LINEAR DOUBLE STRANDED DNA COUPLED TO A SINGLE SUPPORT OR A TAG AND METHODS FOR PRODUCING SAID LINEAR DOUBLE STRANDED DNA

Abstract

The present invention is concerned with linear double stranded DNA, which is coupled to a single support or a tag at the 3′ end of its non-coding strand and methods for producing said linear double stranded DNA. The present invention further relates to the use of said linear double stranded DNA in an RNA in vitro transcription reaction and also to a method for producing RNA in vitro. The present invention also relates to a bioreactor for RNA in vitro transcription.

Description

Claims

1. A linear double stranded DNA comprising a coding strand and a non-coding strand, wherein said DNA comprises (i) a coding sequence element encoded by the coding strand in the direction of from 5′ to 3′ of the coding strand and (ii) an RNA polymerase promotor sequence element upstream of the coding sequence element, wherein said non-coding strand is coupled at its 3′ end to a support or a tag, and wherein said support or tag is the only support or tag coupled to said DNA.

2. The linear double stranded DNA according to claim 1, wherein said non-coding strand is coupled at its 3′ end to a support or a tag via a triazole.

3. The linear double stranded DNA according to claim 1, wherein said tag is biotin.

4. The linear double stranded DNA according to claim 3, wherein said biotin is associated with streptavidin, preferably a streptavidin coated bead, most preferably a streptavidin coated magnetic bead.

5. The linear double stranded DNA according to claim 1, wherein the coding sequence element is flanked by a 5′ UTR and/or a 3′ UTR element.

6. A method for producing linear double stranded DNA comprising a coding strand and a non-coding strand, wherein said non-coding strand is coupled at its 3′ end to a support or a tag, comprising the steps of: (a) providing linear double stranded DNA comprising a coding sequence element encoded by the coding strand, followed at the 3′ end by a restriction site element;(b) incubating said DNA with (i) a modified deoxynucleotide and (ii) an enzyme capable of adding said modified deoxynucleotide at a 3′ end of a strand in order to provide linear double stranded DNA with a modified deoxynucleotide at the 3′ end of each strand;(c) incubating the DNA obtained in step (b) with a restriction endonuclease recognizing said restriction site element in order to obtain linear double stranded DNA with a modified deoxynucleotide at the 3′ end of the non-coding strand;(d) coupling the DNA obtained in step (c) via its modified deoxynucleotide to a support or a tag in order to provide linear double stranded DNA, wherein the non-coding strand of said DNA is coupled at its 3′ end to a support or a tag.

7. A method for producing linear double stranded DNA comprising a coding strand and a non-coding strand, wherein said non-coding strand is coupled at its 3′ end to a support or a tag, comprising the steps of: (a) providing linear double stranded DNA comprising a coding sequence element encoded by the coding strand, followed at the 3′ end by a restriction site element;(b) incubating said DNA with (i) a modified deoxynucleotide and (ii) an enzyme capable of adding said modified deoxynucleotide at a 3′ end of a strand in order to provide linear double stranded DNA with a modified deoxynucleotide at the 3′ end of each strand;(c) coupling the DNA obtained in step (b) via the modified deoxynucleotide at the 3′ end of each strand to a support or a tag;(d) incubating the DNA obtained in step (c) with a restriction endonuclease recognizing said restriction element in order to provide linear double stranded DNA, wherein the non-coding strand of said DNA is coupled at its 3′ end to a support or a tag.

8. The method according to claim 6, wherein the modified deoxynucleotide is selected from the group consisting of an alkyne deoxynucleotide, an azide deoxynucleotide, an azadibenzocyclooctyne deoxynucleotide, a trans-cyclooctene deoxynucleotide, and a vinyl deoxynucleotide.

9. The method according to claim 6, wherein the enzyme capable of adding a modified deoxynucleotide at the 3′ end of a strand in step (b) is a DNA polymerase.

10-39. (canceled)

40. A method for producing RNA in vitro comprising the steps of: (a) providing the double stranded linear DNA according to claim 1 as template DNA;(b) providing (i) ribonucleoside triphosphates and (ii) a DNA-dependent RNA polymerase;(c) incubating the DNA provided in step (a) with (i) and (ii) provided in step (b) under suitable conditions in order to produce RNA.

41. The method according to claim 40, wherein the DNA-dependent RNA polymerase is a bacteriophage RNA polymerase, preferably a T3, T7 or SP6 DNA-dependent RNA polymerase.

42. The method according to claim 40, wherein a cap analogue is additionally provided in step (b).

43. The method according to claim 40, wherein a ribonuclease inhibitor is additionally provided in step (b).

44. The method according to claim 40 3, wherein pyrophosphatase is additionally provided in step (b).

45. The method according to claim 40, wherein MgCl2 is additionally provided in step (b).

46. The method according to claim 40, wherein the DNA is incubated in step (c) in a buffer suitable for producing RNA in vitro.

47. The method according to claim 40, wherein the DNA provided in step (a) is re-used in at least one further RNA in vitro production cycle.

48. A bioreactor for RNA in vitro transcription comprising (a) a reaction vessel (13) comprising the linear double stranded DNA according to claim 1;(b) a vessel (14) comprising ribonucleoside triphosphates and DNA-dependent RNA polymerase, wherein said vessel is connected to the reaction vessel; and(c) a product vessel (15) for collecting the RNA product, wherein said vessel is also connected to the reaction vessel.

49-62. (canceled)

63. A kit comprising (a) a tag-linked deoxynucleotide;(b) a Thermus aquaticus DNA polymerase capable of adding said tag-linked deoxynucleotide to the 3′ end of a strand at a blunt DNA end;(c) a counterpart of said tag associating in a highly specific manner with said tag.