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EP-4735593-A1 - POLYNUCLEOTIDE SYNTHESIS METHOD, KIT AND SYSTEM

EP4735593A1EP 4735593 A1EP4735593 A1EP 4735593A1EP-4735593-A1

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.

Inventors

  • CHAPMAN, Tarun Stephen
  • Clarke, David Edward
  • CLARKE, JAMES ANTHONY
  • COOK, Imogen Helen
  • MÜLLER, Carolin Anne
  • OGAKI, Ryosuke
  • WHITE, James Philip

Assignees

  • Oxford Nanopore Technologies PLC

Dates

Publication Date
20260506
Application Date
20240627

Claims (20)

  1. 1. An in vitro method of synthesising a double-stranded polynucleotide having a predefined sequence, the method comprising performing cycles of synthesis, wherein each cycle comprises: (A) providing an acceptor polynucleotide which is blunt-ended and double-stranded; (B) providing a blunt-ended donor polynucleotide having first and second strands, and comprising a polynucleotide payload comprising one or more nucleotide pairs of the predefined sequence and a cleavage site; (C) performing a single-stranded ligation reaction to form a ligated polynucleotide, the reaction comprising ligating only the first strands of the acceptor and donor polynucleotides, or ligating only the second strands of the acceptor and donor polynucleotides; 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. 2. A method according to claim 1, wherein: step (A) comprises: providing an acceptor polynucleotide comprising first and second polynucleotide strands and first and second terminal ends, wherein the first terminal end is ligatable and blunt-ended; step (B) comprises: providing a donor polynucleotide comprising: (i) first and second polynucleotide strands, (ii) first and second terminal ends; wherein the first terminal end is ligatable, blunt-ended and comprises a polynucleotide payload comprising one or more nucleotide pairs of the predefined sequence, the terminal nucleotide pair being the first pair of the payload; and (iii) a cleavage site adjacent to the polynucleotide payload; step (C) comprises: ligating the first terminal end of the donor polynucleotide to the first terminal end of the acceptor polynucleotide; and step (D) comprises: cleaving the ligated polynucleotide at the cleavage site, separating the cleaved donor polynucleotide from the acceptor polynucleotide, generating a blunt end at the cleaved first terminal end of the acceptor polynucleotide, thereby extending the cleaved first terminal end of the acceptor polynucleotide with the polynucleotide payload and generating a new blunt- ended ligatable first terminal end of the acceptor polynucleotide for ligation in the next cycle.
  3. 3. A method according to any one of the preceding claims, wherein the terminal nucleotide of the first strand at the ligatable end of the acceptor polynucleotide of step (A): (i) comprises a 5’ phosphate group; or (ii) lacks a 5’ phosphate group.
  4. 4. A method according to claim 3(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.
  5. 5. A method according to claim 4, 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.
  6. 6. A method according to claim 3(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.
  7. 7. A method according to claim 6, 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.
  8. 8. A method according to claim 3(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.
  9. 9. A method according to claim 8, 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.
  10. 10. A method according to claim 3(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: both polynucleotide strands of the second terminal end of the donor polynucleotide are connected together by a polynucleotide hairpin loop; 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; and before step (D) the method further comprises breaking the bonds between nucleotides of the first and second strands of the donor polynucleotide, thereby generating a single stranded donor polynucleotide ligated to the first strand of the acceptor polynucleotide, and synthesising a new second strand in the donor polynucleotide using the nucleotides of the first strand and original second strand as templates, preferably by the action of an enzyme having polymerase activity, thereby re-forming and retaining donor nucleotide pairs in the ligated polynucleotide including the one or more payload nucleotide pairs and the cleavage site.
  11. 11. A method according to claim 10, wherein both polynucleotide strands of the second terminal end of the donor polynucleotide of step (B) are connected together by a polynucleotide hairpin loop which encodes one strand of the cleavage site, and wherein the step of synthesising a new second strand in the donor polynucleotide before step (D) comprises using the nucleotides of the hairpin as templates to generate a complete double stranded cleavage site.
  12. 12. A method according to claim 3(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: 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 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.
  13. 13. A method according to claim 12, wherein the cleaved donor polynucleotide is separated from the acceptor polynucleotide: (i) before the incorporation step; or (ii) during the incorporation step.
  14. 14. A method according to claim 3(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.
  15. 15. A method according to claim 14 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.
  16. 16. A method according to: (I) claim 13(ii); or (II) claim 15(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.
  17. 17. A method according to any one of claims 1 to 11 and 14 to 16, 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
  18. 18. A method according to any one of claims 1, 2, 3, 14, 15 or 16(11), 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; and 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.
  19. 19. A method according to any one of claims 1-11, wherein step (D) comprises: I. cleaving the ligated polynucleotide at a site in the first strand of the donor polynucleotide and cleaving the ligated polynucleotide at a site in the second 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 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.
  20. 20. 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.

Description

POLYNUCLEOTIDE SYNTHESIS METHOD, KIT AND SYSTEM FIELD OF THE INVENTION 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. BACKGROUND TO THE INVENTION Various methods exist for the synthesis and assembly of polynucleotide molecules, particularly DNA. 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. 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). 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 posses reversible blocking groups to prevent promiscuous extension. 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. The inventors have developed new methodologies by which single- and doublestranded polynucleotide molecules can be synthesised de novo in a stepwise manner without the need to copy a pre-existing template molecule. Such methods also avoid the extreme conditions associated with phosphoramidite chemistry techniques and in contrast are carried out under mild, aqueous conditions around neutral pH. SUMMARY OF THE INVENTION The invention provides in vitro methods of synthesising a double-stranded polynucleotide having a predefined sequence. The invention is further defined in the section below. Aspects of the Invention. 1. An in vitro method of synthesising a double-stranded polynucleotide having a predefined sequence, the method comprising performing cycles of synthesis, wherein each cycle comprises: (A) providing an acceptor polynucleotide which is blunt-ended and double-stranded; (B) providing a blunt-ended donor polynucleotide having first and second strands, and comprising a polynucleotide payload comprising one or more nucleotide pairs of the predefined sequence and a cleavage site; (C) performing a single-stranded ligation reaction to form a ligated polynucleotide, the reaction comprising ligating only the first strands of the acceptor and donor polynucleotides, or ligating only the second strands of the acceptor and donor polynucleotides; 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 aspect 1, wherein: step (A) comprises: providing an acceptor polynucleotide comprising first and second polynucleotide strands and first and second terminal ends, wherein the first terminal end is ligatable and blunt-ended; step (B) comprises: providing a donor polynucleotide comprising: (i) first and second polynucleotide strands, (ii) first and second terminal ends; wherein the first terminal end is ligatable, blunt-ended and comprises a polynucleotide payload comprising one or more nucleotide pairs of the predefined sequence, the terminal nucleotide pair being the first pair of the payload; and (iii) a cleavage site adjacent to