Polynucleotide synthesis methods, supports and systems

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. The invention further relates to supports and systems for synthesising a double-stranded polynucleotide having a predefined sequence.

Description

[0001] POLYNUCLEOTIDE SYNTHESIS METHODS, SUPPORTS AND SYSTEMS

[0002] FIELD OF THE INVENTION

[0003] The invention relates to 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. The invention further relates to supports and systems for synthesising a double-stranded polynucleotide having a predefined sequence.

[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 and refined new methodologies by which single- and double-stranded 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 a support comprising:

[0017] (i) at least one acceptor polynucleotide having first and second strands, wherein one end is immobilised on the support and the opposite end is free, and wherein the free end is blunt ended; and (ii) at least one tether polynucleotide adjacent the acceptor polynucleotide, wherein one end is immobilised on the support and the opposite end is free, wherein the free end comprises a first hybridisation arm (hyb arm) which is a single-stranded sequence region;

[0018] (B) providing at least one donor polynucleotide having first and second strands and having free first and second terminal ends, wherein the first terminal end is blunt-ended and comprises a polynucleotide payload sequence comprising one or more nucleotide pairs of the predefined sequence and a cleavage site adjacent the payload sequence, and wherein the second terminal end comprises a second hyb arm which is a single-stranded region comprising sequence which is complementary with the sequence region of the first hyb arm;

[0019] (C) contacting the at least one donor polynucleotide with the at least one tether polynucleotide, whereupon the first and second hyb arms hybridize to form a double-stranded region, and ligating the first terminal end of the donor polynucleotide with the free end of the acceptor polynucleotide to form a ligated polynucleotide; and

[0020] (D) cleaving the ligated polynucleotide and generating a cleaved blunt end, thereby extending the acceptor polynucleotide with the polynucleotide payload at the cleaved end, and generating a new blunt-ended double-stranded acceptor polynucleotide for ligation and extension in the next cycle.

[0021] 2. A method according to embodiment 1, wherein

[0022] (i) the tether polynucleotide is single-stranded, wherein one end is immobilised on the support and the opposite end is free, wherein the free end comprises the first hybridisation arm (hyb arm) which is a singlestranded sequence region; or

[0023] (ii) the tether polynucleotide is double-stranded, wherein one end is immobilised on the support and the opposite end is free, wherein the free end comprises a first hybridisation arm (hyb arm) which is a singlestranded sequence region.

[0024] 3. A method according to embodiment 2, wherein: (i) the method is performed by the method of embodiment 2(i), and wherein step (C) comprises performing a single-stranded ligation wherein:

[0025] (a) the first strand of the donor polynucleotide is ligated to the acceptor polynucleotide, or

[0026] (b) the second strand of the donor polynucleotide is ligated to the acceptor polynucleotide; or

[0027] (ii) the method is performed by the method of embodiment 2(i) or 2(ii), and wherein step (C) comprises performing a single-stranded ligation wherein:

[0028] (a) only the first strand of the donor polynucleotide is ligated to the first strand of the acceptor polynucleotide, or

[0029] (b) wherein only the second strand of the donor polynucleotide is ligated to the second strand of the acceptor polynucleotide; or

[0030] (iii) the method is performed by the method of embodiment 2(ii), and wherein step (C) comprises performing a double-stranded ligation wherein the first strand of the donor polynucleotide is ligated to the first strand of the acceptor polynucleotide, and the second strand of the donor polynucleotide is ligated to the second strand of the acceptor polynucleotide.

[0031] 4. A method according to embodiment 2(ii), embodiment 3 (ii) or embodiment

[0032] 3 (iii), wherein at the free end of the tether polynucleotide the terminal nucleotide of the strand which does not comprise the first hyb arm is provided as a non-ligatable nucleotide, such that the donor polynucleotide cannot be ligated to the tether polynucleotide in step (D).

[0033] 5. A method according to any one of the preceding embodiments, wherein the terminal nucleotide of the strand comprising the first hyb arm of the tether polynucleotide forms part of the sequence region of the first hyb arm, and / or wherein the terminal nucleotide of the strand comprising the second hyb arm of the donor polynucleotide forms part of the sequence region of the second hyb arm.

[0034] 6. A method according to any one of the preceding embodiments, wherein the sequence region of the first hyb arm and / or the sequence region of the second hyb arm is 15 or more nucleotides in length, 20 or more nucleotides in length, 25 or more nucleotides in length, 30 or more nucleotides in length, 35 or more nucleotides in length, 40 or more nucleotides in length, 45 or more nucleotides in length or 50 or more nucleotides in length.

[0035] 7. A method according to any one of the preceding embodiments, wherein

[0036] (a) the number of nucleotides in the sequence regions of the first and second hyb arms is the same; or

[0037] (b) the second hyb arm comprises one sequence region, and the sequence region of the first hyb arm comprises two or more copies of the sequence region of the second hyb arm, preferably four, five or six copies.

[0038] 8. A method according to any one of the preceding embodiments, wherein the first hyb arm of the tether polynucleotide comprises two or more repeat sequences, and the second hyb arm of the donor polynucleotide comprises the same number of two or more repeat sequences as the tether polynucleotide, wherein the repeat sequences of the first hyb arm are complementary with the repeat sequences of the second hyb arm.

[0039] 9. A method according to embodiment 8, wherein the hyb arms have 3 to 7 repeat sequences, preferably 4 to 6 repeat sequences.

[0040] 10. A method according to any one of the preceding embodiments, wherein the support comprises a population of tether polynucleotides immobilised on the support and a population of acceptor polynucleotides immobilised on the support, and wherein the relative amount of acceptor polynucleotides to tether polynucleotides is from 5% to 25% acceptor polynucleotides to 75% to 95% tether polynucleotides, e.g. 5% acceptor polynucleotides to 95% tether polynucleotides, 10% acceptor polynucleotides to 90% tether polynucleotides, 15% acceptor polynucleotides to 85% tether polynucleotides, 20% acceptor polynucleotides to 80% tether polynucleotides, or 25% acceptor polynucleotides to 75% tether polynucleotides.

[0041] 11. A method according to any one of the preceding embodiments, wherein in step (A) the support has immobilised on it two or more populations of tether polynucleotides, wherein the sequences in the first hyb arms of each population is different, and step (B) comprises providing the same number of two or more populations of donor polynucleotides as provided for the tether polynucleotides in step (A), wherein the sequences in the second hyb arms of each population of donor polynucleotide is different but complementary with the sequences in the first hyb arms of each respective population of tether polynucleotides.

[0042] 12. A method according to any one of the preceding embodiments, wherein after the cleavage step (D) the method further comprises raising the temperature above the melting temperature of the region of complementarity between the sequences of the first and second hyb arms, thereby detaching the donor polynucleotide from the tether polynucleotide, if attached, by separating the first and second hyb arms, optionally wherein the method further comprises washing the support to remove any unligated donor polynucleotide.

[0043] 13. A method according to any one of embodiments 2(ii), 3(ii) and 4 to 12, wherein the terminal nucleotide of the first strand at the free end of the acceptor polynucleotide of step (A):

[0044] (i) comprises a 5’ phosphate group; or

[0045] (ii) lacks a 5’ phosphate group.

[0046] 14. A method according to embodiment 13 (ii), wherein the terminal nucleotide of the second strand of the donor polynucleotide at the first terminal end comprises a 5’ phosphate group and wherein: step (C) comprises: (i) joining the donor and acceptor polynucleotides at their first terminal ends by ligating the second strand of the donor polynucleotide at its first terminal end with the second strand of the acceptor polynucleotide at its first terminal end; wherein the first strands of the donor and acceptor polynucleotides at their first terminal ends are not joined and are separated by a nick; and (ii) joining the first strands of the donor and acceptor polynucleotides at their first terminal ends; and following step (D) the 5’ phosphate group joined to the terminal nucleotide of the first strand of the cleaved acceptor polynucleotide is removed, preferably by the action of an enzyme having phosphatase activity.

[0047] 15. A method according to embodiment 14, wherein step C(ii), comprises adding a phosphate group to the first strand of the acceptor polynucleotide at its first terminal end, preferably by the action of an enzyme having kinase activity, such as polynucleotide kinase (PNK); and joining the donor and acceptor polynucleotides at their first terminal ends by ligating the first strand of the donor polynucleotide with the first strand of the acceptor polynucleotide.

[0048] 16. A method according to embodiment 13 (ii), wherein the terminal nucleotide of the second strand of the donor polynucleotide at the first terminal end comprises a 5’ phosphate group and wherein: step (C) comprises joining the donor and acceptor polynucleotides at their first terminal ends by ligating the second strand of the donor polynucleotide at its first terminal end with the second strand of the acceptor polynucleotide at its first terminal end; wherein the first strands of the donor and acceptor polynucleotides at their first terminal ends are not joined and are separated by a nick; following step (C) and before step (D) the method further comprises performing an incorporation step to extend the first strand of the donor polynucleotide at its first terminal end at the nick site, the step comprising synthesising new nucleotides in the first strand of the acceptor polynucleotide using the nucleotides of the second strand as templates, preferably by the action of an enzyme having polymerase activity, thereby synthesising a new first strand of the acceptor polynucleotide and re-forming the nucleotide pairs between the first and second strands of the acceptor polynucleotide; and following step (D) the 5’ phosphate group joined to the terminal nucleotide of the first strand of the cleaved acceptor polynucleotide is removed, preferably by the action of an enzyme having phosphatase activity. 17. A method according to embodiment 16, wherein the incorporation step is performed:

[0049] (a) by the action of an enzyme having polymerase activity, and wherein the polymerase displaces the original first strand of the acceptor polynucleotide when synthesising the new first strand; or

[0050] (b) by the action of an enzyme having polymerase activity which possesses 5’ to 3’ exonuclease activity, and wherein the polymerase digests the original first strand of the acceptor polynucleotide when synthesising the new second strand.

[0051] 18. A method according to embodiment 13(i), wherein the terminal nucleotide of the second strand of the donor polynucleotide at the first terminal end lacks a 5’ phosphate group and wherein: step (C) comprises: (i) 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; wherein the second strands of the donor and acceptor polynucleotides at their first terminal ends are not joined and are separated by a nick; and (ii) joining the second strands of the donor and acceptor polynucleotides at their first terminal ends.

[0052] 19. A method according to embodiment 18, wherein step C(ii), comprises adding a phosphate group to the second strand of the donor polynucleotide at its first terminal end, preferably by the action of an enzyme having kinase activity, such as polynucleotide kinase (PNK); and joining the donor and acceptor polynucleotides at their first terminal ends by ligating the second strand of the donor polynucleotide with the second strand of the acceptor polynucleotide.

[0053] 20. A method according to embodiment 13 (i), wherein the terminal nucleotide of the second strand of the donor polynucleotide at the first terminal end lacks a 5’ phosphate group and wherein: step (C) 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, and wherein the second strands of the donor and acceptor polynucleotides at their first terminal ends are not joined and are separated by a nick; and wherein step (D) comprises:

[0054] I. cleaving both the first and second strands of the donor polynucleotide to generate a blunt end at the cleaved first terminal end of the acceptor polynucleotide; or

[0055] II. cleaving the ligated polynucleotide at a site in the first strand of the donor polynucleotide, thereby retaining the nucleotides of the first strand of the polynucleotide payload at the cleaved first terminal end of the acceptor polynucleotide and thereby generating a 5’ overhang at the cleaved first terminal end of the acceptor polynucleotide with the nucleotides of the first strand of the polynucleotide payload overhanging the second strand of the acceptor polynucleotide, wherein the terminal nucleotide of the overhang is the final nucleotide of the payload; and following step (D) the method further comprises separating the cleaved donor polynucleotide from the acceptor polynucleotide and performing an incorporation step comprising extending the second strand of the acceptor polynucleotide at the nick site with new payload nucleotides 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.

[0056] 21. A method according to embodiment 20, wherein the cleaved donor polynucleotide is separated from the acceptor polynucleotide:

[0057] (i) before the incorporation step; or

[0058] (ii) during the incorporation step.

[0059] 22. A method according to embodiment 13(i), wherein the terminal nucleotide of the second strand of the donor polynucleotide at the first terminal end lacks a 5’ phosphate group and wherein: step (C) 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; wherein the second strands of the donor and acceptor polynucleotides at their first terminal ends are not joined and are separated by a nick; following step (C) and before step (D) the method further comprises performing a first incorporation step to extend the second strand of the acceptor polynucleotide from the nick site, the step comprising synthesising new nucleotides in the second strand using the nucleotides of the first strand as templates, preferably by the action of an enzyme having polymerase activity, thereby synthesising a new second strand of the donor polynucleotide and reforming and retaining the nucleotide pairs in the ligated polynucleotide including the one or more payload nucleotide pairs and the cleavage site.

[0060] 23. A method according to embodiment 22 wherein the original second strand of the donor polynucleotide is separated from the first strand:

[0061] (i) before the incorporation step; or

[0062] (ii) during the incorporation step. 24. A method according to embodiment 22 or embodiment 23, 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 step and copying of the first strand the mismatch is corrected thereby generating a cleavable cleavage site.

[0063] 25. A method according to embodiment 24, wherein:

[0064] (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

[0065] (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.

[0066] 26. A method according to: (I) embodiment 21 (ii); or (II) embodiment 23 (ii), wherein incorporation steps are performed:

[0067] (a) by the action of an enzyme having polymerase activity, and wherein the polymerase displaces the second strand when synthesising the new second strand; or (b) by the action of an enzyme having polymerase activity which possesses 5’ to 3’ exonuclease activity, and wherein the polymerase digests the second strand when synthesising the new second strand.

[0068] 27. A method according to any one of embodiments 2(ii), 3 (iii) and 4 to 12, wherein in step (A) the terminal nucleotide of the first strand at the free end of the acceptor polynucleotide of comprises a 5’ phosphate group and the terminal nucleotide of the second strand at the first terminal end of the donor polynucleotide comprises a 5’ phosphate group.

[0069] 28. A method according to any one of the preceding embodiments, wherein

[0070] (a) the nucleotides of the hyb arm of the tether polynucleotide and / or the hyb arm of the donor polynucleotide are continuous with the remaining nucleotides of the same strand of the polynucleotide; or

[0071] (b) the nucleotides of the hyb arm of the tether polynucleotide and / or the hyb arm of the donor polynucleotide are joined to the remainder of the nucleotides of the same strand of the polynucleotide by a linker / spacer, optionally a 18-atom hexa-ethyleneglycol spacer.

[0072] 29. A method according to any one of the preceding embodiments, wherein

[0073] (a) the hyb arms of the tether polynucleotide and the hyb arms of the donor polynucleotide are comprised in the first strands of the polynucleotide; or

[0074] (b) the hyb arms of the tether polynucleotide and the hyb arms of the donor polynucleotide are comprised in the second strands of the polynucleotide.

[0075] 30. A method according to any one of embodiments 1 to 19, and 22 to 25, wherein step (D) comprises: cleaving both strands of the ligated polynucleotide to form a blunt end at the cleaved 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 terminal nucleotides of the cleaved first terminal end are the final pair of nucleotides of the polynucleotide payload 31. A method according to any one of embodiments 1 to 12, 20 and 21, wherein step (D) comprises:

[0076] I. cleaving the ligated polynucleotide at a site in the first strand of the donor polynucleotide, thereby retaining the nucleotides of the first strand of the polynucleotide payload at the cleaved first terminal end of the acceptor polynucleotide and thereby generating a 5’ overhang at the cleaved first terminal end of the acceptor polynucleotide with the nucleotides of the first strand of the polynucleotide payload overhanging the second strand of the acceptor polynucleotide, wherein the terminal nucleotide of the overhang is the final nucleotide of the payload; and

[0077] II. performing an incorporation step comprising extending the second strand of the cleaved acceptor polynucleotide with new payload nucleotides using the payload nucleotides of the first strand as templates, preferably by the action of an enzyme having polymerase activity, thereby reforming 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 and retained 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.

[0078] 32. A method according to any one of the preceding embodiments, wherein cleaving a strand of the ligated polynucleotide in step (D) comprises cleaving the sugarphosphate backbone of the strand.

[0079] 33. A method according to embodiment 30 or embodiment 32, wherein the cleavage site in the donor polynucleotide is adjacent to the polynucleotide payload and comprises a recognition sequence for a type IIS restriction enzyme, preferably the wherein the cleavage site is an Mfyl cleavage site. 34. A method according to embodiment 31 or embodiment 32, wherein cleavage comprises cleaving the sugar-phosphate backbone of the first strand of the donor polynucleotide and breaking the hydrogen bonds between the one or more payload nucleotide pairs.

[0080] 35. A method according to embodiments 31 or embodiment 32, wherein cleaving is performed by the action of an enzyme having overhang cleavage function, preferably a type IIS restriction enzyme, optionally BspQI.

[0081] 36. 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.

[0082] 37. A method according to embodiment 36, 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.

[0083] 38. A method according to embodiment 36, 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.

[0084] 39. A method according to embodiment 36, 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.

[0085] 40. A method according to any one of embodiments 30 to 39, 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.

[0086] 41. A method according to embodiment 40, wherein the first step is performed with a nucleotide-excising enzyme.

[0087] 42. A method according to embodiment 41, wherein the nucleotide-excising enzyme is a 3 -methyladenine DNA glycosylase enzyme.

[0088] 43. A method according to embodiment 42, wherein the nucleotide-excising enzyme is: i. human alkyladenine DNA glycosylase (hAAG); or ii. uracil DNA glycosylase (UDG).

[0089] 44. A method according to any one of embodiments 34 to 43, wherein the second step is performed with a chemical which is a base.

[0090] 45. A method according to embodiment 44, wherein the base is NaOH. 46. A method according to any one of embodiments 34 to 43, wherein the second step is performed with an organic chemical having abasic site cleavage activity.

[0091] 47. A method according to embodiment 46, wherein the organic chemical is N,N’- dimethylethylenediamine.

[0092] 48. A method according to any one of embodiments 34 to 43, wherein the second step is performed with an enzyme having abasic site lyase activity, optionally wherein the enzyme having abasic site lyase activity is.

[0093] (i) AP Endonuclease 1 ;

[0094] (ii) Endonuclease III (Nth); or

[0095] (iii) Endonuclease VIII.

[0096] 49. A method according to any one of embodiments 30 to 33, wherein each cleavage step comprises a one step cleavage process comprising removing the universal nucleotide with a cleavage enzyme wherein the enzyme is

[0097] (i) Endonuclease III;

[0098] (ii) Endonuclease VIII;

[0099] (iii) formamidopirimidine DNA glycosylase (Fpg); or

[0100] (iv) 8-oxoguanine DNA glycosylase (hOGGl).

[0101] 50. A method according to any one of embodiments 30 to 33, wherein the cleavage step comprises cleaving the first strand of the donor polynucleotide with an enzyme.

[0102] 51. A method according to embodiment 50, wherein the enzyme cleaves the first strand of the donor polynucleotide between nucleotide positions n+1 and n.

[0103] 52. A method according to embodiment 50 or embodiment 51, wherein the enzyme is Endonuclease V. 53. A method according to embodiment 34 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.

[0104] 54. A method according to any one of the preceding embodiments, wherein ligation is performed by the action of an enzyme having nucleotide ligase activity.

[0105] 55. A method according to embodiment 54, 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.

[0106] 56. A method according to any one of the preceding embodiments, wherein before step (D) the method further comprises:

[0107] (i) performing a treatment step with an enzyme having phosphatase activity, such as calf intestinal phosphatase (CIP); or

[0108] (ii) performing a nuclease treatment step with an enzyme having 5’ to 3’ exonuclease activity, such as Exonuclease III, T5 exonuclease or T7 exonuclease.

[0109] 57. 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.

[0110] 58. A method according to embodiment 57, 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. 59. A method according to any one of embodiments 1 to 58, wherein both strands of the acceptor and donor polynucleotide comprises DNA.

[0111] 60. A method according to any one of embodiments 1 to 58, wherein the first strands of the acceptor and donor polynucleotide comprise RNA and the second strands of the acceptor and donor polynucleotide comprise DNA.

[0112] 61. A method according to embodiment 60, 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.

[0113] 62. A method according to any one of the preceding embodiments, wherein each cycle does not involve a step of incorporation of a polynucleotide having a reversible terminator group and an additional step of deprotection to remove the reversible terminator group.

[0114] 63. A method according to any one of the preceding embodiments, wherein

[0115] (i) the first and second strands of the acceptor polynucleotide at the second terminal end are each tethered to a surface; or

[0116] (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

[0117] (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

[0118] (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.

[0119] 64. A method according to embodiment 63(i), embodiment 63(iii) or embodiment 63(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.

[0120] 65. A method according to embodiment 63 (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.

[0121] 66. A method according to embodiment 64 or embodiment 65, wherein the cleavable linker is a UV cleavable linker.

[0122] 67. A method according to any one of embodiments 63 to 66, wherein the surface is a particle, optionally a microparticle.

[0123] 68. A method according to any one of embodiments 63 to 66, wherein the surface is a planar surface.

[0124] 69. A method according to embodiment 68, wherein the surface comprises a gel.

[0125] 70. A method according to embodiment 69, 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.

[0126] 71. A method according to any one of embodiments 61 to 68, 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.

[0127] 72. A method according to embodiment 69, 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. 73. A method according to embodiment 70, 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).

[0128] 74. A method according to any one of the preceding embodiments, wherein synthesis cycles are performed in droplets within a microfluidic system.

[0129] 75. A method according to embodiment 72, wherein the microfluidic system is an electrowetting system.

[0130] 76. A method according to embodiment 73, wherein the microfluidic system is an electrowetting-on-dielectric system (EWOD).

[0131] 77. 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.

[0132] 78. 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.

[0133] 79. 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.

[0134] 80. A method according to embodiment 79 wherein the first polynucleotide and the one or more additional polynucleotides are double-stranded. 81. A method according to embodiment 80 wherein the first polynucleotide and the one or more additional polynucleotides are single-stranded.

[0135] 82. A method according to any one of embodiments 79 to 81, wherein the first polynucleotide and the one or more additional polynucleotides are cleaved to create compatible termini and joined together, preferably by ligation.

[0136] 83. A method according to embodiment 82, wherein the first polynucleotide and the one or more additional polynucleotides are cleaved by a restriction enzyme at a cleavage site.

[0137] 84. A method according to any one of embodiments 77 to 83, wherein the synthesis and / or assembly steps are performed in droplets within a microfluidic system.

[0138] 85. A method according to embodiment 84, 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.

[0139] 86. A method according to embodiment 85 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 scaffold polynucleotide in accordance with the steps of the synthesis cycles.

[0140] 87. A method according to embodiment 86, 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.

[0141] 88. A method according to embodiment 86 and 87, wherein the microfluidic system is an electrowetting system. 89. A method according to embodiment 88, wherein the microfluidic system is an electrowetting-on-dielectric system (EWOD).

[0142] 90. A method according to any one of embodiments 86 to 89, wherein synthesis and assembly steps are performed within the same system.

[0143] 91. A support for synthesising a double-stranded polynucleotide having a predefined sequence, the support comprising:

[0144] (A) a population of acceptor polynucleotides, wherein each acceptor polynucleotide has a first strand and a second strand and wherein one end is immobilised on the support and the opposite end is free, and wherein the free end is blunt ended; and

[0145] (B) a population of tether polynucleotides disbursed amongst the acceptor polynucleotides, wherein each tether polynucleotide has one end immobilised on the support and wherein the opposite end is free, and wherein the free end comprises a first hybridisation arm (hyb arm) which is a single-stranded sequence region.

[0146] 92. A support according to embodiment 91, wherein

[0147] (i) the tether polynucleotide is single-stranded, wherein one end is immobilised on the support and the opposite end is free, wherein the free end comprises the first hybridisation arm (hyb arm) which is a singlestranded sequence region; or

[0148] (ii) the tether polynucleotide is double-stranded, wherein one end is immobilised on the support and the opposite end is free, wherein the free end comprises a first hybridisation arm (hyb arm) which is a singlestranded sequence region.

[0149] 93. A support according to embodiment 92(ii), wherein at the free end of the tether polynucleotide the terminal nucleotide of the strand which does not comprise the first hyb arm is provided as a non-ligatable nucleotide. 94. A support according to any one of embodiments 91 to 93, wherein the terminal nucleotide of the strand comprising the first hyb arm of the tether polynucleotide forms part of the sequence region of the first hyb arm, and / or wherein the terminal nucleotide of the strand comprising the second hyb arm of the donor polynucleotide forms part of the sequence region of the second hyb arm.

[0150] 95. A support according to any one of embodiments 91 to 94, wherein the sequence region of the first hyb arm and / or the sequence region of the second hyb arm is 15 or more nucleotides in length, 20 or more nucleotides in length, 25 or more nucleotides in length, 30 or more nucleotides in length, 35 or more nucleotides in length, 40 or more nucleotides in length, 45 or more nucleotides in length or 50 or more nucleotides in length.

[0151] 96. A support according to any one of embodiments 91 to 95, wherein the number of nucleotides in the sequence regions of the first and second hyb arms is the same.

[0152] 97. A support according to any one of embodiments 91 to 96, wherein the first hyb arm of the tether polynucleotide comprises two or more repeat sequences, and the second hyb arm of the donor polynucleotide comprises the same number of two or more repeat sequences as the tether polynucleotide, wherein the repeat sequences of the first hyb arm are complementary with the repeat sequences of the second hyb arm.

[0153] 98. A support according to embodiment 97, wherein the hyb arms have 3 to 7 repeat sequences, preferably wherein the number of repeats is 4 to 6.

[0154] 99. A support according to any one of embodiments 91 to 98, wherein the relative amount of acceptor polynucleotides to tether polynucleotides is from 5% to 25% acceptor polynucleotides to 75% to 95% tether polynucleotides, e.g. 5% acceptor polynucleotides to 95% tether polynucleotides, 10% acceptor polynucleotides to 90% tether polynucleotides, 15% acceptor polynucleotides to 85% tether polynucleotides, 20% acceptor polynucleotides to 80% tether polynucleotides, or 25% acceptor polynucleotides to 75% tether polynucleotides. 100. A support according to any one of embodiments 91 to 99, wherein the support has immobilised on it two or more populations of tether polynucleotides, wherein the sequences in the first hyb arms of each population is different.

[0155] 101. A support according to any one of embodiments 91 to 100, wherein at least one tether polynucleotide is bound to a donor polynucleotide, wherein the donor polynucleotide has first and second strands and first and second terminal ends, wherein the first terminal end is blunt-ended and comprises a polynucleotide payload sequence comprising one or more nucleotide pairs of the predefined sequence and a cleavage site adjacent the payload sequence, wherein the second terminal end comprises a second hyb arm which is a single-stranded region comprising sequence which is complementary with the sequence region of the first hyb arm, and wherein the first and second hyb arms are hybridized together.

[0156] 102. A system for synthesising a double-stranded polynucleotide having a predefined sequence, the system comprising the support as defined in any one of embodiments 91 to 101.

[0157] BRIEF DESCRIPTION OF THE DRAWINGS

[0158] Figure 1. Depicts illustrative, but non-limiting, acceptor, tether, and donor polynucleotides. The acceptor and tether polynucleotides are immobilised on a support. Although the tether polynucleotide shown has a double stranded region, single stranded tether polynucleotides may also be used as illustrated in Figure 3. The hybridisation (hyb) arms of the tether and donor polynucleotide include complementary regions meaning that the two can hybridize to each other.

[0159] Figure 2. Depicts an illustrative, but non-limiting embodiment using tether polynucleotides in synthesis. The support has both acceptor (A) and tether (T) polynucleotides immobilised on it, with the acceptor polynucleotides first being activated (2). The tether polynucleotides comprise hyb arms that are complementary to the hyb arms of the donor polynucleotides. The tether polynucleotides therefore hybridize to donor polynucleotides (3) which helps to localise the donor polynucleotide in the vicinity of the acceptor polynucleotides, which in turn promotes acceptor: donor ligation over acceptor: acceptor ligation (4). The tether and donor polynucleotides cannot ligate to each other because even after the two hybridize a gap is present between the end of the hyb arm of the tether and the end of the helper strand of the donor polynucleotide. After raising the temperature to remove unligated donor polynucleotide (5), the payload sequence is delivered by cleavage of the donor: acceptor ligation product and filling in (6). Successive cycles of payload sequence delivery (not shown) then progressively build up the chosen sequence. The acceptor or the predefined sequence may comprise a cleavage sequence to allow recovery of the desired sequence.

[0160] Figure 3. Shows illustrative, but non-limiting, donor and tether polynucleotide sequences. Both double-stranded and single-stranded tether polynucleotides are depicted and include a hyb arm representing a region comprising a sequence complementary to the hyb arm of the donor polynucleotide.

[0161] Figure 4. Shows the results of ligation with varying amounts of acceptor and spacer polynucleotides immobilised on beads after a first and a second ligation. The spacer polynucleotides help increase the distance between acceptor polynucleotides and therefore reduce ligation of acceptor polynucleotides to each other, but the spacers do not include a hyb arm and so do not help localise donor polynucleotides near the acceptor polynucleotides. The gel analysis allows visualisation of the relative amount of acceptor: acceptor (A: A) and acceptor: donor (A:D) ligation products formed as the two are different sizes. The first ligation is after ligation of the acceptor and donor polynucleotides for the first time, the second ligation is after subsequent cleavage, filling in, and ligation of the next donor polynucleotide.

[0162] Figure 5. The top panel shows the results of ligation with varying amounts of acceptor and tether polynucleotides immobilised on beads after a first and second ligation. The tether polynucleotides include hyb arms with a complementary region to a sequence in the hyb arm of the donor polynucleotide. The bottom panel shows quantification of the relative amount of A:D to A:A ligation products after the second ligation. Figure 6. Shows the impact of reducing the concentration of the donor polynucleotide via serial dilution on the relative amounts of A:D and A: A ligation products for beads with immobilised acceptor and tether polynucleotides at a ratio respectively of 15% to 85%. The top panel shows the results of gel analysis and the bottom panel the results of gel band quantification to allow the relative amounts of A:D and A: A ligation products produced with different concentrations of donor determined.

[0163] Figure 7. Shows the results for a support with 20% acceptor and 80% tether molecule immobilised on it. Panel A) Fluorescence intensity readings following each reaction step. From left to right: before activation the acceptor molecule has an Alexa Fluor TM 488 NHS ester label (first column); the acceptor is activated via Smal cleavage resulting in removal of the labelled portion of the acceptor (second column); the first T4 mediated ligation adds the labelled donor polynucleotide (third column); BspQI cleavage and filling in leaves the payload sequence and removes the labelled portion of the previous ligation product (fourth column); and addition then ligation of a new fluorescently labelled donor polynucleotide (fifth column). Panel B) Rsal-digested acceptor after ligation to Alexa Fluor™ 488 donor (lane 1). The ligation products are cleaved by BspQI and blunted by Q5® polymerase (lane 2). The blunt-ended acceptorpayload molecules from the first cycle can yield ligation products in the presence of double-stranded donor after a second ligation step (lane 3).

[0164] Figure 8. The results shown on the right-hand side are those also shown in Figure 7 employing tether polynucleotides. The results on the left-hand side are an experiment where, rather than tether molecules, spacer molecules were used lacking a hybridisation arm. The spacers therefore physically distance the acceptors from each other, but do not provide a way of localising donor polynucleotide near the acceptor. The the right-hand experiment uses ten times the amount of acceptor immobilised on the support as the higher amount of acceptor allows gel assessment.

[0165] Figure 9. Depicts symbols and terminology used in the schematics of the chemistries of the specific exemplary structures and synthesis methods outlined in Figures 10 to 16. Figure 10. Depicts the chemistry and methology of the specific exemplary synthesis method version 1. N.B. one polynucleotide strand of the donor is show is shown as comprising the hyb arm on one polynucleotide, but it can equally form part of the other polynucleotide strand of the donor instead.

[0166] Figure 11. Depicts the chemistry and methology of the specific exemplary synthesis method version 2. N.B. one polynucleotide strand of the donor is show is shown as comprising the hyb arm on one polynucleotide, but it can equally form part of the other polynucleotide strand of the donor instead.

[0167] Figure 12. Depicts the chemistry and methology of the specific exemplary synthesis method version 3. N.B. one polynucleotide strand of the donor is show is shown as comprising the hyb arm on one polynucleotide, but it can equally form part of the other polynucleotide strand of the donor instead.

[0168] Figure 13. Depicts the chemistry and methology of the specific exemplary synthesis method version 4. N.B. one polynucleotide strand of the donor is show is shown as comprising the hyb arm on one polynucleotide, but it can equally form part of the other polynucleotide strand of the donor instead.

[0169] Figure 14. Depicts the chemistry and methology of the specific exemplary synthesis method version 5. N.B. one polynucleotide strand of the donor is show is shown as comprising the hyb arm on one polynucleotide, but it can equally form part of the other polynucleotide strand of the donor instead.

[0170] Figure 15. Depicts various cleavage mechanisms in methods where the cleavage site is defined by a universal nucleotide. N.B. one polynucleotide strand of the donor is show is shown as comprising the hyb arm on one polynucleotide, but it can equally form part of the other polynucleotide strand of the donor instead.

[0171] Figure 16. Presents schemes showing examples of surface chemistries for attaching polynucleotides to surfaces. 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.

[0172] Figure 17. Presents schemes showing examples of surface chemistries for attaching polynucleotides to surfaces. 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.

[0173] Figure 18. Depicts and explains structural features relating to non-limiting exemplary embodiments. N.B the second terminus of the donor polynucleotide comprising a hyb arm is not shown, but the donor polynucleotide will comprise a hyb arm on one of the two strands.

[0174] DETAILED DESCRIPTION OF THE INVENTION

[0175] 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.

[0176] 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. The methods employ a support onto which acceptor polynucleotides are immobilised at one end, with the other end being free and blunt-ended. The methods involve successive cycles. Each cycle comprises ligation of a donor polynucleotide comprising a payload sequence and a cleavage site to the acceptor polynucleotide, followed by cleavage and, if necessary, filling-in to leave the acceptor polynucleotide with the payload sequence added. The payload sequence can vary with each cycle, meaning that the desired sequence can be progressively built-up.

[0177] The inventors have identified that the blunt-ended acceptor polynucleotides ligate not just to donor polynucleotides. Ligation of acceptor polynucleotides to each other generates unwanted acceptor: acceptor (A: A) ligation products, rather than the desired acceptor: donor ligation product (A:D). In order to address the issue, the inventors first immobilised both acceptor polynucleotides and spacer polynucleotides, where the spacer polynucleotides were unable themselves to ligate to the donor polynucleotides, but increased the physical distance between immobilised acceptor polynucleotides, thereby decreasing the chance of acceptor polynucleotides ligating to each other, rather than donor polynucleotides. Whilst the use of spacers reduced the proportion of A: A ligation products relative to A:D ligation products formed, the overall amount (i.e. the yield) of ligation products formed dropped substantially which would render the synthesis process less efficient.

[0178] The inventors therefore immobilised tether polynucleotides together with acceptor polynucleotides on the support. The tether polynucleotides, and donor polynucleotides, were each provided with single-stranded hybridisation (hyb) arms including a region complementary to a portion of the hyb arm of the other. The inventors unexpectedly found that the use of such tether polynucleotides reduced the formation of unwanted A: A ligation products formed compared to the amount of the desired A:D ligation products, but without the substantial drop in yield seen when spacer polynucleotides with no hyb arms were employed.

[0179] It is believed that the tether polynucleotides may act by helping to bring the donor polynucleotides into closer proximity to the acceptor polynucleotides, effectively increasing the local concentration of donor polynucleotide and thereby promoting more efficient ligation of acceptor and donor polynucleotides. The use of tether polynucleotides may also mean that it is possible to use lower concentrations of reagents in the synthesis and in particular a lower concentration of donor polynucleotide and ligase.

[0180] Figure 2 provides a non-limiting depiction of one embodiment using tether polynucleotides which helps illustrate how tether polynucleotides may be employed. Referring to the numbered parts of Figure 2:

[0181] (1) A support is provided with immobilised acceptor (A) and tether (T) polynucleotides.

[0182] (2) The acceptor polynucleotides are activated by cleavage leaving a free blunt end available for ligation.

[0183] (3) Donor polynucleotide is added. The tether and donor polynucleotides each have a single stranded region referred to as a hybridisation arm (hyb arm) which include complementary sequences to each other. The two can therefore hybridize to each other bringing donor polynucleotides into closer proximity with acceptor polynucleotides.

[0184] (4) Ligation is carried out. At least one strand of the donor polynucleotide is ligated to one strand of the acceptor polynucleotide in a single-stranded ligation. Alternatively, both strands the donor polynucleotide may be ligated to both strands of the acceptor polynucleotide in a double-stranded ligation.

[0185] The tether polynucleotides cannot ligate to each other because the sequences of the hyb arms are trpically the same, non-complementary and thus cannot hybridize together. The donor polynucleotides cannot ligate to each other for the same reasons. Although not necessary, the terminal nucleotide of the free end of the tether polynucleotide in the strand which does not comprise the hyb arm may be provided as a non-ligatable nucleotide so as to prevent ligation of the donor polynucleotide to the tether polynucleotide during the ligation step.

[0186] (5) The temperature is raised above the TM of the annealed hyb arms to remove unligated donor polynucleotide, which can be washed out.

[0187] (6) The acceptor: donor polynucleotide ligation product is cleaved and, where necessary, filled in to leave the acceptor polynucleotide with the payload sequence added.

[0188] Cycles of steps (4) to (6) are repeated to progressively to add new payload sequences to the end of the acceptor polynucleotide and build up the sequence of choice. The payload sequence in each cycle of steps (4) to (6) may be the same or different depending on what sequence it is desired to add each time.

[0189] In each cycle of synthesis in these exemplary synthesis methods the donor polynucleotide carries a polynucleotide payload. The donor polynucleotide is ligated to an acceptor polynucleotide to form a ligated polynucleotide. 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 cleaved molecule. In the next cycle of synthesis, ligation of a further donor polynucleotide to the same terminal end of the new acceptor polynucleotide followed by a further cleavage step results in a further polynucleotide payload being incorporated into the acceptor polynucleotide immediately adjacent the polynucleotide payload of the previous cycle. Accordingly, by performing multiple cycles of synthesis, a polynucleotide molecule having the predefined sequence may be synthesised.

[0190] The tether polynucleotide is not ligated to the donor polynucleotide, with hybridized donor polynucleotide removed from the tether polynucleotide by heating and washing. That means that the tether polynucleotide remains unchanged and is able to perform the same function in each cycle of the method provided that the hyb arm of the tether polynucleotide is able to hybridize to a hyb arm of the donor polynucleotide.

[0191] Accordingly, there is provided in vitro method of synthesising a double-stranded polynucleotide having a predefined sequence, the method comprising performing cycles of synthesis, wherein each cycle comprises:

[0192] (A) providing a support comprising:

[0193] (i) at least one acceptor polynucleotide having first and second strands, wherein one end is immobilised on the support and the opposite end is free, and wherein the free end is blunt ended; and

[0194] (ii) at least one tether polynucleotide adjacent the acceptor polynucleotide, wherein one end is immobilised on the support and the opposite end is free, wherein the free end comprises a first hybridisation arm (hyb arm) which is a single-stranded sequence region;

[0195] (B) providing at least one donor polynucleotide having first and second strands and having free first and second terminal ends, wherein the first terminal end is blunt-ended and comprises a polynucleotide payload sequence comprising one or more nucleotide pairs of the predefined sequence and a cleavage site adjacent the payload sequence, and wherein the second terminal end comprises a second hyb arm which is a single-stranded region comprising sequence which is complementary with the sequence region of the first hyb arm;

[0196] (C) contacting the at least one donor polynucleotide with the at least one tether polynucleotide, whereupon the first and second hyb arms hybridize to form a double-stranded region, and ligating the first terminal end of the donor polynucleotide with the free end of the acceptor polynucleotide to form a ligated polynucleotide; and

[0197] (D) cleaving the ligated polynucleotide and generating a cleaved blunt end, thereby extending the acceptor polynucleotide with the polynucleotide payload at the cleaved end, and generating a new blunt-ended double-stranded acceptor polynucleotide for ligation and extension in the next cycle.

[0198] 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.

[0199] The invention provides various advantages. The use of a tether polynucleotide will typically increase the efficiency of the method compared to performance of the same method without the tether polynucleotide. In one embodiment, the method will result in a higher ratio of A:D to A: A ligation products being formed in each cycle, particularly without a significant drop in overall yield of ligation products compared to if the method was performed without tether polynucleotides. Other advantages include that the use of tether polynucleotides may reduce the concentration of donor polynucleotide needed.

[0200] Other advantages include that 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. A further advantage arises from a non-limiting feature requiring the single-stranded ligation, in step (C), of only the first strands of the acceptor and donor polynucleotides, or the single-stranded ligation of only the second strands of the acceptor and donor polynucleotides. Such single-stranded ligation steps are therefore deliberately directed to ligate only the first strands and not the second strands, or only the second strands and not the first strands. Such single-stranded ligation steps are therefore not random or indiscriminate as between the pairs of first and second strands. This allows a donor polynucleotide to be provided in step (B) such that a terminal end which is ligatable to the first terminal end of an acceptor polynucleotide in step (C) may at the same time not be self-ligatable. Avoiding self-ligation of donor polynucleotides minimises reagent loss and increases the efficiency of synthesis reactions. Moreover, the provision of blunt-ended acceptor and donor polynucleotides in steps (A) and (B) provides flexibility in terms of the number of donor polynucleotide species required to deliver payloads for the generation of a double-stranded polynucleotide having a given predefined sequence.

[0201] Reaction Conditions

[0202] In one aspect the invention provides an in vitro method for synthesising a double-stranded polynucleotide having a predefined sequence.

[0203] Synthesis is carried out under conditions suitable for hybridization of nucleotides within double-stranded polynucleotides. 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 et al., 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)).

[0204] Ligation of polynucleotides can be carried out under suitable conditions, for example using a ligase (e.g., T4 DNA ligase) 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 4.4 mM Tris-HCl, 7mM MgCh, 0.7mM dithiothreitol, 0.7mM ATP, 5% polyethylene glycol (PEG6000).

[0205] After ligation, the methods may include a raising of temperature above the melting temperature of the two hyb arms of the tether polynucleotide and the donor polynucleotide so that the two are no longer hybridized. The donor polynucleotide which is neither ligated to the acceptor polynucleotide nor hybridized to the tether polynucleotide can then be removed by washing.

[0206] Cleavage of polynucleotides can be carried out under suitable conditions, for example 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.

[0207] 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 incoprorate 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 MgSC and 0.01% Triton® X-100.

[0208] Polynucleotide Molecule Having a Predefined Sequence

[0209] 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.

[0210] Acceptor Polynucleotide

[0211] 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.

[0212] 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 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.

[0213] A general scheme for an acceptor polynucleotide is depicted visually in Figure 1.

[0214] The acceptor polynucleotide is blunt-ended and double-stranded.

[0215] 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.

[0216] The first terminal end of the acceptor polynucleotide is ligatable and blunt- ended. By referring to a terminal end of an acceptor polynucleotide as “ligatable” it is meant that it is capable of being ligated to the first 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”.

[0217] The first terminal end is free, i.e. neither the first strand nor the second strand is joined to or otherwise attached to any other structure.

[0218] The terminal nucleotide of the first strand at the ligatable first terminal end of the acceptor polynucleotide may or may not comprise a 5’ phosphate group. Whether or not a 5’ phosphate group is present will depend upon the specific chemistry method employed.

[0219] 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 may or may not comprise a 3’ hydroxyl group. Whether or not a 3’ hydroxyl group is present will depend upon the specific chemistry method employed at the preference of the user.

[0220] The second terminal end of the acceptor polynucleotide is non-ligatable. By referring to the second terminal end of the acceptor polynucleotide as “non-ligatable” it is meant that it is not capable of being ligated to another nucleic acid molecule, including to the first terminal end of a donor polynucleotide as described and defined further herein. The second terminal end of the acceptor polynucleotide is non-ligatable since this end is immobilised on the support, such as depicted in Figure 1. The support may comprise a surface which may be any suitable surface as described and defined elsewhere herein. The second terminal end may be joined to the support due to the second strand of the acceptor polynucleotide being joined to the support whilst the first strand of the acceptor polynucleotide is not joined to the support, such as depicted in Figure 1. Alternatively, the second terminal end may be joined the support due to the first strand of the acceptor polynucleotide being joined to the support, whilst the second strand of the acceptor polynucleotide is not joined to the support. Alternatively still, the second terminal end may be joined to the support due to the first and second strands of the acceptor polynucleotide being joined to the support. Where both the first and second strands of the acceptor polynucleotide are joined to the support, each strand may be independently joined to the support. 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 joined to the support.

[0221] 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. More typically however, 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.

[0222] 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, provided that the first and second strands of the acceptor polynucleotide are suitable to facilitate ligation as described further herein, and, if required, to prime new polynucleotide synthesis as described further herein in relation to specific non-limiting exemplary method versions.

[0223] The first and second strands of the acceptor polynucleotide may comprise nucleotides, nucleotide analogues / derivatives and / or non-nucleotides.

[0224] 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, and which are capable of priming new polynucleotide synthesis as described further herein if desired. At the first terminal end of the acceptor polynucleotide which is to be extended, mismatches between strands should be avoided, GC- and AT-rich regions should be 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.

[0225] 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.

[0226] In an acceptor polynucleotide, the first strand is hybridized to the corresponding region of the second strand. 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 ligation as described herein, or capable of priming new polynucleotide synthesis as described further herein if desired. 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.

[0227] 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.

[0228] In some cases, the acceptor polynucleotides may be immobilised on a support such that the immobilised acceptor poynucleotide is immediately ready for the first cycle of the synthesis. In other cases, immobilised acceptor polynucleotides may be activated prior to the first cycle of the synthesis. One possibility is that the acceptor polynucleotide is immobilised on the spport and then a cleavage carried out to generate the blunt ended first terminal end ready for the first cycle of the synthesis. It may be that the acceptor polynucleotide includes a restriction enzyme recognition site allowing for such cleavage to generate acceptor polynucleotides ready for the first cycle. One possible enzyme is Smal as it generates a blunt ended clevage product. It may be that the acceptor polynucleotide prior to activation comprises a label which is removed when the acceptor polynucleotide is cleaved to activate it. The use of such a label may help in determining the presence or amount of immobilised acceptor polynucleotide on the support. Non-limiting exemplary embodiments of an acceptor polynucleotide is provided in the Examples below.

[0229] Tether Polynucleotide

[0230] Each one of the specific exemplary methods of the present invention involves the use of a tether polynucleotide which is also immobilised on the support with the acceptor polynucleotides. Tether 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.

[0231] A tether polynucleotide may be formed from a first and second strand to form a double-stranded portion and a single-stranded portion, with the single-stranded portion comprising the hyb arm of the tether polynucleotide, the hyb arm being complementary to the hyb arm of the donor polynucleotide.

[0232] Alternatively, a tether polynucleotide may be wholly single-stranded, wherein a portion of the single-stranded polynucleotide includes sequence which is complementary to the hyb arm of the donor polynucleotide and therefore represents the hyb arm of the tether polynucleotide.

[0233] In some cases where the tether polynucleotide comprises a double-stranded and single-stranded portion, it may also comprise a linker joining the double stranded and single stranded regions. Any suitable spacer may be used. One example of a spacer is isp 18 which is an 18-atom hexa-ethyleneglycol spacer.

[0234] A tether polynucleotide has a first terminal end comprising the single stranded hyb arm, as well as a second terminal end which is the end immobilised on the support.

[0235] Where the tether polynucleoptide is single-stranded, the first terminal end is free and the second terminal end is immobilised on the support.

[0236] In cases where the tether polynucleotide is double-stranded, both strands of the first terminal end are free and at least one strand of the second terminal end of the tether polynucleotide is immobilised on the support. In one case one strand is immobilised. Alternatively, both strands of the second terminal end may be immobilised on the support.

[0237] A general scheme for a tether polynucleotide comprising a double-stranded region is depicted visually in Figure 1. The tether polynucleotide depicted in Figure 1 comprises a double stranded region which is made up of a 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 double-stranded polynucleotide region. It may be that the immobilised strand, e.g. the second strand, of the tether polynucleotide is the longer strand and therefore comprises the hyb arm at the first terminal end. Alternatively, it may be that the first strand, e.g. the non-immobilised strand, is the longer and therefore comprises the hyb arm at its first terminal end.

[0238] Whilst a tether polynucleotide is able to hybridize to a donor polynucleotide it may not be able to ligate to it. It may be that when the hyb arms of a tether polynucleotide and donor polynucleotide are hybridized to each other there is a gap between the end of the annealed single stranded hyb arm and the strand of the other molecule, such that the two cannot be ligated to each other. Alternatively, it may be that the tether polynucleotide cannot be ligated to the donor polynucleotide because the terminal nucleotide of the strand of the tether polynucleotide that does not comprise the hyb arm is provided as a non-ligatable nucleotide.

[0239] It is believed that a tether polynucleotide can bring the donor polynucleotide into closer proximity to the acceptor polynucleotide promoting the formation of acceptor: donor polynucleotide ligation products. It may be therefore that a tether polynucleotide is able to increase the local concentration of the donor polynucleotide by hybridising to it.

[0240] After ligation of acceptor and donor polynucleotides, the temperature of the support may be raised so that the hyb arms of the tether polynucleotide and the donor polynucleotide are no longer hybridized, which may be followed by washing to remove any unligated donor polynucleotide.

[0241] 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. In contrast, at the end of each cycle the tether polynucleotide should be unchanged and hence be able to perform the same role in the next cycle of the synthesis.

[0242] 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 tether polynucleotide beyond that the tether polynucleotide includes a single stranded-region representing a hyb arm which comprises a sequence which is complementary to a sequence in the hyb arm of the donor polynucleotide. It is possible to compare the impact of tether polynucleotides with the corresponding polynucleotide lacking a hyb arm, referred to herein as a spacer polynucleotide. The use of a tether polynucleotide may result in a greater yield of acceptor: donor ligation products compared to the same experiment where a spacer polynucleotide lacking a hyb arm is used instead of a tether polynucleotide. The use of a tether polynucleotide may also be compared to the same experiment except for the support lacking any tether polynucleotide or spacer polynucleotide immobilised on it. It may be that such a comparison shows a higher ratio of acceptor: donor ligation products compared to acceptor: acceptor products.

[0243] The first and, if present, second strands of the tether polynucleotide may comprise nucleotides, nucleotide analogues / derivatives and / or non-nucleotides. The skilled person is readily able to construct a tether polynucleotide which will be suitable to facilitate DNA synthesis.

[0244] Tether polynucleotides may act by increasing the local concentration of donor polynucleotide for the acceptor polynucleotide. The use of tether polynucleotide may also reduce the concentration of a particular reagent needed, for instance it may reduce the concentration of donor polynucleotide needed. It may also reduce the amount of DNA ligase needed. Such reductions may help in reducing the cost of the DNA synthesis.

[0245] In the invention it may be that more than one type of tether polynucleotide is present immobilised on the support. It may be that as well as a tether polynucleotide for a donor polynucleotide a second type of tether polynucleotide is present to bring a different donor polynucleotide into proximity with the acceptor polynucleotide. It may be in one case that a second type of tether polynucleotide is immobilised and is used to bring an enzyme for cleaving the synthesised polynucleotide from the support at the end of the cycles of synthesis. Using such an approach may reduce the amount of cleavage enzyme needed. Alternatively, a second type of tether polynucleotide may be used to bring a different donor polynucleotide into proximity with the acceptor polynucleotide. Donor Polynucleotide and Polynucleotide Payload

[0246] 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.

[0247] As its name implies, a donor polynucleotide acts to donate a double-stranded polynucleotide payload consisting of nucletotides of the predefined sequence. The polynucleotide payload is further described herein. Whilst the donor polynucleotide comprises a blunt-ended double stranded region, it also comprises at the other end a single stranded portion which represents the hybridisation (hyb) arm which comprises a sequence which is complementary to a sequence present in the hyb arm of the acceptor polynucleotide.

[0248] General schemes for a donor polunucleotide are depicted visually in Figure 1.

[0249] The donor polynucleotide comprises first and second polynucleotide strands and first and second terminal ends. The first and second strands are of different lengths. The first and second polynucleotide strands are connected by hybridization, via standard Watson-Crick base pairing, to form a double-stranded polynucleotide region, with the donor polynucleotide also comprising a single-stranded region. The first terminal end is at the end of the double-stranded region of the donor polynucleotide formed by the hybridized region of the first and second strands. The first terminal end of the donor polynucleotide is blunt-ended. The donor polynucleotide also comprises a portion which is single-stranded referred to as the hybridisation arm, or hyb arm, and which is found at the second terminal end of the donor polynucleotide.

[0250] A first terminal end of a donor polynucleotide is “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. Further details are provided with reference to the specific method versions described further herein. In embodiments involving single-stranded ligation, the strand of the donor polynucleotide that is to be ligated to the acceptor polynucleotide may be referred to as the ligation strand and the other strand of the donor polynucleotide as the helper strand. It may be that the ligation strand comprises the portion which will represent the single-stranded hybridisation arm when the ligation and helper strands are hybridized together. Alternatively, it may be the helper strand which is longer and provides the hybridisation arm when the ligation and helper strands are annealed to each other.

[0251] A first 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 first terminal end of a donor polynucleotide may be “non-self-ligatable”. A skilled person will readily appreciate how a first 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. It may be that one strand of the first terminal end of the donor polynucleotide may be ligatable to one strand of the first terminal end of the acceptor poynucleotide, but that the other strand of the first terminal end of the donor polynucleotide is not ligatable to the other strand of the first terminal end of the acceptor polynucleotide. It may be that the first strand of the first terminal end of the donor polynucleotide is the strand which is ligatable. It may be that the second strand of the first terminal end of the donor polynucleotide is the strand which is ligatable.

[0252] A second terminal end of a donor polynucleotide is “non-ligatable”. By referring to a second terminal end of a donor polynucleotide as “non-ligatable” it is meant that it is not capable of being ligated to a tether polynucleotide, acceptor polynucleotide, or another donor polynucleotide. A skilled person will readily appreciate how a second terminal end of a donor polynucleotide may be structured so as to be incapable of being ligated to a tether polynucleotide, acceptor polynucleotide, or another donor polynucleotide. The second terminal end of a donor polynucleotide may not self-ligate with the second terminal end of a donor polynucleotide because the sequences of the hyb arms are typically identical, non-complementary and therefore will not hybridize together. It may be that ligation of the second terminal end of a donor polynucleotide to the free end of a tether polynucleotide is prevented by the presence of a gap between the strands of the donor and tether polynucleotides when the two hybridisation arms are hybridized to each other. It may be that the terminal nucleotide of the free end of the tether polynucleotide in the strand which does not comprise the hyb arm may be provided as a non-ligatable nucleotide so as to prevent ligation of the donor polynucleotide to the tether polynucleotide during the ligation step. Further details are provided with reference to the specific method versions described further herein.

[0253] 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 adjacent the payload sequence, wherein the first terminal end is blunt-ended and ligatable.

[0254] 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 adjacent the payload sequence, wherein the first terminal end is blunt-ended, ligatable and non-self- ligatable.

[0255] 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 adjacent the payload sequence, wherein the first terminal end is blunt-ended, ligatable and non-self- ligatable and the second terminal end comprises a single stranded hyb arm with neither of the first and second strands at the second terminal end are ligatable to another donor polynucleotide, an acceptor polynucleotide, or a tether polynucleotide.

[0256] The first terminal end is free, i.e. neither the first strand nor the second strand is attached to any other structure. The terminal nucleotide of the first 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.

[0257] 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.

[0258] The terminal nucleotide of the first or second second strand at the ligatable first terminal end of the donor polynucleotide may or may not comprise a hydroxyl group. Whether or not a hydroxyl group is present will depend upon the specific chemistry method employed at the preference of the user.

[0259] The second terminal end of the donor polynucleotide is not tethered to a surface. The second terminal end of the donor polynucleotide comprises a single stranded hyb arm which is able to hybridize to the hyb arm of the tether polynucleotide.

[0260] The second terminal end of the donor polynucleotide is non-ligatable, for instance 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 when the donor polynucleotide is hybridized to a tether polynucleotide be unable to ligate to the strands of the tether polynucleotide because a gap is present between the strands.

[0261] 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 and subsequently cleavage. The terminal nucleotide of the first strand at the ligatable first terminal end of the donor polynucleotide and the terminal nucleotide of the second strand at the ligatable first terminal end of the donor polynucleotide 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 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 a nucleotide pair, and this pair is the second pair of nucleotides in the polynucleotide payload, and so on.

[0262] It will be appreciated that a payload may comprise only a single nucleotide pair of the predefined sequence, in this case the payload may be referred to as a nucleotide payload instead of a polynucleotide payload.

[0263] A polynucleotide payload may consist of two or more, or three or more consecutive pairs of nucleotides of the predefined sequence. It may be that a polynucleotide payload comprises two consecutive pairs of nucleotides of the predefined sequence. It may be that it comprises three consecutive pairs 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. The payload may consist of one to 10 consecutive pairs of nucleotides of the predefined sequence. It may consist of 1 to 5 consecutive pairs of nucleotides of the predefined sequence. The payload may consist of two nucleotides.

[0264] 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. Multiple cleavage mechanisms are described further herein.

[0265] 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, and also that one strand of the donor polynucleotide comprises a single stranded hyb arm allowing it to hybridize to the complementary hyb arm of the tether polynucleotide.

[0266] The first and second strands of the donor polynucleotide may comprise nucleotides, nucleotide analogues / derivatives and / or non-nucleotides. 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.

[0267] 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.

[0268] 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.

[0269] In a donor polynucleotide, the first strand is hybridized to the corresponding region of the second strand. 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 ligation as described herein. Thus, mismatches between the first strand and the corresponding region of the second strand can be tolerated to a degree. When hybridized the first and second strands will be of different length meaning that at the second terminal end of the donor polynucleotide a single-stranded region is present which is able to act as a hyb arm.

[0270] Hybridisation Arms

[0271] Both tether and donor polynucleotides have a stretch of single-stranded nucleic acid which represents a hybridisation (hyb) arm. The hybridisation arms of a tether polynucleotide and donor polynucleotide each comprise a region that is complementary to the other, meaning that the two hyb arms are able to hybridize to each other. That is the hyb arms of the tether and polynucleotide are able to hybridize to each other via WatsomCrick base pairing to form a double-stranded region.

[0272] Each one of the specific exemplary methods of the present invention involves the use of tether and acceptor polynucleotides comprising hyb arms. Hyb arms are described extensively with respect to the specific chemistry methods of the invention set out below and non-limiting exemplary donor polynucleotides are depicted visually in the corresponding Figures.

[0273] A hyb arm may comprise a repeating sequence where each unit or copy of the repeating sequence is able to hybridize to the hyb arm of the other polynucleotide. It may be that both the hyb arms of the tether polynucleotide and the donor polynucleotide comprise a repeating sequence where each repeat of the repeating sequence is able to hybridize to a repeat of the repeating sequence of the other polynucleotide. It may be that there are one to five repeats of the repeating sequence. The hyb arms have 1-4 repeat sequences, 3 to 7 repeat sequences, preferably 4 to 6 repeat sequences. If the hyb arms of the tether polynucleotide and the donor polynucleotide comprise repeat sequences, it is preferable that they both comprise the same number of repeat sequences.

[0274] The skilled person is readily able to design appropriate hyb arms that allow hybridisation between tether and donor polynucleotides. The skilled person will be able to determine that hyb arms employed result in decreased acceptor: acceptor ligation production formation compared to acceptor: donor ligation product. In one embodiment the hyb arms will each be 5 to 50 nucleotides in length. It may be that the hyb arms are each 10 to 30 nucleotides in length. A hyb arm may be 15 or more nucleotides in length, 20 or more nucleotides in length, 25 or more nucleotides in length, 30 or more nucleotides in length, 35 or more nucleotides in length, 40 or more nucleotides in length, 45 or more nucleotides in length or 50 or more nucleotides in length.. The hyb arms of the tether polynucleotide and the donor polynucleotide may have the same number of nucleotides and therefore will have same length. The hyb arms of the tether polynucleotide and the donor polynucleotide may have a different number of nucleotides and therefore will not have same length, provided that there is sufficient complementary sequence between the two hyb arms such that they will hybridize in the synthesis methods described and defined herein.

[0275] It may be that the whole length of the hyb arm is complementary to the hyb arm it hybridizes to. Illustrative hyb arm sequences are provided in Figure 3 and Table 3.

[0276] The hyb arm of the tether or donor polynucleotide may include a repeating sequence, wherein each repeat of the sequence is complementary to a sequence in the hyb arm of the other of the tether or donor. The hyb arm of the donor may be A:T rich. For example, it may be that the complementary regions in the tether and donor polynucleotide hyb arms only comprise A and T bases. The hyb arm of a tether polynucleotide will be at the first terminal end of the tether polynucleotide as illustrated, for instance, by Figure 1. Hence, the hyb arm of a tether polynucleotide will be at the opposite end to that immobilised to the support. The hyb arm of a donor polynucleotide will be at the second terminal end of the donor polynucleotide as illustrated, for instance, by Figure 1. Hence, the hyb arm of a donor polynucleotide will be at the opposite end to the payload of the donor ploynucleotide.

[0277] The sequence of the hyb arms of the tether and donor polynucleotides may be designed to have a particular sequence to ensure a particular melting temperature (Tm) for the two when hybridized to each other. A method of the invention may comprise raising the temperature after ligation so that the hyb arms of the tether and donor polynucleotides no longer anneal. After the temperature is raised so that the hyb arms are no longer annealed the unligated donor polynucleotide may be removed by washing.

[0278] Blocking Groups

[0279] Polynucleotides may comprise blocking groups. A blocking group may prevent one polynucleotide strand ligating to another polynucleotide strand. For instance, blocking groups may be used to prevent ligation of a tether polynucleotide to a donor polynucleotide. A blocking group may be attached to or configured in one or both strands of the second terminal end of the donor polynucleotide in order to prevent such ligation. Alternatively, it may be that a tether polynucleotide comprises blocking groups to prevent ligation to donor polynucleotides. Alterantively, as illustrated by the Examples of the present application, it may be that when the tether and donor polynucleotides are hybridized to each other that there is a gap between the strands preventing ligation.

[0280] 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.

[0281] A blocking group may be preferably 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.

[0282] 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.

[0283] Cleavage of Ligated Polynucleotide

[0284] 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.

[0285] The cleavage step may comprise 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 blunt end at the cleaved 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 terminal nucleotides of the cleaved first terminal end are the final pair of nucleotides of the polynucleotide payload in that cycle.

[0286] Cleavage is performed such that following cleavage the first terminal end of the acceptor polynucleotide comprising the polynucleotide payload is ligatable. Following cleavage, the terminal nucleotide of the first strand at the ligatable first terminal end of the acceptor polynucleotide (i.e. one of the nucleotides of the final pair of nucleotides of the polynucleotide payload) may or may not comprise a phosphate group. Whether or not the terminal nucleotide of the first strand at the ligatable first terminal end of the acceptor polynucleotide comprises a phosphate group will depend upon the specific chemi sty of the method employed at the preference of the user. Specific methods are described further herein. Following cleavage, the terminal nucleotide of the second strand at the ligatable first terminal end of the acceptor polynucleotide (i.e. the second nucleotide of the final pair of nucleotides of the polynucleotide payload) may or may not comprise a hydroxyl group. Wherther or not the terminal nucleotide of the second strand at the ligatable first terminal end comprises a hydroxyl group will depend upon the specific chemisty of the method employed at the preference of the user. Specific methods are described further herein.

[0287] The cleavage step can be performed by any suitable means for creating the blunt-ended structure described above. The specific type of cleavage will depend upon the specific chemisty of the method employed at the preference of the user.

[0288] Cleavage may comprise a double-stranded cleavage reaction wherein both the first and second strands are cleaved. In such a cleavage step both the first and second strands are cleaved at the same positions in a symmetrical cleavage reaction. In each strand cleavage is performed at the position between the final nucleotide in the polynucleotide 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. This generates a cleaved polynucleotide wherein the first terminal end of the remainder of the donor polynucleotide is blunt-ended and wherein the first terminal end of the acceptor polynucleotide is blunt-ended. The terminal nucleotides of the first and second strands at the first terminal end of the acceptor polynucleotide are the final pair of nucleotides of the polynucleotide payload.

[0289] In certain methods a nick site is introduced into one strand, such as described in the examples herein. A nick site is a single-stranded break or gap between nucleotides of a nucleotide strand.

[0290] If a symmetrical cleavage reaction is performed in a method where a nick site is introduced into one strand, following cleavage the original nucleotides of the polynucleotide payload of the nicked strand may remain attached only via interaction (e.g. hydrogen bonding) with the original nucleotides of the polynucleotide payload of the opposite strand. These original nucleotides of the polynucleotide payload of the nicked strand may be removed and replaced by incorporation of new nucleotides of the polynucleotide payload by extension from the nick site.

[0291] The original nucleotides of the polynucleotide payload of the nicked strand may be separated from the opposite strand: (i) before the incorporation step; or

[0292] (ii) during the incorporation step.

[0293] Where the original nucleotides of the polynucleotide payload of the nicked strand are separated from the opposite strand during the incorporation step, incorporation steps may be performed:

[0294] (a) by the action of an enzyme having polymerase activity, and wherein the polymerase displaces the original nucleotides of the polynucleotide payload of the nicked strand when synthesising the new strand; or

[0295] (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 nicked strand when synthesising the new strand.

[0296] Specific steps for performing such methods are described in detail further herein.

[0297] In certain methods described herein cleavage may be performed in such a way that the first strand is cleaved at a different relative position compared to the second strand. Such a cleavage step consequently results in an asymmetrical cleavage. This can be achieved in two ways.

[0298] In certain methods, both the first and second strands are cleaved, and at different relative positions. This generages a staggered / asymmetrical break.

[0299] In certain alternative methods, a nick site is introduced into one strand only and the opposite strand is cleaved. The opposite strand is cleaved at a different position relative to the nick site. This also generages a staggered / asymmetrical break.

[0300] Thus, the cleavage step may comprise cleaving only the first strand of the ligated polynucleotide where the second strand comprises a nick site. Alternatively, the cleavage step may comprise cleaving only the second strand of the ligated polynucleotide where the first strand comprises a nick site.

[0301] In the case of an asymmetrical cleavage reaction wherein both strands are cleaved, the first strand is cleaved immediately above the nucleotides of the polynucleotide payload i.e. cleavage of the first strand is performed at the position between the final nucleotide in the polynucleotide 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. The second strand is cleaved below the final nucleotide in the polynucleotide payload in the direction proximal to the second terminal end of the acceptor polynucleotide / distal 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. The nucleotides of the polynucleotide payload in the second strand remain attached to the donor polynucleotide following cleavage. Following cleavage, such a method further comprises separating the remainder of the donor polynucleotide from the acceptor polynucleotide. Accordingly, because the original nucleotides of the polynucleotide payload in the second strand remain attached to the donor polynucleotide following cleavage, they are consequently discarded. These steps generate an overhang at the cleaved first terminal end of the acceptor polynucleotide. The nucleotides of the first strand of the polynucleotide payload overhang the second strand of the acceptor polynucleotide, wherein the terminal nucleotide of the overhang is the final nucleotide of the polynucleotide payload. Following such an asymmetrical cleavage reaction, after separating the cleaved donor polynucleotide from the acceptor polynucleotide, the methods further comprise performing an incorporation step comprising extending the second strand of the acceptor polynucleotide by incorporating new payload nucleotides using the original payload nucleotides of the first strand in the overhang as templates, thereby re-forming the payload nucleotides in the second strand, thereby re-forming the payload nucleotide pairs in the ligated 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. Preferably, incorporation of new payload nucleotides is performed by the action of an enzyme having polymerase activity.

[0302] In an asymmetrical cleavage reaction, cleavage of the first strand of the ligated polynucleotide is performed in such a way that a 5’ phosphate group may be retained on the terminal nucleotide of the first strand at the cleaved first terminal end of the acceptor polynucleotide, i.e. on the final nucleotide of the polynucleotide payload in the first strand. This is achieved as a consequence of any standard cleavage reaction. Depending on the specific method employed, the 5’ phosphate group may be removed in a dephosphorylation step. Furthermore, incorporation is performed in such a way that a 3’ hydroxyl group is retained on the terminal nucleotide of the second strand at the cleaved first terminal end of the acceptor polynucleotide, i.e. on the final nucleotide of the polynucleotide payload in the second strand. Thus, such a method is performed wherein following cleavage and following incorporation steps, the first terminal end of the cleaved acceptor polynucleotide comprising the polynucleotide payload is ligatable in the method employed and is thus competent to be ligated to a further donor polynucleotide in the next cycle of synthesis.

[0303] The cleavage step can be performed by any suitable means for creating the cleaved structures described above and herein.

[0304] As described above, cleavage may comprise a double-stranded cleavage wherein both the first and second strands are cleaved. Such a cleavage step is consequently performed as a symmetrical cleavage, so as to form a blunt end at the cleaved first terminal end of the donor polynucleotide. Cleavage may comprise cleaving the sugarphosphate backbone of the first and second strands of the donor polynucleotide molecule. 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, the type IIS restriction enzyme may be Mlyl. The user will readily be able to structure the cleavage site in the donor polynucleotide in a manner that allows the required structure described above to be formed following cleavage.

[0305] As described above, cleavage may comprise a single-stranded cleavage wherein only the first strand is cleaved. Cleavage of only the first strand can be performed in view of the nick site introduced previously into the second strand.

[0306] A single-stranded cleavage may comprises cleaving the sugar-phosphate backbone of the first strand of the donor polynucleotide and breaking the hydrogen bonds between the one or more payload nucleotide pairs.

[0307] Single-stranded cleavage may thus be performed by the action of an enzyme having overhang cleavage function, preferably a type IIS restriction enzyme, such as BspQI. Single-stranded cleavage may alternatively be performed by the action of an enzyme having nicking cleavage function, preferably a type IIS restriction enzyme, optionally Nt.As / .

[0308] Single-stranded cleavage may alternatively be performed using a method wherein the 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.

[0309] Single-stranded cleavage may alternatively be performed using a method 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. The universal nucleotide may be inosine or any other universal nucleotide described herein. Cleavage mechanisms using universal nucleotides are described elsewhere herein.

[0310] Different cleavage configurations may be employed when the cleavage site is defined by a universal nucleotide positioned in the first strand of the donor polynucleotide.

[0311] In such methods, a nucleotide position in the first strand may be referred to as positon “n”. The final nucleotide in the polynucleotide payload, i.e. the final nucleotide of the polynucleotide payload that is most proximal to the second terminal end of the donor polynucleotide, always occupies position n. Starting from position n, the nucleotide which occupies the next nucleotide position in the first strand in the direction distal to the second terminal end of the donor polynucleotide always occupies position n-1. In the case of polynucleotide payloads which comprise more than one nucleotide pair, the nucleotide at position n-1 will be the penultimate nucleotide in the polynucleotide payload. Starting from position n, the nucleotide which occupies the next nucleotide position in the first strand in the direction proximal to the second terminal end of the donor polynucleotide always occupies position n+1. This arrangement of position numbering is depicted visually in Figure 15. Accordingly, in a first cleavage mechanism using a universal nucleotide, 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. Such a method comprises cleaving the first strand between nucleotide positions n and n+1. Such a cleavage mechanism is depicted visually in Figure 15 A.

[0312] Alternatively, in a further cleavage mechanism 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. Such a method comprises cleaving the first strand between nucleotide positions n and n+1. Such a cleavage mechanism is depicted visually in Figure 15B. Alternatively still, in further cleavage mechanisms 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. Such methods comprises cleaving the first strand between nucleotide positions n and n+1. Such cleavage mechanisms are depicted visually in Figure 15C.

[0313] In the methods of the invention, the selection of the reagent to perform the cleavage step will depend upon the particular method employed. Configuration of the desired cleavage site and selection of the appropriate cleavage reagent will therefore depend upon the specific chemistry employed in the method, as will readily be apparent by reference to the exemplary methods described herein.

[0314] Some examples of DNA cleaving enzymes that may be used are shown in Table 1 below.

[0315] Table 1 Following the cleavage step, a new acceptor polynucleotide is created. 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. 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.

[0316] Acceptor Depletion

[0317] 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.

[0318] 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.

[0319] 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.

[0320] 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.

[0321] 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.

[0322] In methods which incorporate a depletion step it may be that the depletion step has no impact on tether polynucleotides. As discussed above, ideally a tether polynucleotide at the end of a cycle is no longer hybridized to a donor polynucleotide and is able to play the same role in the next cycle of the method.

[0323] Nucleotides

[0324] In certain embodiments of the methods described herein it is desirable to perform an incorporation / extension reaction to incorporate nucleotides into polynucleotides, for example to fill in an overhanging end to create a blunt-ended polynucleotide.

[0325] 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.

[0326] 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.

[0327] 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.

[0328] 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).

[0329] 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.

[0330] 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.

[0331] 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.

[0332] 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.

[0333] 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.

[0334] 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.

[0335] 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: Modified Nucleotides

[0336] 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.

[0337] Examples of epigenetic bases which may be incorporated include the following:

[0338] Examples of modified bases which may be incorporated include the following:

[0339] Examples of halogenated bases which may be incorporated include the following: where R1 = F, Cl, Br, I, alkyl, aryl, fluorescent label, aminopropargyl, aminoallyl.

[0340] 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.

[0341] Examples of modified bases, which may be useful in e.g. click chemistry, which may be incorporated include the following:

[0342] Examples of biotin-modified bases which may be incorporated include the following: where base = A, T, G or C with alkyne or alkene linker. Examples of bases bearing fluorophores and quenchers which may be incorporated include the following:

[0343] Universal Nucleotides

[0344] In certain methods of the invention a universal nucleotide may be used to define a cleavage site, as described further herein.

[0345] 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.

[0346] 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- aminobenzimidazole 2' deoxyribonucleoside, 4-aminobenzimidazole ribonucleoside, phenyl C-ribonucleoside or phenyl C-2’-deoxyribosyl nucleoside.

[0347] Some examples of universal bases are shown below:

[0348] 8 h thi 2 azah oxanthine 8 aminohypoxanthine 2-oxopurine ol 7-nitrobenzimidazole

[0349] 3-formylindol pyrrolopyridine benzimidazole 5-benzimidazole nitroindole derivatives

[0350] 3 -nitropyrrol 4-nitroimidazole 4-nitropyrazole 2-nitrobenzene 6-nitropiperonyl nitropyrrol and nitrobenzene derivatives nucleoside analogue

[0351] Universal nucleotides incorporating cleavable bases may also be used, including photo- and enzymatically-cleavable bases, some examples of which are shown below. Photocleavable bases:

[0352] 7-nitroindol 2-nitrophenol 6-nitropiperonyl nucleoside analogue

[0353] 5 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 trans-'] -carbamoyl- 5-hydroxy- 5-hydroxy- (cis & trans) dihydrocytosine hydantoin 2-oxo-4,5-dihydroxy- cytosine uracil imidazolidine

[0354] Base analogues cleavable by Formamidopyrimidine DNA glycosylase (Fpg):

[0355] 7,8-dihydro-8-oxo- 7,8-dihydro-8-oxo- 7,8-dihydro-8-oxo- 7,8-dihydro-8-oxo- nebularine

[0356] 4,6-diamino-5- 2,6-diamino-4-hydroxy- 2,6-diamino-4-hydroxy- 5-hydroxycytosine 5-hydroxyuracil formamidopyrimidine 5-formamidopyrimidine 5-N-methylformamido- (Fapy-adenine) (Fapy-guanine) pyrimidine

[0357] Base analogues cleavable by 8-oxoguanine DNA glycosylase (hOGGl):

[0358] 8-oxoguanine Base analogues cleavable by hNeil 1 : guanidinohydantoin spiroiminodihydantoin 5-hydroxy- thymine glycol Gh Sp uracil (cis & trans)

[0359] Base analogues cleavable by Thymine DNA glycosylase (TDG):

[0360] 5-formylcytosine 5-carboxycytosine Base analogues cleavable by Human Alkyladenine DNA glycosylase (hAAG):

[0361] 3-methyladenine 3-methylguanine 7-methylguanine 7-(2-chloroethyl)- 7-(2-hydroxyethyl)-

[0362] 7-(2-ethoxyethyl)- 1 ,2-bis-(7-guanyl)-ethane 1 ,N6-etheno- 1 , / V2-etheno- guanine adenine guanine

[0363] M2,3-etheno- A / 2,3-ethano- 5-formyluracil 5-hydroxymethyl- hypoxanthine guanine guanine uracil Bases cleavable by uracil DNA glycosylase:

[0364] Bases cleavable by Human single-strand-selective monofunctional uracil-DNA Glycosylase (SMUG1): Bases cleavable by 5-methylcytosine DNA glycosylase (ROS1):

[0365] (see S. S. David, S. D. Williams Chemical reviews 1998, 98, 1221-1262 and M. I. Ponferrada-Marin, T. Roldan-Arjona, R. R. Ariza’ Nucleic Acids Res 2009 ,37, 4264- 4274).

[0366] A preferred universal nucleotide is 2’ -deoxyinosine.

[0367] Nucleotide-Incorporating Enzymes

[0368] In any of the 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.

[0369] In certain situations, it may be desirable to incorporate modified nucleotides, such as nucleotides having attached reversible terminator groups, as described herein, in which case polymerase enzymes may be chosen based on their ability to incorporate modified nucleotides.

[0370] Thus, the polymerase may be a modified polymerase having an enhanced ability to incorporate a nucleotide comprising a reversible terminator group compared to an unmodified polymerase. The polymerase is more preferably a genetically engineered variant of the native DNA polymerase from Thermococcus species 9°N, preferably species 9°N-7. Examples of modified polymerases are Therminator IX DNA polymerase and Therminator X DNA polymerase available from New England BioLabs. This enzyme has an enhanced ability to incorporate 3’-O-modified dNTPs. Examples of other polymerases that can be used for incorporation of reversible terminator dNTPs in any of the methods of the invention are Deep Vent (exo-), Vent (Exo-), 9°N DNA polymerase, Therminator DNA polymerase, Therminator IX DNA polymerase, Therminator X DNA polymerase, KI enow fragment (Exo-), Bst DNA polymerase, Bsu DNA polymerase, Sulfolobus DNA polymerase I, and Taq Polymerase.

[0371] 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.

[0372] To copy a template strand comprising DNA, a DNA polymerase may be used. 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 (KI enow) fragment, DNA Pol I large (KI enow) 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.

[0373] 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.

[0374] In any of the methods described herein it may be desirable to perform one or more additional method steps to extend one or both strands of the acceptor polynucleotide as part of the process of extending the acceptor polynucleotide by the methods of the invention, e.g. before, during or after a process of extending the acceptor polynucleotide using the ligase-mediated methods of the invention. It may be desirable to extend one or both strands as part of a double stranded acceptor polynucleotide. It may also be desirable to extend one or both strands as single stranded polynucleotides following separation of the two strands of the acceptor polynucleotide. In any such additional method step the enzyme may have a terminal transferase activity, e.g. the enzyme may be a terminal nucleotidyl transferase, or terminal deoxynucleotidyl transferase, and wherein the acceptor polynucleotide is extended to form a polynucleotide molecule comprising DNA or RNA, preferably DNA. Any of these enzymes may be used in the methods of the invention wherein such extension of an acceptor polynucleotide is required.

[0375] One such enzyme is a terminal nucleotidyl transferase enzyme, such as terminal deoxynucleotidyl transferase (TdT) (see e.g. Motea et al, 2010; Minhaz Ud-Dean, Syst. Synth. Biol., 2008, 2(3-4), 67-73). TdT is capable of catalysing the addition to a polynucleotide of a nucleotide molecule (nucleoside monophosphate) from a nucleoside triphosphate substrate (NTP or dNTP). TdT is capable of catalysing the addition of natural and non-natural nucleotides. It is also capable of catalysing the addition of nucleotide analogues (Motea et al, 2010). Pol lambda and pol micro enzymes may also be used (Ramadan K, et al., J. Mol. Biol., 2004, 339(2), 395-404), as may 29 DNA polymerase.

[0376] Techniques for the extension of a single-stranded polynucleotide molecule, both DNA and RNA, in the absence of a template by the action of a terminal transferase enzyme (e.g. terminal deoxynucleotidyl transferase; TdT) to create an artificially- synthesised single-stranded polynucleotide molecule has been extensively discussed in the art. Such techniques are disclosed in, for example, Patent application publications WO20 16 / 034807, WO 2016 / 128731, WO2016 / 139477 and WO2017 / 009663, as well as US2014 / 0363852, US2016 / 0046973, US2016 / 0108382, and US2016 / 0168611. These documents describe the controlled extension of a single-stranded polynucleotide synthesis molecule by the action of TdT to create an artificially-synthesised singlestranded polynucleotide molecule. Extension by natural and non-natural / artificial nucleotides using such enzymes is described, as is extension by modified nucleotides, for example, nucleotides incorporating blocking groups. Any of the terminal transferase enzymes disclosed in these documents may be applied to methods of the present invention, as well as any enzyme fragment, derivative, analogue or functional equivalent thereof provided that the terminal transferase function is preserved in the enzyme.

[0377] Directed evolution techniques, conventional screening, rational or semi-rational engineering / mutagenesis methods or any other suitable methods may be used to alter any such enzyme to provide and / or optimise the required function. Any other enzyme which is capable of extending a single-stranded polynucleotide molecule portion, such as a molecule comprising DNA or RNA, or one strand of a blunt-ended molecule with a nucleotide without the use of a template may be used. Thus, in any of the methods defined herein a single stranded portion of a polynucleotide comprising DNA or blunt-ended double-stranded polynucleotide comprising DNA may be extended by an enzyme which has template-independent enzyme activity, such as template-independent polymerase or transferase activity. The enzyme may have nucleotidyl transferase enzyme activity, e.g. a deoxynucleotidyl transferase enzyme, such as terminal deoxynucleotidyl transferase (TdT), or an enzyme fragment, derivative, analogue or functional equivalent thereof. A polynucleotide extended by the action of such an enzyme comprises DNA.

[0378] A single stranded portion of an acceptor polynucleotide comprising RNA, or blunt-ended double-stranded acceptor polynucleotide comprising RNA may be extended by an enzyme which has nucleotidyl transferase enzyme (e.g. including TdT), or an enzyme fragment, derivative, analogue or functional equivalent thereof. An acceptor polynucleotide extended by the action of such an enzyme may comprise RNA. For the synthesis of a single stranded polynucleotide molecule comprising RNA, or a single stranded portion of a polynucleotide molecule comprising RNA, any suitable nucleotidyl transferase enzyme may be used. Nucleotidyl transferase enzymes such as poly (U) polymerase and poly(A) polymerase (e.g. from E. coli) are capable of template-independent addition of nucleoside monophosphate units to polynucleotide synthesis molecules. Any of these enzymes may be applied to methods described herein, as well as any enzyme fragment, derivative, analogue or functional equivalent thereof provided that the nucleotidyl transferase function is preserved in the enzyme. Directed evolution techniques, conventional screening, rational or semi-rational engineering / mutagenesis methods or any other suitable methods may be used to alter any such enzyme to provide and / or optimise the required function.

[0379] Ligation and Ligase Enzymes

[0380] 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 ligase may be a T3 DNA ligase or a T4 DNA ligase

[0381] The enzyme may be human DNA ligase III, T3 DNA ligase, T4 DNA ligase, a T4 DNA ligase which has improved thermal stability compared to wild-type T4 DNA ligase, or a T4 DNA ligase which has improved salt tolerance compared to wild-type T4 DNA ligase. The ligase may a blunt TA ligase. For example, a blunt TA ligase is available from New England BioLabs (NEB). This is a ready -to-use master mix solution of T4 DNA Ligase, ligation enhancer, and optimized reaction buffer specifically formulated to improve ligation. 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. Molecules, enzymes, chemicals and methods for ligating (joining) single- and double-stranded polynucleotides are well known to the skilled person.

[0382] Synthetic Polynucleotide

[0383] 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.

[0384] 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 hybridizes in 6xSSC at 45° C, followed by one or more washes in O. lxSSC, 0.2% SDS at 68° C.

[0385] 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)).

[0386] 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).

[0387] 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.

[0388] 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.

[0389] RNA Synthesis

[0390] The methods described herein may be adapted for the synthesis of RNA.

[0391] 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 hybridized 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. Solid Phase Synthesis

[0392] 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 are known in the art and may be used. Before initiating synthesis of a new double-stranded polynucleotide of predefined sequence, acceptor polynucleotides may be immobilised 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 immobilised to a gel-based material such as e.g. polyacrylamide, and wherein the gel -based material is bound to a supporting substrate such as glass.

[0393] Polynucleotides may be immobilised to surfaces directly or indirectly. For example they may be attached directly to surfaces by chemical bonding. They may be, for instance, covalently joined to the surface. They may be indirectly joined 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 immobilised.

[0394] In such methods a double-stranded acceptor polynucleotide may be immobilised to a surface prior to the incorporation of the first payload. Such an immobilised 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.

[0395] Only one strand of such a double-stranded acceptor polynucleotide may be immobilised to the surface at the same end of the molecule (for example as depicted schematically in Figure 3). Alternatively, both strands of a double-stranded acceptor polynucleotide may each be immobilised to the surface at the same end of the molecule. Solid Phase Synthesis on Planar Surfaces

[0396] 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 joined to a surface. Such methods may also be used to synthesise and join polynucleotides to a surface.

[0397] Pre-formed polynucleotides can be joined 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.

[0398] 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 immobilise preformed acceptor polynucleotides.

[0399] 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). 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.

[0400] It may be that the acceptor and tether polynucleotides are immobilised on the surface at the same time. It may be that the two are mixed together at a chosen ratio and then immobilised onto the support at the same time. Alternatively, it may be that one type of polynucleotide is immobilised on the support and then the other type of polynucleotide is, for instance the acceptor polynucleotides may be immobilised or synthesised first on the surface and then the tether polynucleotides also immobilised on it. Alterantively, it may be that the tether polynucleotides are immobilised or synthesized on the support and then the acceptor polynucleotides are then immobilised on the support.

[0401] An example of how acceptor polynucleotides may be joined to the support is a 5”-thiophosphate-Sp9-Sp-Sp9 group. Sp9 is a tri ethylene glycol chain that is 9 atoms long (6 carbons + 3 oxygens). Such a group may also be used to join tether polynucleotides to a support.

[0402] One option to aid in recovery of the recovery of the predefined sequence is that an acceptor polynucleotide may comprise a cleavage site between the second terminal end of the acceptor and the start of the payload sequence that has been built up, to allow for eventual cleavage and recovery of the synthesized sequence. Any suitable cleavage site may be used.

[0403] Reversible Immobilization

[0404] 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.).

[0405] 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.

[0406] Microparticles and beads may be allowed to move freely within a reaction solution and then reversibly immobilised, 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.

[0407] Thus, before initiating synthesis of a new double-stranded polynucleotide of predefined sequence, acceptor and tether polynucleotides in accordance with the invention can be synthesised and then reversibly immobilised to such binding surfaces. It may be the acceptor and tether polynucleotides are synthesised separately, mixed, and then immobilised on the support. The two may be mixed in a chosen ratio. Polynucleotides synthesised by methods of the invention can be synthesised whilst reversibly immobilised to such binding surfaces.

[0408] Microfluidic techniques and systems

[0409] 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 immobilise polynucleotides during and after synthesis. Polynucleotides having a predefined sequence may be synthesised in solid phase by methods described herein, wherein polynucleotides are immobilised 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.

[0410] 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.

[0411] 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 1pm 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)

[0412] Sequencing of Intermediate or Final Synthesis Products

[0413] 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.

[0414] Surface Attachment Chemistries

[0415] 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.

[0416] 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.

[0417] Some examples of chemistries suitable for attaching polynucleotides to surfaces are shown in Figures 16 and 17.

[0418] In any of the methods described herein, polynucleotides may be immobilised 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).

[0419] 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.

[0420] A linker may be a linear linker or a branched linker.

[0421] 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)-).

[0422] 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.

[0423] 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.

[0424] 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.

[0425] 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. The same chemistries may be used to join the tether polynucleotides to the support. It may be that the acceptor and tether polynucleotides are joined to the support at the same time.

[0426] 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. The same approach may be used for a tether polynucleotide.

[0427] 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. The same approach may be used for the tether polynucleotide.

[0428] 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. The same approach may be used for the tether polynucleotide.

[0429] 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. The same approach may be used for the tether polynucleotide.

[0430] Some non-limiting exemplary branching nucleotides are shown below. linker to 5" end

[0431] 5-methylC brancher nucleotide

[0432]

[0433] 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

[0434] 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.

[0435] 3' direction to 3' direction to brancher brancher point 5"endpoint 5" end 3' direction to brancher point 5" end

[0436] Sp18 unit

[0437] A linker may comprise one or more ethylene glycol units.

[0438] A linker may comprise an oligonucleotide, wherein multiple units are nucleotides.

[0439] 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.

[0440] In any of the methods described herein:

[0441] (i) the first and second strands of the acceptor polynucleotide at the second terminal end may each be tethered to a surface; or

[0442] (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

[0443] (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

[0444] (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.

[0445] In any of the methods described herein:

[0446] (i) the first and second strands of the tether polynucleotide at the second terminal end may each be joined to a surface or immobilised on it; or (ii) the first and second strands of the tether polynucleotide at the second terminal end may connected together by a polynucleotide hairpin loop and tethered to a surface; or

[0447] (iii) the first strand of the tether polynucleotide at the second terminal end may be joined to a surface and the second strand of the tether polynucleotide at the second terminal end may not be joined to the surface; or

[0448] (iv) the second strand of the acceptor polynucleotide at the second terminal end may be joined to a surface and the first strand of the acceptor polynucleotide at the second terminal end may be untethered.

[0449] 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.

[0450] 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.

[0451] The cleavable linker may be a UV cleavable linker.

[0452] The strand(s) joined or immobilised on the support 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.

[0453] It may be that the same approach is used for immobilisation of both the acceptor and tether polynucleotides. It may though be where there is an eventual cleavage step to release the synthesized polynucleotide sequence that the acceptor polynucleotide comprises the cleavage site allowing for release of the synthesized sequence but that the tether polynucleotide does not comprise such a cleavage site. Microarrays

[0454] 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 and tether 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.

[0455] 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.

[0456] 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 and tether polynucleotides may be joined to, or immobilised on, surfaces using known surface attachment methods and chemistries, including those described herein.

[0457] Following synthesis of the polynucleotides of predefined sequence, there may be provided a final cleavage step to release the desired sequence synthesised so also remove any unwanted polynucleotide sequence from the acceptor polynucleotides with the desired sequence joined to it.

[0458] It may be that the desired sequence synthesised includes a cleavage site within that sequence such as a restriction enzyme recognition site. The cleavage site may be at the start of the predefined sequence synthesised. It may be that the support comprises not just the tether polynucleotides that hybridize to the donor polynucleotides, but also a second tether polynucleotide that acts to localise an enzyme for cleavage and release of the predefined sequence. Such a further tether polynucleotide may help increase the local concentration of the enzyme meaning less of it is needed.

[0459] 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 joining of only one of the two strands of the acceptor polynucleotide at the second terminal end to the support may mean that the desired single strand can be recovered more readily. Separation of strands may be performed by conventional methods, such as heat treatment.

[0460] Assembly of Synthetic Polynucleotides

[0461] 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.

[0462] 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.

[0463] 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 immobilised 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 immobilised 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. 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.

[0464] 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.

[0465] 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.

[0466] Examples of cleavage methods involving enzymatic cleavage are shown in the Examples.

[0467] Thus, polynucleotides having a predefined sequence may be synthesised whilst immobilised 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. Supports, Systems and Kits

[0468] The invention also provides supports and 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.

[0469] Synthesis cycle reactions will be carried out by incorporating nucleotides of predefined sequence into acceptor polynucleotide molecules which are immobilised on a support by means described and defined herein. The support may be any suitable surface as described and defined herein.

[0470] Accordingly there is provided a support for synthesising a double-stranded polynucleotide having a predefined sequence, the support comprising:

[0471] (A) a population of acceptor polynucleotides, wherein each acceptor polynucleotide has a first strand and a second strand and wherein one end is immobilised on the support and the opposite end is free, and wherein the free end is blunt ended; and

[0472] (B) a population of tether polynucleotides disbursed amongst the acceptor polynucleotides, wherein each tether polynucleotide has one end immobilised on the support and wherein the opposite end is free, and wherein the free end comprises a first hybridisation arm (hyb arm) which is a single-stranded sequence region.

[0473] 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.

[0474] 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.

[0475] 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.

[0476] 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 give rise to synthetic polynucleotide molecules having the same or substantially the same nucleotide sequence.

[0477] In one embodiment a synthesis system for carrying out any of the synthesis methods described and defined herein may comprise any surface described or defined herein or 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.

[0478] A system having any surface described or defined herein or 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.

[0479] 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.

[0480] In cases where the invention employs a support with areas with different acceptor polynucleotides each area may comprise a different tether polynucleotide, so that the pair of acceptor and tether polynucletide for each area are specific for each other, but different to those in the other areas. 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, tether 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, 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.

[0481] The present invention also provides a support with the chosen acceptor and donor polynucleotides already immobilised on the support. Such a support may be provided in the form of a kit that also comprises donor polynucleotides and optionally any of the other reagents set out herein for performing a method of the invention. Such a kit may further comprise the ligase. It may also comprise the polymerase.

[0482] Data Storage

[0483] 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. 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.

[0484] 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.

[0485] 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.

[0486] 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.

[0487] Reversible Terminator

[0488] 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.

[0489] 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.

[0490] Examples of reversible terminators are provided below.

[0491] Propargyl reversible terminators:

[0492] Allyl reversible terminators: Cyclooctene reversible terminators:

[0493] Cyanoethyl reversible terminators:

[0494] Nitrobenzyl reversible terminators: Disulfide reversible terminators: Azidomethyl reversible therminators:

[0495] Aminoalkoxy reversible therminators:

[0496] 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

[0497] X = O, S, NH, CH2Z = bulky group

[0498] Exemplary Methods

[0499] The methods set out below may be preceded by an activation step or other steps not forming part of the cycle for payload delivery. For example, Figure 2 illustrates prior to starting cycles of synthesis there may be an activation step to cleave the acceptor molecules so that they have blunt first terminal ends ready for ligation.

[0500] In the methods set out below as well as immobilised acceptor polynucleotide, the support will also have immobilised tether polynucleotide. The immobilised tether polynucleotide can be the same for all of the exemplary methods set out below namely that the tether polynucleotides can hybridize, but not ligate, to the donor polynucleotide in the cycles. Given that tether polynucleotides will play the same role in the methods set out below, the tether polynucleotides will be described first before setting out the exemplary synthesis methods. The tether polynucleotides and their function will not be set out in detail for each method given that they are typically the same for each method.

[0501] The tether polynucleotide will either be single or double stranded. In either case it will comprise a single stranded portion at the first terminal end representing a hyb arm which is complementary to the hyb arm of the donor polynucleotide. In the case that the tether polynucleotide is single stranded, the second terminal end, i.e. that at the opposite end to the hyb arm, will be immobilised on the support. In the case where the tether polynucleotide is double stranded, at least one strand at the second terminal end will be immobilised on, or joined to, the support. In some cases, both strands of the tether polynucleotide will be joined to, or immobilised on, the support at the second terminal end of the tether polynucleotide. In others embodiment, only one strand will be joined to the support.

[0502] The tether polynucleotides will be present as a chosen ratio to the acceptor polynucleotides in terms of the number of tether polynucleotides compared to the number of acceptor polynucleotides. The ratio will be one effective so that the amount of acceptor: donor ligation product formed relative to acceptor: acceptor ligation product is improved relative to in the absence of the tether polynucleotides and so with acceptor polynucleotides, without a substantial drop in the yield of acceptor: donor ligation products. This can be optimized by the user to fit the circumstances.

[0503] For the purposes of describing the methods set out below, the terms “suface” and “support” are used interchangeably.

[0504] Method version 1

[0505] Provision of acceptor polynucleotide

[0506] To initiate a cycle of synthesis an acceptor polynucleotide is first provided. As discussed above, the surface will also have a tether polynucleotide provided immobilised on it helping to localize the donor polynucleotide near to the acceptor polynucleotide without the tether and donor ligating to each other.

[0507] 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.

[0508] The first terminal end of the acceptor polynucleotide is ligatable and blunt- ended. The first terminal end is free, i.e. neither the first strand nor the second strand is joined to or otherwise attached to any other structure.

[0509] 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 does not comprise a 5’ phosphate group. The (3’) terminal nucleotide of the second strand at the ligatable first terminal end may or may not comprise a 3’ hydroxyl group. The second terminal end of the acceptor polynucleotide is preferably non- ligatable.

[0510] The second terminal end of the acceptor polynucleotide is immobilised on the surface. Such a surface may be any suitable surface as described and defined elsewhere herein. The second terminal end may be joined to a surface due to the second strand of the acceptor polynucleotide being joined to the surface whilst the first strand of the acceptor polynucleotide is not so joined to the surface. Alternatively, the second terminal end may be joined to a surface due to the first strand of the acceptor polynucleotide being joined to the surface whilst the second strand of the acceptor polynucleotide is not joined or immobiled to the surface. Alternatively still, the second terminal end may be joined to a surface due to the first and second strands of the acceptor polynucleotide being joined to the surface. Where both the first and second strands of the acceptor polynucleotide are joined to the surface, each strand may be independently joined 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 joined to the surface.

[0511] 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 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.

[0512] Provision of donor polynucleotide

[0513] To initiate a cycle of synthesis a donor polynucleotide is also provided.

[0514] 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 region, with a single stranded polynucleotide region representing a hyb arm that is able to hybridize to the hyb arm of the tether polynucleotide, but where the two cannot ligate to each other.

[0515] The first terminal end of the donor polynucleotide is ligatable and blunt-ended. The first terminal end is free, i.e. neither the first strand nor the second strand is joined to or otherwise attached to any other structure and in particular not to a surface.

[0516] The second terminal end is free, i.e. neither the first strand nor the second strand is attached to any other structure. The second terminal end includes a single stranded region representing the hyb arm which is complementary to the hyb arm of the tether polynucleotide.

[0517] 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 does not comprise a 5’ phosphate group. The (3’) terminal nucleotide of the first strand at the ligatable first terminal end comprises a 3’ hydroxyl group.

[0518] The second terminal end of the donor polynucleotide will not ligate to other donor polynucleotide molecules. It will also not ligate to the tether polynucleotide when hybridized to it. It may be that it will not ligate to the tether polynucleotide because when the two are hybridized there is a gap at the termini between the strand of the hyb arm of the tether and the terminal nucleotide of the same sense strand of the donor, as well as gap at the termini of the strand of the hyb arm of the donor and the terminal nucleotide of the same sense strand of the tether. Alternatively, blocking groups may be used to prevent ligation. 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.

[0519] 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 terminal nucleotide 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 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 a nucleotide pair, and this pair is the second pair of nucleotides in the polynucleotide payload, and so on.

[0520] 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.

[0521] Ligation of acceptor and donor polynucleotides

[0522] As discussed above, the tether polynucleotides present hybridize to the donor polynucleotides but do not ligate with them, serving to bring donor polynucleotides into closer proximity with the acceptor polynucleotides and reduce the rate of acceptor: acceptor ligation product formation.

[0523] 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 ligating the blunt ends of the acceptor and donor polynucleotides to form a ligated polynucleotide. In particular, the ligation step comprises ligating the first terminal end of the donor polynucleotide to the first terminal end of the acceptor polynucleotide.

[0524] 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, and 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 wherein the first strands of the donor and acceptor polynucleotides are ligated together at their first terminal ends 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.

[0525] The step of ligating the donor and acceptor polynucleotides creates a ligated polynucleotide.

[0526] Donor polynucleotide wash-out (optional)

[0527] After ligation a washout may be performed to removed donor polynucleotides that are either free or hybridized to tether polynucleotides but not ligated to the acceptor polynucleotides. The temperature may be increased above the melting temperature of the hybridized hyb arms of the tether and donor polynucleotides so that hybridized donor polynucleotides are released. The donor polynucleotides may then be washed out to remove them.

[0528] Acceptor depletion (optional)

[0529] 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.

[0530] 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. 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.

[0531] 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.

[0532] 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. Cleavage

[0533] 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.

[0534] 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.

[0535] In method version 1, the cleavage step may comprise cleaving the ligated polynucleotide at sites in both the first and second strands of the donor polynucleotide. Alternatively, the cleavage step may comprise cleaving the ligated polynucleotide at a site in only the first strand of the donor polynucleotide.

[0536] The cleavage step may comprise cleaving the first strand of the ligated polynucleotide and cleaving the second strand of the ligated polynucleotide at the same relative position in each strand. Such a cleavage step is consequently performed as a symmetrical cleavage reaction, so as to form a blunt end at the cleaved first terminal end of the acceptor polynucleotide, wherein initially all pairs of nucleotides of the polynucleotide payload are retained 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 in that cycle.

[0537] In the case of a symmetrical cleavage reaction, the nucleotides of the polynucleotide payload of the second strand at the first terminal end of the cleaved acceptor polynucleotide are separated from the donor polynucleotide and initially remain attached to the cleaved acceptor polynucleotide. As a consequence of the nick site previously introduced into the second strand, the nucleotides of the polynucleotide payload of the second strand remain attached only via interaction (e.g. hydrogen bonding) with the nucleotides of the polynucleotide payload of the first strand. The nucleotides of the polynucleotide payload of the second strand are subsequently discarded. These steps in effect generate a 5’ overhang at the cleaved first terminal end of the acceptor polynucleotide.

[0538] In method version 1, the cleavage step may alternatively comprise cleaving the first strand of the ligated polynucleotide only. A nick site is already present in the second strand. Cleavage is performed in such a way that the first strand is cleaved at a different relative position compared to the nick site in the second strand. Such a cleavage step consequently results in an asymmetrical cleavage.

[0539] 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 nucleotides of the polynucleotide payload (in the direction proximal to the second terminal end of the acceptor polynucleotide). Accordingly, the nucleotides of the polynucleotide payload in the second strand remain attached to the donor polynucleotide following cleavage. Following asymmetrical cleavage, method version 1 further comprises separating the cleaved donor polynucleotide from the acceptor polynucleotide. Accordingly, because the original nucleotides of the polynucleotide payload in the second strand remain attached to the donor polynucleotide following cleavage, they are consequently discarded. These steps generate the 5’ overhang at the cleaved first terminal end of the acceptor polynucleotide. The nucleotides of the first strand of the polynucleotide payload overhang the second strand of the acceptor polynucleotide, wherein the terminal nucleotide of the overhang is the final nucleotide of the polynucleotide payload.

[0540] Following an asymmetrical cleavage reaction, after separating the cleaved donor polynucleotide from the acceptor polynucleotide, method version 1 further comprises performing an incorporation step comprising extending the second strand of the acceptor polynucleotide by incorporating new payload nucleotides using the original payload nucleotides of the first strand in the overhang as templates, thereby re-forming the payload nucleotides in the second strand, thereby re-forming the payload nucleotide pairs in the ligated 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. Preferably, incorporation of new payload nucleotides is performed by the action of an enzyme having polymerase activity.

[0541] In an asymmetrical cleavage reaction, cleavage of the first strand of the ligated polynucleotide is performed in such a way that a 5’ phosphate group is retained on the terminal nucleotide of the first strand at the cleaved first terminal end of the acceptor polynucleotide, i.e. on the final nucleotide of the polynucleotide payload in the first strand. This is achieved as a consequence of any standard cleavage reaction. Furthermore, incorporation is performed in such a way that a 3’ hydroxyl group is retained on the terminal nucleotide of the second strand at the cleaved first terminal end of the acceptor polynucleotide, i.e. on the final nucleotide of the polynucleotide payload in the second strand. Thus, method version 1 is performed such that following cleavage and following incorporation steps, the first terminal end of the cleaved acceptor polynucleotide comprising the polynucleotide payload is ligatable and is thus competent to be ligated to a further donor polynucleotide in the next cycle of synthesis.

[0542] As described previously, if a symmetrical cleavage reaction is performed, following cleavage the original nucleotides of the polynucleotide payload of the second strand remain attached only via interaction (e.g. hydrogen bonding) with the original nucleotides of the polynucleotide payload of the first strand. The original nucleotides of the polynucleotide payload of the second strand may be separated from the first strand:

[0543] (i) before the incorporation step; or

[0544] (ii) during the incorporation step.

[0545] 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:

[0546] (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

[0547] (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.

[0548] The cleavage step can be performed by any suitable means for creating the cleaved structures described above.

[0549] As described above, cleavage may comprise a double-stranded cleavage wherein both the first and second strands are cleaved. Cleavage may comprise cleaving the sugar-phosphate backbone of the first and second strands of the donor polynucleotide molecule. In such a cleavage step both the first and second strands are cleaved at the same positions in a symmetric cleavage reaction. This generates a cleaved donor polynucleotide wherein the first terminal end is blunt-ended. 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, the type IIS restriction enzyme may be Mlyl. The user will readily be able to structure the cleavage site in the donor polynucleotide in a manner that allows the required structure described above to be formed following cleavage.

[0550] As described above, cleavage may comprise a single-stranded cleavage wherein only the first strand is cleaved. Cleavage of only the first strand can be performed in view of the nick site introduced previously into the second strand. In the case of a single-stranded cleavage reaction, the nucleotides of the second strand of the polynucleotide payload at the first terminal end of the donor polynucleotide remain attached to the donor polynucleotide at its first terminal end and are consequently discarded. These steps generate the 5’ overhang at the cleaved first terminal end of the acceptor polynucleotide.

[0551] A single-stranded cleavage may comprises cleaving the sugar-phosphate backbone of the first strand of the donor polynucleotide and breaking the hydrogen bonds between the one or more payload nucleotide pairs.

[0552] Single-stranded cleavage may thus be performed by the action of an enzyme having overhang cleavage function, preferably a type IIS restriction enzyme, such as BspQI. Single-stranded cleavage may alternatively be performed by the action of an enzyme having nicking cleavage function, preferably a type IIS restriction enzyme, optionally Nt.As / .

[0553] Single-stranded cleavage may alternatively be performed using a method 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. The universal nucleotide may be inosine. Cleavage mechanisms using universal nucleotides are described elsewhere herein.

[0554] Single-stranded cleavage may alternatively be performed using a method wherein the 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.

[0555] Following the cleavage and incorporation steps, a new acceptor polynucleotide is created. 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 double-stranded polynucleotide having a predefined sequence, by allowing multiple polynucleotide payloads to be successively joined together.

[0556] During this step of the method the tether polynucleotide should typically play no role and simply be present ready for the next cycle of the method in the same form as it was present at the start of the previous cycle because no payload sequence is added to the tether polynucleotide. Method version 2

[0557] Provision of acceptor polynucleotide

[0558] To initiate a cycle of synthesis an acceptor polynucleotide is first provided. As discussed above, the surface will also have tether polynucleotide provided immobilised on it helping to localize donor polynucleotide near to the acceptor polynucleotides without the tether and donor ligating to each other.

[0559] 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.

[0560] The first terminal end of the acceptor polynucleotide is ligatable and blunt- ended. The first terminal end is free, i.e. neither the first strand nor the second strand is joined to or otherwise attached to any other structure.

[0561] 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 does not comprise a 5’ phosphate group. The (3’) terminal nucleotide of the second strand at the ligatable first terminal end may or may not comprise a 3’ hydroxyl group.

[0562] The second terminal end of the acceptor polynucleotide is preferably non- ligatable.

[0563] The second terminal end of the acceptor polynucleotide is preferably joined to a surface. Such a surface may be any suitable surface as described and defined elsewhere herein. The second terminal end may be joined to a surface due to the second strand of the acceptor polynucleotide being joined to the surface whilst the first strand of the acceptor polynucleotide is unjoined. Alternatively, the second terminal end may be joined to a surface due to the first strand of the acceptor polynucleotide being joined to the surface whilst the second strand of the acceptor polynucleotide is not joined. Alternatively still, the second terminal end may be joined to a surface due to the first and second strands of the acceptor polynucleotide being joined to the surface. Where both the first and second strands of the acceptor polynucleotide are joined 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 joined to the surface.

[0564] 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 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.

[0565] Provision of donor polynucleotide

[0566] To initiate a cycle of synthesis a donor polynucleotide is also provided. 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 double-stranded polynucleotide region and with a single stranded polynucleotide region representing a hyb arm that is able to hybridize to the hyb arm of the tether, but where the two cannot ligate to each other.

[0567] The first terminal end of the donor polynucleotide is ligatable and blunt-ended. The first terminal end is free, i.e. neither the first strand nor the second strand is joined to or otherwise attached to any other structure.

[0568] The second terminal end is free, i.e. neither the first strand nor the second strand is attached to any other structure. The second terminal end includes a single stranded region representing the hyb arm which is complementary to the hyb arm of the tether polynucleotide.

[0569] 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 does not comprise 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 will not ligate to other donor polynucleotide molecules. It will also not ligate to the tether polynucleotide when hybridized to it. It may be that it will not ligate to the tether polynucleotide because when the two are hybridized there is a gap at the termini between the strand of the hyb arm of the tether and the terminal nucleotide of the same sense strand of the donor, as well as gap at the termini of the strand of the hyb arm of the donor and the terminal nucleotide of the same sense strand of the tether. Alternatively, blocking groups may be used to prevent ligation. 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.

[0570] 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 terminal nucleotide 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 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 a nucleotide pair, and this pair is the second pair of nucleotides in the polynucleotide payload, and so on.

[0571] 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. Ligation of acceptor and donor polynucleotides and first incorporation step

[0572] As discussed above, the tether polynucleotides present hybridize to the donor polynucleotides but do not ligate with them, serving to bring donor polynucleotides into closer proximity with the acceptor polynucleotides and reduce the rate of acceptor: acceptor ligation product formation.

[0573] 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 ligating the blunt ends of the acceptor and donor polynucleotides to form a ligated polynucleotide. In particular, the ligation step comprises ligating the first terminal end of the donor polynucleotide to the first terminal end of the acceptor polynucleotide.

[0574] In method version 2, 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, and 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 2, the ligation step comprises a single-stranded ligation wherein the first strands of the donor and acceptor polynucleotides are ligated together at their first terminal ends but the second strands of the donor and acceptor polynucleotides are not ligated together at their first terminal ends.

[0575] 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.

[0576] The step of ligating the donor and acceptor polynucleotides creates a ligated polynucleotide.

[0577] In method version 2, following the ligation step and before the cleavage step, the method further comprises performing a first incorporation step to extend the second strand of the acceptor polynucleotide from the nick site. The first incorporation step comprises synthesizing new nucleotides in the second strand using the nucleotides of the first strand as templates, preferably by the action of an enzyme having polymerase activity, thereby synthesizing a new second strand of the donor polynucleotide and reforming the nucleotide pairs in the ligated polynucleotide including the one or more payload nucleotide pairs and the cleavage site.

[0578] The original second strand of the donor polynucleotide may be separated from the first strand:

[0579] (i) before the first incorporation step; or

[0580] (ii) during the first incorporation step.

[0581] Where the original second strand of the donor polynucleotide is separated from the first strand during the first incorporation step, incorporation steps may be performed:

[0582] (a) by the action of an enzyme having polymerase activity, and wherein the polymerase displaces the second strand when synthesising the new second strand; or

[0583] (b) by the action of an enzyme having polymerase activity which possesses 5’ to 3’ exonuclease activity, and wherein the polymerase digests / degrades the second strand when synthesising the new second strand.

[0584] Donor polynucleotide wash-out (optional)

[0585] After ligation a washout may be performed to removed donor polynucleotides that are either free or hybridized to tether polynucleotides but not ligated to the acceptor polynucleotides. The temperature may be increased above the melting temperature of the hybridized hyb arms of the tether and donor polynucleotides so that hybridized donor polynucleotides are released. The donor polynucleotides may then be washed out to remove them.

[0586] Acceptor depletion (optional)

[0587] 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.

[0588] 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.

[0589] 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.

[0590] 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.

[0591] 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.

[0592] Cleavage

[0593] 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, whether part of the original donor polynucleotide, or newly synthesized (as described further below), 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 donor polynucleotide from the ligated 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.

[0594] 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.

[0595] In method version 2, the cleavage step comprises cleaving the ligated polynucleotide at sites in both the first and second strands of the donor polynucleotide.

[0596] The cleavage step may comprise cleaving the first strand of the ligated polynucleotide and cleaving the second strand of the ligated polynucleotide at the same relative position in each strand. Such a cleavage step is consequently performed as a symmetrical cleavage, so as to form a blunt end at the cleaved first terminal end of the acceptor polynucleotide, wherein all pairs of nucleotides of the polynucleotide payload are retained 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 in that cycle. In such a cleavage step, cleavage is performed such that following cleavage the first terminal end of the acceptor polynucleotide comprising the polynucleotide payload is ligatable. Following cleavage, the terminal nucleotide of the first strand at the ligatable first terminal end of the acceptor polynucleotide (i.e. one of the nucleotides of the final pair of nucleotides of the polynucleotide payload) comprises a 5’ phosphate group. The (3’) terminal nucleotide of the second strand at the ligatable first terminal end (i.e. the second nucleotide of the final pair of nucleotides of the polynucleotide payload) does not comprise a 5’ phosphate group. The (3’) terminal nucleotide of the second strand at the ligatable first terminal end comprises a 3’ hydroxyl group. This is achieved as a consequence of any standard symmetrical cleavage reaction.

[0597] In method version 2, the cleavage step may alternatively comprise cleaving the first strand of the ligated polynucleotide and cleaving the second strand of the ligated polynucleotide at different relative positions in each strand. Such a cleavage step is performed as an asymmetrical cleavage, so as to form a 5 ’overhang at the cleaved first terminal end of the acceptor polynucleotide. Following asymmetrical cleavage, the nucleotides of the first strand of the polynucleotide payload are retained at the cleaved first terminal end of the acceptor polynucleotide. The nucleotides of the first strand of the polynucleotide payload overhang the second strand of the acceptor polynucleotide, wherein the terminal nucleotide of the overhang is the final nucleotide of the polynucleotide payload.

[0598] Following asymmetrical cleavage, method version 2 further comprises separating the cleaved donor polynucleotide from the acceptor polynucleotide.

[0599] 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 cleaved acceptor polynucleotide is the final nucleotide of the polynucleotide payload. In an asymmetrical cleavage reaction, the second strand is cleaved below the nucleotides of the polynucleotide payload (in the direction proximal to the second terminal end of the acceptor polynucleotide). Accordingly, the nucleotides of the polynucleotide payload in the second strand remain attached to the donor polynucleotide following cleavage, and are consequently discarded. These steps generate the 5’ overhang at the cleaved first terminal end of the acceptor polynucleotide.

[0600] Following an asymmetrical cleavage reaction, after separating the cleaved donor polynucleotide from the acceptor polynucleotide, method version 2 further comprises performing a second incorporation step comprising extending the second strand of the acceptor polynucleotide by incorporating new payload nucleotides using the payload nucleotides of the first strand in the overhang as templates, thereby re-forming the payload nucleotides in the second strand, thereby re-forming the payload nucleotide pairs in the ligated 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. Preferably, incorporation of new payload nucleotides is performed by the action of an enzyme having polymerase activity.

[0601] In an asymmetrical cleavage reaction, cleavage of the first strand of the ligated polynucleotide is performed in such a way that a 5’ phosphate group is retained on the terminal nucleotide of the first strand at the cleaved first terminal end of the acceptor polynucleotide, i.e. on the final nucleotide of the polynucleotide payload in the first strand. This is achieved as a consequence of any standard cleavage reaction. Furthermore, incorporation is performed in such a way that a 3’ hydroxyl group is retained on the terminal nucleotide of the second strand at the cleaved first terminal end of the acceptor polynucleotide, i.e. on the final nucleotide of the polynucleotide payload in the second strand. Thus method version 2 is performed such that following cleavage and following incorporation steps, the first terminal end of the cleaved acceptor polynucleotide comprising the polynucleotide payload is ligatable and is thus competent to be ligated to a further donor polynucleotide in the next cycle of synthesis. The cleavage step can be performed by any suitable means for creating the cleaved structures described above.

[0602] As described above, cleavage may comprise cleaving the first strand of the ligated polynucleotide and cleaving the second strand of the ligated polynucleotide at the same relative position in each strand. Such a cleavage step is consequently performed as a symmetrical cleavage, so as to form a blunt end at the cleaved first terminal end of the acceptor polynucleotide. Cleavage may comprise cleaving the sugar-phosphate backbone of the first and second strands of the donor polynucleotide molecule. 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, the type IIS restriction enzyme may be Mlyl. The user will readily be able to structure the cleavage site in the donor polynucleotide in a manner that allows the required structure described above to be formed following cleavage.

[0603] As described above, cleavage may comprise an asymmetrical cleavage reaction, wherein the first strand of the ligated polynucleotide and the second strand of the ligated polynucleotide are cleaved at different relative positions in each strand. In the case of an asymmetrical cleavage reaction, the nucleotides of the second strand of the polynucleotide payload at the first terminal end of the donor polynucleotide remain attached to the donor polynucleotide at its first terminal end and are consequently discarded. These steps generate the 5’ overhang at the cleaved first terminal end of the acceptor polynucleotide.

[0604] An asymmetrical cleavage reaction may comprises cleaving the sugar-phosphate backbone of the first and second strands of the ligated polynucleotide and breaking the hydrogen bonds between the one or more payload nucleotide pairs.

[0605] Asymmetrical cleavage may thus be performed by the action of an enzyme having overhang cleavage function, preferably a type IIS restriction enzyme, such as BspQI.

[0606] Asymmetrical cleavage may alternatively be performed using a method 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. The universal nucleotide may be inosine. Cleavage mechanisms using universal nucleotides are described elsewhere herein.

[0607] Asymmetrical cleavage may alternatively be performed using a method wherein the 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.

[0608] Following the incorporation and cleavage steps, a new acceptor polynucleotide is created. 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 double-stranded polynucleotide having a predefined sequence, by allowing multiple polynucleotide payloads to be successively joined together.

[0609] During this step of the method the tether polynucleotide should typically play no role and simply be present ready for the next cycle of the method in the same form as it was present at the start of the previous cycle because no payload sequence is added to the tether polynucleotide.

[0610] Method version 3

[0611] Provision of acceptor polynucleotide

[0612] To initiate a cycle of synthesis an acceptor polynucleotide is first provided. As discussed above, the surface will also have tether polynucleotide provided immobilised on it helping to localize donor polynucleotide near to the acceptor polynucleotides without the tether and donor ligating to each other.

[0613] 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.

[0614] The first terminal end of the acceptor polynucleotide is ligatable and blunt- ended. The first terminal end is free, i.e. neither the first strand nor the second strand is joined to or otherwise attached to any other structure.

[0615] 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 does not comprise a 5’ phosphate group. The (3’) terminal nucleotide of the second strand at the ligatable first terminal end may or may not comprise a 3’ hydroxyl group.

[0616] The second terminal end of the acceptor polynucleotide is preferably non- ligatable.

[0617] 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.

[0618] 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 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.

[0619] Provision of donor polynucleotide

[0620] To initiate a cycle of synthesis a donor polynucleotide is also provided.

[0621] 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 region and with a single stranded polynucleotide region representing a hyb arm that is able to hybridize to the hyb arm of the tether polynucleotide, but where the two cannot ligate to each other.

[0622] The first terminal end of the donor polynucleotide is ligatable and blunt-ended. The first terminal end is free, i.e. neither the first strand nor the second strand is joined to or otherwise attached to any other structure.

[0623] The second terminal end is typically also free, i.e. neither the first strand nor the second strand is joined to or otherwise attached to any other structure. The second terminal end includes a single stranded region representing the hyb arm which is complementary to the hyb arm of the tether polynucleotide.

[0624] 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 does not comprise a 5’ phosphate group. The (3’) terminal nucleotide of the first strand at the ligatable first terminal end comprises a 3’ hydroxyl group.

[0625] The second terminal end of the donor polynucleotide will not ligate to other donor polynucleotide molecules. It will also not ligate to the tether polynucleotide when hybridized to it. It may be that it will not ligate to the tether polynucleotide because when the two are hybridized there is a gap at the termini between the strand of the hyb arm of the tether and the terminal nucleotide of the same sense strand of the donor, as well as gap at the termini of the strand of the hyb arm of the donor and the terminal nucleotide of the same sense strand of the tether. Alternatively, blocking groups may be used to prevent ligation. 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.

[0626] 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 terminal nucleotide 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 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 a nucleotide pair, and this pair is the second pair of nucleotides in the polynucleotide payload, and so on.

[0627] 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.

[0628] Ligation of acceptor and donor polynucleotides

[0629] As discussed above, the tether polynucleotides present hybridize to the donor polynucleotides but do not ligate with them, serving to bring donor polynucleotides into closer proximity with the acceptor polynucleotides and reduce the rate of acceptor: acceptor ligation product formation.

[0630] 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 ligating the blunt ends of the acceptor and donor polynucleotides to form a ligated polynucleotide. In particular, the ligation step comprises ligating the first terminal end of the donor polynucleotide to the first terminal end of the acceptor polynucleotide.

[0631] 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, and 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 4, the ligation step comprises a single-stranded ligation wherein the first strands of the donor and acceptor polynucleotides are ligated together at their first terminal ends but the second strands of the donor and acceptor polynucleotides are not ligated together at their first terminal ends.

[0632] In method version 4, after the step of joining the donor and acceptor polynucleotides at their first terminal ends, e.g. after a first ligation step comprising a single-stranded ligation wherein the first strands of the donor and acceptor polynucleotides are ligated together at their first terminal ends, a second step of joining the donor and acceptor polynucleotides at their first terminal ends is performed. A second ligation step may be performed comprising a second single-stranded ligation step wherein the second strands of the donor and acceptor polynucleotides are ligated together at their first terminal ends. Preferably, but not essentially, the second strands of the donor and acceptor polynucleotides are ligated together by steps comprising adding a phosphate group to the second strand of the donor polynucleotide at its first terminal end, preferably by the action of an enzyme having kinase activity, such as polynucleotide kinase (PNK); and joining the donor and acceptor polynucleotides at their first terminal ends by ligating the second strand of the donor polynucleotide with the second strand of the acceptor polynucleotide.

[0633] 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.

[0634] The step of ligating the donor and acceptor polynucleotides creates a ligated polynucleotide. Donor polynucleotide wash-out (optional)

[0635] After ligation a washout may be performed to removed donor polynucleotides that are either free or hybridized to tether polynucleotides but not ligated to the acceptor polynucleotides. The temperature may be increased above the melting temperature of the hybridized hyb arms of the tether and donor polynucleotides so that hybridized donor polynucleotides are released. The donor polynucleotides may then be washed out to remove them.

[0636] 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.

[0637] 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.

[0638] 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.

[0639] 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.

[0640] 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.

[0641] Cleavage

[0642] 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.

[0643] 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.

[0644] In method version 4, 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, at the end of the cycle of synthesis, a blunt end at the cleaved 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 terminal nucleotides of the cleaved first terminal end are the final pair of nucleotides of the polynucleotide payload in that cycle.

[0645] In method version 4, cleavage is performed such that following cleavage the first terminal end of the acceptor polynucleotide comprising the polynucleotide payload is ligatable. Following cleavage, the terminal nucleotide of the first strand at the ligatable first terminal end of the acceptor polynucleotide (i.e. one of the nucleotides of the final pair of nucleotides of the polynucleotide payload) comprises a 5’ phosphate group. The (3’) terminal nucleotide of the second strand at the ligatable first terminal end (i.e. the second nucleotide of the final pair of nucleotides of the polynucleotide payload) does not comprise a 5’ phosphate group. The (3’) terminal nucleotide of the second strand at the ligatable first terminal end comprises a 3’ hydroxyl group.

[0646] The cleavage step can be performed by any suitable means for creating the blunt-ended structure described above. Cleavage may comprise a double-stranded cleavage reaction wherein both the first and second strands are cleaved. In such a cleavage step both the first and second strands are cleaved at the same positions in a symmetric cleavage reaction. This generates a cleaved donor polynucleotide wherein the first terminal end is blunt-ended. As noted previously, 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, the type IIS restriction enzyme may be Mlyl. The user will readily be able to structure the cleavage site in the donor polynucleotide in a manner that allows the blunt-ended structure described above to be formed following cleavage.

[0647] Although not depicted in the non-limiting schematic shown in Figure 7, the cleavage step can also be performed via an asymmetrical cleavage reaction as described above for method versions 2 and 3, including any further incorporation steps required to reconstitute the polynucleotide payload and the blunt end at the first terminal end of the acceptor polynucleotide. An asymmetrical cleavage reaction may be performed using any suitable reagents and methods as described for method versions 2 and 3 as described above.

[0648] Following the phosphorylation, ligation and cleavage steps, a new acceptor polynucleotide is created. 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 double-stranded polynucleotide having a predefined sequence, by allowing multiple polynucleotide payloads to be successively joined together.

[0649] During this step of the method the tether polynucleotide should typically play no role and simply be present ready for the next cycle of the method in the same form as it was present at the start of the previous cycle because no payload sequence is added to the tether polynucleotide.

[0650] Method version 4

[0651] Provision of acceptor polynucleotide

[0652] To initiate a cycle of synthesis an acceptor polynucleotide is first provided. As discussed above, the surface will also have tether polynucleotide provided immobilised on it helping to localize donor polynucleotide near to the acceptor polynucleotides without the tether and donor ligating to each other.

[0653] 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.

[0654] The first terminal end of the acceptor polynucleotide is ligatable and blunt- ended. 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.

[0655] The terminal nucleotide of the first strand at the ligatable first terminal end of the acceptor polynucleotide lacks a 5’ phosphate group. 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 comprises a 3’ hydroxyl group.

[0656] The second terminal end of the acceptor polynucleotide is preferably non- ligatable.

[0657] The second terminal end of the acceptor polynucleotide is preferably joined, or immobilised, to a surface. Such a surface may be any suitable surface as described and defined elsewhere herein. The second terminal end may be joined, or immobilised, to a surface due to the second strand of the acceptor polynucleotide being joined, or immobilised, to the surface whilst the first strand of the acceptor polynucleotide is untethered. Alternatively, the second terminal end may be joined, or immobilised, to a surface due to the first strand of the acceptor polynucleotide being joined, or immobilised, to the surface whilst the second strand of the acceptor polynucleotide is untethered. Alternatively still, the second terminal end may be joined, or immobilised, 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 joined, or immobilised, 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.

[0658] 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 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.

[0659] Provision of donor polynucleotide

[0660] To initiate a cycle of synthesis a donor polynucleotide is also provided.

[0661] 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 region and with a single stranded polynucleotide region representing a hyb arm that is able to hybridize to the hyb arm of the tether polynucleotide, but where the two cannot ligate to each other.

[0662] The first terminal end of the donor polynucleotide is ligatable and blunt-ended. The first terminal end is free, i.e. neither the first strand nor the second strand is joined, immobilised, or otherwise attached to any other structure.

[0663] The second terminal end is typically also free, i.e. neither the first strand nor the second strand is joined, immobilised, or otherwise attached to any other structure. The second terminal end includes a single stranded region representing the hyb arm which is complementary to the hyb arm of the tether polynucleotide.

[0664] The terminal nucleotide of the second strand at the ligatable first terminal end of the donor polynucleotide comprises a 5’ phosphate group. The (3’) terminal nucleotide of the first strand at the ligatable first terminal end does not comprise a 5’ phosphate group. The (3’) terminal nucleotide of the first strand at the ligatable first terminal end comprises a 3’ hydroxyl group.

[0665] The second terminal end of the donor polynucleotide will not ligate to other donor polynucleotide molecules. It will also not ligate to the tether polynucleotide when hybridized to it. It may be that it will not ligate to the tether polynucleotide because when the two are hybridized there is a gap at the termini between the strand of the hyb arm of the tether and the terminal nucleotide of the same sense strand of the donor, as well as gap at the termini of the strand of the hyb arm of the donor and the terminal nucleotide of the same sense strand of the tether. Alternatively, blocking groups may be used to prevent ligation. 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.

[0666] 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 terminal nucleotide 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 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 a nucleotide pair, and this pair is the second pair of nucleotides in the polynucleotide payload, and so on.

[0667] 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.

[0668] Ligation of acceptor and donor polynucleotides

[0669] As discussed above, the tether polynucleotides present hybridize to the donor polynucleotides but do not ligate with them, serving to bring donor polynucleotides into closer proximity with the acceptor polynucleotides and reduce the rate of acceptor: acceptor ligation product formation.

[0670] 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 ligating the blunt ends of the acceptor and donor polynucleotides to form a ligated polynucleotide. In particular, the ligation step comprises ligating the first terminal end of the donor polynucleotide to the first terminal end of the acceptor polynucleotide.

[0671] In method version 4, the ligation step comprises joining the donor and acceptor polynucleotides at their first terminal ends by ligating the second strand of the donor polynucleotide at its first terminal end with the second strand of the acceptor polynucleotide at its first terminal end, and wherein the first 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 4, the ligation step comprises a single-stranded ligation wherein the second strands of the donor and acceptor polynucleotides are ligated together at their first terminal ends but the first strands of the donor and acceptor polynucleotides are not ligated together at their first terminal ends.

[0672] In method version 4, after the step of joining the donor and acceptor polynucleotides at their first terminal ends, e.g. after a first ligation step comprising a single-stranded ligation wherein the second strands of the donor and acceptor polynucleotides are ligated together at their first terminal ends, a second step of joining the donor and acceptor polynucleotides at their first terminal ends is performed. A second ligation step may be performed comprising a second single-stranded ligation step wherein the first strands of the donor and acceptor polynucleotides are ligated together at their first terminal ends. Preferably, but not essentially, the first strands of the donor and acceptor polynucleotides are ligated together by steps comprising adding a phosphate group to the first strand of the donor polynucleotide at its first terminal end, preferably by the action of an enzyme having kinase activity, such as polynucleotide kinase (PNK); and joining the donor and acceptor polynucleotides at their first terminal ends by ligating the first strand of the donor polynucleotide with the first strand of the acceptor polynucleotide.

[0673] 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.

[0674] The step of ligating the donor and acceptor polynucleotides creates a ligated polynucleotide. Donor polynucleotide wash-out (optional)

[0675] After ligation a washout may be performed to removed donor polynucleotides that are either free or hybridized to tether polynucleotides but not ligated to the acceptor polynucleotides. The temperature may be increased above the melting temperature of the hybridized hyb arms of the tether and donor polynucleotides so that hybridized donor polynucleotides are released. The donor polynucleotides may then be washed out to remove them.

[0676] 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.

[0677] 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.

[0678] 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.

[0679] 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.

[0680] 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.

[0681] Cleavage

[0682] 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.

[0683] 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.

[0684] In method version 4, 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 blunt end at the cleaved 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 terminal nucleotides of the cleaved first terminal end are the final pair of nucleotides of the polynucleotide payload in that cycle.

[0685] The cleavage step can be performed by any suitable means for creating the blunt-ended structure described above. Cleavage may comprise a double-stranded cleavage reaction wherein both the first and second strands are cleaved. In such a cleavage step both the first and second strands are cleaved at the same positions in a symmetric cleavage reaction. This generates a cleaved donor polynucleotide wherein the first terminal end is blunt-ended. As noted previously, 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, the type IIS restriction enzyme may be Mlyl. The user will readily be able to structure the cleavage site in the donor polynucleotide in a manner that allows the blunt-ended structure described above to be formed following cleavage.

[0686] Although not depicted in the non-limiting schematic shown in Figure 14, the cleavage step can also be performed via an asymmetrical cleavage reaction as described above for method versions 2 and 3, including any further incorporation steps required to reconstitute the polynucleotide payload and the blunt end at the first terminal end of the acceptor polynucleotide. An asymmetrical cleavage reaction may be performed using any suitable reagents and methods as described for method versions as described above. In method version 4, following cleavage, the terminal nucleotide of the first strand at the first terminal end of the acceptor polynucleotide (i.e. one of the nucleotides of the final pair of nucleotides of the polynucleotide payload) comprises a 5’ phosphate group. The (3’) terminal nucleotide of the second strand at the first terminal end (i.e. the second nucleotide of the final pair of nucleotides of the polynucleotide payload) does not comprise a 5’ phosphate group. The (3’) terminal nucleotide of the second strand at the first terminal end comprises a 3’ hydroxyl group.

[0687] In method version 5, following cleavage, the 5’ phosphate group joined to the terminal nucleotide of the first strand of the cleaved acceptor polynucleotide is removed, preferably by the action of an enzyme having phosphatase activity, such as calf intestinal phosphatase (CIP).

[0688] Following the cleavage and de-phosphorylation steps, a new acceptor polynucleotide is created. 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 double-stranded polynucleotide having a predefined sequence, by allowing multiple polynucleotide payloads to be successively joined together.

[0689] During this step of the method the tether polynucleotide should typically play no role and simply be present ready for the next cycle of the method in the same form as it was present at the start of the previous cycle because no payload sequence is added to the tether polynucleotide.

[0690] Method version 5

[0691] Provision of acceptor polynucleotide

[0692] To initiate a cycle of synthesis an acceptor polynucleotide is first provided. As discussed above, the surface will also have tether polynucleotide provided immobilised on it helping to localize donor polynucleotide near to the acceptor polynucleotides without the tether and donor ligating to each other. 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.

[0693] The first terminal end of the acceptor polynucleotide is ligatable and blunt- ended. 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.

[0694] The terminal nucleotide of the first strand at the ligatable first terminal end of the acceptor polynucleotide lacks a 5’ phosphate group. 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 comprises a 3’ hydroxyl group.

[0695] The second terminal end of the acceptor polynucleotide is preferably non- ligatable.

[0696] 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.

[0697] 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 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.

[0698] Provision of donor polynucleotide

[0699] To initiate a cycle of synthesis a donor polynucleotide is also provided.

[0700] 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 region and with a single stranded polynucleotide region representing a hyb arm that is able to hybridize to the hyb arm of the tether polynucleotide, but where the two cannot ligate to each other.

[0701] The first terminal end of the donor polynucleotide is ligatable and blunt-ended. 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.

[0702] 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 includes a single stranded region representing the hyb arm which is complementary to the hyb arm of the tether polynucleotide.

[0703] The terminal nucleotide of the second strand at the ligatable first terminal end of the donor polynucleotide comprises a 5’ phosphate group. The (3’) terminal nucleotide of the first strand at the ligatable first terminal end does not comprise a 5’ phosphate group. The (3’) terminal nucleotide of the first strand at the ligatable first terminal end comprises a 3’ hydroxyl group.

[0704] The second terminal end of the donor polynucleotide will not ligate to other donor polynucleotide molecules. It will also not ligate to the tether polynucleotide when hybridized to it. It may be that it will not ligate to the tether polynucleotide because when the two are hybridized there is a gap at the termini between the strand of the hyb arm of the tether and the terminal nucleotide of the same sense strand of the donor, as well as gap at the termini of the strand of the hyb arm of the donor and the terminal nucleotide of the same sense strand of the tether. Alternatively, blocking groups may be used to prevent ligation. 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.

[0705] 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 terminal nucleotide 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 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 a nucleotide pair, and this pair is the second pair of nucleotides in the polynucleotide payload, and so on.

[0706] 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.

[0707] Ligation of acceptor and donor polynucleotides

[0708] As discussed above, the tether polynucleotides present hybridize to the donor polynucleotides but do not ligate with them, serving to bring donor polynucleotides into closer proximity with the acceptor polynucleotides and reduce the rate of acceptor: acceptor ligation product formation.

[0709] 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 ligating the blunt ends of the acceptor and donor polynucleotides to form a ligated polynucleotide. In particular, the ligation step comprises ligating the first terminal end of the donor polynucleotide to the first terminal end of the acceptor polynucleotide.

[0710] In method version 5, the ligation step comprises joining the donor and acceptor polynucleotides at their first terminal ends by ligating the second strand of the donor polynucleotide at its first terminal end with the second strand of the acceptor polynucleotide at its first terminal end, and wherein the first 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 5, the ligation step comprises a single-stranded ligation wherein the second strands of the donor and acceptor polynucleotides are ligated together at their first terminal ends but the first strands of the donor and acceptor polynucleotides are not ligated together at their first terminal ends.

[0711] 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.

[0712] The step of ligating the donor and acceptor polynucleotides creates a ligated polynucleotide.

[0713] In method version 5, after the step of joining the donor and acceptor polynucleotides at their first terminal ends, e.g. after a first ligation step comprising a single-stranded ligation wherein the second strands of the donor and acceptor polynucleotides are ligated together at their first terminal ends, a further step is performed before the cleavage step. The further step comprises performing an incorporation step to extend the first strand of the donor polynucleotide at its first terminal end at the nick site. The step comprises synthesising new nucleotides in the first strand of the acceptor polynucleotide using the nucleotides of the second strand as templates, preferably by the action of an enzyme having polymerase activity, thereby synthesising a new first strand of the acceptor...

Claims

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 a support comprising:(i) at least one acceptor polynucleotide having first and second strands, wherein one end is immobilised on the support and the opposite end is free, and wherein the free end is blunt ended; and(ii) at least one tether polynucleotide adjacent the acceptor polynucleotide, wherein one end is immobilised on the support and the opposite end is free, wherein the free end comprises a first hybridisation arm (hyb arm) which is a single-stranded sequence region;(B) providing at least one donor polynucleotide having first and second strands and having free first and second terminal ends, wherein the first terminal end is blunt-ended and comprises a polynucleotide payload sequence comprising one or more nucleotide pairs of the predefined sequence and a cleavage site adjacent the payload sequence, and wherein the second terminal end comprises a second hyb arm which is a single-stranded region comprising sequence which is complementary with the sequence region of the first hyb arm;(C) contacting the at least one donor polynucleotide with the at least one tether polynucleotide, whereupon the first and second hyb arms hybridize to form a double-stranded region, and ligating the first terminal end of the donor polynucleotide with the free end of the acceptor polynucleotide to form a ligated polynucleotide; and(D) cleaving the ligated polynucleotide and generating a cleaved blunt end, thereby extending the acceptor polynucleotide with the polynucleotide payload at the cleaved end, and generating a new blunt-ended double-stranded acceptor polynucleotide for ligation and extension in the next cycle.

2. A method according to claim 1, wherein(i) the tether polynucleotide is single-stranded, wherein one end is immobilised on the support and the opposite end is free, wherein the freeend comprises the first hybridisation arm (hyb arm) which is a singlestranded sequence region; or(ii) the tether polynucleotide is double-stranded, wherein one end is immobilised on the support and the opposite end is free, wherein the free end comprises a first hybridisation arm (hyb arm) which is a singlestranded sequence region.

3. A method according to claim 2, wherein:(i) the method is performed by the method of claim 2(i), and wherein step (C) comprises performing a single-stranded ligation wherein:(a) the first strand of the donor polynucleotide is ligated to the acceptor polynucleotide, or(b) the second strand of the donor polynucleotide is ligated to the acceptor polynucleotide; or(ii) the method is performed by the method of claim 2(i) or 2(ii), and wherein step (C) comprises performing a single-stranded ligation wherein:(a) only the first strand of the donor polynucleotide is ligated to the first strand of the acceptor polynucleotide, or(b) wherein only the second strand of the donor polynucleotide is ligated to the second strand of the acceptor polynucleotide; or(iii) the method is performed by the method of claim 2(ii), and wherein step (C) comprises performing a double-stranded ligation wherein the first strand of the donor polynucleotide is ligated to the first strand of the acceptor polynucleotide, and the second strand of the donor polynucleotide is ligated to the second strand of the acceptor polynucleotide.

4. A method according to claim 2(ii), claim 3 (ii) or claim 3 (iii), wherein at the free end of the tether polynucleotide the terminal nucleotide of the strand which does not comprise the first hyb arm is provided as a non-ligatable nucleotide, such that the donor polynucleotide cannot be ligated to the tether polynucleotide in step (D).

5. A method according to any one of the preceding claims, wherein the terminal nucleotide of the strand comprising the first hyb arm of the tether polynucleotide formspart of the sequence region of the first hyb arm, and / or wherein the terminal nucleotide of the strand comprising the second hyb arm of the donor polynucleotide forms part of the sequence region of the second hyb arm.

6. A method according to any one of the preceding claims, wherein the sequence region of the first hyb arm and / or the sequence region of the second hyb arm is 15 or more nucleotides in length, 20 or more nucleotides in length, 25 or more nucleotides in length, 30 or more nucleotides in length, 35 or more nucleotides in length, 40 or more nucleotides in length, 45 or more nucleotides in length or 50 or more nucleotides in length.

7. A method according to any one of the preceding claims, wherein(a) the number of nucleotides in the sequence regions of the first and second hyb arms is the same; or(b) the second hyb arm comprises one sequence region, and the sequence region of the first hyb arm comprises two or more copies of the sequence region of the second hyb arm, preferably four, five or six copies.

8. A method according to any one of the preceding claims, wherein the first hyb arm of the tether polynucleotide comprises two or more repeat sequences, and the second hyb arm of the donor polynucleotide comprises the same number of two or more repeat sequences as the tether polynucleotide, wherein the repeat sequences of the first hyb arm are complementary with the repeat sequences of the second hyb arm.

9. A method according to claim 8, wherein the hyb arms have 3 to 7 repeat sequences, preferably 4 to 6 repeat sequences.

10. A method according to any one of the preceding claims, wherein the support comprises a population of tether polynucleotides immobilised on the support and a population of acceptor polynucleotides immobilised on the support, and wherein the relative amount of acceptor polynucleotides to tether polynucleotides is from 5% to 25% acceptor polynucleotides to 75% to 95% tether polynucleotides, e.g. 5% acceptorpolynucleotides to 95% tether polynucleotides, 10% acceptor polynucleotides to 90% tether polynucleotides, 15% acceptor polynucleotides to 85% tether polynucleotides, 20% acceptor polynucleotides to 80% tether polynucleotides, or 25% acceptor polynucleotides to 75% tether polynucleotides.

11. A method according to any one of the preceding claims, wherein in step (A) the support has immobilised on it two or more populations of tether polynucleotides, wherein the sequences in the first hyb arms of each population is different, and step (B) comprises providing the same number of two or more populations of donor polynucleotides as provided for the tether polynucleotides in step (A), wherein the sequences in the second hyb arms of each population of donor polynucleotide is different but complementary with the sequences in the first hyb arms of each respective population of tether polynucleotides.

12. A method according to any one of the preceding claims, wherein after the cleavage step (D) the method further comprises raising the temperature above the melting temperature of the region of complementarity between the sequences of the first and second hyb arms, thereby detaching the donor polynucleotide from the tether polynucleotide, if attached, by separating the first and second hyb arms, optionally wherein the method further comprises washing the support to remove any unligated donor polynucleotide.

13. A method according to any one of claims 2(ii), 3(ii) and 4 to 12, wherein the terminal nucleotide of the first strand at the free end of the acceptor polynucleotide of step (A):(i) comprises a 5’ phosphate group; or(ii) lacks a 5’ phosphate group.

14. A method according to claim 13 (ii), wherein the terminal nucleotide of the second strand of the donor polynucleotide at the first terminal end comprises a 5’ phosphate group and wherein:step (C) comprises: (i) joining the donor and acceptor polynucleotides at their first terminal ends by ligating the second strand of the donor polynucleotide at its first terminal end with the second strand of the acceptor polynucleotide at its first terminal end; wherein the first strands of the donor and acceptor polynucleotides at their first terminal ends are not joined and are separated by a nick; and (ii) joining the first strands of the donor and acceptor polynucleotides at their first terminal ends; and following step (D) the 5’ phosphate group joined to the terminal nucleotide of the first strand of the cleaved acceptor polynucleotide is removed, preferably by the action of an enzyme having phosphatase activity.

15. A method according to claim 14, wherein step C(ii), comprises adding a phosphate group to the first strand of the acceptor polynucleotide at its first terminal end, preferably by the action of an enzyme having kinase activity, such as polynucleotide kinase (PNK); and joining the donor and acceptor polynucleotides at their first terminal ends by ligating the first strand of the donor polynucleotide with the first strand of the acceptor polynucleotide.

16. A method according to claim 13 (ii), wherein the terminal nucleotide of the second strand of the donor polynucleotide at the first terminal end comprises a 5’ phosphate group and wherein: step (C) comprises joining the donor and acceptor polynucleotides at their first terminal ends by ligating the second strand of the donor polynucleotide at its first terminal end with the second strand of the acceptor polynucleotide at its first terminal end; wherein the first strands of the donor and acceptor polynucleotides at their first terminal ends are not joined and are separated by a nick; following step (C) and before step (D) the method further comprises performing an incorporation step to extend the first strand of the donor polynucleotide at its first terminal end at the nick site, the step comprising synthesising new nucleotides in the first strand of the acceptor polynucleotide using thenucleotides of the second strand as templates, preferably by the action of an enzyme having polymerase activity, thereby synthesising a new first strand of the acceptor polynucleotide and re-forming the nucleotide pairs between the first and second strands of the acceptor polynucleotide; and following step (D) the 5’ phosphate group joined to the terminal nucleotide of the first strand of the cleaved acceptor polynucleotide is removed, preferably by the action of an enzyme having phosphatase activity.

17. A method according to claim 16, wherein the incorporation step is performed:(a) by the action of an enzyme having polymerase activity, and wherein the polymerase displaces the original first strand of the acceptor polynucleotide when synthesising the new first strand; or(b) by the action of an enzyme having polymerase activity which possesses 5’ to 3’ exonuclease activity, and wherein the polymerase digests the original first strand of the acceptor polynucleotide when synthesising the new second strand.

18. A method according to claim 13(i), wherein the terminal nucleotide of the second strand of the donor polynucleotide at the first terminal end lacks a 5’ phosphate group and wherein: step (C) comprises: (i) 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; wherein the second strands of the donor and acceptor polynucleotides at their first terminal ends are not joined and are separated by a nick; and (ii) joining the second strands of the donor and acceptor polynucleotides at their first terminal ends.

19. A method according to claim 18, wherein step C(ii), comprises adding a phosphate group to the second strand of the donor polynucleotide at its first terminal end, preferably by the action of an enzyme having kinase activity, such as polynucleotide kinase (PNK); and joining the donor and acceptor polynucleotides attheir first terminal ends by ligating the second strand of the donor polynucleotide with the second strand of the acceptor polynucleotide.

20. A method according to claim 13(i), wherein the terminal nucleotide of the second strand of the donor polynucleotide at the first terminal end lacks a 5’ phosphate group and wherein: step (C) 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, and wherein the second strands of the donor and acceptor polynucleotides at their first terminal ends are not joined and are separated by a nick; and wherein step (D) comprises:II. cleaving both the first and second strands of the donor polynucleotide to generate a blunt end at the cleaved first terminal end of the acceptor polynucleotide; orII. cleaving the ligated polynucleotide at a site in the first strand of the donor polynucleotide, thereby retaining the nucleotides of the first strand of the polynucleotide payload at the cleaved first terminal end of the acceptor polynucleotide and thereby generating a 5’ overhang at the cleaved first terminal end of the acceptor polynucleotide with the nucleotides of the first strand of the polynucleotide payload overhanging the second strand of the acceptor polynucleotide, wherein the terminal nucleotide of the overhang is the final nucleotide of the payload; and following step (D) the method further comprises separating the cleaved donor polynucleotide from the acceptor polynucleotide and performing an incorporation step comprising extending the second strand of the acceptorpolynucleotide at the nick site with new payload nucleotides 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.

21. A method according to claim 20, wherein the cleaved donor polynucleotide is separated from the acceptor polynucleotide:(i) before the incorporation step; or(ii) during the incorporation step.

22. A method according to claim 13(i), wherein the terminal nucleotide of the second strand of the donor polynucleotide at the first terminal end lacks a 5’ phosphate group and wherein: step (C) 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; wherein the second strands of the donor and acceptor polynucleotides at their first terminal ends are not joined and are separated by a nick; following step (C) and before step (D) the method further comprises performing a first incorporation step to extend the second strand of the acceptor polynucleotide from the nick site, the step comprising synthesising new nucleotides in the second strand using the nucleotides of the first strand as templates, preferably by the action of an enzyme having polymerase activity, thereby synthesising a new second strand of the donor polynucleotide and re-forming and retaining the nucleotide pairs in the ligated polynucleotide including the one or more payload nucleotide pairs and the cleavage site.

23. A method according to claim 22 wherein the original second strand of the donor polynucleotide is separated from the first strand:(i) before the incorporation step; or(ii) during the incorporation step.

24. A method according to claim 22 or claim 23, 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 step and copying of the first strand the mismatch is corrected thereby generating a cleavable cleavage site.

25. A method according to claim 24, 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.

26. A method according to: (I) claim 21(ii); or (II) claim 23 (ii), wherein incorporation steps are performed:(a) by the action of an enzyme having polymerase activity, and wherein the polymerase displaces the second strand when synthesising the new second strand; or(b) by the action of an enzyme having polymerase activity which possesses 5’ to 3’ exonuclease activity, and wherein the polymerase digests the second strand when synthesising the new second strand.

27. A method according to any one of claims 2(ii), 3 (iii) and 4 to 12, wherein in step (A) the terminal nucleotide of the first strand at the free end of the acceptor polynucleotide of comprises a 5’ phosphate group and the terminal nucleotide of the second strand at the first terminal end of the donor polynucleotide comprises a 5’ phosphate group.

28. A method according to any one of the preceding claims, wherein(a) the nucleotides of the hyb arm of the tether polynucleotide and / or the hyb arm of the donor polynucleotide are continuous with the remaining nucleotides of the same strand of the polynucleotide; or(b) the nucleotides of the hyb arm of the tether polynucleotide and / or the hyb arm of the donor polynucleotide are joined to the remainder of the nucleotides of the same strand of the polynucleotide by a linker / spacer, optionally a 18-atom hexa-ethyleneglycol spacer.

29. A method according to any one of the preceding claims, wherein(a) the hyb arms of the tether polynucleotide and the hyb arms of the donor polynucleotide are comprised in the first strands of the polynucleotide; or(b) the hyb arms of the tether polynucleotide and the hyb arms of the donor polynucleotide are comprised in the second strands of the polynucleotide.

30. A method according to any one of claims 1 to 19, and 22 to 25, wherein step (D) comprises: cleaving both strands of the ligated polynucleotide to form a blunt end at thecleaved 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 terminal nucleotides of the cleaved first terminal end are the final pair of nucleotides of the polynucleotide payload31. A method according to any one of claims 1 to 12, 20 and 21, wherein step (D) comprises:I. cleaving the ligated polynucleotide at a site in the first strand of the donor polynucleotide, thereby retaining the nucleotides of the first strand of the polynucleotide payload at the cleaved first terminal end of the acceptor polynucleotide and thereby generating a 5’ overhang at the cleaved first terminal end of the acceptor polynucleotide with the nucleotides of the first strand of the polynucleotide payload overhanging the second strand of the acceptor polynucleotide, wherein the terminal nucleotide of the overhang is the final nucleotide of the payload; andII. performing an incorporation step comprising extending the second strand of the cleaved acceptor polynucleotide with new payload nucleotides using the payload nucleotides of the first strand as templates, preferably by the action of an enzyme having polymerase activity, thereby reforming 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 and retained 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.

32. A method according to any one of the preceding claims, wherein cleaving a strand of the ligated polynucleotide in step (D) comprises cleaving the sugar-phosphate backbone of the strand.

33. A method according to claim 30 or claim 32, wherein the cleavage site in the donor polynucleotide is adjacent to the polynucleotide payload and comprises a recognition sequence for a type IIS restriction enzyme, preferably the wherein the cleavage site is an Mfyl cleavage site.

34. A method according to claim 31 or claim 32, wherein cleavage comprises cleaving the sugar-phosphate backbone of the first strand of the donor polynucleotide and breaking the hydrogen bonds between the one or more payload nucleotide pairs.

35. A method according to claims 31 or claim 32, wherein cleaving is performed by the action of an enzyme having overhang cleavage function, preferably a type IIS restriction enzyme, optionally BspQI.

36. 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.

37. A method according to claim 36, 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.

38. A method according to claim 36, wherein the final nucleotide of the polynucleotide payload in the first strand occupies nucleotide position n, thepenultimate 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.

39. A method according to claim 36, 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.

40. A method according to any one of claims 30 to 39, 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.

41. A method according to claim 40, wherein the first step is performed with a nucleotide-excising enzyme.

42. A method according to claim 41, wherein the nucleotide-excising enzyme is a 3- methyladenine DNA glycosylase enzyme.

43. A method according to claim 42, wherein the nucleotide-excising enzyme is: i. human alkyladenine DNA glycosylase (hAAG); or ii. uracil DNA glycosylase (UDG).

44. A method according to any one of claims 34 to 43, wherein the second step is performed with a chemical which is a base.

45. A method according to claim 44, wherein the base is NaOH.

46. A method according to any one of claims 34 to 43, wherein the second step is performed with an organic chemical having abasic site cleavage activity.

47. A method according to claim 46, wherein the organic chemical is N,N’- dimethylethylenediamine.

48. A method according to any one of claims 34 to 43, 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.

49. A method according to any one of claims 30 to 33, 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).

50. A method according to any one of claims 30 to 33, wherein the cleavage step comprises cleaving the first strand of the donor polynucleotide with an enzyme.

51. A method according to claim 50, wherein the enzyme cleaves the first strand of the donor polynucleotide between nucleotide positions n+1 and n.

52. A method according to claim 50 or claim 51, wherein the enzyme isEndonuclease V.

53. A method according to claim 34 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.

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

55. A method according to claim 54, 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.

56. 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.

57. 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.

58. A method according to claim 57, 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.

59. A method according to any one of claims 1 to 58, wherein both strands of the acceptor and donor polynucleotide comprises DNA.

60. A method according to any one of claims 1 to 58, wherein the first strands of the acceptor and donor polynucleotide comprise RNA and the second strands of the acceptor and donor polynucleotide comprise DNA.

61. A method according to claim 60, 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.

62. A method according to any one of the preceding claims, wherein each cycle does not involve a step of incorporation of a polynucleotide having a reversible terminator group and an additional step of deprotection to remove the reversible terminator group.

63. 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.

64. A method according to claim 63(i), claim 63(iii) or claim 63(iv), wherein the tethered strand(s) at the second terminal end comprises a cleavable linker(s), whereinthe linker(s) may be cleaved to detach the double-stranded polynucleotide from the surface following synthesis.

65. A method according to claim 63 (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.

66. A method according to claim 64 or claim 65, wherein the cleavable linker is a UV cleavable linker.

67. A method according to any one of claims 63 to 66, wherein the surface is a particle, optionally a microparticle.

68. A method according to any one of claims 63 to 66, wherein the surface is a planar surface.

69. A method according to claim 68, wherein the surface comprises a gel.

70. A method according to claim 69, 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.

71. A method according to any one of claims 61 to 68, 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.

72. A method according to claim 69, 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.

73. A method according to claim 70, 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).

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

75. A method according to claim 72, wherein the microfluidic system is an electrowetting system.

76. A method according to claim 73, wherein the microfluidic system is an electrowetting-on-dielectric system (EWOD).

77. 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.

78. 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.

79. 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.

80. A method according to claim 79 wherein the first polynucleotide and the one or more additional polynucleotides are double-stranded.

81. A method according to claim 80 wherein the first polynucleotide and the one or more additional polynucleotides are single-stranded.

82. A method according to any one of claims 79 to 81, wherein the first polynucleotide and the one or more additional polynucleotides are cleaved to create compatible termini and joined together, preferably by ligation.

83. A method according to claim 82, wherein the first polynucleotide and the one or more additional polynucleotides are cleaved by a restriction enzyme at a cleavage site.

84. A method according to any one of claims 77 to 83, wherein the synthesis and / or assembly steps are performed in droplets within a microfluidic system.

85. A method according to claim 84, 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.

86. A method according to claim 85 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 scaffold polynucleotide in accordance with the steps of the synthesis cycles.

87. A method according to claim 86, 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.

88. A method according to claim 86 and 87, wherein the microfluidic system is an electrowetting system.

89. A method according to claim 88, wherein the microfluidic system is an electrowetting-on-dielectric system (EWOD).

90. A method according to any one of claims 86 to 89, wherein synthesis and assembly steps are performed within the same system.

91. A support for synthesising a double-stranded polynucleotide having a predefined sequence, the support comprising:(A) a population of acceptor polynucleotides, wherein each acceptor polynucleotide has a first strand and a second strand and wherein one end is immobilised on the support and the opposite end is free, and wherein the free end is blunt ended; and(B) a population of tether polynucleotides disbursed amongst the acceptor polynucleotides, wherein each tether polynucleotide has one end immobilised on the support and wherein the opposite end is free, and wherein the free end comprises a first hybridisation arm (hyb arm) which is a single-stranded sequence region.

92. A support according to claim 91, wherein(i) the tether polynucleotide is single-stranded, wherein one end is immobilised on the support and the opposite end is free, wherein the free end comprises the first hybridisation arm (hyb arm) which is a singlestranded sequence region; or(ii) the tether polynucleotide is double-stranded, wherein one end is immobilised on the support and the opposite end is free, wherein the free end comprises a first hybridisation arm (hyb arm) which is a singlestranded sequence region.

93. A support according to claim 92(ii), wherein at the free end of the tether polynucleotide the terminal nucleotide of the strand which does not comprise the first hyb arm is provided as a non-ligatable nucleotide.

94. A support according to any one of claims 91 to 93, wherein the terminal nucleotide of the strand comprising the first hyb arm of the tether polynucleotide forms part of the sequence region of the first hyb arm, and / or wherein the terminal nucleotide of the strand comprising the second hyb arm of the donor polynucleotide forms part of the sequence region of the second hyb arm.

95. A support according to any one of claims 91 to 94, wherein the sequence region of the first hyb arm and / or the sequence region of the second hyb arm is 15 or more nucleotides in length, 20 or more nucleotides in length, 25 or more nucleotides in length, 30 or more nucleotides in length, 35 or more nucleotides in length, 40 or more nucleotides in length, 45 or more nucleotides in length or 50 or more nucleotides in length.

96. A support according to any one of claims 91 to 95, wherein the number of nucleotides in the sequence regions of the first and second hyb arms is the same.

97. A support according to any one of claims 91 to 96, wherein the first hyb arm of the tether polynucleotide comprises two or more repeat sequences, and the second hyb arm of the donor polynucleotide comprises the same number of two or more repeat sequences as the tether polynucleotide, wherein the repeat sequences of the first hyb arm are complementary with the repeat sequences of the second hyb arm.

98. A support according to claim 97, wherein the hyb arms have 3 to 7 repeat sequences, preferably wherein the number of repeats is 4 to 6.

99. A support according to any one of claims 91 to 98, wherein the relative amount of acceptor polynucleotides to tether polynucleotides is from 5% to 25% acceptor polynucleotides to 75% to 95% tether polynucleotides, e.g. 5% acceptor polynucleotides to 95% tether polynucleotides, 10% acceptor polynucleotides to 90% tether polynucleotides, 15% acceptor polynucleotides to 85% tether polynucleotides, 20% acceptor polynucleotides to 80% tether polynucleotides, or 25% acceptor polynucleotides to 75% tether polynucleotides.

100. A support according to any one of claims 91 to 99, wherein the support has immobilised on it two or more populations of tether polynucleotides, wherein the sequences in the first hyb arms of each population is different.

101. A support according to any one of claims 91 to 100, wherein at least one tether polynucleotide is bound to a donor polynucleotide, wherein the donor polynucleotide has first and second strands and first and second terminal ends, wherein the first terminal end is blunt-ended and comprises a polynucleotide payload sequence comprising one or more nucleotide pairs of the predefined sequence and a cleavage site adjacent the payload sequence, wherein the second terminal end comprises a second hyb arm which is a single-stranded region comprising sequence which is complementary with the sequence region of the first hyb arm, and wherein the first and second hyb arms are hybridized together.

102. A system for synthesising a double-stranded polynucleotide having a predefined sequence, the system comprising the support as defined in any one of claims 91 to 101.

Images