Methods and compositions for preparing capped RNA
The present invention concerns methods and compositions for increasing the yield of capped and full-length RNA transcripts produced in in vitro transcription reactions. Such methods and compositions can be used for cost-efficient, large scale production of capped full-length RNA transcripts that can be subsequently translated. Methods and compositions involve reaction conditions that promote such production, and includes the implementation of fed-batch introduction of GTP, which competes with a cap analog.
1 . A method for producing capped RNA comprising:
a) incubating components for a transcription and capping reaction under conditions to promote transcription and capping, wherein the components include a cap analog, a nucleotide that competes with the cap analog, and non-competing nucleotides; and,
b) supplementing the reaction with the competing nucleotide to maintain the concentration of the competing nucleotide in the reaction at a ratio between about 1:1 and about 1:50 relative to the concentration of the cap analog in the reaction.
2 . The method of claim 1 , wherein the reaction is supplemented to maintain the concentration of the competing nucleotide in the reaction at a ratio between about 1:4 and about 1:25 relative to the concentration of the cap analog in the reaction.
3 . The method of claim 1 , wherein the reaction is supplemented intermittently by a fed batch process.
4 . The method of claim 1 , wherein the competing nucleotide is GTP or a GTP analog.
5 . The method of claim 1 , wherein the concentration of the cap analog in the reaction is between about 1 mM and about 10 mM.
6 . The method of claim 5 , wherein the concentration of the cap analog in the reaction is between about 2 mM and about 6 mM.
7 . The method of claim 3 , wherein the reaction is supplemented at least two times by the fed-batch process.
8 . The method of claim 1 , wherein the reaction is supplemented with the competing nucleotide to maintain the concentration of the competing nucleotide in the reaction between about 0.1 mM and about 2.0 mM.
9 . The method of claim 3 , wherein each supplementation by the fed-batch process adds between about 0.1 mM and about 2.0 mM of the competing nucleotide to the reaction.
10 . The method of claim 9 , wherein each supplementation by the fed-batch process adds between about 0.2 mM and about 1 mM of the competing nucleotide to the reaction.
11 . The method of claim 1 , wherein the reaction is supplemented with other components of the reaction but not all components of the reaction.
12 . The method of claim 1 , wherein the reaction yields between about 1 mg/ml and about 10 mg/ml of capped transcript.
13 . The method of claim 12 , wherein the reaction yields between about 4 and about 7 mg/ml of capped transcript.
14 . The method of claim 3 , wherein the supplementation is periodic.
15 . The method of claim 1 , wherein the supplementation is continuous during most of the reaction.
16 . The method of claim 15 , wherein the supplementation of the competing nucleotide is at a rate of about 10 μM per minute to about 200 μM per minute.
17 . A method for producing capped RNA comprising introducing to a transcription and capping reaction GTP or a GTP analog by a fed-batch process.
18 . The method of claim 17 , wherein other reaction components, except a cap analog, are introduced to the reaction by the fed-batch process.
19 . The method of claim 17 , wherein the concentration of GTP introduced into the reaction depends on the initial concentration of a cap analog in the reaction.
20 . The method of claim 19 , wherein the concentration of GTP introduced into the reaction is determined based on a ratio of the concentration of GTP to the concentration of the cap analog, wherein the ratio is between about 1:1 and about 1:50.
21 . (canceled)
22 . The method of claim 17 , wherein the amount of GTP or a GTP analog introduced in the reaction by the fed-batch process increases the concentration of GTP or GTP analog in the reaction by less than about 4 mM after each introduction.
23 - 24 . (canceled)
25 . The method of claim 17 , wherein the initial concentration of a cap analog in the reaction is between about 1 mM and about 10 mM.
26 - 30 . (canceled)
31 . The method of claim 17 , wherein the cap analog is selected from the group consisting of m7GpppG; m7GpppA; m7GpppC; GpppG; m2,7GpppG; m2,2,7GpppG; m7Gpppm7G; ARCA; and, m7,2′OmeGpppG, m72′dGpppG, m7,3′OmeGpppG, m7,3′dGpppG and their tetraphosphate derivatives.
32 - 33 . (canceled)
34 . The method of claim 17 , wherein the initial reaction volume is at least about 100 μl.
35 - 37 . (canceled)
38 . The method of claim 17 , wherein one or more of the following components is also introduced by the fed-batch process: polymerase, pyrophosphatase, a magnesium salt, or a ribonuclease inhibitor.
39 . The method of claim 17 , wherein GTP or a GTP analog are introduced into the reaction by a fed-batch process so as to maintain the concentration of GTP or a GTP analog in the reaction less than about 1 mM.
40 . The method of claim 17 , wherein introduction of GTP or a GTP analog by the fed-batch process is intermittent or periodic.
41 - 46 . (canceled)
47 . The method of claim 17 , wherein one of more reaction components are immobilized.
48 . The method of claim 47 , wherein template is immobilized.
49 - 60 . (canceled)
61 . A method for producing transcripts with a nonextending nucleotide at the 5′ end comprising introducing a nucleotide that competes with the nonextending nucleotide by a fed-batch process to a transcription reaction comprising RNA polymerase and the non-extending nucleotide.
62 - 65 . (canceled)