# US 5,700,667 A · Strategy for the production of RNA from immobilized templates

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- Number: US 5,700,667 A
- Document ID: US5700667A
- Status: Granted
- Kind: A
- Application number: 08/443,958
- Filed: 1995-05-18
- Granted: 1997-12-23
- Earliest claimed: 1993-09-20
- Assignees: The Regents of the University of Colorado
- Inventors: Herbert A. Marble; Robert H. Davis
- Web page: https://patentgrep.com/patent/US5700667A

## CPC

- C12P 19/34: Polynucleotides, e.g. nucleic acids, oligoribonucleotides
- C12Q 1/6865: Promoter-based amplification, e.g. nucleic acid sequence amplification [NASBA], self-sustained sequence replication [3SR] or transcription-based amplification system [TAS]
- B01F 33/45: Magnetic mixers; Mixers with magnetically driven stirrers
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- B01F: MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING (mixing paints B44D3/06)

## 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.

## Claims

### Claim 1

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 (depends on 1)

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 3 (depends on 1)

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 4 (depends on 1)

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 5 (depends on 1)

The method of producing RNA of claim 1 wherein the 5'-end of said noncoding DNA strand is immobilized.

### Claim 6 (depends on 1)

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 7 (depends on 1)

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 8 (depends on 1)

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 9 (depends on 1)

The method of producing RNA of claim 1 wherein said spacer sequence comprises a plurality of promoter sequences.

### Claim 10 (depends on 1)

The method of producing RNA of claim 1 wherein said spacer sequence comprises a 5'-upstream gene region.

### Claim 11 (depends on 1)

The method of producing RNA of claim 1 wherein said spacer sequence comprises a plurality of 5'-upstream gene regions.

### Claim 12 (depends on 1)

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 13 (depends on 1)

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 14 (depends on 1)

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 15 (depends on 1)

The method of producing RNA of claim 1 wherein said coding strand is replaced by a new DNA strand comprising multiple coding strand.

### Claim 16 (depends on 1)

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 17 (depends on 1)

The method of producing RNA of claim 1 wherein said solid support is streptavidin-coated agarose beads.

### Claim 18 (depends on 1)

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 19 (depends on 1)

The method of producing RNA of claim 1 wherein said nucleoside triphosphates are nucleoside triphosphate analogs.

### Claim 20 (depends on 1)

The method of producing RNA of claim 1 wherein said method operates in a semi-batch mode.

### Claim 21 (depends on 1)

The method of producing RNA of claim 1 wherein said method operates in a continuous mode.

### Claim 22 (depends on 1)

The method of producing RNA of claim 1 wherein said bioreactor is a continuous flow stir-cell transcription reactor.

### Claim 23 (depends on 1)

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 24 (depends on 1)

The method of producing RNA of claim 1 wherein said coding strand is present in excess over said noncoding strand.

### Claim 25 (depends on 2)

The method of producing RNA of claim 2 wherein said nucleotide sequence is between 2 and 20 bp in length.

### Claim 26 (depends on 1)

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 27 (depends on 3)

The method of producing RNA of claim 3 wherein said noncovalent linkage is a biotin-streptavidin linkage.

### Claim 28 (depends on 3)

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 29 (depends on 12)

The method of producing RNA of claim 12 wherein said biotin linked to a DNA is a biotin-DNA with a hydrophilic tether.

### Claim 30 (depends on 18)

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 31 (depends on 19)

The method of producing RNA of claim 19 wherein said nucleoside triphosphate analogs are selected from the group consisting of .alpha.-thio-nucleoside triphosphate, 2'-amino-nucleoside triphosphate and 2'-fluoro-nucleoside triphosphate.

### Claim 32 (depends on 22)

The method of producing RNA of claim 22 wherein said stir-cell transcription reactor comprises a filtration membrane.

### Claim 33 (depends on 22)

The method of producing RNA of claim 22 wherein said stir-cell transcription reactor comprises an inflow feed line and an exit port.

### Claim 34 (depends on 33)

The method of producing RNA of claim 33 wherein said exit port comprises a filtration membrane.

### Claim 35 (depends on 34)

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 36

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 37 (depends on 36)

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 38 (depends on 36)

The method of producing RNA of claim 36 wherein said DNA template is immobilized through a noncovalent linkage.

### Claim 39 (depends on 36)

The method of producing RNA of claim 36 wherein said DNA template is immobilized through a covalent linkage.

### Claim 40 (depends on 38)

The method of producing RNA of claim 38 wherein said DNA template is immobilized through a biotin-streptavidin linkage.

### Claim 41 (depends on 39)

The method of producing RNA of claim 39 wherein said DNA template is immobilized through a succinyl-3'-0-ester linkage.

### Claim 42

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 43 (depends on 42)

The method of producing RNA of claim 42 wherein said DNA template is chemically synthesized on said controlled pore glass support.

### Claim 44 (depends on 42)

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 45 (depends on 42)

The method of producing RNA of claim 42 wherein said immobilized DNA template is reused in successive rounds of transcription reaction.

### Claim 46 (depends on 42)

The method of producing RNA of claim 42 wherein said DNA template comprises two DNA strands.