EP-4735631-A2 - CELL-FREE REPLICATION OF A NUCLEIC ACID CONSTRUCT

Abstract

The present disclosure provides methods for cell free replication of nucleic acid constructs from template circular nucleic acid constructs that comprises rolling circle amplification of the template circular nucleic acid constructs to produce a replication product. In some instances, the present disclosure provides methods of producing a replicated circular DNA construct from the replication product using a recombinase. In some instances, the present disclosure provides methods of producing a replicated linear RNA construct from the replication product by digesting the replication product and performing in vitro transcription of the digested replication product. The disclosure also provides kits for cell-free replication of said nucleic acid constructs.

Inventors

• WEINSTOCK, Matthew

Assignees

• Novel Biotechnology USA Inc.

Dates

Publication Date

20260506

Application Date

20240628

Claims (1)

1. CLAIMS WHAT IS CLAIMED IS: 1 . Au in vitro method of producing a replicated circular DN A. construct or a replicated linear RNA construct from a template circular nucleic acid constnict in a cell -free system, wherein the template nucleic acid construct comprises: G) a target sequence; (ii) cue or more enzyme recognition sites flanking one or more ends of the target sequence, and (ni) a backbone sequence. the method comprising: (a) contacting the template circular nucleic acid construct with a. DNA polymerase configured to perform rolling circle amplification of the template circular nucleic acid via the DNA polymerase, thereby fonning a replication product that comprises concatemeric replications of the target sequence and the backbone sequence joined together; and (b) contacting the replication product with an enzyme, thereby producing the replicated circular DNA construct or the replicated linear RNA construct from the replication product; wherein: (i) when the one or more enzyme recognition sites comprise recombination sites that flank each end of the target sequence and the enzyme is a reeombma.se, the contacting results in recombination of the replication product, which removes the backbone sequence from the replication product and joins the ends of the target sequence, thereby producing the replicated, circular DNA construct; and (») when the one or more enzyme recognition sues compose one or more restriction sites banking one or more ends of the target sequence and the enzyme is a restriction enzyme that cleaves the replication product at the one or more restriction sites, the contacting results m formation of a restriction enzyme-digested replication product, and the method further comprises contacting the restriction enzyme-digested replication product with an RNA polymerase configured to perform in iriff’o transcription, thereby producing the replicated, linear RNA construct. The method of claim I, wherein the DNA. polymerase is a <p29, a. Bst, or a Vent exo- DNA. polymerase. 3. The method of claim 2, wherein the DMA polymerase is a. Bst polymerase. 4. The method of claim 2, wherein the DMA polymerase is a q?29 polymerase. 5. The method of claim 2, wherein the DNA polymerase is a Vent exo-DNA polymerase. 6. The method of claim 1 , wherem the one or more enzyme recognition sites comprise recombination sites that hank each end of the target sequence, and wherein the enzyme is the recombinase, thereby producing the replicated circular DMA construct. 7. The method of claim 6, wherein the recombination sites compri se a LoxP sequence or functional variant thereof 8. The method of claim 7, wherein the recombination sites comprise a Lox66 sequence, a I,ox71 sequence, or fractional variant the reof 9. The method of claim 6, wherein recombination sites comprise a. FRT sequence or functional variant tiiereof 10. 'Die method of claim 6, wherein the recombinase is a. Cre recombinase, an FLP recombinase, a. lambda phage integrase, a. TP901 -1 lactococcal phage recombinase, a Bxbl integrase, an R4 integrase, a Xer recombinase, a Dre recombinase, or a pC3 i integrase. 1 1 . The method of claim 10, wherein the recombinase is a Cre recombinase. 12. The method of claim 10. wherein the recombinase is a FLP recombinase. 13. The method of claim 6, further comprising contacting the replication product with a restriction enzyme that selecti vely cleaves the backbone sequence prior to contacting the replication product with the recombinase, thereby forming a digested replication product for contacting with the recombinase. 14. The method of claim 6, further comprising contacting the replication product with an exonuclease after contacting the replication product with the recombinase, wherein the exonuclease selectively digests the cleaved backbone sequence. 15. The method of claim 6, further comprising contacting the replicated circular DMA construct with a composition that comprises one or more of (a) a second DNA polymerase, (b) a DNA. ligase, (c) a restriction enzyme, or (d) a gyrase, wherem the contacting is in an amount and duration sufficient to repair nicks in the replicated circular DM A construct and introduce supercoiling in the replicated circular DNA construct. 16 The method of claim 6. further comprising; (a) contacting the replicated circular DNA construct with a composition that comprises a second DNA polymerase, and a DNA ligase, wherein the contacting is in an amount and duration sufficient to repair nicks in the replicated circular DNA construct; and (b) contacting the replicated circular' DNA construct with a composition that comprises a gyrase, wherein the contacting is in an amount and duration sufficient to introduce supercoiling in the replicated circular DNA constnict. 17. The method of claim 1, wherein the one or more enzyme recognition sites comprise one or more restriction sites that flank one or more ends of the target sequence, and wherein the enzyme is the restriction enzyme. thereby producing the replicated linear RNA construct. 18. The method of claim 17, wherein the template circular nucleic acid sequence further comprises a Poly-A tail sequence adjacent to the 3” end of the target sequence. 19. The method of claim 18, wherein the one or more restriction sites are present on the Poly- A tail sequence. 20. The method of claim 17, wherein the template circular nucleic acid sequence further comprises a promoter configured to interface with the RN A polymerase. 21. The method of claim 20. wherein the RN A polymerase is a T7 RN A polymerase, and wherein the promote?' is a T7 promoter. 22. A replicated circular DNA constnict formed by the method of any one of claims 1-16. 23 A replicated linear RN A construct formed, by the method of any one of claims I -5 or 17- 21. 24. A composition comprising the replicated circular DNA construct of claim 22 or the raphcated linear RN A construct of claim 23, and an excipient, diluen t, or carrier. 25. The composi tion of claim 24, wherein the composition comprises a diluen t, wherein the diluent is a storage buffer, 26. A kit for replicating a circular DNA construct in a cell-free system, tire kit comprising: (a) a template circular nucleic acid construct that comprises: (i) a target sequence flanked by recombination sites on each end, wherein the target sequence is configured to form the circular nucleic acid construct upon recombination, and (il) a backbone sequence: (b) a <p29, a Bst, or a Vent exo-DNA polymerase; and (c) a recombinase. 27 The kit of claim 26, further comprising one or more of (a) an exonuclease; (b) a DMA polymerase; (c) a DM A ligase; (d) a restriction enzyme; and (e) a gyrase. 28. The kit of claim 26 or 27. wherein the recombinase is a Cre recombinase, an FLP recombinase, a. lambda phage integrase, a. T.P901 -I lactococcal phage recombinase, a Bxbl integrase, an R4 integrase, a Xer recombinase, a Dre recombinase, or a φ :-C3 l integrase. 29. A kit for replicating a linear RN A construct in a cell-free system, the kit comprising: (a) a templa te circular nucleic acid construct that comprises: (n a target sequence, (it) a 17 promoter adjacent to the 5’ end of the target sequence, arid (th) a Poly- A tail adjacent to the 3 end of the target sequence, wherein the ' Poly-A tail comprises one or more restriction enzyme sites, (b) a (p29, a Bst, or a Vent exo-DNA polymerase; (c) a restriction enzyme configured to cleave the template circular nucleic acid, construct at the one or more restnotion enzyme sites; and (d) a T7 RM A polymerase . 30. An in vfiro method of producing a supercoiled replicated circular DMA construct from a template circular nucleic acid, construct m a cell-free system , wherein the template circular nucleic acid, construct comprises: (0 a target sequence, wherein the target sequence is configured to form tlie circular DM A construct upon recombination; (u) recombination sites that flank each end of the target sequence; and (hi) a backbone sequence; the method comprising: (a) contacting the template circular nucleic acid construct with a Bst polymerase and performing roiling circle amplification of the template circular nucleic acid via the Bst polymerase, thereby forming a replication product that comprises concatemeric replications of the target sequence and the backbone sequence joined together; (b) contacting the replication product with a recombinase, resulting in recombination of the replication product, thereby removing the backbone sequence from the replication product, joining the ends of the target sequence, mid producing a replicated circular DNA constnict; (c) contacting the backbone sequence with an exonuclease, wherein the exonuclease selectively digests the cleaved backbone sequence; (d) purifying the replicated circular DNA construct, thereby removing the backbone sequence, and (e) contacting the purified replicated circular DMA constnict with a composition thin comprises one or mote of the following: a DNA polymerase, a DNA ligase, and a gyrase, thereby repairing nicks in the replicated circular DN A construct and producing the supercoiled replicated circular DNA constnict. 31 . The method of claim 3d. further comprising contacting the replication product with a. restfiction enzyme prior to contacting the replication product with the recombinase, thereby fbnning a digested replication product wherein the restriction enzyme selectively cleaves the backbone sequence. 32. The method of claim 30, wherein the recombinase is a Cre recombinase, an FTP recombinase, a lambda phage integrase, a TP901- 1 lactococcal phage recombinase, a Bxbl mtegra.se, an R4 integrase, a .Xer recombinase, a Dre recombinase, or a <?C31 integrase . 33. The method of claim 32, wherein the recombinase is a Cre recombinase. 34. 'Die method of claim 32, wherein the recombinase is a FLP recombinase. 35. ’The method, of claim 32, wherein one or more of the recombination sites comprise a LoxP sequence or a. functional variant thereof 36. The method of claim 35, wherein one or more of the recombination sites comprise a I,ox66 sequence, a Lox? I sequence, or a functional variant thereof 37. The method of claim 32. wherein one or more of the recombination sites comprise a FRT sequence or functional variant thereof 38. An In vdro method of producing a supercoiled replicated circular DNA constnict from a template circular nucleic acid, constnict m a cell-free system, wherein the template circular nucleic acid, constnict comprises: (0 a target sequence, wherein the target sequence is configured to form rhe circular DNA construct upon recombination; Ui) recombination sites that flank each end of the target sequence; and (hi) a backbone sequence; the method comprising: (a) contacting the template circular nucleic acid construct with a <g29 polymerase and performing rolling circle amplification of the template circular nucleic acid via the ip29 polymerase, thereby forming a replication product that comprises concatemeric replications ofthe target sequence and the backbone sequence joined together; (b) contacting the replication product with a recombinase, resulting in recombination of the replication product, thereby removing the backbone sequence from the replication product, joining the ends of the target sequence, mid producing a replicated circular DMA construct; (c) contacting the backbone sequence with an exonuclease, wherein the exonuclease selectively digests the cleaved backbone sequence; (d) purifying the replicated circular DNA construct, thereby removing the backbone sequence, and (e) contacting the purified replicated circular DMA construct with a composition that comprises one or more of the following: a DNA polymerase, a DNA ligase, and a gyrase, thereby repairing nicks in the replicated circular DN A construct and producing the supercoiled replicated circular DNA construct. 39. Die method of claim 38. further comprising contacting the replication product witii a restriction enzyme prior to contacting the replication product with the recombinase, thereby forming a digested replication product, wherein the restriction enzyme selectively cleaves the backbone sequence. 40. The method of claim 38, wherein the recombinase is a. Cre recombinase, an FLP recombinase, a lambda phage integrase, a TP9G M lactococcal phage recombinase, a Bxbl mtegra.se, an R4 integrase, a .Xer recombinase, a Dre recombinase, or a <?C31 mtegra.se . 41 . The method of claim 40, wherein the recombinase is a Cre recombinase. 42. Die method of claim 40, wherein the recombinase is a FLP recombinase. 43 The method, of claim 38, wherein one or more ofthe recombination sites comprise a LoxP sequence or functional variant thereof 44. The method of claim 43, wherein one or more of the recombination sites comprise a Lox66 sequence, a Lox71 sequence, or functional variant thereof 45. The method of claim 38, wherein one or more of the recombination sites comprise a F.R.T sequence or functional variant thereof. 46. An in v/fro method of producing a supercoiled replicated circular DNA construct from a template circular nucleic acid construct m a. cell-free system, wherein the template circular nucleic acid construct composes: (i) a target sequence, wherein the target sequence is configured to form the circular DNA construct upon recombination: 01) recombination sites that flank each end of the target sequence, and (til) a backbone sequence; the method comprising: (a) contacting the template circular nucleic acid construct with a Vent exo-DNA polymerase and performing rolling circle amplification of the template circular nucleic acid via the Vent exo-DNA polymerase, drereby forming a replication product that comprises concatenieric replications of the target sequence and the backbone sequence joined together: (b) contacting the replication product with a recombinase, resulting m recombination of the replication product, thereby removing the backbone sequence from the replication product, joining the ends of the target sequence, and producing a replicated circular DNA construct; (c) contacting the backbone sequence with an exonuclease, wherein the exonuclease selectively digests the cleaved backbone sequence; (d) purifying the replicated circular DNA construct, thereby removing the backbone sequence: and. (e) contacting the purified replicated circular DNA construct with a composition that comprises one or more of the following: a DNA polymerase, a DNA ligase, and a. gyrase, thereby repai ring nicks m the replicated, circular DN A construct and producing the supereoiled replicated circular DNA construct 47. The method of claim 46, further comprising contacting the replication product with a. restriction enzyme prior to contacting the replication product with the recombinase, thereby forming a digested replication product wherein the restriction enzyme selectively cleaves the backbone sequence 48. The method of claim 46, wherein the recombinase is a Cre recombinase, an FLP recombinase, a lambda phage integrase, a TP901- 1 lactococcal phage recombinase, a Bxbl mtegra.se, an R4 integrase, a Xer recombinase, a Dre recombinase, or a <pC31 integrase. 49. 'Die method of claim 48, wherein the recombinase is a. Cre recombinase. 59 The method of claim 48, wherein the recombinase is a FLP recombinase. 51. The method of claim 46, wherein one or more recombination sites comprises a LoxP sequence or functional variant thereof 52. 'The method of claim 51 , wherein the one or more recombination sites are a Lox.66 sequence. a Lox71 sequence, or fiinctional vanant thereof 53. Tiie method of claim 46, wherein one or more recombination sites comprises a FRT sequence or fiinctional variant thereof 54. A cell-free method of producing a replicated circular DNA construct from a template circular nucleic acid constmct m a cell-free system, wherein the template circular nucleic acid construct comprises: (i) a target sequence, wherein the target sequence is configured to form the circular DNA construct upon recombination; (fo recombination sites that flank each end of the target sequence: and (lii) a backbone sequence; the method comprising: (a) contacting the template circular nucleic acid construct with a DNA polymerase configured to perforin rolling circle amplification of the template circular nucleic acid via the DNA polymerase, thereby fonning a replication product that comprises concatemenc replications of the target sequence and the backbone sequence joined together; (b) contacting the replication product with a recombinase, resulting in recombination of the replication product, thereby removing the backbone sequence from the replication product, joining the ends of the target sequence, and. producing a replicated circular DNA constiuct; (c) purifying the replicated, circular DNA construct; wherein the purified replicated circular DNA construct produced by the cell-free system comprises less endotoxin tiian a comparable amount of replicated circular DNA construct produced m /.'. co/z, as measured by an amebocyte lysate assay . 55 . The method of claim 54, wherein the template circular nucleic acid, consfruct is a singlestranded template circular DNA construct. 56. The method of claim 55, wherein the method further comprises producing the singlestranded template circular DNA construct from a template linear DNA construct prior to ia). 57. The method of claim 56, wherein the template linear DNA constiuct comprises a telRL sequence on each side of the target sequence, and wherein the single-stranded template circular DNA construct is produced from the template linear DNA construct prior to (a) by contacting the template linear DNA construct with a telN protelomerase. 58. The method of claim 54, wherem the template circular nucleic acid construct is a doublestranded template circular DNA construct 59. The method of claim 58, wherem the method ft other comprises producing the doublestranded template circular DNA construct from a template linear DNA construct prior to (a). 60. The method of claim 59, wherein the double-stranded template circular DNA construct is produced from the template linear DNA construct prior to (a) by ligation of the ends of the template linear DNA construct. 61 . The method of claim 59. wherem the double-stranded template circular DNA construct is produced from the template linear DNA construct prior to (a) by Gibson or GoldenGate assembly. 62. An m w’rro method of producing a replicated linear RNA construct from a templa te circular nucleic acid construct; m a cell-free system, wherein the template circular nucleic acid construct comprises: (i) a target sequence: (ii) a T7 promoter adjacent to the 5 ? end of the target sequence, and (Hit a Poly-A tail adjacent to the 3’ end of the target sequence, wherein the Poly-A tail comprises one or more restriction enzyme sites; the method comprising; (a) contacting the template circular nucleic acid construct with a. DNA polymerase configured to perform roiling circle amplification of the template circular nucleic acid via the DNA polymerase, thereby fonning a replication product that comprises concatemeric replications of the target sequence; (b) contacting the replication product with restriction enzyme configured to cleave the template circular nucleic acid construct at the one or more restriction enzyme sites, thereby forming a digested replication prod.net; (c) purifying the digested, replication product; and. (d) contacting the digested replication product with an RN A polymerase, thereby performing m vfow transcription of the purified digested replication product and producing tire replicated RNA construct. 63. The method of claim 62, wherein the template circular nucleic acid construct is a singlestranded template circular DNA construct. 64. The method of claim 63, wherein the method further comprises producing the singlestranded template circular DNA construct from a template linear DNA construct prior to ia). 65. ’lire method of claim 64. wherein the template linear DNA. construct comprises a teIRL sequence on each side of the target sequence, and wherein the single-stranded template circular DNA construct is produced from the template linear DNA construct prior to (a) by contacting the template linear DN A construct with a telN protelomera.se. 66. The method of claim 62, wherein the template circular nucleic acid construct is a doublestranded template circular DNA construct. 67. The method of claim 66, wherein the method further comprises producing the doublestranded template circular DN A construct from a template linear DNA construct prior to (a). 68. The method of claim 67, wherein the double "Stranded template circular DNA construct is produced from the templa te linear DN A con struct prior to (a.) by ligation of the ends of the template linear DNA construct. 69. The method of claim 67, wherein the double -stranded template circular DNA construct is produced from the template linear DN A construct prior to (a) by Gibson or GoldenGate assembly.

Description

CELL-FREE REPLICATION OF A NUCLEIC ACID CONSTRUCT CROSS REFERENCE 10001J This application claims the benefit of US Provisional Application No. 63/511,373, filed on June 30, 2023, which is incorporated by reference in ns entirety’. SUMMARY [0002j Disclosed herein are in vitro methods of replicating a nucleic acid, construct (such as a DNA construct or an RNA construct) from a template circular nucleic acid construct. |0003l One aspect the invention features an re v/reo method of producing a replicated circular DNA construct or a replicated linear RNA construct from a template circular nucleic acid, construct m a cell-free system, where the template nucleic acid construct comprises a target sequence, one or more enzyme recognition sites flanking one or more ends of the target sequence, and a backbone sequence ’The method comprising contacting tire template circular nucleic acid construct with a DNA polymerase configured to perform rolling circle amplification of the template circular nucleic acid via. the DNA polymerase, thereby forming a replication product that comprises concatemenc replications of the target sequence and the backbone sequence joined together and contacting the replication product with an enzyme, thereby producing the replicated circular DNA construct or the replicated linear RNA construct from the replication product. When the one or more enzyme recognition sites comprise recombination sites that flank each end oft.be target sequence and the enzyme is a recombinase, the contacting results in recombination of the replication product, which removes the backbone sequence from the replication product and joins the ends of the target sequence, thereby producing the replicated circular DN A construct and when the one or more enzyme recognition sites comprise one or more restriction sites flanking one or more ends of the target sequence and. the enzyme is a restriction enzyme that cleaves the replication product at the one or more restriction sites, the contacting results in formation of a restriction enzyme-digested replication product. 'Die method further comprises contacting the restriction enzyme-digested replication product with an RN A polymerase configured to perform in vitro transcription, thereby producing the replicated linear RNA construct. In some embodiments, the method further comprises contacting the replicated circular DN A construct with a composition that comprises one or more of a second DNA polymerase, a DNA. ligase, a restriction enzyme, or a gyrase, where the contacting is in an amount and duration sufficient to repair nicks in the replicated circular DNA construct and. introduce supercoiling in the replicated circular DNA construct. In some embodiments, the step contacting the replicated circular DNA construct with a composition that comprises gyrase is performed after the step of contacting the replicated circular DNA construct with a composition that comprises one or more of a second DNA polymerase, a DNA ligase, or a restriction enzyme. [0004] In some embodiments, the DN A polymerase is a Bst poiymerase. in some embodiments, the DNA polymerase is a q>29 polymerase. In some embodiments, the DNA polymerase is a Vent exo-DNA polymerase. In some embodiments, the method further comprises contacting tire replication product with as exonuclease after contacting the replication product with tire recombinase, wherein tire exonuclease selectively digests the cleaved backbone sequence. In some embodiments, the method furtiter comprises purifying the replicated circular DNA construct, tiiereby removing die backbone sequence. In some embodiments, the one or more enzyme recognition sites comprise one or more restriction sites that flank one or more ends of the target sequence, and. wherein the enzym e is tire restriction enzyme, thereby producing a replicated linear RNA construct. In some embodiments, the one or more enzyme recognition sites comprise one or more restriction sites that Sank one or more ends of the target sequence, and wherein tire enzyme is the restriction enzyme, thereby producing a replicated linear DNA construct. In some embodiments, the template circular nucleic acid sequence further comprises a Poly-A tail sequence adjacent to the 3’ end of the target sequence. In some embodiments, the one or more restriction sites are present near the Poly-A tail sequence. In some embodiments, the template circular nucleic acid sequence further comprises a promoter configured to interface with the RNA polymerase. In some embodiments, the RNA polymerase is a T7 RNA polymerase, and wherein the promoter is a 1’7 promoter. [0005] Another aspect of the invention features an fe vfoo method of producing a supercoiled replicated, circular DNA construct from a template circular nucleic acid construct in a. cell-free system, where the template circular nucleic acid construct comprises a target sequence, where the target sequence is configured to form the circular DNA construct upon recom