SELECTING AND STABILIZING dsRNA CONSTRUCTS

Abstract

The invention provides methods for selecting nucleotide sequences that yield dsRNA-mediated gene suppression in a target organism and enable their uptake by the target organism. The invention further provides expression constructs that confer stabilized expression of such sequences in a transgenic host cell, and methods for their use. Also provided are organisms, cells and tissues prepared by a method of the invention.

1 . A method of obtaining a nucleic acid segment providing a desired level of suppression of a target gene, comprising: 
 a) obtaining a starting nucleic acid molecule substantially complementary to a target gene;    b) preparing a plurality of nucleic acid segments from the starting nucleic acid molecule;    c) assaying the nucleic acid segments for the ability to suppress expression of the target gene when expressed as a dsRNA in a cell comprising the target gene; and    d) identifying at least a first nucleic acid segment from the plurality of nucleic acid segments that provides a desired level of suppression of the target gene when expressed as a dsRNA.    
     
     
         2 . The method of  claim 1 , wherein the nucleic acid segments comprise from about 21 to about 26 contiguous nucleotide portions of said starting nucleic acid molecule.  
     
     
         3 . The method of  claim 1 , wherein the nucleic acid segments comprise overlapping portions of said starting nucleic acid molecule.  
     
     
         4 . The method of  claim 1 , wherein the nucleic acid segments each comprise from about 0.1% to about 98% of said target gene.  
     
     
         5 . The method of  claim 1 , further comprising the step of: 
 e) ranking the nucleic acid segments according to the level of suppression of the target gene obtained when the nucleic acid segments are expressed as dsRNA.    
     
     
         6 . The method of  claim 1 , wherein the desired level of suppression of the target gene is from about 1% to about 100% suppression of the expression of said target gene.  
     
     
         7 . The method of  claim 1 , wherein the desired level of suppression is complete suppression of the target gene.  
     
     
         8 . The method of  claim 1 , wherein the desired level of suppression is incomplete suppression of the target gene.  
     
     
         9 . The method of  claim 1 , wherein the target gene is from a plant, insect, fungal, bacterial or vertebrate organism.  
     
     
         10 . The method of  claim 1 , wherein the target gene is a plant gene.  
     
     
         11 . The method of  claim 1 , wherein the target gene is a crop pest or pathogen gene.  
     
     
         12 . The method of  claim 1 , wherein assaying the nucleic acid segments for the ability to suppress the target gene comprises expressing the segments as a dsRNA in a cell comprising the target gene and determining the level of suppression of the target gene.  
     
     
         13 . The method of  claim 1 , wherein assaying the nucleic acid segments for the ability to suppress the target gene comprises expressing the segments as a dsRNA in a cell; allowing a pest comprising the target gene to feed on the plant cell; and determining the level of suppression of the target gene.  
     
     
         14 . The method of  claim 1 , wherein assaying the nucleic segments for the ability to suppress the target gene comprises calculating a Reynolds score for the nucleic acid segments.  
     
     
         15 . The method of  claim 1 , wherein assaying the nucleic acid segments for the ability to suppress the target gene comprises providing said segments as dsRNA molecules in the diet of an organism comprising the target gene and determining the level of suppression of the target gene.  
     
     
         16 . The method of  claim 15 , wherein determining the level of suppression of the target gene comprises observing morbidity, mortality, or stunting of said organism.  
     
     
         17 . A method of suppressing the expression of a target gene in a cell comprising 
 a) obtaining a nucleic acid segment according to the method of  claim 1;  and    b) providing a dsRNA expressed from the nucleic acid to a host cell comprising the target gene to suppress the expression of the target gene.    
     
     
         18 . The method of  claim 17 , wherein providing the dsRNA expressed from the nucleic acid segment to the host cell comprises expressing the nucleic acid segment in the host cell in sense and antisense orientation.  
     
     
         19 . The method of  claim 17 , wherein providing the dsRNA expressed from the nucleic acid segment to the host cell comprises providing a diet comprising the dsRNA to the cell or an organism comprising the cell and allowing the cell to take up the dsRNA.  
     
     
         20 . The method of  claim 17 , wherein the host cell is a pest cell and wherein providing the dsRNA expressed from the nucleic acid to the pest cell comprises expressing the dsRNA in a plant cell and allowing a pest comprising the host cell to feed on the plant cell.  
     
     
         21 . The method of  claim 20 , wherein suppressing the expression of the target gene in the pest cell is manifested by a phenotypic effect on said cell or the pest comprising the cell.  
     
     
         22 . The method of  claim 21 , wherein the phenotypic effect is programmed cell death.  
     
     
         23 . A method for modulating the expression of at least a first gene in an organism comprising 
 a) providing as a dsRNA at least a first nucleic acid segment obtained by the method of  claim 1  to said organism, wherein said dsRNA segment is specific for said gene in said organism; and    b) observing a phenotypic effect in said organism.    
     
     
         24 . The method of  claim 23 , wherein the phenotypic effect is selected from the group consisting of cessation of vegetative growth, cessation of reproductive growth, cessation of feeding, mortality, morbidity, stunting, paralysis, inhibition of sexual reproduction, molt inhibition, flightless, and failure to emerge from pupal stage.  
     
     
         25 . A method for modulating the level of expression of a gene in a plant pest comprising providing in the diet of said pest at least a first dsRNA molecule, and observing a phenotypic effect of suppression of one or more genes in said pest, wherein said dsRNA molecule is produced from a nucleotide sequence that exhibits substantial homology with a corresponding DNA sequence of one or more essential genes in said pest, and wherein said nucleotide sequence is a nucleic acid segment identified according to  claim 1 .  
     
     
         26 . A method for inhibiting plant pest infestation comprising expressing at least a first dsRNA molecule obtained according to  claim 1  in a transgenic plant and providing the plant or a part or tissue thereof to one or more pests comprising said target gene, and observing a phenotypic effect in said organism, wherein the phenotypic effect is sufficient to inhibit infestation of said transgenic plant by said pest.  
     
     
         27 . A method for protecting a plant from pest infestation comprising expressing a dsRNA molecules obtained according to  claim 1  in a transgenic plant, providing said plant or a part or tissue thereof to one or more pests comprising said target gene, and observing a phenotypic effect in the organism, wherein the phenotypic effect is sufficient to inhibit infestation of the transgenic plant by the pest.  
     
     
         28 . The method of  claim 27 , further comprising expressing in said plant at least a first protein selected from the group consisting of a patatin, a  Bacillus thuringiensis  insecticidal protein, a  Xenorhabdus  insecticidal protein, a  Photorhabdus  insecticidal protein, a  Bacillus laterosporus  insecticidal protein, and a  Bacillus sphaericus  insecticidal protein.  
     
     
         29 . A method for protecting a plant or a seed thereof from pest infestation, comprising contacting the plant or seed thereof with a dsRNA molecule obtained according to  claim 1 .  
     
     
         30 . The method of  claim 29 , further comprising contacting the plant or seed thereof with a patatin, a  Bacillus thuringiensis  insecticidal protein, a  Xenorhabdus  insecticidal protein, a  Photorhabdus  insecticidal protein, a  Bacillus laterosporus  insecticidal protein, a  Bacillus sphaericus  insecticidal protein, a biocontrol agent or an insecticide.  
     
     
         31 . A cell of a plant protected from pest infestation according to the method of  claim 23 .  
     
     
         32 . A plant regenerated from the plant cell of  claim 31 .  
     
     
         33 . A seed or progeny produced from the plant of  claim 32 , wherein said seed comprises said nucleotide sequence.  
     
     
         34 . A method of producing an expression construct for expressing a dsRNA with reduced transgene silencing in a plant cell, comprising: 
 a) preparing an expression construct comprising a first sequence, a second sequence, and a third polynucleotide sequence, wherein the third polynucleotide sequence is linked to the first polynucleotide sequence by the second polynucleotide sequence and the third polynucleotide sequence is substantially the reverse complement of the first polynucleotide sequence; and    b) introducing an intron into at least one of the first and third polynucleotide sequences or introducing said expression construct into the intron, wherein the first and third polynucleotide sequences hybridize when transcribed into RNA and form a dsRNA molecule stabilized by the second polynucleotide sequence after intron splicing, and wherein the expression construct exhibits reduced transgene silencing in a plant cell transformed with the expression construct relative to an expression construct that lacks the intron.    
     
     
         35 . The method of  claim 34 , wherein the intron is introduced into at least one of the first and third polynucleotide sequences.  
     
     
         36 . The method of  claim 34 , wherein the intron is introduced into the first and third polynucleotide sequences.  
     
     
         37 . The method of  claim 34 , wherein the expression construct is introduced into the intron.  
     
     
         38 . A method of controlling feeding by a target crop pest or pathogen or progeny thereof on a plant comprising introducing into the plant an expression construct prepared by the method of  claim 34 .  
     
     
         39 . An expression construct prepared according to the method of  claim 34 .  
     
     
         40 . A plant cell transformed with the expression construct of  claim 39 .  
     
     
         41 . A method of increasing the pest or pathogen-inhibitory activity of a dsRNA, comprising 
 a) obtaining a first nucleic acid segment that when expressed as a dsRNA and taken up by a target crop pest or pathogen inhibits feeding by the target crop pest or pathogen or progeny thereof; and    b) linking the first nucleic acid segment to a second nucleic acid segment to create a longer nucleic acid segment, wherein the second nucleic acid segment is a nucleic acid that does not inhibit feeding by the target crop pest or pathogen or progeny thereof when expressed as a dsRNA, and wherein a dsRNA expressed from the longer nucleic acid exhibits increased potency of inhibition of feeding by the target crop pest or pathogen or progeny thereof relative to the dsRNA expressed from the first nucleic acid segment alone.    
     
     
         42 . The method of  claim 41 , wherein the first nucleic acid segment is obtained by a method comprising the steps of: 
 I) obtaining a starting nucleic acid molecule that when expressed as a dsRNA and taken up by a target crop pest or pathogen inhibits feeding by the target crop pest or pathogen or progeny thereof; and    II) selecting at least a first portion of the starting nucleic acid molecule that inhibits feeding by a target crop pest or pathogen or a progeny thereof following uptake of a dsRNA expressed from said portion; and    III) employing the portion as said the first nucleic acid segment in step a).    
     
     
         43 . The method of  claim 42 , wherein the starting nucleic acid molecule is a cDNA.  
     
     
         44 . The method of  claim 42 , wherein step II) comprises preparing a series of overlapping or consecutive portions from the starting nucleic acid molecule and identifying from said portions at least a first portion that inhibits feeding by a target crop pest or pathogen or a progeny thereof when expressed as a dsRNA and taken up by the target crop pest or pathogen.  
     
     
         45 . The method of  claim 41 , further comprising producing a recombinant vector comprising a first, a second and a third polynucleotide sequence, wherein the first polynucleotide sequence comprises the longer nucleotide segment and wherein the third polynucleotide sequence is linked to the first polynucleotide sequence by the second polynucleotide sequence, and wherein the third polynucleotide sequence is substantially the reverse complement of the first polynucleotide sequence such that the first and the third polynucleotide sequences hybridize when transcribed into a ribonucleic acid to form the double stranded ribonucleotide molecule stabilized by the linked second ribonucleotide sequence.  
     
     
         46 . The method of  claim 41 , wherein the second nucleotide segment is not substantially complementary to a nucleotide sequence of the target crop pest or pathogen.  
     
     
         47 . The method of  claim 41 , wherein one or both of the first nucleic acid segment and the third nucleic acid segment comprises an intron.  
     
     
         48 . The method of  claim 47 , comprising introducing an intron into said first nucleic acid segment.  
     
     
         49 . The method of  claim 41 , wherein the first nucleic acid segment comprises about 19 to about 80 contiguous bases substantially complementary to a coding sequence of the target crop pest or pathogen.  
     
     
         50 . The method of  claim 41 , wherein the first nucleic acid segment comprises about 19 to about 50 contiguous bases substantially complementary to a coding sequence of the target crop pest or pathogen.  
     
     
         51 . The method of  claim 41 , wherein the first nucleic acid segment comprises about 21 to about 30 contiguous bases substantially complementary to a coding sequence of the target crop pest or pathogen.  
     
     
         52 . The method of  claim 41 , wherein the longer nucleic acid segment comprises at least about 80 bases.  
     
     
         53 . The method of  claim 41 , wherein the longer nucleic acid segment comprises at least about 100 bases.  
     
     
         54 . The method of  claim 41 , wherein the longer nucleic acid segment comprises from about 80 bp to about 250 bases.  
     
     
         55 . The method of  claim 41 , where the target crop pest or pathogen is an insect.  
     
     
         56 . The method of  claim 55 , wherein the insect is selected from the group consisting of a Coleopteran, a Lepidopteran, a Hemipteran, and a Homopteran insect.  
     
     
         57 . The method of  claim 55 , wherein the target crop pest or pathogen is a  Diabrotica  spp.  
     
     
         58 . The method of  claim 41 , wherein target crop pest or pathogen is a nematode.  
     
     
         59 . An expression construct comprising the longer nucleic acid segment prepared according to the method of  claim 41  and the reverse complement thereof operably linked to a promoter.  
     
     
         60 . A method of controlling feeding by a target crop plant pest or pathogen or progeny thereof on a plant comprising introducing into the plant cell the expression construct of  claim 59 .  
     
     
         61 . A dsRNA expressed by the longer nucleic acid prepared according to the method of  claim 41 .  
     
     
         62 . A plant cell transformed with the expression construct of  claim 59 .  
     
     
         63 . A transgenic plant comprising the expression construct of  claim 59 .  
     
     
         64 . A method of producing an expression construct for expressing a dsRNA with increased specificity of pest or pathogen-inhibitory activity comprising: 
 a) obtaining a starting nucleic acid molecule substantially complementary to at least a first coding sequence of a target crop pest or pathogen;    b) selecting a region within the starting nucleic acid molecule that when expressed as a dsRNA inhibits feeding by the target crop pest or pathogen or progeny thereof following uptake of the dsRNA expressed from the region by the target crop pest or pathogen;    c) linking the region to a second nucleic acid molecule to produce an expression construct, wherein the second nucleic acid molecule when expressed as a dsRNA does not inhibit feeding by a target crop pest or pathogen or progeny thereof following uptake of the dsRNA.    
     
     
         65 . The method of  claim 64 , wherein selecting a region within the starting molecule comprises screening a series of overlapping or consecutive regions from the starting nucleic acid molecule and identifying from said regions at least a first region that inhibits feeding by a target crop pest or pathogen or a progeny thereof when expressed as a dsRNA and taken up by the target crop pest or pathogen.  
     
     
         66 . The method of  claim 64 , wherein the starting nucleic acid molecule is a cDNA from the target crop pest or pathogen.  
     
     
         67 . The method of  claim 64  where the target crop pest or pathogen is an insect.  
     
     
         68 . The method of  claim 67 , wherein the insect is selected from the group consisting of a Coleopteran, a Lepidopteran, a Hemipteran, and a Homopteran insect.  
     
     
         69 . The method of  claim 67 , wherein the insect is selected from the group consisting of:  D. virgifera virgifera; D. virgifera zeae; D. undecimpunctata; D. balteata; D. barberi ; and  D. speciosa.    
     
     
         70 . The method of  claim 64 , wherein target crop pest or pathogen is a nematode.  
     
     
         71 . The method of  claim 64 , wherein the target crop pest or pathogen is a  Diabrotica  spp.  
     
     
         72 . The method of  claim 64 , wherein the region comprises from about 19 bp to about 50 bp substantially complementary to a coding sequence of the target crop pest or pathogen.  
     
     
         73 . The method of  claim 72 , wherein the region comprises from about 21 bp to about 30 bp substantially complementary to a coding sequence of the target crop pest or pathogen.  
     
     
         74 . The method of  claim 64 , comprising identifying at least a second region within the starting molecule that when expressed as a dsRNA inhibits feeding by the target crop pest or pathogen or progeny thereof and linking the second region to the second nucleic acid molecule or a third nucleic acid molecule that when expressed as a dsRNA does not inhibit feeding by a target crop pest or pathogen or progeny thereof following uptake of the dsRNA expressed from the third nucleic acid molecule by the target plant pest or pathogen.  
     
     
         75 . The method of  claim 64 , wherein the region is not substantially complementary to a nucleic acid of a non-target crop pest or pathogen.  
     
     
         76 . The method of  claim 64 , wherein the region is complementary to a nucleic acid unique to the species in which the target crop pest or pathogen is classified.  
     
     
         77 . The method of  claim 64 , wherein the region is complementary to a nucleic acid unique to the genus in which the target crop pest or pathogen is classified.  
     
     
         78 . The method of  claim 64 , wherein the region is unique to  Diabrotica  spp.  
     
     
         79 . The method of  claim 78 , wherein the region is unique to a  Diabrotica  spp. selected from the group consisting of  Diabrotica undecimpunctata howardii  (Southern Corn Rootworm (SCR)),  Diabrotica virgifera virgifera  (Western Corn Rootworm (WCR)),  Diabrotica barberi  (Northern Corn Rootworm (NCR)),  Diabrotica virgifera zeae  (Mexican Corn Rootworm (MCR)),  Diabrotica balteata, Diabrotica viridula , and  Diabrotica speciosa  (Brazilian Corn Rootworm (BZR)).  
     
     
         80 . A method of controlling feeding by a target crop plant pest or pathogen or progeny thereof on a plant comprising introducing into the plant an expression construct prepared by the method of  claim 64 .  
     
     
         81 . A dsRNA expressed by an expression construct prepared by the method of  claim 64 .  
     
     
         82 . A plant cell transformed with an expression construct prepared by the method of  claim 64 .  
     
     
         83 . A method of enhancing the control of a target crop pest or pathogen in a plant comprising expressing in the cells of the plant at least two dsRNA sequences that function upon uptake by the pest or pathogen to inhibit the expression of at least a first target coding sequence within the target crop pest or pathogen, wherein the two dsRNA sequences are substantially complementary to two non-contiguous portions of the first target coding sequence or to two different coding sequences of the target crop pest or pathogen.  
     
     
         84 . The method of  claim 83 , wherein the two dsRNA sequences comprises about 19 bp to about 80 bp.  
     
     
         85 . The method of  claim 83 , wherein the two dsRNA sequences comprises about 19 bp to about 50 bp.  
     
     
         86 . The method of  claim 83 , wherein the two dsRNA sequences comprises about 21 bp to about 30 bp.  
     
     
         87 . The method of  claim 83 , wherein the two dsRNA sequences are substantially complementary to at least two target coding sequences of the target crop pest or pathogen.  
     
     
         88 . The method of  claim 87 , further comprising expressing in the cells of the plant at least a third dsRNA sequence that functions upon uptake by the pest or pathogen to inhibit the expression of a third target coding sequence within the target crop pest or pathogen, wherein the third dsRNA sequence is substantially complementary to a portion of the third target coding sequence.  
     
     
         89 . The method of  claim 83 , wherein the two dsRNA sequences are expressed from regions selected from a starting nucleic acid molecule that when expressed as a dsRNA inhibits feeding by a target crop pest or pathogen or progeny thereof following uptake of the dsRNA by the target crop pest or pathogen.  
     
     
         90 . The method of  claim 89 , wherein the starting nucleic acid molecule is a cDNA from the target crop pest or pathogen.  
     
     
         91 . The method of  claim 83 , further comprising expressing a polynucleotide sequence in the cell selected from the group consisting of a patatin, a  Bacillus thuringiensis  insecticidal protein, a  Xenorhabdus  insecticidal protein, a  Photorhabdus  insecticidal protein, a  Bacillus laterosporus  insecticidal protein, and a  Bacillus sphaericus  insecticidal protein.  
     
     
         92 . The method of  claim 91 , wherein the  Bacillus thuringiensis  insecticidal protein is selected from the group consisting of a Cry1, a Cry3, a TIC851, a CryET70, a Cry2, ET29, ET37, a binary insecticidal protein CryET33 and CryET34, a binary insecticidal protein CryET80 and CryET76, a binary insecticidal protein TIC100 and TIC101, a binary insecticidal protein ET29 and TIC810, a binary insecticidal protein ET37 and TIC812, and a binary insecticidal protein PS149B1.  
     
     
         93 . The method of  claim 83 , wherein the target coding sequence encodes a protein, the predicted function of which is selected from the group consisting of muscle formation, juvenile hormone formation, juvenile hormone regulation, ion regulation and transport, digestive enzyme synthesis, maintenance of cell membrane potential, feeding site formation, feeding site development, feeding site maintenance, infection, molting, amino acid biosynthesis, amino acid degradation, sperm formation, pheromone synthesis, pheromone sensing, antennae formation, wing formation, leg formation, development and differentiation, egg formation, larval maturation, digestive enzyme formation, haemolymph synthesis, haemolymph maintenance, neurotransmission, cell division, energy metabolism, respiration, and apoptosis.  
     
     
         94 . The method of  claim 87 , wherein the two target coding sequences perform at least two functions essential for target crop pest or pathogen survival that are suppressed by the dsRNA sequences, the functions being selected from the group consisting of feeding by the pest or pathogen, cell apoptosis, cell differentiation and development, capacity or desire for sexual reproduction, muscle formation, muscle twitching, muscle contraction, juvenile hormone formation, juvenile hormone regulation, ion regulation and transport, maintenance of cell membrane potential, amino acid biosynthesis, amino acid degradation, sperm formation, pheromone synthesis, pheromone sensing, antennae formation, wing formation, leg formation, egg formation, larval maturation, digestive enzyme formation, haemolymph synthesis, haemolymph maintenance, neurotransmission, larval stage transition, pupation, emergence from pupation, cell division, energy metabolism, respiration, and formation of cytoskeletal structure.  
     
     
         95 . The method of  claim 83 , wherein the target crop pest is a corn rootworm selected from the group consisting of  Diabrotica undecimpunctata howardii  (Southern Corn Rootworm (SCR)),  Diabrotica virgifera virgifera  (Western Corn Rootworm (WCR)),  Diabrotica barberi  (Northern Corn Rootworm (NCR)),  Diabrotica virgifera zeae  (Mexican Corn Rootworm (MCR)),  Diabrotica balteata, Diabrotica viridula , and  Diabrotica speciosa  (Brazilian Corn Rootworm (BZR)).  
     
     
         96 . A method for managing crop pest resistance to an agent for controlling the crop pest, comprising contacting the crop pest with a nucleic acid segment produced by the method of  claim 1  and one additional agent selected from the group consisting of a patatin, a  Bacillus thuringiensis  insecticidal protein, a  Xenorhabdus  insecticidal protein, a  Photorhabdus  insecticidal protein, a  Bacillus laterosporus  insecticidal protein, a  Bacillus sphaericus  insecticidal protein, a biocontrol agent, and an insecticide.  
     
     
         97 . The method of  claim 96 , wherein the insecticide is selected from the group consisting of a carbaryl insecticide, fenvalerate, esfenvalerate, malathion, a carbofuran insecticide, chloropyrifos, fonophos, phorate, terbufos, permethrin, a neonicotinoid, and tefluthrin.  
     
     
         98 . The method of  claim 96 , wherein the additional agent is provided as a seed treatment.