Polynucleotide synthesis methods

Abstract

The invention relates to new methods for synthesising polynucleotide molecules according to a predefined nucleotide sequence. The invention also relates to methods for the assembly of synthetic polynucleotides following synthesis.

Description

[0001] POLYNUCLEOTIDE SYNTHESIS METHODS

[0002] FIELD OF THE INVENTION

[0003] The invention relates to new methods for synthesizing polynucleotide molecules according to a predefined nucleotide sequence. The invention also relates to methods for the assembly of synthetic polynucleotides following synthesis.

[0004] BACKGROUND TO THE INVENTION

[0005] Various methods exist for the synthesis and assembly of polynucleotide molecules, particularly DNA.

[0006] Phosphoramidite chemistry is a synthetic approach involving the assembly of monomers of chemically activated T, C, A or G into oligonucleotides of approximately 100 / 150 bases in length via a stepwise process. The chemical reaction steps are highly sensitive and the conditions alternate between fully anhydrous (complete absence of water), aqueous oxidative and acidic conditions (Roy and Caruthers, Molecules, 2013, 18, 14268-14284). If the reagents from the previous reaction step have not been completely removed this will be detrimental to future steps of synthesis. Accordingly, this synthesis method is limited to the production of polynucleotides of length of approximately 100 nucleotides.

[0007] The Polymerase Synthetic approach uses a polymerase to synthesise a complementary strand to a DNA template using T, C, A and G triphosphates. The reaction conditions are aqueous and mild and this approach can be used to synthesise DNA polynucleotides which are many thousands of bases in length. The main disadvantage of this method is that single- and double-stranded DNA cannot be synthesised de novo by this method, it requires a DNA template from which a copy is made, thus limiting its utility (Kosuri and Church, Nature Methods, 2014, 11, 499-507).

[0008] Template-independent synthesis methods have also been described, particularly using terminal deoxynucleotidyl transferase (TdT) (Schott and Schrade, Eur. J. Biochem, 1984, 143, 613-620). This enzyme can be used to extend a single-stranded oligonucleotide in a 5’ to 3’ direction in a controlled manner. The synthesised singlestranded oligonucleotide can subsequently be converted to a double-stranded molecule using the synthesised single-stranded oligonucleotide as a template. Although a starting template is not required, these methods can suffer from a number of drawbacks, including the tendency of the TdT enzyme to more efficiently incorporate certain nucleotides compared to others, and a requirement for incorporated nucleotides to possess reversible blocking groups to prevent promiscuous extension.

[0009] Accordingly, the previous methods described above cannot be used to synthesise double-stranded DNA de novo without the aid of some pre-existing template molecule which is copied.

[0010] The inventors have developed new methodologies by which single- and doublestranded polynucleotide molecules can be synthesised de novo in a stepwise manner without the need to copy a pre-existing template molecule. Such methods also avoid the extreme conditions associated with phosphoramidite chemistry techniques and in contrast are carried out under mild, aqueous conditions around neutral pH.

[0011] SUMMARY OF THE INVENTION

[0012] The invention provides in vitro methods of synthesising a double-stranded polynucleotide having a predefined sequence.

[0013] The invention is further defined in the section below.

[0014] Embodiments of the Invention.

[0015] 1. An in vitro method of synthesising a double-stranded polynucleotide having a predefined sequence, the method comprising performing cycles of synthesis, wherein each cycle comprises:

[0016] (A) providing an acceptor polynucleotide having first and second strands and comprising a single or multiple nucleotide overhang at one terminal end; (B) providing a donor polynucleotide having first and second strands, and at one terminal end comprises a cleavage site and a polynucleotide payload comprising one or more nucleotide pairs of the predefined sequence and a single or multiple nucleotide overhang;

[0017] (C) performing a single-stranded ligation reaction to form a ligated polynucleotide, the reaction comprising ligating only the first strands of the acceptor and donor polynucleotides at said terminal ends; and

[0018] (D) cleaving the ligated polynucleotide and generating a cleaved terminal end having a single or multiple nucleotide overhang, thereby extending the acceptor polynucleotide with the polynucleotide payload at the cleaved end, and generating a new double-stranded acceptor polynucleotide comprising a single or multiple nucleotide overhang for ligation and extension in the next cycle.

[0019] 2. A method according to embodiment 1, wherein in each cycle:

[0020] (1) step (A) comprises providing a donor polynucleotide having first and second strands and first and second terminal ends, wherein the first terminal end comprises the cleavage site and the polynucleotide payload comprising one or more nucleotide pairs of the predefined sequence, wherein the payload comprises a 5’ or a 3’ single or multiple nucleotide overhang;

[0021] (2) step (B) comprises providing an acceptor polynucleotide having first and second strands and having a 5’ or 3’ single or multiple nucleotide overhang at a first terminal end, wherein the number of nucleotides in the overhang of the acceptor polynucleotide is the same as the number of nucleotides in the overhang of the donor polynucleotide, and wherein the nucleotides of the single or multiple nucleotide overhang of the acceptor polynucleotide are payload partner nucleotides for the corresponding payload nucleotides of the single or multiple nucleotide overhang of the donor polynucleotide, and wherein: (i) if the acceptor polynucleotide has a 5’ overhang, the overhang of the payload is a 5’ overhang; or

[0022] (ii) if the acceptor polynucleotide has a 3’ overhang, the overhang of the payload is a 3’ overhang;

[0023] (3) step (C) comprises performing a single-stranded ligation reaction, wherein following ligation the one or more nucleotide pairs of the payload and the nucleotides of the single or multiple nucleotide overhangs of the acceptor and donor polynucleotides form nucleotide pairs of the predefined sequence; and

[0024] (4) step (D) comprises cleaving the polynucleotide produced in step (C) at the cleavage site by the action of an enzyme having cleavage activity to generate a cleaved terminal end with a 5’ or 3’ single or multiple nucleotide overhang, thereby extending the acceptor polynucleotide with the polynucleotide payload at the cleaved end, and generating a new double-stranded acceptor polynucleotide having a 5’ or 3’ single or multiple nucleotide overhang for ligation and extension in the next cycle.

[0025] 3. A method according to embodiment 1 or embodiment 2, wherein the cleavage step of step (D) is the only cleavage step performed in each cycle of synthesis.

[0026] 4. A method according to any one of the preceding embodiments, wherein in each cycle there is no step of incorporation of a polynucleotide having a reversible terminator group and no additional step of deprotection to remove the reversible terminator group.

[0027] 5. A method according to any one of the preceding embodiments, wherein the 5’ or 3’ overhangs of the acceptor and donor polynucleotides in each cycle are single nucleotide overhangs.

[0028] 6. A method according to any one of embodiments 2 to 5 wherein in step (C) the terminal 5’ nucleotide of the payload is non-ligatable and step (C) comprises ligating the terminal 3’ nucleotide of the payload of the donor polynucleotide to the terminal 5’ nucleotide of the first terminal end of the acceptor polynucleotide, and whereupon a single-stranded nick is formed between the non-ligatable terminal 5’ nucleotide of the payload and the terminal 3 ’ nucleotide of the first terminal end of the acceptor polynucleotide.

[0029] 7. A method according to embodiment 6, wherein in step (A) the terminal 5’ nucleotide of the payload of the donor polynucleotide is provided without a phosphate group and is thereby non-ligatable.

[0030] 8. A method according to embodiment 6 or embodiment 7, wherein after step (C) and before step (D) the method further comprises either:

[0031] (i) performing a nucleotide incorporation reaction, by the action of an enzyme having polymerase activity, initiated at the free 3’ hydroxyl group of the unligated second strand of the acceptor polynucleotide, thereby extending said second strand and using the ligated first strand as a template; or

[0032] (ii) converting the non-ligatable terminal 5’ nucleotide of the payload of the donor polynucleotide into a ligatable nucleotide followed by ligating the second strands of the acceptor and donor polynucleotides.

[0033] 9. A method according to embodiment 8(i), wherein the incorporation reaction is performed:

[0034] (i) by the action of an enzyme having polymerase activity, and wherein the polymerase displaces the original second strand of the donor polynucleotide when synthesising a new second strand; or

[0035] (ii) by the action of an enzyme having polymerase activity which possesses 5’ to 3’ exonuclease activity, and wherein the polymerase digests the original second strand of the donor polynucleotide when synthesising a new second strand. 10. A method according to embodiment 9, wherein in each cycle the cleavage site of the donor polynucleotide provided in step (A) comprises a non-cleavable cleavage site, wherein at least one nucleotide in the cleavage site in the second strand of the donor polynucleotide does not match the consensus sequence comprising the cleavage recognition site, but the corresponding nucleotides in the cleavage site in the first strand match the consensus sequence comprising the cleavage recognition site, and wherein following the incorporation reaction and copying of the first strand the mismatch is corrected thereby generating a cleavable cleavage site.

[0036] 11. A method according to any one of embodiments 8(i), 9 and 10, wherein:

[0037] (i) a single reaction fluid is used to perform step (C) and the incorporation reaction, and the reaction fluid comprises the enzyme having ligase activity and the enzyme having polymerase activity, wherein the enzyme having polymerase activity is a heat-activatable polymerase and is inactive at the temperature used to perform step (C), and wherein after step (C) and before step (D)(i) the method comprises raising the temperature of the reaction fluid to activate the polymerase; or

[0038] (ii) a single reaction fluid is used to perform step (C), the incorporation reaction and step (D), and the reaction fluid comprises the enzyme having ligase activity and the enzyme having polymerase activity and the enzyme having cleavage activity, wherein the enzyme having polymerase activity is a heat- activatable polymerase and is inactive at the temperature used to perform step (C), and wherein after step (C) and before step (D)(i) the method comprises raising the temperature of the reaction fluid to activate the polymerase.

[0039] 12. A method according to any one of embodiments 8(i), 9, 10 and 11, wherein following step (D) the method further comprises separating the cleaved donor polynucleotide from the acceptor polynucleotide and performing an incorporation reaction comprising extending the second strand of the acceptor polynucleotide at the previous nick site with new payload nucleotide(s) using the payload nucleotides of the first strand as templates, preferably by the action of an enzyme having polymerase activity, thereby re-forming the payload nucleotide pairs in the cleaved polynucleotide and thereby forming a ligatable blunt end at the first end of the acceptor polynucleotide, whereupon all pairs of nucleotides of the polynucleotide payload are incorporated at the cleaved first terminal end of the acceptor polynucleotide, and wherein the terminal nucleotides of the cleaved first terminal end are the final pair of nucleotides of the polynucleotide payload.

[0040] 13. A method according to embodiment 8(ii), wherein the step of converting the terminal 5’ non-ligatable nucleotide of the payload of the donor polynucleotide into a ligatable nucleotide comprises a performing a phosphorylation reaction comprising phosphorylating the terminal 5’ nucleotide.

[0041] 14. A method according to embodiment 13, wherein the phosphorylation reaction is performed by the action of an enzyme having kinase activity, such as by polynucleotide kinase (PNK).

[0042] 15. A method according to any one of embodiments 2 to 5, wherein in step (C) the terminal 3’ nucleotide of the payload is non-ligatable and step (C) comprises ligating the terminal 5’ nucleotide of the payload of the donor polynucleotide to the terminal 3’ nucleotide of the first terminal end of the acceptor polynucleotide, and whereupon a single-stranded nick is formed between the non-ligatable terminal 3’ nucleotide of the payload and the terminal 5’ nucleotide of the first terminal end of the acceptor polynucleotide.

[0043] 16. A method according to embodiment 15, wherein in step (A) the terminal 3’ nucleotide of the payload of the donor polynucleotide is a non-ligatable 2’,3’- dideoxynucleotide or a 2’-deoxynucleotide, or any other suitable non-ligatable nucleotide. 17. A method according to embodiment 15 or embodiment 16, wherein after step (C) and before step (D) the method further comprises converting the non-ligatable terminal 3’ nucleotide of the payload of the donor polynucleotide into a ligatable nucleotide followed by ligating the second strands of the acceptor and donor polynucleotides.

[0044] 18. A method according to embodiment 17, wherein the step of converting the non- ligatable terminal 3’ nucleotide of the payload of the donor polynucleotide into a ligatable nucleotide comprises reconstituting the 3’ hydroxy group of the ligatable terminal 3’ nucleotide.

[0045] 19. A method according to embodiment 15, wherein the terminal 3’ nucleotide of the payload comprises a reversible blocking group which prevents ligation, and wherein after step (C) and before step (D) the method further comprises removing the blocking group.

[0046] 20. A method according to any one of embodiments 15 to 19, wherein a single reaction fluid is used to perform step (C) and the incorporation reaction, and the reaction fluid comprises the enzyme having ligase activity and the enzyme having polymerase activity, wherein the enzyme having polymerase activity is a heat- activatable polymerase and is inactive at the temperature used to perform step (C), and wherein after step (C) and before step (D)(i) the method comprises raising the temperature of the reaction fluid to activate the polymerase.

[0047] 21. A method according to any one of the preceding embodiments, wherein before step (D) the method further comprises:

[0048] (i) performing a treatment step with an enzyme having phosphatase activity, such as calf intestinal phosphatase (CIP); or

[0049] (ii) performing a nuclease treatment step with an enzyme having 5’ to 3’ exonuclease activity, such as Exonuclease III, T5 exonuclease or T7 exonuclease, thereby depleting or rendering non-ligatable any acceptor polynucleotides that have failed to ligate to a donor polynucleotide.

[0050] 22. A method according to any one of the preceding embodiments, wherein the donor polynucleotide is asymmetrical, whereby only a first terminal end of the donor polynucleotide comprises a cleavage site and a polynucleotide payload comprising a 5’ or 3’ single or multiple nucleotide overhang.

[0051] 23. A method according to embodiment 22, wherein the second terminal end of the donor polynucleotide is non-ligatable and wherein:

[0052] (i) the terminal nucleotide of the first and / or second strands of the second terminal end of the donor polynucleotide comprises a blocking group; or

[0053] (ii) both polynucleotide strands of the second terminal end of the donor polynucleotide are connected together, preferably by a polynucleotide hairpin loop; or

[0054] (iii) the donor polynucleotide is blunt ended at the second terminal end and the 5’ terminal nucleotide at the second terminal end is dephosphorylated.

[0055] 24. A method according to any one of embodiments 1 to 21, wherein the second terminal end of the donor polynucleotide comprises a second polynucleotide payload, wherein:

[0056] (i) the payload nucleotide sequence of the first strand at the second terminal end of the donor polynucleotide in the 5’ to 3’ direction is the same as the payload nucleotide sequence of the second strand at the first terminal end of the donor polynucleotide in the 5’ to 3’ direction;

[0057] (ii) the payload nucleotide sequence of the second strand at the second terminal end of the donor polynucleotide in the 3’ to 5’ direction is the same as the payload nucleotide sequence of the first strand at the first terminal end of the donor polynucleotide in the 3’ to 5’ direction. 25. A method according to any one of the preceding embodiments, wherein the polynucleotide payload consists of two or more, or three or more consecutive pairs of nucleotides of the predefined sequence, as well as the nucleotide(s) of the overhang.

[0058] 26. A method according to embodiment 25, wherein the polynucleotide payload consists of four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more consecutive pairs of nucleotides of the predefined sequence, as well as the nucleotide(s) of the overhang.

[0059] 27. A method according to any one of embodiments 1 to 26, wherein both strands of the acceptor and donor polynucleotide comprises DNA.

[0060] 28. A method according to any one of embodiments 1 to 26, wherein the first strands of the acceptor and donor polynucleotide comprise RNA and the first strands of the acceptor and donor polynucleotide comprise DNA.

[0061] 29. A method according to embodiment 28, wherein following completion of cycles of synthesis the method further comprises separating the first strand of the acceptor polynucleotide comprising the nucleotides of the predefined sequence to form a singlestranded RNA polynucleotide molecule having the predefined sequence.

[0062] 30. A method according to any one of the preceding embodiments, wherein cleavage comprises cleaving the sugar-phosphate backbone of the first strand of the donor polynucleotide.

[0063] 31. A method according to any one of the preceding embodiments, wherein cleaving is performed by the action of an enzyme having overhang cleavage function, preferably a type IIS restriction enzyme, optionally BspQI. 32. A method according to any one of the preceding embodiments, wherein the cleavage site is defined by a universal nucleotide positioned in the first strand of the donor polynucleotide, wherein following cleavage the terminal nucleotide in the first strand of the cleaved first terminal end of the acceptor polynucleotide is the final nucleotide of the polynucleotide payload, optionally wherein the universal nucleotide is inosine.

[0064] 33. A method according to embodiment 32, wherein the final nucleotide of the polynucleotide payload in the first strand occupies nucleotide position n, the penultimate nucleotide of the polynucleotide payload in the first strand occupies nucleotide position n-1 and the universal nucleotide in the first strand occupies nucleotide position n+1; the method further comprising cleaving the first strand between nucleotide positions n and n+1.

[0065] 34. A method according to embodiment 32, wherein the final nucleotide of the polynucleotide payload in the first strand occupies nucleotide position n, the penultimate nucleotide of the polynucleotide payload in the first strand occupies nucleotide position n-1 and the universal nucleotide in the first strand occupies nucleotide position n+2; the method further comprising cleaving the first strand between nucleotide positions n and n+1.

[0066] 35. A method according to embodiment 32, wherein the final nucleotide of the polynucleotide payload in the first strand occupies nucleotide position n, the penultimate nucleotide of the polynucleotide payload in the first strand occupies nucleotide position n-1 and the universal nucleotide in the first strand occupies nucleotide position n+2+x, wherein x is a whole number from 1 to 10 or more; the method further comprising cleaving the first strand between nucleotide positions n and n+1. 36. A method according to any one of embodiments 32 to 35, wherein each cleavage step comprises a two-step cleavage process wherein each cleavage step comprises a first step comprising removing the universal nucleotide to form an abasic site, and a second step comprising cleaving the first strand of the donor polynucleotide at the abasic site.

[0067] 37. A method according to embodiment 36, wherein the first step is performed with a nucleotide-excising enzyme.

[0068] 38. A method according to embodiment 37, wherein the nucleotide-excising enzyme is a 3 -methyladenine DNA glycosylase enzyme.

[0069] 39. A method according to embodiment 38, wherein the nucleotide-excising enzyme is: i. human alkyladenine DNA glycosylase (hAAG); or ii. uracil DNA glycosylase (UDG).

[0070] 40. A method according to any one of embodiments 36 to 39, wherein the second step is performed with a chemical which is a base.

[0071] 41. A method according to embodiment 40, wherein the base is NaOH.

[0072] 42. A method according to any one of embodiments 36 to 39, wherein the second step is performed with an organic chemical having abasic site cleavage activity.

[0073] 43. A method according to embodiment 42, wherein the organic chemical is N,N’- dimethylethylenediamine.

[0074] 44. A method according to any one of embodiments 36 to 39, wherein the second step is performed with an enzyme having abasic site lyase activity, optionally wherein the enzyme having abasic site lyase activity is. (i) AP Endonuclease 1 ;

[0075] (ii) Endonuclease III (Nth); or

[0076] (iii) Endonuclease VIII.

[0077] 45. A method according to any one of embodiments 32 to 35, wherein each cleavage step comprises a one step cleavage process comprising removing the universal nucleotide with a cleavage enzyme wherein the enzyme is

[0078] (i) Endonuclease III;

[0079] (ii) Endonuclease VIII;

[0080] (iii) formamidopirimidine DNA glycosylase (Fpg); or

[0081] (iv) 8-oxoguanine DNA glycosylase (hOGGl).

[0082] 46. A method according to any one of embodiments 32 to 35, wherein the cleavage step comprises cleaving the first strand of the donor polynucleotide with an enzyme.

[0083] 47. A method according to embodiment 46, wherein the enzyme cleaves the first strand of the donor polynucleotide between nucleotide positions n+1 and n.

[0084] 48. A method according to embodiment 46 or embodiment 47, wherein the enzyme is Endonuclease V.

[0085] 49. A method according to embodiment 30, wherein the cleavage site is defined by a uracil nucleotide positioned in the first strand of the donor polynucleotide, wherein cleavage is performed by an enzyme having uracil DNA glycosylase activity and DNA glycosylase-lyase activity e.g. Endonuclease VIII activity, and wherein following cleavage the terminal nucleotide of first strand at the cleaved first terminal end of the acceptor polynucleotide is the final nucleotide of the polynucleotide payload. 50. A method according to any one of the preceding embodiments, wherein ligation is performed by the action of an enzyme having nucleotide ligase activity.

[0086] 51. A method according to embodiment 50, wherein the enzyme is human DNA ligase III, T3 DNA ligase, T4 DNA ligase, optionally T4 DNA ligase which has improved thermal stability compared to wild-type T4 DNA ligase, preferably wherein the enzyme is a T3 DNA ligase or a T4 DNA ligase which has improved salt tolerance compared to wild-type T4 DNA ligase.

[0087] 52. A method according to any one of the preceding embodiments, wherein

[0088] (i) the first and second strands of the acceptor polynucleotide at the second terminal end are each tethered to a surface; or

[0089] (ii) the first and second strands of the acceptor polynucleotide at the second terminal end are connected together by a polynucleotide hairpin loop and are tethered to a surface; or

[0090] (iii) the first strand of the acceptor polynucleotide at the second terminal end is tethered to a surface and the second strand of the acceptor polynucleotide at the second terminal end is untethered; or

[0091] (iv) the second strand of the acceptor polynucleotide at the second terminal end is tethered to a surface and the first strand of the acceptor polynucleotide at the second terminal end is untethered.

[0092] 53. A method according to embodiment 52(i), embodiment 52(iii) or embodiment 52(iv), wherein the tethered strand(s) at the second terminal end comprises a cleavable linker(s), wherein the linker(s) may be cleaved to detach the double-stranded polynucleotide from the surface following synthesis.

[0093] 54. A method according to embodiment 52(ii), wherein the hairpin loop at the second terminal end is tethered to a surface via a cleavable linker, wherein the linker may be cleaved to detach the double-stranded polynucleotide from the surface following synthesis. 55. A method according to embodiment 53 or embodiment 54, wherein the cleavable linker is a UV cleavable linker.

[0094] 56. A method according to any one of embodiments 52 to 55, wherein the surface is a particle, optionally a microparticle.

[0095] 57. A method according to any one of embodiments 52 to 55, wherein the surface is a planar surface.

[0096] 58. A method according to embodiment 57, wherein the surface comprises a gel.

[0097] 59. A method according to embodiment 58, wherein the surface comprises a polyacrylamide surface, such as about 2% polyacrylamide, preferably wherein the polyacrylamide surface is coupled to a solid support such as glass.

[0098] 60. A method according to any one of embodiments 52 to 59, wherein the first and second strands of the acceptor polynucleotide at the second terminal end are tethered to the surface via one or more covalent bonds.

[0099] 61. A method according to embodiment 60, wherein the one or more covalent bonds is formed between a functional group on the surface and a functional group on the acceptor polynucleotide, wherein the functional group on the acceptor polynucleotide is an amine group, a thiol group, a thiophosphate group or a thioamide group.

[0100] 62. A method according to embodiment 61, wherein the functional group on the surface is a bromoacetyl group, optionally wherein the bromoacetyl group is provided on a polyacrylamide surface derived using N- (5-bromoacetamidylpentyl) acrylamide (BRAPA). 63. A method according to any one of the preceding embodiments, wherein synthesis cycles are performed in droplets within a microfluidic system.

[0101] 64. A method according to embodiment 63, wherein the microfluidic system is an electrowetting system.

[0102] 65. A method according to embodiment 64, wherein the microfluidic system is an electrowetting-on-dielectric system (EWOD).

[0103] 66. A method according to any one of the preceding embodiments, wherein following synthesis the strands of the double-stranded polynucleotide having a predefined sequence are separated to provide a single-stranded polynucleotide having a predefined sequence.

[0104] 67. A method according to any one of the preceding embodiments, wherein following synthesis the double-stranded polynucleotide having a predefined sequence, or a region thereof, is amplified, preferably by PCR.

[0105] 68. A method of assembling a polynucleotide having a predefined sequence, the method comprising performing the method of any one of the preceding embodiments to synthesise a first polynucleotide having a predefined sequence and one or more additional polynucleotides having a predefined sequence and joining together the first and one or more additional polynucleotides.

[0106] 69. A method according to embodiment 68 wherein the first polynucleotide and the one or more additional polynucleotides are double-stranded.

[0107] 70. A method according to embodiment 69 wherein the first polynucleotide and the one or more additional polynucleotides are single-stranded. 71. A method according to any one of embodiments 68 to 70, wherein the first polynucleotide and the one or more additional polynucleotides are cleaved to create compatible termini and joined together, preferably by ligation.

[0108] 72. A method according to embodiment 71, wherein the first polynucleotide and the one or more additional polynucleotides are cleaved by a restriction enzyme at a cleavage site.

[0109] 73. A method according to any one of embodiments 64 to 72, wherein the synthesis and / or assembly steps are performed in droplets within a microfluidic system.

[0110] 74. A method according to embodiment 73, wherein the assembly steps comprise providing a first droplet comprising a first synthesised polynucleotide having a predefined sequence and a second droplet comprising an additional one or more synthesised polynucleotides having a predefined sequence, wherein the droplets are brought in contact with each other and wherein the synthesised polynucleotides are joined together thereby assembling a polynucleotide comprising the first and additional one or more polynucleotides.

[0111] 75. A method according to embodiment 74 wherein the synthesis steps are performed by providing a plurality of droplets each droplet comprising reaction reagents corresponding to a step of the synthesis cycle, and sequentially delivering the droplets to the acceptor polynucleotide in accordance with the steps of the synthesis cycles.

[0112] 76. A method according to embodiment 75, wherein following delivery of a droplet and prior to the delivery of a next droplet, a washing step is carried out to remove excess reaction reagents.

[0113] 77. A method according to embodiment 75 and 76, wherein the microfluidic system is an electrowetting system. 78. A method according to embodiment 77, wherein the microfluidic system is an electrowetting-on-dielectric system (EWOD).

[0114] 79. A method according to any one of embodiments 75 to 78, wherein synthesis and assembly steps are performed within the same system.

[0115] BRIEF DESCRIPTION OF THE DRAWINGS

[0116] Figure 1

[0117] Figure 1 depicts symbols and terminology used in the schematics of the chemistries of the specific exemplary structures and synthesis methods outlined in the later Figures.

[0118] Figure 2

[0119] Figure 2 depicts a general non-limiting exemplary structure for acceptor and donor polynucleotides for each of the 5’ and 3’ overhang approaches which use singlestranded ligation owing to the donor polynucleotide lacking 5’ phosphate groups at the terminal ends of the donor. The donor polynucleotides depicted are symmetrical, but asymmetrical donor polynucleotides, including those with a hairpin loop, may also be used. Furthermore, the donor polynucleotides depicted lack 5’ phosphate groups at their first terminal end, but as described herein as well as single-stranded ligation approaches, double-stranded ligation approaches are also possible wherein the donor polynucleotide will have a 5’ phosphate group at the first terminal end.

[0120] Figure 3

[0121] Figure 3 depicts illustrative symmetrical and symmetrical donor polynucleotides each having single nucleotide 5’ overhangs. Another possible donor polynucleotide format, which is not depicted, is a donor polynucleotide with a hairpin loop at the second terminal end (i.e. the other end to the payload).

[0122] Figure 4

[0123] Figure 4 depicts an illustrative, nonlimiting 5’ overhang synthesis approach, with the Figure depicting a single cycle with ligation, nucleotide incorporation, and cleavage steps. Single-stranded ligation occurs owing to only the 5’ end of the first strand of the acceptor having a free 5’ phosphate group, but not the 5’ end of the second strand of the donor at the first terminal end of the donor, meaning that a nick is present between the unligated second strands of the donor and acceptor. That is then followed by nucleotide incorporation at the nick in the unligated second strand using the free 3 ’OH group of the second strand of the acceptor for the incorporation and the ligated first strands of the acceptor and donor as a template. After incorporation, cleavage then results in the generation of a new acceptor polynucleotide which incorporates the payload nucleotides and has a fresh 5’ overhang ready for the start of the next cycle.

[0124] Figure 5

[0125] Figure 5 depicts an illustrative, nonlimiting 3’ overhang synthesis approach, with the Figure depicting a single cycle with ligation, nucleotide incorporation, and cleavage steps. Single-stranded ligation occurs owing to only the 5’ end of the first strand of the acceptor at the first terminal end having a 5’ phosphate group, but not the second strand of the donor at the first terminal end of the donor, meaning that a nick is present between the unligated second strands of the donor and acceptor. That is then followed by nucleotide incorporation at the nick in the unligated second strand using the 3 ’OH group of the second strand of the acceptor for the incorporation and the ligated first strands of the acceptor and donor as a template. After incorporation, cleavage then results in the generation of a new acceptor polynucleotide which incorporates the payload nucleotides and has a fresh 3’ overhang at its first terminal end ready for the start of the next cycle. Figure 6

[0126] Figure 6 depicts one advantage common to both the 5’ overhang and 3’ overhang approaches in terms of eliminating, or reducing, unwanted acceptor: acceptor and donordonor ligations. The Figure illustrates the advantage with reference to the 5’overhang method, but it is also an advantage of the 3’ overhang method. As shown, the complementary 5’ overhangs of the acceptor anneal through standard Watson-Crick base pairing allowing for subsequent ligation. In contrast, acceptor: acceptor and donor donor ligations are reduced because of the absence of complementary overhangs that can anneal through Watson-Crick base pairing and in the case of the donor polynucleotides the absence of either, or both, of a complementary overhang and 5’ phosphate groups.

[0127] Figure 7

[0128] Figure 7 illustrates a further advantage common to both the 5’ and 3’ approaches which is that a hot start polymerase can be used meaning that the ligase and polymerase can be added in a single fluid. The Figure depicts the advantage in relation to the 5’ overhang approach, but it is also applicable to 3’ overhang approach. The ligase and hot start polymerase are added together in a single fluid, ligation is carried out and then the temperature is raised to activate the hot start polymerase allowing incorporation of nucleotides. The rise in temperature may also inactivate the ligase. The ability to add both enzymes in a single fluid reduces the number of fluid addition and washing steps enhancing further the efficiency of the method.

[0129] Figure 8

[0130] Figure 8 illustrates an advantage specific to the 5’ overhang method including nucleotide incorporation in promoting correction of erroneous ligations. That is because the incorporation uses the ligated first stands of the acceptor and donor polynucleotides as a template leading to the replacement of the incorrect nucleotide responsible for the mismatch with the correct nucleotide.

[0131] Figure 9

[0132] Figure 9 illustrates an advantage specific to the 5’ overhang method including nucleotide incorporation in terms of eliminating, or at least minimising, the carrying over of unligated acceptor polynucleotide into subsequent cycles, as in the incorporation the overhang of the acceptor is effectively filled in to leave a blunt ended acceptor lacking a complementary overhang to participate in the next cycle. That helps ensure that polynucleotide sequences are not synthesised where the payload for a particular cycle is absent. As discussed herein, there are also other methods that can be used instead, or in addition to, in order to help minimise carryover of unligated acceptor polynucleotide into future cycles.

[0133] Figure 10

[0134] Figure 10 illustrates a method employing the 3’ overhang method with a mismatch in the second strand of the donor at the cleavage site together with a host start polymerase. When the mismatch is corrected owing to the incorporation using the ligated first strands of the donor and acceptor as a template the cleavage site has the correct sequence and can be cleaved. That, together with the use of a hot start polymerase, means that the ligation, incorporation, and cleavage can be carried out after the addition of a single fluid comprising all the enzymes necessary. That therefore reduces fluid addition and washing steps. The advantage illustrated is specific to the 3’ overhang method.

[0135] Figure 11

[0136] Figure 11 depicts a variant of the 5’ overhang method wherein, rather than incorporation after single stranded ligation, the nucleotide at the 5’ end of the donor polynucleotide at the nick between second strands of the donor and acceptor is phosphorylated using a kinase. The second strands of the acceptor and donor can then be ligated in a second ligation. The ligation product can then be cleaved to generate an acceptor with a 5’ overhang at the first terminal end of the acceptor ready for the next cycle with the acceptor incorporating the payload sequence from the previous cycle or cycles. Although the method is shown in relation to a 5’ overhang approach it is also applicable to the 3’ overhang approach.

[0137] Figure 12

[0138] Figure 12 provides a summary of various different methods using different 5’ and 3’ overhang-based methods as outlined herein.

[0139] Figure 13

[0140] Figure 13 presents schemes showing examples of surface chemistries for attaching polynucleotides to a surface. The examples show double-stranded embodiments wherein both strands are connected via a hairpin, but the same chemistries may be used for attaching one or both strands of a double-stranded polynucleotide where the strands are not connected via a hairpin. The surface chemistries may be used, for instance, for attaching acceptor polynucleotides to a support.

[0141] Figure 14

[0142] Figure 14 presents schemes showing examples of surface chemistries for attaching polynucleotides to surfaces. The examples show double-stranded embodiments where both strands are connected via a hairpin, but the same chemistries may be used for attaching one or both strands of a double-stranded polynucleotide where the strands are not connected via a hairpin. The surface chemistries may be used, for instance, for attaching acceptor polynucleotides to a support. Figure 15

[0143] Figure 15 depicts and explains structural features relating to the Examples which are non-limiting and exemplary.

[0144] Figure 16

[0145] Figure 16 shows the results of the gel analysis and visualisation for the experiment described in Example 1 involving immobilisation of double stranded acceptor polynucleotide precursor onto beads, enzyme digestion to activate the acceptor so that it has a 5’ overhang, followed by two cycles of the DNA synthesis method. Going from left to right in respect of the gel the results shown are for: acceptor starting material (28 bp in length, lane 1) which is then Neil digested, leaving a 20 bp fragment with a single-base 5’ overhang ready to act as an acceptor (lane 2); TVcz'I-digested acceptor after one-sided ligation to a 30 bp donor with a single-base overhang yields a 50 bp ligation product (lane 3); nick extension and Bed cleavage results in the generation of an acceptor polynucleotide for the next cycle with the acceptor including the nucleotide payload from the previous cycle (lane 4); and the acceptors are then ligated to a donor with complementary 5’ overhang in the second cycle (lane 5).

[0146] Figure 17

[0147] Figure 17 shows the results obtained in Example 2. Figure 17A shows the results of fluorescence-based analysis of a 5’ overhang synthesis method where both the initial acceptor prior to activation and the donor polynucleotide are fluorescently labelled. Going from left to right in the graph the columns are: Column 1 shows fluorescence resulting from the immobilised acceptor prior to cleavage to generate the 5’ overhang which also releases the label. Column 2 shows the fluorescence after such cleavage. Column 3 shows fluorescence after the activated acceptor has been ligated to the labelled donor. Column 4 shows the fluorescence after that ligation product has been cleaved to generate the acceptor with the payload incorporated, but with no label, which is then ready for the next cycle. Column 5 shows the fluorescence after that acceptor has been ligated in the next cycle to a new donor polynucleotide. Figure 17B shows the results of gel electrophoresis analysis for the same experiment where the acceptor is cleaved from the support and then analysed. Lanes 1 and 3 show the results after the ligations to generate acceptor: donor ligation products in the first and second cycles. Lane 2 shows the product after the cleavage step of the first cycle.

[0148] DETAILED DESCRIPTION OF THE INVENTION

[0149] The present invention provides methods for the de novo synthesis of polynucleotide molecules according to a predefined nucleotide sequence. Synthesised polynucleotides are preferably DNA and are preferably double-stranded polynucleotide molecules.

[0150] Certain embodiments of the synthesis methods of the invention will be described in more general detail herein by reference to exemplary synthesis method versions of the invention. These methods and their advantages are depicted schematically in Figures 2 to 11. Figure 12 provides a summary of a number of illustrative different methods which are discussed further below.

[0151] In each cycle of synthesis in these exemplary synthesis methods the present inventors have utilised a donor polynucleotide which carries a polynucleotide payload. The donor polynucleotide is ligated to an acceptor polynucleotide to form a ligated polynucleotide. Single-stranded ligation is employed. Single-stranded ligation is either followed by: (i) nucleotide incorporation at the single-stranded nick site present between the unligated strands; or (ii) gap repair of the unligated strands to allow a second ligation reaction to ligate the second strands of the acceptor and donor. Thus, ultimately both first and second strands are present without a nick after (i) or (ii). The ligated polynucleotide is then cleaved. Cleavage is structured such that the polynucleotide payload, which was originally part of the donor polynucleotide, is retained as part of the acceptor polynucleotide. Following cleavage, the remainder of the donor polynucleotide is released thus forming a new acceptor polynucleotide which now incorporates the polynucleotide payload at one terminal end of the molecule. The acceptor and the donor polynucleotides used in the methods provided have complementary overhangs that can pair by Watson-Crick base pairing. The overhangs may be 5’ or 3’ overhangs, with both the acceptor and the donor polynucleotide having the same type of overhang, i.e. both either have a 5’ overhang or both have a 3’ overhang. Typically, the number of nucleotides in the overhang of the donorpolynucleotide is the same as the number of nucleotides in the overhang of the acceptorpolynucleotide. In one embodiment, the acceptor and donor polynucleotides both have a 5’ overhang with the overhangs having complementary sequence to each other allowing them to hybridise (anneal) to each other. In another case, the acceptor and donor polynucleotide both have a 3’ overhang with the overhangs having complementary sequence to each other allowing them to hybridise (anneal) to each other. In any of the embodiments set out herein the 5’ or 3’ overhangs may be a single nucleotide in length.

[0152] The use of such 5’ and 3’ overhangs serves as a convenient way to help bring together the acceptor polynucleotide and the donor polynucleotide comprising the payload prior to ligation. At the end of a cycle of the synthesis cleavage results in the generation of a product which comprises the payload sequence and also a 5’ or 3’ overhang meaning that is able to act as an acceptor polynucleotide in the next cycle. Successive cycles allow the desired predefined sequence to be progressively built-up. Accordingly, by performing multiple cycles of synthesis, a polynucleotide molecule having the predefined sequence may be synthesised.

[0153] It is to be understood that all exemplary methods, including the exemplary method versions of the invention and variants thereof, and Figures depicting these methods, are not intended to be limiting on the invention.

[0154] Specific methods described herein are provided as embodiments of the invention.

[0155] Preferably, the methods are performed to synthesise a DNA polynucleotide having a predefined sequence. The methods may also be performed to synthesise an RNA polynucleotide having a predefined sequence. The methods may be performed to synthesise a double-stranded polynucleotide having a predefined sequence. The methods may be performed to synthesise a double-stranded polynucleotide having a predefined sequence wherein one strand is a DNA strand and the other strand is an RNA strand. The two strands of a double-stranded polynucleotide synthesised in accordance with the methods of the invention may be separated to form a single-stranded polynucleotide having a predefined sequence. The methods of the invention may therefore be performed to form a single-stranded DNA polynucleotide having a predefined sequence, or a single-stranded RNA polynucleotide having a predefined sequence. The invention is not limited to synthesising exclusively DNA or RNA molecules, and other forms of polynucleotide may be synthesised as discussed further herein.

[0156] The invention provides advantages compared with existing synthesis methods. For example, all reaction steps may be performed in aqueous conditions at mild pH, extensive protection and deprotection procedures are not required. Furthermore, synthesis is not dependent upon the copying of a pre-existing template strand comprising the predefined nucleotide sequence.

[0157] All of the methods share the common advantage of using overhangs to help bring together the donor and acceptor. The various forms of the overhang-based methods may also have further advantages which are outlined herein.

[0158] For example, some of the methods provided use 5’ or 3’ overhangs in conjunction with only single-stranded ligation, with single-stranded ligation being between the first strands of the donor and acceptor polynucleotides, whereas the second strands of the donor and acceptor do not ligate because the nucleotide at the 5’ end of the second strand of the donor lacks a free phosphate group. Such approaches have the advantage that they reduce the amount of unwanted acceptor: acceptor and donordonor ligations.

[0159] The 5’ and 3’ overhang methods using nucleotide incorporation in each cycle can be designed to use a ligase in conjunction with a “hot start” polymerase which only becomes active when the temperature is raised. This means that the ligase and polymerase can be added in a single reaction fluid helping reduce the number of fluid addition and washing steps needed.

[0160] The 5’ overhang methods using nucleotide incorporation in each cycle have the advantage that the incorporation may also “fill-in” the 5’ overhangs of unligated acceptor polynucleotides helping to reduce or eliminate carryover of unused acceptor polynucleotides from one cycle to the next and therefore reduce sequence errors in the synthesised sequence.

[0161] The 3’ overhang method using nucleotide incorporation can employ donor polynucleotides with a mismatch in the recognition site for the enzyme responsible for the cleavage, with the incorrect nucleotide in the second strand. That can allow all three of a ligase, hot start polymerase, and the cleavage enzyme to be added in a single fluid, reducing still further the amount of fluid additions and washing steps needed.

[0162] The methods are outlined further below.

[0163] Reaction Conditions

[0164] In one aspect the invention provides an in vitro method for synthesising a double-stranded polynucleotide having a predefined sequence.

[0165] Synthesis is carried out under conditions suitable for hybridization of nucleotides within double-stranded polynucleotides. The conditions are such as to allow the overhangs of the acceptor and donor polynucleotides which have complementary sequence to hybridise to each other. Polynucleotides are typically contacted with reagents under conditions which permit the hybridization of nucleotides to complementary nucleotides. Conditions that permit hybridization are well-known in the art (for example, Sambrook etal., 2001, Molecular Cloning: a laboratory manual, 3rd edition, Cold Spring Harbour Laboratory Press; and Current Protocols in Molecular Biology, Greene Publishing and Wiley-lnterscience, New York (1995)).

[0166] Ligation of polynucleotides can be carried out under suitable conditions, for example using a ligase (e.g., human ligase III) at a temperature that is compatible with the enzyme (e.g., room temperature) in the presence of a suitable buffered solution. In one embodiment, the buffered solution can comprise Ligation Buffer (50.5 mM Tris- HC1 pH 7.5, 10 mM MgC12, 10 mM NaCl, 10 pM ATP, 2.5% PEG8000). Alternatively, another ligase that may be used is T4 ligase. An example of a buffer suitable for T4 ligase is a ligation buffer comprising 4.4 mM Tris-HCl, 7mM MgCh, 0.7mM dithiothreitol, 0.7mM ATP, 5% polyethylene glycol (PEG6000). When necessary for incorporation / extension reactions, incorporation of nucleotides into polynucleotides can be carried out under suitable conditions, for example using a polymerase (e.g., Therminator IX polymerase) or a terminal deoxynucleotidyl transferase (TdT) enzyme or functional variant thereof to incorporate nucleotides at a suitable temperature (e.g.,~65°C) in the presence of a suitable buffered solution. In one embodiment, the buffered solution can comprise 2 mM Tris-HCl, 1 mM (NH4)2SO4, 1 mM KC1, 0.2 mM MgSCU and 0.01% Triton® X-100. One example of a polymerase that may be used for incorporation is DNA polymerase I, Large (Klenow) fragment). An example of a buffer that may be used for the incorporation is 500 mM Sodium Chloride, 100 mM Tris-HCl, 100 mM Magnesium Chloride, 10 mM DTT and pH 7.9 at 25°C.

[0167] Cleavage of polynucleotides can be carried out using a polynucleotide cleaving enzyme (e.g., endonuclease) at a temperature that is compatible with the enzyme (e.g., 37°C) in the presence of a suitable buffered solution. In one embodiment, the buffered solution can comprise 5 mM potassium acetate, 2 mM Tris-acetate, 1 mM magnesium acetate and 0.1 mM DTT. In another embodiment, the buffer used is 500 mM Potassium Acetate, 200 mM Tris-acetate, 100 mM Magnesium Acetate, 1 ng / ml Recombinant Albumin and pH 7.9 at 25°C. Given that different restriction enzymes show optimal activity in different buffers, the buffer suitable for the specific enzyme will be typically employed.

[0168] Polynucleotide Molecule Having a Predefined Sequence

[0169] The methods of the invention involve synthesising a double-stranded polynucleotide molecule having a predefined sequence. By “predefined sequence” it is meant that the nucleotide sequence of the polynucleotide molecule is determined by the user before the method is performed. The method is therefore performed in a manner that results in the final de novo synthesised polynucleotide molecule having the nucleotide sequence that was determined by the user before synthesis. As will be apparent from the description of the methods set out further herein, the methods do not require the “copying”, via complementary Watson-Crick base-pairing, of a “template” polynucleotide strand that existed before the method was performed.

[0170] Acceptor Polynucleotide

[0171] Each one of the specific exemplary methods of the present invention involves the use of an acceptor polynucleotide. Acceptor polynucleotides are described extensively with respect to the specific chemistry methods of the invention set out below and are depicted visually in the corresponding figures.

[0172] As its name implies, an acceptor polynucleotide acts to accept a double-stranded polynucleotide payload consisting of nucleotides of the predefined sequence. The polynucleotide payload is further described herein. Successive cycles of synthesis lead to the stepwise addition of multiple polynucleotide payloads to the acceptor polynucleotide leading to the generation of the polynucleotide having a predefined sequence. Accordingly, the acceptor polynucleotide acts as a scaffold on which the polynucleotide having a predefined sequence is synthesised.

[0173] A general scheme for an acceptor polynucleotide is depicted visually in Figure 2.

[0174] As shown in Figure 2, the first strand of the acceptor will be orientated so that the 5’ end is at the first terminal end. The 3’ end of the first strand of the acceptor will be therefore at second terminal end. The second strand of the acceptor will be orientated such that the 3’ end is at the first terminal end of the acceptor, with the 5’ end at the second terminal end of the acceptor.

[0175] The acceptor polynucleotide is double-stranded and has a 5’ or 3’ single stranded overhang at the first terminal end. In one case, the overhang at the first terminal end of the acceptor is a 5’ overhang. In another case, the overhang at the first terminal end of the acceptor is a 3’ overhang. In any of the embodiments set out herein, it may be that the overhang of the acceptor and donor polynucleotides is a single nucleotide in length.

[0176] The acceptor polynucleotide comprises first and second polynucleotide strands and first and second terminal ends. The first and second polynucleotide strands are connected by hybridization, via standard Watson-Crick base pairing, to form a doublestranded polynucleotide molecule. The acceptor polynucleotide comprises the overhang at the first terminal end of the acceptor. It may be, for instance, that the overhang is from one to five nucleotides in length. In one case, the overhang is, for instance, one to three nucleotides in length. In one case, the overhang is a single nucleotide in length.

[0177] In some cases, the second terminal end of the acceptor will comprise a single stranded region, for instance because the strand which is attached to the support is longer than the other strand. Such a single stranded region at the second terminal end of the acceptor will not be considered as an overhang that participates in the synthesis.

[0178] The first terminal end of the acceptor polynucleotide is ligatable. By referring to a terminal end of an acceptor polynucleotide as “ligatable” it is meant that it is capable of being ligated to a ligatable terminal end of a donor polynucleotide, as described and defined further herein. Thus a “ligatable” terminal end of an acceptor polynucleotide may be interpreted as, or explicitly referred to as, “donor ligatable” or “donor polynucleotide ligatable”. It may be ligatable in the sense that a single-stranded ligation can take place between the first strand of the acceptor polynucleotide and the first strand of the donor polynucleotide, but the second strand of the acceptor and the second strand of the donor are not ligated. In the case of a single stranded ligation, the unligated strands of the acceptor and the donor may be said to be separated by a nick.

[0179] Prior to ligation, the first terminal end of the acceptor is free, i.e. neither the first strand nor the second strand is tethered to, or otherwise attached to, any other structure.

[0180] The terminal nucleotide of the first strand at the ligatable first terminal end of the acceptor polynucleotide comprises a 5’ phosphate group to allow the ligation to take place.

[0181] The 3’ terminal nucleotide of the second strand at the ligatable first terminal end does not comprise a 5’ phosphate group. The 3’ terminal nucleotide of the second strand at the ligatable first terminal end typically comprises a 3’ hydroxyl group.

[0182] The second terminal end of the acceptor polynucleotide is preferably tethered to a surface, such as depicted in Figure 2. Such a surface may be any suitable surface as described and defined elsewhere herein. The second terminal end may be tethered to a surface due to the second strand of the acceptor polynucleotide being tethered to the surface whilst the first strand of the acceptor polynucleotide is untethered, such as depicted in Figure 2. Alternatively, the second terminal end may be tethered to a surface due to the first strand of the acceptor polynucleotide being tethered to the surface whilst the second strand of the acceptor polynucleotide is untethered. Alternatively still, the second terminal end may be tethered to a surface due to the first and second strands of the acceptor polynucleotide being tethered to the surface. Where both the first and second strands of the acceptor polynucleotide are tethered to the surface, each strand may be independently tethered to the surface. Alternatively, the first and second strands at the second terminal end of the acceptor polynucleotide may be connected together via a connector, such as via a hairpin loop, and the connector, or any other part of the second terminal end, may be tethered to the surface.

[0183] The acceptor polynucleotide may initially be provided without comprising any of the nucleotides of the predefined sequence which the user wishes to synthesise. This may be the case, for example, before commencing the very first cycle of synthesis. The sequence of the overhang of the acceptor at the start of the very first cycle may not necessarily be considered to be part of the polynucleotide payload. However, in all subsequent cycles the sequence of the overhang of the acceptor will form part of the polynucleotide payload for a given cycle. Thus, other than in the very first cycle, the acceptor polynucleotide, when provided, will already include pairs of nucleotides of the predefined sequence at the first terminal end, for example where the acceptor polynucleotide is the product of a previous cycle of synthesis.

[0184] When initially provided, prior to the commencement of any cycles of synthesis, there are no special requirements for the parameters of length, sequence and structure of the first or second strand of the acceptor polynucleotide, other than when hybridised they give an acceptor with a 5’ or 3’ overhang at the first terminal end, and that they are able to participate in the specific steps of the method.

[0185] The first and second strands of the acceptor polynucleotide may comprise nucleotides, nucleotide analogues / derivatives and / or non-nucleotides.

[0186] The skilled person is readily able to construct an acceptor polynucleotide comprising first and second strands which will be suitable to facilitate ligation, as described further herein. In certain cases the method provided comprises nucleotide incorporation that will be capable of priming new polynucleotide synthesis, as described further herein. At the first terminal end of the acceptor polynucleotide which is to be extended, mismatches between strands should be avoided, particularly in the region immediately before the overhang, GC- and AT-rich regions should be also avoided, and in addition regions of secondary structure such as hairpins or bulges which might interfere with ligation and / or other extension should be avoided.

[0187] Prior to the initiation of synthesis the length of the first and second strands of the acceptor polynucleotide can be chosen by the skilled person depending on preference and the ligase enzyme to be used and also to ensure the presence of the desired overhang.

[0188] In an acceptor polynucleotide, the first strand is hybridized to the corresponding region of the second strand and an overhang at the first terminal end of the acceptor is present. It is not essential that the entirety of the first strand is hybridized to the corresponding region of the second strand, provided that the method is still capable of being performed. Thus, mismatches between the first strand and the corresponding region of the second strand can be tolerated to a degree. Preferably, the region of sequence of the first and second strands at the end of the acceptor polynucleotide to be extended should comprise nucleobases which are complementary to corresponding nucleobases in the opposite strand, apart from the 5’ or 3’ overhang which is singlestranded. The single stranded overhang should though be complementary to that of the donor polynucleotide. In one embodiment, the overhang is a single nucleotide in length.

[0189] The first strand may be connected to the corresponding region of the second strand at the end of the acceptor polynucleotide which is not to be extended, i.e. the second terminal end, e.g. via a hairpin.

[0190] In some cases, immediately before the first cycle, a method of the invention may comprise an initial activation step wherein a precursor of the acceptor is treated to give the 5’ or 3’ overhang needed for the first cycle. Alternatively, it may be that the acceptor is immobilised on the support already with the necessary overhangs so cleavage to form the initial overhang does not form part of a method provided.

[0191] A non-limiting exemplary embodiment of an acceptor polynucleotide is provided in the Examples below. Donor Polynucleotide and Polynucleotide Payload

[0192] Each one of the specific exemplary methods of the present invention involves the use of a donor polynucleotide. Donor polynucleotides are described extensively with respect to the specific chemistry methods of the invention set out below and nonlimiting exemplary donor polynucleotides are depicted visually in the corresponding Figures.

[0193] As its name implies, a donor polynucleotide acts to donate a polynucleotide payload consisting of nucleotides of the predefined sequence. The donated payload nucleotides consist of the payload nucleotide(s) of the overhang of the donor polynucleotide as well as the one or more pairs of nucleotides of the payload in the first and second strands of the donor polynucleotide. The polynucleotide payload is further described herein. Effectively, at the end of the cycle the nucleotide(s) of the overhang at the first terminal end of the cleaved acceptor polynucleotide forms part of the payload for the next cycle, as it or they will pair with corresponding nucleotides from the overhang of the payload of the donor in the next cycle.

[0194] General schemes for a donor polynucleotide are depicted visually in Figure 2.

[0195] As shown in Figure 2, the first strand of the donor will be orientated so that the 3’ end is at the first terminal end. The 5’ end of the first strand of the donor will be therefore at second terminal end. The second strand of the donor will be orientated such that the 5’ end is at the first terminal end of the acceptor, with the 3’ end at the second terminal end of the acceptor. As donor polynucleotide may be symmetrical, the first and second terminal ends of the donor will be defined with reference to the end which will be ligated to the first terminal end of the acceptor, with that end defined as the first terminal end of the donor.

[0196] The donor polynucleotide is double-stranded, but comprises at least one end with a single-stranded 5’ or 3’ overhang present. The overhang of the donor polynucleotide has a complementary sequence to the overhang of the acceptor polynucleotide. That means the two overhangs are able to hybridise (anneal) to each other via standard Watson-Crick base pairing. In cases where the acceptor has a 5’ overhang, the donor will also have a 5’ overhang. In cases where the acceptor has a 3’ overhang, the donor will also have a 3 ’ overhang. Typically, when the donor and the acceptor hybridise to each other through their complementary overhangs, there will be no remaining single-stranded region, i.e. no absence of a nucleotide pair or no presence of an abasic site. Hence, the length of the overhang for the acceptor and the donor will typically be the same. In any of the embodiments set out herein it may be that the overhang of both the donor and the acceptor is a single nucleotide.

[0197] The donor polynucleotide comprises first and second polynucleotide strands and first and second terminal ends. The first and second polynucleotide strands are connected by hybridization, via standard Watson-Crick base pairing, to form a doublestranded polynucleotide molecule with the 5’ or 3’ overhang present at least at one end.

[0198] A terminal end of a donor polynucleotide may be “ligatable”. By referring to a terminal end of a donor polynucleotide as “ligatable” it is meant that it is capable of being ligated to the first terminal end of an acceptor polynucleotide, as described and defined further herein. Thus, a “ligatable” terminal end of a donor polynucleotide may interpreted as, or explicitly referred to as, “acceptor ligatable” or “acceptor polynucleotide ligatable”. A skilled person will readily appreciate how a terminal end of a donor polynucleotide may be structured so as to be capable of being ligated to the first terminal end of an acceptor polynucleotide, hence the end of the acceptor which is not attached to the support.

[0199] A terminal end of a donor polynucleotide may be structured so that only one of the two strands of the donor is able to ligate to a strand of the acceptor polynucleotide. For instance, it may be that at the first terminal end of the donor polynucleotide, whilst the first strand ends in a free 3’ hydroxyl group meaning it can be ligated to the first strand of the acceptor polynucleotide, it is the case that the second strand of the donor polynucleotide may lack a 5’ phosphate group so that it is not ligated to the second strand of the acceptor in the ligation step. This may mean that the ligation is a singlestranded ligation, with the other strands not ligated, there being a nick between the two termini of the strands. Alternatively, in those embodiments where the first ligation is a ligation of both strands of the acceptor and donor polynucleotides, it may be that the donor does comprise a second strand with a free 5’ phosphate group at the first terminal end of the donor polynucleotide. Further details are provided with reference to the specific method versions described further herein.

[0200] A terminal end of a donor polynucleotide may be “ligatable” and at the same time it may be structured so that it cannot be ligated to another donor polynucleotide, e.g. another donor polynucleotide of the same structure (excepting for variations in the payload sequence), in a self-ligation reaction. Accordingly, a terminal end of a donor polynucleotide may be “non-self-ligatable”. A skilled person will readily appreciate how a terminal end of a donor polynucleotide may be structured so as to be incapable of being ligated to another donor polynucleotide. Further details are provided with reference to the specific method versions described further herein. In one case, a donor polynucleotide may be non-self ligatable as the free 5’ ends of the donor lack a free phosphate group.

[0201] A terminal end of a donor polynucleotide may be “non-ligatable”. By referring to a terminal end of a donor polynucleotide as “non-ligatable” it is meant that it is not capable of being ligated to the first terminal end of an acceptor polynucleotide and it is also not capable of being ligated to another donor polynucleotide, i.e. it is also “non- self-ligatable”. A skilled person will readily appreciate how a terminal end of a donor polynucleotide may be structured so as to be incapable of being ligated to the first terminal end of an acceptor polynucleotide and to another donor polynucleotide. Further details are provided with reference to the specific method versions described further herein. One option for a non-ligatable second terminal end is for the second terminal end to be a hairpin and so having no free 5’ or 3’ free ends.

[0202] In the methods described and defined herein, only the second terminal end of a donor polynucleotide may be non-ligatable. The second terminal end of a donor polynucleotide may be non-ligatable, such as in the asymmetrical donor polynucleotide depicted in Figure 3. Alternatively, the second terminal end of a donor polynucleotide may be ligatable, such as in the symmetrical donor polynucleotide depicted in Figure 3.

[0203] Accordingly, in any of the methods of the invention described and defined herein, step (B) may comprise providing a donor polynucleotide having first and second strands, and first and second terminal ends, and comprising a polynucleotide payload comprising one or more nucleotide pairs of the predefined sequence and a cleavage site, wherein the both the acceptor and the donor polynucleotides comprise an overhang at their first terminal ends which are complementary to each other. It may be that they both comprise a 5’ overhang. Alternatively, they may both comprise a 3’ overhang. In such an embodiment, the first terminal end of both the acceptor and donor polynucleotides will be ligatable at least to each other in respect of at least one strand. In one case, the first terminal end of the donor will be non-self-ligatable.

[0204] In any of the methods of the invention described and defined herein, step (B) may comprise providing a donor polynucleotide having first and second strands, and first and second terminal ends, and comprising a polynucleotide payload comprising one or more nucleotide pairs of the predefined sequence and a cleavage site, wherein the first terminal end has a 5’ or 3’ overhang, is ligatable, but is non-self-ligatable.

[0205] In any of the methods of the invention described and defined herein, step (B) may comprise providing a donor polynucleotide having first and second strands, and first and second terminal ends, and comprising a polynucleotide payload comprising one or more nucleotide pairs of the predefined sequence and a cleavage site, wherein the first terminal end has a 5’ or 3’ overhang, is ligatable, but is non-self-ligatable, wherein the second terminal end of the donor polynucleotide either: (i) has the same 5’ or 3’ overhang, is ligatable and non-self-ligatable; or (ii) is non-ligatable.

[0206] The first terminal end of a donor polynucleotide is free, i.e. neither the first strand nor the second strand is tethered to, or otherwise attached to, any other structure.

[0207] The terminal nucleotide of the second strand at the ligatable first terminal end of the donor polynucleotide may or may not comprise a phosphate group. Whether or not a phosphate group is present will depend upon the specific chemistry method employed. It may be that the 5’ terminal nucleotide of the second strand does not comprise a phosphate group.

[0208] The terminal nucleotide of the first strand at the ligatable first terminal end of the donor polynucleotide will comprise a free 3’ hydroxyl group to allow for ligation to the first strand of the acceptor polynucleotide.

[0209] The second terminal end of the donor polynucleotide is preferably not tethered to a surface, such as the donor polynucleotides depicted in Figure 3. Where the second terminal end of the donor polynucleotide is non-ligatable, the terminal nucleotides of the first and / or second strands at the second terminal end may comprise a blocking group. A blocking group is any blocking group defined elsewhere herein. A blocking group(s) renders the second terminal end non-ligatable. Alternatively, the second terminal end of the donor polynucleotide may be provided without a 5’ phosphate group. Alternatively still, the first and second strands at the second terminal end of the donor polynucleotide may be connected together via a connector, such as via a hairpin loop. Such a connector also renders the second terminal end non-ligatable.

[0210] The donor polynucleotide comprises, at the ligatable first terminal end, a polynucleotide payload comprising one or more nucleotide pairs of the predefined sequence. The polynucleotide payload comprises one or more nucleotide pairs of the predefined sequence that are to be incorporated into the acceptor polynucleotide following ligation. The overhang is also considered part of the polynucleotide payload

[0211] The first nucleotide pair of the payload nucleotides for a given cycle can be considered to be the first pair positioned immediately before the overhang in the donor polynucleotide at the start of the cycle. There may be further nucleotide pairs forming the payload. If the polynucleotide payload comprises more than one nucleotide pair of the predefined sequence, the penultimate nucleotide of the first strand at the ligatable first terminal end prior to the overhang may be considered the next nucleotide pair of the payload, and so on.

[0212] A polynucleotide payload may consist of two or more, or three or more consecutive pairs of nucleotides of the predefined sequence. A polynucleotide payload may consist of four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more consecutive pairs of nucleotides of the predefined sequence.

[0213] The donor polynucleotide is provided with a cleavage site adjacent to the polynucleotide payload. The cleavage site is situated between the polynucleotide payload and the second terminal end of the donor polynucleotide. The exact type of cleavage site and its location within the donor polynucleotide can vary, and may be defined by the user when providing the donor polynucleotide, provided that the cleavage step is performed as described below, and results in the formation of the appropriate 5’ or 3’ overhang for the next cycle.

[0214] When initially provided, prior to the commencement of any cycles of synthesis, there are no special requirements for the parameters of length, sequence and structure of the first or second strands of the donor polynucleotide, provided that the first and second strands of the donor polynucleotide are suitable to facilitate ligation and cleavage as described further herein, e.g. in relation to specific non-limiting exemplary method versions.

[0215] The first and second strands of the donor polynucleotide may comprise nucleotides, nucleotide analogues / derivatives and / or non-nucleotides.

[0216] The skilled person is readily able to construct a donor polynucleotide comprising first and second strands which will be suitable to facilitate ligation, as described further herein.

[0217] The polynucleotide payload sequence may be any sequence desired by the user. It is not a requirement that nucleotide pairs are formed of pairs having perfect Watson- Crick complementarity. Mismatches between nucleotides at the same position in the first and second strands can be tolerated. GC- and AT -rich regions may be included if desired. Regions of secondary structure such as hairpins or bulges which might interfere with ligation should however be avoided.

[0218] Prior to the initiation of synthesis, the length of the first and second strands of the donor polynucleotide can be chosen by the user depending on preference and the ligase enzyme to be used, as well as to ensure the necessary overhangs.

[0219] In a donor polynucleotide, the first strand is hybridized to the corresponding region of the second strand. As well as having a first terminal end with a 5’ or 3’ nucleotide overhang, it is not essential that the entirety of the first strand is hybridized to the corresponding region of the second strand, provided that first and second strands are suitable for the methods as described herein. Thus, mismatches between the first strand and the corresponding region of the second strand can be tolerated to a degree.

[0220] In embodiments of the invention, one possibility is to provide a reaction fluid comprising more than one donor polynucleotide. For instance, where the donor polynucleotides are not self-ligatable that may allow donor polynucleotides to be pooled having the four different possible sequence single nucleotide 5’ overhangs, i.e. adenosine, thymine, guanine, or a cytosine nucleotide as the single nucleotide overhang, but with all four donor polynucleotides having the same payload polynucleotide pairs immediately after the single nucleotide overhang. Such a pooling approach means the number of pools of donor polynucleotides is a quarter of the number of individual donor polynucleotides which can be used to reduce the number of different fluids needed to synthesize a given sequence.

[0221] Blocking Groups

[0222] Donor polynucleotides may comprise blocking groups. A blocking group with respect to a donor polynucleotides functions to prevent two or more donor polynucleotides ligating together in a self-ligation reaction. If this were to occur it could reduce the efficiency of and interfere with the ligation step. A blocking group is typically attached to or configured in one or both strands of the second terminal end of the donor polynucleotide.

[0223] A blocking group may be attached to the 5’ terminal nucleotide of the second terminal end of the donor polynucleotide and may be 2'-3 '-dideoxy cytidine, inverted deoxythymidine or a spacer, such as an ethylene glycol based spacer e.g. hexanediol. The 5’ terminal nucleotide of the second terminal end of the donor polynucleotide may alternatively be dephosphorylated as a means to block self-ligation.

[0224] A blocking group may preferably be attached to the 3’ terminal nucleotide of the second terminal end of the donor polynucleotide and may be a phosphate group, 2'-3 dideoxycytidine, inverted deoxythymidine or a spacer, such as an ethylene glycol based spacer e.g. hexanediol.

[0225] Where the 3’ terminal nucleotide of the second terminal end of the donor polynucleotide comprises a phosphate group, the 5’ terminal nucleotide may have no blocking group.

[0226] A blocking group may be a hairpin loop, or other type of secondary structure, which connects the first and second strands of the donor polynucleotide at the second terminal end of the donor polynucleotide. In one embodiment, blocking groups are employed at the second terminal end of donor polynucleotides, particularly in those methods where the first ligation is a doublestranded ligation.

[0227] Ligation and Ligase Enzymes

[0228] The methods provided comprise a ligation step where the acceptor is ligated to the donor. The ligation may be a single-stranded ligation, specifically where the first strand of the acceptor is ligated to the first strand of the donor, with the ligation occurring at the first terminal ends of the acceptor and donor. In such methods, typically the second strand of the acceptor and the donor polynucleotides do not ligate to each other because the 5’ end of the second strand of the donor lacks a phosphate group to participate in ligation. The unligated second strands means that there is a nick in the ligation product. Alternatively, in some methods both strands of the acceptor and donor may be ligated to each other in the ligation so that the ligation is double stranded. Further alternatively, it may be that there is an initial single stranded ligation, but then the free 5’ end of the unligated second strand of the donor is phosphorylated using a kinase allowing the second strands to then be ligated.

[0229] In the methods of the invention ligation may be achieved using any suitable means. Preferably, the ligation step will be performed by a ligase enzyme. The enzyme may be human DNA ligase III

[0230] The ligase may, for instance, be selected from T3, T4, Salt-T4, Hi-T4, and hLig3 ligases. The ligase may be a T3 DNA ligase or a T4 DNA ligase

[0231] The enzyme may be human DNA ligase III, T3 DNA ligase, T4 DNA ligase, or a T4 DNA ligase which has improved salt tolerance compared to wild-type T4 DNA ligase. Preferably the enzyme is a T3 DNA ligase or a T4 DNA ligase which has improved salt tolerance compared to wild-type T4 DNA ligase.

[0232] In methods comprising incorporation of nucleotides, it may be that the enzyme has a lower optimal temperature for its activity than the polymerase. For example, it may be that the temperature of the reaction is varied so that first the temperature is optimal for the ligase, but it is then raised so that it is optimal for the polymerase. One option is to use a hot start polymerase together with a ligase which is inactivated when the temperature is increased to the optimal temperature of the polymerase. The use of such combinations of ligase and hot start polymerase may mean that the two enzymes are added in a single fluid, thereby reducing the number of fluid addition and washing steps needed.

[0233] Molecules, enzymes, chemicals and methods for ligating (joining) single- and double-stranded polynucleotides are well known to the skilled person.

[0234] Nucleotides and Nucleotide Incorporation

[0235] In certain embodiments of the methods described herein, it is desirable to perform an incorporation / extension reaction to incorporate nucleotides into polynucleotides. In particular, single-stranded ligation of the acceptor and donor polynucleotides means that the unligated second strands of the acceptor and donor will be separated by nick with the 3’ OH group of the unligated second strand of the acceptor polynucleotide able to serve as a starting point for nucleotide incorporation.

[0236] Nucleotides which can be incorporated into synthetic polynucleotides or provided in polynucleotide payloads by any of the methods described herein may be nucleotides, nucleotide analogues and modified nucleotides.

[0237] Nucleotides may comprise natural nucleobases or non-natural nucleobases. Nucleotides may contain a natural nucleobase, a sugar and a phosphate group. Natural nucleobases comprise adenosine (A), thymine (T), uracil (U), guanine (G) and cytosine (C). One of the components of the nucleotide may be further modified.

[0238] Nucleotide analogues are nucleotides that are modified structurally either in the base, sugar or phosphate or combination therein and that are still acceptable to a polymerase enzyme as a substrate for incorporation into an oligonucleotide strand.

[0239] A non-natural nucleobase may be one which will bond, e.g. hydrogen bond, to some degree to all of the nucleobases in the target polynucleotide. A non-natural nucleobase is preferably one which will bond, e.g. hydrogen bond, to some degree to nucleotides comprising the nucleosides adenosine (A), thymine (T), uracil (U), guanine (G) and cytosine (C). A non-natural nucleotide may be a peptide nucleic acid (PNA), a locked nucleic acid (LNA) and an unlocked nucleic acid (UNA), a bridged nucleic acid (BNA) or a morpholino, a phosphorothioate or a methylphosphonate.

[0240] A non-natural nucleotide may comprise a modified sugar and / or a modified nucleobase. Modified sugars include but are not limited to 2’-(9-methylribose sugar. Modified nucleobases include but are not limited to methylated nucleobases. Methylation of nucleobases is a recognised form of epigenetic modification which has the capability of altering the expression of genes and other elements such as microRNAs. Methylation of nucleobases occurs at discrete loci which are predominately dinucleotide consisting of a CpG motif, but may also occur at CHH motifs (where H is A, C, or T). Typically, during methylation a methyl group is added to the fifth carbon of cytosine bases to create methylcytosine. Thus, modified nucleobases include but are not limited to 5-methylcytosine. In some cases during synthesis modified nucleotides may be converted back to the conventional nucleotide. In one embodiment, such modification may mean that a cleavage site can be cleaved. An example of that is the conversion of methylcytosine to deoxycytidine. The invention may use protected enzyme cleavage sites with one way to achieve such protection being the use of modified bases.

[0241] Nucleotides of the predefined sequence may be incorporated opposite partner nucleotides to form a nucleotide pair. A partner nucleotide may be a complementary nucleotide. A complementary nucleotide is a nucleotide which is capable of bonding, e.g. hydrogen bonding, to some degree to the nucleotides of the predefined sequence. A complementary nucleotide will typically be one that is able to pair by Watson-Crick base pairing.

[0242] Typically, a nucleotide of the predefined sequence is positioned opposite a naturally complementary partner nucleobase. Thus adenosine may be incorporated opposite thymine and vice versa. Guanine may be incorprated opposite cytosine and vice versa. Alternatively, a nucleotide of the predefined sequence may be positioned opposite a partner nucleobase to which it will bond, e.g. hydrogen bond, to some degree. Alternatively, a partner nucleotide may be a non-complementary nucleotide. A non-complementary nucleotide is a nucleotide which is not capable of bonding, e.g. hydrogen bonding, to the nucleotide of the predefined sequence. Thus, a nucleotide of the predefined sequence may be incorporated opposite a partner nucleotide to form a mismatch, provided that the synthesised polynucleotide overall is double-stranded and wherein the first strand is attached to the second strand by hybridization. In one embodiment such a mismatch may be present in the recognition site for the cleavage enzyme. It may be that incorporation of nucleotides leads to correction of the mismatch and the consensus cleavage sequence being restored to allow cleavage.

[0243] The term “opposite” is to be understood as relating to the normal use of the term in the field of nucleic acid biochemistry, and specifically to conventional Watson- Crick base-pairing. Thus, a first nucleic acid molecule of sequence 5’-ACGA-3’ may form a duplex with a second nucleic acid molecule of sequence 5’-TCGT-3’ wherein the G of the first molecule will be positioned opposite the C of the second molecule and will hydrogen bond therewith. A first nucleic acid molecule of sequence 5’-ATGA-3’ may form a duplex with a second nucleic acid molecule of sequence 5’-TCGT-3’, wherein the T of the first molecule will mismatch with the G of the second molecule but will still be positioned opposite therewith and will act as a partner nucleotide. This principle applies to any nucleotide partner pair relationship disclosed herein, including partner pairs comprising universal nucleotides.

[0244] The methods of the invention employ acceptor and donor polynucleotides which have complementary 5’ or 3’ overhangs. The nucleotide or nucleotides in the overhang of the acceptor polynucleotide are able to base pair with those of the complementary overhang in the donor polynucleotide. Single nucleotide overhangs in particular may be employed with the single nucleotide in the acceptor being complementary to the single nucleotide in the donor.

[0245] Nucleotides and nucleotide analogues may preferably be provided as nucleoside triphosphates. Thus, in any of the methods of the invention which require an incorporation / extension step, nucleotides may be incorporated from 2’- deoxyribonucleoside-5’-(9-triphosphates (dNTPs), e.g. preferably via the action of a DNA polymerase enzyme or e.g. via the action of an enzyme having deoxynucleotidyl terminal transferase activity as described herein. Triphosphates can be substituted by tetraphosphates or pentaphosphates (generally oligophosphate). These oligophosphates can be substituted by other alkyl or acyl groups.

[0246] Modified Nucleotides

[0247] In certain embodiments of the methods described herein it may be desirable to perform an incorporation / extension reaction to incorporate one or more modified nucleotides into polynucleotides, or a polynucleotide payload may comprise one or more modified nucleotides.

[0248] Examples of epigenetic bases which may be incorporated include the following:

[0249] Examples of modified bases which may be incorporated include the following:

[0250] Examples of halogenated bases which may be incorporated include the following: where R1 = F, Cl, Br, I, alkyl, aryl, fluorescent label, aminopropargyl, aminoallyl. Examples of amino-modified bases, which may be useful in e.g. attachment / linker chemistry, which may be incorporated include the following: where base = A, T, G or C with alkyne or alkene linker.

[0251] Examples of modified bases, which may be useful in e.g. click chemistry, which may be incorporated include the following:

[0252] Examples of biotin-modified bases which may be incorporated include the following: where base = A, T, G or C with alkyne or alkene linker.

[0253] Examples of bases bearing fluorophores and quenchers which may be incorporated include the following:

[0254] Universal Nucleotides

[0255] In certain methods of the invention a universal nucleotide may be used to define a cleavage site, as described further herein.

[0256] A universal nucleotide is one wherein the nucleobase will bond, e.g. hydrogen bond, to some degree to the nucleobase of any nucleotide of the predefined sequence. A universal nucleotide is preferably one which will bond, e.g. hydrogen bond, to some degree to nucleotides comprising the nucleosides adenosine (A), thymine (T), uracil (U), guanine (G) and cytosine (C). The universal nucleotide may bond more strongly to some nucleotides than to others. For instance, a universal nucleotide (I) comprising the nucleoside, 2’-deoxyinosine, will show a preferential order of pairing of I-C > I-A > I-G approximately = I-T.

[0257] Examples of possible universal nucleotides are inosines or nitro-indoles. The universal nucleotide preferably comprises one of the following nucleobases: hypoxanthine, 4-nitroindole, 5-nitroindole, 6-nitroindole, 3 -nitropyrrole, nitroimidazole, 4-nitropyrazole, 4-nitrobenzimidazole, 5-nitroindazole, 4-aminobenzimidazole or phenyl (C6-aromatic ring. The universal nucleotide more preferably comprises one of the following nucleosides: 2'-deoxyinosine, inosine, 7-deaza-2’-deoxyinosine, 7-deaza- inosine, 2-aza-deoxyinosine, 2-aza-inosine, 4-nitroindole 2'-deoxyribonucleoside, 4- nitroindole ribonucleoside, 5-nitroindole 2' deoxyribonucleoside, 5-nitroindole ribonucleoside, 6-nitroindole 2' deoxyribonucleoside, 6-nitroindole ribonucleoside, 3- nitropyrrole 2' deoxyribonucleoside, 3 -nitropyrrole ribonucleoside, an acyclic sugar analogue of hypoxanthine, nitroimidazole 2' deoxyribonucleoside, nitroimidazole ribonucleoside, 4-nitropyrazole 2' deoxyribonucleoside, 4-nitropyrazole ribonucleoside, 4-nitrobenzimidazole 2' deoxyribonucleoside, 4-nitrobenzimidazole ribonucleoside, 5- nitroindazole 2' deoxyribonucleoside, 5 -nitroindazole ribonucleoside, 4- ami nobenzimidazole 2' deoxyribonucleoside, 4-aminobenzimidazole ribonucleoside, phenyl C-ribonucleoside or phenyl C-2’-deoxyribosyl nucleoside.

[0258] Some examples of universal bases are shown below:

[0259] 8-aminohypoxanthine 2-oxopurine hypoxanthine 8-azahypoxanthine 2-azahypoxanthine inosine base analogues

[0260] 5-nitroindol 6-nitrobenzimidazole 7-nitroindol 7-nitrobenzimidazole

[0261] 3-formylindol pyrrolopyridine benzimidazole 5-benzimidazole nitroindole derivatives

[0262] 3 -nitropyrrol 4-nitroimidazole 4-nitropyrazole 2-nitrobenzene 6-nitropiperonyl nitropyrrol and nitrobenzene derivatives nucleoside analogue Universal nucleotides incorporating cleavable bases may also be used, including photo- and enzymatically-cleavable bases, some examples of which are shown below.

[0263] Photocleavable bases:

[0264] 7-nitroindol 2-nitrophenol 6-nitropiperonyl nucleoside analogue

[0265] Base analogues cleavable by Endonuclease III: urea thymine glycol methyl tartonyl 5-hydroxy-5- 5,6-dihydro 5-hydroxy-6- 5-hydroxy-6- 5,6- (cis & trans) urea methyhydantoin thymine hydrothymine hydrouracil dihydrouracil alloxan uracil glycol 6-hydroxy-5,6- 5-hydroxy frans-1-carbamoyl- 5-hydroxy- 5-hydroxy- (cis & trans) dihydrocytosine hydantoin 2-oxo-4,5-dihydroxy- cytosine uracil

[0266] 10 imidazolidine

[0267] Base analogues cleavable by Formamidopyrimidine DNA glycosylase (Fpg):

[0268] 7,8-dihydro-8-oxo- 7,8-dihydro-8-oxo- 7,8-dihydro-8-oxo- 7,8-dihydro-8-oxo- guanine inosine adenine nebularine

[0269] 4,6-diamino-5- 2,6-diamino-4-hydroxy- 2,6-diamino-4-hydroxy- 5-hydroxycytosine 5-hydroxyuracil formamidopyrimidine 5-formamidopyrimidine 5-M-methylformamido- (Fapy-adenine) (Fapy-guanine) pyrimidine

[0270] Base analogues cleavable by 8-oxoguanine DNA glycosylase (hOGGl):

[0271] 8-oxoguanine

[0272] Base analogues cleavable by hNeill : guanidinohydantoin spiroiminodihydantoin 5-hydroxy- thymine glycol Gh Sp uracil (c / s & trans)

[0273] Base analogues cleavable by Thymine DNA glycosylase (TDG):

[0274] 5-formylcytosine 5-carboxycytosine Base analogues cleavable by Human Alkyladenine DNA glycosylase (hAAG):

[0275] 3-methyladenine 3-methylguanine 7-methylguanine 7-(2-chloroethyl)- 7-(2-hydroxyethyl)-

[0276] 7-(2-ethoxyethyl)- 1 ,2-bis-(7-guanyl)-ethane 1 ,N6-etheno- 1 , / V2-etheno- guanine adenine guanine

[0277] A / 2,3-etheno- A / 2, 3-ethano- 5-formyluracil 5-hydroxymethyl- hypoxanthine guanine guanine uracil Bases cleavable by uracil DNA glycosylase:

[0278] Bases cleavable by Human single-strand-selective monofunctional uracil-DNA Glycosylase (SMUG1): Bases cleavable by 5-methylcytosine DNA glycosylase (ROS1):

[0279] (see S. S. David, S. D. Williams Chemical reviews 1998, 98, 1221-1262 and M. I.

[0280] Ponferrada-Marin, T. Roldan-Arjona, R. R. Ariza’ Nucleic Acids Res 2009 ,37, 4264- 4274).

[0281] A preferred universal nucleotide is 2’ -deoxyinosine.

[0282] Nucleotide-

[0283] In methods described herein it may be desirable to copy the whole or a portion of one or both strands as the context dictates. For example, first and second strands may be separated. One strand may be discarded and the other strand may be copied to provide a copied strand which has a nucleotide sequence which is complementary to the template strand which is copied. It may be desirable to copy both strands, such as in an amplification reaction e.g. PCR, or any alternative method as described further herein. In any such method any suitable enzyme may be provided to copy the template strand, such as a polymerase enzyme.

[0284] Methods of the invention may comprise a nucleotide incorporation step following the ligation, in particular where single-strand ligation has taken place and the method involves extending from the free 3 ’ OH of the unligated second strand of the acceptor.

[0285] Examples of polymerases that can be used to copy a template strand are T3 RNA polymerase, T7 RNA polymerase, SP6 RNA polymerase, pol lambda, pol micro or 29 DNA polymerase. In some embodiments, the polymerase used is DNA Pol I large Klenow Fragment (KF), Taq, or Deep Vent. Polymerases which are heat resistant or have high optimal temperatures for activity may be employed. Such polymerases may allow the polymerase to be added in a fluid with another enzyme or enzymes. For example, a hot start polymerase and ligase may be added in a single fluid, with a ligation performed first, then the temperature raised to both inactivate the ligase and allow the polymerase to act. In some cases, the single fluid may further comprise the enzyme for cleavage. To copy a template strand comprising DNA, a DNA polymerase may be used.

[0286] Any suitable DNA polymerase may be used. The DNA polymerase may be for example Bst DNA polymerase full length, Bst DNA polymerase large fragment, Bsu DNA polymerase large fragment, E. coli DNA polymerase DNA Pol I large (Klenow) fragment, M-MuLV reverse transcriptase, phi29 DNA polymerase, Sulfolobus DNA polymerase IV, Taq DNA polymerase, T4 DNA polymerase, T7 DNA polymerase and enzymes having reverse transcriptase activity, for example M-MuLV reverse transcriptase. The DNA polymerase may lack 3’ to 5’ exonuclease activity. Any such suitable polymerase enzyme may be used. Such a DNA polymerase may be, for example, Bst DNA polymerase full length, Bst DNA polymerase large fragment, Bsu DNA polymerase large fragment, DNA Pol I large (Klenow) fragment (3’— >5’ exo-), M-MuLV reverse transcriptase, Sulfolobus DNA polymerase IV, Taq DNA polymerase. The DNA polymerase may possess strand displacement activity. Any such suitable polymerase enzyme may be used. Such a DNA polymerase may be, for example, Bst DNA polymerase large fragment, Bsu DNA polymerase large fragment, DNA Pol I large (Klenow) fragment (3’— >5’ exo-), M-MuLV reverse transcriptase, phi29 DNA polymerase. The DNA polymerase may lack 3’ to 5’ exonuclease activity and may posess strand displacement activity. Any such suitable polymerase enzyme may be used. Such a DNA polymerase may be, for example, Bst DNA polymerase large fragment, Bsu DNA polymerase large fragment, E. coli DNA polymerase DNA Pol I large (Klenow) fragment, M-MuLV reverse transcriptase. The DNA polymerase may lack 5’ to 3’ exonuclease activity. Any such suitable polymerase enzyme may be used. Such a DNA polymerase may be, for example, Bst DNA polymerase large fragment, Bsu DNA polymerase large fragment, DNA Pol I large (Klenow) fragment, DNA Pol I large (Klenow) fragment (3’— >5’ exo-), M-MuLV reverse transcriptase, phi29 DNA polymerase, Sulfolobus DNA polymerase IV, T4 DNA polymerase, T7 DNA polymerase. The DNA polymerase may lack both 3’ to 5’ and 5’ to 3’ exonuclease activities and may possess strand displacement activity. Any such suitable polymerase enzyme may be used. Such a DNA polymerase may be, for example, Bst DNA polymerase large fragment, Bsu DNA polymerase large fragment, DNA Pol I large (Klenow) fragment (3’— >5’ exo-), M-MuLV reverse transcriptase. The DNA polymerase may also be a genetically engineered variant. For example, the DNA polymerase may be a genetically engineered variant of the native DNA polymerase from Thermococcus species 9°N, such as species 9°N-7. One such example of a modified polymerase is Therminator IX DNA polymerase or Therminator X DNA polymerase available from New England BioLabs. Other engineered or variant DNA polymerases include Deep Vent (exo-), Vent (Exo-), 9°N DNA polymerase, Therminator DNA polymerase, Klenow fragment (Exo-), Bst DNA polymerase, Bsu DNA polymerase, Sulfolobus DNA polymerase I, and Taq Polymerase.

[0287] To copy a template strand comprising RNA, any suitable enzyme may be used. For example, an RNA polymerase may be used. Any suitable RNA polymerase may be used. The RNA polymerase may be T3 RNA polymerase, T7 RNA polymerase, SP6 RNA polymerase, E. coli RNA polymerase holoenzyme.

[0288] Those methods provided which comprise a nucleotide incorporation step in the cycle will typically use nucleotide incorporation which is template based, in particular using the ligated first strands of the acceptor polynucleotide and donor polynucleotide as a template. The methods provided may alternatively, or in addition, at some point during the overall method comprise non-template based nucleotide incorporation, for instance at the end of a method.

[0289] Phosphorylation and Second Ligation

[0290] In some embodiments, instead of nucleotide incorporation the nick between the second strands of the acceptor and the donor polynucleotide is ligated by a second ligation reaction. The non-ligatable nucleotide of the donor polynucleotide which forms the nick site is converted to a ligatable nucleotide. Typically, the non-ligatable nucleotide is of the second strand of the donor polynucleotide is provided without a free 5’ phosphate group and the method comprises phosphorylating the non-ligatable nucleotide followed by performing the second ligation raction. Such a method may be used for both 5’ and 3’ overhangs. Typically, phosphorylation is performed bya kinase enzyme. In one embodiment, the kinase used is T4 polynucleotide kinase.

[0291] In one embodiment, the kinase and ligase are added in a single reaction fluid to help streamline the method by using less fluid addition and washing steps.

[0292] Cleavage of Ligated Polynucleotide

[0293] The cleavage step functions to cleave the ligated polynucleotide such that the one or more nucleotide pairs of the predefined sequence comprising the polynucleotide payload that was previously part of the donor polynucleotide becomes incorporated into the acceptor polynucleotide at the ligatable first terminal end of the acceptor polynucleotide. At the same time, the cleavage step functions to separate the polynucleotide payload from the remainder of the donor polynucleotide. Thus, the remainder of the donor polynucleotide can then be removed from the ligated polynucleotide, leaving behind the acceptor polynucleotide with the polynucleotide payload incorporated at the ligatable first terminal end of the acceptor polynucleotide.

[0294] The cleavage step comprises cleaving the first strand of the ligated polynucleotide and cleaving the second strand of the ligated polynucleotide. Cleavage is performed so as to form a new 5’ or 3’ overhang at the first terminal end of the acceptor polynucleotide, whereupon all pairs of nucleotides of the polynucleotide payload are retained at the cleaved first terminal end of the acceptor polynucleotide, and wherein the nucleotide or nucleotides present in the new overhang form part of the nucleotide payload for the cycle being completed. The 5’ or 3’ overhang generated by the cleavage means that a new acceptor for the next cycle is effectively generated, but including the payload or payloads from the previous cycle or cycles.

[0295] In any of the methods described herein, cleavage may be cleavage of a double stranded molecule with no nick. Hence, in any of the embodiments set out herein, cleavage may be of both strands of the target to generate an overhang. Cleavage will typically be of the ligation product of the acceptor and donor polynucleotides after any incorporation or second ligation step.

[0296] Cleavage is performed such that following cleavage the first terminal end of the resulting acceptor polynucleotide attached to the support comprises the desired 5’ or 3’ overhang and also so that the first strand has a 5’ phosphate group meaning that the acceptor polynucleotide generated comprising the polynucleotide payload is ligatable.

[0297] The cleavage step can be performed by any suitable means for creating the desired overhang whilst allowing the acceptor to retain the polynucleotide payload from that cycle. Cleavage is therefore a double-stranded cleavage reaction wherein the first and second strands are cleaved at different positions in an asymmetrical cleavage reaction. In the strand which is cleaved to give the overhang cleavage is between the final nucleotide of the payload and the next nucleotide in the strand in the direction proximal to the second terminal end of the donor polynucleotide / distal to the second terminal end of the acceptor polynucleotide. In the strand which is cleaved to form the non-overhanging strand of the acceptor, the cleavage is effectively between nucleotides in the payload such the resulting terminal nucleotide of that strand forms part of the first nucleotide pair of the payload which has been delivered, but which is not the final portion of the payload because that is provided by the overhang.

[0298] As the 5’ or 3’ overhang generated by cleavage will be used for the next cycle of the synthesis, a method of the invention will not usually comprise any filling of the overhang generated after cleavage. Hence, for any of the embodiments set out herein it may be that there is no nucleotide incorporation after cleavage.

[0299] The cleavage step can be performed by any suitable means for creating the cleaved structures described above and herein where the cleavage product has a 5’ or 3’ overhang. Cleavage may comprise cleaving the sugar-phosphate backbone of the first and second strands of the donor polynucleotide molecule. In one embodiment, the overhang generated by cleavage is a single nucleotide overhang.

[0300] Preferably, cleavage may be performed by a restriction enzyme. Preferably, but not essentially, cleavage may be performed by a type IIS restriction enzyme. Preferably, but not essentially, cleavage may be performed by a type IIS restriction enzyme which generates cleavage products which have a single nucleotide overhang.

[0301] The enzyme will be chosen to given the desired overhang. Hence, where a 5’ overhang is desired a type IIS restriction enzyme which cleaves to give 5’ overhangs may be used. Examples of type IIS restriction enzymes which may be used that give cleavage products with 5’ overhangs include Alwl, and Bccl. Examples of type IIS restriction enzymes which may be used that give cleavage products with 3’ overhangs include MnII and BciVI.

[0302] Other approaches for cleavage include those which utilise a specific base nucleotide incorporated into a strand of the ligation product. One such example is wherein a cleavage site is defined by a uracil nucleotide and cleavage is performed by the combined action of a Uracil DNA glycosylase enzyme and a DNA glycosylase- lyase enzyme such as Endonuclease VIII. The Uracil DNA glycosylase enzyme catalyses the excision of the uracil base, thus forming an abasic (apyrimidinic) site while at the same time leaving the phosphodiester backbone intact. The DNA glycosylase-lyase enzyme activity creates a break in the phosphodiester backbone at the 3' and 5' sides of the abasic site, thus generating a single-strand break. Other cleavage mechanisms using a universal nucleotide may be employed. The universal nucleotide may be inosine or any other universal nucleotide described herein. Cleavage mechanisms using universal nucleotides are described elsewhere herein.

[0303] Some further examples of DNA cleaving enzymes that may be used are shown in the Table below.

[0304] Overall, following the cleavage step, a new acceptor polynucleotide is created with the chosen 5’ or 3’ overhang. The new acceptor polynucleotide comprises the old acceptor polynucleotide, that was provided at the start of the cycle, with a polynucleotide payload incorporated at the first terminal end of the acceptor polynucleotide, and the desired 5’ or 3’ overhang. The new acceptor polynucleotide is thus competent to act as an acceptor polynucleotide to accept a new polynucleotide payload to be incorporated during the next cycle. This facilitates the stepwise synthesis of the double-stranded polynucleotide having a predefined sequence, by allowing multiple polynucleotide payloads to be successively joined together.

[0305] The methods of the invention may also include a cleavage step after the predefined sequence has been synthesised in order to release it from the support.

[0306] In any of the embodiments set out herein, it may be that after cleavage has taken place that there is no filling-in of the 5’ or 3’ overhang present after cleavage and which will be utilised in the next cycle.

[0307] In one embodiment, it may be that cleavage is dependent on the preceding nucleotide incorporation step. In particular, a cleavage enzyme recognition site with a mismatch may be included, with the incorrect nucleotide in the strand with the nick, so that subsequent nucleotide incorporation based on the sequence of the ligated first and second strands of the acceptor and donor results in correction of the mismatch meaning that the sequence can be successfully recognised by the enzyme performing the cleavage and cleaved. In one embodiment, the use of such mismatches means that cleavage is made dependent on nucleotide incorporation. It may also allow the polymerase and cleavage enzyme to be added in the same fluid. In a further embodiment, all three of ligase, a hot start polymerase, and a cleavage enzyme may be added. Such mistmatch based approaches are in particular used where the overhangs are 3’ overhangs.

[0308] Acceptor Depletion

[0309] Before the cleavage step a depletion step may be performed. Such a step is optional and not essential. The depletion step allows for any acceptor polynucleotide to be depleted, if that acceptor polynucleotide has for some reason failed to join with a donor polynucleotide to form a ligated polynucleotide. Such an acceptor polynucleotide may be referred to as an unreacted acceptor polynucleotide. Failure to react may occur for various reasons including a failure to ligate with a donor polynucleotide. Depletion of any unreacted acceptor polynucleotide may be desirable so that in the next round of synthesis it does not act as acceptor polynucleotide for a subsequent polynucleotide payload. If this were to occur, the double-stranded polynucleotide having a predefined sequence would not be synthesized correctly in that particular acceptor polynucleotide structure.

[0310] In the depletion step an unreacted acceptor polynucleotide is rendered inert, i.e. incapable of acting as an acceptor polynucleotide in the next and subsequent cycles of synthesis.

[0311] Since the terminal nucleotide of the first strand at the ligatable first terminal end of the acceptor polynucleotide comprises a 5’ phosphate group, the unreacted acceptor polynucleotide can be rendered inert by removing the 5’ phosphate group so that it cannot be ligated to a donor polynucleotide in any further synthesis cycle. Accordingly, a depletion step may comprise performing a treatment step to remove the 5’ phosphate group from the terminal nucleotide of the first strand at the ligatable first terminal end of the acceptor polynucleotide. Such a step may comprise performing a treatment step with an enzyme having phosphatase activity, such as calf intestinal phosphatase (CIP). If the terminal nucleotide in the first strand at the second terminal end of the donor polynucleotide comprises a 5’ phosphate group, removal of this group at the same time will have no effect, since this part of the donor molecule will be removed following the cleavage step.

[0312] In an alternative variant method, the unreacted acceptor polynucleotide may act as a substrate for a nuclease enzyme, which can act to render an unreacted acceptor polynucleotide inert by degrading the first strand of the unreacted acceptor polynucleotide. Since the terminal nucleotide of the first strand at the ligatable first terminal end of the acceptor polynucleotide comprises a 5’ phosphate group, an unreacted acceptor polynucleotide can act as a substrate for an enzyme having 5’ to 3’ exonuclease activity, which can degrade the first strand. Accordingly, a depletion step may comprise performing a nuclease treatment step to degrade the first strand of the acceptor polynucleotide. A depletion step may comprise performing a nuclease treatment step with an enzyme having 5’ to 3’ exonuclease activity to degrade the first strand of the acceptor polynucleotide.

[0313] In methods which incorporate a depletion step before the cleavage step using an enzyme having 5’ to 3’ exonuclease activity, the donor polynucleotide for use in that cycle of synthesis should be provided such that the second terminal end of the donor polynucleotide cannot act as a substrate for an enzyme having 5’ to 3’ exonuclease activity. This can be achieved by providing the donor polynucleotide without a 5’ phosphate group at its second terminal end, or with a blocking group at its second terminal which prevents an enzyme having 5’ to 3’ exonuclease activity from degrading the first strand of the donor polynucleotide when it is ligated to the acceptor polynucleotide.

[0314] One advantage of the use of acceptors polynucleotides, and hence donor polynucleotides, with 5’ overhangs, together with incorporation of nucleotides following ligation is that the incorporation may also render unreacted acceptor polynucleotides inert because the polymerase employed can fill-in the 5’ overhangs of unligated acceptor polynucleotides. That advantage of the 5’ overhang approach with a nucleotide incorporation step is illustrated in Figure 9. In one embodiment, such a method will not include other means for depletion of unligated acceptor polynucleotide as the incorporation step will render the unreacted acceptor polynucleotides inert. Alternatively, a method employing 5’ overhangs and an incorporation step may also include other means of depleting unligated acceptor as well to increase the level of depletion.

[0315] Synthetic Polynucleotide

[0316] The polynucleotide having a predefined sequence synthesised according to the methods described herein is double-stranded. The synthesised polynucleotide overall is double-stranded and wherein the first strand is attached to the second strand by hybridization. Mismatches and regions of non-hybridization may be tolerated, provided that overall the first strand is attached to the second strand by hybridization. The strands may be separated as required to form single-stranded molecules.

[0317] Hybridisation may be defined by moderately stringent or stringent hybridisation conditions. A moderately stringent hybridisation condition uses a prewashing solution containing 5x sodium chloride / sodium citrate (SSC), 0.5% SDS, 1.0 mM EDTA (pH 8.0), hybridisation buffer of about 50% formamide, 6xSSC, and a hybridisation temperature of 55° C (or other similar hybridisation solutions, such as one containing about 50% formamide, with a hybridisation temperature of 42° C), and washing conditions of 60° C, in 0.5xSSC, 0.1% SDS. A stringent hybridisation condition hybridises in 6xSSC at 45° C, followed by one or more washes in 0. IxSSC, 0.2% SDS at 68° C.

[0318] The double-stranded polynucleotide having a predefined sequence synthesised according to the methods described herein may be retained as a double-stranded polynucleotide. Alternatively, the two strands of the double-stranded polynucleotide may be separated to provide a single-stranded polynucleotide having a predefined sequence. Conditions that permit separation of two strands of a double-stranded polynucleotide (melting) are well-known in the art (for example, Sambrook el al.. 2001, Molecular Cloning: a laboratory manual, 3rd edition, Cold Spring Harbour Laboratory Press; and Current Protocols in Molecular Biology, Greene Publishing and Wiley - Interscience, New York (1995)).

[0319] The double-stranded polynucleotide having a predefined sequence synthesised according to the methods described herein may be amplified following synthesis. Any region of the double-stranded polynucleotide may be amplified. The whole or any region of the double-stranded polynucleotide may be amplified. Conditions that permit amplification of a double-stranded polynucleotide are well-known in the art (for example, Sambrook etal., 2001, Molecular Cloning: a laboratory manual, 3rd edition, Cold Spring Harbour Laboratory Press; and Current Protocols in Molecular Biology, Greene Publishing and Wiley-lnterscience, New York (1995)). Thus any of the synthesis methods described herein may further comprise an amplification step wherein the double-stranded polynucleotide having a predefined sequence, or any region thereof, is amplified as described above. Amplification may be performed by any suitable method, such as polymerase chain reaction (PCR), polymerase spiral reaction (PSR), loop mediated isothermal amplification (LAMP), nucleic acid sequence based amplification (NASBA), self-sustained sequence replication (3 SR), rolling circle amplification (RCA), strand displacement amplification (SDA), multiple displacement amplification (MDA), ligase chain reaction (LCR), helicase dependant amplification (HD A), ramification amplification method (RAM), recombinase polymerase amplification (RPA) etc. Preferably, amplification is performed by polymerase chain reaction (PCR).

[0320] In any of the methods described and defined herein, the first and / or second strands of the acceptor polynucleotide at the second terminal end may consist of a polynucleotide sequence which is complementary to the polynucleotide sequence of a first primer oligonucleotide. The first primer oligonucleotide may be used to prime an amplification reaction to amplify all or a portion of the double-stranded polynucleotide having a predefined sequence. The first primer oligonucleotide may be used together with a second primer oligonucleotide to prime the amplification reaction to amplify all or a portion of the double-stranded polynucleotide having a predefined sequence. The second primer oligonucleotide consists of a polynucleotide sequence which is complementary to the polynucleotide sequence of a portion of the double-stranded polynucleotide having a predefined sequence to be amplified. The first and second primer oligonucleotides bind to different sites on the double-stranded polynucleotide having a predefined sequence to be amplified, thereby allowing amplicons of any desired length to be generated. The amplification reaction may be any suitable amplification reaction, such as PCR. Amplicons generated from the amplification reaction may consequently be released from the template polynucleotide, which may remain tethered to a surface.

[0321] The double-stranded or single-stranded polynucleotide having a predefined sequence synthesised according to the methods described herein can be any length. For example, the polynucleotides can be at least 10, at least 50, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450 or at least 500 nucleotides or nucleotide pairs in length. For example, the polynucleotides may be from about 10 to about 100 nucleotides or nucleotide pairs, about 10 to about 200 nucleotides or nucleotide pairs, about 10 to about 300 nucleotides or nucleotide pairs, about 10 to about 400 nucleotides or nucleotide pairs and about 10 to about 500 nucleotides or nucleotide pairs in length. The polynucleotides can be up to about 1000 or more nucleotides or nucleotide pairs, up to about 5000 or more nucleotides or nucleotide pairs in length or up to about 100000 or more nucleotides or nucleotide pairs in length.

[0322] RNA Synthesis

[0323] The methods described herein may be adapted for the synthesis of RNA.

[0324] The donor polynucleotide may be provided as an RNA / DNA hybrid polynucleotide. More specifically, the first strand of the donor polynucleotide may be provided as RNA and the second strand of the donor polynucleotide hybridised to the first strand may be provided as DNA. Following cycles of synthesis the resulting synthetic polynucleotide will itself be an RNA / DNA hybrid polynucleotide. Following synthesis it is then possible to separate the RNA and DNA strands of the hybrid polynucleotide. The DNA strand can be separated and the RNA strand can be retained for further use, e.g. in single- stranded form.

[0325] In such embodiments, it may be that rather than restriction enzymes, other cleavage means are used to generate the overhangs, for example by positioning nucleotides for cleavage in both strands such that cleavage results in the desired 5’ or 3’ overhang. Solid Phase Synthesis

[0326] Synthetic polynucleotides produced in accordance with the synthesis methods of the invention may preferably be synthesised using solid phase or reversible solid phase techniques. A variety of such techniques is known in the art and may be used. Before initiating synthesis of a new double-stranded polynucleotide of predefined sequence, acceptor polynucleotides may be immobilized to a surface e.g. a planar surface such as glass, a gel-based material, or the surface of a microparticle such as a bead or functionalised quantum dot. The material comprising the surface may itself be bound to a substrate. For example, acceptor polynucleotides may be immobilized to a gel -based material such as e.g. polyacrylamide, and wherein a gel-based material is bound to a supporting substrate such as glass.

[0327] In one embodiment, the support may be a bead. One example of a support is Dynabeads™ MyOne™ Streptavidin Cl beads.

[0328] Polynucleotides may be immobilized or tethered to surfaces directly or indirectly. For example they may be attached directly to surfaces by chemical bonding. They may be indirectly tethered to surfaces via an intermediate surface, such as the surface of a microparticle or bead e.g. as in SPRI or as in electrowetting systems, as described below. Cycles of synthesis may then be initiated and completed whilst the acceptor polynucleotide incorporating the newly-synthesised polynucleotide is immobilized.

[0329] In such methods a double-stranded acceptor polynucleotide may be immobilized to a surface prior to the incorporation of the first payload. Such an immobilized doublestranded acceptor polynucleotide may therefore act as an anchor to tether the doublestranded polynucleotide of the predefined sequence to the surface during and after synthesis.

[0330] Only one strand of such a double-stranded acceptor polynucleotide may be immobilized to the surface at the same end of the molecule (for example as depicted schematically in Figure 2). Alternatively, both strands of a double-stranded acceptor polynucleotide may each be immobilized to the surface at the same end of the molecule. A double-stranded acceptor polynucleotide may be provided with each strand connected at adjacent ends, such as via a hairpin loop at the second terminal end, i.e. the opposite end to the site of initiation of new synthesis, and connected ends may be immobilized on a surface.

[0331] Solid Phase Synthesis on Planar Surfaces

[0332] Before initiating synthesis of a new double-stranded polynucleotide of predefined sequence synthetic acceptor polynucleotides can be synthesised by methods known in the art, including those described herein, and tethered to a surface.

[0333] Pre-formed polynucleotides can be tethered to surfaces by methods commonly employed to create nucleic acid microarrays attached to planar surfaces. For example, acceptor polynucleotides may be created and then spotted or printed onto a planar surface. Acceptor polynucleotides may be deposited onto surfaces using contact printing techniques. For example, solid or hollow tips or pins may be dipped into solutions comprising pre-formed acceptor polynucleotides and contacted with the planar surface. Alternatively, oligonucleotides may be adsorbed onto micro-stamps and then transferred to a planar surface by physical contact. Non-contact printing techniques include thermic printing or piezoelectric printing wherein sub-nanolitre size microdroplets comprising pre-formed acceptor polynucleotides may be ejected from a printing tip using methods similar to those used in inkjet and bubblejet printing.

[0334] Single-stranded oligonucleotides may be synthesised directly on planar surfaces such as using so-called “on-chip” methods employed to create microarrays. Such single-stranded oligonucleotides may then act as attachment sites to immobilize preformed acceptor polynucleotides.

[0335] On-chip techniques for generating single-stranded oligonucleotides include photolithography which involves the use of UV light directed through a photolithographic mask to selectively activate a protected nucleotide allowing for the subsequent incorporation of a new protected nucleotide. Cycles of UV-mediated deprotection and coupling of pre-determined nucleotides allows the in situ generation of an oligonucleotide having a desired sequence. As an alternative to the use of a photolithographic mask, oligonucleotides may be created on planar surfaces by the sequential deposition of nucleobases using inkjet printing technology and the use of cycles of coupling, oxidation and deprotection to generate an oligonucleotide having a desired sequence (for a review see Kosuri and Church, Nature Methods, 2014, 11, 499- 507).

[0336] In any of the synthesis methods described herein, including methods involving reversible immobilisation as described below, surfaces can be made of any suitable material. Typically, a surface may comprise silicon, glass or polymeric material. A surface may comprise a gel surface, such as a polyacrylamide surface, such as about 2% polyacrylamide, optionally a polyacrylamide surface derived using N- (5- bromoacetamidylpentyl) acrylamide (BRAPA), preferably the polyacrylamide surface is coupled to a solid support, such as glass.

[0337] Reversible Immobilization

[0338] Synthetic polynucleotides having a predefined sequence can be synthesised in accordance with the invention using binding surfaces and structures, such as particles (e.g. microparticles) and beads, which facilitate reversible immobilization. Solid phase reversible immobilization (SPRI) methods or modified methods are known in the art and may be employed (e.g. see DeAngelis M. M. et al. (1995) Solid-Phase Reversible Immobilization for the Isolation of PCR Products, Nucleic Acids Research, 23(22): 4742-4743.).

[0339] Surfaces can be provided in the form of microparticles, such as paramagnetic beads. Paramagnetic beads can agglomerate under the influence of a magnetic field. For example, paramagnetic surfaces can be provided with chemical groups, e.g. carboxyl groups, which in appropriate attachment conditions will act as binding moieties for nucleic acids, as described in more detail below. Nucleic acids can be eluted from such surfaces in appropriate elution conditions. Surfaces of microparticles and beads can be provided with UV-sensitive polycarbonate. Nucleic acids can be bound to the activated surface in the presence of a suitable immobilization buffer. Microparticles and beads may be allowed to move freely within a reaction solution and then reversibly immobilized, e.g. by holding the bead within a microwell or pit etched into a surface. A bead can be localised as part of an array e.g. by the use of a unique nucleic acid “barcode” attached to the bead or by the use of colour-coding.

[0340] Thus, before initiating synthesis of a new double-stranded polynucleotide of predefined sequence, acceptor polynucleotides in accordance with the invention can be synthesised and then reversibly immobilized to such binding surfaces. Polynucleotides synthesised by methods of the invention can be synthesised whilst reversibly immobilized to such binding surfaces.

[0341] Acceptor Polynucleotide Activation

[0342] In some embodiments the acceptor polynucleotide for the first cycle is provided immobilised on a support, but requires cleavage to generate the necessary 5’ or 3’ overhang for the first ligation. For example, it may be that the acceptor polynucleotide immobilised on the support is blunt ended and therefore needs to be cleaved to generate the necessary 5’ or 3’ overhang for the first cycle of the synthesis. Thus, the methods provided may comprise such an initial step of cleavage so that the acceptor polynucleotide has the necessary overhangs. In other embodiments, the method provided does not comprise such an initial activation step either because it has already been performed or it is unnecessary as the acceptor polynucleotide immobilised on the support already has the necessary overhangs.

[0343] Microfluidic Techniques and Systems

[0344] The surface may be part of an electrowetting-on-dielectric system (EWOD). EWOD systems provide a dielectric-coated surface which facilitates microfluidic manipulation of very small liquid volumes in the form of microdroplets (e.g. see Chou, W-L., et al. (2015) Recent Advances in Applications of Droplet Microfluidics, Micromachines, 6: 1249-1271.). Droplet volumes can programmably be created, moved, partitioned and combined on-chip by electro wetting techniques. Thus, electrowetting systems provide alternative means to reversibly immobilize polynucleotides during and after synthesis.

[0345] Polynucleotides having a predefined sequence may be synthesised in solid phase by methods described herein, wherein polynucleotides are immobilized on an EWOD surface and required steps in each cycle facilitated by electrowetting techniques. For example, reagents required for each step, as well as for any required washing steps to remove used and unwanted reagent, can be provided in the form of microdroplets transported under the influence of an electric field via electrowetting techniques.

[0346] Other microfluidic platforms are available which may be used in the synthesis methods of the invention. For example, the emulsion-based microdroplet techniques which are commonly employed for nucleic acid manipulation can be used. In such systems microdroplets are formed in an emulsion created by the mixing of two immiscible fluids, typically water and an oil. Emulsion microdroplets can be programmably be created, moved, partitioned and combined in microfluidic networks. Hydrogel systems are also available. In any of the synthesis methods described herein microdroplets may be manipulated in any suitable compatible system, such as EWOD systems described above and other microfluidic systems, e.g. microfluidic systems comprising architectures based on components comprising elastomeric materials.

[0347] Microdroplets may be of any suitable size, provided that they are compatible with the synthesis methods herein. Microdroplet sizes will vary depending upon the particular system employed and the relevant architecture of the system. Sizes may thus be adapted as appropriate. In any of the synthesis methods described herein droplet diameters may be in the range from about 150nm to about 5mm. Droplet diameters below 1 pm may be verified by means known in the art, such as by techniques involving capillary jet methods, e.g. as described in Ganan-Calvo et al. (Nature Physics, 2007, 3, pp737-742)

[0348] Sequencing of Intermediate or Final Synthesis Products.

[0349] The intermediate products of synthesis or assembly, or the final polynucleotide synthesis products may be sequenced as a quality control check to determine whether the desired polynucleotide or polynucleotides have been correctly synthesised or assembled. The polynucleotide or polynucleotides of interest can be removed from the solid phase synthesis platform and sequenced by any one of a number of known commercially available sequencing techniques such as nanopore sequencing using a MinlON™ device sold by Oxford Nanopore Technologies Ltd. In a particular example, the sequencing may be carried out on the solid phase platform itself, removing the need to transfer the polynucleotide to a separate synthesis device. Sequencing may be conveniently carried out on the same electrowetting device, such as an EWOD device as used for synthesis whereby the synthesis device comprises one or more measurement electrode pairs. A droplet comprising the polynucleotide of interest can be contacted with one of the electrodes of the electrode pair, the droplet forming a droplet interface bilayer with a second droplet in contact with the second electrode of the electrode pair wherein the droplet bilayer interface comprises a nanopore in an amphipathic membrane. The polynucleotide can be caused to translocate the nanopore for example under enzyme control and ion current flow through the nanopore can be measured under a potential difference between the electrode pair during passage of the polynucleotide through the nanopore. The ion current measurements over time can be recorded and used to determine the polynucleotide sequence. Prior to sequencing, the polynucleotide may be subjected to one or more sample preparation steps in order to optimise it for sequencing such as disclosed in patent application no. PCT / GB2015 / 050140. Examples of enzymes, amphipathic membranes and nanopores which may be suitably employed are disclosed in patent application nos. PCT / GB2013 / 052767 and PCT / GB2014 / 052736. The necessary reagents for sample preparation of the polynucleotide, nanopores, amphipathic membranes and so on may be supplied to the EWOD device via sample inlet ports. The sample inlet ports may be connected to reagent chambers.

[0350] Surface Attachment Chemistries

[0351] Although polynucleotides will typically be attached chemically, they may also be attached to surfaces by indirect means such as via affinity interactions. For example, polynucleotides may be functionalised with biotin and bound to surfaces coated with avidin or streptavidin.

[0352] For the immobilization of polynucleotides to surfaces (e.g. planar surfaces), microparticles and beads etc., a variety of surface attachment methods and chemistries are available. Surfaces may be functionalised or derivatized to facilitate attachment. Such functionalisations are known in the art. For example, a surface may be functionalised with a polyhistidine-tag (hexa histidine-tag, 6xHis-tag, His6 tag or His- tag®), Ni-NTA, streptavidin, biotin, an oligonucleotide, a polynucleotide (such as DNA, RNA, PNA, GNA, TNA or LNA), carboxyl groups, quaternary amine groups, thiol groups, azide groups, alkyne groups, DIBO, lipid, FLAG-tag (FLAG octapeptide), polynucleotide binding proteins, peptides, proteins, antibodies or antibody fragments. The surface may be functionalised with a molecule or group which specifically binds to the acceptor polynucleotide.

[0353] Some examples of chemistries suitable for attaching polynucleotides to surfaces are shown in Figures 13 and 14.

[0354] In any of the methods described herein polynucleotides may be tethered to a common surface via one or more covalent bonds. The one or more covalent bonds may be formed between a functional group on the common surface and a functional group on the polynucleotides molecule. The functional group on the polynucleotide molecule may be e.g. an amine group, a thiol group, a thiophosphate group or a thioamide group. The functional group on the common surface may be a bromoacetyl group, optionally wherein the bromoacetyl group is provided on a polyacrylamide surface derived using N- (5- bromoacetamidylpentyl) acrylamide (BRAPA).

[0355] In any of the methods of the invention a polynucleotide may be attached to a surface, either directly or indirectly, via a linker. Any suitable linker which is biocompatible and hydrophilic in nature may be used.

[0356] A linker may be a linear linker or a branched linker.

[0357] A linker may comprise a hydrocarbon chain. A hydrocarbon chain may comprise from 2 to about 2000 or more carbon atoms. The hydrocarbon chain may comprise an alkylene group, e.g. C2 to about 2000 or more alkylene groups. The hydrocarbon chain may have a general formula of -(CH2)n- wherein n is from 2 to about 2000 or more. The hydrocarbon chain may be optionally interrupted by one or more ester groups (i.e. -C(O)-O-) or one or more amide groups (i.e. -C(O)-N(H)-).

[0358] Any linker may be used selected from the group comprising PEG, polyacrylamide, poly(2 -hydroxyethyl methacrylate), Poly-2-methyl-2-oxazoline (PMOXA), zwitterionic polymers, e.g. poly(carboxybetaine methacrylate) (PCBMA), poly[ N -(3 -sulfopropyl)- N -methacryloxyethyl- N , N dimethyl ammonium betaine] (PSBMA), glycopolymers, and polypeptides.

[0359] A linker may comprise a polyethylene glycol (PEG) having a general formula of -(CH2-CH2-O)n-, wherein n is from 1 to about 600 or more.

[0360] A linker may comprise oligoethylene glycol-phosphate units having a general formula of -[(CH2-CH2-O)n-PO2 -O]m- where n is from 1 to about 600 or more and m could be 1-200 or more.

[0361] Any of the above-described linkers may be attached at one end of the linker to an acceptor molecule as described herein, and at the other end of the linker to a first functional group wherein the first functional group may provide a covalent attachment to a surface. The first functional group may be e.g. an amine group, a thiol group, a thiophosphate group or a thioamide group as further described herein. The surface may be functionalised with a further functional group to provide a covalent bond with the first functional group. The further functional group may be e.g. a 2-bromoacetamido group as further described herein. Optionally a bromoacetyl group is provided on a polyacrylamide surface derived using N- (5- bromoacetamidylpentyl) acrylamide (BRAPA). The further functional group on the surface may be a bromoacetyl group, optionally wherein the bromoacetyl group is provided on a polyacrylamide surface derived using N- (5- bromoacetamidylpentyl) acrylamide (BRAPA) and the first functional group may be e.g. an amine group, a thiol group, a thiophosphate group or a thioamide group as appropriate. The surface to which polynucleotides are attached may comprise a gel. The surface comprises a polyacrylamide surface, such as about 2% polyacrylamide, preferably the polyacrylamide surface is coupled to a solid support such as glass.

[0362] In any of the methods of the invention an acceptor polynucleotide may optionally be attached to a linker via a branching nucleotide incorporated into the acceptor polynucleotide. Any suitable branching nucleotide may be used with any suitable compatible linker.

[0363] Prior to initiating synthesis cycles of the invention, acceptor polynucleotides may be synthesised with one or more branching nucleotides incorporated into the polynucleotide. The exact position at which the one or more branching nucleotides are incorporated into the polynucleotide, and thus where a linker may be attached, may vary and may be chosen as desired. The position may e.g. be at the terminal end of a strand or e.g. in the loop region which connects first and second strands in embodiments which comprise a hairpin loop.

[0364] During synthesis of the acceptor polynucleotide the one or more branching nucleotides may be incorporated into the acceptor polynucleotide with a blocking group which blocks a reactive group of the branching moiety. The blocking group may then be removed (deblocked) prior to the coupling to the branching moiety of the linker, or a first unit (molecule) of the linker if a linker comprises multiple units.

[0365] During synthesis of the acceptor polynucleotide the one or more branching nucleotides may be incorporated into the polynucleotide with a group suitable for use in a subsequent “click chemistry” reaction to couple to the branching moiety the linker, or a first unit of the linker if a linker comprises multiple units. An example of such a group is an acetylene group.

[0366] Some non-limiting exemplary branching nucleotides are shown below. linker to 5" end

[0367] 5-methylC brancher nucleotide

[0368]

[0369] A linker may optionally comprise one or more spacer molecules (units), such as e.g. an Sp9 spacer, wherein the first spacer unit is attached to the branching nucleotide. The linker may comprise one or more further spacer groups attached to the first spacer group. For example, the linker may comprise multiple e.g. Sp9 spacer groups. A first spacer group is attached to the branching moiety and then one or more further spacer groups are sequentially added to extend a spacer chain comprising multiple spacer units connected with phosphate groups therebetween. Shown below are some non-limiting examples of spacer units (Sp3, Sp9 and

[0370] Spl3) which could comprise the first spacer unit attached to a branching nucleotide, or a further spacer unit to be attached to an existing spacer unit already attached to the branching nucleotide.

[0371] 3' direction to 3' direction to brancher brancher point5„endpoint 5" end 3' direction to brancher point 5" end

[0372] Sp18 unit

[0373] A linker may comprise one or more ethylene glycol units.

[0374] A linker may comprise an oligonucleotide, wherein multiple units are nucleotides.

[0375] In the structures depicted above the term 5” is used to differentiate from the 5’ end of the nucleotide to which the branching moiety is attached, wherein 5’ has its ordinary meaning in the art. By 5” it is intended to mean a position on the nucleotide from which a linker can be extended. The 5” position may vary. The 5” position is typically a position in the nucleobase of the nucleotide. The 5” position in the nucleobase may vary depending on the nature of the desired branching moiety, as depicted in the structures above.

[0376] In any of the methods described herein:

[0377] (i) the first and second strands of the acceptor polynucleotide at the second terminal end may each be tethered to a surface; or

[0378] (ii) the first and second strands of the acceptor polynucleotide at the second terminal end may connected together by a polynucleotide hairpin loop and tethered to a surface; or

[0379] (iii) the first strand of the acceptor polynucleotide at the second terminal end may be tethered to a surface and the second strand of the acceptor polynucleotide at the second terminal end may be untethered; or

[0380] (iv) the second strand of the acceptor polynucleotide at the second terminal end may be tethered to a surface and the first strand of the acceptor polynucleotide at the second terminal end may be untethered. The tethered strand(s) at the second terminal end may comprise a cleavable linker(s), wherein the linker(s) may be cleaved to detach the double-stranded polynucleotide from the surface following synthesis.

[0381] In methods using a hairpin loop at the second terminal end, the hairpin loop may be tethered to a surface via a cleavable linker, wherein the linker may be cleaved to detach the double-stranded polynucleotide from the surface following synthesis.

[0382] The cleavable linker may be a UV cleavable linker.

[0383] The tethered strand(s) at the second terminal end may include a recognition site, which may be used to cleave the double-stranded polynucleotide and thereby detach the double-stranded polynucleotide from the surface following synthesis. The recognition site may be a recognition site for an endonuclease. The recognition site may be a recognition site for a restriction enzyme. The recognition site may comprise a cleavage site defined by a uracil nucleotide positioned in either the first or second strands of the acceptor polynucleotide, wherein cleavage may be performed by an enzyme having uracil DNA glycosylase activity and DNA glycosylase-lyase activity e.g. Endonuclease VIII activity.

[0384] Microarrays

[0385] Any of the polynucleotide synthesis methods described herein may be used to manufacture a polynucleotide microarray (Trevino, V. et al., Mol. Med. 2007 13, pp527-541). Thus, acceptor polynucleotides may be tethered to a plurality of individually addressable reaction sites on a surface and polynucleotides having a predefined sequence may be synthesised in situ on the microarray.

[0386] Following synthesis, at each reaction area the polynucleotide of predefined sequence may be provided with a unique sequence. The polynucleotides may be provided with barcode sequences to facilitate identification.

[0387] Other than the method of synthesising the polynucleotides of predefined sequence, microarray manufacture may be performed using techniques commonly used in this technical field, including techniques described herein. For example, acceptor polynucleotides may be tethered to surfaces using known surface attachment methods and chemistries, including those described herein.

[0388] Following synthesis of the polynucleotides of predefined sequence, there may be provided a final cleavage step to remove any unwanted polynucleotide sequence from untethered ends.

[0389] Polynucleotides of predefined sequence may be provided at reaction sites in double-stranded form. Alternatively, following synthesis double-stranded polynucleotides may be separated and one strand removed, leaving single-stranded polynucleotides at reaction sites. Selective tethering of strands may be provided to facilitate this process as described elsewhere herein. Separation of strands may be performed by conventional methods, such as heat treatment.

[0390] Assembly of Synthetic Polynucleotides

[0391] A polynucleotide having a predefined sequence synthesised by methods described herein, and optionally amplified by methods described herein, may be joined to one or more other such polynucleotides to create larger synthetic polynucleotides.

[0392] Joining of multiple polynucleotides can be achieved by techniques commonly known in the art. A first polynucleotide and one or more additional polynucleotides synthesised by methods described herein may be cleaved to create compatible termini and then polynucleotides joined together by ligation. Cleavage can be achieved by any suitable means. Typically, restriction enzyme cleavage sites may be created in polynucleotides and then restriction enzymes used to perform the cleavage step, thus releasing the synthesised polynucleotides from any other undesirable polynucleotide sequence. Cleavage sites could be designed as part of the synthesised polynucleotides. Alternatively, cleavage sites could be created within the newly-synthesised polynucleotide as part of the predefined nucleotide sequence.

[0393] Assembly of polynucleotides is preferably performed using solid phase methods. For example, following synthesis a first polynucleotide may be subject to a single cleavage at a suitable position distal to the site of surface immobilization. The first polynucleotide will thus remain immobilized to the surface, and the single cleavage will generate a terminus compatible for joining to another polynucleotide. An additional polynucleotide may be subject to cleavage at two suitable positions to generate at each terminus a compatible end for joining to other polynucleotides, and at the same time releasing the additional polynucleotide from surface immobilization. The additional polynucleotide may be compatibly joined with the first polynucleotide thus creating a larger immobilized polynucleotide having a predefined sequence and having a terminus compatible for joining to yet another additional polynucleotide. Thus, iterative cycles of joining of preselected cleaved synthetic polynucleotides may create much longer synthetic polynucleotide molecules. The order of joining of the additional polynucleotides will be determined by the required predefined sequence.

[0394] Thus, the assembly methods of the invention may allow the creation of synthetic polynucleotide molecules having lengths in the order of one or more Mb.

[0395] The assembly and / or synthesis methods of the invention may be performed using apparatuses known in the art. Techniques and apparatuses are available which allow very small volumes of reagents to be selectively moved, partitioned and combined with other volumes in different locations of an array, typically in the form of droplets. Electro wetting techniques, such as electrowetting-on-dielectric (EWOD), may be employed, as described above. Suitable electrowetting techniques and systems that may be employed in the invention that are able to manipulate droplets are disclosed for example in US8653832, US8828336, US20140197028 and US20140202863.

[0396] Cleavage from the solid phase may be achieved by providing cleavable linkers in one or both the primer strand portion and the portion of the support strand hybridized thereto. The cleavable linker may be e.g. a UV cleavable linker.

[0397] Thus, polynucleotides having a predefined sequence may be synthesised whilst immobilized to an electrowetting surface, as described above. Synthesised polynucleotides may be cleaved from the electrowetting surface and moved under the influence of an electric field in the form of a droplet. Droplets may be combined at specific reaction sites on the surface where they may deliver cleaved synthesised polynucleotides for ligation with other cleaved synthesised polynucleotides. Polynucleotides can then be joined, for example by ligation. Using such techniques populations of different polynucleotides may be synthesised and attached in order according to the predefined sequence desired. Using such systems, a fully automated polynucleotide synthesis and assembly system may be designed. The system may be programmed to receive a desired sequence, supply reagents, perform synthesis cycles and subsequently assemble desired polynucleotides according to the predefined sequence desired.

[0398] Systems and Kits

[0399] The invention also provides polynucleotide synthesis systems for carrying out any of the synthesis methods described and defined herein, as well as any of the subsequent amplification and assembly steps described and defined herein.

[0400] Typically, synthesis cycle reactions will be carried out by incorporating nucleotides of predefined sequence into acceptor polynucleotide molecules which are tethered to a surface by means described and defined herein. The surface may be any suitable surface as described and defined herein.

[0401] In one embodiment, reactions to incorporate nucleotides of predefined sequence into an acceptor polynucleotide molecule involve performing any of the synthesis methods on an acceptor polynucleotide within a reaction area.

[0402] A reaction area is any area of a suitable substrate to which an acceptor polynucleotide molecule is attached and wherein reagents for performing the synthesis methods may be delivered.

[0403] In one embodiment a reaction area may be a single area of a surface comprising a single acceptor polynucleotide molecule wherein the single acceptor polynucleotide molecule can be addressed with reagents.

[0404] In another embodiment a reaction area may be a single area of a surface comprising multiple acceptor polynucleotide molecules, wherein the acceptor polynucleotide molecules cannot be individually addressed with reagent in isolation from each other. Thus, in such an embodiment the multiple acceptor polynucleotide molecules in the reaction area are exposed to the same reagents and conditions and may thus give rise to synthetic polynucleotide molecules having the same or substantially the same nucleotide sequence. In one embodiment a synthesis system for carrying out any of the synthesis methods described and defined herein may comprise multiple reaction areas, wherein each reaction area comprises one or more attached acceptor polynucleotide molecules and wherein each reaction area may be individually addressed with reagent in isolation from each of the other reaction areas. Such a system may be configured e.g. in the form of an array, e.g. wherein reaction areas are formed upon a substrate, typically a planar substrate.

[0405] A system having a substrate comprising a single reaction area or comprising multiple reaction areas may be comprised within e.g. an EWOD system or a microfluidic system and the systems configured to deliver reagents to the reaction site. EWOD and microfluidic systems are described in more detail herein. For example an EWOD system may be configured to deliver reagents to the reaction site(s) under electrical control. A microfluidic system, such as comprising microfabricated architecture e.g. as formed from elastomeric or similar material, may be configured to deliver reagents to the reaction site(s) under fluidic pressure and / or suction control or by mechanical means. Reagents may be delivered by any suitable means, for example via carbon nanotubes acting as conduits for reagent delivery. Any suitable system may be envisaged.

[0406] EWOD, microfluidic and other systems may be configured to deliver any other desired reagents to reaction sites, such as enzymes for cleaving a synthesised doublestranded polynucleotide from the acceptor polynucleotide following synthesis, and / or reagents for cleaving a linker to release an entire polynucleotide from the substrate and / or reagents for amplifying a polynucleotide molecule following synthesis or any region or portion thereof, and / or reagents for assembling larger polynucleotide molecules from smaller polynucleotide molecules which have been synthesised according to the synthesis methods of the invention.

[0407] The invention also provides kits for carrying out any of the synthesis methods described and defined herein. A kit may contain any desired combination of reagents for performing any of the synthesis and / or assembly methods of the invention described and defined herein. For example, a kit may comprise any one or more volume(s) of reaction reagents comprising acceptor polynucleotides, donor polynucleotides, volume(s) of reaction reagents corresponding to any one or more steps of the synthesis cycles described and defined herein, volume(s) of reaction reagents comprising nucleotides comprising reversible blocking groups or reversible terminator groups, volume(s) of reaction reagents for amplifying one or more polynucleotide molecules following synthesis or any region or portion thereof, volume(s) of reaction reagents for assembling larger polynucleotide molecules from smaller polynucleotide molecules which have been synthesised according to the synthesis methods of the invention, volume(s) of reaction reagents for cleaving a double- stranded polynucleotide, and volume(s) of reaction reagents for cleaving one or more linkers e.g. to release polynucleotides from a substrate. A kit may contain any number of, and type of donor polynucleotides sufficient for the synthesis of a polynucleotide molecules of any given predetermined sequence, including mixtures of any desired length and sequence of polynucleotide payload, as required by the user.

[0408] Data Storage

[0409] Polynucleotide molecules are naturally capable of storing information encoded within them due to differences in the identity and sequences of nucleobases forming the structure of the polynucleotide molecule. The natural data storage function of polynucleotide molecules can be exploited for the storage of new information by synthesising new polynucleotide molecules according to a specific nucleobase sequence which can thus encode new information within the polynucleotide molecule which can later be accessed or “read” to retrieve the information.

[0410] New information can, for example, be encoded into a polynucleotide molecule in a digital form. Thus, the invention additionally provides methods of storing data in digital form in a polynucleotide molecule, thereby generating a nucleotide sequence in the polynucleotide synthesis molecule indicative of the “0” or “1” state of a bit of digital information.

[0411] A nucleotide sequence can be incorporated into a polynucleotide synthesis molecule to be indicative of the “0” or “1” state of a bit of digital information in any suitable way. For example, bits of digital information can be created using two different species of nucleotide. For example, a polynucleotide can be extended so as to generate an adenine (A) - thymine (T) pair in a first cycle of synthesis followed by extension so as to generate a cytosine (C) - guanine (G) pair in a second subsequent cycle. The presence of the A-T pair in the polynucleotide molecule can thus be indicative of the “0” or “1” state of a bit of digital information. The presence of the C- G pair juxtaposed adjacent to the A-T pair can thus be indicative of the opposite state of the bit. Incorporation of multiple A-T and C-G pairs of nucleobases in sequence can therefore allow for digital information to be encoded into the polynucleotide in bit form. A-T and C-G are provided as examples only. Any nucleobases can be used provided they can be distinguished from each other.

[0412] Incorporation of single nucleobases of alternating species is one way of generating a bit of digital information. Bits can alternatively be generated by the incorporation of two or more, i.e. a first string, of nucleobases of the same or indistinguishable species in the same or successive cycles of synthesis which can thus be indicative of the “0” or “1” state of a bit of digital information. This can then be followed by the incorporation of two or more, i.e. a second string, of nucleobases of the same or indistinguishable species in the same or successive cycles of synthesis which can thus be indicative of the opposite state of the bit to that previously generated. Any nucleobases can be used provided that the nucleobases of the first string can be distinguished from the nucleobases of the second string. First and second strings need not consist of the same number of nucleobases since the transition between first and second string is indicative of the transition between the “0” or “1” state of the bit of digital information and the opposite state of the bit.

[0413] Any such method of data storage may be performed using any of the in vitro methods of synthesising a double-stranded polynucleotide molecule as described and defined herein. Any such method of data storage may be performed using any of the apparatus, devices and systems described and defined herein. Reversible Terminator Groups

[0414] In any of the synthesis methods of the invention defined and described herein, the method may not involve a step of incorporation of a polynucleotide having a reversible terminator group (reversible blocking group) and an additional step of deprotection to remove the reversible terminator group.

[0415] A reversible terminator group is a chemical group which is incorporated into a nucleic acid strand and which acts to prevent further extension of the strand by an enzyme, such as a polymerase enzyme.

[0416] Examples of reversible terminators are provided below.

[0417] Propargyl reversible terminators:

[0418] Allyl reversible terminators: Cyclooctene reversible terminators:

[0419] Cyanoethyl reversible terminators:

[0420] Nitrobenzyl reversible terminators: Disulfide reversible terminators: Azidomethyl reversible therminators:

[0421] Aminoalkoxy reversible therminators:

[0422] Nucleoside triphosphates with bulky groups attached to the base can serve as substitutes for a reversible terminator group on 3 ’-hydroxy group and can block further incorporation. guanine adenine

[0423] X = O, S, NH, CH2Z = bulky group

[0424] Exemplary Methods

[0425] The following set out examples of particular illustrative synthesis methods of the present invention. Figure 12 sets out an overview summary of the different methods. The following description of the methods sets out an individual cycle of the synthesis methods, with such cycles being repeated to build-up the chosen predefined sequence. Prior to the first cycle or the end of the last cycle the various methods for provided the acceptor and recovering the final synthesized sequence set out above may be employed.

[0426] The methods set out should be viewed as non -limiting. For instance, it may be that a specific DNA synthesis method combines cycles with 5’ overhangs with other cycles with 3’ overhangs.

[0427] Method 1 - 5’ overhans with nucleotide incorporation

[0428] An illustrative example of such a synthesis method is depicted in Figure 4. The 5’ overhang with nucleotide incorporation of method version 1 can have a number of advantages. Those including helping to limit self-ligation of acceptors polynucleotides to each other, as well as limiting self-ligation of donor polynucleotides to each other as illustrated in Figure 6. A further advantage is that the method may be useful in helping to correct erroneous ligations as illustrated by Figure 8. A further advantage is that the combination of 5’ overhangs with incorporation that uses a polymerase able to fill in such overhangs, is that such filling-in may help render unligated acceptor polynucleotides inert. That advantage is illustrated in Figure 9.

[0429] Provision of acceptor polynucleotide

[0430] To initiate a cycle of synthesis an acceptor polynucleotide is first provided.

[0431] The acceptor polynucleotide comprises first and second polynucleotide strands and first and second terminal ends. The first and second polynucleotide strands are connected by hybridization, via standard Watson-Crick base pairing, to form a doublestranded polynucleotide molecule.

[0432] The first terminal end of the acceptor polynucleotide is ligatable and the first strand has a 5’ overhang. In one embodiment, the 5’ overhang of the first strand is a single nucleotide in length. The first terminal end is free, i.e. neither the first strand nor the second strand is tethered to or otherwise attached to any other structure.

[0433] The terminal nucleotide of the first strand at the ligatable first terminal end of the acceptor polynucleotide comprises a 5’ phosphate group. The 3’ terminal nucleotide of the second strand at the ligatable first terminal end comprises a 3’ hydroxyl group.

[0434] The second terminal end of the acceptor polynucleotide is preferably non- ligatable.

[0435] The second terminal end of the acceptor polynucleotide is preferably tethered to a surface. Such a surface may be any suitable surface as described and defined elsewhere herein. The second terminal end may be tethered to a surface due to the second strand of the acceptor polynucleotide being tethered to the surface whilst the first strand of the acceptor polynucleotide is untethered. Alternatively, the second terminal end may be tethered to a surface due to the first strand of the acceptor polynucleotide being tethered to the surface whilst the second strand of the acceptor polynucleotide is untethered. Alternatively still, the second terminal end may be tethered to a surface due to the first and second strands of the acceptor polynucleotide being tethered to the surface. Where both the first and second strands of the acceptor polynucleotide are tethered to the surface, each strand may be independently tethered to the surface. Alternatively, the first and second strands at the second terminal end of the acceptor polynucleotide may be connected together via a connector, such as via a hairpin loop, and the connector, or any other part of the second terminal end, may be tethered to the surface.

[0436] The acceptor polynucleotide may be initially provided without comprising any of the nucleotides of the predefined sequence which the user wishes to synthesize. This may be the case, for example, before commencing the very first cycle of synthesis. More typically however, the acceptor polynucleotide, when provided, will already include the polynucleotide payload of the previous cycle with the 5’ overhang forming part of the payload of the previous cycle of synthesis.

[0437] Provision of donor polynucleotide

[0438] To initiate a cycle of synthesis a donor polynucleotide is also provided.

[0439] The donor polynucleotide comprises first and second polynucleotide strands and first and second terminal ends. The first and second polynucleotide strands are connected by hybridization, via standard Watson-Crick base pairing, to form a doublestranded polynucleotide molecule.

[0440] The first terminal end of the donor polynucleotide is ligatable and has a 5’ overhang. Hence, as show in Figure 2, the first terminal end of the donor polynucleotide has a 5’ overhang because the second strand has such an overhang. The 5’ overhang has a complementary sequence to the 5’ overhang of the acceptor polynucleotide. For instance, wherein the acceptor polynucleotide has a 5’ overhang with adenosine as the base in the overhanging nucleotide, the donor polynucleotide will have a 5’ with thymine as the base in the overhanging nucleotide. As the overhangs are complementary they will be able to hybridise to each other via Watson-Crick base-pairing. The first terminal end is free, i.e. neither the first strand nor the second strand is tethered to or otherwise attached to any other structure.

[0441] The second terminal end is typically also free, i.e. neither the first strand nor the second strand is tethered to or otherwise attached to any other structure. The second terminal end may be non-ligatable or it may be ligatable (i.e. in the case of a symmetrical donor, as described below).

[0442] The terminal nucleotide of the second strand at the ligatable first terminal end of the donor polynucleotide is non-ligatable, e.g. lacks a 5’ phosphate group. The 3’ terminal nucleotide of the first strand at the ligatable first terminal end comprises a 3’ hydroxyl group.

[0443] The second terminal end of the donor polynucleotide may be non-ligatable. This helps avoid the problem of multiple donor polynucleotides ligating together. Where the second terminal end of the donor polynucleotide is non-ligatable, the terminal nucleotides of the first and / or second strands at the second terminal end may comprise a blocking group. A blocking group is any blocking group defined elsewhere herein. A blocking group(s) renders the second terminal end non-ligatable. Alternatively, the second terminal end of the donor polynucleotide may be provided without a 5’ phosphate group. Alternatively, still, the first and second strands at the second terminal end of the donor polynucleotide may be connected together via a connector, such as via a hairpin loop. Such a connector also renders the second terminal end non-ligatable.

[0444] The second terminal end of the donor polynucleotide may be ligatable with respect to an acceptor polynucleotide, but it may be structured so that it cannot ligate to another donor polynucleotide. For example, the donor polynucleotide may be a symmetrical donor polynucleotide. The second terminal end of the donor polynucleotide may comprise a second polynucleotide payload, wherein:

[0445] (i) the payload nucleotide sequence of the first strand at the second terminal end of the donor polynucleotide in the 5’ to 3’ direction is the same as the payload nucleotide sequence of the second strand at the first terminal end of the donor polynucleotide in the 5’ to 3’ direction;

[0446] (ii) the payload nucleotide sequence of the second strand at the second terminal end of the donor polynucleotide in the 3’ to 5’ direction is the same as the payload nucleotide sequence of the first strand at the first terminal end of the donor polynucleotide in the 3’ to 5’ direction;

[0447] (iii) the second terminal end of the donor polynucleotide comprises a 3 ’ hydroxyl group and lacks a 5’ phosphate group; and (iv) the first strand of the donor polynucleotide at the second terminal end comprises a 5’ overhang which has the same sequence as the overhang at the first terminal end.

[0448] In such a donor polynucleotide e.g. both the first and the second terminal ends lack 5’ phosphate groups. Accordingly, a first donor polynucleotide cannot be ligated to a second donor polynucleotide in a standard ligation reaction, thus avoiding the problem of self-ligation between donor polynucleotides. As the two overhangs of a symmetrical donor are not designed to be complementary to each other that two will make it less likely that such symmetrical donor polynucleotides will ligate to each other. Nevertheless, since both the first and the second terminal ends comprise a 3’ hydroxyl group, either end of the donor polynucleotide is capable of ligating to an acceptor polynucleotide as described below. Such a donor polynucleotide therefore has the advantage of possessing two identical ligatable (with respect to an acceptor polynucleotide) ends. By having more ligatable ends available per reaction, the efficiency of ligation can be improved.

[0449] The donor polynucleotide comprises, at the ligatable first terminal end, a polynucleotide payload comprising one or more nucleotide pairs of the predefined sequence. The polynucleotide payload comprises one or more nucleotide pairs of the predefined sequence that are to be incorporated into the acceptor polynucleotide following ligation. The 5’ overhang of the donor polynucleotide also constitutes part of the payload for that cycle.

[0450] The first nucleotide before the overhang of the first strand at the ligatable first terminal end and the terminal nucleotide of the second strand at the ligatable first terminal end form a nucleotide pair, and this pair is the first pair of nucleotides in the polynucleotide payload. If the polynucleotide payload comprises more than one nucleotide pair of the predefined sequence, the penultimate nucleotide before the overhang of the first strand at the ligatable first terminal end and the penultimate nucleotide of the second strand at the ligatable first terminal end form the next nucleotide pair of the payload, and this pair is the second pair of nucleotides in the polynucleotide payload, and so on, with any nucleotides that will form part of the acceptor after cleavage at the end of the cycle forming the final part of the nucleotide payload.

[0451] The donor polynucleotide is provided with a cleavage site adjacent to the polynucleotide payload. The cleavage site is situated between the polynucleotide payload and the second terminal end of the donor polynucleotide. The exact type of cleavage site and its location within the donor polynucleotide can vary, and may be defined by the user when providing the donor polynucleotide, provided that the cleavage step is performed as described below. The cleavage site though will be chosen so that cleavage results in a fresh overhang that can be used in the next cycle.

[0452] Ligation of acceptor and donor polynucleotides

[0453] The ligation step functions to physically join the donor polynucleotide to the acceptor polynucleotide as a first step to facilitate the transfer of the polynucleotide payload from the donor polynucleotide to the acceptor polynucleotide. The ligation step comprises a single stranded ligation so that the first strand of the acceptor polynucleotide and the first strand of the donor polynucleotide are ligated together at was originally the first terminal end of each, where the second strand of the acceptor polynucleotide and the donor polynucleotide not being ligated together to leave a nick with the free 3 ’OH of the first terminal end of the second strand of the acceptor polynucleotide being free for nucleotide incorporation in the next step.

[0454] In method version 1, the ligation step comprises joining the donor and acceptor polynucleotides at their first terminal ends by ligating the first strand of the donor polynucleotide at its first terminal end with the first strand of the acceptor polynucleotide at its first terminal end, but wherein the second strands of the donor and acceptor polynucleotides at their first terminal ends are not joined and are separated by a nick. Accordingly, in method version 1, the ligation step comprises a single-stranded ligation of the first strands of the donor and acceptor polynucleotides, but the second strands of the donor and acceptor polynucleotides are not ligated together at their first terminal ends. The ligation step can be performed by any suitable means for physically joining together polynucleotide strands that were previously not joined together. Preferably the ligation step is performed by the action of an enzyme having nucleotide ligase activity, such as any ligase enzyme described elsewhere herein and which can perform the required ligase function for this particular method version.

[0455] The step of ligating the donor and acceptor polynucleotides creates a ligated polynucleotide which has a nick.

[0456] Acceptor depletion (optional)

[0457] Before the cleavage step a depletion step may be performed. Such a step is optional and not essential. The depletion step allows for any acceptor polynucleotide to be depleted, if that acceptor polynucleotide has for some reason failed to join with a donor polynucleotide to form a ligated polynucleotide. Such an acceptor polynucleotide may be referred to as an unreacted acceptor polynucleotide. Failure to react may occur for various reasons including a failure to ligate with a donor polynucleotide. Depletion of any unreacted acceptor polynucleotide may be desirable so that in the next round of synthesis it does not act as acceptor polynucleotide for a subsequent polynucleotide payload. If this were to occur, the double-stranded polynucleotide having a predefined sequence would not be synthesized correctly in that particular acceptor polynucleotide structure.

[0458] In the depletion step an unreacted acceptor polynucleotide is rendered inert, i.e. incapable of acting as an acceptor polynucleotide in the next and subsequent cycles of synthesis.

[0459] Since the terminal nucleotide of the first strand at the ligatable first terminal end of the acceptor polynucleotide comprises a 5’ phosphate group, the unreacted acceptor polynucleotide can be rendered inert by removing the 5’ phosphate group so that it cannot be ligated to a donor polynucleotide in any further synthesis cycle. Accordingly, a depletion step may comprise performing a treatment step to remove the 5’ phosphate group from the terminal nucleotide of the first strand at the ligatable first terminal end of the acceptor polynucleotide. Such a step may comprise performing a treatment step with an enzyme having phosphatase activity, such as calf intestinal phosphatase (CIP). If the terminal nucleotide in the first strand at the second terminal end of the donor polynucleotide comprises a 5’ phosphate group, removal of this group at the same time will have no effect, since this part of the donor molecule will be removed following the cleavage step.

[0460] One advantage of approaches using 5’ overhangs and nucleotide incorporation is that a separate depletion step may be unnecessary because as well as acting on the nicked ligation product, the polymerase used may effectively fill-in the 5’ overhang of unligated acceptor polynucleotides leaving them blunt ended and lacking the overhang needed for the next cycle. Hence, in one embodiment, a synthesis method 1 does not comprise a separate acceptor depletion step. In another embodiment though, it may comprise such an acceptor depletion step which is in addition to the ability of the polymerase used to fill-in unligated acceptors. If that is the case, then any of the acceptor depletion methods set out herein may be employed.

[0461] Incorporation

[0462] Following-on from the ligation step, and optionally any depletion step, the ligation product formed by ligating the first strands of the acceptor and donor polynucleotides has a nick between the second strands with the 3’ terminus of the second strand having a free 3 ’OH group able to act as a substrate for a polymerase and hence nucleotide incorporation. The ligated first strands of the acceptor and donor polynucleotides therefore effectively serve as a template for the nucleotide incorporation. As nucleotides are incorporated that may displace the unligated second strand of the donor polynucleotide. The incorporation step effectively removes the nick present in the ligation step and means that the ligation product is ready for cleavage.

[0463] The original nucleotides of the polynucleotide payload of the second strand may be separated from the first strand:

[0464] (i) before the incorporation step; or

[0465] (ii) during the incorporation step. Where the original nucleotides of the polynucleotide payload of the second strand are separated from the first strand during the incorporation step, incorporation steps may be performed:

[0466] (a) by the action of an enzyme having polymerase activity, and wherein the polymerase displaces the original nucleotides of the polynucleotide payload of the second strand when synthesising the new second strand; or

[0467] (b) by the action of an enzyme having polymerase activity which possesses 5’ to 3’ exonuclease activity, and wherein the polymerase digests / degrades the original nucleotides of the polynucleotide payload of the second strand when synthesising the new second strand.

[0468] Cleavage

[0469] The cleavage step functions to cleave the ligated polynucleotide to generate a new acceptor polynucleotide comprising the payload from the previous cycle or cycles and a new 5’ overhang ready for the next cycle. Thus, cleavage is such that the one or more nucleotide pairs of the predefined sequence and the overhang now formed were previously part of the donor polynucleotide and instead have become incorporated into the acceptor polynucleotide at the ligatable first terminal end of the acceptor polynucleotide. At the same time, the cleavage step functions to separate the polynucleotide payload from the remainder of the donor polynucleotide. The remainder of the donor polynucleotide can then be removed from the ligated polynucleotide, leaving behind the acceptor polynucleotide with the polynucleotide payload incorporated at the ligatable first terminal end of the acceptor polynucleotide.

[0470] In each cycle of synthesis, each polynucleotide payload comprises one or more nucleotide pairs of the predefined sequence that the user wishes to synthesize. Successive cycles therefore provide for the stepwise synthesis of the double-stranded polynucleotide having a predefined sequence, by successively joining together multiple polynucleotide payloads. In method version 1, the cleavage is in both strands to generate an asymmetrical acceptor with the desired 5’ overhang.

[0471] In the case of an asymmetrical cleavage reaction, the first strand is cleaved immediately above the nucleotides of the polynucleotide payload (in the direction proximal to the second terminal end of the donor polynucleotide). Accordingly, the nucleotides of the first strand of the polynucleotide payload are retained at the cleaved first terminal end of the acceptor polynucleotide in the first strand, wherein the terminal nucleotide of the first strand of the acceptor polynucleotide is the final nucleotide of the polynucleotide payload. In an asymmetrical cleavage reaction, the second strand is cleaved below the final nucleotide or nucleotides of the polynucleotide payload (in the direction proximal to the second terminal end of the acceptor polynucleotide) such that the second strand is shorter than the first, meaning the desired 5’ overhang is formed. The first strand of the resulting cleavage product able to act as an acceptor polynucleotide in the next cycle will have a 5’ phosphate group at the first terminal end. The second strand of the resulting cleavage product able to act as an acceptor polynucleotide in the next cycle will have a free 3’ OH group at the first terminal end.

[0472] Any suitable means may be used for the cleavage which results in the desired 5’ overhang may be used for the cleavage. In one embodiment, the cleavage is performed with a restriction enzyme. In particular, the cleavage is performed with a Type IIS endonuclease that means the first terminal end of the resultant acceptor for the next cycle has the desired 5’ overhang. An example of such a restriction enzyme is AlwI. A further example of such a restriction enzyme is Bccl.

[0473] Following cleavage, method version 1 further comprises separating the cleaved donor polynucleotide from the acceptor polynucleotide. Washing may, for instance, be performed to remove the unwanted cleavage product, leaving the acceptor polynucleotide ready for the next cycle. Following the cleavage and incorporation steps, a new acceptor polynucleotide is therefore created with a new 5’ overhang. The new acceptor polynucleotide comprises the old acceptor polynucleotide, that was provided at the start of the cycle, with the polynucleotide payload incorporated at the first terminal end of the acceptor polynucleotide. The new acceptor polynucleotide is thus competent to act as an acceptor polynucleotide to accept a new polynucleotide payload to be incorporated during the next cycle. This facilitates the stepwise synthesis of the doublestranded polynucleotide having a predefined sequence, by allowing multiple polynucleotide payloads to be successively joined together.

[0474] Method 2 - 5’ overhang with nucleotide incorporation and hot start polymerase

[0475] Method 2 is effectively a variant of method 1 in that a specific type of polymerase is used in the incorporation step, in particular a polymerase that has a higher optimal temperature than the ligase is used, a so-called hot start polymerase. Hence, method 2 still comprises cycles with ligation, nucleotide incorporation, and cleavage steps as set out for method 1 and also uses 5’ overhangs, but the host start polymerases means the ligase and polymerase can be added in a single fluid. Figure 7 depicts an illustrative example for synthesis method 2.

[0476] The ligase and polymerase are added in a single fluid. The ligation step of a cycle is then performed at a temperature at which the ligase is active, but the polymerase is not. The temperature is then raised for the nucleotide incorporation step to a temperature at which the ligase is no longer active, but the polymerase is, allowing the nucleotide incorporation step to take place. In one embodiment, the ligase may actually be inactivated at the second temperature.

[0477] One important advantage of method 2 is that because the ligase and polymerase can be added in a single fluid, the number of fluid addition and washing stages needed can be reduced making the method more streamlined.

[0478] Method 3 — 5’ overhang with nick repair

[0479] Method 3 is another 5’ overhang synthesis method which corresponds to method 1 except that the nucleotide incorporation step of each cycle is replaced instead with a step comprising joining the two unligated second strands of the acceptor and donor polynucleotides. The terminal 5’ nucleotide of the payload of the donor polynucleotide (i.e. at the 5’ end of the second strand at the first terminal end of the donor polynucleotide) is provided as a non-ligatable nucleotide and the method comprises converting this non-ligatable nucleotide into a ligatable nucleotide followed by a second ligation reaction to repair the nick. For example, the terminal 5’ nucleotide of the payload of the donor polynucleotide is provided without a free phosphate group and the step of converting this non-ligatable nucleotide into a ligatable nucleotide comprises phosphorylating the nucleotide and then sealing the nick by performing a second ligation. An illustrative embodiment of a method 3 is depicted in Figure 11.

[0480] Thus, the initial (first) ligation step of each cycle and the cleavage step of each cycle are the same as in method 1. However, in between those steps of each cycle there is conversion step e.g. phosphorylation and a second ligation (also known as nick sealing, nick repair or gap repair) to seal the nick left at the end of the first ligation.

[0481] Hence, after the first ligation, the first strand of the acceptor and the donor polynucleotides have been ligated to each other at the first terminal end of the donor and acceptor, but the second strands of the donor and acceptor have not, thus leaving a nick between those strands. In an illustrative example, the 5’ end of the second strand of the donor polynucleotide, which is at the first terminal end of the donor polynucleotide, lacks a phosphate group at the end of the first ligation. A kinase is used to add a phosphate group to it, followed by a second ligation or nick sealing of the 3’ end of the second strand of the acceptor and the 5’ end of the second strand of the donor polynucleotide. Acordingly, the nick at the second strand at the first terminal end of the donor and acceptor polynucleotides is ligated and consequently the resultant ligation product is ready for the cleavage step.

[0482] Method 4- 3’ overhang with nucleotide incorporation

[0483] An illustrative example of such a synthesis method is depicted in Figure 5.

[0484] The 3’ overhang with nucleotide incorporation of method version 1 can have a number of advantages. Those including helping to limit self-ligation of acceptors polynucleotides to each other, as well as limiting self-ligation of donor polynucleotides. That is illustrated in Figure 6 for the 5’ approach, but is also equally applicable to the 3’ approach. Provision of acceptor polynucleotide

[0485] To initiate a cycle of synthesis an acceptor polynucleotide is first provided.

[0486] The acceptor polynucleotide comprises first and second polynucleotide strands and first and second terminal ends. The first and second polynucleotide strands are connected by hybridization, via standard Watson-Crick base pairing, to form a doublestranded polynucleotide molecule.

[0487] The first terminal end of the acceptor polynucleotide is ligatable and the second strand has a 3’ overhang. In one embodiment, the 3’ overhang of the second strand is a single nucleotide in length. The first terminal end is free, i.e. neither the first strand nor the second strand is tethered to or otherwise attached to any other structure.

[0488] The terminal nucleotide of the first strand at the ligatable first terminal end of the acceptor polynucleotide comprises a 5’ phosphate group. The 3’ terminal nucleotide of the second strand at the ligatable first terminal end comprises a 3’ hydroxyl group.

[0489] The second terminal end of the acceptor polynucleotide is preferably non- ligatable.

[0490] The second terminal end of the acceptor polynucleotide is preferably tethered to a surface. Such a surface may be any suitable surface as described and defined elsewhere herein. The second terminal end may be tethered to a surface due to the second strand of the acceptor polynucleotide being tethered to the surface whilst the first strand of the acceptor polynucleotide is untethered. Alternatively, the second terminal end may be tethered to a surface due to the first strand of the acceptor polynucleotide being tethered to the surface whilst the second strand of the acceptor polynucleotide is untethered. Alternatively still, the second terminal end may be tethered to a surface due to the first and second strands of the acceptor polynucleotide being tethered to the surface. Where both the first and second strands of the acceptor polynucleotide are tethered to the surface, each strand may be independently tethered to the surface. Alternatively, the first and second strands at the second terminal end of the acceptor polynucleotide may be connected together via a connector, such as via a hairpin loop, and the connector, or any other part of the second terminal end, may be tethered to the surface. The acceptor polynucleotide may initially be provided without comprising any of the nucleotides of the predefined sequence which the user wishes to synthesize. This may be the case, for example, before commencing the very first cycle of synthesis. More typically however, the acceptor polynucleotide, when provided, will already include the polynucleotide payload of the previous cycle with the 5’ overhang forming part of the payload of the previous cycle of synthesis.

[0491] Provision of donor polynucleotide

[0492] To initiate a cycle of synthesis a donor polynucleotide is also provided.

[0493] The donor polynucleotide comprises first and second polynucleotide strands and first and second terminal ends. The first and second polynucleotide strands are connected by hybridization, via standard Watson-Crick base pairing, to form a doublestranded polynucleotide molecule.

[0494] The first terminal end of the donor polynucleotide is ligatable and has a 3’ overhang. Hence, as show in Figure 2, the first terminal end of the donor polynucleotide has a 3’ overhang because the first strand has such an overhang. The 3’ overhang has a complementary sequence to the 3’ overhang of the acceptor polynucleotide. For instance, wherein the acceptor polynucleotide has a 3’ overhang with adenosine as the base, the donor polynucleotide will have a 3’ with thymine as the base. As the overhangs are complementary they will be able to hybridise to each other via Watson- Crick base-pairing. The first terminal end is free, i.e. neither the first strand nor the second strand is tethered to or otherwise attached to any other structure.

[0495] The second terminal end is typically also free, i.e. neither the first strand nor the second strand is tethered to or otherwise attached to any other structure. The second terminal end may be non-ligatable or it may be ligatable (i.e. in the case of a symmetrical donor, as described below).

[0496] The terminal nucleotide of the second strand at the ligatable first terminal end of the donor polynucleotide lacks a 5’ phosphate group. The 3’ terminal nucleotide of the first strand at the ligatable first terminal end comprises a 3’ hydroxyl group. The second terminal end of the donor polynucleotide may be non-ligatable. This avoids the problem of multiple donor polynucleotides ligating together. Where the second terminal end of the donor polynucleotide is non-ligatable, the terminal nucleotides of the first and / or second strands at the second terminal end may comprise a blocking group. A blocking group is any blocking group defined elsewhere herein. A blocking group(s) renders the second terminal end non-ligatable. Alternatively, the second terminal end of the donor polynucleotide may be provided without a 5’ phosphate group. Alternatively, still, the first and second strands at the second terminal end of the donor polynucleotide may be connected together via a connector, such as via a hairpin loop. Such a connector also renders the second terminal end non-ligatable.

[0497] The second terminal end of the donor polynucleotide may be ligatable with respect to an acceptor polynucleotide, but it may be structured so that it cannot ligate to another donor polynucleotide. For example, the donor polynucleotide may be a symmetrical donor polynucleotide. The second terminal end of the donor polynucleotide may comprise a second polynucleotide payload, wherein:

[0498] (i) the payload nucleotide sequence of the first strand at the second terminal end of the donor polynucleotide in the 5’ to 3’ direction is the same as the payload nucleotide sequence of the second strand at the first terminal end of the donor polynucleotide in the 5’ to 3’ direction;

[0499] (ii) the payload nucleotide sequence of the second strand at the second terminal end of the donor polynucleotide in the 3’ to 5’ direction is the same as the payload nucleotide sequence of the first strand at the first terminal end of the donor polynucleotide in the 3’ to 5’ direction;

[0500] (iii) the second terminal end of the donor polynucleotide comprises a 3 ’ hydroxyl group and lacks a 5’ phosphate group; and

[0501] (iv) the second strand of the donor polynucleotide at the second terminal end comprises a 3’ overhang which has the same sequence as the overhang at the first terminal end.

[0502] In such a donor polynucleotide both the first and the second terminal ends lack 5’ phosphate groups. Accordingly, a first donor polynucleotide cannot be ligated to a second donor polynucleotide in a standard ligation reaction, thus avoiding the problem of self-ligation between donor polynucleotides. As the two overhangs of a symmetrical donor are not designed to be complementary to each other that two will make it less likely that such symmetrical donor polynucleotides will ligate to each other.

[0503] Nevertheless, since both the first and the second terminal ends comprise a 3’ hydroxyl group, either end of the donor polynucleotide is capable of ligating to an acceptor polynucleotide as described below. Such a donor polynucleotide therefore has the advantage of possessing two identical ligatable (with respect to an acceptor polynucleotide) ends. By having more ligatable ends available per reaction, the efficiency of ligation can be improved.

[0504] The donor polynucleotide comprises, at the ligatable first terminal end, a polynucleotide payload comprising one or more nucleotide pairs of the predefined sequence. The polynucleotide payload comprises one or more nucleotide pairs of the predefined sequence that are to be incorporated into the acceptor polynucleotide following ligation. The 3’ overhang of the first strand donor polynucleotide also constitutes part of the payload.

[0505] The first nucleotide before the overhang of the first strand at the ligatable first terminal end and the terminal nucleotide of the second strand at the ligatable first terminal end form a nucleotide pair, and this pair is the first pair of nucleotides in the polynucleotide payload. If the polynucleotide payload comprises more than one nucleotide pair of the predefined sequence, the penultimate nucleotide before the overhang of the first strand at the ligatable first terminal end and the penultimate nucleotide of the second strand at the ligatable first terminal end form the next nucleotide pair of the payload, and this pair is the second pair of nucleotides in the polynucleotide payload, and so on, with any nucleotides that will form part of the overhang of the acceptor after cleavage at the end of the cycle form the final part of the nucleotide payload.

[0506] The donor polynucleotide is provided with a cleavage site adjacent to the polynucleotide payload. The cleavage site is situated between the polynucleotide payload and the second terminal end of the donor polynucleotide. The exact type of cleavage site and its location within the donor polynucleotide can vary, and may be defined by the user when providing the donor polynucleotide, provided that the cleavage step is performed as described below. The cleavage site though will be chosen so that cleavage results in a fresh 3’ overhang that can be used in the next cycle.

[0507] Ligation of acceptor and donor polynucleotides

[0508] The ligation step functions to physically join the donor polynucleotide to the acceptor polynucleotide as a first step to facilitate the transfer of the polynucleotide payload from the donor polynucleotide to the acceptor polynucleotide. The ligation step comprises a single stranded ligation so that the first strand of the acceptor polynucleotide and the first strand of the donor polynucleotide are ligated together at was originally the first terminal end of each, where the second strand of the acceptor polynucleotide and the donor polynucleotide not being ligated together to leave a nick with the free 3 ’ hydroxy group of the first terminal end of the second strand of the acceptor polynucleotide being free for nucleotide incorporation in the next step.

[0509] In method version 4, the ligation step comprises joining the donor and acceptor polynucleotides at their first terminal ends by ligating the first strand of the donor polynucleotide at its first terminal end with the first strand of the acceptor polynucleotide at its first terminal end, but wherein the second strands of the donor and acceptor polynucleotides at their first terminal ends are not joined and are separated by a nick. Accordingly, in method version 1, the ligation step comprises a single-stranded ligation of the first strands of the donor and acceptor polynucleotides, but the second strands of the donor and acceptor polynucleotides are not ligated together at their first terminal ends.

[0510] The ligation step can be performed by any suitable means for physically joining together polynucleotide strands that were previously not joined together. Preferably the ligation step is performed by the action of an enzyme having nucleotide ligase activity, such as any ligase enzyme described elsewhere herein and which can perform the required ligase function for this particular method version.

[0511] The step of ligating the donor and acceptor polynucleotides creates a ligated polynucleotide which has a nick. Acceptor depletion (optional)

[0512] Before the cleavage step a depletion step may be performed. Such a step is optional and not essential. The depletion step allows for any acceptor polynucleotide to be depleted, if that acceptor polynucleotide has for some reason failed to join with a donor polynucleotide to form a ligated polynucleotide. Such an acceptor polynucleotide may be referred to as an unreacted acceptor polynucleotide. Failure to react may occur for various reasons including a failure to ligate with a donor polynucleotide. Depletion of any unreacted acceptor polynucleotide may be desirable so that in the next round of synthesis it does not act as acceptor polynucleotide for a subsequent polynucleotide payload. If this were to occur, the double-stranded polynucleotide having a predefined sequence would not be synthesized correctly in that particular acceptor polynucleotide structure.

[0513] In the depletion step an unreacted acceptor polynucleotide is rendered inert, i.e. incapable of acting as an acceptor polynucleotide in the next and subsequent cycles of synthesis.

[0514] Since the terminal nucleotide of the first strand at the ligatable first terminal end of the acceptor polynucleotide comprises a 5’ phosphate group, the unreacted acceptor polynucleotide can be rendered inert by removing the 5’ phosphate group so that it cannot be ligated to a donor polynucleotide in any further synthesis cycle. Accordingly, a depletion step may comprise performing a treatment step to remove the 5’ phosphate group from the terminal nucleotide of the first strand at the ligatable first terminal end of the acceptor polynucleotide. Such a step may comprise performing a treatment step with an enzyme having phosphatase activity, such as calf intestinal phosphatase (CIP). If the terminal nucleotide in the first strand at the second terminal end of the donor polynucleotide comprises a 5’ phosphate group, removal of this group at the same time will have no effect, since this part of the donor molecule will be removed following the cleavage step. Incorporation

[0515] Following-on from the ligation step, and optionally any depletion step, the ligation product formed by ligating the first strands of the acceptor and donor polynucleotides has a nick between the second strands with the 3’ terminus of the second strand having a free 3 ’OH group able to act as a substrate for a polymerase and hence nucleotide incorporation. The ligated first strands of the acceptor and donor polynucleotides therefore effectively serve as a template for the nucleotide incorporation. As nucleotides are incorporated that may displace the unligated second strand of the donor polynucleotide. The incorporation step effectively removes the nick present in the ligation step and means that the ligation product is ready for cleavage.

[0516] The original nucleotides of the polynucleotide payload of the second strand may be separated from the first strand:

[0517] (i) before the incorporation step; or

[0518] (ii) during the incorporation step.

[0519] Where the original nucleotides of the polynucleotide payload of the second strand are separated from the first strand during the incorporation step, incorporation steps may be performed:

[0520] (a) by the action of an enzyme having polymerase activity, and wherein the polymerase displaces the original nucleotides of the polynucleotide payload of the second strand when synthesising the new second strand; or

[0521] (b) by the action of an enzyme having polymerase activity which possesses 5’ to 3’ exonuclease activity, and wherein the polymerase digests / degrades the original nucleotides of the polynucleotide payload of the second strand when synthesising the new second strand. Cleavage

[0522] The cleavage step functions to cleave the ligated polynucleotide to generate a new acceptor polynucleotide comprising the payload from the previous cycle or cycles and a new 3’ overhang ready for the next cycle. Thus, cleavage is such that the one or more nucleotide pairs of the predefined sequence and the overhang now formed were previously part of the donor polynucleotide and instead have become incorporated into the acceptor polynucleotide at the ligatable first terminal end of the acceptor polynucleotide. At the same time, the cleavage step functions to separate the polynucleotide payload from the remainder of the donor polynucleotide. The remainder of the donor polynucleotide can then be removed from the ligated polynucleotide, leaving behind the acceptor polynucleotide with the polynucleotide payload incorporated at the ligatable first terminal end of the acceptor polynucleotide.

[0523] In each cycle of synthesis, each polynucleotide payload comprises one or more nucleotide pairs of the predefined sequence that the user wishes to synthesize. Successive cycles therefore provide for the stepwise synthesis of the double-stranded polynucleotide having a predefined sequence, by successively joining together multiple polynucleotide payloads.

[0524] In method version 4, the cleavage is in both strands to generate an asymmetrical acceptor with the desired 3’ overhang.

[0525] In the case of an asymmetrical cleavage reaction, the first strand is cleaved immediately above the nucleotides of the polynucleotide payload (in the direction proximal to the second terminal end of the donor polynucleotide). Accordingly, the nucleotides of the first strand of the polynucleotide payload are retained at the cleaved first terminal end of the acceptor polynucleotide in the first strand, wherein the terminal nucleotide of the first strand of the acceptor polynucleotide is the final nucleotide of the polynucleotide payload. As the cleavage reaction is asymmetrical, the second strand is longer than the first strand after cleavage to give the 3’ overhang with the nucleotide or nucleotides of the 3’ overhang constituting part of the payload of the cycle that has just finished. The first strand of the resulting cleavage product able to act as an acceptor polynucleotide in the next cycle will have a 5’ phosphate group at the first terminal end. The second strand of the resulting cleavage product able to act as an acceptor polynucleotide in the next cycle will have a free 3’ OH group at the first terminal end and have the desired 3’ overhang.

[0526] Any suitable means may be used for the cleavage which results in the desired 3’ overhang may be used for the cleavage. In one embodiment, the cleavage is performed with a restriction enzyme. In particular, the cleavage is performed with a Type IIS endonuclease that means the first terminal end of the resultant acceptor for the next cycle has the desired 3’ overhang. An example of such a restriction enzyme is MnII. A further example of such a restriction enzyme is BciVI.

[0527] Following cleavage, method version 4 further comprises separating the cleaved donor polynucleotide from the acceptor polynucleotide. Washing may, for instance, be performed to remove the unwanted cleavage product, leaving the acceptor polynucleotide ready for the next cycle. Following the cleavage and incorporation steps, a new acceptor polynucleotide is therefore created with a new 5’ overhang. The new acceptor polynucleotide comprises the old acceptor polynucleotide, that was provided at the start of the cycle, with the polynucleotide payload incorporated at the first terminal end of the acceptor polynucleotide. The new acceptor polynucleotide is thus competent to act as an acceptor polynucleotide to accept a new polynucleotide payload to be incorporated during the next cycle. This facilitates the stepwise synthesis of the doublestranded polynucleotide having a predefined sequence, by allowing multiple polynucleotide payloads to be successively joined together.

[0528] Method 5 - 3 overhang with nucleotide incorporation and hot start polymerase

[0529] Method 5 is effectively a variant of method 4 in that a specific type of polymerase is used in the incorporation step, in particular a polymerase that has a higher optimal temperature than the ligase is used, a so-called hot start polymerase. Hence, method 5 still comprises cycles with ligation, nucleotide incorporation, and cleavage steps as set out for method 1 and also uses 3’ overhangs, but the host start polymerases means the ligase and polymerase can be added in a single fluid. Figure 7 depicts an illustrative example for using such an approach using 5’ overhangs (synthesis method 2), but the method is equally applicable to 3; overhangs.

[0530] The ligase and polymerase are added in a single fluid. The ligation step of a cycle is then performed at a temperature at which the ligase is active, but the polymerase is not. The temperature is then raised for the nucleotide incorporation step to a temperature at which the ligase is no longer active, but the polymerase is, allowing the nucleotide incorporation step to take place. In one embodiment, the ligase may actually be inactivated at the second temperature.

[0531] One important advantage of method 5 is that because the ligase and polymerase can be added in a single fluid, the number of fluid addition and washing stages needed can be reduced making the method more streamlined.

[0532] Method 6- 3’ overhang with nucleotide incorporation and hot start polymerase, wherein ligase, polymerase, and restriction enzyme are added in a single fluid

[0533] Method 6 is effectively a variant of method 5 in that the ligase, hot start polymerase, and cleavage enzyme are all added in a single fluid. An illustrative embodiment of method 6 is shown in Figure 10. Method 6 differs in a further way from method 5 in that the donor polynucleotide includes a nucleotide mismatch in the consensus sequence for the cleavage enzyme, with the first strand of the donor polynucleotide having the correct nucleotide for the consensus recognition sequence and the second strand of the donor polynucleotide having the incorrect nucleotide for the consensus recognition sequence for the cleavage enzyme.

[0534] During the nucleotide incorporation step the first strand of the donor polynucleotide is used as the template for the nucleotide incorporation resulting in the nucleotide mismatch being corrected so the correct consensus recognition sequence for the cleavage enzyme is present. Overall, that means the ligase, hot start polymerase, and the cleavage enzyme may all be added in a single fluid. In one embodiment, the cleavage enzyme is also heat resistant. Hence, in the ligation step the ligase is active, the host start polymerase is not active as the temperature is not high enough, and the consensus sequence for the cleavage enzyme still includes the mismatch as the ligation and nucleotide incorporation have yet to occur. After ligation the temperature is raised so that the hot start polymerase is active and nucleotide incorporation takes place, resulting in correction of the mismatch so that cleavage can take place.

[0535] One advantage of synthesis method 6 is that all three of the ligase, hot start polymerase, and the cleavage enzyme can be added in a single fluid further reducing the number of fluid addition and washing steps needed helping to streamline the method.

[0536] Method 7— 3’ overhang with nick repair

[0537] Method 7 is another 3’ overhang synthesis method which corresponds to method 4 except that the nucleotide incorporation step of each cycle is replaced instead with a step comprising joining the two unligated second strands of the acceptor and donor polynucleotides

[0538] The terminal 5’ nucleotide of the payload of the donor polynucleotide (i.e. at the 5’ end of the second strand at the first terminal end of the donor polynucleotide) is provided as a non-ligatable nucleotide and the method comprises converting this non- ligatable nucleotide into a ligatable nucleotide followed by a second ligation reaction to repair the nick. For example, the terminal 5’ nucleotide of the payload of the donor polynucleotide is provided without a free phosphate group and the step of converting this non-ligatable nucleotide into a ligatable nucleotide comprises phosphorylating the nucleotide and then sealing the nick by performing a second ligation. An illustrative embodiment of 5’ overhang and nick repair method is depicted in Figure 11 and the method shown is equally applicable to 3’ overhangs.

[0539] Thus, the initial (first) ligation step of each cycle and the cleavage step of each cycle are the same as in method 4. However, in between those steps of each cycle there is a conversin reaction e.g. phosphorylation and a second ligation (also known as nick sealing) to seal the nick left at the end of the first ligation. Hence, after the first ligation, the first strand of the acceptor and the donor polynucleotides have been ligated to each other at the first terminal end of the donor and acceptor, but the second strands of the donor and acceptor have not leaving a nick between those strands. In an illustrative embodiment the 5’ end of the second strand of the donor polynucleotide, which is at the first terminal end of the donor polynucleotide, lacks a phosphate group at the end of the first ligation. A kinase is used to add a phosphate group to it, followed by a second ligation or nick sealing of the 3’ end of the second strand of the acceptor and the 5’ end of the second strand of the donor polynucleotide. Accordingly, the nick at the second strand at the first terminal end of the donor and acceptor polynucleotides is ligated and consequently the resultant ligation product is ready for the cleavage step.

[0540] EXAMPLES

[0541] The invention is further illustrated by the following Examples. The Examples are presented merely to illustrate the invention, and should not be construed as limiting on the invention.

[0542] For the purposes of Examples 1 and 2 below, certain terminology used in the Examples associated with structural features is explained in Figure 15. For the purposes of Examples 1 and 2 below, the strand of the acceptor polynucleotide shown in Figure 15 marked “5”’ corresponds to the first strand of the acceptor polynucleotide as described elsewhere herein. The strand of the acceptor polynucleotide shown in Figure 15 marked “3”’ corresponds to the second strand of the acceptor polynucleotide as described elsewhere herein. The terminal end of the acceptor polynucleotide shown in Figure 15 which is tethered to a surface corresponds with the second terminal end of the acceptor polynucleotide as described elsewhere herein.

[0543] For the purposes of Examples 1 and 2 below, the strand of the donor polynucleotide shown in Figure 15 marked “ligation strand” corresponds to the first strand of the donor polynucleotide as described elsewhere herein. The strand of the donor polynucleotide shown in Figure 15 marked “helper strand” corresponds to the second strand of the donor polynucleotide as described elsewhere herein. The terminal end of the donor polynucleotide shown in Figure 15 which comprises the payload corresponds with the first terminal end of the donor polynucleotide as described elsewhere herein. The structure marked “payload” corresponds with the polynucleotide payload as described elsewhere herein. Example 1: Chemistry with single-base 5’ overhang ligation, incorporation., and cleavage leaving single-base 5’ overhang

[0544] 5 Introduction

[0545] This Example describes the synthesis of polynucleotides that takes place on a substrate using cycles that each comprise:

[0546] 1. One-sided ligation of a nucleotide payload onto a double-stranded acceptor 0 DNA. The strand of the acceptor that participates in the one-side ligation is referred to as the ligation or first strand. The strand of the acceptor that does not participate in the ligation may be referred to as the helper or second strand. The acceptor and donor polynucleotides used have complementary 5’ overhangs of a single nucleotide length. The 5’ overhang of the acceptor comprises a phosphate group, whereas the 5’ overhang 5 of the acceptor does not. That means only the first (ligation) strands of the donor and acceptor polynucleotides are ligated to each other, whereas the second (helper) strands are not ligated leaving a nick.

[0547] 2. dNTP incorporation extending the second (helper) strand of the acceptor from the nick using the first strand (ligation strand) of the donor polynucleotide as a template. 0 3. Cleavage to generate a new acceptor polynucleotide incorporating the payload sequence and a new 5’ overhang ready for the next cycle.

[0548] Oligonucleotide sequences

[0549] Oligonucleotides were designed in-house and synthesised by Integrated DNA 5 Technologies (see Table 1 below for sequences).

[0550] Table 1

[0551] Preparation of acceptor with 5’ overhang immobilised on support

[0552] 5 Overview

[0553] Prior to the cycles of the synthesis method being performed the acceptor polynucleotide was immobilised on the substrate and then activated by enzymatic cleavage to give 5’ single nucleotide overhangs. 0

[0554] Immobilisation

[0555] Dynabeads™ MyOne™ Streptavidin Cl beads. Double-stranded acceptor DNA was immobilised on the beads using the following method: 5

[0556] 1. Dynabeads™ MyOne™ Streptavidin Cl beads were vortexed for 30 sec.

[0557] 2. 15 pl of lOmg / ml beads were transferred into a 1.5 ml Eppendorf tube.

[0558] 3. The tube was placed into a magnetic rack (DynaMag-2, Invitrogen) and beads were left to pellet for 2 - 3 minutes. 0 4. Supernatant was removed and discarded using a pipette.

[0559] 5. The tube was removed from the magnet and 1 ml of Binding and washing buffer (5 mM Tris-HCl, 0.5 mM EDTA, 1 M NaCl) was added. 6. Beads were resuspended by vortexing, before returning the tube back on the magnet for 2 - 3 minutes and removing the supernatant using a pipette.

[0560] 7. Steps 5 - 6 were repeated twice, for a total of three washes.

[0561] 8. Beads were resuspended in 7.5 pl of 2x Binding and washing buffer (10 mM Tris-

[0562] HC1, 1 mM EDTA, 20 mM NaCl).

[0563] 9. 5 pl of sterile water and 1.5 pl of 50 pM acceptor were added to the beads.

[0564] 10. The tube was placed on a rotator for 15 min at room temperature.

[0565] 11. After the 15 min, the tube was placed into a magnetic rack (DynaMag-2, Invitrogen) and beads were left to pellet for 2 - 3 minutes.

[0566] 12. Supernatant was removed and discarded using a pipette.

[0567] 13. The tube was removed from the magnet and 1 ml of Binding and washing buffer (5 mM Tris-HCl, 0.5 mM EDTA, 1 M NaCl) was added.

[0568] 14. Beads were resuspended by vortexing, before returning the tube back on the magnet for 2 - 3 minutes and then removing the supernatant using a pipette.

[0569] 15. Steps 13 - 14 were repeated twice, for a total of three washes.

[0570] 16. The bead pellet was resuspended in 100 pl sterile water (ELGA VEOLIA).

[0571] 17. The tube was vortexed briefly, centrifuged at 1,000 rpm for 5 seconds.

[0572] 18. 20 pl of bead solution was removed for gel electrophoresis.

[0573] 19. The remaining 80 pl of bead solution are carried forward to the overhang generation step.

[0574] Based on DNA quantification methods (gel electrophoresis and Qubit readings) there was approximately 75 fmol of immobilised acceptor per pl of bead solution.

[0575] Generation of 5 ’ single nucleotide overhangs

[0576] The single-base 5’ overhang on the immobilised acceptor were next generated via endonuclease cleavage reaction using the following method: 1. The tube containing 80 pl of beads with immobilised acceptor was placed into a magnetic rack (DynaMag-2, Invitrogen) and beads were left to pellet for 2 - 3 minutes before the supernatant was removed using a pipette.

[0577] 2. The bead pellet was resuspended in 20 pl restriction enzyme solution containing 2 pl of lOx rCutSmart buffer NEB (500 mM Potassium Acetate, 200 mM Trisacetate, 100 mM Magnesium Acetate, 1 ng / ml Recombinant Albumin and pH 7.9 at 25°C), 2 pl of Neil (20,000 U / ml) and 16 pl of sterile water.

[0578] 3. Reaction mixture was then briefly mixed on a vortexer, centrifuged at 1,000 rpm for 5seconds and incubated at 37°C for 30 minutes.

[0579] 4. After the incubation time had elapsed, the tube was centrifuged at 1,000 rpm for 5 seconds and placed into a magnetic rack for 2 - 3 minutes before removing the supernatant with a pipette.

[0580] 5. The tube was removed from the magnet, and beads were resuspended in 800 pl of lOx TE with 0,05% Tween 20.

[0581] 6. The tube was vortexed briefly, centrifuged at 1,000 rpm for 5 seconds and placed into a magnetic rack for 2 - 3 minutes before removing the supernatant with a pipette.

[0582] 7. The tube was removed from the magnet, and beads were again resuspended in 800 pl of lOx TE with 0,05% Tween 20.

[0583] 8. The tube was vortexed briefly and then incubated on a rotator for 20 minutes at room temperature.

[0584] 9. The tube was centrifuged at 1,000 rpm for 5 seconds and placed into a magnetic rack for 2 - 3 minutes before removing the supernatant with a pipette.

[0585] 10. The tube was removed from the magnet, and beads were resuspended in 800 pl of O.lx TE.

[0586] 11. The tube was vortexed briefly, centrifuged at 1,000 rpm for 5 seconds and placed into a magnetic rack for 2 - 3 minutes before removing the supernatant with a pipette.

[0587] 12. The tube was removed from the magnet and beads were resuspended in 800 pl of sterile water. 13. The tube was vortexed briefly, centrifuged at 1,000 rpm for 5 seconds and placed into a magnetic rack for 2 - 3 minutes before removing the supernatant with a pipette.

[0588] 14. Steps 31 and 32 were repeated once.

[0589] 15. After the second water wash, the bead pellet was resuspended in 80 pl sterile water.

[0590] 16. The tube was vortexed briefly, centrifuged at 1,000 rpm for 5 seconds.

[0591] 17. 20 pl of bead solution was removed for gel electrophoresis.

[0592] 18. The remaining 60 pl of bead solution are carried forward to the ligation step.

[0593] Once the initial acceptor polynucleotide with 5’ overhangs had been generated the first cycle of the synthesis of the defined sequence was then performed.

[0594] Cycle Step 1: Ligation

[0595] Overview

[0596] The first step describes the ligation of polynucleotides with DNA ligase. A diagrammatic illustration is shown in Figure 1. As shown in Figure 1, the acceptor polynucleotide has a 5’ overhang with a free phosphate group available for ligation whereas the 5’ overhang of the donor polynucleotide lacks a phosphate group meaning the second (helper) strands of the acceptor and donor polynucleotides are not ligated in the single stranded ligation.

[0597] Materials

[0598] The main reagents were as follows:

[0599] I. The oligonucleotides were diluted to a stock concentration of 1 mM using 0. lx TE (1 mM Tris-HCl, 0.1 mM EDTA).

[0600] II. Dynabeads™ MyOne™ Streptavidin Cl (Invitrogen) with immobilised, singlebase overhang acceptor.

[0601] III. Human Ligase III (14 pM) obtained from NEB.

[0602] IV. Thermal Liable Proteinase K (120 U / ml, NEB) Methods

[0603] A ligation reaction on oligonucleotides was carried out using the procedure below:

[0604] 1. The tube containing 60 pl of bead solution after 5’ single-base overhang generation of the acceptor was placed into a magnetic rack (DynaMag-2, Invitrogen) and beads were left to pellet for 2 - 3 minutes.

[0605] 2. Supernatant was removed and discarded using a pipette.

[0606] 3. 20 pl of ligation reaction solution containing Ligation Buffer (50.5 mM Tris-HCl pH 7.5, 10 mM MgCh, 10 mM NaCl, 10 pM ATP, 2.5% PEG8000), 25 pM annealed donor and 1.4 pM Human Ligase III was then added into the same Eppendorf tube.

[0607] 4. Reaction mixture was then briefly mixed by resuspension on a vortexer, centrifuged at 1,000 rpm for 5 seconds and incubated at room temperature for 45 minutes with rotation.

[0608] 5. After the incubation time had elapsed, the tube was centrifuged at 1,000 rpm for 5 seconds and placed into a magnetic rack and beads were left to pellet for 2 - 3 minutes before the supernatant was removed using a pipette.

[0609] 6. The tube was removed from the magnet, and beads were resuspended in 800 pl of lOx TE, briefly vortexed, then centrifuged at 1,000 rpm for 5 seconds and placed into a magnetic rack for 2 - 3 minutes before removing the supernatant with a pipette.

[0610] 7. The tube was removed from the magnet, and beads were again resuspended in 800 pl of lOx TE with 0.05% Tween 20.

[0611] 8. The tube was vortexed briefly and then incubated on a rotator for 20 minutes at room temperature.

[0612] 9. The tube was centrifuged at 1,000 rpm for 5 seconds and placed into a magnetic rack for 2 - 3 minutes before removing the supernatant with a pipette.

[0613] 10. The tube was removed from the magnet, and beads were resuspended in 800 pl of O.lx TE. 11. The tube was vortexed briefly, centrifuged at 1,000 rpm for 5 seconds and placed into a magnetic rack for 2 - 3 minutes before removing the supernatant with a pipette.

[0614] 12. The tube was removed from the magnet, and beads were resuspended in 800 pl of sterile water.

[0615] 13. The tube was vortexed briefly, centrifuged at 1,000 rpm for 5 seconds and placed into a magnetic rack for 2 - 3 minutes before removing the supernatant with a pipette.

[0616] 14. Beads were resuspended in 20 pl of a proteinase solution containing 2 pl of lOx rCutSmart buffer NEB (500 mM Potassium Acetate, 200 mM Tris-acetate, 100 mM Magnesium Acetate, 1 ng / ml Recombinant Albumin and pH 7.9 at 25°C), 2 pl of Thermal Labile Proteinase K (120 U / ml) and 16 pl of sterile water.

[0617] 15. Reaction mixture was then briefly mixed on a vortexer, centrifuged at 1,000 rpm for 5 seconds and incubated at 37°C for 15 minutes.

[0618] 16. The reaction mixture was transferred to 55°C and incubated for 10 minutes.

[0619] 17. After the incubation time had elapsed, the tube was centrifuged at 1,000 rpm for 5 seconds and placed into a magnetic rack for 2 - 3 minutes before removing the supernatant with a pipette.

[0620] 18. The tube was removed from the magnet, and beads were resuspended in 800 pl of lOx TE with 0,05% Tween 20.

[0621] 19. The tube was vortexed briefly, centrifuged at 1,000 rpm for 5 seconds and placed into a magnetic rack for 2 - 3 minutes before removing the supernatant with a pipette.

[0622] 20. The tube was removed from the magnet, and beads were again resuspended in 800 pl of lOx TE with 0,05% Tween 20.

[0623] 21. The tube was vortexed briefly and then incubated on a rotator for 20 minutes at room temperature.

[0624] 22. The tube was centrifuged at 1,000 rpm for 5 seconds and placed into a magnetic rack for 2 - 3 minutes before removing the supernatant with a pipette.

[0625] 23. The tube was removed from the magnet, and beads were resuspended in 800 pl of O.lx TE. 24. The tube was vortexed briefly, centrifuged at 1,000 rpm for 5 seconds and placed into a magnetic rack for 2 - 3 minutes before removing the supernatant with a pipette.

[0626] 25. The tube was removed from the magnet, and beads were resuspended in 800 pl of sterile water.

[0627] 26. The tube was vortexed briefly, centrifuged at 1,000 rpm for 5 seconds and placed into a magnetic rack for 2 - 3 minutes before removing the supernatant with a pipette.

[0628] 27. Steps 25 and 26 were repeated once.

[0629] 28. After the second water wash, the bead pellet was resuspended in 60 pl sterile water.

[0630] 29. The tube was vortexed briefly, centrifuged at 1,000 rpm for 5 seconds.

[0631] 30. 20 pl of bead solution was removed for gel electrophoresis.

[0632] 31. The remaining 40 pl of bead solution are carried forward to the incorporation step.

[0633] Cycle Step 2: Incorporation

[0634] Overview

[0635] The second step involves the nick extension on the donor helper strand using the donor ligation strand as template for enzymatic dNTP incorporation by a DNA polymerase. A diagrammatic illustration is shown in Figure 1.

[0636] Materials

[0637] The main reagents were as follows:

[0638] I. Deoxynucleotide (dNTP) Solution Mix with equimolar solutions (10 mM) of dATP, dCTP, dGTP and dTTP was obtained from NEB.

[0639] II. DNA Polymerase I, Large (Klenow) Fragment and accompanying buffer were obtained from NEB.

[0640] III. Dynabeads™ MyOne™ Streptavidin Cl (Invitrogen) with immobilised acceptor, ligated to a donor from the ligation step (step 1). Methods dNTP incorporation reactions were carried out using the procedure below:

[0641] 1. The tube containing 40 pl of bead solution was placed into a magnetic rack (DynaMag-2, Invitrogen) and beads were left to pellet for 2 - 3 minutes.

[0642] 2. Supernatant was removed and discarded using a pipette.

[0643] 3. The bead pellet was resuspended in 17 pl sterile water (ELGA VEOLIA).

[0644] 4. 2 pl of lOx NEBuffer 2 (500 mM Sodium Chloride, 100 mM Tris-HCl, 100 mM Magnesium Chloride, 10 mM DTT and pH 7.9 at 25°C) was then added into the same Eppendorf tube.

[0645] 5. 0.5 pl of dNTPs (10 mM) were added into the same tube.

[0646] 6. 0.5 pl of DNA Polymerase I, Large (Klenow) Fragment (5 units / pl) was added to the tube.

[0647] 7. Reaction mixture was then briefly mixed on a vortexer, centrifuged at 1,000 rpm for 5 seconds and incubated at 37°C for 5 minutes.

[0648] 8. After the incubation time had elapsed, the tube was centrifuged at 1,000 rpm for 5 seconds and placed into a magnetic rack and beads were left to pellet for 2 - 3 minutes before the supernatant was removed using a pipette.

[0649] 9. The tube was removed from the magnet, and beads were resuspended in 800 pl of lOx TE, briefly vortexed, then centrifuged at 1,000 rpm for 5 seconds and placed into a magnetic rack for 2 - 3 minutes before removing the supernatant with a pipette.

[0650] 10. The tube was vortexed briefly, centrifuged at 1,000 rpm for 5 seconds and placed into a magnetic rack for 2 - 3 minutes before removing the supernatant with a pipette.

[0651] 11. The tube was removed from the magnet, and beads were again resuspended in 800 pl of lOx TE with 0.05% Tween 20.

[0652] 12. The tube was vortexed briefly and then incubated on a rotator for 20 minutes at room temperature.

[0653] 13. The tube was centrifuged at 1,000 rpm for 5 seconds and placed into a magnetic rack for 2 - 3 minutes before removing the supernatant with a pipette. 14. The tube was removed from the magnet, and beads were resuspended in 800 pl of O.lx TE.

[0654] 15. The tube was vortexed briefly, centrifuged at 1,000 rpm for 5 seconds and placed into a magnetic rack for 2 - 3 minutes before removing the supernatant with a pipette.

[0655] 16. The tube was removed from the magnet, and beads were resuspended in 800 pl of sterile water.

[0656] 17. The tube was vortexed briefly, centrifuged at 1,000 rpm for 5 seconds and placed into a magnetic rack for 2 - 3 minutes before removing the supernatant with a pipette.

[0657] 18. Steps 16 and 17 were repeated once.

[0658] 19. After the second water wash, the bead pellet was resuspended in 40 pl sterile water and carried forward to the Bed cleavage step.

[0659] Cycle Step 3: Cleavage

[0660] Overview

[0661] The third step describes the cleavage of the phosphodiester backbone on the donor above the payload, leaving a new 5’ single-base overhang. A diagrammatic illustration is shown in Figure 1.

[0662] Materials

[0663] The main reagents used were as follows:

[0664] I. NEB Bed Endonuclease (10,000 U / ml), supplied with lOx NEB rCutSmart Buffer and 1.0 M DTT solution in ultrapure water.

[0665] II. Dynabeads™ MyOne™ Streptavidin Cl (Invitrogen) with immobilised acceptor, ligated to a donor, and nick-repair by dNTP incorporation.

[0666] Methods

[0667] Cleavage reaction on oligonucleotides was carried out using the procedure below: 1. The tube containing 40 pl of bead solution after dNTP incorporation was placed into a magnetic rack (DynaMag-2, Invitrogen) and beads were left to pellet for 2 - 3 minutes.

[0668] 2. Supernatant was removed and discarded using a pipette.

[0669] 3. The bead pellet was resuspended in 7.8 pl sterile water.

[0670] 4. 1 pl of lOx rCutSmart buffer NEB (500 mM Potassium Acetate, 200 mM Trisacetate, 100 mM Magnesium Acetate, 1 ng / ml Recombinant Albumin and pH 7.9 at 25°C), 0.02 pl of IM DTT (NEB) and 1 pl of Bcc\ (NEB) were then added into the same Eppendorf tube.

[0671] 5. Reaction mixture was then briefly mixed on a vortexer, centrifuged at 1,000 rpm for

[0672] 5seconds and incubated at 37°C for 15 minutes.

[0673] 6. After the incubation time had elapsed, the tube was centrifuged at 1,000 rpm for 5 seconds and placed into a magnetic rack and beads were left to pellet for 2 - 3 minutes before the supernatant was removed using a pipette.

[0674] 7. The tube was removed from the magnet, and beads were resuspended in 800 pl of lOx TE, briefly vortexed, then centrifuged at 1,000 rpm for 5 seconds and placed into a magnetic rack for 2 - 3 minutes before removing the supernatant with a pipette.

[0675] 8. The tube was vortexed briefly, centrifuged at 1,000 rpm for 5 seconds and placed into a magnetic rack for 2 - 3 minutes before removing the supernatant with a pipette.

[0676] 9. The tube was removed from the magnet, and beads were again resuspended in 800 pl of lOx TE with 0,05% Tween 20.

[0677] 10. The tube was vortexed briefly and then incubated on a rotator for 20 minutes at room temperature.

[0678] 11. The tube was centrifuged at 1,000 rpm for 5 seconds and placed into a magnetic rack for 2 - 3 minutes before removing the supernatant with a pipette.

[0679] 12. The tube was removed from the magnet, and beads were resuspended in 800 pl of

[0680] O.lx TE. 13. The tube was vortexed briefly, centrifuged at 1,000 rpm for 5 seconds and placed into a magnetic rack for 2 - 3 minutes before removing the supernatant with a pipette.

[0681] 14. The tube was removed from the magnet, and beads were resuspended in 800 pl of sterile water.

[0682] 15. The tube was vortexed briefly, centrifuged at 1,000 rpm for 5 seconds and placed into a magnetic rack for 2 - 3 minutes before removing the supernatant with a pipette.

[0683] 16. Steps 14 and 15 were repeated once.

[0684] 17. After the second water wash, the bead pellet was resuspended in 40 pl sterile water.

[0685] 18. The tube was vortexed briefly, centrifuged at 1,000 rpm for 5 seconds.

[0686] 19. 20 pl of bead solution was removed for gel electrophoresis.

[0687] 20. The remaining 20 pl of bead solution are carried forward to a second ligation step

[0688] (Step 1). This second ligation step was done to verify the completion of the cycle, using a donor comprising of Ligation Strand 2 and Helper Strand 2.

[0689] Gel Electrophoresis and DNA Visualization:

[0690] Overview

[0691] In order to analyse the results of the synthesis method DNA was cleaved from the beads, followed by gel electrophoresis and DNA visualisation being performed for analysis.

[0692] Materials

[0693] The main reagents used were as follows:

[0694] I. NEB Rsa Endonuclease (10,000 U / ml), supplied with lOx NEB rCutSmart Buffer.

[0695] IE Samples of Dynabeads™ MyOne™ Streptavidin Cl (Invitrogen) with immobilised acceptor taken after each step described above. Methods

[0696] Cleavage reaction to remove acceptor from Dynabeads™ MyOne™ Streptavidin Cl beads and subsequent gel electrophoresis was carried out using the procedure below:

[0697] 1. Tubes containing 20 pl of bead solution were placed into a magnetic rack (DynaMag-2, Invitrogen) and beads were left to pellet for 2 - 3 minutes before removing the supernatant with a pipette.

[0698] 2. The bead pellets were resuspended in 5 pl solution containing 0.5 pl of lOx rCutSmart buffer NEB (500 mM Potassium Acetate, 200 mM Tris-acetate, 100 mM Magnesium Acetate, 1 ng / ml Recombinant Albumin and pH 7.9 at 25°C), 0.5 pl of Rsa (10,000 U / ml) and 4 pl of sterile water.

[0699] 3. Reaction mixture was then briefly mixed on a vortexer, centrifuged at 1,000 rpm for 5 seconds and incubated at 37°C for 15 minutes.

[0700] 4. The tube was placed into a magnetic rack and beads were left to pellet for 2 - 3 minutes.

[0701] 5. 5 pl of supernatant were transferred to a sterile 0.2 ml PCR tube.

[0702] 6. 1 pl of 6X Purple Gel Loading Dye NEB (15% Ficoll®-400, 60 mM EDTA, 19.8 mM Tris-HCl, 0.48% SDS, 0.12% Dye 1, 0.006% Dye 2 and pH 8 at 25°C) was then added into the same Eppendorf tube.

[0703] 7. The reaction was loaded into the wells of a on polyacrylamide gel (20%) submersed in TBE buffer.

[0704] 8. X-cell sure lock module (Novex) was fastened in place and electrophoresis performed at constant 200 V for 75 minutes at room temperature.

[0705] 9. The gel was incubated in SYBR Gold Nucleic Acid Gel Stain for 10 minutes, and then in water for 5 min with gentle agitation.

[0706] 10. The gel was visualized by ChemiDoc MP (BioRad) using SYBR Gold settings.

[0707] 11. Visualization and analysis were carried out on the Image lab 2.0 platform. Results and Conclusions

[0708] The results of gel electrophoresis and DNA visualisation are presented in Figure 16. Ligation between a single-base overhang acceptor (Figure 16, lane 2) and a donor featuring a complementary overhang with Human Ligase III at room temperature (25°C) resulted in high yield obligated product (Figure 16, lane 3). After nick extension, the majority of acceptor-donor product is susceptible to Bed cleavage (Figure 16, lane 4). The cleavage product increased in length compared to the starting acceptor, confirming the incorporation of the payload (comparing Figure 16, lanes 2 and 4). Furthermore, most of the cleavage product ligates to a second single-base overhang donor (Figure 16, lane 5), confirming that the products of the incorporation and cleavage step featured a single-base 5’ overhang.

[0709] Example 2: Cycle chemistry on a 2D surface with a 5’ single base overhang ligation, followed by incorporation and cleavage to leave a single-base 5’ overhang

[0710] Introduction

[0711] This Example describes the synthesis of polynucleotides using a 3-step cycle chemistry: one-sided ligation of the payload onto double-stranded acceptor DNA, nick extension via dNTP incorporation, followed by cleavage to produce a single-base 5’ overhang. The specific method performed used a double-stranded donor, comprising of an Alexa Fluor™ 488 labelled Ligation strand and the Helper strand with a one base single overhang that facilitates ligation to the one base overhang on the acceptor. All reactions were carried out on glass bottom 96 well plate surface (Greiner Sensoplate™) which had been previously coated with a polyacrylamide-BRAPA copolymer. Oligonucleotide sequences

[0712] The first (ligation) and second (help) strand sequences of the acceptor and donor polynucleotides employed are set out in the Table below.

[0713] Table 2

[0714] Preparation of acceptor with 5’ overhang immobilised on support Overview Prior to performing the first synthesis cycle double stranded acceptor was first immobilised on a support followed by enzymatic cleavage to generate 5’ overhangs.

[0715] Immobilisation

[0716] The double-stranded acceptor DNA was immobilised and quantified on the prepared surfaces, using the following method:

[0717] 1. Stock solutions of Acceptor dsDNA were diluted to 50 pM in a 10 mM phosphate buffer (pH 7.0) containing 5 mM TCEP and left at room temperature for 2 hours.

[0718] 2. The surfaces of the wells were primed with 50 pl immobilisation buffer (10 mM phosphate buffer, 5% glycerol, pH 7.0) for 10 mins.

[0719] 3. The priming buffer was removed from the wells.

[0720] 4. The surfaces were exposed to a 25 pl solution of immobilisation buffer containing dsDNA Acceptor at a total concentration of 5 pM and left to incubate for 1 hr (covered).

[0721] 5. The surfaces were washed three times with 200 pl of wash buffer (10 mM Tris- HC1, 1 mM EDTA, pH 8.0) followed by 3 times with 200 pl ultrapure water.

[0722] 6. The surfaces were then exposed to 200 pl of a solution containing 10 mM Tris- HC1, 1 mM EDTA, IM NaCl and 0.05% Tween20, and left to incubate (covered) for 1 hr.

[0723] 7. The solution was removed, and the wells washed a further 3 times with 200 pl of wash buffer.

[0724] 8. After the final wash cycle, the buffer was removed and replaced with 50 pl TE (10 mM Tris-HCl, 1 mM EDTA, pH 8.0).

[0725] 9. The fluorescence intensity of the immobilised acceptor DNA was then recorded with a BMG VANTAstar plate reader in Fluorescein channel before being stored in the refrigerator for future use.

[0726] From the fluorescence intensity reading, each well was calculated to contain approximately 200 fmol of immobilised acceptor dsDNA. Generation of 5 ’ acceptor overhangs

[0727] Following on from the immobilisation, the surface immobilised acceptors were next activated via a cleavage reaction to generate a single base overhang with 5’ phosphate group using the following method:

[0728] 1. 50 pl TE storage buffer was removed from the wells.

[0729] 2. Each well was then exposed to a 30 pl solution containing 3 pl of lOx NEB rCutSmart Buffer (500 mM Potassium Acetate, 200 mM Tris-acetate, 100 mM Magnesium Acetate, 1 ng / ml Recombinant Albumin and pH 7.9 at 25°C), 3 pl of Neil (20,000 U / ml, NEB) and 24 pl ultrapure water.

[0730] 3. After being added to each well the solution was mixed up and down three times with a pipette

[0731] 4. The well plate was then placed on top of an Eppendorf ThermoMixer PCR plate set to 37°C and shaken at 300 rpm for 1 hour (covered).

[0732] 5. The reaction solution was removed from each well before being washed 3 times with 200 pl wash buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0).

[0733] 6. The surfaces were exposed to 200 pl of a solution containing 10 mM Tris-HCl, 1 mM EDTA, IM NaCl and 0.05% Tween20, and left to incubate (covered) for 10 minutes.

[0734] 7. The solution was then removed, and the wells were washed a further 3 times with 200 pl of wash buffer.

[0735] 8. The wash buffer was removed and replaced with 50 pl TE (10 mM Tris-HCl, 1 mM EDTA, pH 8.0).

[0736] 9. The fluorescence intensity of the immobilised acceptor DNA was then recorded with a BMG VANTAstar plate reader in the Fluorescein channel to verify the generation of 5’ phosphate groups and single base 5’ overhang (via a decrease in fluorescence). Cycle Step 1: Ligation

[0737] Overview

[0738] The first step describes the ligation of polynucleotides with DNA ligase. A diagrammatic illustration is shown in Figure 1. As shown in Figure 1, the acceptor polynucleotide has a 5’ overhang with a free phosphate group available for ligation whereas the 5’ overhang of the donor polynucleotide lacks a phosphate group meaning the second (helper) strands of the acceptor and donor polynucleotides are not ligated in the single stranded ligation.

[0739] Materials

[0740] The main reagents employed were

[0741] I. Greiner Sensoplate™ glass bottom 96 well plate surface previously immobilised with dsDNA Acceptor (see Table 2 for sequences).

[0742] II. Human Ligase III (14 pM) obtained from NEB.

[0743] III. Thermal Liable Proteinase K (120 U / ml, NEB).

[0744] Methods

[0745] Ligation reaction on oligonucleotides was carried out using the procedure below:

[0746] 1. The 50 pl TE storage buffer was then removed from the wells.

[0747] 2. 30 pl of a ligation reaction solution containing a Ligation Buffer (50.5 mM Tris- HC1 pH 7.5, 10 mM MgCh, 10 mM NaCl, 10 pM ATP, 2.5% PEG8000), 2 pM annealed donor dsDNA (2 pM Ligation Strand 1:2 pM Helper Strand I) and 3 pl Human Ligase III suspended in ultrapure water was pipetted into each well. 3. The solution was mixed up and down three times, before being placed on an Eppendorf ThermoMixer PCR plate set to 25°C and shaken at 300 rpm for 15 minutes (covered). . The reaction solution was removed from each well before being washed 2 times with 200 pl wash buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0).

[0748] 5. To remove any excess Human Ligase III, the well surfaces were exposed to 30 pl of a solution containing 3 pl Thermal Liable Proteinase K (120 U / ml, NEB) and 27 pl TE (10 mM Tris-HCl, 1 mM EDTA, pH 8.0).

[0749] 6. The well plate was then placed on top of an Eppendorf ThermoMixer PCR plate set to 37°C and shaken at 300 rpm for 20 minutes (covered).

[0750] 7. The reaction solution was removed from each well before being washed with 200 pl wash buffer.

[0751] 8. Another 200 ul was added to each well and placed on an Eppendorf ThermoMixer PCR plate set to 55°C and shaken at 300 rpm for 10 minutes.

[0752] 9. The solution was then removed, and the wells were washed a further 2 times with 200 pl of wash buffer.

[0753] 10. The surfaces were exposed to 200 pl of a solution containing 10 mM Tris-HCl, 1 mM EDTA, IM NaCl and 0.05% Tween20, and left to incubate (covered) for 10 minutes.

[0754] 11. The solution was then removed, and the wells were washed a further 3 times with 200 pl of wash buffer.

[0755] 12. The wash buffer was removed and replaced with 50 pl TE

[0756] 13. The fluorescence intensity of the wells was then recorded with a BMG VANTAstar plate reader in the Alexa Fluor™ 488 channel to verify the ligation reaction and formation of the acceptor-donor conjugate (via an increase in fluorescence).

[0757] 14. Following the fluorescence reading, a set of wells were reserved for further quantification and visualisation via gel electrophoresis. Cycle Step 2: Incorporation

[0758] Overview

[0759] The second step involves the nick extension on the donor helper strand using the donor ligation strand as template for enzymatic dNTP incorporation by a DNA polymerase. A diagrammatic illustration is shown in Figure 1.

[0760] Materials

[0761] The main reagents employed were:

[0762] I. Greiner Sensoplate™ glass bottom 96 well plate surface immobilised with dsDNA acceptor-donor oligomers (from Step 1).

[0763] II. Deoxynucleotide (dNTP) Solution Mix obtained from NEB (premixed solution containing 10 mM of each dATP, dCTP, dGTP and dTTP)

[0764] III. DNA Polymerase I, Large (Klenow) Fragment (5,000 U / ml) and accompanying NEB 2 Reaction buffer obtained from NEB.

[0765] Methods

[0766] The following method was used for the incorporation:

[0767] 1. 50 pl TE storage buffer was removed from the wells.

[0768] 2. Each well was then exposed to a 30 pl solution containing 3 pl lOx NEB 2 Reaction buffer, 0.6 pl of DNA Polymerase I, Large (Klenow) Fragment (5,000 U / ml), 0.75 pl NEB dNTP Solution Mix (10 mM), and 25.65 pl ultrapure water.

[0769] 3. After being added to each well the solution was mixed up and down three times with a pipette.

[0770] 4. The well plate was then placed on top of an Eppendorf ThermoMixer PCR plate set to 37°C and shaken at 300 rpm for 15 minutes (covered).

[0771] 5. The reaction solution was removed from each well before being washed 3 times with 200 pl wash buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0).

[0772] 6. The surfaces were exposed to 200 pl of a solution containing 10 mM Tris-HCl, 1 mM EDTA, IM NaCl and 0.05% Tween20, and left to incubate (covered) for 10 minutes. 7. The solution was then removed, and the wells were washed a further 6 times with 200 JJ.1 of wash buffer.

[0773] 8. The wash buffer was removed and replaced with 50 pl TE.

[0774] Cycle Step 3: Cleavage

[0775] Overview

[0776] The third cycle step describes a double strand cleavage of the phosphodiester backbone above the payload, yielding a 5’ single base overhang.

[0777] Materials

[0778] The following main reagents were employed:

[0779] I. Greiner Sensoplate™ glass bottom 96 well plate surface immobilised with dsDNA acceptor-donor oligomers (from Step 2).

[0780] II. NEB Bed Endonuclease (10,000 U / ml).

[0781] III. 1 Ox NEB rCutSmart Buffer.

[0782] IV. 1.0 M DTT solution in ultrapure water received from NEB.

[0783] Methods

[0784] The cleavage on the surface ligated acceptor-donor oligonucleotides was carried out using the procedure below:

[0785] 1. 50 pl TE storage buffer was removed from the wells.

[0786] 2. Each well was then exposed to a 30 pl solution containing 3 pl of lOx NEB rCutSmart Buffer (500 mM Potassium Acetate, 200 mM Tris-acetate, 100 mM Magnesium Acetate, 1 ng / ml Recombinant Albumin and pH 7.9 at 25°C), 0.75 pl of Bed (10,000 U / ml, NEB), 0.06 pl DDT (1.0 M) and 26.2 pl ultrapure water. 3. After being added to each well the solution was mix up and down three times with a pipette

[0787] 4. The well plate was then placed on top of an Eppendorf ThermoMixer PCR plate set to 37°C and shaken at 300 rpm for 30 minutes (covered).

[0788] 5. The reaction solution was removed from each well before being washed 6 times with 200 pl wash buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0).

[0789] 6. The surfaces were exposed to 200 pl of a solution containing 10 mM Tris-HCl, 1 mM EDTA, IM NaCl and 0.05% Tween20, and left to incubate (covered) for 10 minutes.

[0790] 7. The solution was then removed, and the wells were washed a further 6 times with 200 pl of wash buffer.

[0791] 8. The wash buffer was removed and replaced with 50 pl TE (10 mM Tris-HCl, 1 mM EDTA, pH 8.0).

[0792] 9. The fluorescence intensity of the immobilised acceptor DNA was recorded with a BMG VANTAstar plate reader in the Alexa Fluor™ 488 to verify the cleavage of the acceptor-donor oligomers (via a decrease in fluorescence).

[0793] 10. Following the fluorescence reading, a set of wells were reserved for further quantification and visualisation via gel electrophoresis.

[0794] 11. To verify the completion of the cycle, the ligation step (Step 1) was repeated using a different 2 pM annealed donor dsDNA, which was formulated from Ligation Strand II and Helper Strand II.

[0795] 12. Similarly, a fluorescence intensity reading of the ligation products was recorded using a BMG VANTAstar plate reader in the Alexa Fluor™ 488 channel and a set of wells were reserved for further quantification and visualisation via gel electrophoresis.

[0796] Fluorescence analysis during synthesis method

[0797] Both the precursor acceptor immobilised on the support and the donor polynucleotide included a fluorescent label. The fluorescent label therefore allowed the uncleaved precursor immobilised on the support and the ligation products resulting from acceptor: donor ligations to be detected and quantified. The results obtained are shown in Figure 17A.

[0798] Ligation of dsDNA acceptor with Human Ligase III at room temperature (25°C) in the presence of double-stranded donors resulted in an increase in fluorescence intensity relative to the immobilised acceptor fluorescence following the incubation with Neil (Figure 17A). Figure 17A shows the results of two cycles, the acceptor is first immobilised (lane 1); then cleaved with Neil to generate a 5’ overhang which also results in removal of the label (lane 2); then the labelled donor is ligated (lane 3) and nucleotides incorporated, followed by cleavage with BccI to generate a new acceptor with the payload incorporated and a 5’ overhang (lane 4); and the ligation for the next cycle is performed generation the next ligation product (lane 5).

[0799] Gel Electrophoresis and DNA Visualization:

[0800] Overview

[0801] Gel electrophoresis and DNA visualisation was used to assess the synthesis method by cleaving the acceptor from the support at various points in the synthesis method.

[0802] Methods

[0803] The following method was used for cleavage from the support followed by gel electrophoresis and DNA visualisation:

[0804] 1. 50 pl TE storage buffer was removed from the reserved wells for each step described,

[0805] 2. 20 pl of a solution containing 2 pl of lOx NEB rCutSmart Buffer, 2 pl of Rsal (10,000 U / ml) and 16 pl ultrapure water was added to the reserved wells from each reaction step.

[0806] 3. The well plate was then placed on top of an Eppendorf ThermoMixer PCR plate set to 37°C and shaken at 300 rpm for 45 minutes (covered). 4. The supernatants were removed from each well and transferred to sterile 0.2 ml PCR tubes.

[0807] 5. 3 pl of 6X Purple Gel Loading Dye NEB (15% Ficoll®-400, 60 mM EDTA, 19.8 mM Tris-HCl, 0.48% SDS, 0.12% Dye 1, 0.006% Dye 2 and pH 8 at 25°C) was then added into each tube.

[0808] 6. 20 pl of each sample was then loaded into the wells of a polyacrylamide gel (20%) TBE buffer 1.0mm x 10 well (Invitrogen).

[0809] 7. X-cell sure lock module (Novex) was fastened in place and electrophoresis performed at constant 200 V for 75 minutes at room temperature.

[0810] 8. The gel was incubated in SYBR Gold Nucleic Acid Gel Stain for 10 minutes, and then in water for 5 min with gentle agitation.

[0811] 9. The gel was visualized by ChemiDoc MP (BioRad) using SYBR Gold settings.

[0812] 10. Visualization and analysis were carried out on the Image lab 2.0 platform.

[0813] Results and Conclusions

[0814] Figure 17B shows the results obtained. Lane 1 shows the result for the first ligation involving the activated acceptor and donor with 5’ overhangs ligated to form a ligation product. Lane 2 shows the result for the subsequent cleavage of the acceptor: donor product. Lane 3 shows the result for the next cycle and the ligation production for the second ligation between the regenerated acceptor (now including payload) and the donor.

Claims

1. CLAIMS:

1. An in vitro method of synthesising a double-stranded polynucleotide having a predefined sequence, the method comprising performing cycles of synthesis, wherein each cycle comprises:(A) providing an acceptor polynucleotide having first and second strands and comprising a single or multiple nucleotide overhang at one terminal end;(B) providing a donor polynucleotide having first and second strands, and at one terminal end comprises a cleavage site and a polynucleotide payload comprising one or more nucleotide pairs of the predefined sequence and a single or multiple nucleotide overhang;(C) performing a single-stranded ligation reaction to form a ligated polynucleotide, the reaction comprising ligating only the first strands of the acceptor and donor polynucleotides at said terminal ends; and(D) cleaving the ligated polynucleotide and generating a cleaved terminal end having a single or multiple nucleotide overhang, thereby extending the acceptor polynucleotide with the polynucleotide payload at the cleaved end, and generating a new double-stranded acceptor polynucleotide comprising a single or multiple nucleotide overhang for ligation and extension in the next cycle.

2. A method according to claim 1, wherein in each cycle:(1) step (A) comprises providing a donor polynucleotide having first and second strands and first and second terminal ends, wherein the first terminal end comprises the cleavage site and the polynucleotide payload comprising one or more nucleotide pairs of the predefined sequence, wherein the payload comprises a 5’ or a 3’ single or multiple nucleotide overhang;(2) step (B) comprises providing an acceptor polynucleotide having first and second strands and having a 5’ or 3’ single or multiple nucleotide overhang at a first terminal end, wherein the number of nucleotides in the overhang of the acceptor polynucleotide is the same as the number of nucleotides in the overhang of the donor polynucleotide, and wherein the nucleotides of the single or multiple nucleotide overhang of the acceptor polynucleotide are payload partner nucleotides for the corresponding payload nucleotides of the single or multiple nucleotide overhang of the donor polynucleotide, and wherein:(i) if the acceptor polynucleotide has a 5’ overhang, the overhang of the payload is a 5’ overhang; or(ii) if the acceptor polynucleotide has a 3’ overhang, the overhang of the payload is a 3’ overhang;(3) step (C) comprises performing a single-stranded ligation reaction, wherein following ligation the one or more nucleotide pairs of the payload and the nucleotides of the single or multiple nucleotide overhangs of the acceptor and donor polynucleotides form nucleotide pairs of the predefined sequence; and(4) step (D) comprises cleaving the polynucleotide produced in step (C) at the cleavage site by the action of an enzyme having cleavage activity to generate a cleaved terminal end with a 5’ or 3’ single or multiple nucleotide overhang, thereby extending the acceptor polynucleotide with the polynucleotide payload at the cleaved end, and generating a new double-stranded acceptor polynucleotide having a 5’ or 3’ single or multiple nucleotide overhang for ligation and extension in the next cycle.

3. A method according to claim 1 or claim 2, wherein the cleavage step of step (D) is the only cleavage step performed in each cycle of synthesis.

4. A method according to any one of the preceding claims, wherein in each cycle there is no step of incorporation of a polynucleotide having a reversible terminator group and no additional step of deprotection to remove the reversible terminator group.

5. A method according to any one of the preceding claims, wherein the 5’ or 3’ overhangs of the acceptor and donor polynucleotides in each cycle are single nucleotide overhangs.

6. A method according to any one of claims 2 to 5 wherein in step (C) the terminal 5’ nucleotide of the payload is non-ligatable and step (C) comprises ligating the terminal 3’ nucleotide of the payload of the donor polynucleotide to the terminal 5’ nucleotide of the first terminal end of the acceptor polynucleotide, and whereupon a single-stranded nick is formed between the non-ligatable terminal 5’ nucleotide of the payload and the terminal 3 ’ nucleotide of the first terminal end of the acceptor polynucleotide.

7. A method according to claim 6, wherein in step (A) the terminal 5’ nucleotide of the payload of the donor polynucleotide is provided without a phosphate group and is thereby non-ligatable.

8. A method according to claim 6 or claim 7, wherein after step (C) and before step (D) the method further comprises either:(i) performing a nucleotide incorporation reaction, by the action of an enzyme having polymerase activity, initiated at the free 3’ hydroxyl group of the unligated second strand of the acceptor polynucleotide, thereby extending said second strand and using the ligated first strand as a template; or(ii) converting the non-ligatable terminal 5’ nucleotide of the payload of the donor polynucleotide into a ligatable nucleotide followed by ligating the second strands of the acceptor and donor polynucleotides.

9. A method according to claim 8(i), wherein the incorporation reaction is performed:(i) by the action of an enzyme having polymerase activity, and wherein the polymerase displaces the original second strand of the donor polynucleotide when synthesising a new second strand; or(ii) by the action of an enzyme having polymerase activity which possesses 5’ to 3’ exonuclease activity, and wherein the polymerase digests the original second strand of the donor polynucleotide when synthesising a new second strand.

10. A method according to claim 9, wherein in each cycle the cleavage site of the donor polynucleotide provided in step (A) comprises a non-cleavable cleavage site, wherein at least one nucleotide in the cleavage site in the second strand of the donor polynucleotide does not match the consensus sequence comprising the cleavage recognition site, but the corresponding nucleotides in the cleavage site in the first strand match the consensus sequence comprising the cleavage recognition site, and wherein following the incorporation reaction and copying of the first strand the mismatch is corrected thereby generating a cleavable cleavage site.

11. A method according to any one of claims 8(i), 9 and 10, wherein:(i) a single reaction fluid is used to perform step (C) and the incorporation reaction, and the reaction fluid comprises the enzyme having ligase activity and the enzyme having polymerase activity, wherein the enzyme having polymerase activity is a heat-activatable polymerase and is inactive at the temperature used to perform step (C), and wherein after step (C) and before step (D)(i) the method comprises raising the temperature of the reaction fluid to activate the polymerase; or(ii) a single reaction fluid is used to perform step (C), the incorporation reaction and step (D), and the reaction fluid comprises the enzyme having ligase activity and the enzyme having polymerase activity and the enzyme having cleavage activity, wherein the enzyme having polymerase activity is a heat-activatable polymerase and is inactive at the temperature used to perform step (C), and wherein after step (C) and before step (D)(i) the method comprises raising the temperature of the reaction fluid to activate the polymerase.

12. A method according to any one of claims 8(i), 9, 10 and 11, wherein following step (D) the method further comprises separating the cleaved donor polynucleotide from the acceptor polynucleotide and performing an incorporation reaction comprising extending the second strand of the acceptor polynucleotide at the previous nick site with new payload nucleotide(s) using the payload nucleotides of the first strand as templates, preferably by the action of an enzyme having polymerase activity, thereby re-forming the payload nucleotide pairs in the cleaved polynucleotide and thereby forming a ligatable blunt end at the first end of the acceptor polynucleotide, whereupon all pairs of nucleotides of the polynucleotide payload are incorporated at the cleaved first terminal end of the acceptor polynucleotide, and wherein the terminal nucleotides of the cleaved first terminal end are the final pair of nucleotides of the polynucleotide payload.

13. A method according to claim 8(ii), wherein the step of converting the terminal 5’ non-ligatable nucleotide of the payload of the donor polynucleotide into a ligatable nucleotide comprises a performing a phosphorylation reaction comprising phosphorylating the terminal 5’ nucleotide.

14. A method according to claim 13, wherein the phosphorylation reaction is performed by the action of an enzyme having kinase activity, such as by polynucleotide kinase (PNK).

15. A method according to any one of claims 2 to 5, wherein in step (C) the terminal 3’ nucleotide of the payload is non-ligatable and step (C) comprises ligating the terminal 5’ nucleotide of the payload of the donor polynucleotide to the terminal 3’ nucleotide of the first terminal end of the acceptor polynucleotide, and whereupon a single-stranded nick is formed between the non-ligatable terminal 3’ nucleotide of thepayload and the terminal 5’ nucleotide of the first terminal end of the acceptor polynucleotide.

16. A method according to claim 15, wherein in step (A) the terminal 3’ nucleotide of the payload of the donor polynucleotide is a non-ligatable 2’,3’-dideoxynucleotide or a 2’-deoxynucleotide, or any other suitable non-ligatable nucleotide.

17. A method according to claim 15 or claim 16, wherein after step (C) and before step (D) the method further comprises converting the non-ligatable terminal 3’ nucleotide of the payload of the donor polynucleotide into a ligatable nucleotide followed by ligating the second strands of the acceptor and donor polynucleotides.

18. A method according to claim 17, wherein the step of converting the non- ligatable terminal 3’ nucleotide of the payload of the donor polynucleotide into a ligatable nucleotide comprises reconstituting the 3’ hydroxy group of the ligatable terminal 3’ nucleotide.

19. A method according to claim 15, wherein the terminal 3’ nucleotide of the payload comprises a reversible blocking group which prevents ligation, and wherein after step (C) and before step (D) the method further comprises removing the blocking group.

20. A method according to any one of claims 15 to 19, wherein a single reaction fluid is used to perform step (C) and the incorporation reaction, and the reaction fluid comprises the enzyme having ligase activity and the enzyme having polymerase activity, wherein the enzyme having polymerase activity is a heat-activatable polymerase and is inactive at the temperature used to perform step (C), and wherein after step (C) and before step (D)(i) the method comprises raising the temperature of the reaction fluid to activate the polymerase.

21. A method according to any one of the preceding claims, wherein before step (D) the method further comprises:(i) performing a treatment step with an enzyme having phosphatase activity, such as calf intestinal phosphatase (CIP); or(ii) performing a nuclease treatment step with an enzyme having 5’ to 3’ exonuclease activity, such as Exonuclease III, T5 exonuclease or T7 exonuclease, thereby depleting or rendering non-ligatable any acceptor polynucleotides that have failed to ligate to a donor polynucleotide.

22. A method according to any one of the preceding claims, wherein the donor polynucleotide is asymmetrical, whereby only a first terminal end of the donor polynucleotide comprises a cleavage site and a polynucleotide payload comprising a 5’ or 3’ single or multiple nucleotide overhang.

23. A method according to claim 22, wherein the second terminal end of the donor polynucleotide is non-ligatable and wherein:(i) the terminal nucleotide of the first and / or second strands of the second terminal end of the donor polynucleotide comprises a blocking group; or(ii) both polynucleotide strands of the second terminal end of the donor polynucleotide are connected together, preferably by a polynucleotide hairpin loop; or(iii) the donor polynucleotide is blunt ended at the second terminal end and the 5’ terminal nucleotide at the second terminal end is dephosphorylated.

24. A method according to any one of claims 1 to 21, wherein the second terminal end of the donor polynucleotide comprises a second polynucleotide payload, wherein:(i) the payload nucleotide sequence of the first strand at the second terminal end of the donor polynucleotide in the 5’ to 3’ direction is the same asthe payload nucleotide sequence of the second strand at the first terminal end of the donor polynucleotide in the 5’ to 3’ direction;(ii) the payload nucleotide sequence of the second strand at the second terminal end of the donor polynucleotide in the 3’ to 5’ direction is the same as the payload nucleotide sequence of the first strand at the first terminal end of the donor polynucleotide in the 3’ to 5’ direction.

25. A method according to any one of the preceding claims, wherein the polynucleotide payload consists of two or more, or three or more consecutive pairs of nucleotides of the predefined sequence, as well as the nucleotide(s) of the overhang.

26. A method according to claim 25, wherein the polynucleotide payload consists of four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more consecutive pairs of nucleotides of the predefined sequence, as well as the nucleotide(s) of the overhang.

27. A method according to any one of claims 1 to 26, wherein both strands of the acceptor and donor polynucleotide comprises DNA.

28. A method according to any one of claims 1 to 26, wherein the first strands of the acceptor and donor polynucleotide comprise RNA and the first strands of the acceptor and donor polynucleotide comprise DNA.

29. A method according to claim 28, wherein following completion of cycles of synthesis the method further comprises separating the first strand of the acceptor polynucleotide comprising the nucleotides of the predefined sequence to form a singlestranded RNA polynucleotide molecule having the predefined sequence.

30. A method according to any one of the preceding claims, wherein cleavage comprises cleaving the sugar-phosphate backbone of the first strand of the donor polynucleotide.

31. A method according to any one of the preceding claims, wherein cleaving is performed by the action of an enzyme having overhang cleavage function, preferably a type IIS restriction enzyme, optionally BspQI.

32. A method according to any one of the preceding claims, wherein the cleavage site is defined by a universal nucleotide positioned in the first strand of the donor polynucleotide, wherein following cleavage the terminal nucleotide in the first strand of the cleaved first terminal end of the acceptor polynucleotide is the final nucleotide of the polynucleotide payload, optionally wherein the universal nucleotide is inosine.

33. A method according to claim 32, wherein the final nucleotide of the polynucleotide payload in the first strand occupies nucleotide position n, the penultimate nucleotide of the polynucleotide payload in the first strand occupies nucleotide position n-1 and the universal nucleotide in the first strand occupies nucleotide position n+1; the method further comprising cleaving the first strand between nucleotide positions n and n+1.

34. A method according to claim 32, wherein the final nucleotide of the polynucleotide payload in the first strand occupies nucleotide position n, the penultimate nucleotide of the polynucleotide payload in the first strand occupies nucleotide position n-1 and the universal nucleotide in the first strand occupies nucleotide position n+2; the method further comprising cleaving the first strand between nucleotide positions n and n+1.

35. A method according to claim 32, wherein the final nucleotide of the polynucleotide payload in the first strand occupies nucleotide position n, the penultimate nucleotide of the polynucleotide payload in the first strand occupiesnucleotide position n-1 and the universal nucleotide in the first strand occupies nucleotide position n+2+x, wherein x is a whole number from 1 to 10 or more; the method further comprising cleaving the first strand between nucleotide positions n and n+1.

36. A method according to any one of claims 32 to 35, wherein each cleavage step comprises a two-step cleavage process wherein each cleavage step comprises a first step comprising removing the universal nucleotide to form an abasic site, and a second step comprising cleaving the first strand of the donor polynucleotide at the abasic site.

37. A method according to claim 36, wherein the first step is performed with a nucleotide-excising enzyme.

38. A method according to claim 37, wherein the nucleotide-excising enzyme is a 3- methyladenine DNA glycosylase enzyme.

39. A method according to claim 38, wherein the nucleotide-excising enzyme is: i. human alkyladenine DNA glycosylase (hAAG); or ii. uracil DNA glycosylase (UDG).

40. A method according to any one of claims 36 to 39, wherein the second step is performed with a chemical which is a base.

41. A method according to claim 40, wherein the base is NaOH.

42. A method according to any one of claims 36 to 39, wherein the second step is performed with an organic chemical having abasic site cleavage activity.

43. A method according to claim 42, wherein the organic chemical is N,N’- dimethylethylenediamine.

44. A method according to any one of claims 36 to 39, wherein the second step is performed with an enzyme having abasic site lyase activity, optionally wherein the enzyme having abasic site lyase activity is.(i) AP Endonuclease 1 ;(ii) Endonuclease III (Nth); or(iii) Endonuclease VIII.

45. A method according to any one of claims 32 to 35, wherein each cleavage step comprises a one step cleavage process comprising removing the universal nucleotide with a cleavage enzyme wherein the enzyme is(i) Endonuclease III;(ii) Endonuclease VIII;(iii) formamidopirimidine DNA glycosylase (Fpg); or(iv) 8-oxoguanine DNA glycosylase (hOGGl).

46. A method according to any one of claims 32 to 35, wherein the cleavage step comprises cleaving the first strand of the donor polynucleotide with an enzyme.

47. A method according to claim 46, wherein the enzyme cleaves the first strand of the donor polynucleotide between nucleotide positions n+1 and n.

48. A method according to claim 46 or claim 47, wherein the enzyme is Endonuclease V.

49. A method according to claim 30, wherein the cleavage site is defined by a uracil nucleotide positioned in the first strand of the donor polynucleotide, wherein cleavage is performed by an enzyme having uracil DNA glycosylase activity and DNA glycosylase- lyase activity e.g. Endonuclease VIII activity, and wherein following cleavage the terminal nucleotide of first strand at the cleaved first terminal end of the acceptor polynucleotide is the final nucleotide of the polynucleotide payload.

50. A method according to any one of the preceding claims, wherein ligation is performed by the action of an enzyme having nucleotide ligase activity.

51. A method according to claim 50, wherein the enzyme is human DNA ligase III, T3 DNA ligase, T4 DNA ligase, optionally T4 DNA ligase which has improved thermal stability compared to wild-type T4 DNA ligase, preferably wherein the enzyme is a T3 DNA ligase or a T4 DNA ligase which has improved salt tolerance compared to wildtype T4 DNA ligase.

52. A method according to any one of the preceding claims, wherein(i) the first and second strands of the acceptor polynucleotide at the second terminal end are each tethered to a surface; or(ii) the first and second strands of the acceptor polynucleotide at the second terminal end are connected together by a polynucleotide hairpin loop and are tethered to a surface; or(iii) the first strand of the acceptor polynucleotide at the second terminal end is tethered to a surface and the second strand of the acceptor polynucleotide at the second terminal end is untethered; or(iv) the second strand of the acceptor polynucleotide at the second terminal end is tethered to a surface and the first strand of the acceptor polynucleotide at the second terminal end is untethered.

53. A method according to claim 52(i), claim 52(iii) or claim 52(iv), wherein the tethered strand(s) at the second terminal end comprises a cleavable linker(s), wherein the linker(s) may be cleaved to detach the double-stranded polynucleotide from the surface following synthesis.

54. A method according to claim 52(ii), wherein the hairpin loop at the second terminal end is tethered to a surface via a cleavable linker, wherein the linker may becleaved to detach the double-stranded polynucleotide from the surface following synthesis.

55. A method according to claim 53 or claim 54, wherein the cleavable linker is a UV cleavable linker.

56. A method according to any one of claims 52 to 55, wherein the surface is a particle, optionally a microparticle.

57. A method according to any one of claims 52 to 55, wherein the surface is a planar surface.

58. A method according to claim 57, wherein the surface comprises a gel.

59. A method according to claim 58, wherein the surface comprises a polyacrylamide surface, such as about 2% polyacrylamide, preferably wherein the polyacrylamide surface is coupled to a solid support such as glass.

60. A method according to any one of claims 52 to 59, wherein the first and second strands of the acceptor polynucleotide at the second terminal end are tethered to the surface via one or more covalent bonds.

61. A method according to claim 60, wherein the one or more covalent bonds is formed between a functional group on the surface and a functional group on the acceptor polynucleotide, wherein the functional group on the acceptor polynucleotide is an amine group, a thiol group, a thiophosphate group or a thioamide group.

62. A method according to claim 61, wherein the functional group on the surface is a bromoacetyl group, optionally wherein the bromoacetyl group is provided on a polyacrylamide surface derived using N- (5-bromoacetamidylpentyl) acrylamide (BRAPA).

63. A method according to any one of the preceding claims, wherein synthesis cycles are performed in droplets within a microfluidic system.

64. A method according to claim 63, wherein the microfluidic system is an electrowetting system.

65. A method according to claim 64, wherein the microfluidic system is an electrowetting-on-dielectric system (EWOD).

66. A method according to any one of the preceding claims, wherein following synthesis the strands of the double-stranded polynucleotide having a predefined sequence are separated to provide a single-stranded polynucleotide having a predefined sequence.

67. A method according to any one of the preceding claims, wherein following synthesis the double-stranded polynucleotide having a predefined sequence, or a region thereof, is amplified, preferably by PCR.

68. A method of assembling a polynucleotide having a predefined sequence, the method comprising performing the method of any one of the preceding claims to synthesise a first polynucleotide having a predefined sequence and one or more additional polynucleotides having a predefined sequence and joining together the first and one or more additional polynucleotides.

69. A method according to claim 68 wherein the first polynucleotide and the one or more additional polynucleotides are double-stranded.

70. A method according to claim 69 wherein the first polynucleotide and the one or more additional polynucleotides are single-stranded.

71. A method according to any one of claims 68 to 70, wherein the first polynucleotide and the one or more additional polynucleotides are cleaved to create compatible termini and joined together, preferably by ligation.

72. A method according to claim 71, wherein the first polynucleotide and the one or more additional polynucleotides are cleaved by a restriction enzyme at a cleavage site.

73. A method according to any one of claims 64 to 72, wherein the synthesis and / or assembly steps are performed in droplets within a microfluidic system.

74. A method according to claim 73, wherein the assembly steps comprise providing a first droplet comprising a first synthesised polynucleotide having a predefined sequence and a second droplet comprising an additional one or more synthesised polynucleotides having a predefined sequence, wherein the droplets are brought in contact with each other and wherein the synthesised polynucleotides are joined together thereby assembling a polynucleotide comprising the first and additional one or more polynucleotides.

75. A method according to claim 74 wherein the synthesis steps are performed by providing a plurality of droplets each droplet comprising reaction reagents corresponding to a step of the synthesis cycle, and sequentially delivering the droplets to the acceptor polynucleotide in accordance with the steps of the synthesis cycles.

76. A method according to claim 75, wherein following delivery of a droplet and prior to the delivery of a next droplet, a washing step is carried out to remove excess reaction reagents.

77. A method according to claim 75 and 76, wherein the microfluidic system is an electrowetting system.

78. A method according to claim 77, wherein the microfluidic system is an electrowetting-on-dielectric system (EWOD).

79. A method according to any one of claims 75 to 78, wherein synthesis and assembly steps are performed within the same system.

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