Compositions and methods for the improved production and delivery of rna by efficient transcription termination

Abstract

Compositions and methods for efficiently producing and delivering double stranded RNA (dsRNA) are provided. Vector constructs useful for in vitro and in vivo expression of dsRNA are described. Also described are cell expression systems for efficient and cost-effective production of dsRNA in living cells and methods and compositions for providing the expressed dsRNA to target organisms. The described compositions and methods can be used to produce RNA molecules for screening or other uses, and to amplify RNA sequences for analysis.

1 .- 34 . (canceled) 
     
     
         35 . A vector comprising an engineered expression construct comprising:
 a. a promoter;   b. a first nucleic acid sequence positioned transcriptionally downstream of the promoter, wherein the first nucleic acid sequence encodes a double-stranded RNA (dsRNA) or a protein; and   c. a second nucleic acid sequence, positioned 3′ to the first nucleic acid sequence, wherein the second nucleic acid sequence comprises a rrn BT2 transcription terminator sequence, a first PET transcription terminator sequence, a PTH transcription terminator sequence, and a second PET transcription terminator sequence;   wherein the first nucleic acid sequence and the second nucleic acid sequence are operably linked to the promoter, and   
       wherein the vector is a plasmid vector. 
     
     
         36 . The vector of  claim 35 , wherein
 a. the rrn BT2 transcription terminator sequence comprises SEQ ID NO: 9;   b. the PTH transcription terminator sequence comprises a sequence selected from the group consisting of SEQ ID NOs: 6 and 7; or   c. the first PET transcription terminator sequence, the second PET transcription terminator sequence, or both, comprises SEQ ID NO: 5.   
     
     
         37 . The vector of  claim 35 , wherein the rrn BT2 transcription terminator sequence, the first and second PET transcription terminator sequences, and the PTH transcription terminator sequence each comprise a hairpin. 
     
     
         38 . The vector of  claim 37 , wherein the hairpins are separated by a spacer region comprising ten or fewer nucleotides. 
     
     
         39 . The vector of  claim 37 , wherein the hairpins comprise a stem region with fewer than three unpaired nucleotides. 
     
     
         40 . The vector of  claim 35 , wherein the promoter is selected from the group consisting of a T7 promoter, a T3 promoter, a SV40 promoter, a T5 promoter, a β-lactamase promoter, an  Escherichia coli  galactose promoter, an arabinose promoter, an alkaline phosphatase promoter, a tryptophan promoter, a lactose (lac) operon promoter, a lacUV5 promoter, a trc promoter, and a tac promoter. 
     
     
         41 . The vector of  claim 35 , wherein the promoter is selected from the group consisting of an RNA Pol I promoter, an RNA Pol II promoter, and an RNA Pol III promoter. 
     
     
         42 . The vector of  claim 35 , wherein the promoter is an RNA Pol III promoter that is selected from the group consisting of a U6 promoter, a tRNA promoter, a retroviral LTR promoter, an adenovirus VA1 promoter, a 5S rRNA promoter, a 7SK RNA promoter, a 7SL RNA promoter, and an H1 RNA promoter. 
     
     
         43 . The vector of  claim 35 , wherein transcription of the construct achieves a termination efficiency of at least 60%. 
     
     
         44 . A bacterial host cell comprising the vector of  claim 35 . 
     
     
         45 . The bacterial host cell of  claim 44 , wherein the bacterial host cell does not express RNase A. 
     
     
         46 . A cell culture system for in vivo synthesis of dsRNA or protein comprising the bacterial host cell of  claim 44  and growth media. 
     
     
         47 . The cell culture system of  claim 46 , wherein the growth media comprises 3.2% Tryptone, 2% Yeast Extract, 0.5% NaCl, 1% glycerol, 0.1% glucose, 0.4% alpha-lactose, 50 mM (NH4)2SO4, 10 mM KH2PO4, 40 mM Na2HPO4, and 2 mM MgSO4. 
     
     
         48 . A composition for controlling an invertebrate pest infestation comprising the bacterial host cell of  claim 44 , wherein the bacterial host cell is dead and un-lysed. 
     
     
         49 . A method for controlling an invertebrate pest infestation comprising applying the composition of  claim 48  to a plant. 
     
     
         50 . A composition for controlling an invertebrate pest infestation comprising a lysate of the bacterial host cell of  claim 44 . 
     
     
         51 . A method for controlling an invertebrate pest infestation comprising applying the composition of  claim 50  to a plant. 
     
     
         52 . A composition for inhibiting the spread of a viral disease in a population of plants comprising a lysate of the bacterial host cell of  claim 44 . 
     
     
         53 . A method for inhibiting the spread of a viral disease in a population of plants comprising applying the composition of  claim 52  to a plant, wherein the plant is a food source for an insect or nematode vector of the virus.