Strategy for the production of RNA from immobilized templates
AssigneeThe Regents of the University of Colorado
InventorsHerbert A. Marble, Robert H. Davis
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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.
Claims46
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).
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).
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.
Dependencies are inferred from the claim text.