METHODS OF NUCLEIC ACID SYNTHESIS

Abstract

The invention relates to improved methods of enzymatic solid-supported nucleic acid synthesis that make use of terminal deoxynucleotidyl transferase (TdT) enzymes or modified terminal deoxynucleotidyl transferase (TdT) enzymes on polyacrylamide type supports. The invention further relates to the use of kits comprising said enzymes in a method of solid-supported nucleic acid synthesis.

Claims

1. A method of nucleic acid synthesis, wherein the method comprises the steps of: (a1) providing a solid support comprising particles coated with a pre-polymerised material, wherein the pre-polymerised material comprises a co-polymer to which an initiator oligonucleotide is to be attached, wherein the co-polymer is a co-polymer of one or more first co-monomer(s) selected from acrylamide, methacrylamide, N-methylacrylamide, N,N′-dimethylacrylamide, N-(hydroxylmethyl)acrylamide, N-(hydroxyethyl)acrylamide, N-[tris(hydroxymethyl)methyl]acrylamide, hydroxyethyl methacrylate and N-vinyl pyrrolidinone and a second co-monomer which attaches to the initiator; (a2) coupling an initiator oligonucleotide to said co-polymer to form a solid-supported initiator oligonucleotide; (b) adding a 3ʹ-blocked nucleoside triphosphate to said initiator oligonucleotide in the presence of a terminal deoxynucleotidyl transferase (TdT) enzyme or modified terminal deoxynucleotidyl transferase (TdT) enzyme; (c) cleaving the blocking group from the 3ʹ-blocked nucleoside triphosphate in the presence of a cleaving agent; and (d) repeating steps (b) and (c) to synthesise an extended nucleic acid.

2. The method according to claim 1, wherein the first co-monomer is acrylamide.

3. The method according to any one of claims 1 or 2, wherein the solid support is coated with a pre-polymerised material comprising a monomer selected from acrylamide, methacrylamide, N-methylacrylamide, N,N′-dimethylacrylamide, N-(hydroxylmethyl)acrylamide, N-(hydroxyethyl)acrylamide, N-[tris(hydroxymethyl)methyl]acrylamide, hydroxyethyl methacrylate and N-vinyl pyrrolidinone and a second co-monomer containing a reactive moiety selected from haloacetamide, carboxylic acid, alkyne, azide, amine or thiol.

4. The method according to claim 3, wherein the second co-monomer is a haloacetamide-containing monomer.

5. The method according to any one of claims 1 to 4, wherein the second co-monomer is a monomer of formula (3) or (3a): ##STR00013## ##STR00014## wherein X is Cl, Br or I; Y is NR.sup.1 or 0; Z is an optionally substituted C.sub.1-50 alkyl bridge; and R.sup.1 is H or an optionally substituted C.sub.1-5 alkyl group.

6. The method according to claim 5, wherein X is Br.

7. The method according to claim 5 or 6, wherein Y is NH.

8. The method according to any one of claims 5 to 7, wherein Z is a C5 alkyl bridge.

9. The method according to any one of claims 1 to 8, wherein the initiator oligonucleotide is coupled via a phosphorothioate moiety.

10. The method according to any one of claims 1 to 8, wherein the initiator oligonucleotide is coupled via click chemistry between an azide and an alkyne.

11. The method according to any one of claims 1 to 10, wherein the extended nucleic acid is detached from the solid support.

12. The method according to any one of claims 1 to 11, wherein the initiator contains a uracil moiety and nucleic acid is detached by removing the uracil base and cleaving the abasic site.

13. The method according to any one of claims 1 to 12, wherein the particles are magnetic.

14. The method according to any one of claims 1 to 13, wherein the particles are silica coated.

15. The method as defined in any one of claims 1 to 14, wherein the 3ʹ-blocked nucleoside triphosphate is blocked a group selected from 3ʹ-O-azidomethyl, 3ʹ-aminooxy, 3ʹ-O-allyl, 3ʹ-O-cyanoethyl, 3ʹ-O-acetyl, 3ʹ-O-nitrate, 3ʹ-O-phosphate, 3ʹO-acetyl levulinic ester, 3ʹ-O-tert butyl dimethyl silane, 3ʹ-O-trimethyl(silyl)ethoxymethyl, 3ʹ-O-ortho-nitrobenzyl, or 3ʹ-O-para-nitrobenzyl.

16. The method according to any one of claims 1 to 15, wherein the cleaving agent is tris(2-carboxyethyl)phosphine (TCEP), a palladium complex, an organic or inorganic base, sodium nitrite or a photoactivated transition metal complex.

17. The method according to any one preceding claim, wherein the solid support contains a plurality of coagulated magnetic particles.

18. The method according to any one preceding claim, wherein the pre-polymerisation is carried out for at least 90 minutes prior to exposure to the surface being coated.

19. A kit comprising: (i) a solid support comprising particles coated with a pre-polymerised material, wherein the pre-polymerised material comprises a co-polymer to which an initiator oligonucleotide is attached or is to be attached, wherein the co-polymer is a co-polymer of one or more first co-monomer(s) selected from acrylamide, methacrylamide, N-methylacrylamide, N,N′-dimethylacrylamide, N-(hydroxylmethyl)acrylamide, N-(hydroxyethyl)acrylamide, N-[tris(hydroxymethyl)methyl]acrylamide, hydroxyethyl methacrylate and N-vinyl pyrrolidinone and a second co-monomer which attaches to or is attached to the initiator; (ii) a 3ʹ-blocked nucleoside triphosphate; (iii) a terminal deoxynucleotidyl transferase (TdT) enzyme or modified terminal deoxynucleotidyl transferase (TdT) enzyme and optionally; (iv) a cleaving agent.

20. The kit according to claim 19 further comprising an initiator oligonucleotide.

21. The kit according to claim 19, wherein the initiator oligonucleotide is attached to the coated support.

Description

BRIEF DESCRIPTION OF THE FIGURES

Annotated PAGE Gel Image for N+1 Addition Reactions

[0016] FIG. 1 shows denaturing polyacrylamide gel electrophoresis (PAGE) analysis of oligonucleotides prepared using the methods of the invention. The importance of polymer preparation is clearly shown.

Variation in Oligo Loading per Bead Type

[0017] FIG. 2 shows a quantitative analysis of the PAGE image in FIG. 1.

[0018] FIG. 3 shows denaturing polyacrylamide gel electrophoresis (PAGE) analysis of oligonucleotides.

[0019] FIG. 3 shows the PAGE image for N+12 addition reactions performed on different PAC-BRAC polymer coated bead types (PCM02-07).

[0020] FIG. 4. X-ray photoelectron spectroscopy (XPS) of coated and uncoated silica beads. PAC-BRAC polymerisation and coating was performed in situ with silica paramagnetic particles. Subsequently, X-ray photoelectron spectroscopy (XPS) was used to analyse the surface chemistry of silica beads with (Panel B) and without (Panel A) a PAC-BRAC coating. The atomic % of oxygen (O), nitrogen (N), carbon (C), and silicon (Si) was determined and used to establish the presence of a polyacrylamide coating on the surface of the silica paramagnetic particles. Comparing the atomic % of carbon and nitrogen between the uncoated and coated samples (Panel C), as well as the ratio of carbon and nitrogen, clearly shows the presence of a polyacrylamide-based surface coating.

[0021] FIG. 5. Denaturing polyacrylamide gel electrophoresis analysis of oligonucleotides prepared using the methods of the invention. Cleaned silica paramagnetic particles were coated with pre-polymerised PAC-BRAC for ninety minutes with periodic vortexing. The coated particles were washed with 10 mM potassium phosphate pH 7. A phosphorothioate-containing oligonucleotide of SEQ 2 was coupled to the PAC-BRAC surface in a 60 minute incubation at 52° C. The oligonucleotide-coated particles were split into three aliquots and washed with different solutions to remove non-specifically bound oligonucleotide. Finally, the particles were exposed to a solution containing uracil DNA glycosylase (UDG) and N,N′-dimethylethylenediamine (DMED) to cleave the oligonucleotide at the U base. Subsequent analysis was via denaturing polyacrylamide gel electrophoresis, which was visualised by virtue of the cyanine 3 (cy3) dye present in the oligonucleotide.

[0022] Lane 1: Control lane. Oligonucleotide was exposed to the UDG/DMED cleavage solution. The higher molecular weight band is uncut oligonucleotide and the lower molecular weight band is cut oligonucleotide. Lane 2: PAC-BRAC beads washed with 1 M aqueous sodium chloride, 0.1 % tween-20, and 20 mM HEPES KOH. The presence of bands in the gel shows that DNA was bound to the particles. The presence of full-length (uncut) oligonucleotide suggests some non-specific binding was present. Lane 3: PAC-BRAC beads washed with 20 mM aqueous sodium hydroxide. The presence of bands in the gel shows that DNA was bound to the particles. The absence of any full-length (uncut) oligonucleotide demonstrates that no non-specific binding was present. Lane 4: PAC-BRAC beads washed with 50% formamide. The presence of bands in the gel shows that DNA was bound to the particles. The presence of full-length (uncut) oligonucleotide suggests some non-specific binding was present, though at a reduced level to that seen in Lane 1.

[0023] FIG. 6. Denaturing polyacrylamide gel electrophoresis (PAGE) analysis of DNA products synthesised from a selection of solid supports. DNA synthesis was performed enzymatically using an engineered terminal deoxynucleotidyl transferase (TdT) to incorporate reversibly terminated nucleotides into a growing single stranded DNA (ssDNA) product. For each surface, 0, 10, 20, 30, 40, and 50 cycles of synthesis were performed in separate experiments. Synthesis initiators were grafted to the solid supports as follows: (1) 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) coupling to carboxyl surface, followed by capping with tris(hydroxymethyl)aminomethane (Carboxy-T) or ethanolamine (Carboxy-EA); (2) affinity interaction between a biotinylated initiator and a neutravidin surface (Neutraividin); (3) reaction between a phosphorothioate-containing initiator and a haloacetimide containing surface (PAC-BRAC). (H) and (L) indicate high and low initiator loading densities respectively. The synthesis process is markedly more efficient when performed on the PAC-BRAC surface, as evidenced by the primary product being the target product in all experiments (in contrast to other surfaces) and the significant reduction in N-1, N-2, etc deletion products.

[0024] FIG. 7. The effect of first co-monomer identity on polymer performance. xPAC-BRAC (where xPAC indicates that the first co-monomer was varied) was-pre-polymerised and coated onto silica paramagnetic particles. The first co-monomer was selected from acrylamide, N-(hydroxylmethyl)acrylamide, N-(hydroxyethyl)acrylamide, N,N′-dimethyl acrylamide, and N-[tris(hydroxymethyl)methyl]acrylamide. At 100% alternative monomer, there was complete replacement of acrylamide, while at 50% alternative monomer 50% acrylamide was present. Synthesis initiators were then grafted to the polymer and 17 cycles of DNA synthesis were performed using an engineered terminal deoxynucleotidyl transferase and reversibly terminated nucleotides. The synthesised DNA was then cleaved from the solid support and prepared into a library and sequenced on an Illumina iSeq. The percentage of reads with the correct length and sequence identity was calculated for each sample. All samples were then normalised to the value obtained when using acrylamide as the first co-monomer. The data clearly shows that the identity of the first co-monomer can be varied to alter the properties of the polymer coating on a solid support. Indeed, certain compositions exceed the performance obtained when using acrylamide as the first co-monomer. Note that values shown are the average of two technical duplicates.

[0025] FIG. 8. Denaturing polyacrylamide gel electrophoresis (PAGE) analysis of oligonucleotides prepared using the methods of the invention in a frit-retained beads format. Polymer coated beads were prepared using the method described herein. Beads were loaded into either wells of a 96-well plate with a 0.22 micron frit or 384-well plate with a 0.45 micron frit (Millipore HTS plates). DNA synthesis was performed enzymatically using an engineered terminal deoxynucleotidyl transferase (TdT) to incorporate reversibly terminated nucleotides into a growing single stranded DNA (ssDNA) product. Introduction of addition solution (NAM), wash solution, nitrite deblock solution (NDS) was accomplished by dispensing liquid into the wells with a liquid handling robot and removal of solution was accomplished by application of a vacuum to draw liquid through the filter frit of the plate. The polymer coated silica particles were retained in the wells due to their size (> 1 micron) being greater than the frit pore size of 0.22 micron or 0.45 micron. Lane 1 of the gel shows the DNA initiator (N); lane 2 shows the product after 8 cycle of enzymatic DNA synthesis (N+8) on a 96-well plate with 0.22 micron frit; lane 3 shows the product after 8 cycles of enzymatic DNA synthesis (N+8) on a 384-well plate with 0.45 frit. The major product in lanes 2 and 3 is the N+8 oligonucleotide. The fainter bands below (N+7) and above (N+8) arise due to incomplete enzymatic addition and multiple addition within one cycle respectively.

[0026] FIG. 9. Denaturing polyacrylamide gel electrophoresis (PAGE) analysis of oligonucleotides prepared using the methods of the invention in a polymer coated frit format. The glass fibre frits of a multiwell plate were cleaned with 80% Decon, 1 M NaOH, and 0.1M HCl in sequence. The frit was coated with prepolymerised PAC-BRAC polymer by repeated 60 second incubations and vacuum solution removal. Oligonucleotide with phosphorothioate linkages was then grafted to the polymer to act as an initiator for enzymatic DNA synthesis. DNA synthesis was performed enzymatically using an engineered terminal deoxynucleotidyl transferase (TdT) to incorporate reversibly terminated nucleotides into a growing single stranded DNA (ssDNA) product. Introduction of addition solution (NAM), wash solutions, and deblock solution (NDS) was accomplished by dispensing liquid into the wells with a liquid handling robot and removal of solutions was accomplished by application of a vacuum to draw liquid through the polymer coated frit of the plate. Synthesised oligonucleotide was cleaved from the polymer coated frit and analysed by polyacrylamide gel electrophoresis (PAGE). Lanes 1-3 contain DNA initiator (N); lanes 4-6 contain the products after 8 cycles of enzymatic DNA synthesis. The major product in lanes 4-6 is N+8 corresponding to the full-length synthesis product.

[0027] FIG. 10. Schematic showing various types of solid support

[0028] Part A shows coated particles. The particles are optionally magnetic.

[0029] Part B shows the particles retained in a frit.

[0030] Part C shows a coated frit.

[0031] Part D shows a plurality of coagulated particles.

[0032] FIG. 11. Particle coagulation The same volume (10 .Math.L) of 1 mg/mL magbead suspensions (in 20 mM HEPES 50 mM NaCl 0.1% v/v Tween 20 pH 7.2 buffer) were applied to the surface of a glass microscope slide and sealed with a glass cover slip. Slides were prepared using suspensions of uncoated (1 .Math.m silica coated paramagnetic particles, panel A), polymer-coated (PAC-BRAC, Panel B) and oligo-coupled (oligo = 43 mer with 5ʹ phosphorothioate conjugation modification, Panel C) magbeads were taken on a microscope (x25 objective). An approximate 10 .Math.m scale bar is given in each panel for reference. A circle has been drawn on each panel as a region of interest to highlight populations of particles. The uncoated particles are generally monodisperse and form a well separated suspension. Coated beads form large multi-particle aggregates of heterogeneous size typically >50 .Math.m. Oligo coupled bead suspensions appear to exist in smaller aggregated populations, within a nominal size range of 10-50 .Math.m. This data demonstrates the effect polymer coating and oligo coupling to the particle surface has on their aggregative behaviour.

DETAILED DESCRIPTION OF THE INVENTION

[0119] Described herein is a method of nucleic acid synthesis, wherein the method comprises the steps of: [0120] (a) providing a solid support, wherein the solid support comprises a co-polymer to which an initiator oligonucleotide is attached, wherein the co-polymer is a co-polymer of one or more first co-monomer(s) selected from acrylamide, methacrylamide, N-methylacrylamide, N,N′-dimethylacrylamide, N-(hydroxylmethyl)acrylamide, N-(hydroxyethyl)acrylamide, N-[tris(hydroxymethyl)methyl]acrylamide, bisacrylamide, hydroxyethyl methacrylate and N-vinyl pyrrolidinone and a second co-monomer which attaches to or is attached to the initiator; and [0121] (b) adding a 3ʹ-blocked nucleoside triphosphate to said initiator oligonucleotide in the presence of a terminal deoxynucleotidyl transferase (TdT) enzyme or modified terminal deoxynucleotidyl transferase (TdT) enzyme.

[0122] Inventors have appreciated that the process of coating surfaces can be improved by [0123] a) using a non cross-linked ‘linear’ polymer [0124] b) not covalently attaching the polymer to the support [0125] c) pre-polymerising the material before exposing the polymerized material to the support.

[0126] Inventors have further appreciated that whilst coating a population of beads with polymer, discreet units of solid support can be obtained, each containing a plurality of the smaller beads. The support can comprise a plurality of optionally magnetic particles held within a polymerised material. Magnetic beads of for example 1 .Math.m in size can be bound together within a polymerized material, thereby forming discreet magnetic particles. The size of, and number of magnetic particles within, the aggregated solid supports can be controlled by the polymerization conditions, including choice of monomers, concentration of monomers and length or polymerization time. For example the solid support may contain at least 10 magnetic particles or at least 100 magnetic particles.

[0127] The solid support may have an average particle diameter of at least 50 .Math.m. The magnetic particles within the solid support have be beads of size approximately 1 .Math.m diameter. The magnetic particles within the solid support have be beads having an average size distribution of less than 2 .Math.m.

[0128] Described herein is a method of nucleic acid synthesis, wherein the method comprises the steps of: [0129] (a) providing a solid support, wherein the solid support comprises a co-polymer to which an initiator oligonucleotide is attached, wherein the co-polymer is a co-polymer of one or more first co-monomer(s) selected from acrylamide, methacrylamide, N-methylacrylamide, N,N′-dimethylacrylamide, N-(hydroxylmethyl)acrylamide, N-(hydroxyethyl)acrylamide, N-[tris(hydroxymethyl)methyl]acrylamide, bisacrylamide, hydroxyethyl methacrylate and N-vinyl pyrrolidinone and a second co-monomer which attaches the initiator; and [0130] (b) adding a 3ʹ-blocked nucleoside triphosphate to said initiator oligonucleotide in the presence of a terminal deoxynucleotidyl transferase (TdT) enzyme or modified terminal deoxynucleotidyl transferase (TdT) enzyme.

[0131] The method may further comprise the step:

[0132] (c) cleaving the blocking group from the 3ʹ-blocked nucleoside triphosphate in the presence of a cleaving agent.

[0133] In the methods described herein, further nucleotides may be added by repeating steps (b) and (c).

[0134] Thus the methods of the invention may comprise the steps of: [0135] (a) providing a solid support, wherein the solid support is coated with a co-polymer to which an initiator oligonucleotide is attached, wherein the co-polymer is a co-polymer of a first co-monomer selected from acrylamide, methacrylamide, N-methylacrylamide, N,N′-dimethylacrylamide, hydroxyethyl methacrylate and N-vinyl pyrrolidinone and a second co-monomer which attaches the initiator; [0136] (b) adding a 3ʹ-blocked nucleoside triphosphate to said initiator oligonucleotide in the presence of a terminal deoxynucleotidyl transferase (TdT) enzyme or modified terminal deoxynucleotidyl transferase (TdT) enzyme; [0137] (c) cleaving the blocking group from the 3ʹ-blocked nucleoside triphosphate in the presence of a cleaving agent; [0138] (d) repeating steps (b) and (c) one or more times.

[0139] In the methods described herein, step (a) may comprise the steps of: [0140] (a1) providing a solid support, wherein the solid support is coated with a co-polymer of a first co-monomer and a second co-monomer, wherein the first co-monomer is acrylamide, methacrylamide, N-methylacrylamide, N,N′-dimethylacrylamide, hydroxyethyl methacrylate or N-vinyl pyrrolidinone and the second co-monomer enables coupling of an initiator oligonucleotide; [0141] (a2) coupling an initiator oligonucleotide to said co-polymer to form a solid-supported initiator oligonucleotide.

[0142] Thus the methods of the invention may comprise the steps of: [0143] (a1) providing a solid support, wherein the solid support is coated with a co-polymer of a first co-monomer and a second co-monomer, wherein the first co-monomer is acrylamide, methacrylamide, N-methylacrylamide, N,N′-dimethylacrylamide, hydroxyethyl methacrylate or N-vinyl pyrrolidinone and the second co-monomer enables coupling of an initiator oligonucleotide; [0144] (a2) coupling an initiator oligonucleotide to said co-polymer to form a solid-supported initiator oligonucleotide; [0145] (b) adding a 3ʹ-blocked nucleoside triphosphate to said initiator oligonucleotide in the presence of a terminal deoxynucleotidyl transferase (TdT) enzyme or modified terminal deoxynucleotidyl transferase (TdT) enzyme; [0146] (c) cleaving the blocking group from the 3ʹ-blocked nucleoside triphosphate in the presence of a cleaving agent; [0147] (d) repeating steps (b) and (c) one or more times.

[0148] In some of the methods described herein, the solid support coatings comprise a co-polymer to which an initiator oligonucleotide is attached. Said coatings, when applied to solid supports may create a polymer surface which is effectively immobilised to the solid support. The coatings comprise a co-polymer of one or more first co-monomers and a second co-monomer, wherein the first co-monomer may be selected from acrylamide, methacrylamide, N-methylacrylamide, N,N′-dimethylacrylamide, N-(hydroxylmethyl)acrylamide, N-(hydroxyethyl)acrylamide, N-[tris(hydroxymethyl)methyl]acrylamide, bisacrylamide, hydroxyethyl methacrylate or N-vinyl pyrrolidinone. The first co-monomer may be acrylamide. In some embodiments linear polymers are preferred.

[0149] The second co-monomer serves to provide functionalisation in the form of a chemically reactive group on the support such that the resultant co-polymer allows coupling of the co-polymer to an initiator oligonucleotide, thereby immobilizing the initiator oligonucleotide. Thus the second co-monomer can be selected from any co-monomer that is suitable for allowing coupling of the resultant co-polymer to an initiator oligonucleotide. The second co-monomer can for example be selected from a thiol-containing monomer, an amine-containing monomer, an acid-containing monomer, a haloacetamide-containing monomer, an alkyne-containing monomer and an azide-containing monomer. The second co-monomer can be a haloacetamide-containing monomer. The second co-monomer can be a bromoacetamide-containing monomer. The second co-monomer can be N-(5-bromoacetamidylpentyl) acrylamide (BRAC). The second co-monomer may contain a reactive moiety selected from haloacetamide, carboxylic acid, alkyne, azide, amine or thiol.

[0150] The second co-monomer can be a monomer of formula (1a) or (1b):

##STR00002##

##STR00003##

wherein Q is any group suitable to allow coupling of the resultant co-polymer to an initiator oligonucleotide;

[0151] and V is a linker group.

[0152] Alternatively Q can be attached to the initiator oligonucleotide, for example in the form of a methacrylate group.

[0153] When the second co-monomer is a monomer of formula (1a) or formula (1b), Q can be a group suitable to allow coupling of the resultant co-polymer to an initiator oligonucleotide. Q can be a group comprising an azide, haloacetamide, alkyne, amine, carboxylic acid or thiol moiety. Q can be a group comprising a haloacetamide moiety. Q can be selected from C(O)CH.sub.2Br, NH.sub.2, N.sub.3, CO.sub.2H, NHC(O)CH.sub.2Br, C≡CH and SH. Q can be NHC(O)CH.sub.2Br. V can be any suitable linker group. V can be selected from an optionally substituted alkyl linker, an optionally substituted alkoxy linker or an optionally substituted polyethylene glycol linker. V can be optionally substituted C.sub.1-50 alkyl. V can be optionally substituted C(O)C.sub.1-50 alkyl. V can be -(OCH.sub.2CH.sub.2)-.sub.n where n is 1 to 20.

[0154] The second co-monomer can be a monomer of formula (2a) or (2b):

##STR00004##

##STR00005##

wherein Q is any group suitable to allow coupling of the resultant co-polymer to an initiator oligonucleotide;

[0155] Y is NR.sup.1 or O;

[0156] Z is an optionally substituted C.sub.1-50 alkyl bridge;

[0157] and R.sup.1 is H or an optionally substituted C.sub.1-5 alkyl group.

[0158] Alternatively Q can be attached to the initiator oligonucleotide, for example in the form of a methacrylate group.

[0159] When the second co-monomer is a monomer of formula (2a) or formula (2b), Q can be a group suitable to allow coupling of the resultant co-polymer to an initiator oligonucleotide. Q can be a group comprising an azide, haloacetamide, alkyne, amine, carboxylic acid or thiol moiety. Q can be a group comprising a haloacetamide moiety. Q can be selected from C(O)CH.sub.2Br, NH.sub.2, N.sub.3, CO.sub.2H, NHC(O)CH.sub.2Br, C≡CH and SH. Q can be NHC(O)CH.sub.2Br. V can be any suitable linker group. V can be selected from an optionally substituted alkyl linker, an optionally substituted alkoxy linker or an optionally substituted polyethylene glycol linker. V can be optionally substituted C.sub.1-50 alkyl. V can be optionally substituted C(O)C.sub.1-50 alkyl. V can be -(OCH.sub.2CH.sub.2)-.sub.n where n is 1 to 20.

[0160] Y can be NH. Y can be O. R.sup.1 can be H. R.sup.1 can be an optionally substituted C.sub.1-5 alkyl group. Z can be an optionally substituted C.sub.1-20 alkyl bridge. Z can be an optionally substituted C.sub.1-10 alkyl bridge. Z can be an optionally substituted C.sub.5 alkyl bridge. Z can be a C.sub.5 alkyl bridge.

[0161] The second co-monomer can be a monomer of formula (3) or (3a):

##STR00006##

##STR00007##

wherein X is Cl, Br or I;

[0162] Y is NR.sup.1 or O;

[0163] Z is an optionally substituted C.sub.1-50 alkyl bridge;

[0164] and R.sup.1 is H or an optionally substituted C.sub.1-5 alkyl group.

[0165] When the second co-monomer is a monomer of formula (3) or (3a), X can be Br. X can be Cl. X can be I. Y can be NH. Y can be O. R.sup.1 can be H. R.sup.1 can be an optionally substituted C.sub.1-5 alkyl group. Z can be an optionally substituted C.sub.1-20 alkyl bridge. Z can be an optionally substituted C.sub.1-10 alkyl bridge. Z can be an optionally substituted C.sub.5 alkyl bridge. Z can be a C.sub.5 alkyl bridge.

[0166] The second co-monomer can be a monomer of formula (4) or (4a):

##STR00008##

##STR00009##

wherein X is Cl, Br or I;

[0167] Y is NR.sup.1 or O;

[0168] Z is an optionally substituted C.sub.1-50 alkyl bridge;

[0169] and R.sup.1 is H or an optionally substituted C.sub.1-5 alkyl group.

[0170] When the second co-monomer is a monomer of formula (4) or (4a), X can be Br. X can be Cl. X can be I. Y can be NH. Y can be O. R.sup.1 can be H. R.sup.1 can be an optionally substituted C.sub.1-5 alkyl group. n can be 2 to 10. n can be 5.

[0171] In the methods described herein, the polymerisation reaction between the first and second co-monomer may be carried out in the presence or absence of the solid support. When carried out in the presence of the solid support, the coating is applied to the solid support as the polymerisation reaction progresses. Accordingly the coating may be applied to the solid support during polymerisation of the first and second co-monomers. Alternatively the polymerisation reaction may be carried out independently of the solid support. Therefore the resultant co-polymer may be coated on to the solid support separately to the polymerisation process. Accordingly the coating may be applied to the solid support as a pre-polymerised polymer mixture of the first and second co-monomers.

[0172] In the polymerisation reaction between the first and second co-monomer, the first co-monomer is used in a molar excess relative to the second co-monomer. For example, the second co-monomer may be present in an amount of 1 mol% or less relative to the total molar quantity of co-monomers. The second co-monomer may be present in an amount of 1 mol% or greater relative to the total molar quantity of co-monomers. The second co-monomer may be present in an amount of 2 mol% or greater relative to the total molar quantity of co-monomers.

[0173] In the methods herein, the initiator oligonucleotide is coupled to the solid support co-polymer coating to form a solid-supported initiator oligonucleotide. The initiator oligonucleotide may be coupled to the solid support co-polymer coating using any suitable method. The initiator oligonucleotide may for example be coupled to the solid support co-polymer coating via a phosphorothioate moiety or via click chemistry between an azide and an alkyne. Preferably the initiator oligonucleotide is coupled via a phosphorothioate moiety. In one embodiment the second co-monomer is a haloacetamide-containing monomer and the initiator oligonucleotide is coupled to the solid support co-polymer coating via a phosphorothioate moiety, wherein the phosphorothioate moiety couples to the co-polymer by displacement of halide from the haloacetamide-containing portion of the co-polymer.

[0174] Prior to coupling to the solid support co-polymer coating the initiator oligonucleotide may comprise a moiety of formula (5):

##STR00010##

wherein, U is O, S or NR.sup.2;

[0175] T is O, S or an optionally substituted C.sub.1-10 alkyl group;

[0176] W is O or S;

[0177] R.sup.2 is H or an optionally substituted C.sub.1-10 alkyl group.

[0178] Where the initiator oligonucleotide is coupled to the solid support co-polymer coating via a phosphorothioate moiety, prior to coupling to the solid support co-polymer coating the initiator oligonucleotide may comprise a moiety of formula:

##STR00011##

[0179] The phosphorothioate can be on the terminus of the oligonucleotide, as shown above, or can be internal to the sequence. Coupling can be performed using a moiety of formula:

##STR00012##

where R.sub.1 is an oligo fragment and R.sup.2 is an oligo fragment.

[0180] The methods described herein are not limited by the format, size, geometry or the material of the solid support itself. The solid support may comprise any material suited to the methods of the invention. The solid support may be a silica based solid support. The solid support may be a silica based solid support wherein said silica is fused silica. The solid support may be a polystyrene based support. The solid support may be a plastic well or a plastic slide. The solid support may comprise silica beads, magnetic beads, paramagnetic beads, superparamagnetic beads, glass fibres or a glass slide. The solid support may be silica beads. The solid support may be silica superparamagnetic beads. The solid support may be polystyrene beads. The solid support may be a non-silica based support. The solid support may comprise silica beads, paramagnetic beads, glass fibres, a glass slide, a plastic well or a plastic slide. The beads may be silica beads or comprise a silica shell.

[0181] As used herein, the term “beads” covers any particle of suitable size. The solid support particles may have a diameter within the range of 0.1-200 .Math.m. The solid support particles may have a diameter within the range of 0.1-50 .Math.m. The solid support particles may have a diameter within the range of 100-5000 nm.

[0182] The nucleic acid strands may include a cleavage site to enable cleavage from the solid support. The cleavage site may be a base or base sequence recognisable by an enzyme. A base recognised by an enzyme, such as a glycosylase, may be removed to generate an abasic site which may be cleaved by chemical or enzymatic means. An example of such a glycosylase system includes the presence of a uracil base in the initiator sequence, which may be excised with uracil DNA glycosylase (UDG) to leave an abasic site which may be cleaved with, for example, basic solutions, organic amines, or an endonuclease (such as endonuclease VIII), to release a nucleic acid bearing a 5ʹ-phosphate into solution. A base sequence may be recognised and cleaved by a restriction enzyme.

[0183] In the methods of the invention 3ʹ-blocked nucleoside triphosphates are added in the presence of a terminal deoxynucleotidyl transferase (TdT) enzyme which results in extension of the solid-supported sequence by one nucleotide unit. The 3ʹ-blocking group present on the 3ʹ-blocked nucleoside triphosphates prevents further incorporation of nucleotides. Upon cleavage of the blocking group present on the 3ʹ-blocked nucleoside triphosphate, the solid-supported nucleotide sequence may be further extended by adding a further 3ʹ-blocked nucleoside triphosphate in the presence of a terminal deoxynucleotidyl transferase (TdT) enzyme. Cleavage of the blocking group and addition of a further 3ʹ-blocked nucleoside triphosphate in the presence of a terminal deoxynucleotidyl transferase (TdT) enzyme may be repeated any number of times as desired to synthesise a target sequence.

[0184] The 3ʹ-blocked nucleoside 5ʹ-triphosphate can be blocked by any chemical group that can be unmasked to reveal a 3ʹ-OH. For example, the 3ʹ-blocked nucleoside triphosphate can be blocked by a 3ʹ-O-azidomethyl, 3ʹ-aminooxy, 3ʹ-O-allyl group, 3ʹ-O-cyanoethyl, 3ʹ-O-acetyl, 3ʹ-O-nitrate, 3ʹ-O-phosphate, 3ʹ-O-acetyl levulinic ester, 3ʹ-O-tert butyl dimethyl silane, 3ʹ-O-aminoxy oxime, 3ʹ-O-trimethyl(silyl)ethoxymethyl, 3ʹ-O-ortho-nitrobenzyl, and 3ʹ-O-para-nitrobenzyl. The 3ʹ-blocking group may be selected from 3ʹ-O-azidomethyl, 3ʹ-aminooxy, 3ʹ-O-cyanoethyl and a 3ʹ-O-allyl group. The 3ʹ-blocked nucleoside 5ʹ-triphosphate can also be blocked by any chemical group that can be directly utilized in chemical ligations, such as copper-catalyzed or copper-free azide-alkyne click reactions and tetrazine-alkene click reactions. The 3ʹ-blocked nucleoside triphosphate can include chemical moieties containing an azide, alkyne, alkene, and tetrazine. In another embodiment the 3ʹ-blocked nucleoside 5ʹ-triphosphate can be blocked by a chemical group that can be unmasked to reveal a 3ʹ-O-NH.sub.2, which can subsequently be unmasked to reveal a 3ʹ-OH. The 3ʹ-blocked nucleoside triphosphate can be blocked by a 3ʹ-O-NC(CH.sub.3).sub.2.

[0185] In the methods of the invention, the blocking group of the 3ʹ-blocked nucleoside triphosphate is cleaved in the presence of a cleaving agent. The cleaving agent used will depend on the 3ʹ-blocking group present and may be any cleaving agent suitable for cleaving the 3ʹ-blocking group. For instance, tris(2-carboxyethyl)phosphine (TCEP) or tris(hydroxypropyl)phosphine (THPP) can be used to cleave a 3ʹ-O-azidomethyl group, palladium complexes can be used to cleave a 3ʹ-O-allyl group, or sodium nitrite can be used to cleave a 3ʹ-aminoxy group. In one embodiment, the cleaving agent is selected from: tris(2- carboxyethyl)phosphine (TCEP), a palladium complex or sodium nitrite. The cleaving agent may be selected from tris(2-carboxyethyl)phosphine (TCEP), tris(hydroxypropyl)phosphine (THPP), a palladium complex, an organic or inorganic base, sodium nitrite and a photoactivated transition metal complex. In one embodiment the photoactivated transition metal complex is tris(2,2ʹ-bipyridyl)ruthenium(II)).

[0186] 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.

[0187] The methods described herein rely on terminal deoxynucleotidyl transferase (TdT) enzymes or modified terminal deoxynucleotidyl transferase (TdT) enzymes. Sequences described herein are modified from the sequence of the Spotted Gar, but the corresponding changes can be introduced into the homologous sequences from other species. Terminal transferases are ubiquitous in nature and are present in many species. Many known TdT sequences have been reported in the NCBI database htt://www.ncbi.nlm.nih.gov/. The inventors have identified a number of modified TdT enzymes with improved properties. Any such TdT enzyme or modified TdT enzyme or a truncated version thereof may be used in the methods described herein.

[0188] The inventors have modified the terminal transferase from Lepisosteus oculatus TdT (spotted gar) (shown below). However the corresponding modifications can be introduced into the analogous terminal transferase sequences from any other species, including the sequences listed above in the various NCBI entries. Suitable enzymes are described in other patents, for example WO2020/161480 and GB2012542.3.

[0189] The amino acid sequence of the spotted gar ( Lepisosteusoculatus) is shown below

TABLE-US-00002 MLHIPIFPPIKKRQKLPESRNSCKYEVKFSEVAIFLVERKMGSSRRKFLTNLARSKGFRIEDVLSDAVTHVVAEDNSADELWQWLQNSSLGDLSKIEVLDISWFTECMGAGKPVQVEARHCLVKSCPVIDQYLEPSTVETVSQYACQRRTTMENHNQIFTDAFAILAENAEFNESEGPCLAFMRAASLLKSLPHAISSSKDLEGLPCLGDQTKAVIEDILEYGQCSKVQDVLCDDRYQTIKLFTSVFGVGLKTAEKWYRKGFHSLEEVQADNAIHFTKMQKAGFLYYDDISAAVCKAEAQAIGQIVEETVRLIAPDAIVTLTGGFRRGKECGHDVDFLITTPEMGKEVWLLNRLINRLQNQGILLYYDIVESTFDKTRLPCRKFEAMDHFQKCFAIIKLKKELAAGRVQKDWKAIRVDFVAPPVDNFAFALLGWTGSRQFERDLRRFARHERKMLLDNHALYDKTKKIFLPAKTEEDIFAHLGLDYIDPWQRNA

[0190] The inventors have identified various regions in the amino acid sequence having improved properties. Certain regions improve the solubility and handling of the enzyme. Certain other regions improve the ability to incorporate nucleotides with modifications at the 3ʹ-position.

[0191] Modifications which improve the solubility include a modification within the amino acid region WLLNRLINRLQNQGILLYYDIV shown highlighted in the sequence below.

TABLE-US-00003 MLHIPIFPPIKKRQKLPESRNSCKYEVKFSEVAIFLVERKMGSSRRKFLTNLARSKGFRIEDVLSDAVTHVVAEDNSADELWQWLQNSSLGDLSKIEVLDISWFTECMGAGKPVQVEARHCLVKSCPVIDQYLEPSTVETVSQYACQRRTTMENHNQIFTDAFAILAENAEFNESEGPCLAFMRAASLLKSLPHAISSSKDLEGLPCLGDQTKAVIEDILEYGQCSKVQDVLCDDRYQTIKLFTSVFGVGLKTAEKWYRKGFHSLEEVQADNAIHFTKMQKAGFLYYDDISAAVCKAEAQAIGQIVEETVRLIAPDAIVTLTGGFRRGKECGHDVDFLITTPEMGKEVWLLNRLINRLQNQGILLYYDIVESTFDKTRLPCRKFEAMDHFQKCFAIIKLKKELAAGRVQKDWKAIRVDFVAPPVDNFAFALLGWTGSRQFERDLRRFARHERKMLLDNHALYDKTKKIFLPAKTEEDIFAHLGLDYIDPWQRNA

[0192] Modifications which improve the incorporation of modified nucleotides can be at one or more of selected regions shown below. The second modification can be selected from one or more of the amino acid regions VAIF, EDN, MGA, ENHNQ, FMRA, HAl, TKA, FHS, QADNA, MQK, SAAVCK, EAQA, TVR, KEC, TPEMGK, DHFQ, LAAG, APPVDN, FARHERKMLLDNHA, and YIDP shown highlighted in the sequence below.

TABLE-US-00004 MLHIPIFPPIKKRQKLPESRNSCKYEVKFSEVAIFLVERKMGSSRRKFLTNLARSKGFRIEDVLSDAVTHVVAEDNSADELWQWLQNSSLGDLSKIEVLDISWFTECMGAGKPVQVEARHCLVKSCPVIDQYLEPSTVETVSQYACQRRTTMENHNQIFTDAFAILAENAEFNESEGPCLAFMRAASLLKSLPHAISSSKDLEGLPCLGDQTKAVIEDILEYGQCSKVQDVLCDDRYQTIKLFTSVFGVGLKTAEKWYRKGFHSLEEVQADNAIHFTKMQKAGFLYYDDISAAVCKAEAQAIGQIVEETVRLIAPDAIVTLTGGFRRGKECGHDVDFFLITTPEMGKEVWLLNRLINRLQNQGILLYYDIVESTFDKTRLPCRKFEAMDHFQKCFAIIKLKKELAAGRVQKDWKAIRVDFVAPPVDNFAFALLGWTGSRQFERDLRRFARHERKMLLDNHALYDKTKKIFLPAKTEEDIFAHLGLDYIDPWQRNA

[0193] Modified terminal deoxynucleotidyl transferase (TdT) enzymes comprising at least one amino acid modification when compared to a wild type sequence SEQ ID NO 1 or the homologous amino acid sequence of a terminal deoxynucleotidyl transferase (TdT) enzyme in other species may be preferable for use in the methods described herein, wherein the modification is selected from one or more of the amino acid regions WLLNRLINRLQNQGILLYYDI, VAIF, EDN, MGA, ENHNQ, FMRA, HAI, TKA, FHS, QADNA, MQK, SAAVCK, EAQA, TVR, KEC, TPEMGK, DHFQ, LAAG, APPVDN, FARHERKMLLDNHA, and YIDP of the sequence of SEQ ID NO 1 or the homologous regions in other species.

[0194] Homologous refers to protein sequences between two or more proteins that possess a common evolutionary origin, including proteins from superfamilies in the same species of organism as well as homologous proteins from different species. Such proteins (and their encoding nucleic acids) have sequence homology, as reflected by their sequence similarity, whether in terms of percent identity or by the presence of specific residues or motifs and conserved positions. A variety of protein (and their encoding nucleic acid) sequence alignment tools may be used to determine sequence homology. For example, the Clustal Omega multiple sequence alignment program provided by the European Molecular Biology Laboratory (EMBL) can be used to determine sequence homology or homologous regions.

[0195] Preferable sequences can contain both modifications, namely [0196] a. a first modification is within the amino acid region WLLNRLINRLQNQGILLYYDI of the sequence of SEQ ID NO 1 or the homologous region in other species; and [0197] b. a second modification is selected from one or more of the amino acid regions VAIF, EDN, MGA, ENHNQ, FMRA, HAl, TKA, FHS, QADNA, MQK, SAAVCK, EAQA, TVR, KEC, TPEMGK, DHFQ, LAAG, APPVDN, FARHERKMLLDNHA, and YIDP of the sequence of SEQ ID NO 1 or the homologous regions in other species.

[0198] As a comparison with other species, the sequence of Bostaurus (bovine) TdT is shown below:

TABLE-US-00005 MDPLCTASSGPRKKRPRQVGASMASPPHDIKFQNLVLFILEKKMGTTRRNFLMELARRKGFRVENELSDSVTHIVAENNSGSEVLEWLQVQNIRASSQLELLDVSWLIESMGAGKPVEITGKHQLVVRTDYSATPNPGFQKTPPLAVKKISQYACQRKTTLNNYNHIFTDAFEILAENSEFKENEVSYVTFMRAASVLKSLPFTIISMKDTEGIPCLGDKVKCIIEEIIEDGESSEVKAVLNDERYQSFKLFTSVFGVGLKTSEKWFRMGFRSLSKIMSDKTLKFTKMQKAGFLYYEDLVSCVTRAEAEAVGVLVKEAVWAFLPDAFVTMTGGFRRGKKIGHDVDFLITSPGSAEDEEQLLPKVINLWEKKGLLLYYDLVESTFEKFKLPSRQVDTLDHFQKCFLILKLHHQRVDSSKSNQQEGKTWKAIRVDLVMCPYENRAFALLGWTGSRQFERDIRRYATHERKMMLDNHALYDKTKRVFLKAESEEEIFAHLGLDYIEPWERNA

[0199] Modifications which improve the solubility include a modification within the amino acid region QLLPKVINLWEKKGLLLYYDLV shown highlighted in the sequence below.

TABLE-US-00006 MDPLCTASSGPRKKRPRQVGASMASPPHDIKFQNLVLFILEKKMGTTRRNFLMELARRKGFRVENELSDSVTHIVAENNSGSEVLEWLQVQNIRASSQLELLDVSWLIESMGAGKPVEITGKHQLVVRTDYSATPNPGFQKTPPLAVKKISQYACQRKTTLNNYNHIFTDAFEILAENSEFKENEVSYVTFMRAASVLKSLPFTIISMKDTEGIPCLGDKVKCIIEEIIEDGESSEVKAVLNDERYQSFKLFTSVFGVGLKTSEKWFRMGFRSLSKIMSDKTLKFTKMQKAGFLYYEDLVSCVTRAEAEAVGVLVKEAVWAFLPDAFVTMTGGFRRGKKIGHDVDFLITSPGSAEDEEQLLPKVINLWEKKGLLLYYDLVESTFEKFKLPSRQVDTLDHFQKCFLILKLHHQRVDSSKSNQQEGKTWKAIRVDLVMCPYENRAFALLGWTGSRQFERDIRRYATHERKMMLDNHALYDKTKRVFLKAESEEEIFAHLGLDYIEPWERNA

[0200] Modifications which improve the incorporation of modified nucleotides can be at one or more of selected regions shown below. The second modification can be selected from one or more of the amino acid regions LVLF, ENN, MGA, NNYNH, FMRA, FTI, VKC, FRS, MSDKT, MQK, EAEA, AVW, KKI, SPGSAE, DHFQ, MCPYEN, YATHERKMMLDNHA, and YIEP shown highlighted in the sequence below.

TABLE-US-00007 MDPLCTASSGPRKKRPRQVGASMASPPHDIKFQNLVLFILEKKMGTTRRNFLMELARRKGFRVENELSDSVTHIVAENNSGSEVLEWLQVQNIRASSQLELLDVSWLIESMGAGKPVEITGKHQLVVRTDYSATPNPGFQKTPPLAVKKISQYACQRKTTLNNYNHIFTDAFEILAENSEFKENEVSYVTFMRAASVLKSLPFTIISMKDTEGIPCLGDKVKCIIEEIIEDGESSEVKAVLNDERYQSFKLFTSVFGVGLKTSEKWFRMGFRSLSKIMSDKTLKFTKMQKAGFLYYEDLVSCVTRAEAEAVGVLVKEAVWAFLPDAFVTMTGGFRRGKKIGHDVDFLITSPGSAEDEEQLLPKVINLWEKKGLLLYYDLVESTFEKFKLPSRQVDTLDHFQKCFLILKLHHEGKTWKAIRVDLVMCPYENRAFALLGWTGSRFERDIRRYATHERKMLDNHALYDKTKRVFLKAESEEEIFAHLGLDYIEPWERNA

[0201] As a comparison with other species, the sequence of Musmusculus (mouse) TdT is shown below:

TABLE-US-00008 MDPLQAVHLGPRKKRPRQLGTPVASTPYDIRFRDLVLFILEKKMGTTRRAFLMELARRKGFRVENELSDSVTHIVAENNSGSDVLEWLQLQNIKASSELELLDISWLIECMGAGKPVEMMGRHQLVVNRNSSPSPVPGSQNVPAPAVKKISQYACQRRTTLNNYNQLFTDALDILAENDELRENEGSCLAFMRASSVLKSLPFPITSMKDTEGIPCLGDKVKSIIEGIIEDGESSEAKAVLNDERYKSFKLFTSVFGVGLKTAEKWFRMGFRTLSKIQSDKSLRFTQMQKAGFLYYEDLVSCVNRPEAEAVSMLVKEAVVTFLPDALVTMTGGFRRGKMTGHDVDFLITSPEATEDEEQQLLHKVTDFWKQQGLLLYCDILESTFEKFKQPSRKVDALDHFQKCFLILKLDHGRVHSEKSGQQEGKGWKAIRVDLVMCPYDRRAFALLGWTGSRQFERDLRRYATHERKMMLDNHALYDRTKGKTVTISPLDGKVSKLQKALRVFLEAESEEEIFAHLGLDYIEPWERNA

[0202] Modifications which improve the solubility include a modification within the amino acid region QLLHKVTDFWKQQGLLLYCDIL shown highlighted in the sequence below:

TABLE-US-00009 MDPLQAVHLGPRKKRPRQLGTPVASTPYDIRFRDLVLFILEKKMGTTRRAFLMELARRKGFRVENELSDSVTHIVAENNSGSDVLEWLQLQNIKASSELELLDISWLIECMGAGKPVEMMGRHQLVVNRNSSPSPVPGSQNVPAPAVKKISQYACQRRTTLNNYNQLFTDALDILAENDELRENEGSCLAFMRASSVLKSLPFPITSMKDTEGIPCLGDKVKSIIEGIIEDGESSEAKAVLNDERYKSFKLFTSVFGVGLKTAEKWFRMGFRTLSKIQSDKSLRFTQMQKAGFLYYEDLVSCVNRPEAEAVSMLVKEAVVTFLPDALVTMTGGFRRGKMTGHDVDFLITSPEATEDEEQQLLHKVTDFWKQQGLLYCDILESTFEKFKQPSRKVDALDHFQKCFLILKLDHGRVHSEKSGQQEGKGWKAIRVDLVMCPYDRRAFALLGWTGSRQFERDLRRYATHERKMMLDNHALYDRTKGKTVTISPLDGKVSKLQKALRVFLEAESEEEIFAHLGLDYIEPWERNA

[0203] Modifications which improve the incorporation of modified nucleotides can be at one or more of selected regions shown below. The second modification can be selected from one or more of the amino acid regions LVLF, ENN, MGA, NNYNQ, FMRA, FPI, VKS, FRT, QSDKS, MQK, VSCVNR, EAEA, AVV, KMT, SPEATE, DHFQ, MCPYDR, YATHERKMMLDNHA, and YIEP shown highlighted in the sequence below.

TABLE-US-00010 MDPLQAVHLGPRKKRPRQLGTPVASTPYDIRFRDLVLFILEKKMGTTRRAFLMELARRKGFRVENELSDSVTHIVAENNSGSDVLEWLQLQNIKASSELELLDISWLIECMGAGKPVEMMGRHQLVVNRNSSPSPVPGSQNVPAPAVKKISQYACQRRTTLNNYNQLFTDALDILAENDELRENEGSCLAFMRASSVLKSLPFPITSMKDTEGIPCLGDKVKSIIEGIIEDGESSEAKAVLNDERYKSFKLFTSVFGVGLKTAEKWFRMGFRTLSKIQSDKSLRFTQMQKAGFLYYEDLVSCNRPEAEAVSMLVKEAVVTFLPDALVTMTGGFRRGKMTGHDVDFLITSPEATEDEEQQLLHKVTDFWKQQGLLLYCDILESTFEKFKQPSRKVDALDHFQKCFLILKLDHGRVHSEKSGQQEGKGWKAIRVDLVMCPYDRRAFALLGWTGSRQFERDLRRYATHERKMMLDNHALYDRTKGKTVTISPLDGKVSKLQKALRVFLEAESEEEIFAHLGLDYIEPWERNA

[0204] Thus by a process of aligning sequences, it is immediately apparent which regions in the sequences of terminal transferases from other species correspond to the sequences described herein with respect to the spotted gar sequence shown in SEQ ID NO 1.

[0205] Modified terminal deoxynucleotidyl transferase (TdT) enzymes that may be used in the methods of the invention may comprise at least one amino acid modification when compared to a wild type sequence SEQ ID NO 1 or the homologous amino acid sequence of a terminal deoxynucleotidyl transferase (TdT) enzyme in other species, wherein the modification is selected from one or more of the amino acid regions WLLNRLINRLQNQGILLYYDI, VAIF, EDN, MGA, ENHNQ, FMRA, HAl, TKA, FHS, QADNA, MQK, SAAVCK, EAQA, TVR, KEC, TPEMGK, DHFQ, LAAG, APPVDN, FARHERKMLLDNHA, and YIDP of the sequence of SEQ ID NO 1 or the homologous regions in other species.

[0206] Furthermore, TdT enzymes that may be used in the methods of the invention include a modified TdT enzyme comprising at least two amino acid modifications when compared to a wild type sequence SEQ ID NO 1 or the homologous amino acid sequence of a terminal deoxynucleotidyl transferase (TdT) enzyme in other species, wherein; [0207] a. a first modification is within the amino acid region WLLNRLINRLQNQGILLYYDIV of the sequence of SEQ ID NO 1 or the homologous region in other species; and [0208] b. a second modification is selected from one or more of the amino acid regions VAIF, EDN, MGA, ENHNQ, FMRA, HAl, TKA, FHS, QADNA, MQK, SAAVCK, EAQA, TVR, KEC, TPEMGK, DHFQ, LAAG, APPVDN, FARHERKMLLDNHA, and YIDP of the sequence of SEQ ID NO 1 or the homologous regions in other species.

[0209] When compared to the sequence of Bostaurus (bovine) TdT; SEQ ID NO 2, [0210] a. a first modification is within the amino acid region QLLPKVINLWEKKGLLLYYDLV of the sequence of SEQ ID NO 2 or the homologous region in other species; and [0211] b. a second modification is selected from one or more of the amino acid regions LVLF, ENN, MGA, NNYNH, FMRA, FTI, VKC, FRS, MSDKT, MQK, EAEA, AVW, KKI, SPGSAE, MCP, YATHERKMMLDNHA, and YIEP of the sequence of SEQ ID NO 2 or the homologous regions in other species.

[0212] When compared to the sequence of Musmusculus (mouse) TdT; SEQ ID NO 3, [0213] a. a first modification is within the amino acid region QLLHKVTDFWKQQGLLLYCDIL of the sequence of SEQ ID NO 3 or the homologous region in other species; and [0214] b. a second modification is selected from one or more of the amino acid regions LVLF, ENN, MGA, NNYNQ, FMRA, FPI, VKS, FRT, QSDKS, MQK, VSCVNR, EAEA, AVV, KMT, SPEATE, DHFQ, MCPYDR, YATHERKMMLDNHA, and YIEP of the sequence of SEQ ID NO 3 or the homologous regions in other species.

[0215] The modifications can be chosen from any amino acid that differs from the wild type sequence. The amino acid can be a naturally occurring amino acid. The modified amino acid can be selected from ala, arg, asn, asp, cys, gln, glu, gly, his, ile, leu, lys, met, phe, pro, ser, thr, trp, val, and sec.

[0216] For the purposes of brevity, the modifications are further described in relation to SEQ ID NO 1, but the modifications are applicable to the sequences from other species, for example those sequences listed above having sequences in the NCBI database.

[0217] The sequences can be modified at positions in addition to those regions described. Embodiments of the invention may include for example methods that make use of sequences having modifications to amino acids outside the defined positions, providing those sequences retain terminal transferase activity. Embodiments of the invention may include for example methods that make use of sequences having truncations of amino acids outside the defined positions, providing those sequences retain terminal transferase activity. For example the sequences may be BRCT truncated as described in application WO2018215803 where amino acids are removed from the N-terminus whilst retaining or improving activity. Alterations, additions, insertions or deletions or truncations to amino acid positions are therefore within the scope of the methods of the invention.

[0218] The modification within the region WLLNRLINRLQNQGILLYYDIV or the corresponding region from other species help improve the solubility of the enzyme. The modification within the amino acid region WLLNRLINRLQNQGILLYYDIV can be at one or more of the underlined amino acids.

[0219] Particular changes can be selected from W-Q, N-P, R-K, L-V, R-L, L-W, Q-E, N-K, Q-K or I-L. The sequence WLLNRLINRLQNQGILLYYDIV can be altered to QLLPKVINLWEKKGLLLYYDLV.

[0220] The second modification improves incorporation of nucleotides having a modification at the 3’ position in comparison to the wild type sequence. The second modification can be selected from one or more of the amino acid regions VAIF, EDN, MGA, ENHNQ, FMRA, HAI, TKA, FHS, QADNA, MQK, SAAVCK, EAQA, TVR, KEC, TPEMGK, DHFQ, LAAG, APPVDN, FARHERKMLLDNHA, and YIDP of the sequence of SEQ ID NO 1 or the homologous regions in other species. The second modification can be selected from two or more of the amino acid regions VAIF, EDN, MGA, ENHNQ, FMRA, HAI, TKA, FHS, QADNA, MQK, SAAVCK, EAQA, TVR, KEC, TPEMGK, DHFQ, LAAG, APPVDN, FARHERKMLLDNHA, and YIDP of the sequence of SEQ ID NO 1 or the homologous regions in other species shown highlighted in the sequence below.

TABLE-US-00011 MLHIPIFPPIKKRQKLPESRNSCKYEVKFSEVAIFLLVERKMGSSRRKFLTNLARSKGFRIEDVLSDAVTHVVAEDNSADELWQWLQNSSLGDLSKIEVLDISWFTECMGAGKPVQVEARHCLVKSCPVIDQYLEPSTVETVSQYACQRRTTMENHNQIFTDAFAILAENAEFNESEGPCLAFMRAASLLKSLPHAISSSKDLEGLPCLGDQTKAVIEDILEYGQCSKVQDVLCDDRYQTIKLFTSVFGVGLKTAEKWYRKGFHSLEEVQADNAIHFTKMQKAGFLYYDDISAAVCKAEAQAIGQIVEETVRLIAPDAIVTLTGGFRRGKECGHDVDFLITTPEMGKEVWLLNRLINRLQNQGILLYYDIVESTFDKTRLPCRKFEAMDHFQKCFAIIKLKKELAAGRVQKDWKAIRVDFVAPPVDNFAFALLGWTGSRQFERDLRRFARHERKMLLDNHALYDKTKKIFLPAKTEEDIFAHLGLDYIDPWQRNA

[0221] The identified positions commence at positions V32, E74, M108, F182, T212, D271, M279, E298, A421, L456, Y486. Modifications disclosed herein contain at least one modification at the defined positions.

[0222] The modified amino acid can be in the region FMRA. The modified amino acid can be in the region QADNA. The modified amino acid can be in the region EAQA. The modified amino acid can be in the region APP. The modified amino acid can be in the region LDNHA. The modified amino acid can be in the region YIDP. The region FARHERKMLLDNHA is advantageous for removing substrate biases in modifications. The FARHERKMLLDNHA region appears highly conserved across species.

[0223] The modification selected from one or more of the amino acid regions FMRA, QADNA, EAQA, APP, FARHERKMLLDNHA, and YIDP can be at the underlined amino acid(s).

[0224] The positions for modification can include A53, V68, V71, D75, E97, 1101, G109, Q115, V116, S125, T137, Q143, N154, H155, Q157, I158, 1165, G177, L180, A181, M183, A195, K200, T212, K213, A214, E217, T239, F262, S264, Q269, N272, A273, K281, S291, K296, Q300, T309, R311, E330, T341, E343, G345, N352, N360, Q361, 1363, Y367, H389, L403, G406, D411, A421, P422, V424, N426, R438, F447, R452, L455, and/or D488.

[0225] Amino acid changes include any one of A53G, V68I, V71I, D75N, D75Q, E97A, I101V, G109E, G109R, Q115E, V116I, V116S, S125R, T137A, Q143P, N154H, H155C, Q157K, Q157R, I158M, I165V, G177D, L180V, A181E, M183R, A195P, K200R, T212S, K213S, A214R, E217Q, T239S, F262L, S264T, Q269K, N272K, A273S, A273T, K281R, S291N, K296R, Q300D, T309A, R311W, E330N, T341S, E343Q, G345R, N352Q, N360K, Q361K, I363L, Y367C, H389A, L403R, G406R, D411N, A421L, A421M, A421V, P422A, P422C, V424Y, N426R, R438K, F447W, R452K, L455I, and/or D488P.

[0226] Amino acid changes include any two or more of A53G, V68I, V71I, D75N, D75Q, E97A, I101V, G109E, G109R, Q115E, V116I, V116S, S125R, T137A, Q143P, N154H, H155C, Q157K, Q157R, I158M, I165V, G177D, L180V, A181E, M183R, A195P, K200R, T212S, K213S, A214R, E217Q, T239S, F262L, S264T, Q269K, N272K, A273S, A273T, K281R, S291N, K296R, Q300D, T309A, R311W, E330N, T341S, E343Q, G345R, N352Q, N360K, Q361K, I363L, Y367C, H389A, L403R, G406R, D411N, A421L, A421M, A421V, P422A, P422C, V424Y, N426R, R438K, F447W, R452K, L455Iand/or D488P.

[0227] The modification of QADNA to KADKA, QADKA, KADNA, QADNS, KADNT, or QADNT is advantageous for the incorporation of 3ʹ-O-modified nucleoside triphosphates to the 3ʹ-end of nucleic acids and removing substrate biases during the incorporation of modified nucleoside triphosphates. The modification of APPVDN to MCPVDN, MPPVDN, ACPVDR, VPPVDN, LPPVDR, ACPYDN, LCPVDN, or MAPVDN is advantageous for the incorporation of 3ʹ-O-modified nucleoside triphosphates to the 3ʹ-end of nucleic acids and removing substrate biases during the incorporation of modified nucleoside triphosphates. The modification of FARHERKMLLDRHA to WARHERKMILDNHA, FARHERKMILDNHA, WARHERKMLLDNHA, FARHERKMLLDRHA, or FARHEKKMLLDNHA is also advantageous for the incorporation of 3ʹ-O-modified nucleoside triphosphates to the 3ʹ-end of nucleic acids and removing substrate biases during the incorporation of modified nucleoside triphosphates.

[0228] The modification can be selected from one or more of the following sequences FRRA, QADKA, EADA, MPP, FARHERKMLLDRHA, and YIPP. Included is a modified terminal deoxynucleotidyl transferase (TdT) enzyme wherein the second modification is selected from two or more of the following sequences FRRA, QADKA, EADA, MPP, FARHERKMLLDRHA, and YIPP. Included is a modified terminal deoxynucleotidyl transferase (TdT) enzyme wherein the second modification contains each of the following sequences FRRA, QADKA, EADA, MPP, FARHERKMLLDRHA, and YIPP.

[0229] References herein to ‘nucleoside 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 nucleoside triphosphates that contain deoxyribose are: deoxyadenosine triphosphate (dATP), deoxyguanosine triphosphate (dGTP), deoxycytidine triphosphate (dCTP) or deoxythymidine triphosphate (dTTP). Examples of nucleoside 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 nucleoside triphosphates, such as naturally occurring modified nucleosides and artificial nucleosides.

[0230] Therefore, references herein to ‘3ʹ-blocked nucleoside triphosphates’ refer to nucleoside triphosphates (e.g., dATP, dGTP, dCTP or dTTP) which have an additional group on the 3ʹ end which prevents further addition of nucleotides, i.e., by replacing the 3ʹ-OH group with a protecting group.

[0231] It will be understood that references herein to ‘3ʹ-block’, ‘3ʹ-blocking group’ or ‘3ʹ-protecting group’ refer to the group attached to the 3ʹ end of the nucleoside triphosphate which prevents further nucleotide addition. The present method uses reversible 3ʹ-blocking groups which can be removed by cleavage to allow the addition of further nucleotides. By contrast, irreversible 3ʹ-blocking groups refer to dNTPs where the 3ʹ-OH group can neither be exposed nor uncovered by cleavage.

[0232] References herein to ‘cleaving agent’ refer to a substance which is able to cleave the 3ʹ- blocking group from the 3ʹ-blocked nucleoside triphosphate. In one embodiment, the cleaving agent is a chemical cleaving agent. In an alternative embodiment, the cleaving agent is an enzymatic cleaving agent.

[0233] References herein to an ‘initiator oligonucleotide’ refer to a short oligonucleotide with a free 3ʹ-end which the 3ʹ-blocked nucleoside 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.

[0234] References herein to a ‘DNA initiator sequence’ refer to a short DNA oligonuleotide with a free 3ʹ-end which the 3ʹ-blocked nucleoside triphosphate can be attached to, i.e., DNA will be synthesised from the end of the DNA initiator sequence.

[0235] In one embodiment, the initiator oligonucleotide is between 5 and 50 nucleotides long, such as between 5 and 30 nucleotides long (i.e. between 10 and 30), in particular between 5 and 20 nucleotides long (i.e., approximately 20 nucleotides long), more particularly 5 to 15 nucleotides long, for example 10 to 15 nucleotides long, especially 12 nucleotides long.

[0236] In one embodiment, the initiator oligonucleotide is single-stranded. In an alternative embodiment, the initiator oligonucleotide is double-stranded. It will be understood by persons skilled in the art that a 3ʹ-overhang (i.e., a free 3ʹ-end) allows for efficient addition.

[0237] The initiator oligonucleotide is immobilised on a solid support. This allows TdT and the cleaving agent to be removed between cycles of sequence extension without washing away the synthesised nucleic acid. The methods described herein may be carried out under aqueous conditions so that the methods can be easily performed via a flow setup.

[0238] In one embodiment, the terminal deoxynucleotidyl transferase (TdT) 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.sup.+, K.sup.+, Mg.sup.2+, Mn.sup.2+, Cu.sup.2+, Zn.sup.2+, Co.sup.2+, etc. all with appropriate counterions, such as Cl) and inorganic pyrophosphatase (e.g., the Saccharomycescerevisiae or Escherichiacoli 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 nucleoside triphosphate hydrolysis by TdT. Therefore, the use of an inorganic pyrophosphatase has the advantage of reducing the rate of (1) backwards reaction and (2) TdT strand dismutation.

[0239] In one embodiment, step (b) is performed at a pH range between 5 and 10. Therefore, it will be understood that any buffer with a buffering range of pH 5-10 could be used, for example cacodylate, Tris, HEPES or Tricine, in particular cacodylate or Tris.

[0240] In one embodiment, step (c) is performed at a temperature less than 99° C., such as less than 95° C., 90° C., 85° C., 80° C., 75° C., 70° C., 65° C., 60° C., 55° C., 50° C., 45° C., 40° C., 35° C., or 30° C. 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.

[0241] In one embodiment, after steps (b) and (c) a wash solution is applied. 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 and recycled as extension solution in step (b) when the method steps are repeated.

[0242] In another embodiment, the wash solution contains agents to abolish secondary structure or protein-nucleic acid interactions. Suitable agents are known in the art to include monovalent salts, divalent salts, chaotropic agents such as guanidinium chloride, proteinase K, detergents, and surfactants.

[0243] Also disclosed is a kit suitable for carrying out the methods of the invention, wherein the kit comprises: [0244] (i) a solid support, wherein the solid support is coated with a co-polymer to which an initiator oligonucleotide is attached, wherein the co-polymer is a co-polymer of a first co-monomer selected from acrylamide, methacrylamide, N-methylacrylamide, N,N′-dimethylacrylamide, hydroxyethyl methacrylate and N-vinyl pyrrolidinone and a second co-monomer which attaches the initiator oligonucleotide; [0245] (ii) a 3ʹ-blocked nucleoside triphosphate; [0246] (iii) a terminal deoxynucleotidyl transferase (TdT) enzyme or modified terminal deoxynucleotidyl transferase (TdT) enzyme and optionally; [0247] (iv) a cleaving agent.

[0248] Also disclosed is a kit comprising: [0249] (i) a solid support, wherein the solid support comprises a co-polymer to which an initiator oligonucleotide is attached, wherein the co-polymer is a co-polymer of one or more first co-monomer(s) selected from acrylamide, methacrylamide, N-methylacrylamide, N,N′-dimethylacrylamide, N-(hydroxylmethyl)acrylamide, N-(hydroxyethyl)acrylamide, N-[tris(hydroxymethyl)methyl]acrylamide, hydroxyethyl methacrylate and N-vinyl pyrrolidinone and a second co-monomer which attaches the initiator; [0250] (ii) a 3ʹ-blocked nucleoside triphosphate; [0251] (iii) a terminal deoxynucleotidyl transferase (TdT) enzyme or modified terminal deoxynucleotidyl transferase (TdT) enzyme and optionally; [0252] (iv) a cleaving agent.

[0253] The method can be performed on a microfluidic device such as a digital microfluidic device.

[0254] Digital microfluidics (DMF) refers to a two-dimensional planar surface platform for lab-on-a-chip systems that is based upon the manipulation of microdroplets. Droplets can be dispensed, moved, stored, mixed, reacted, or analyzed on a platform with a set of insulated electrodes. Digital microfluidics can be used together with analytical analysis procedures such as mass spectrometry, colorimetry, electrochemical, and electrochemiluminescense.

[0255] The droplet can be moved using any means of electrokinesis. The aqueous droplet can be moved using electrowetting-on-dielectric (EWoD). Electrowetting on a dielectric (EWOD) is a variant of the electrowetting phenomenon that is based on dielectric materials. During EWoD, a droplet of a conducting liquid is placed on a dielectric layer with insulating and hydrophobic properties. Upon activation of the electrodes the dielectric layer becomes less hydrophobic, thus causing the droplet to spread onto the surface.

[0256] The electrical signal on the EWoD or optically-activated amorphous silicon (a-Si) EWoD device can be delivered through segmented electrodes, active-matrix thin-film transistors or digital micromirrors. Optically-activated s-Si EWoD devices are well known in the art for actuating droplets (J. Adhes. Sci. Technol., 2012, 26, 1747-1771).

[0257] The oil in the device can be any water immiscible or hydrophobic liquid. The oil can be mineral oil, silicone oil, an alkyl-based solvent such as decane or dodecane, or a fluorinated oil. The air in the device can be any humidified gas.

[0258] The droplets can be actuated on a hydrophobic surface on the digital microfluidic device (ACS Nano 2018, 12, 6, 6050-6058). The hydrophobic surface can be a hydrophobic surface such as polytetrafluoroethylene (PTFE), Teflon AF (DuPont Inc), CYTOP (AGC Chemicals Inc), or FluoroPel (Cytonix LLC). The hydrophobic surface may be modified in such a way to reduce biofouling, especially biofouling resulting from exposure to CFPS reagents or nucleic acid reagents. The hydrophobic surface may also be superhydrophobic, such as NeverWet (NeverWet LLC) or Ultra-Ever

[0259] Dry (Flotech Performance Systems Ltd). Superhydrophobic surfaces prevent biofouling compared with typical fluorocarbon-based hydrophobic surfaces. Superhydrophobic surfaces thus prolong the capability of digital microfluidic devices to move CFPS droplets and general solutions containing biopolymers (RSC Adv., 2017, 7, 49633-49648). The hydrophobic surface can also be a slippery liquid infused porous surface (SLIPS), which can be formed by infusing Krtox-103 oil (DuPont) with porous PTFE film (Lab Chip, 2019, 19, 2275).

[0260] Magnetic particles of the invention can be used on a EWoD device and be retained in place on the device using magnets. Thus for example the beads can be retained on the surface of the support whilst the surrounding liquid is exchanged. Thus the immobilised solid support can be exposed to cycles of different reagents.

Example 1

[0261] Comparison of different PAC-BRAC polymer coating strategies and their influence on enzymatic DNA synthesis efficiency

Overview

[0262] Silica magbeads were subjected to a series of coating treatments with the same PAC-BRAC polymer formulation to determine whether the coating time, or the period of pre-polymerisation prior to coating, affected the following set of factors:

[0263] 1. The efficiency of oligo immobilization to the coated magbeads.

[0264] 2. The efficiency of N+1 enzymatic addition to oligos immobilized to the coated magbeads.

[0265] 3. The efficiency of N+12 enzymatic addition to oligos immobilized to the coated magbeads.

[0266] A graphical overview of the experimental layout is shown in Scheme 1.

[0267] Scheme 1. Experimental layout embedded image

Method

[0268] Magnetic particles: Silica-coated 1 .Math.m magnetic particles (Alpha Nanotech, 50 mg/mL suspension) were used for all of the conditions described.

[0269] Particle washing: 7 mg (140 .Math.L) of silica-coated 1 .Math.m magnetic particles (Si-beads) were washed prior to PAC-BRAC polymer coating by sequentially washing in Decon90, milliQ water, NaOH, HCI, and milliQ water.

[0270] PAC-BRAC polymerisation: The PAC-BRAC polymer solution used to coat the washed Si-beads was prepared as follows. 10 mL of 0.8% acrylamide solution (Sigma PN: A4058-100ML) was degassed before addition of 165 .Math.L 10% BRAC (N-(5-bromoacetamidylpentyl)acrylamide) in DMF. To this solution was added 11.5 .Math.L TEMED (Sigma PN: T9281-25ML). Polymerisation was initiated by addition of 100 .Math.L 5% potassium persulfate solution (Sigma PN: 216224). Solution was left to stand at RT until required for coating.

[0271] Si-bead coating with PAC-BRAC polymer solution: The MilliQ supernatant was removed and discarded from Si-beads. Si-beads were coated with PAC-BRAC polymer solution as outlined in Table 1. 167 .Math.L aliquots of PAC-BRAC solution were used to resuspend each of the pellets, either immediately after PAC-BRAC polymerisation initiation (t0) or after a delay following initiation as shown in Table 1.

TABLE-US-00012 Si-bead PAC-Brac coating conditions (PCM = Polymer Coated Magbead) Aliquot Coating start (mins) Coating incubation (mins) PCM01 t0 30 PCM02 t0 90 PCM03 t0 240 PCM04 t0 330 PCM05 t0+30 90 PCM06 t0+90 90 PCM07 t0+240 90

[0272] Washing of PAC-BRAC coated Si-beads: Each aliquot of coated PAC-BRAC-Si-beads (PMC01-07) was washed with phosphate buffer pH 7.0.

[0273] Oligo immobilization to PAC-BRAC-Si-beads: PAC-BRAC-Si-bead aliquots were resuspended in a 10 .Math.M solution of oligo D197 in 10 mM KPi pH 7.0. The resulting bead suspension was incubated at 52° C. and 1000 rpm for 60 minutes.

TABLE-US-00013 Oligo D197 sequence 5ʹ T*T*T*TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTUTTTT/icy3/TTTTT 3ʹ * = internal phosphorothioate bond U = UDG-cleavable 2dU base

[0274] Washing of oligo-PAC-BRAC-Si-beads: Each aliquot of oligo-PAC-BRAC-Si-beads was washed with a high salt buffer. Washed pellets were resuspended in 50% formamide/milliQ water and the suspension incubated at 37° C. for 30 minutes. After formamide treatment, oligo-immobilized bead aliquots were washed with a high salt buffer and finally resuspended in HEPES-KOH pH 7.2 ready for use in enzymatic synthesis reactions.

[0275] N+1 enzymatic synthesis reactions: Enzymatic 3ʹ-oxyamine N+1 nucleotide addition reactions were conducted on each of the oligo-PAC-BRAC-Si-bead aliquots; reactions were performed in duplicate for each bead type. 150 .Math.g of each oligo-PAC-BRAC-Si-magbead type (PCM01-07) was pipetted into wells of a 96 well plate. Beads were pelleted and resuspended in nucleotide addition mix (NAM) before incubation at 37 for 5 minutes. Each well was washed twice with a high salt buffer to stop the synthesis reaction and remove reaction components. Oligonucleotide was removed from the bead with uracil DNA glycosylase (UDG). The oligonucleotide was analysed by PAGE and visualised on a Typhoon scanner.

[0276] N+12 enzymatic synthesis reactions: Enzymatic 3ʹ-oxyamine N+12 (ATCGATCGATCG) nucleotide addition reactions were conducted on each of the oligo-PAC-BRAC-Si-bead aliquots; reactions were conducted in duplicate for each bead-type. 150 .Math.g of each oligo-PAC-BRAC-Si-magbead type (PCM01-07) was pipetted into wells of a 96 well plate. Beads were pelleted and resuspended in nucleotide addition mix (NAM) before incubation at 37 for 5 minutes. Each well was washed twice with a high salt buffer to stop the synthesis reaction and remove reaction components. Beads were pelleted and resuspended in nitrite deprotection solution (NDS) before incubation at room temperature for 5 minutes. Each well waas washed twice with a low salt buffer to stop the deprotection reaction and remove reaction components. The NAM, washing, NDS, washing process was repeated 12 times. Oligonucleotide was then recovered from the beads with uracil DNA glycosylase (UDG). The oligonucleotide was analysed by PAGE and visualised on a Typhoon scanner.

Results

[0277] N+1 enzymatic synthesis reactions: FIG. 2 shows the PAGE image for N+1 addition reactions performed on different PAC-BRAC polymer coated bead types (PCM01-07). The intensity of bands clearly shows that certain bead treatments are beneficial. Plotting the N-band intensity of each bead type (FIG. 3) aids interpretation of the gel image.

Example 2: Varying the Identity of the First Co-Monomer

[0278] Comparison of polymers formed from varying the first co-monomer while maintaining the identity of the second co-monomer.

OVERVIEW

[0279] Silica paramagnetic particles were subjected to a series of coating treatments with different xPAC-BRAC polymer formulations, where xPAC indicates that different acrylamide monomers were used in the polymer preparation. The different coatings were performed to determine:

[0280] 1. The ability to form polymer coats on solid supports with a range of monomer identities and combinations.

[0281] 2. The performance of the various polymer coats in a method of enzymatic DNA synthesis.

Method

[0282] Solid support preparation: The polymerisation and coating of solid support was performed as described in other examples, with the exception that some or all (0-100%) of the acrylamide co-monomer was replaced with either N-(hydroxylmethyl)acrylamide, N-(hydroxyethyl)acrylamide, N,N′-dimethyl acrylamide, or N-[tris(hydroxymethyl)methyl]acrylamide. Oligonucleotide initiator grafting was performed as described in a previous example.

[0283] N+17 enzymatic synthesis reactions: Enzymatic N+17 (CTTCATGACGTAAGGCC) nucleotide addition reactions were conducted on each of the oligo-xPAC-BRAC-Si-bead aliquots; reactions were conducted in duplicate for each bead-type. 150 .Math.g of each oligo-PAC-BRAC-Si-magbead type was pipetted into wells of a 96 well plate. Beads were pelleted and resuspended in nucleotide addition mix (NAM; containing engineered TdT, 3ʹ-aminooxy nucleotide, divalent metal ion, inorganic pyrophosphatase, and buffered to pH 7.2) before incubation at 37° C. for 5 minutes. Each well was washed twice with a high salt buffer to stop the synthesis reaction and remove reaction components. Beads were pelleted and resuspended in nitrite deprotection solution (NDS) before incubation at room temperature for 5 minutes. Each well was washed twice with a low salt buffer to stop the deprotection reaction and remove reaction components. The NAM, washing, NDS, washing process was repeated 17 times. Oligonucleotide was then recovered from the beads with uracil DNA glycosylase (UDG). The samples were prepared into indexed libraries and sequenced on an Illumina iSeq. For each sample, the percentage of reads that showed synthesis of the correct length and sequence identity was calculated.

Results

[0284] FIG. 7 shows the effect of altering the first co-monomer identity on polymer performance in a method of multicycling enzymatic synthesis. Complete replacement of acrylamide as the first co-monomer with N-[tris(hydroxymethyl)methyl]acrylamide or N,N′-dimethyl acrylamide reduces the synthesis performance obtained on the surface; meanwhile complete replacement with N-(hydroxylmethyl)acrylamide or N-(hydroxyethyl)acrylamide yielded a result comparable to the parent polymer (100% acrylamide as the first co-monomer). Surprisingly, using a mixed species first co-polymer (e.g. 50% acrylamide and 50% N-(hydroxymethyl)acrylamide) generates a polymer coating that exceeds the performance of the parent polymer. This situation can occur with even a small percentage addition of an alternative first co-monomer. Overall, this work demonstrates that the properties of the polymer coating can be finely tuned by either altering the identity of the first co-monomer or using a mixture of first co-monomers (i.e. a mixture of compounds that form the polymer but do not serve as grafting points for an initiator oligonucleotide).

Example 3. Preparation of Oligonucleotides Using the Methods of the Invention in a Frit-Retained Beads Format

[0285] The oligo-PAC-BRAC-Si-beads can be used in a method of enzymatic synthesis whereby the solutions are exchanged by means of pelleting the beads using a magnet. However, there are other ways of exchanging the solutions. In this example, the oligo-PAC-BRAC-Si-beads are used in a method of enzymatic synthesis whereby the solutions are exchanged by means of retaining the polymer coated beads with a frit in a well and applying a vacuum to remove the solutions through the frit.

Method

[0286] PAC-BRAC-Si-beads (100% acrylamide as first co-monomer) were prepared and had oligonucleotide initiators grafted to them as described in Example 1. Enzymatic DNA synthesis was performed on a Tecan Freedom Evo 200 liquid handling system using the following automated workflow (T-NAM = nucleotide addition mix [containing an engineered terminal deoxynucleotidyl transferase, divalent metal ion, inorganic pyrophosphatase, reversibly terminated dTTP nucleotide, and buffered to pH 7.2], wash 1 = high salt wash buffer, NDS = nitrite deprotection solution, wash 2 = low salt buffer):

TABLE-US-00014 Step Description Stage 1 aliquot 100 .Math.L bead solution (1.5 ug/ml) per well Preparation 2 Apply 350 mBar pressure for 10 sec 3 Apply 60 .Math.L T-NAM per well onto filter plate and mix 5 times Addition 4 Incubate at 37° C. for 15 minutes on shaker 5 Pipette mix 5 times 6 Apply 350 mBar pressure for 10 sec 7 Apply 180 .Math.L wash 1 per well onto filter plate and mix 5 times 8 Apply 350 mBar pressure for 10 sec 9 Repeat steps 7-8 twice 10 Apply 180 .Math.L wash 2 per well onto filter plate and mix 5 times 11 Apply 350 mBar pressure for 10 sec 12 Apply 60 .Math.L NDS per well onto filter plate and mix 5 times Deblock 13 Incubate at 25° C. for 5 min on shaker 14 Apply 350 mBar vacuum pressure for 10 sec 15 Pipette mix 5 times 16 Apply 180 .Math.L wash 2 per well onto filter plate and mix 5 times 17 Apply 350 mBar pressure for 10 sec 18 Repeat steps 19-20 twice Repeat addition and deblock stages (i.e. steps 3-18) as required.

[0287] The synthesised oligonucleotides were then cleaved from the solid support using uracil DNA glycosylase (UDG) and alkaline cleavage of the resulting abasic site. The cleaved oligonucleotides were analysed by polyacrylamide gel electrophoresis (PAGE) and visualized by virtue of an internal TAMRA dye on a Typhoon Biomolecular Imager.

Results

[0288] FIG. 8 shows the resulting gel image from representative wells from the 96-well and 384-well fritted plates. An initiator control was run in lane 1 as an N marker. The increase in molecular weight corresponding to the TdT-mediated addition of 8 nucleotides causes the product to migrate slower down the gel giving the major N+8 band (initiator + TTTTTTTT). These results clearly show that polymer coated beads support enzymatic synthesis in a fritted column format. Each well of the multiwell plate has a frit as the bottom surface that retains the polymer coated beads while solutions are dispensed into the wells and then removed by application of a vacuum. This set up is analogous to packed bed reactors commonly used in the chemical and biochemical industries.

Example 4. Preparation of Oligonucleotides Using the Methods of the Invention in a Polymer Coated Glass Fibre Frit Format

[0289] The oligo-PAC-BRAC-Si-beads can be used in a method of enzymatic synthesis whereby the solutions are exchanged by means of pelleting the beads using a magnet. However, there are other ways of exchanging the solutions. In this example, the glass fibre frit of a multiwell plate is directly coated with pre-polymerised PAC-BRAC and subsequently oligonucleotide initiators are attached. Addition, wash, and deblock solutions are then conveniently exchanged by means of applying a vacuum.

Method

[0290] Pre-polymerisation: The PAC-BRAC polymer solution was prepared as follows. First solution A was formed by combining 125 .Math.L 40% w/v acrylamide solution with 10 mL of ultrapure water. Solution A was then degassed with nitrogen for 20 minutes. Solution B was prepared by dissolving 108.8 mg BRAC in 1080 .Math.L dimethylformamide. Solution C was prepared by dissolving 177 mg potassium persulfate in 3527 .Math.L ultrapure water. Solution B (800 .Math.L) was added to solution A and vortexed to mix. To the combined solution A+B was added 11.5 .Math.L tetramethylethylenediamine following by mixing. Polymerisation was initiated by addition of 100 .Math.L of solution C and brief mixing. The polymerising solution was sealed and kept in the dark for 18 hours. Centrifugation was performed prior to decanting the polymer into a fresh tube.

Glass Fibre Frit Preparation, Polymer Coating, and Initiator Grafting

[0291] Wells of an AcroPrep 384-well Glass Fibre Filter Plate (5073W) were treated in turn with 80% Decon, 1 M NaOH, 0.1 M HCl, and ultrapure water to clean the glass fibre frits. The pre-polymerised PAC-BRAC was applied to the glass fibre frits in 80 .Math.L portions and incubated for 60 seconds before application of vacuum to remove the solution. This coating step was repeated four times. The polymer coated glass fibre frit was then washed with 80 .Math.L potassium phosphate (10 mM, pH 7) three times. Oligonucleotide initiator containing three internal phosphorothioate linkages was grafted to the polymer through 30 minutes of incubation at 37° C. Excess initiator was washed from the frit with 20 mM NaOH. Finally the plates were reconditioned with potassium phosphate buffer (10 mM, pH 7).

Enzymatic DNA Synthesis

[0292] Enzymatic DNA synthesis was performed on a Tecan Freedom Evo 200 liquid handling system using the following automated workflow (T-NAM = nucleotide addition mix [containing an engineered terminal deoxynucleotidyl transferase, divalent metal ion, inorganic pyrophosphatase, reversibly terminated dTTP nucleotide, and buffered to pH 7.2], wash 1 = high salt wash buffer, NDS = nitrite deprotection solution, wash 2 = low salt buffer):

TABLE-US-00015 Step Description Stage 1 Apply 30 .Math.L T-NAM per well onto filter plate Addition 2 Apply 690 mBar vacuum for 0.1 sec 3 Incubate at 37° C. for 15 4 Apply 250 mBar pressure for 20 sec 5 Apply 80 .Math.L wash 1 per well onto filter plate 6 Apply 250 mBar pressure for 20 sec 7 Repeat steps 7-8 8 Apply 80 .Math.L wash 2 per well onto filter plate 9 Apply 250 mBar pressure for 60 sec 10 Repeat steps 10-11 12 Apply 50 .Math.L NDS per well onto filter plate Deblock 13 Apply 690 mBar vaccum for 0.05 sec 14 Incubate at room temperature for 3 minutes 15 Apply to 250 mBar for 20 sec 16 Repeat 12-15 twice 17 Repeat steps 7-8 twice 18 Repeat steps 8-9 Repeat add t on and debloc< stages (i.e. steps 3-18) as required.

[0293] The synthesised oligonucleotides were then cleaved from the solid support using uracil DNA glycosylase (UDG) and N,N′-dimethylethylenediamine (DMED). The cleaved oligonucleotides were analysed by polyacrylamide gel electrophoresis (PAGE) and visualized by virtue of an internal TAMRA dye on a Typhoon Biomolecular Imager.

Results

[0294] FIG. 9 shows the resulting gel image from representative wells from the 384-well glass fibre fritted plates. An initiator control was run in lane 1 as an N marker. The increase in molecular weight corresponding to the TdT-mediated addition of 8 nucleotides causes the product to migrate slower down the gel giving the major N+8 band (initiator + TTTTTTTT). These results clearly show that polymer coated beads support enzymatic synthesis in a fritted column format. Each well of the multicell plate has a frit as the bottom surface that maintains the polymer coated beads while solutions are dispensed into the wells and then removed by application of a vacuum. This set up is analogous to the packed bed reactors commonly used in the chemical and biochemical industries.

METHODS OF NUCLEIC ACID SYNTHESIS

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Abstract

Claims

Description