deoxynucleotidyl transferase (TdT) enzyme comprising an amino acid sequence selected from either: (a) any one of SEQ ID NOS: <NUM> to <NUM> or a functional equivalent or fragment thereof having at least <NUM>% sequence homology to said amino acid sequence; or (b) a modified derivative of SEQ ID NO: <NUM>; in a method of nucleic acid synthesis.

The present invention relates to the identification of never before studied terminal deoxynucleotidyl transferases that surprisingly have the ability to incorporate deoxynucleotide triphosphates with large <NUM>'-O reversibly terminating moieties.

Since commercially available recombinant TdT sourced from calf thymus does not readily incorporate <NUM>'-O reversibly terminated nucleotides, it is most unexpected that the present inventors have located a terminal deoxynucleotidyl transferase, which is a DNA polymerase, to accept a <NUM>'-O reversibly terminating nucleotide, such as dNTPs modified with a <NUM>'-O-azidomethyl.

Furthermore, the present invention relates to engineered terminal deoxynucleotidyl transferases, which achieve a substantial increase in incorporation rates of dNTPs containing <NUM>'-O reversibly terminating moieties to be useful for the controlled de novo synthesis of single-stranded DNA.

As controlled de novo single-stranded DNA synthesis is an additive process, coupling efficiency is extremely important to obtaining practically useful yields of final single-stranded DNA product for use in applications such as gene assembly or hybridization microarrays. Thus, the present invention relates to the identification of TdT orthologs with the capability to add <NUM>'-O reversibly terminated nucleotides, and also an engineered variant of the TdT ortholog that adds <NUM>'-O reversibly terminated nucleotides in a quantitative fashion that is practically useful for a single-stranded DNA synthesis process.

The use described herein has significant advantages, such as the ability to rapidly produce long lengths of DNA while still maintaining high yields and without using any toxic organic solvents.

The terminal deoxynucleotidyl transferase (TdT) enzyme may comprise an amino acid sequence selected from any one of SEQ ID NOS: <NUM> to <NUM> and <NUM> or a functional equivalent or fragment thereof having at least <NUM>% sequence homology to said amino acid sequence.

Further, the terminal deoxynucleotidyl transferase (TdT) enzyme may comprise an amino acid sequence selected from any one of SEQ ID NOS: <NUM> to <NUM> or a functional equivalent or fragment thereof having at least <NUM>% sequence homology to said amino acid sequence.

Further, the terminal deoxynucleotidyl transferase (TdT) enzyme may comprise an amino acid sequence selected from SEQ ID NO: <NUM>. The amino acid sequence of SEQ ID NO: <NUM> is the terminal deoxynucleotidyl transferase (TdT) sequence from Sarcophilus harrisii (UniProt: G3VQ55). Sarcophilus harrisii (also known as the Tasmanian devil) is a carnivorous marsupial of the family Dasyuridae, now found in the wild only on the Australian island state of Tasmania.

Further, the terminal deoxynucleotidyl transferase (TdT) enzyme may comprise an amino acid sequence selected from SEQ ID NO: <NUM>. The amino acid sequence of SEQ ID NO: <NUM> is the terminal deoxynucleotidyl transferase (TdT) sequence from Lepisosteus oculatus (UniProt: W5MK82). Lepisosteus oculatus (also known as the spotted gar) is a primitive freshwater fish of the family Lepisosteidae, native to North America from the Lake Erie and southern Lake Michigan drainages south through the Mississippi River basin to Gulf Slope drainages, from lower Apalachicola River in Florida to Nueces River in Texas, USA.

Further, the terminal deoxynucleotidyl transferase (TdT) enzyme may comprise an amino acid sequence selected from SEQ ID NO: <NUM>. The amino acid sequence of SEQ ID NO: <NUM> is the terminal deoxynucleotidyl transferase (TdT) sequence from Chinchilla lanigera (NCBI Reference Sequence: XP_005407631. <NUM>; http://www. gov/protein/<NUM>). Chinchilla lanigera (also known as the long-tailed chinchilla, Chilean, coastal, common chinchilla, or lesser chinchilla), is one of two species of rodents from the genus Chinchilla, the other species being Chinchilla chinchilla.

Further, the terminal deoxynucleotidyl transferase (TdT) enzyme may comprise an amino acid sequence selected from SEQ ID NO: <NUM>. The amino acid sequence of SEQ ID NO: <NUM> is the terminal deoxynucleotidyl transferase (TdT) sequence from Otolemur gamettii (UniProt: A4PCE6). Otolemur gamettii (also known as the northern greater galago, Garnett's greater galago or small-eared greater galago), is a nocturnal, arboreal primate endemic to Africa.

Further, the terminal deoxynucleotidyl transferase (TdT) enzyme may comprise an amino acid sequence selected from SEQ ID NO: <NUM>. The amino acid sequence of SEQ ID NO: <NUM> is the terminal deoxynucleotidyl transferase (TdT) sequence from Sus scrofa (UniProt: F1 SBG2). Sus scrofa (also known as the wild boar, wild swine or Eurasian wild pig) is a suid native to much of Eurasia, North Africa and the Greater Sunda Islands.

In a one embodiment of the invention, the terminal deoxynucleotidyl transferase (TdT) enzyme comprises an amino acid sequence selected from SEQ ID NO: <NUM>. The amino acid sequence of SEQ ID NO: <NUM> is a variant of SEQ ID NO: <NUM> which has been engineered for improved activity by alteration of the amino acid sequence. Data are provided in Example <NUM> and <FIG>, which demonstrate the benefits of engineered variants, such as SEQ ID NO: <NUM>, over the wild-type SEQ ID NO: <NUM>.

In a further embodiment of the disclosure, the terminal deoxynucleotidyl transferase (TdT) enzyme comprises an amino acid sequence selected from SEQ ID NOS: <NUM>, <NUM> or <NUM>.

Further, the terminal deoxynucleotidyl transferase (TdT) enzyme may comprise an amino acid sequence selected from SEQ ID NO: <NUM> or <NUM>. Data are provided in Example <NUM> and <FIG>, which demonstrate beneficial results over the natural, recombinant TdT enzyme from Bos taurus.

Alternatively, the terminal deoxynucleotidyl transferase (TdT) enzyme may comprise an amino acid sequence selected from a modified derivative of SEQ ID NO: <NUM> (i.e. a non-natural, mutated derivative of SEQ ID NO: <NUM>). The amino acid sequence of SEQ ID NO: <NUM> is the terminal deoxynucleotidyl transferase (TdT) sequence from Bos taurus (UniProt: P06526). Bos taurus (also known as cattle, or colloquially cows) are the most common type of large domesticated ungulates. They are a prominent modern member of the subfamily Bovinae, are the most widespread species of the genus Bos.

References herein to "TdT" refer to a terminal deoxynucleotidyl transferase (TdT) enzyme and include references to purified and recombinant forms of said enzyme. TdT is also commonly known as DNTT (DNA nucleotidylexotransferase) and any such terms should be used interchangeably.

References herein to a "method of nucleic acid synthesis" include methods of synthesising lengths of DNA (deoxyribonucleic acid) or RNA (ribonucleic acid) wherein a strand of nucleic acid (n) is extended by adding a further nucleotide (n+<NUM>). In one embodiment, the nucleic acid is DNA. In an alternative embodiment, the nucleic acid is RNA.

References herein to "method of DNA synthesis" refer to a method of DNA strand synthesis wherein a DNA strand (n) is extended by adding a further nucleotide (n+<NUM>). The method described herein provides a novel use of the terminal deoxynucleotidyl transferases of the invention and <NUM>'-reversibly blocked nucleotide triphosphates to sequentially add nucleotides in de novo DNA strand synthesis which has several advantages over the DNA synthesis methods currently known in the art.

Current synthetic methods for coupling nucleotides to form sequence-specific DNA have reached asymptotic length limits, therefore a new method of de novo DNA synthesis is required. Synthetic DNA synthesis methods also have the disadvantage of using toxic organic solvents and additives (e.g., acetonitrile, acetic anhydride, trichloroacetic acid, pyridine, etc.), which are harmful to the environment.

An alternative, enzymatic method of nucleic acid synthesis is desirable. Natural enzymes such as DNA polymerases are able to add <NUM>,<NUM> nucleotides before disassociation. However, DNA polymerases require a template strand, thereby defeating the purpose of de novo strand synthesis.

However, a DNA polymerase, called TdT, capable of template-independent DNA synthesis is found in vertebrates. Given a free <NUM>'-end and nucleotide triphosphates, recombinant TdTs from Bos taurus and Mus musculus were shown to add ten to several hundred nucleotides onto the <NUM>'-end of a DNA strand. As shown in a paper by <NPL>) TdT will uncontrollably add nucleotide triphosphates to the <NUM>'-end of a DNA strand. However, this uncontrolled addition is unsuitable for controlled de novo strand synthesis where a sequence-specific oligonucleotide is required. Thus, commercially available recombinant TdT is used primarily as a tool for molecular biologists to label DNA with useful chemical tags.

The present inventors have discovered several orthologs of Bos taurus TdT that, coupled with <NUM>'-reversibly protected nucleotide triphosphates, are able to synthesise DNA in a controlled manner. Bos taurus TdT is not efficient at incorporating nucleotide triphosphates with <NUM>'-protecting groups, likely due to steric issues in the TdT active site. Data is presented herein in Example <NUM> and <FIG> which demonstrates that orthologs of Bos taurus TdT, such as the purified recombinant TdTs of the first aspect of the invention (in particular SEQ ID NOS: <NUM> and <NUM>), are far more efficient at incorporating <NUM>'-OH blocked nucleotide triphosphates, thereby enabling template-independent, sequence-specific synthesis of nucleic acid strands.

This enzymatic approach means that the method has the particular advantage of being able to produce DNA strands beyond the <NUM>-<NUM> nucleotide limit of current synthetic DNA synthesis methods. Furthermore, this enzymatic method avoids the need to use strong organic solvents which may be harmful to the environment.

It will be understood that the term 'functional equivalent' refers to the polypeptides which are different to the exact sequence of the TdTs of the first aspect of the invention, but can perform the same function, i.e., catalyse the addition of a nucleotide triphosphate onto the <NUM>'-end of a DNA strand in a template dependent manner.

In one embodiment, the terminal deoxynucleotidyl transferase (TdT) is a non-natural derivative of TdT, such as a functional fragment or homolog of the TdTs of the first aspect of the invention.

References herein to 'fragment' include, for example, functional fragments with a C-terminal truncation, or with an N-terminal truncation. Fragments are suitably greater than <NUM> amino acids in length, for example greater than <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> or <NUM> amino acids in length.

It will be appreciated that references herein to "homology" are to be understood as meaning the percentage identity between two protein sequences, e.g.: SEQ ID NO: X and SEQ ID NO: Y, which is the sum of the common amino acids between aligned sequences SEQ ID NO: X and SEQ ID NO: Y, divided by the shorter length of either SEQ ID NO: X or SEQ ID NO: Y, expressed as a percentage.

The terminal deoxynucleotidyl transferase (TdT) may have at least <NUM>% homology with the TdTs of the first aspect of the invention, such as at least <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>% or <NUM>% homology.

According to the disclosure, there is provided a method of nucleic acid synthesis, which comprises the steps of:.

In one embodiment, step (c) comprises removal of deoxynucleotide triphosphates and TdT, such as TdT. Thus, according to one particular aspect of the disclosure, there is provided a method of nucleic acid synthesis, which comprises the steps of:.

It will be understood that steps (b) to (e) of the method may be repeated multiple times to produce a DNA or RNA strand of a desired length. Therefore, in one embodiment, greater than <NUM> nucleotide is added to the initiator sequence, such as greater than <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> or <NUM> nucleotides are added to the initiator sequence by repeating steps (b) to (e). In a further embodiment, greater than <NUM> nucleotides are added, such as greater than <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> or <NUM> nucleotides.

References herein to 'nucleotide triphosphates' refer to a molecule containing a nucleoside (i.e. a base attached to a deoxyribose or ribose sugar molecule) bound to three phosphate groups. Examples of nucleotide triphosphates that contain deoxyribose are: deoxyadenosine triphosphate (dATP), deoxyguanosine triphosphate (dGTP), deoxycytidine triphosphate (dCTP) or deoxythymidine triphosphate (dTTP). Examples of nucleotide triphosphates that contain ribose are: adenosine triphosphate (ATP), guanosine triphosphate (GTP), cytidine triphosphate (CTP) or uridine triphosphate (UTP). Other types of nucleosides may be bound to three phosphates to form nucleotide triphosphates, such as naturally occurring modified nucleosides and artificial nucleosides.

Therefore, references herein to '<NUM>'-blocked nucleotide triphosphates' refer to nucleotide triphosphates (e.g., dATP, dGTP, dCTP or dTTP) which have an additional group on the <NUM>' end which prevents further addition of nucleotides, i.e., by replacing the <NUM>'-OH group with a protecting group.

It will be understood that references herein to '<NUM>'-block', '<NUM>'-blocking group' or '<NUM>'-protecting group' refer to the group attached to the <NUM>' end of the nucleotide triphosphate which prevents further nucleotide addition. The present method uses reversible <NUM>'-blocking groups which can be removed by cleavage to allow the addition of further nucleotides. By contrast, irreversible <NUM>'-blocking groups refer to dNTPs where the <NUM>'-OH group can neither be exposed nor uncovered by cleavage.

There exist several documented reversible protecting groups, such as azidomethyl, aminoxy, and allyl, which can be applied to the method described herein. Examples of suitable protecting groups are described in <NPL>).

In one embodiment, the <NUM>'-blocked nucleotide triphosphate is blocked by a reversible protecting group. In an alternative embodiment of the disclosure, the <NUM>'-blocked nucleotide triphosphate is blocked by an irreversible protecting group.

Therefore, in one embodiment of the disclosure, the <NUM>'-blocked nucleotide triphosphate is blocked by either a <NUM>'-O-methyl, <NUM>'-azido, <NUM>'-O-azidomethyl, <NUM>'-aminoxy or <NUM>'-O-allyl group. In a further embodiment of the invention, the <NUM>'-blocked nucleotide triphosphate is blocked by either a <NUM>'-O-azidomethyl, <NUM>'-aminoxy or <NUM>'-O-allyl group. Alternatively, the <NUM>'-blocked nucleotide triphosphate is blocked by either a <NUM>'-O-methyl or <NUM>'-azido group.

References herein to 'cleaving agent' refer to a substance which is able to cleave the <NUM>'-blocking group from the <NUM>'-blocked nucleotide triphosphate.

The <NUM>'-blocking groups described herein may all be quantitatively removed in aqueous solution with documented compounds which may be used as cleaving agents (for example, see: Wuts, P. & Greene, T. (<NUM>) 4th Ed. , John Wiley & Sons;<NPL>; <CIT> and <CIT>).

In one embodiment, the cleaving agent is a chemical cleaving agent. In an alternative embodiment, the cleaving agent is an enzymatic cleaving agent.

It will be understood by the person skilled in the art that the selection of cleaving agent is dependent on the type of <NUM>'-nucleotide blocking group used. For instance, tris(<NUM>-carboxyethyl)phosphine (TCEP) can be used to cleave a <NUM>'-O-azidomethyl group, palladium complexes can be used to cleave a <NUM>'-O-allyl group, or sodium nitrite can be used to cleave a <NUM>'-aminoxy group. Therefore, in one embodiment, the cleaving agent is selected from: tris(<NUM>-carboxyethyl)phosphine (TCEP), a palladium complex or sodium nitrite.

In one embodiment, the cleaving agent is added in the presence of a cleavage solution comprising a denaturant, such as urea, guanidinium chloride, formamide or betaine. The addition of a denaturant has the advantage of being able to disrupt any undesirable secondary structures in the DNA. In a further embodiment, the cleavage solution comprises one or more buffers. It will be understood by the person skilled in the art that the choice of buffer is dependent on the exact cleavage chemistry and cleaving agent required.

References herein to an 'initiator sequence' refer to a short oligonucleotide with a free <NUM>'-end which the <NUM>'-blocked nucleotide triphosphate can be attached to. In one embodiment, the initiator sequence is a DNA initiator sequence. In an alternative embodiment, the initiator sequence is an RNA initiator sequence.

References herein to a 'DNA initiator sequence' refer to a small sequence of DNA which the <NUM>'-blocked nucleotide triphosphate can be attached to, i.e. DNA will be synthesised from the end of the DNA initiator sequence.

In one embodiment, the initiator sequence is between <NUM> and <NUM> nucleotides long, such as between <NUM> and <NUM> nucleotides long (i.e. between <NUM> and <NUM>), in particular between <NUM> and <NUM> nucleotides long (i.e., approximately <NUM> nucleotides long), more particularly <NUM> to <NUM> nucleotides long, for example <NUM> to <NUM> nucleotides long, especially <NUM> nucleotides long.

In one embodiment, the initiator sequence has the following sequence: <NUM>'-CGTTAACATATT-<NUM>' (SEQ ID NO: <NUM>).

In one embodiment, the initiator sequence is single-stranded. In an alternative embodiment, the initiator sequence is double-stranded. It will be understood by persons skilled in the art that a <NUM>'-overhang (i.e., a free <NUM>'-end) allows for efficient addition.

In one embodiment, the initiator sequence is immobilised on a solid support. This allows TdT and the cleaving agent to be removed (in steps (c) and (e), respectively) without washing away the synthesised nucleic acid. The initiator sequence may be attached to a solid support stable under aqueous conditions so that the method can be easily performed via a flow setup.

In one embodiment, the initiator sequence is immobilised on a solid support via a reversible interacting moiety, such as a chemically-cleavable linker, an antibody/immunogenic epitope, a biotin/biotin binding protein (such as avidin or streptavidin), or glutathione-GST tag. Therefore, in a further embodiment, the method additionally comprises extracting the resultant nucleic acid by removing the reversible interacting moiety in the initiator sequence, such as by incubating with proteinase K.

In a further embodiment, the initiator sequence is immobilised on a solid support via a chemically-cleavable linker, such as a disulfide, allyl, or azide-masked hemiaminal ether linker. Therefore, in one embodiment, the method additionally comprises extracting the resultant nucleic acid by cleaving the chemical linker through the addition of tris(<NUM>-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.

In one embodiment, the resultant nucleic acid is extracted and amplified by polymerase chain reaction using the nucleic acid bound to the solid support as a template. The initiator sequence could therefore contain an appropriate forward primer sequence and an appropriate reverse primer could be synthesised.

In an alternative embodiment, the immobilised initiator sequence contains at least one restriction site. Therefore, in a further embodiment, the method additionally comprises extracting the resultant nucleic acid by using a restriction enzyme.

The use of restriction enzymes and restriction sites to cut nucleic acids in a specific location is well known in the art. The choice of restriction site and enzyme can depend on the desired properties, for example whether 'blunt' or 'sticky' ends are required. Examples of restriction enzymes include: Alul, BamHI, EcoRl, EcoRII, EcoRV, Haell, Hgal, Hindlll, Hinfl, NotI, PstI, Pvull, Sall, Sau3A, Scal, Smal, Taql and Xbal.

In an alternative embodiment, the initiator sequence contains at least one uridine. Treatment with uracil-DNA glycosylase (UDG) generates an abasic site. Treatment on an appropriate substrate with an apurinic/apyrimidinic (AP) site endonuclease will extract the nucleic acid strand.

In one embodiment, the terminal deoxynucleotidyl transferase (TdT) of the invention is added in the presence of an extension solution comprising one or more buffers (e.g., Tris or cacodylate), one or more salts (e.g., Na+, K+, Mg<NUM>+, Mn<NUM>+, Cu<NUM>+, Zn<NUM>+, Co<NUM>+, etc., all with appropriate counterions, such as Cl-) and inorganic pyrophosphatase (e.g., the Saccharomyces cerevisiae homolog). It will be understood that the choice of buffers and salts depends on the optimal enzyme activity and stability.

The use of an inorganic pyrophosphatase helps to reduce the build-up of pyrophosphate due to nucleotide triphosphate hydrolysis by TdT. Therefore, the use of an inorganic pyrophosphatase has the advantage of reducing the rate of (<NUM>) backwards reaction and (<NUM>) TdT strand dismutation. Thus, according to a further aspect of the invention, there is provided the use of inorganic pyrophosphatase in a method of nucleic acid synthesis. Data is presented herein in Example <NUM> and <FIG> which demonstrates the benefit of the use of inorganic pyrophosphatase during nucleic acid synthesis. In one embodiment, the inorganic pyrophosphatase comprises purified, recombinant inorganic pyrophosphatase from Saccharomyces cerevisiae.

In one embodiment, step (b) is performed at a pH range between <NUM> and <NUM>. Therefore, it will be understood that any buffer with a buffering range of pH <NUM>-<NUM> could be used, for example cacodylate, Tris, HEPES or Tricine, in particular cacodylate or Tris.

In one embodiment, step (d) is performed at a temperature less than <NUM>, such as less than <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or <NUM>. It will be understood that the optimal temperature will depend on the cleavage agent utilised. The temperature used helps to assist cleavage and disrupt any secondary structures formed during nucleotide addition.

In one embodiment, steps (c) and (e) are performed by applying a wash solution. In one embodiment, the wash solution comprises the same buffers and salts as used in the extension solution described herein. This has the advantage of allowing the wash solution to be collected after step (c) and recycled as extension solution in step (b) when the method steps are repeated.

In one embodiment, the method is performed within a flow instrument as shown in <FIG>, such as a microfluidic or column-based flow instrument. The method described herein can easily be performed in a flow setup which makes the method simple to use. It will be understood that examples of commercially available DNA synthesisers (e.g., MerMade 192E from BioAutomation or H-<NUM> SE from K&A) may be optimised for the required reaction conditions and used to perform the method described herein.

In one embodiment, the method is performed on a plate or microarray setup. For example, nucleotides may be individually addressed through a series of microdispensing nozzles using any applicable jetting technology, including piezo and thermal jets. This highly parallel process may be used to generate hybridization microarrays and is also amenable to DNA fragment assembly through standard molecular biology techniques.

In one embodiment, the method additionally comprises amplifying the resultant nucleic acid. Methods of DNA/RNA amplification are well known in the art. For example, in a further embodiment, the amplification is performed by polymerase chain reaction (PCR). This step has the advantage of being able to extract and amplify the resultant nucleic acid all in one step.

The template independent nucleic acid synthesis method described herein has the capability to add a nucleic acid sequence of defined composition and length to an initiator sequence. Therefore, it will be understood by persons skilled in the art, that the method described herein may be used as a novel way to introduce adapter sequences to a nucleic acid library.

If the initiator sequence is not one defined sequence, but instead a library of nucleic acid fragments (for example generated by sonication of genomic DNA, or for example messenger RNA) then this method is capable of de novo synthesis of 'adapter sequences' on every fragment. The installation of adapter sequences is an integral part of library preparation for next-generation library nucleic acid sequencing methods, as they contain sequence information allowing hybridisation to a flow cell/solid support and hybridisation of a sequencing primer.

Currently used methods include single-stranded ligation, however this technique is limited because ligation efficiency decreases strongly with increasing fragment length. Consequently, current methods are unable to attach sequences longer than <NUM> nucleotides in length. Therefore, the method described herein allows for library preparation in an improved fashion to that which is currently possible.

Therefore, in one embodiment, an adapter sequence is added to the initiator sequence. In a further embodiment, the initiator sequence may be a nucleic acid from a library of nucleic acid fragments.

There is also provided the use of a kit in a method of nucleic acid synthesis, wherein said kit comprises a TdT as defined in the first or second aspects of the disclosure optionally in combination with one or more components selected from: an initiator sequence, one or more <NUM>'-blocked nucleotide triphosphates, inorganic pyrophosphatase, such as purified, recombinant inorganic pyrophosphatase from Saccharomyces cerevisiae, and a cleaving agent; further optionally together with instructions for use of the kit in accordance with any of the methods defined herein.

Suitably a kit according to the invention may also contain one or more components selected from the group: an extension solution, a wash solution and/or a cleaving solution as defined herein; optionally together with instructions for use of the kit in accordance with any of the methods defined herein.

According to a further aspect of the disclosure, there is provided the use of a <NUM>'-blocked nucleotide triphosphate in a method of template independent nucleic acid synthesis, wherein the <NUM>'-blocked nucleotide triphosphate is selected from a compound of formula (I), (II), (III) or (IV):
<CHM>
<CHM>
wherein.

References herein to a "template independent nucleic acid synthesis method" refer to a method of nucleic acid synthesis which does not require a template DNA/RNA strand, i.e. the nucleic acid can be synthesised de novo.

In one embodiment, the nucleic acid is DNA. References herein to a "template independent DNA synthesis method" refer to a method of DNA synthesis which does not require a template DNA strand, i.e. the DNA can be synthesised de novo. In an alternative embodiment, the nucleic acid is RNA.

It will be understood that 'PPP' in the structures shown herein represents a triphosphate group.

References to the term 'C<NUM>-<NUM> alkyl' as used herein as a group or part of a group refers to a linear or branched saturated hydrocarbon group containing from <NUM> to <NUM> carbon atoms. Examples of such groups include methyl, ethyl, butyl, n-propyl, isopropyl and the like.

References to the term 'C<NUM>-<NUM> alkoxy' as used herein refer to an alkyl group bonded to oxygen via a single bond (i.e. R-O). Such references include those with straight and branched alkyl chains containing <NUM> to <NUM> carbon atoms, such as methoxy (or methyloxy), ethyloxy, n-propyloxy, iso-propyloxy, n-butyloxy and <NUM>-methylpropyloxy.

References to the term 'allyl' as used herein refer to a substituent with the structural formula RCH<NUM>-CH=CH<NUM>, where R is the rest of the molecule. It consists of a methyl group (-CH<NUM>-) attached to a vinyl group (-CH=CH<NUM>).

References to the term 'COOH' or 'CO<NUM>H' refer to a carboxyl group (or carboxy) which consists of a carbonyl (C=O) and a hydroxyl (O-H) group. References to the term 'COH' refer to a formyl group which consists of a carbonyl (C=O) group bonded to hydrogen.

The term 'N<NUM>' (drawn structurally as -N=N+=N-) refers to an azido group.

In one embodiment of the disclosure, Ra and Rb both represent hydrogen (i.e. R<NUM> represents NH<NUM>).

Alternatively, Ra represents hydrogen and Rb represents methyl (i.e. R<NUM> represents NHCH<NUM>).

In one embodiment of the disclosure, R<NUM> represents hydrogen, methyl or methoxy. In a further embodiment of the disclosure, R<NUM> represents hydrogen. Alternatively, R<NUM> represents methyl. In a yet further alternative embodiment of the disclosure, R<NUM> represents methoxy.

In one embodiment of the disclosure, X represents -OR<NUM>, and R<NUM> represents C<NUM>-<NUM> alkyl, CH<NUM>N<NUM>, NH<NUM> or allyl.

Alternatively, X represents C<NUM>-<NUM> alkyl (such as methyl) or N<NUM>.

In one embodiment of the disclosure, Y represents hydrogen or hydroxyl.

In one embodiment of the disclosure, Y represents hydrogen.

Alternatively, Y represents halogen, such as fluorine.

In one embodiment of the disclosure, Z represents N.

In an alternative embodiment of the disclosure, Z represents CR<NUM>.

In one embodiment of the disclosure, R<NUM> represents C<NUM>-<NUM> alkyl, C<NUM>-<NUM>alkoxy, COH or COOH.

In a further embodiment of the disclosure, R<NUM> represents methoxy, COOH or COH. In a yet further embodiment of the disclosure, R<NUM> represents methoxy. Alternatively, R<NUM> represents COOH. In a yet further alternative embodiment of the disclosure, R<NUM> represents COH.

In one embodiment of the disclosure, the <NUM>'-blocked nucleotide triphosphate is selected from:.

The following studies and protocols illustrate embodiments of the methods described herein:.

A single-stranded DNA initiator (SEQ ID NO: <NUM>) was incubated with <NUM> U Bos taurus TdT (Thermo Scientific), required salts (<NUM> potassium acetate, <NUM> tris acetate pH <NUM>, <NUM> cobalt chloride), and <NUM>'-O-Methyl dTTP (TriLink) at <NUM> for up to one hour. The <NUM>'-irreversibly blocked nucleotide triphosphate was at a concentration of <NUM> and the DNA initiator at <NUM> pM for a <NUM>:<NUM> ratio to encourage nucleotide addition. The reaction was stopped with EDTA (<NUM>) at various intervals and the results are shown in <FIG>.

The experiment was repeated with the exception that <NUM>'-Azido dTTP was used as the <NUM>'-irreversibly blocked nucleotide triphosphate instead of <NUM>'-O-Methyl dTTP. The reaction was stopped with EDTA (<NUM>) at various intervals and the results are shown in <FIG>.

These studies show that the commercially available Bos taurus TdT adds irreversibly blocked nucleotides, specifically <NUM>'-O-Methyl dTTP and <NUM>'-Azido dTTP onto a DNA initiator strand (<FIG>). Furthermore, the addition of a <NUM>'-blocked nucleotide triphosphate to a single-stranded DNA initiator is completed to greater than <NUM>% conversion of the n strand to the n+<NUM> strand.

A single-stranded DNA initiator (SEQ ID NO: <NUM>) was incubated with purified, recombinant TdT orthologs, required salts (<NUM> potassium acetate, <NUM> tris acetate pH <NUM>, <NUM> cobalt chloride, Saccharomyces cerevisiae inorganic pyrophosphatase), and <NUM>'-O-azidomethyl dTTP at <NUM> for <NUM>. The <NUM>'-blocked nucleotide triphosphate was at a concentration of <NUM> and the DNA initiator at <NUM> and analysed via capillary electrophoresis as shown in <FIG>. Reactions were quantified after <NUM> and results are shown in <FIG>.

The experiment was repeated with the exception that both <NUM>'-O-azidomethyl dTTP and <NUM>'-O-azidomethyl dCTP was used as the <NUM>'-blocked nucleotide triphosphate. The reaction was stopped after <NUM> and analysed via capillary electrophoresis. Quantified reactions are shown in <FIG>.

Bos taurus TdT only efficiently adds irreversibly blocked nucleotides, which is not useful for controlled, enzymatic single-stranded DNA synthesis. This example demonstrates that naturally occurring TdT orthologs other than Bos taurus TdT perform significantly better at adding <NUM>'-reversibly blocked nucleotide triphosphates. Better performance, which is judged by the N+<NUM> addition rate (<FIG>), results in longer achievable lengths and greater control of nucleic acid sequence specificity.

A single-stranded DNA initiator (SEQ ID NO: <NUM>) was incubated with either a purified wild-type Lepisosteus oculatus TdT or a purified, recombinant engineered form of Lepisosteus oculatus TdT (SEQ ID NO: <NUM>), required salts, cobalt chloride, Saccharomyces cerevisiae inorganic pyrophosphatase, and <NUM>'-O-azidomethyl dTTP at <NUM> for <NUM>.

The wild-type Lepisosteus oculatus TdT was outperformed by the engineered variant (SEQ ID NO: <NUM>), as demonstrated by improved ability to convert the initiator strand of length n to a strand of length n+<NUM>, when supplied with <NUM>'-reversibly blocked dCTP, dGTP or dTTP.

A single-stranded DNA initiator (SEQ ID NO: <NUM>) was incubated with a purified, recombinant engineered form of Lepisosteus oculatus TdT, required salts, cobalt chloride, Saccharomyces cerevisiae inorganic pyrophosphatase, and <NUM>'-O-azidomethyl dTTP at <NUM> for <NUM>.

Bos Taurus TdT was outperformed by an engineered variant of Lepisosteus oculatus TdT, as demonstrated by the denaturing PAGE gel in <FIG>. This example demonstrates an engineered form of Lepisosteus oculatus TdT incorporates <NUM>'-reversibly blocked nucleotides (<NUM>) better than the wild-type Lepisosteus oculatus TdT, and (<NUM>) much better than Bos Taurus TdT (see <FIG>).

A single-stranded DNA initiator (SEQ ID NO: <NUM>) was incubated with Bos taurus TdT at <NUM> for <NUM> under the reaction buffer shown above with the exception that the concentration of Saccharomyces cerevisiae inorganic pyrophosphatase was varied. When dideoxyTTP (ddTTP) was used, the concentration of the NTP was <NUM>. Reactions were analysed by PAGE and are shown in <FIG>.

Claim 1:
A method of nucleic acid synthesis, comprising the steps of:
(a) providing an initiator sequence immobilised on a solid support, wherein the initiator sequence is an oligonucleotide with a free <NUM>'-end and is between <NUM> and <NUM> nucleotides long;
(b) adding a <NUM>'-blocked nucleoside triphosphate to said initiator sequence in the presence of a terminal deoxynucleotidyl transferase (TdT) having at least <NUM>% sequence identity to SEQ ID NO: <NUM> or a functional fragment thereof having terminal deoxynucleotidyl transferase activity and comprising an N-terminal or a C-terminal truncation, wherein the nucleoside is blocked by either a <NUM>'-O-azidomethyl, <NUM>'-aminoxy or <NUM>'-O-allyl group;
(c) removal of all reagents from the initiator sequence;
(d) cleaving the blocking group from the <NUM>'-blocked nucleoside in the presence of a cleaving agent; and
(e) removal of the cleaving agent.