Linear double stranded DNA coupled to a single support or a tag and methods for producing said linear double stranded DNA
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.
1. A method for producing RNA in vitro comprising
(I) producing a linear double stranded DNA comprising the steps of:
(a) providing linear double stranded DNA comprising a coding strand and a non-coding strand, said coding strand comprising an RNA polymerase promotor sequence element and a coding sequence element followed 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;
(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 and wherein said support is the only support coupled to said DNA
(II) providing (i) ribonucleoside triphosphates and (ii) a DNA-dependent RNA polymerase; and
(III) incubating the DNA provided in step (I)(d) with (i) and (ii) provided in step (II) under suitable conditions in order to produce RNA.
2. The method according to claim 1 , 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.
3. The method according to claim 1 , wherein the enzyme capable of adding a modified deoxynucleotide at the 3′ end of a strand in step (b) is a DNA polymerase.
4. The method according to claim 1 , wherein the DNA-dependent RNA polymerase is a bacteriophage RNA polymerase.
5. The method of claim 4 , wherein the bacteriophage RNA polymerase is SP6 polymerase.
6. The method of claim 4 , wherein the bacteriophage RNA polymerase is T7 polymerase.
7. The method of claim 4 , wherein said non-coding strand is coupled at its 3′ end to a support via a triazole.
8. The method according to claim 4 , wherein said modified deoxynucleotide comprises biotin.
9. The method according to claim 8 , wherein said biotin is associated with streptavidin.
10. The method according to claim 4 , wherein the coding sequence element is flanked by a 5′ UTR and/or a 3′ UTR element.
11. The method of claim 10 , wherein the linear double stranded DNA comprises (i) an RNA polymerase promotor sequence element; (ii) a 5′ UTR sequence; (iii) the coding sequence element; (iv) a 3′UTR sequence; and (v) a poly-A sequence, followed at the 3′ end by a restriction site element.
12. The method of claim 11 , wherein the poly-A sequence is at 50 nucleotides in length.
13. The method according to claim 1 , wherein a cap analogue is additionally provided in step (II).
14. The method according to claim 1 , wherein a ribonuclease inhibitor is additionally provided in step (II).
15. The method according to claim 1 , wherein pyrophosphatase is additionally provided in step (II).
16. The method according to claim 1 , wherein MgCl 2 is additionally provided in step (II).
17. The method according to claim 1 , wherein step (II) includes at least one ribonucleoside triphosphate analog.
18. The method according to claim 1 , wherein the DNA provided in step (I)(d) is re-used in at least two further RNA in vitro production cycles, defined is steps (II) and (III).
19. The method according to claim 1 , wherein the DNA provided in step (I)(d) is re-used in at least one further RNA in vitro production cycle, defined is steps (II) and (III).