US5700667A
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Strategy for the production of RNA from immobilized templates
Assignee: University of Colorado Boulder
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Filing: 1995-05-18
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Granted: 1997-12-23
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Expires: 2015-05-18
Abstract
Abstract The instant invention provides a method for producing RNA enzymatically in a semi-batch or continuous-flow bioreactor using a DNA template immobilized to a solid support through a linkage having a single-stranded overhang extending from a noncoding strand. The coding strand remains dissociable from the immobilized noncoding strand. The immobilized RNA template is reused in at least sixteen rounds of transcription reaction. The coding strand can be replaced with a fresh DNA strand encoding the same or a different RNA.
Inventors
- 👤 Herbert A. Marble
- 👤 Robert H. Davis
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-06 20:25
CPC Classifications
C; C12; C12P; C12P19/00; C12P19/26; C12P19/28; C12P19/30; C12P19/34; C12Q; C12Q1/00; C12Q1/68; C12Q1/6844; C12Q1/6865; B; B01; B01F; B01F33/00; B01F33/45
Claims Analysis
Total Claims: 49
Claim 1
Independent Claim
123 words
A method of producing RNA enzymatically in a bioreactor comprising the steps of: (a) immobilizing a DNA template, comprising a noncoding strand and a coding strand comprising a double-stranded promoter sequence and a double-stranded spacer sequence upstream of said promoter sequence, to a solid support through a linkage comprising a single-stranded overhang extending from the 5'-end of said noncoding strand and such that said coding strand remains dissociable from said noncoding strand; (b) contacting said immobilized DNA template of step (a) with a transcription reaction mixture comprising a buffer, nucleoside triphosphates and an RNA polymerase such that RNA having a sequence complementary to said coding strand is produced; (c) removing said RNA produced in step (b); and, (d) repeating steps (b) and (c).
Claim 2
Independent Claim
123 words
A method of producing RNA enzymatically in a bioreactor comprising the steps of: (a) immobilizing a DNA template, comprising a noncoding strand and a coding strand comprising a double-stranded promoter sequence and a double-stranded spacer sequence upstream of said promoter sequence, to a solid support through a linkage comprising a single-stranded overhang extending from the 5'-end of said noncoding strand and such that said coding strand remains dissociable from said noncoding strand; (b) contacting said immobilized DNA template of step (a) with a transcription reaction mixture comprising a buffer, nucleoside triphosphates and an RNA polymerase such that RNA having a sequence complementary to said coding strand is produced; (c) removing said RNA produced in step (b); and, (d) repeating steps (b) and (c).
Claim 3
Dependent Claim
24 words
The method of producing RNA of claim 1 wherein said single-stranded overhang is a nucleotide sequence of between 1 and 50 bp in length.
Claim 4
Dependent Claim
24 words
The method of producing RNA of claim 1 wherein said immobilization of said DNA template to said solid support occurs through a noncovalent linkage.
Claim 5
Dependent Claim
24 words
The method of producing RNA of claim 1 wherein said immobilization of said DNA template to said solid support occurs through a covalent linkage.
Claim 6
Dependent Claim
18 words
The method of producing RNA of claim 1 wherein the 5'-end of said noncoding DNA strand is immobilized.
Claim 7
Dependent Claim
32 words
The method of producing RNA of claim 1 wherein said immobilized DNA template comprises a two-stranded duplex comprising a spacer sequence and a promoter sequence followed by a single stranded coding region.
Claim 8
Dependent Claim
32 words
The method of producing RNA of claim 1 wherein said immobilized DNA template comprises a two-stranded duplex comprising a spacer sequence and a promoter sequence followed by two strands of coding region.
Claim 9
Dependent Claim
38 words
The method of producing RNA of claim 1 wherein said DNA template is obtained from a source of DNA selected from the group consisting of a chemically synthesized oligonucleotide, an isolated DNA fragment and an amplified DNA oligonucleotide.
Claim 10
Dependent Claim
18 words
The method of producing RNA of claim 1 wherein said spacer sequence comprises a plurality of promoter sequences.
Claim 11
Dependent Claim
17 words
The method of producing RNA of claim 1 wherein said spacer sequence comprises a 5'-upstream gene region.
Claim 12
Dependent Claim
19 words
The method of producing RNA of claim 1 wherein said spacer sequence comprises a plurality of 5'-upstream gene regions.
Claim 13
Dependent Claim
54 words
The method of producing RNA of claim 1 wherein said noncoding strand is modified at one end with a biotin moiety such that said biotin linked to DNA is selected from the group consisting of a biotin-DNA with a hydrophobic tether, a biotin-DNA with a hydrophilic tether and a biotin-DNA with an amphipathic tether.
Claim 14
Dependent Claim
32 words
The method of producing RNA of claim 1 further comprising: (e) replacing said coding strand with a new DNA strand comprising said coding region; and (f) repeating steps (b), (c) and (d).
Claim 15
Dependent Claim
27 words
The method of producing RNA of claim 1 wherein said coding strand is replaced by a new DNA strand comprising a modified or a different coding strand.
Claim 16
Dependent Claim
23 words
The method of producing RNA of claim 1 wherein said coding strand is replaced by a new DNA strand comprising multiple coding strand.
Claim 17
Dependent Claim
39 words
The method of producing RNA of claim 1 wherein said solid support is selected from the group consisting of polystyrene, latex microsphere, streptavidin-coated agarose beads, 3M EMPHASE supports, streptavidin-coated paramagnetic particles, acrylic beads, SEPHAROSE, TOYOPEARL, and controlled pore glass.
Claim 18
Dependent Claim
16 words
The method of producing RNA of claim 1 wherein said solid support is streptavidin-coated agarose beads.
Claim 19
Dependent Claim
39 words
The method of producing RNA of claim 1 wherein said RNA polymerase is selected from the group consisting of T7, T3, and SP6 RNA polymerases and said promoter sequence is a promoter of T7, T3, or SP6 bacteriophage, respectively.
Claim 20
Dependent Claim
16 words
The method of producing RNA of claim 1 wherein said nucleoside triphosphates are nucleoside triphosphate analogs.
Claim 21
Dependent Claim
16 words
The method of producing RNA of claim 1 wherein said method operates in a semi-batch mode.
Claim 22
Dependent Claim
16 words
The method of producing RNA of claim 1 wherein said method operates in a continuous mode.
Claim 23
Dependent Claim
18 words
The method of producing RNA of claim 1 wherein said bioreactor is a continuous flow stir-cell transcription reactor.
Claim 24
Dependent Claim
34 words
The method of producing RNA of claim 1 wherein said coding strand is dissociated from said immobilized DNA template, is removed from said transcription reaction mix and is replaced with a new coding strand.
Claim 25
Dependent Claim
20 words
The method of producing RNA of claim 1 wherein said coding strand is present in excess over said noncoding strand.
Claim 26
Dependent Claim
20 words
The method of producing RNA of claim 2 wherein said nucleotide sequence is between 2 and 20 bp in length.
Claim 27
Dependent Claim
24 words
The method of producing RNA of claim 1 wherein said single-stranded overhang is a nucleotide sequence of between 1 and 500 bp in length.
Claim 28
Dependent Claim
16 words
The method of producing RNA of claim 3 wherein said noncovalent linkage is a biotin-streptavidin linkage.
Claim 29
Dependent Claim
25 words
The method of producing RNA of claim 3 wherein said noncovalent linkage is selected from a group consisting of a poly(A)-poly(T) linkage and poly(G)-poly(C) linkage.
Claim 30
Dependent Claim
22 words
The method of producing RNA of claim 12 wherein said biotin linked to a DNA is a biotin-DNA with a hydrophilic tether.
Claim 31
Dependent Claim
28 words
The method of producing RNA of claim 18 wherein said RNA polymerase is expressed in a bacterium selected from the group consisting of Escherichia, Bacillus, Salmonella, and Pseudomonas.
Claim 32
Dependent Claim
27 words
The method of producing RNA of claim 19 wherein said nucleoside triphosphate analogs are selected from the group consisting of α-thio-nucleoside triphosphate, 2'-amino-nucleoside triphosphate and 2'-fluoro-nucleoside triphosphate.
Claim 33
Dependent Claim
17 words
The method of producing RNA of claim 22 wherein said stir-cell transcription reactor comprises a filtration membrane.
Claim 34
Dependent Claim
22 words
The method of producing RNA of claim 22 wherein said stir-cell transcription reactor comprises an inflow feed line and an exit port.
Claim 35
Dependent Claim
16 words
The method of producing RNA of claim 33 wherein said exit port comprises a filtration membrane.
Claim 36
Dependent Claim
24 words
The method of producing RNA of claim 34 wherein said filtration membrane is selected from the group consisting of a microfiltration and ultrafiltration membrane.
Claim 37
Independent Claim
85 words
A method of producing RNA enzymatically in a bioreactor comprising the steps of: (a) immobilizing from the 5'-end a single-stranded, hairpin DNA template, comprising a single-stranded overhang, a spacer sequence a promoter sequence and a coding sequence, to a solid support; (b) contacting said immobilized DNA template of step (a) with a transcription reaction mixture comprising a buffer, nucleoside triphosphates and an RNA polymerase such that RNA is produced; (c) removing said RNA produced in step (b); and, (d) optionally, repeating steps (b) and (c).
Claim 38
Independent Claim
85 words
A method of producing RNA enzymatically in a bioreactor comprising the steps of: (a) immobilizing from the 5'-end a single-stranded, hairpin DNA template, comprising a single-stranded overhang, a spacer sequence a promoter sequence and a coding sequence, to a solid support; (b) contacting said immobilized DNA template of step (a) with a transcription reaction mixture comprising a buffer, nucleoside triphosphates and an RNA polymerase such that RNA is produced; (c) removing said RNA produced in step (b); and, (d) optionally, repeating steps (b) and (c).
Claim 39
Dependent Claim
39 words
The method of producing RNA of claim 36 wherein said single-stranded hairpin DNA template is immobilized to a solid support at a nucleotide position selected from a group consisting of the 5'-end, the 3'-end and an internal nucleotide position.
Claim 40
Dependent Claim
18 words
The method of producing RNA of claim 36 wherein said DNA template is immobilized through a noncovalent linkage.
Claim 41
Dependent Claim
18 words
The method of producing RNA of claim 36 wherein said DNA template is immobilized through a covalent linkage.
Claim 42
Dependent Claim
18 words
The method of producing RNA of claim 38 wherein said DNA template is immobilized through a biotin-streptavidin linkage.
Claim 43
Dependent Claim
18 words
The method of producing RNA of claim 39 wherein said DNA template is immobilized through a succinyl-3'-0-ester linkage.
Claim 44
Independent Claim
77 words
A method of producing RNA enzymatically comprising the steps of: (a) immobilizing a single-stranded DNA template, to a controlled pore glass support such that the 3'-end of said DNA template is covalently linked to said controlled pore glass support using succinyl-3'-0-ester chemistry; (b) contacting said immobilized single-stranded DNA template of step (a) with a transcription reaction mixture comprising a complementary spacer and promoter strand, a buffer, nucleoside triphosphates and an RNA polymerase such that RNA is produced.
Claim 45
Independent Claim
77 words
A method of producing RNA enzymatically comprising the steps of: (a) immobilizing a single-stranded DNA template, to a controlled pore glass support such that the 3'-end of said DNA template is covalently linked to said controlled pore glass support using succinyl-3'-0-ester chemistry; (b) contacting said immobilized single-stranded DNA template of step (a) with a transcription reaction mixture comprising a complementary spacer and promoter strand, a buffer, nucleoside triphosphates and an RNA polymerase such that RNA is produced.
Claim 46
Dependent Claim
21 words
The method of producing RNA of claim 42 wherein said DNA template is chemically synthesized on said controlled pore glass support.
Claim 47
Dependent Claim
29 words
The method of producing RNA of claim 42 wherein said method is operated in a mode selected from a group consisting of a semi-batch mode or a continuous-flow mode.
Claim 48
Dependent Claim
21 words
The method of producing RNA of claim 42 wherein said immobilized DNA template is reused in successive rounds of transcription reaction.
Claim 49
Dependent Claim
16 words
The method of producing RNA of claim 42 wherein said DNA template comprises two DNA strands.
References Cited (Backward Citations)
No citation records indexed.
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