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EP4396348A2 - High molecular weight modified dsrna compositions - Google Patents

High molecular weight modified dsrna compositions

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Publication number
EP4396348A2
EP4396348A2 EP22783223.5A EP22783223A EP4396348A2 EP 4396348 A2 EP4396348 A2 EP 4396348A2 EP 22783223 A EP22783223 A EP 22783223A EP 4396348 A2 EP4396348 A2 EP 4396348A2
Authority
EP
European Patent Office
Prior art keywords
seq
mrna
formula
compound
mdsrna
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP22783223.5A
Other languages
German (de)
French (fr)
Inventor
Darren H. Wakefield
Grace B. Arhancet
Vladimir Trubetskoy
Juan P. Arhancet
Jason Klein
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Nanosur LLC
Original Assignee
Nanosur LLC
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Filing date
Publication date
Application filed by Nanosur LLC filed Critical Nanosur LLC
Publication of EP4396348A2 publication Critical patent/EP4396348A2/en
Pending legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/60—Isolated nucleic acids
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
    • A01P23/00—Chemosterilants
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
    • A01P7/00—Arthropodicides
    • A01P7/02—Acaricides
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
    • A01P7/00—Arthropodicides
    • A01P7/04—Insecticides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2330/00—Production
    • C12N2330/30—Production chemically synthesised

Definitions

  • alkenyl as used herein are acyclic branched or unbranched hydrocarbons having one carbon-carbon double bond and the general formula -CnEbn-i. One or more of the hydrogen atoms can be substituted.
  • An alkyl group can be straight or branched chain and include ethenyl, propenyl, isopropenyl, butenyl, isobutenyl, hexenyl, and the like.
  • an alkenyl contains from 2 to 25 carbon atoms in the principal chain.
  • aryl or “Ar” as used herein alone or as part of another group denote optionally substituted homocyclic aromatic groups.
  • Aryl groups can be monocyclic or bicyclic groups containing from 5 to 12 carbons in the ring portion, such as phenyl, biphenyl, naphthyl, substituted phenyl, substituted biphenyl, or substituted naphthyl.
  • acceptable salts refers to salts derived from suitable inorganic and organic acids and inorganic and organic bases that are, within the scope of sound judgment, suitable for use in contact with the tissues of humans, lower animals, and plants without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio.
  • activation agent refers to a compound that increases the nucleophilicity or electrophilicity of target moiety.
  • the change in electron density can be the result of an ionic or covalent bond from the activating group.
  • the activation agents described herein increase the electrophilicity of a target carbonyl.
  • Non-limiting examples of activation agents are lewis acids, protons, and coupling reagents.
  • suitable leaving group refers to a molecular fragment that departs with a pair of electrons in heterolytic bond cleavage.
  • the leaving group helps facilitate the reaction by lowering the energy of activation.
  • suitable leaving groups are halides, tosylated or mesylated alcohols, pseudohalides, amines, heterocycles, and the like.
  • the suitable leaving group is imidazole.
  • MdsRNAs are described having no more than about 30% of all the ribose rings chemically modified at the 2'-OH position with high molecular weight polyalkyloxy polymers, and optionally at least about 2% of all the nucleotides being chemically modified with LMW modified moieties.
  • the MdsRNAs are capable of inhibiting gene expression in a sequence specific manner, such as through RNA interference or antisense mechanisms.
  • composition comprising a post- transcriptionally chemically modified double strand RNA (MdsRNA) wherein the MdsRNA comprises a double strand RNA wherein no more than about 30% of all the nucleotides independently comprise Formula (I):
  • MdsRNA post- transcriptionally chemically modified double strand RNA
  • B is a nucleobase
  • B is a nucleobase
  • R 1 is selected from:
  • b is an integer from 12-1000, and c is an integer from 12-1000; and optionally wherein at least about 2% of all the nucleotides independently comprise
  • B is a nucleobase
  • R 2 is selected from C1-C25 alkyl, C1-C25 alkenyl, C1-C25 alkynyl, C5-C12 aryl or C5-C12 heteroaryl, wherein R 2 is optionally substituted with one or more substituents selected from halo, C1-12 alkyl, C1-C12 aminoalkyl, or Ci-C 12 alkoxy.
  • R 1 is selected from:
  • x is an integer from 80-1000.
  • x is an integer from 50-900.
  • a is an integer from 80-1000.
  • b is an integer from 80-1000.
  • c is an integer from 80-1000.
  • R 1 has a molecular weight between 5,000 and 15,000 Da.
  • R 1 has a molecular weight between 5,000 and 40,000 Da.
  • R 1 is polyethylene glycol (PEG).
  • R 1 is poloxamer 407, 338, 188, 184, 401 or any combination thereof.
  • R 1 is a HMW polymer with an HLB ranging from about 2 to about 30. In another embodiment, R 1 is a HMW polymer with an HLB ranging from about 15 to about 27.
  • lauroyl In an embodiment, lauroyl.
  • R 1 is and R 2 is linoleyl.
  • the ratio of Formula (I) to Formula (III) to unmodified nucleotides is about 3:5:2; 3:6.5:0.5; 1 :7:2; .05:9:0.5; 1.3:4:5.7; 0.01 :4:5.99; 0.06:0:96.4.
  • from about 2% to about 50% of all nucleotides are modified with LMW moieties.
  • the target insect is Diamondback moth (Plutella xylostella), Gypsy moth, Red imported fire ant (Solenopsis invicta), Fall armyworm, Colorado potato beetle, Canola flea beetle, Aedes aegypti, or Western corn root worm (Diabrotica virgifera virgifera).
  • the target insect is Pea aphid (Acyrthosiphon pisum), Soybean aphid, Piezodorus guildinii.
  • the target is an acari, such as Verroa mite.
  • the target is a weed, such as Palmer amaranth.
  • the target is a fungus such as Palmer amaranth, Fusarium graminearum (Gibberella zeae) or Botrytis.
  • the MdsRNA used in the compositions of the disclosure will be a nucleotide sequence that can inhibit the expression of target genes or regions in these insects.
  • the MdsRNA comprises a sequence complementary to a target region in Diamondback moth, such as but not limited to AChE2, P450, P450 CYP6BFlvl, Cytokine receptor DOMELESS, DOUX, Protein MESH transcript variant XI, Venom carboylesterase-6 and VPASE-E.
  • the MdsRNA comprises a sequence complementary to a target region in Fall armyworm, such as but not limited to P450 CYP9A58, Cytokine receptor DOMELESS, Dredd, VPASE, Protein MESH transcript variant XI, P450 CYP321 A8, P450 CYP6B2- like.
  • the MdsRNA comprises a sequence complementary to a target region in Western corn root worm, such as but not limited to SNF7.
  • composition comprising a post- transcriptionally modified double strand RNA (MdsRNA) wherein the MdsRNA comprises a double strand RNA wherein no more than about 30% of all the nucleotides independently comprise Formula (I): or an acceptable salt thereof, wherein:
  • MdsRNA post- transcriptionally modified double strand RNA
  • B is a nucleobase
  • R 1 is a linear or branched polyalkyloxy having a molecular weight between about 400 Da and about 15 kDa; optionally wherein at least about 2% of all the nucleotides independently comprise LMW modified nucleotides of Formula (III): or an acceptable salt thereof, wherein:
  • B is a nucleobase
  • R 2 is selected from C1-C25 alkyl, C1-C25 alkenyl, C1-C25 alkynyl, C5-C12 aryl or C5- C12 heteroaryl, wherein R 2 is optionally substituted with one or more substituents selected from halo, C1-12 alkyl, C1-C12 aminoalkyl, or Ci-C 12 alkoxy.
  • R 1 is a poloxamer.
  • the poloxamer is a triblock polymer comprising poly(ethylene glycol)-block-poly(propylene glycol)-block- poly (ethylene glycol).
  • R 1 is a HMW polymer with an HLB ranging from about 2 to about 30. In another embodiment, R 1 is a HMW polymer with an HLB ranging from about 15 to about 27.
  • R 2 is N-methyl anthranoyl (NMA), N-benzyl anthranoyl (NBA), dimethyl furoyl, -Tyr, -Trp, -Leu, octanoyl, lauroyl, linoleyl, oleyl, nicotinoyl or benzoyl.
  • NMA N-methyl anthranoyl
  • NBA N-benzyl anthranoyl
  • dimethyl furoyl -Tyr, -Trp, -Leu, octanoyl, lauroyl, linoleyl, oleyl, nicotinoyl or benzoyl.
  • the ratio of Formula (I) to Formula (III) to unmodified nucleotides is about 3:5:2; 3:6.5:0.5; 1 :7:2; .05:9:0.5; 1.3:4:5.7; 0.01 :4:5.99; 0.06:0:96.4.
  • from about 2% to about 50% of all nucleotides are modified with LMW moieties.
  • R 1 is selected from: wherein at least 2% of all the nucleotides independently comprise LMW modified nucleotides of Formula (III) as defined above.
  • x is an integer from 50-900.
  • b is an integer from 80-1000.
  • R 1 has a molecular weight between 5,000 and 15,000 Da.
  • R 1 has a molecular weight between 5,000 and 40,000 Da.
  • steps (c) and (d) are present.
  • a target gene is selected such that inhibiting expression of the target gene kills, inhibits growth or appetite of, or slows reproduction of an animal, fungus, or weed. Inhibiting expression of the target gene can control, kill, inhibit growth or appetite of, or slow reproduction of the animal, fungus, or weed.
  • the insect, fungus, or plant is of agricultural significance.
  • an agriculturally significant animal, fungus, or plant is an insect, fungus, or weed.
  • the modified expression reduces the fertility rate of the target insect.
  • the MdsRNA comprises a sequence complementary to the P450, CYP6FBlvl, MESH, AChE2, VPASE, DOMELESS, DOUX or Venom target region in a Lepidopteran.
  • Carrier and excipients include, but are not limited to, absorption enhancers, anti-adherents, anti-foaming agents, anti-oxidants, binders, buffering agents (pH regulating agents), chelating agents, coating agents, colors, delivery enhancers, dextran, dextrose, diluents, disintegrates, dispersants, emulsifiers, extenders, fillers, foam control agents, glidants, humectants, lubricants, oils, pigments, polymers, preservatives, saline, salts, solvents, sugars, surfactants, suspending agents, sustained release matrices, sweeteners, thickening agents, tonicity agents, vehicles, water-repelling agents, and wetting agents.
  • absorption enhancers include, but are not limited to, absorption enhancers, anti-adherents, anti-foaming agents, anti-oxidants, binders, buffering agents (pH regulating agents),
  • the liquid formulation is an aqueous formulation.
  • an aqueous formulation contains only water, the MdsRNA and other agents.
  • additional compounds, solvents, or adjuvants are provided with the aqueous formulation.
  • the agrochemical composition is formulated as a powder or dust.
  • the powder or dust can be granulated to be suitable for applying the powder or dust directly to a crop (i.e., by dusting the crop), or it can be granulated for eventual dissolution in a solvent such as water.
  • the composition is a lyophilisate.
  • the MdsRNA and the other agents are lyophilized together.
  • one or more MdsRNAs and the other agents can be lyophilized separately.
  • an agrochemical composition is formulated as a spray in the form of an aerosol.
  • the composition When formulated as an aerosol spray, the composition is generally charged in a container under pressure together with a propellant.
  • propellants include fluorotrichloromethane and dichlorodifluoromethane.
  • the fungicidal composition is present inside the seed coat, or internal to the seed. In some embodiments, the fungicidal composition is formed over the seed such that it covers the exterior of the seed, either fully or partially. Methods for coating a seed include those known in the art.
  • a composition comprising one or more MdsRNAs can be applied to a plant prior to infection to prevent an insect infection.
  • the composition may also be applied after the appearance of signs of infection to treat an insect infection.
  • the composition can be applied by a variety of methods depending on the plant part to be treated.
  • the composition can be applied to a plant seed prior to planting to prevent insect infection of the seed.
  • the composition can be applied to the soil at the time of planting or just before planting to prevent insect infestation of the newly planted seed (i.e., as a pre- emergent).
  • the composition can be applied to a plant after its germination or to the foliage of the plant after emergence to either treat or prevent insect infestation (i.e., as a post-emergent).
  • the application occurs during the stages of germination, seedling growth, vegetative growth, and reproductive growth. In some embodiments, application occurs during vegetative and reproductive growth stages.
  • Table 1 Modified MdsRNA Compounds Synthesized by Procedure of Example 1.
  • a Input is moles of PEG/mole of RNA nucleotide.
  • b Modifier is the -OC(O)R 1 modification as defined in Formula I.
  • c Sequence is the sequence of nucelobases of the RNA strand backbone.
  • Example 2 Preparation of High Molecular Weight Polyalkyloxy Polymer dsRNA using DCM for the activation reaction.
  • Table 2 Modified MdsRNA Compounds Synthesized by Procedure of Example 2.
  • a Input is moles of PEG/mole of RNA nucleotide.
  • b Modifier is the -OC(O)R 1 modification as defined in Formula I.
  • c Sequence is the sequence of nucelobases of the RNA strand backbone.
  • Example 3 Preparation of High Molecular Weight Polyalkyloxy Polymer dsRNA in DMSO.
  • the polyaloxy polymer was reacted with a linker to introduce a reactive COOH at one end of the polymer chain.
  • One eq. of polyalkyloxy polymer was dissolved in 1,4- dioxane.
  • DMAP 0.5 eq.
  • DIEA 3 eq.
  • the corresponding anhydride 1.5. eq.
  • the reaction mixture was stirred overnight at room temperature.
  • the mixture was diluted with DCM and extracted with sodium bicarbonate, brine.
  • the organic layer was dried over sodium sulfate, and concentrated at reduced pressure.
  • the resulting gum was triturated with fresh DCM and the solid polyalkyloxy-COOH was collected via filtration.
  • Benzyl-TriButyl Ammonium-Chloride was dissolved in DMSO at 200 mM. An aliquot of this solution was added to freeze-dried dsRNA in a reaction tube. The tube was warmed to 65°C in a shaker for 1 hr. Then the temperature was reduced to 55°C and catalytic amount of pyridine was added followed by a 200 pg/pL solution of DMAP. A 200 ug/uL solution of activated polyalkyloxy polymer was added in portions. The mixture was kept at 55 °C for 90 min and then was quenched with 500 mM citrate pH 4.5 and diluted to 40 mL with water.
  • the resulting mixture containing the crude M-dsRNA was purified using tangential flow filtration (TFF), to yield MdsRNA with >90% purity.
  • the MdsRNAs were characterized by gel electrophoresis with non-denaturing agarose gel and denaturing polyacrylamide gel.
  • MdsRNAs targeting Diamondback moth (DBM) (P. xylostella) were tested on DBM larvae using cabbage leaf discs.
  • DBM Diamondback moth
  • the MdsRNA treatments were dissolved in water with an adjuvant and diluted to obtain the desired concentration.
  • the desired treatment solution was sprayed on both sides of fresh cabbage leaf discs (3.5 cm diameter). Each treatment was applied with 3 repetitions and a water only control.
  • the discs were placed on a wet paper towel in a container.
  • P. xylostella eggs (8-12) were transferred to each treated disc and the containers were incubated at 26 °C and 72% relative humidity (RH).
  • b Modifier is the -OC(O)R 1 modification as defined in Formula I.
  • c Sequence is the sequence of nucleobases of the RNA strand backbone.
  • Example 4 Preparation of High Molecular Weight Polyalkyloxy Polymer- NMIA dsRNA.
  • MPEGA- dsAChE2 was prepared and purified according to the procedure described in Example 3 using Methoxy PEG acetic acid 10K at input 0.1. Benzyl-TriBA- C1 was dissolved in DMSO at 200 mM. An aliquot (3.4 mL) of this solution was added to freeze-dried MPEGA-dsAChE (34 mg) in a reaction tube. The tube was warmed to 65°C in a shaker for 1 hr.
  • Example 5 Preparation of High Molecular Weight Polyalkyloxy Polymer- FA dsRNAs. [00267] Following a similar procedure as described in Example 4. MPEGA-P450 5K (115) was reacted with activated lauryl (LAU), oleic (OLE) and linoleic (LIN) fatty acids to yield the MPEGA-FA-P450 materials described in Table 4.
  • LAU lauryl
  • OLE oleic
  • LIN linoleic
  • Cabbage leaves were collected at stage 4-5. Leaves were prepared by washing with tap water using a nozzle to remove insects, dust etc. The leaves were then wiped with paper towels. Leaf discs of 3.5 cm diameter were punched from the cabbage leaves using a metal cutter. An aqueous solution of the desired treatment solution was prepared at 500 ppm or 150 ppm concentrationand 60uL were sprayed on both sides of the disc. After the treatment was applied to both sides of the leaf disc, four 1 cm diameter discs were then punched from the treated 3.5 cm. The resulting treated 1 cm diameter discs were used in the assay. Three P. xylostella egg wassere placed on each treated disc placed in a petri dish.
  • the plates were incubated at 26 °C and 72% room humidity (RH). After 48 hours only one neonate was transferred to a new (2 nd ) treated disc. A third treated disc was provided after 24 hours of the 2 nd treated disc. The larvae were then provided with an untreated disc every day for up to 10 days. Mortality was recorded daily starting on the 2 nd day after incubation. Mortality rate was calculated for each test during the duration of the experiment and cumulative mortality after 5 days and 7 or 9 days was calculated.
  • RH room humidity
  • Benzyl-TriButyl Ammonium-Chloride is dissolved in DMSO at 200 mM. An aliquot of this solution is added to freeze-dried dsRNA in a reaction tube. The tube is warmed to 65°C in a shaker for 1 hr. Then the temperature is reduced to 55C and 110 uL pyridine are added followed by a 200 ug/uL solution of DMAP and two additions of a 200 ug/uL solution of N-methyl isatoic anhydride (NMIA). The mixture is kept at 55°C for 90 min and then is quenched with 500 mM citrate, and diluted with water.
  • NMIA N-methyl isatoic anhydride
  • NMA-dsRNA The resulting mixture containing the crude NMA-dsRNA is purified using tangential flow filtration, TFF, to yield MdsRNA with >90% purity.
  • the following NMA-dsRNA were prepared according to the procedure described above: NMA-P450(l 15) (37% NMA), NMA- VPASE(7) (81% NMA), NMIA-AChE(l) (40%, 80% NMA), NMA-B1(4), NMA-TH(3). This procedure was also used to prepare dsRNA analogs for NBA, dimethyl furoyl, -Tyr, - Trp, -Leu, and octanoyl. This procedure can be used for other modifications such as, but not limited to, lauroyl, linoleyl, and the like.
  • the dsRNA used in this trial is a dsSNF7 sequence that has been previously reported in field dissipation study (Bachman et al., 2020).
  • QuantGene a nucleic acid detection platform marketed by Invitrogen, was the preferred analytical method used to quantify of dsRNA sequences on the cabbage plant leaves after spray.
  • the sequence and corresponding Quantigene probe sets were published previously (Armstrong et al., 2013). The sequence is not directed against Diamondback Moth (DBM) and used here only for analytical purposes. Naive (unmodified) dsSNF7, treatment C2, was used as a comparison.
  • SEQ ID Nos: 1-12 are provided above.
  • ID 1 Plutella xylostella V-type proton ATPase subunit E (LOC 105389010), mRNA. NCBI Reference Sequence. NM 001305532 1 . 51-150 GGCAGCAACCATG GCGC TCAGCGATGCAGA TGTCCAAAAACAGATCAAGCATATGATGGCCTTCA TCGAGCAAGAGGCAAATGAAAAGGCCGAAGAAATC (SEQ ID NO: 13).
  • ID 2 Plutella xylostella V-type proton ATPase subunit E (LOC 105389010), mRNA 151-250 GATGCTAAGGCTGAGGAGGAGTTCAACATCGAGAAGGGGCGTCTGG
  • ID 4 Plutella xylostella V-type proton ATPase subunit E (LOC 105389010), mRNA 351-450 GACGCGCCGCCGCCTCGCCGAGGTGCCCAACGACCAGGGGCTCTACTCCGACC
  • ID 5 Plutella xylostella V-type proton ATPase subunit E (LOC 105389010), mRNA 451-550 ACCGTAACCCTCCGCGTGCGCGAGGCCGACAAGCCGCTGATCGACAGCCTGCT
  • ID 6 Plutella xylostella V-type proton ATPase subunit E (LOC 105389010), mRNA 551-650 CCTTGAAGGTGGACACGGAGCACTACCTGCCGGTGGGCACCTGCGGCGGGAT TGAGTTGGTCGCCGCTAGGGGCCGCATCAAGATCATCAACACCCTGGA (SEQ ID NO: 18).
  • ID 7 Plutella xylostella V-type proton ATPase subunit E (LOCI 05389010), mRNA 651-750
  • ID 8 Plutella xylostella V-type proton ATPase subunit E (LOCI 05389010), mRNA 1501-1600
  • ACATTCCTCTTTCATGTTTCCTGTCCTGGCAATCAAATGTTTCGGCT SEQ ID NO: 20.
  • ID 10 Plutella xylostella V-type proton ATPase subunit E (LOCI 05389010), mRNA 1401-1500 ATACTGTTTACTATCGTGGACTTCCTGGGAATTATTTGATGCTGTAAGGTTTAT
  • ID 25. 1001-1100 PREDICTED: Plutella xylostella venom carboxylesterase-6 (LOCI 05388350), mRNA GGAACTTACACTAAAATACCTATGATCTTCGGATTTGTTGAAAACGAAGGAAC
  • ID 32 601-700 Plutella xylostella prophenoloxidase 1 mRNA, complete cds GACATCGGCATCAACCTGCACCACTGGCACTGGCACCTGGTGTACCCCTTCGA GGCGGCCGACCGCGCCGTGGTGGACAAGGACAGGCGCGGCGAGCTGCGC (SEQ ID NO: 47).
  • ID 40. 1301-1400 Plutella xylostella prophenoloxidase 1 mRNA, complete cds TCTCCTCCATCGCCATCTCGGGCCGCACTCCGAACCAGTTCTCGACGCAGTGG GAGCAGAGTTCAGTGAACCTGGCGCGCGGGCTGGACTTCATGCCGCG (SEQ ID NO: 54).
  • ID 43 1601-1700 Plutella xylostella prophenol oxidase 1 mRNA, complete cds TGCGCCCCGGCAGCAACACCATCCGGCGTCGCAGCATCGACTCCTCAGTTACC ATCCCCTACGAGCGAACATTCCGGGACGAGTCCCAACACCCCGGAGA (SEQ ID NO: 57)
  • ID 46 1901-2000 Plutella xylostella prophenol oxidase 1 mRNA, complete cds GCTACCCGGACCGCAAGCCTATGGGATTCCCCTTCGATAGACCAGCGCCATCT ACCGGCAGTTTGGGAGACTTCTTGACCCCCAACATGACTGTGCAGAA (SEQ ID NO: 60)
  • ID 46-2 Plutella xylostella prophenol oxidase 1 mRNA, complete cds TCAGGTTTACTGATGCGGTCAGGCAGCGTCAGCAGCGGTAGAGTGTGGGAGG GAGACATGGTTCATACGTTACGAGAGTTCTAGCTAAACTTAAACACACAATGA AAGCTGACTGTAACTTTTGTAGATTTTGCCTTCAAAATGACAATTACTTAGGCA CTAATTAGGTTCTAATTTTGTATAATTCATTTTTAGCGACA (SEQ ID NO: 62) [00325] ID 46-3.
  • ID 49 351-450 Plutella xylostella glutathione synthetase (Gss), mRNA CGCCTGATCTCTCAGTACCTACAGGACGCTGGTTACAGGACCCAGATGGTCGG AAAGTGGCACGTCGGTCACGCCTTCTTCGAGCAGCTGCCCACTTACA (SEQ ID NO: 66)
  • ID 50 451-550 Plutella xylostella glutathione synthetase (Gss), mRNA GAGGATTCGAGAACCACTTCGGTGTCCGCGGTGGATTCATCGACTACTACGAA TACAACGCTCAGGAGCA GCTTGACGGCAGGCCAGTCACTGGACTGTG (SEQ ID NO: 67)
  • ID 51 551-650 Plutella xylostella glutathione synthetase (Gss), mRNA TCTGTTCGACGACCTGCAGCCCGACTGGACCACCGAGGGATACATCACCGACG TCTACACCGAGAAGTCCACCACCATCATTGAGAACCACAACGTCTCC (SEQ ID NO: 68)
  • ID 52. 651-750 Plutella xylostella glutathione synthetase (Gss), mRNA GAGCCCCTGTACCTGCTGCTGACCCACCACGCTCCCCACAACGGCAACGAAGA CGCTTCCCTGCAGGCCCCTCCTGAAGAGGTCCGCGCCCAGAGGCACG (SEQ ID NO: 69)
  • ID 53 751-850 Plutella xylostella glutathione synthetase (GSS), mRNA TCGAGCTCCACCCCAGACGTATCTTCGCCGCTATGGTTAAGAAACTGGACGAC AGTATCGGAGAAATCGTCGCTACCCTCGAGAAGAAGGGCATGCTCGA (SEQ ID NO: 70)
  • ID 54 951-1050 Plutella xylostella glutathione synthetase (Gss), mRNA CCCTGGGAGGGAGGTATCCGTGGTAACGCCATGATCTGGGCCGGTCCCGAGGT CGCCCCCGGAAACGCGTGGCGTGGAAAGGTTTACGACGGCAACATGC (SEQ ID NO: 72)
  • ID 57. 1051-1150 Plutella xylostella glutathione synthetase (Gss), mRNA ACGCCGCTGACTGGGTCCCCACTCTGCTTGAGGCCATCGGTGAGAAGATCCCC GCCGGTCTGGACGGTATCCCCATGTGGAGCCACATCATCGAGAACAA (SEQ ID NO: 73)
  • ID 58. 1151-1250 Plutella xylostella glutathione synthetase (Gss), mRNA GCCTTCTCCCCGTACCGAGATCTTCGAGATCGACGACTACTTCAACCACTCCTC TGTCACCCTCGGCCGCCACAAGCTCGTCAAGGGAACCATCGACGAG (SEQ ID NO: 74)
  • ID 59. 1251-1350 Plutella xylostella glutathione synthetase (Gss), mRNA TCTCTCAGCAAGCACTACGGTGAAGACCTCCGTGGCATCATCGGAACTCCCCC AGACTACAAGCAGAAGCTGCGCGACAGCAAGGCATGGGAGTCTCTGG (SEQ ID NO: 75)
  • ID 60. 1351-1450 Plutella xylostella glutathione synthetase (Gss), mRNA AGACCATCGGCATCCCTCTGGACGCTGACGTCATGGCTGACCGCGATGAGGCT ATCGTCACTTGCGGAAATGTCGTCCCCAAGCCTTGCAGTCCTTCTGC (SEQ ID NO: 76)
  • ID 61. 1451-1550 Plutella xylostella glutathione synthetase (Gss), mRNA CGAGTCTTGGTGCCTGTACGACATCATCGAGGACCCTTGTGAGCTTCGTGACC TGTCTGAGGAGCTTCCTCAGCTGGCTCAGATCCTTCTGTACCGTCTG (SEQ ID NO: 77)
  • ID 63. 1651-1750 Plutella xylostella glutathione synthetase (Gss), mRNA CCGTCGAACCCTACTCCGACTCCGAATAGACGAAGCTCAGCTCAAGCGGCGCA GTTCGCCGTGAAAGTTGTAAATGTTGATCCTGGCCTTAATTTCAGTA (SEQ ID NO: 79)
  • ID 63-2 Plutella xylostella glutathione synthetase (Gss), mRNA
  • AAAGTTGTAAATGTTGATCCTGGCCTTAATTTCAGTA (SEQ ID NO: 80) [00343] ID 64.
  • ID 70. 651-751 PREDICTED: Plutella xylostella charged multivesicular body protein 4b (LOC105396929), mRNA
  • ID 71. 751-850 PREDICTED Plutella xylostella charged multivesicular body protein 4b (LOC105396929), mRNA
  • ID 73. 951-1050 PREDICTED: Plutella xylostella charged multivesicular body protein 4b (LOC105396929), mRNA AGTAGAGTGAAAGAACAACTAGCTAATAAAAATAAGGAAGGCCCAAGACAG
  • ID 74. 1051-1150 PREDICTED: Plutella xylostella charged multivesicular body protein 4b (LOC105396929), mRNA
  • ID 76. 826-975 PREDICTED: Plutella xylostella charged multivesicular body protein 4b (LOC105396929), mRNA TAAGTGTTGGAAGCGAAACCGAATACATATGTATAGTCTCCGTTACACATCCA ACTACTTAGCTTATTCTAAGGCTGCGTCCCTGTAGGCAAACAGTATTCTCGTTA CTAGTCTATGGTAATTTGAGTAGAGTGAAAGAACAACTAGCTA (SEQ ID NO:
  • CGCTA (SEQ ID NO: 138) [00401] ID 109-12 1217-1274 Plutella xylostella cytochrome P450 (CYP6BFlvl) mRNA, complete cds
  • TTTGC (SEQ ID NO: 139)
  • ID 109-13 1275-1333 Plutella xylostella cytochrome P450 (CYP6BFlvl) mRNA, complete cds
  • ID 109-14 1334-1390 Plutella xylostella cytochrome P450 (CYP6BFlvl) mRNA, complete cds
  • ID P2. 251-400 Plutella xylostella cytochrome P450 6kl-like
  • ID P4 551-700 Plutella xylostella cytochrome P450 6kl-like
  • ID P5. 701-850 Plutella xylostella cytochrome P450 6kl-like (LOC105392167), mRNA
  • ID P6. 851-1000 Plutella xylostella cytochrome P450 6kl-like (LOC105392167), mRNA
  • ID P7. 751-1050 Plutella xylostella cytochrome P450 6kl-like (LOC105392167), mRNA
  • ID P8. 450-751 Plutella xylostella cytochrome P450 6kl-like (LOC105392167), mRNA
  • AACTA SEQ ID NO: 1557
  • ACATGGGTAAACCAAATGAAAGGCTGAGAAGGA SEQ ID NO: 160 [00423] ID 120. 101-200 PREDICTED: Spodoptera frugiperda cytochrome P450 dislike (LOCI 18273915), mRNA
  • ID 121 201-300 PREDICTED: Spodoptera frugiperda cytochrome P450 6B2- like (LOCI 18273915), mRNA
  • ID 125. 1201-1300 PREDICTED: Spodoptera frugiperda cytochrome P450 6B2-like (LOCI 18273915), mRNA
  • ID 133 801-900 Spodoptera frugiperda cytochrome P450 CY321A8 mRNA, complete cds
  • ID. 1749-2049 Plutella xylostella strain DBM1 Ac-S ABC transporter subfamily H member 1 (ABCH1) mRNA, complete cds Sequence ID: KP260785.1 GACTACAACCCGAAGGTGGGCGACATCCCGATCGACTTCAAGGAGCCCATCT
  • ID 143 351-450 PREDICTED: Spodoptera frugiperda PBAN-type neuropeptides (LOCI 18281022), mRNA
  • ID 190 Spodoptera frugiperda glutathione S-transferase 1-like
  • ID 195 [00494] ID 195. 401-500 PREDICTED: Spodoptera frugiperda protein mesh (LOCI 18271033), transcript variant XI, mRNA GAAATACTAAGTCCAACGGATGATCTGCAAGTAAGAAGCGGGAAATATCAGC TCAATGATGGGCTCGTGGGTGAAGAGCCGATGCCACTAGATGCTGTTA (SEQ ID NO: 231)
  • ID 196 1901-2000 PREDICTED: Spodoptera frugiperda protein mesh (LOCI 18271033), transcript variant XI, mRNA AAGGAGATTACCATCAGACCCCAGCTTGAATACATAGATATTATCGAAATGGG CGTGGCTAACACTGGAGAATATGTGATCAATCCCCAAAACTTTAGGA (SEQ ID NO: 232)
  • ID 204 401-500 PREDICTED: Spodoptera frugiperda uncharacterized LOCI 18263801 (LOCI 18263801), transcript variant XI, mRNA GGTTAAACAAAAAGGGTAAAAATGGCCTTCAAAGGATTCTGTGGCGAAGTGA TCGGGTTTTTCCTGGCTGTGGGTTTTTGCATCATATGTCCGGAATATG (SEQ ID NO: 239)
  • ID 205 701-800 PREDICTED: Spodoptera frugiperda uncharacterized LOCI 18263801 (LOCI 18263801), transcript variant XI, mRNA AACTGCCGAGTCAAGAATTTAGGAAATAAAACGTTAAACATGCAAGTATCGT GGGTACGGCATAGAGACATCCATCTGCTGACAGTCGGCCGGTACACAT (SEQ ID NO: 240)
  • ID 207. 1351-1450 PREDICTED: Spodoptera frugiperda uncharacterized LOCI 18263801 (LOCI 18263801), transcript variant XI, mRNA GTTGGTTGCCTGCCTGTTCATCGCACTCTCTTGACACATACGGATCTTTAACTA AATGTAAATACAAGGATTTTATCAACATCAGCTGCTTATATTGACA (SEQ ID NO: 242)
  • ID 214 1501-1600 Spodoptera frugiperda V-ATPase subunit A mRNA, complete cds
  • ID 220 301-400 Spodoptera frugiperda cytochrome P450 CY321A8 mRNA, complete cds
  • Diabrotica virgifera virgifera charged multivesicular body protein 4b (LOCI 14337301), mRNA Sequence ID: XM_028287710.1 gcaaagaaaaatgcgtcgaaaataaaagagttgcactccaagccctcaaaaagaagaaacgattggaaaagacccaactaca aatagatggaacccttacaactattgaaatgcagagggaagccctcgaaggagctagcacaaatactgctgtattagattctatgaa aatgctgcagatgcccttaagaaagctcataagaatttgaatgtagatgatgttcacgatatcatggat (SEQ ID NO: 264)

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Abstract

Post-transcriptionally chemically modified double strand RNAs (MdsRNAs) having a high molecular weight polyalkyloxy modification at the 2'-OH position are described. This modification allows for greater bioavailability of the compound, better stability of the compound, and allows for greater stability against nucleases. The MdsRNAs can be economically produced in a readily scalable manner. Compositions of MdsRNAs having chemically modified nucleotides are described, such that the MdsRNAs are modified to contain high molecular weight polyalkyloxy polymers. Synthetic methods for efficiently making these MdsRNAs and methods of using MdsRNAs are described.

Description

HIGH MOLECULAR WEIGHT MODIFIED dsRNA COMPOSITIONS
RELATED APPLICATION
[0001] This application claims the benefit of U.S. Provisional Application No. 63/239,165, filed on August 31, 2021. The entire teachings of the above application(s) are incorporated herein by reference.
INCORPORATION BY REFERENCE OF MATERIAL IN XML
[0002] This application incorporates by reference the Sequence Listing contained in the following extensible Markup Language (XML) file being submitted concurrently herewith: a) File name: 55811003001. xml; created August 29, 2022, 278,517 Bytes in size.
BACKGROUND
[0003] Because they dominate all terrestrial environments that support human life, insects are usually humanity’s most important competitors for food, fiber, and other natural resources. Insects have a direct impact on agricultural food production by chewing the leaves of crop plants, sucking out plant juices, boring within the roots, stems or leaves, and spreading plant pathogens. Insects feed on natural fibers, destroy wooden building materials, ruin stored grain, and accelerate the process of decay.
[0004] The economic impact of insects is measured not only by the market value of products they destroy and the cost of damage they inflict but also by the money and resources expended on prevention and control of pest outbreaks. Although dollar values for these losses are nearly impossible to calculate, especially when they affect human health and welfare, economists generally agree that insects consume or destroy around 10% of gross national product in large, industrialized nations and up to 25% of gross national product in some developing countries.
[0005] One possible method for controlling insect populations is the use of RNA interference (RNAi), which is a naturally occurring biological process by which doublestranded ribonucleic acid (dsRNA) silences (knocks down) target gene expression in a sequence specific manner. Cellular enzymes use dsRNA to target and cleave single stranded RNA (ssRNA), including messenger RNA (mRNA) and non-coding RNA. RNAi is known to occur in many eukaryotes, including plants, insects, acari, fungi and animals, and offers great potential for selective and efficient regulation of gene expression. [0006] The dsRNA has an antisense strand containing sequence complementary to a sequence in the mRNA or non-coding RNA and a sense strand containing sequence complementary to the guide strand sequence and substantially identical to the sequence in the mRNA or non-coding RNA. The sense and antisense sequences can be present on separate RNA strands or on a single strand. When present on a single strand, the complementary sequences are connected by a non-hybridizing hairpin or loop sequence. [0007] RNAi-mediated gene suppression on targeted plants, insects, acari, and fungi affecting crops described in the prior art has been achieved using exogenously supplied unmodified dsRNA (UdsRNA) (US Patent 9,121,022; Ivashuta et al. 2015; US Publication No. 20160215290; Koch et al. 2016). It has been found, that when dsRNAs are used to induce RNAi in insects and are supplied in the insects’ diet, 60 base pair (bp) or longer dsRNAs are sometimes required for efficient uptake and processing (Bolognesi et al. 2012).
[0008] Preparation of UdsRNA longer than about 30 base pairs (bp) has been achieved by in vitro transcription (Timmons 2006) and by fermentation (Fire et al. 1998). Commercially feasible large-scale methods for preparation and purification of the UdsRNA has been described (Arhancet et al., US9822361B2). However, UdsRNAs are sensitive to degradation by nucleases in the environment and the host, reducing efficacy of inhibition of gene expression (Baum 2016).
[0009] DsRNA degradation has been addressed in in vitro and in vivo research and for human therapeutics (Ku et al. 2015) by using chemical synthesis of small (<30 bp) interfering dsRNAs (siRNA) with nucleotides modified by chemical means. Preparation of siRNA with chemically modified nucleotides involves sequential protectiondeprotection chemical reactions for each nucleotide added in the elongating single strand RNA (ssRNA) chain (Micura 2002). The complexity and expense of such processes are significantly increased for RNA molecules which trigger RNAi (RNAi triggers) that are longer than about 30 bp. While chemically synthesized siRNAs targeting insects using nucleotides chemically modified at the 2'-OH position of the ribose have been also described (Gong et al. 2013), the cost and synthetic complexity of modified siRNAs is neither economically feasible or sufficiently scalable, for preparation of amounts larger than a few grams, or of chemically modified dsRNA longer than about 30 bp. [0010] Post-transcriptional chemical modification of ssRNA for analytical purposes was also described by Merino (2005). Merino reacted ssRNA in aqueous media containing 10% DMSO with N-methylisatoic anhydride (NMIA) to produce the 2'aO- esters of N-m ethyl-anthranilic acid at single stranded nucleotides. Derivatization by this method was inefficient. Less than 15% of ssRNA chains in a reaction vessel were modified and those that were modified had, on average, a single 2'nO-ester of NMIA per ssRNA chain. Under these conditions, dsRNA reacted more than 80 times less efficiently, with less than 0.18% of nucleotides in a stem region being modified and only within one (1) nucleotide of the end of a stem (e.g. within one nucleotide of a single strand region) Similar results have been observed for reaction of RNA with other reactants (Nodin 2015). l-methyl-7-nitroisatoic anhydride (1M7), benzoyl cyanide (BzCN), 2-methyl-3 -furoic acid imidazolide (FAI), and 2-methylnicotinic acid imidazolide (NAI) have been used to post- transcriptionally produce 2'-ribose esters of RNA, but have a similarly low percentage of the modification, with modification primarily occurring at riboses of unpaired nucleotides or their immediately adjacent paired nucleotides.
[0011] Unmodified poly-ribonucleic acid is an exceptionally unstable molecule. Unlike DNA, it contains a hydroxyl group at the 2' position of the ribose which renders the RNA polymer sensitive to hydrolysis. Deprotonation of 2'-OH and, consequently, nucleophilic attack of 2' oxygen on the backbone phosphorus is the primary molecular mechanism of cleavage of phosphodiester bonds by various nucleolytic enzymes, including RNase A (Elliot and Ladomery, 2011). Stabilization of the nucleic acid backbone against nucleolysis has become a success-defining issue, much the same as for the evolution of siRNA-based therapeutics (Nair et al., 2014). The issue of RNA stability is especially important in intestinal milieu rich in nucleolytic activity with various endonucleases and phosphodiesterases responsible for digestion of nucleic acids (Whitt and Savage, 1988; Liu et al., 2015). A highly effective strategy for stabilizing RNA molecules is the derivatization of 2'-0 group. Various modifications at this position have been demonstrated to improve enzymatic stability of various RNA molecules, including nanoassemblies (Liu et al. 2010) and RNAi silencing molecules (Khvorova and Watts, 2017). These modifications are generally applied during chemical synthesis of small interfering RNAs (siRNAs).
[0012] While many advances have been made in the use of dsRNA, especially those that have chemical modifications (specifically polyalkyloxy polymers) at the 2’ -OH position, there is the need for chemically modified dsRNA compositions that are more efficient, more soluble, and more bioavailable than have been available in the past. There is also a need for synthetic methods of making these chemically modified dsRNA compositions in an efficient manner to allow for mass-scale production.
SUMMARY
[0013] Described herein are post-transcriptionally chemically modified double strand RNAs (MdsRNAs) having a high molecular weight polyalkyloxy modification at the 2’- OH position. This modification allows for greater bioavailability of the compound, better stability of the compound, and allows for greater stability against nucleases. As described herein, these MdsRNAs can be economically produced in a readily scalable manner.
[0014] This disclosure describes compositions of modified double strand RNA (MdsRNA) having chemically modified nucleotides, such that the MdsRNAs are modified to contain high molecular weight polyalkyloxy polymers, optionally further comprising 2’-0 chemically modified nucleotides to contain low molecular weight (LMW) moi eties (LMW modified nucleotides). This disclosure also describes synthetic methods for efficiently making these modified MdsRNAs.
[0015] Thus, in one aspect, the present disclosure provides compositions comprising a post-transcriptionally chemically modified double strand RNA (MdsRNA) wherein the MdsRNA comprises a double strand RNA wherein no more than about 30% of all the nucleotides independently comprise Formula (I): or an acceptable salt thereof, wherein B and R1 are defined herein.
[0016] In an embodiment of this aspect, the post-transcriptional chemical modification of the double strand RNA comprises no more than about 30% of all the nucleotides being modified with high molecular weight polyalkyloxy polymers. In another embodiment, the post-transcriptional chemical modification of the double strand RNA further comprises at least about 2% of all the nucleotides being modified with LMW moieties. In one embodiment, the post-transcriptional chemical modification of the double strand RNA further comprises from about 2% to about 50% of all the nucleotides being modified with LMW moieties. In an embodiment, the post-transcriptional chemical modification of the double strand RNA comprises about a 3:5:2 ratio of high molecular weight modified nucleotides to LMW modified nucleotides to unmodified nucleotides. In another embodiment, the post-transcriptional chemical modification of the double strand RNA comprises about a 3:6.5:0.5 ratio of high molecular weight modified nucleotides to LMW modified nucleotides to unmodified nucleotides. In still another embodiment, the post- transcriptional chemical modification of the double strand RNA comprises about a 1 :7:2 ratio of high molecular weight modified nucleotides to LMW modified nucleotides to unmodified nucleotides. In still another embodiment, the post-transcriptional chemical modification of the double strand RNA comprises about a 0.5:9:0.5 ratio of high molecular weight modified nucleotides to LMW modified nucleotides to unmodified nucleotides. In still another embodiment, the post-transcriptional chemical modification of the double strand RNA comprises about a 0.3:4:5.7 ratio of high molecular weight modified nucleotides to LMW modified nucleotides to unmodified nucleotides. In still another embodiment, the post-transcriptional chemical modification of the double strand RNA comprises about a 0.01 :4:5.99 ratio of high molecular weight modified nucleotides to LMW modified nucleotides to unmodified nucleotides. In still another embodiment, the post-transcriptional chemical modification of the double strand RNA comprises about a 0.06:0:96.4 ratio of high molecular weight modified nucleotides to LMW modified nucleotides to unmodified nucleotides.
[0017] In another aspect, the disclosure provides a method of preparing a composition comprising a post-transcriptionally chemically modified double strand RNA (MdsRNA) wherein the MdsRNA comprises a double strand RNA wherein no more than about 30% of all the nucleotides independently comprise Formula (I): or an acceptable salt thereof, wherein B and R1 are defined herein.
[0018] Methods for making embodiments of the compositions described herein are disclosed. In an embodiment of the methods of preparing compositions, the post- transcriptional chemical modification of the double strand RNA comprises no more than about 30% of all the nucleotides being modified with high molecular weight polyalkyloxy polymers. In another embodiment of the methods of preparing compositions, the post- transcriptional chemical modification of the double strand RNA further comprises at least about 2% of all the nucleotides being modified with LMW moieties. In one embodiment, the post-transcriptional chemical modification of the double strand RNA further comprises from about 2% to about 50% of all the nucleotides being modified with LMW moieties. In an embodiment of the methods of preparing compositions, the post-transcriptional chemical modification of the double strand RNA comprises about a 3:5:2 ratio of high molecular weight modified nucleotides to LMW modified nucleotides to unmodified nucleotides. In another embodiment of the methods of preparing compositions, the post- transcriptional chemical modification of the double strand RNA comprises about a 3:6.5:0.5 ratio of high molecular weight modified nucleotides to LMW modified nucleotides to unmodified nucleotides. In still another embodiment of the methods of preparing compositions, the post-transcriptional chemical modification of the double strand RNA comprises about a 1 :7:2 ratio of high molecular weight modified nucleotides to LMW modified nucleotides to unmodified nucleotides. In still another embodiment of the methods of preparing compositions, the post-transcriptional chemical modification of the double strand RNA comprises about a 0.5:9:0.5 ratio of high molecular weight modified nucleotides to LMW modified nucleotides to unmodified nucleotides. In still another embodiment of the methods of preparing compositions, the post-transcriptional chemical modification of the double strand RNA comprises about a 0.3:4:5.7 ratio of high molecular weight modified nucleotides to LMW modified nucleotides to unmodified nucleotides. In still another embodiment of the methods of preparing compositions, the post-transcriptional chemical modification of the double strand RNA comprises about a 0.01 :4:5.99 ratio of high molecular weight modified nucleotides to LMW modified nucleotides to unmodified nucleotides. In still another embodiment of the methods of preparing compositions, the post-transcriptional chemical modification of the double strand RNA comprises about a 0.06:0:96.4 ratio of high molecular weight modified nucleotides to LMW modified nucleotides to unmodified nucleotides. [0019] In further aspects, the disclosure provides methods of modifying the expression of polynucleotides of interest in an insect, a fungus, a weed or an acari, using any of the compositions, target insects, fungi, weeds or acari, and sequences set forth herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] The foregoing will be apparent from the following more particular description of example embodiments.
[0021] FIG. 1 shows cumulative mortality for Diamondback moth larvae after administration of select compounds of Formula (I).
[0022] FIG. 2 shows decrease of dsRNA content on cabbage leaves in the field for treatment C2, unmodified dsSNF7, and modified dsSNF7 treatments NS2 (PEG-dsSNF7) and NS5 (NMIA-dsSNF7).
[0023] FIG. 3 shows the mortality rate of Diamondback moth field larvae after administration of select compounds of Formula (I) after 3-days (3 DA-A) and 4-days (4 DA-A).
[0024] FIG. 4 shows Agarose gel electrophoresis analysis of 300 bp dsRNA modified with activated 10k PEG. Lines: 1 - DNA ladder; 2 - original dsRNA; 3 - lOkPEG- MdsRNA reaction mixture.
DESCRIPTION
DEFINITIONS
[0025] As used herein the term “about” is used herein to mean approximately, roughly, around, or in the region of. When the term ' about ' is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term “about” is used herein to modify a numerical value above and below the stated value by a variance of 20 percent up or down (higher or lower).
[0026] As used herein, the term “sequence” or “nucleotide sequence” refers to a succession or order of nucleobases, nucleotides, and/or nucleosides, described with a succession of letters using the standard nucleotide nomenclature and the key for modified nucleotides described herein. The sequences as described herein are listed from the 5’ terminus to the 3’ terminus. The skilled artisan would understand that the reverse complementary strand (i.e., described from the 3’ terminus to the 5’ terminus) can be the full reverse complementary strand or a strand comprising stretches of contiguous nucleotides between about 11 nucleotides and about 60 nucleotides long, wherein each of such stretches of about 11 to about 60 contiguous nucleotides are the reverse complementary strand of a stretch of equal length in the sequences as described herein. [0027] In some embodiments, a MdsRNA is at least 40, at least 30, at least 50, at least 70, at least 80, at least 90, or at least 100 base pairs in length. A MdsRNA sense strand contains a sense sequence and a MdsRNA antisense strand contains an antisense sequence. The antisense sequence is 100% (perfectly) complementary or at least 90% (substantially) complementary or at least 80% (partial) complementary to a nucleotide sequence present in a target gene transcribed mRNA or non-coding RNA (i.e., expressed RNA). The sense sequence is 100% (perfectly) complementary or at least 90% (substantially) complementary or at least 80% (partially) complementary the antisense sequence. A sense sequence may also be 100% identical, at least 90% identical, or at least 80% identical to a nucleotide sequence (target sequence) present in a target gene mRNA or non-coding RNA. The sense sequence and a corresponding antisense sequence are partially (at least 80%), substantially (90%), or fully (100%) complementary to each other. In some embodiments, the region of complementarity (antisense sequence) or identity (sense sequence) between the MdsRNA and a corresponding sequence in the target gene transcribed mRNA or noncoding RNA sequence is greater than 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 nucleotides in length. In some embodiments, the antisense sequence contains a contiguous sequence greater than 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 nucleotides in length that is 100% complementary or at least 80% complementary to a corresponding contiguous sequence in the target gene transcribed mRNA or non-coding RNA. MdsRNA sense and antisense sequences can be either the same length or they can be 60 different lengths. Suitable sense and antisense sequences are identified using known methods readily available in the art.
[0028] As used herein, the term “nucleotide” refers to one base pairing unit (e.g., a unit of Formula I or an unmodified ribose) comprising a purine or pyrimidine base pairing moiety. The term may refer to the nucleotide unit with or without the attached intersubunit linkage, although, when referring to a “charged subunit”, the charge typically resides within the intersubunit linkage.
[0029] The purine or pyrimidine base pairing moiety, also referred to herein simply as a “nucleobases,” “base,” or “bases,” may be adenine, cytosine, guanine, uracil, thymine or inosine. Also included are bases such as pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2,4,6-trimethoxy benzene, 3 -methyl uracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidines (e.g., 5-methylcytidine), 5-alkyluridines (e.g., ribothymidine), 5-halouridine (e.g., 5-bromouridine) or 6-azapyrimidines or 6- alkylpyrimidines (e.g. 6-methyluridine), propyne, quesosine, 2-thiouridine, 4-thiouridine, wybutosine, wybutoxosine, 4-acetyltidine, 5-(carboxyhydroxymethyl)uridine, 5'- carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluridine, P-D- galactosylqueosine, 1 -methyladenosine, 1 -methylinosine, 2,2-dimethylguanosine, 3- methylcytidine, 2-methyladenosine, 2-methylguanosine, N6-methyladenosine, 7- methylguanosine, 5-methoxyaminomethyl-2-thiouridine, 5-methylaminomethyluridine, 5- methylcarbonylmethyluridine, 5-methyloxyuridine, 5-methyl-2-thiouridine, 2-methylthio- N6-isopentenyladenosine, 13-D-mannosylqueosine, uridine-5-oxyacetic acid, 2- thiocytidine, threonine derivatives and others (Burgin et al., 1996, Biochemistry, 35: 14090; Uhlman & Peyman, supra). By “modified bases” in this aspect is meant nucleotide bases other than adenine (A), guanine (G), cytosine (C), thymine (T), and uracil (U), as illustrated above; such bases can be used at any position in the antisense molecule. Persons skilled in the art will appreciate that depending on the uses of the oligomers, Ts and Us are interchangeable. For instance, with other antisense chemistries such as 2'-O- methyl antisense oligonucleotides that are more RNA-like, the T bases may be shown as U.
[0030] As used herein, a “modified nucleotide” is a nucleotide other than a ribonucleotide (2'-hydroxyl nucleotide). In some embodiments, at least about 2%, at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% of the nucleotides in a MdsRNA are post-synthetically modified. Modified nucleotides include, but are not limited to, nucleotides having a ribose 2'-OH substitution. It should be understood that “independently” means that the modified nucleotides can each have the same modification or they can have different modifications from each other, selected from high molecular weight polyalkyloxy polymers, and if present, low molecular weight moieties.
[0031] An “unmodified” dsRNA is an RNA molecule that has not been chemically modified.
[0032] As used herein, the term “polyalkoxy” or “polyalkyloxy” as used interchangeably herein refers to a suitable water-soluble polymer characterized by repeating alkoxy units. Select, but non-limiting, examples of such polymers are polyethylene glycol (PEG), polypropylene glycol (PPG), poloxamers, hyaluronic acid, polyvinyl alcohols, polyoxazolines, polyanhydrides, poly(ortho esters), polycarbonates, polyurethanes, polyacrylic acids, polyacrylamides, polyacrylates, polymethacrylates, polyorganophosphazenes, polysiloxanes, polyvinylpyrrolidone, polycyanoacrylates, polyesters, or any derivatives of the foregoing.
[0033] In an embodiment, the term “polyalkoxy” or “polyalkyloxy” used herein refers to a suitable linear or branched polyethylene glycol (PEG) polymer. In some embodiments, the polyalkoxy is a linear or branched polypropylene glycol (PPG) polymer. In some embodiments, the polyalkyloxy is a poloxamer. In some embodiments the polymer is an ethylene glycol-propylene glycol block copolymer poloxamer. In another embodiments the polymer is a poly(ethylene glycol)-block-poly(propylene glycol)-block- poly(ethylene glycol), PEG-PPG-PEG poloxamer. In yet another embodiment the polymer is an ethylene oxi de-propylene oxide triblock copolymer. In some embodiments, the polyalkyloxy has a molecular weight of at least about 400 Da, at least about 1 kDa, at least about 5 kDa, or at least about 10 kDa. In another embodiment, the molecular weight of the polyalkyloxy is between about 400 Da and about 40 kDa or between about 5 kDa and about 40 kDa.
[0034] As used herein, the term “poloxamers” refers to nonionic triblock copolymers composed of a central hydrophobic chain of polyoxypropylene flanked by two hydrophilic chains of polyoxyethylene. Select, but non-limiting, examples of poloxamers are poloxamers 407, 338, 188, 184, and 401 (i.e., F127, F108, L68, L64 and L121 Pluronic®, BASF). The first two digits multiplied by 100 give the approximate molecular mass of the poly oxypropylene core, and the last digit multiplied by 10 gives the percentage polyoxyethylene content. “L” stands for liquid and “F” stands for flake solid.
[0035] The amphiphilic character, that is the presence of both hydrophilic and lipophilic groups, of the resulting polymer modified dsRNAs of the disclosure modulate their physical and chemical properties such as solubility, absorption and permeability through plant tissue and target cell membranes and resistance to nucleases. The polymers that produced the most efficacious MdsRNAs have Hydrophilic-Lipohilic Balance (HLB) numbers ranging from 8 to 27. In one embodiment, the HLB is from about 2 to about 30. In another embodiment, the HLB is from about 15 to about 27.
[0036] As used herein, the term “high molecular weight polyalkyloxy” refers to a compound, substituent, or other chemical moiety comprising a polyalkyloxy group having a molecular weight of at least about 400 Da. In some embodiments, the molecular weight of the polyalkyloxy is at least about 1 kDa. In some embodiments, the molecular weight of the polyalkyloxy is at least about 5 kDa. In another embodiment, the molecular weight of the polyalkyloxy is at least about 10 kDa. In another embodiment, the molecular weight of the polyalkyloxy is between about 400 Da and about 40 kDa. In yet another embodiment, the molecular weight of the polyalkyloxy is between about 5 kDa and about 40 kDa.
[0037] As used herein, a “low molecular weight (LMW) modified nucleotide” is a nucleotide having a 2’ -OH low molecular weight modification. Low molecular weight modifications are moi eties that are 1,000 daltons or less. Examples of modifications, for example as identified herein as R2 in Formula III include, but are not limited to, C1-C25 alkyl, C1-C25 alkenyl, C1-C25 alkynyl, C5-C12 aryl or C5-C12 heteroaryl, wherein any of these is optionally substituted with one or more substituents selected from halo, C1-12 alkyl, C1-C12 aminoalkyl, or C1-C12 alkoxy (i.e., R2 as described herein). In some embodiments, at least about 2%, at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% of the nucleotides in a MdsRNA are post-transcriptionally chemically modified as LMW modified nucleotides. In some embodiments, the chemical modifications are esters of N-m ethyl-anthranilic acid (from modification with N-methylisatoic anhydride (NMIA)), esters of N-benzyl-anthranilic acid (from modifications with N-benzylisatoic anhydride (NBIA)), dimethyl furoyl, esters of a fatty acid (e.g., Cl -Cl 8, such as but not limited to lauryl, oleic, linoleic), acetic acid, propionic acid, esters of amino acids (e.g., tyrosine, tryptophan, leucine), low molecular weight PEG, nitrogen containing moieties.
[0038] Without being limited by theory, it is believed that the presence of the LMW moieties allows the composition to better dissolve in organic solvents, for delivery of the compositions to plants and ultimately to the target insects. The compositions can be made more concentrated, in amounts that are economical for the end-user. Further, the decomposition rate in the field for the compositions of the application has been shown to decrease by filling in the spaces in the MdsRNA that are not modified with the high molecular polyalkyloxy polymers. In other words, the nucleotides that are not modified with high molecular weight polyalkyloxy polymer can be modified with low molecular weight moieties, as described herein. The degree of modification of the nucleotides with low molecular weight moieties will depend on the desired properties of the compositions with respect to decomposition rate, solubility and concentration, among other benefits. [0039] The term “alkyl” or “alkyl group” as used herein describes a univalent group derived from alkanes by removal of a hydrogen atom from any carbon atom -CnEbn+i. An alkyl group can be straight or branched chain or cyclic and include methyl, ethyl, propyl, isopropyl, butyl, hexyl and the like. As used herein, a lower alkyl group contains from 1 to 25 carbon atoms in the principal chain.
[0040] The term “alkenyl” as used herein are acyclic branched or unbranched hydrocarbons having one carbon-carbon double bond and the general formula -CnEbn-i. One or more of the hydrogen atoms can be substituted. An alkyl group can be straight or branched chain and include ethenyl, propenyl, isopropenyl, butenyl, isobutenyl, hexenyl, and the like. As used herein, an alkenyl contains from 2 to 25 carbon atoms in the principal chain.
[0041] The term “alkoxide” or “alkoxy” as used herein is the conjugate base of an alcohol. The alcohol can be straight chain, branched, cyclic, and includes aryloxy compounds and include methoxy, ethoxy, isoproyloxy, butoxy, and the like.
[0042] The term “alkynyl” as used herein are acyclic branched or unbranched hydrocarbons having a carbon-carbon triple bond and the general formula -CnH2n-3. They can be straight or branched chain and include ethynyl, propynyl, butynyl, isobutynyl, hexynyl, and the like. As used herein a lower alkynyl containing from 2 to 25 carbon atoms in the principal chain.
[0043] The terms “aryl” or “Ar” as used herein alone or as part of another group denote optionally substituted homocyclic aromatic groups. Aryl groups can be monocyclic or bicyclic groups containing from 5 to 12 carbons in the ring portion, such as phenyl, biphenyl, naphthyl, substituted phenyl, substituted biphenyl, or substituted naphthyl.
[0044] The term “heteroaryl” as used herein alone or as part of another group denotes optionally substituted aromatic groups having at least one heteroatom in at least one ring. In some embodiments, heteroaromatic group contains 5 or 6 atoms in each ring. In some embodiments, a heteroaromatic group contains 1 or 2 oxygen atoms and/or 1 to 4 nitrogen atoms in the ring, and is bonded to the remainder of the molecule through a carbon. Exemplary groups include furyl, benzofuryl, oxazolyl, isoxazolyl, oxadiazolyl, benzoxazolyl, benzoxadiazolyl, pyrrolyl, pyrazolyl, imidazolyl, triazolyl, tetrazolyl, pyridyl, pyrimidyl, pyrazinyl, pyridazinyl, indolyl, isoindolyl, indolizinyl, benzimidazolyl, indazolyl, benzotriazolyl, tetrazolopyridazinyl, carbazolyl, purinyl, quinolinyl, isoquinolinyl, imidazopyridyl, and the like. Exemplary substituents include one or more of the following groups: hydrocarbyl, substituted hydrocarbyl, alkyl, alkoxy, acyl, acyloxy, alkenyl, alkenoxy, aryl, aryloxy, amino, amido, acetal, carbamyl, carbocyclo, cyano, ester, ether, halogen, heterocyclo, hydroxyl, keto, ketal, phosphor, nitro, and thio. [0045] The term “heteroatom” refers to atoms other than carbon and hydrogen.
[0046] The term “halogen” or “halo” refers to as used herein alone or as part of another group refer to chlorine, bromine, fluorine or iodine.
[0047] As used herein, the term “greater stability” refers to the compounds of the instant application in comparison to unmodified dsRNAs or MdsRNAs modified with low molecular weight polyalkyloxy. In some embodiments, greater stability means increased persistence in agricultural fields, in plant tissue, on plant leaves, etc. In some embodiments, greater stability means increased in vivo half-life. In some embodiments, greater stability means enhanced resistance to physiological conditions. In other embodiments, greater stability means enhanced resistance to nucleases.
[0048] As used herein, “acceptable salts” refers to salts derived from suitable inorganic and organic acids and inorganic and organic bases that are, within the scope of sound judgment, suitable for use in contact with the tissues of humans, lower animals, and plants without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio.
[0049] A salt of a compound of the present disclosure can be synthesized from the parent compound that contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two.
[0050] As used herein, the term “scalable” refers to the processes ability to produce quantities of the target chemical in commercially relevant amounts. For example, the process of the instant application may allow for the production of compounds in the hundreds of milligram scale. In some examples, the process of the instant application may allow for the production of compounds in the gram scale. In some examples, the process of the instant application may allow for the production in the kilogram scale. In some examples, the process of the instant application may allow for the production in the metric ton scale.
[0051] As used herein, the term “activation agent” or “activating agent” refers to a compound that increases the nucleophilicity or electrophilicity of target moiety. The change in electron density can be the result of an ionic or covalent bond from the activating group. In some embodiments, the activation agents described herein increase the electrophilicity of a target carbonyl. Non-limiting examples of activation agents are lewis acids, protons, and coupling reagents. Non-limiting examples of coupling reagents are benzotriazol-l-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP), benzotriazol- 1-yloxytripyrrolidinophosphonium hexafluorophosphate (PyBOP), (2-(lH- benzotriazol- 1 -yl)- 1 , 1 ,3 ,3 -tetramethyluronium hexafluorophosphate, Hexafluorophosphate Benzotriazole Tetramethyl Uronium (HBTU), N,N' -Dicyclohexylcarbodiimide (DCC), carbonyldiimidazole (CDI), and 1- [Bis(dimethylamino)methylene]-lH-l,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate, Hexafluorophosphate Azabenzotriazole Tetramethyl Uronium (HATU).
[0052] As used herein, the term “suitable leaving group” refers to a molecular fragment that departs with a pair of electrons in heterolytic bond cleavage. The leaving group helps facilitate the reaction by lowering the energy of activation. Non-limiting examples of suitable leaving groups are halides, tosylated or mesylated alcohols, pseudohalides, amines, heterocycles, and the like. In a particular embodiment, the suitable leaving group is imidazole.
[0053] As used herein, the term “anhydrous solvent” refers to a solvent that has been physically or chemically treated to reduce the water content of the solvent. Anhydrous solvents may contain less than about 1% by weight water, less than about 0.3% by weight water, or less than about 0.1% by weight water.
COMPOSITIONS
[0054] MdsRNAs are described having no more than about 30% of all the ribose rings chemically modified at the 2'-OH position with high molecular weight polyalkyloxy polymers, and optionally at least about 2% of all the nucleotides being chemically modified with LMW modified moieties. The MdsRNAs are capable of inhibiting gene expression in a sequence specific manner, such as through RNA interference or antisense mechanisms.
[0055] Thus, in an aspect, provided herein is a composition comprising a post- transcriptionally chemically modified double strand RNA (MdsRNA) wherein the MdsRNA comprises a double strand RNA wherein no more than about 30% of all the nucleotides independently comprise Formula (I):
or an acceptable salt thereof, wherein:
B is a nucleobase;
R1 is selected from: wherein y is an integer from 1-8, x is an integer from 12-1000, a is an integer from 12- 1000, b is an integer from 12-1000, and c is an integer from 12-1000.
[0056] In another aspect, provided herein are compositions comprising a post- transcriptionally chemically modified double strand RNA (MdsRNA) wherein the
MdsRNA comprises a double strand RNA wherein no more than about 30% of all the nucleotides independently comprise Formula (I): or an acceptable salt thereof, wherein:
B is a nucleobase;
R1 is selected from:
wherein y is an integer from 1-8, x is an integer from 12-1000, a is an integer from 12-
1000, b is an integer from 12-1000, and c is an integer from 12-1000; and optionally wherein at least about 2% of all the nucleotides independently comprise
LMW modified nucleotides of Formula (III): or an acceptable salt thereof, wherein:
B is a nucleobase;
R2 is selected from C1-C25 alkyl, C1-C25 alkenyl, C1-C25 alkynyl, C5-C12 aryl or C5-C12 heteroaryl, wherein R2 is optionally substituted with one or more substituents selected from halo, C1-12 alkyl, C1-C12 aminoalkyl, or Ci-C 12 alkoxy.
[0057] Embodiments of this aspect of the composition follow. [0058] In an embodiment, R1 is selected from:
[0059] In another embodiment, x is an integer from 80-1000.
[0060] In another embodiment, x is an integer from 50-900.
[0061] In another embodiment, a is an integer from 80-1000.
[0062] In yet another embodiment, b is an integer from 80-1000.
[0063] In a further embodiment, c is an integer from 80-1000.
[0064] In an embodiment, R1 has a molecular weight between 5,000 and 15,000 Da.
[0065] In an embodiment, R1 has a molecular weight between 5,000 and 40,000 Da.
[0066] In an embodiment, R1 is polyethylene glycol (PEG).
[0067] In an embodiment, R1 is poloxamer 407, 338, 188, 184, 401 or any combination thereof.
[0068] In an embodiment, a is 101, b is 56, and c is 101.
[0069] In an embodiment, R1 is a HMW polymer with an HLB ranging from about 2 to about 30. In another embodiment, R1 is a HMW polymer with an HLB ranging from about 15 to about 27.
[0070] In an embodiment, R2 is N-methyl anthranoyl (NMA), N-benzyl anthranoyl (NBA), dimethyl furoyl, -Tyr, -Trp, -Leu, octanoyl, lauroyl, linoleyl, oleyl, nicotinoyl or benzoyl.
[0071] In an embodiment, -methyl anthranoyl.
[0072] In an embodiment, lauroyl.
[0073] In an embodiment, R1 is and R2 is linoleyl.
[0074] In an embodiment, oleyl.
[0075] In embodiments, the ratio of Formula (I) to Formula (III) to unmodified nucleotides is about 3:5:2; 3:6.5:0.5; 1 :7:2; .05:9:0.5; 1.3:4:5.7; 0.01 :4:5.99; 0.06:0:96.4. [0076] In embodiments, from about 2% to about 50% of all nucleotides are modified with LMW moieties.
[0077] In another embodiment, the MdsRNA comprises a sequence complementary to an expressed RNA in a target insect. For example, an efficacious sequence or target gene useful in the products and methods of the disclosure will inhibit the expression of the target gene, act on the mid gut of the insect and increase mortality or induce growth stunting, or stop instar development.
[0078] In an embodiment, the target insect is Diamondback moth (Plutella xylostella), Gypsy moth, Red imported fire ant (Solenopsis invicta), Fall armyworm, Colorado potato beetle, Canola flea beetle, Aedes aegypti, or Western corn root worm (Diabrotica virgifera virgifera). In another embodiment, the target insect is Pea aphid (Acyrthosiphon pisum), Soybean aphid, Piezodorus guildinii. In another embodiment the target is an acari, such as Verroa mite. In another embodiment the target is a weed, such as Palmer amaranth. In yet another embodiment, the target is a fungus such as Palmer amaranth, Fusarium graminearum (Gibberella zeae) or Botrytis. Accordingly, the MdsRNA used in the compositions of the disclosure will be a nucleotide sequence that can inhibit the expression of target genes or regions in these insects. For example, the MdsRNA comprises a sequence complementary to a target region in Diamondback moth, such as but not limited to AChE2, P450, P450 CYP6BFlvl, Cytokine receptor DOMELESS, DOUX, Protein MESH transcript variant XI, Venom carboylesterase-6 and VPASE-E. In another embodiment, the MdsRNA comprises a sequence complementary to a target region in Fall armyworm, such as but not limited to P450 CYP9A58, Cytokine receptor DOMELESS, Dredd, VPASE, Protein MESH transcript variant XI, P450 CYP321 A8, P450 CYP6B2- like.
[0079] In another embodiment, the MdsRNA comprises a sequence complementary to a target region in Western corn root worm, such as but not limited to SNF7.
[0080] In an embodiment, the MdsRNA comprises a sequence selected from one of SEQ ID Nos. 1-269 and its reverse complementary strand. In a particular embodiment, the sequence is selected from one of SEQ ID Nos. 13 or 248-251 and its reverse complementary strand. In an embodiment, the target insect is a Lepidopteran and can be targeted using any one of SEQ ID Nos. 1-269. In an embodiment, the Lepidopteran is targeted using any one of SEQ ID Nos. 13 or 248-251.
[0081] In another aspect, provided herein is a composition comprising a post- transcriptionally modified double strand RNA (MdsRNA) wherein the MdsRNA comprises a double strand RNA wherein no more than about 30% of all the nucleotides independently comprise Formula (I): or an acceptable salt thereof, wherein:
B is a nucleobase;
R1 is a linear or branched polyalkyloxy having a molecular weight between about 400 Da and about 15 kDa; optionally wherein at least about 2% of all the nucleotides independently comprise LMW modified nucleotides of Formula (III): or an acceptable salt thereof, wherein:
B is a nucleobase; and
R2 is selected from C1-C25 alkyl, C1-C25 alkenyl, C1-C25 alkynyl, C5-C12 aryl or C5- C12 heteroaryl, wherein R2 is optionally substituted with one or more substituents selected from halo, C1-12 alkyl, C1-C12 aminoalkyl, or Ci-C 12 alkoxy.
[0082] Embodiments of this aspect of the composition follow.
[0083] In an embodiment, R1 is a linear or branched polyalkyloxy or a poloxamer having a molecular weight between about 1 kDa and about 15 kDa.
[0084] In another embodiment, R1 is a linear or branched polyalkyloxy or a poloxamer having a molecular weight between about 5 kDa and about 15 kDa. [0085] In another embodiment, R1 is a linear or branched polyalkyloxy or a poloxamer having a molecular weight between about 5 kDa and about 10 kDa.
[0086] In an embodiment, R1 is a polyethylene glycol polymer.
[0087] In an embodiment, R1 is a poloxamer. In an embodiment, the poloxamer is a triblock polymer comprising poly(ethylene glycol)-block-poly(propylene glycol)-block- poly (ethylene glycol).
[0088] In an embodiment, R1 is a HMW polymer with an HLB ranging from about 2 to about 30. In another embodiment, R1 is a HMW polymer with an HLB ranging from about 15 to about 27.
[0089] In an embodiment, R2 is C1-C25 alkyl. In an embodiment, the C1-C25 alkyl is substituted with one or more substituents selected from halo, C1-12 alkyl, C1-C12 aminoalkyl, or C1-C12 alkoxy. In an embodiment, the C1-C25 alkyl is substituted with one, two, or three substituents selected from halo, C1-12 alkyl, C1-C12 aminoalkyl, or C1-C12 alkoxy.
[0090] In an embodiment, R2 is N-methyl anthranoyl (NMA), N-benzyl anthranoyl (NBA), dimethyl furoyl, -Tyr, -Trp, -Leu, octanoyl, lauroyl, linoleyl, oleyl, nicotinoyl or benzoyl.
[0091] In embodiments, the ratio of Formula (I) to Formula (III) to unmodified nucleotides is about 3:5:2; 3:6.5:0.5; 1 :7:2; .05:9:0.5; 1.3:4:5.7; 0.01 :4:5.99; 0.06:0:96.4. [0092] In embodiments, from about 2% to about 50% of all nucleotides are modified with LMW moieties.
[0093] In another embodiment, the MdsRNA comprises a sequence complementary to an expressed RNA in target insects, weeds, fungi or acari, as discussed herein, the sequence listing and examples of this disclosure. wherein y is an integer from 1-8, x is an integer from 12-1000, a is an integer from 12-1000, b is an integer from 12-1000, and c is an integer from 12-1000.
[0095] In an embodiment, R1 is selected from: wherein at least 2% of all the nucleotides independently comprise LMW modified nucleotides of Formula (III) as defined above.
[0096] In another embodiment, x is an integer from 80-1000.
[0097] In another embodiment, x is an integer from 50-900.
[0098] In another embodiment, a is an integer from 80-1000.
[0099] In yet another embodiment, b is an integer from 80-1000.
[00100] In a further embodiment, c is an integer from 80-1000.
[00101] In an embodiment, R1 has a molecular weight between 5,000 and 15,000 Da.
[00102] In an embodiment, R1 has a molecular weight between 5,000 and 40,000 Da.
[00103] In an embodiment, R1 is polyethylene glycol (PEG).
[00104] In an embodiment, R1 is poloxamer 407, 338, 188, 184, 401, or any combination thereof.
[00105] In an embodiment, a is 101, b is 56, and c is 101.
[00106] In an embodiment, R1 is a HMW polymer with an HLB ranging from about 2 to about 30. In another embodiment, R1 is a HMW polymer with an HLB ranging from about 15 to about 27.
[00107] In an embodiment, R2 is N-methyl anthranoyl (NMA), N-benzyl anthranoyl (NBA), dimethyl furoyl, -Tyr, -Trp, -Leu, octanoyl, lauroyl, linoleyl, oleyl, nicotinoyl or benzoyl.
[00108] In embodiments, the ratio of Formula (I) to Formula (III) to unmodified nucleotides is about 3:5:2; 3:6.5:0.5; 1 :7:2; .05:9:0.5; 1.3:4:5.7; 0.01 :4:5.99; 0.06:0:96.4. [00109] In embodiments, from about 2% to about 50% of all nucleotides are modified with LMW moieties.
[00110] In an embodiment, the sequence of the MdsRNA is selected from one of 13 or 248-251.
[00111] In another embodiment, the MdsRNA comprises a sequence complementary to an expressed RNA in a target insect, weed, fungi, acari or in any of the targets as discussed herein and the sequence listing and examples of this disclosure.
[00112] In an embodiment, the compound of Formula (I) is or an acceptable salt thereof, wherein each variable is defined above.
[00113] In another embodiment, the compound of Formula (I) is: or an acceptable salt thereof, wherein each variable is defined above.
[00114] In yet another embodiment, the compound of Formula (I) is: or an acceptable salt thereof, wherein each variable is defined above. [00115] In another embodiment, the compound of Formula (I) is: or an acceptable salt thereof, wherein each variable is defined above. In another embodiment, the terminal -H can be substituted with -CH3.
[00116] In a further embodiment, the compound of Formula (I) is: or an acceptable salt thereof, wherein each variable is defined above. In another embodiment, the terminal -H can be substituted with -CH3.
[00117] In another embodiment, the compound of Formula (I) is: or an acceptable salt thereof, wherein each variable is defined above. In another embodiment, the terminal -H can be substituted with -CH3.
[00118] In a further embodiment, the compound of Formula (I) is: or an acceptable salt thereof, wherein each variable is defined above. In another embodiment, the terminal -H can be substituted with -CH3.
[00119] In some embodiment, each B of Formula I is the same.
[00120] In another embodiment, each B of Formula I is different.
[00121] In some embodiment, each R1 of Formula I is the same.
[00122] In some embodiment, each R1 of Formula I is different.
[00123] In some embodiments, the base pairs in the MdsRNA is at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 200, at least about 350, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, or at least about 1,000 in length. In an embodiment, the base pairs in the MdsRNA is between about 40 base pairs and about 1,000 base pairs. In another embodiment, the MdsRNA is between about 50 base pairs and about 900 base pairs. In an embodiment, the MdsRNA is between about 70 base pairs and about 800 base pairs. In another embodiment, the MdsRNA is between about 80 base pairs and about 700 base pairs. In an embodiment, the MdsRNA is between about 90 base pairs and about 600 base pairs. In an embodiment, the MdsRNA is between about 100 nucleotides and about 500 nucleotides. In another embodiment, the MdsRNA is between about 200 base pairs and about 400 base pairs.
[00124] A target sequence useful in the products and methods of this disclosure will be selected to target the lowest guanine-cytosine (GC) content, such as no more than about 60% and preferably about 40% or less of the GC content of the target insect, fungus or weed, to allow for the highest level of modification under mild reaction conditions. In another embodiment, the MdsRNA targets a sequence with a low guanine-cytosine content. In an embodiment, the guanine-cytosine content is no more than about 50%. In an embodiment, the guanine-cytosine content is about 40% or less. In an embodiment, the guanine-cytosine content is between about 30% and about 40%. In an embodiment, guanine-cytosine content is about 30%, about 35%, about 40%, about 50% or about 55%. The degree of modification of the nucleotides will depend upon the GC content of the dsRNA. The degree of modification (e.g., the percentage of nucleotides that can be modified) can range from about 2% to about 90%, with from about 2% to about 30% being high molecular weight polyalkyloxy polymers, and with from about 2% to about 90% being low molecular weight filler moieties.
[00125] Without being bound by theory, it is believed that when the reaction conditions are such that the sense and antisense strands in the dsRNA are only partially dissociated (i.e., only partially unzipped), for example at temperatures below 60°C, or at low concentrations of a solvent or co-solvent capable of dissociating the sense and antisense strands, e.g., at DMSO concentrations below 70%, or at temperatures below 60°C and at lower than 70% DMSO, the riboses in the nucleotides at positions located less than 10 contiguous nucleotides away from the beginning or the end of a perfectly paired stretch of dsRNA, may be preferentially modified, i.e., they may be modified to a larger extent than the riboses in nucleotides located more than 10 contiguous nucleotides away. For example, under such partially unzipping reaction conditions, the riboses in the nucleotides at positions 1 through 10 and 290 through 300 in a 300 bp dsRNA may be modified to ta larger extent than those at positions 11 through 289.
[00126] Post transcriptionally modified double strand RNA (MdsRNA) compounds are significantly less susceptible to degradation by nucleases in the environment and the host. MdsRNA down-regulate expression of polynucleotides present in the target host by the RNAi mechanism. MdsRNA compounds consist of UdsRNA in which some of the H atoms of its 2’OH groups have been replaced with different chemical moieties, for example with, benzoyl, lauroyl, oleyl, linoleyl, N-methyl anthranoyl, nicotinoyl, or furoyl (US Patent 10,131,911). These chemical moieties are generally non-toxic and, in any case, susceptible to eventual degradation in the environment, thereby reducing negative environmental impact. MdsRNAs are expected to be similarly non-toxic as UdsRNA. [00127] In some embodiments, a MdsRNA sense strand is connected to the antisense strand. A sense strand may be connected to an antisense strand via a non-hybridizing hairpin or loop sequence. A loop sequence can be about 4 to about 100 or more nucleotides in length. In some embodiments, a loop is 150 or more nucleotides in length (Hauge et al. 2009). In some embodiments, a MdsRNA further comprises one or more additional sequences including, but not limited to: promoter sequences, 5' sequences, 3' sequences, terminator sequences, and polyA sequences. [00128] A promoter is a region (sequence) of DNA that initiates transcription of a gene. A promoter can be a bacterial promoter, archaea promoter, eukaryotic promoter, or a Pol I, Pol II, or Poll III promoter. In some embodiments, a bacterial promoter comprises the sequence 5'-TTGACA-3' about 35 units upstream from the transcription start site and the sequence 5'-TATAAT-3' about 10 bp upstream from the transcription start site. Other promoters, suitable for use with different expression systems are well known in the art. [00129] Particular embodiments of the current disclosure call for higher molecular polyalkyloxy polymers to be covalently bound to the 2’ -OH position of the intersubunit linkages. Preferred embodiments of the disclosure call for a low percentage of the overall subunits to be substituted with such a polymer (e.g., less than about 30%). In some embodiments, the percentage of the overall subunits to be substituted with such a polymer is about 1%. In some embodiments, the percentage of substituted subunits is about 1% to about 30%. In other embodiments, the percentage of substituted subunits is about 1% to about 5%. In yet other embodiments, the percentage of substituted subunits is less than about 1%. In another embodiment, the percentage of substituted subunits is between about 0.1% and about 1%. The compounds of the disclosure show equal or superior properties to compounds having lower weight polyalkyloxy polymers at the 2’ -OH position.
[00130] Without being bound by any theory, the compounds of the disclosure show equal or superior properties to compounds having lower weight polyalkyloxy polymer compounds because the instant compounds have better solubility, enhanced bioavailability, and are resistant to nucleases. Unexpectedly, while PEG and similar groups are known to prevent crossing of membranes, the instant compounds readily cross the cell-membrane. Also, unexpectedly, the low percentage levels (e.g., about 1% to about 30%) of the polyalkyloxy polymers of the disclosure still impart the beneficial stability properties of other compounds with significantly higher percentages of PEG or PEG-type substitutions (e.g., the instant compounds are resistant to exonucleases at about 1% to about 30%).
[00131] Without being bound by any theory, the compounds of the disclosure (i.e., compounds having high molecular weight polyalkyloxy polymers) are resistant to hydrolysis by carboxylate esterase. This may be due to the polyalkyloxy polymers being too sterically large for the catalytic pocket of the esterase. The larger polyalkyloxy polymers may further hydrogen bond with the 2’ OH groups of the unmodified nucleotides, thus creating further steric hinderance, even at the low levels of modification as described herein. Finally, the modifications of the MdsRNA strands, as described herein, may preferentially occur at the ends of the MdsRNA strands due to the steric hinderance of the molecules. Thus, the MdsRNA strands are resistant to exonucleases while still being available to endonucleases, allowing the compounds of the disclosure to enter into the RNAi process easier.
[00132] Embodiments and aspects in this and the other sections of the disclosure regarding the composition, methods of making the compositions, nucleotide sequences and uses for target insects, fungi, weeds or acari are intended to be covered herein in this disclosure.
Selected Sequences
[00133] In an embodiment, the sequence is Plutella xylostella acetylcholinesterase 2 mRNA, GenBank AY061975.1 nucleotide #s: 512-811; catatcgga ggattgcctc tatttgaaca tatgggtgcc gcagcacttg cgcgtccgtc accatcagga caagccatta accgagcgac cgaaggttcc aatactagtg tggatttacg gcgggggtta catgagtggc acggcgacac ttgatctata taaagccgac ataatggcgt cttcgagtga tgtgatcgta gcctcgatgc agtatagggt tggcgcgttt ggatttttgt accttaacaa atatttttcc cctggtagcg aggaagcggc aggaaatatg ggcttgtggg a (SEQ ID NO: 1).
[00134] In another embodiment, the sequence is Plutella xylostella acetylcholinesterase 1 mRNA, GenBank: AY970293.1 nucleotide #s: 889-1188; tc acaatgtcac attgtttgga gaatcgtccg gtgcagtttc cgtgtcatta cacttactgt ctccgctgtc aagaaacatg ttttctcaag ctattatgca atctgcagcc gcatctgcac cttgggccat catttccaga gaggagagtg tgataagggg catccgcctg gccgaggccg tccactgc (SEQ ID NO: 2).
[00135] In another embodiment, the sequence is Plutella xylostella tyrosine hydroxylase mRNA, GenBank: JN410829.1 nucleotide #s: 301-600; gctgaggtcg gtggaataga cggaaatgca gatgatgatt acaccttgac cgaggaggag gtgatcttgc agaactccgc cagcgagtcc ccggaggccg agcaggcgct gcaacaagcg gctttgcttc tgcgcctgcg cgacggcatg ggctcgctcg cgcgcatcct caagaccatc gacaactaca agggatgcgt tcaacacctc gagactcgcc cctccaacgc caacgacatc caattcgatg ctctcatcaa agtgagcatg tcccgtggca acctgctcca actcatccga (SEQ ID NO: 3).
[00136] In another embodiment, the sequence is Plutella xylostella integrin beta 1 mRNA, GenBank: GQ178290.1 nucleotide #s: 531-830; tgcaggtcaa gccgcagagg gtcaagctgc agctgcgcat gaaccagatg cagaaactag acgtcgccta ttcccaagcc caagactacc cggtggacct gtactacttg atggacctga gtcgttccat gaagaacgac aaggagaagc tcagtacatt gggcagtctg ctgtccagca ctatgaggaa tatcacgccc aacttccgtc ttggcttcgg ctccttcgtg gacaagctcg tcatgcccta cgtgtctact gtgcctaaga atttgatatc cccttgtgat ggctgcgcgg (SEQ ID NO: 4). [00137] In another embodiment, the sequence is Plutella xylostella charged multivesicular body protein 4b-like mRNA (XM_011555904.1) nucleotide #s: 321-620; ccaggaaaca tggcactaag aacaaaagag cggccatcgc tgcacttaaa cgcaagaagc gttacgagaa gcaactcaca cagattgacg gcacgctcag ccagatagag atgcagagag aggcattgga gggcgccaac actaacactc aagtactgaa cacgatgcga gaggccgccg cggctatgaa gctcgctcac aaggatattg acgtagacaa agtgcatgat atcatggacg acatcgctga acaacatgat gtggctcgcg aaatcacgga tgccatcagc aacaatgtgg (SEQ ID NO: 5).
[00138] In another embodiment, the sequence is Plutella xylostella peptidoglycan recognition protein mRNA, GenBank: EU399240.1 nucleotide #s: 31-330; tcagtgt tttgttgttg tgctcatgca gggtgtggcg tggtgaccag acagcagtgg gatgggctgg acccgataca gttggagtac ctgccccggc ccctggggct ggtggtggtc cagcacaccg ccacccccgc gtgtgacact gacgccgcgt gtgtggagct ggtgcagaa atacagacca atcatatgga tgtgctgaag ttttgggata ttggaccgaa cttcctgatt ggt (SEQ ID NO: 6).
[00139] In another embodiment, the sequence is Plutella xylostella mRNA for vacuolar ATP synthethase subunit E, GenBank: AB189032.1 (identical to NM_001305532.1) nucleotide #s: 64-363; gcgctca gcgatgcaga tgtccaaaaa cagatcaagc atatgatggc cttcatcgag caagaggcaa atgaaaaggc cgaagaaatc gatgctaagg ctgaggagga gttcaacatc gagaaggggc gtctggtgca gcagcagcgc ctcaagatca tggagtacta cgagaagaag gagaagcagg tggaactcca gaagaagatc caatcctcca acatgctgaa ccaggcccgt ctgaaggtgc tgaaggtgcg cgaggaccac gtgggccacg tgttggacga gacgcgccgc cgc (SEQ ID NO: 7).
[00140] In another embodiment, the sequence is Diabrotica virgifera virgifera charged multivesicular body protein 4b (LOCI 14337301), mRNA Sequence ID:
XM_028287710.1; cacaactgac agataacgtc agtagttgtc tattttcact ggtgactaat ttttgagaat tagtaattgg tttcgtattt ttttcttaac aaaagggcaa aatgagcttt tttggaaaat tgttcggggg gaaaaaggaa gagatagccc ctagtcctgg ggaggctatt caaaaactca gagagactga agaaatgtta ataaaaaaac aggatttttt agaaaagaag atagaagaat ttaccatggt agcaaagaaa aatgcgtcga aaaataaaag agttgcactc caagccctca aaaagaagaa acgattggaa aagacccaac tacaaataga tggaaccctt acaactattg aaatgcagag ggaagccctc gaaggagcta gcacaaatac tgctgtatta gattctatga aaaatgctgc agatgccctt aagaaagctc ataagaattt gaatgtagat gatgttcacg atatcatgga tgacatagcc gaacaacacg acatagccaa cgaaatcaca aacgctatta gcaatcctgt cggattcacc gacgatctgg atgacgatga attagaaaaa gaattagaag agctcgaaca agaaggattg gaagaagacc tgctccaagt gccaggtcca actcaactgc cggctgtgcc tgctgatgca gttgctacta aaccaatcaa accagcagct aaaaaagttg aagatgatga cgatatgaaa gaattggaag cctgggcctc gtaaaattcc tgaaatctga atatttgtaa acgaaatcac ccatctaaga tgaaaacatt ataaatatat aggtaataac agctaaaaac gtttcaatgt agaacaagct tttgctgaaa aggcgtcttg caaaaaatgt tgataattta gaattctcta tatattatat atttgccctt taaggaaaga tttcttttat agtcatagtt caaccaaacg tttcataaat tagaatacag gggtgtcaga attctacgtg aatttattga aaaaaaagaa ctttccttaa agttggcata atgatatttt tgaaattatg taaataggta tatgtatgct cttactaacg gttttaatat tgggtttaga agcaccttaa ttttattttt atacaaggta gtaacatttt ttcattgtaa tttgttaaaa aatattgtat aacgcaaaaa atggtgtgat aaagcaaaat attattgagt gcttattttc ttattttatt aaaagatctt atttacgtta aatctaaata tattgggtat ctcagaatat gttaacaaag gtttcttgta tcaacagaaa aatacaaact tattcattat tgtggttcat ttgattatgc ttgtgtatat ttaatctgcc ataaacaagt tttgataaat gtcactgctc tgta (SEQ ID NO: 8).
[00141] In another embodiment, the sequence is Acyrthosiphon pisum V-type proton ATPase subunit E Genbank#: XM_008185078.2 nucleotide#s: 540-724; t tagccaacac tggaataaac gtcaaaataa acattgataa aagtattaaa ttaccgactc aagaaatagg aggcgtcgtg gtcacgtcca aagatcgaag ggtacatgtt gaaaatacgc ttgtagtgag attgctctat ctcacccaac aagcaatacc aataatatgc actggactgt ttgg (SEQ ID NO: 9).
[00142] In another embodiment, the sequence is Solenopsis invi eta’s (RIF A) actin muscle (LOC105205816, GenBank: XM_011175337.1 nucleotides# 465-763; gatctc tctccctcga ctctaacacc agegaaagta acagccaatc aagatgtgtg aegatgatgt tgcggcatta gtcgtggaca atgggtccgg tatgtgeaag getggatteg cgggggatga tgcaccacgc gctgtgtttc ccagcatcgt cggtcgtcct cgtcatcagg gtgtgatggt cggtatgggt caaaaagaca gttatgttgg egaegaggeg caaagtaaga gaggtatatt gacactaaag tatcctatag aacatg (SEQ ID NO: 10).
[00143] In yet another embodiment, the sequence is Gibberella zeae isolate NX3 cytochrome P450 5 IB gene GenBank: FJ216402.1 nucleotide positions 804-1023; cagcaag tttgaegagt ccctggccgc tctctaccac gacctcgata tgggcttcac ccccatcaac ttcatgcttc actgggcccc tctcccctgg aaccgtaagc gcgaccacgc ccagcgcact gttgccaaga tctacatgga cactatcaag gagcgccgcg ccaagggcaa caacgaatcc gagcatgaca tgatgaagca ccttatgaac tet (SEQ ID NO: H).
[00144] In another embodiment, the sequence is Fusarium graminearum PH-1 cytochrome P450 51 NCBI Reference Sequence: XM 011327038.1 nucleotide positions 163-400; attggaag caccgtacaa tatggcatcg acccgtacgc ttttttcttc gaetgeagag ataaataegg egaetgettt acctttattc tccttggcaa atcaacgact gtctttcttg gtcccaaggg caatgacttt atcctcaacg gcaaacacgc cgatctcaac gccgaggacg tttatgggaa acttaccacg cccgtgtttg gtgaggaggt tgtttatgac tgctccaatg (SEQ ID NO: 12). Method of Preparation of High-Molecular Weight Polyalkyloxy Polymers by Ionic Solvation
[00145] In another aspect, provided herein are methods of preparing a composition comprising a post-transcriptionally chemically modified double strand RNA (MdsRNA) wherein the MdsRNA comprises a double strand RNA wherein no more than about 30% of all the nucleotides independently comprise Formula (I): or an acceptable salt thereof, wherein:
B is a nucleobase;
R1 is selected from: wherein y is an integer from 1-8, x is an integer from 12-1000, a is an integer from 12- 1000, b is an integer from 12-1000, and c is an integer from 12-1000; and the method comprising:
(a) contacting a compound of Formula (II):
(II), with an activation agent to form a compound of Formula (IIA):
O
X R
(HA), wherein X is a suitable leaving group; and (b) contacting a compound of Formula (IA): with a compound of Formula (IIA) to form a compound of Formula (I).
[00146] In another aspect, provided herein are methods of preparing a composition comprising a post-transcriptionally chemically modified double strand RNA (MdsRNA) wherein the MdsRNA comprises a double strand RNA wherein no more than about 30% of all the nucleotides independently comprise Formula (I): or an acceptable salt thereof, wherein:
B is a nucleobase;
R1 is selected from: wherein y is an integer from 1-8, x is an integer from 12-1000, a is an integer from 12- 1000, b is an integer from 12-1000, and c is an integer from 12-1000; and optionally wherein at least about 2% of all the nucleotides independently comprise
LMW modified nucleotides of Formula (III): or an acceptable salt thereof, wherein:
B is a nucleobase;
R2 is selected from C1-C25 alkyl, C1-C25 alkenyl, C1-C25 alkynyl, C5-C12 aryl or C5-C12 heteroaryl, wherein R2 is optionally substituted with one or more substituents selected from halo, C1-12 alkyl, C1-C12 aminoalkyl, or C1-C12 alkoxy; the method comprising:
(a) contacting a compound of Formula (II):
(II), with an activation agent to form a compound of Formula (IIA):
O XAR |
(IIA), wherein X is a suitable leaving group;
(b) contacting a compound of Formula (IA): with a compound of Formula (IIA) to form a compound of Formula (I);
(c) optionally contacting a compound of Formula (V):
(V), with an activation agent to form a compound of Formula (VA):
O
XAR2
(VA), wherein X is a suitable leaving group;
(d) optionally contacting a compound of Formula (IA): with a compound of Formula (VA) to form a compound of Formula (III).
[00147] In an embodiment, (a) and (b) are carried out in an anhydrous solvent.
[00148] In an embodiment, (c) and (d) are carried out in an anhydrous solvent.
[00149] In an embodiment, the anhydrous solvent is selected from DMSO or DCM.
[00150] In another embodiment, (a) and (b) are carried out without intervening purification.
[00151] In another embodiment, (c) and (d) are carried out without intervening purification.
[00152] In yet another embodiment, there is a purification step between (a) and (b). [00153] In another embodiment, there is a purification step between (c) and (d).
[00154] In an embodiment, an ionic solvent is added after (a).
[00155] In an embodiment, an ionic solvent is added after (c).
[00156] In an embodiment, the ionic solvent is selected from benzyltributyl ammonium chloride or benzyltrimethyl ammonium chloride.
[00157] In another embodiment, the activation agent is carbonyldiimidazole.
[00158] In yet another embodiment, the suitable leaving group is: wherein X / ' represents the covalent point of attachment to carbonyl of Formula (IIA).
[00159] In yet another embodiment, the suitable leaving group is: wherein »AAA/' represents the covalent point of attachment to carbonyl of Formula (VA). [00160] In a further embodiment, (b) has a ratio of less than ten equivalents of the compound of Formula (IIA) per nucleotide of the dsRNA.
[00161] In a further embodiment, (d) has a ratio of between about two equivalents and about fifty equivalents of the compound of Formula (VA) per nucleotide of the dsRNA.
[00162] In another embodiment, (b) has a ratio of less than two equivalents of the compound of Formula (IIA) per nucleotide of the dsRNA.
[00163] In another embodiment, (d) has a ratio of between about four equivalents and about twenty -five equivalents of the compound of Formula (VA) per nucleotide of the dsRNA.
[00164] In another embodiment, the compound of Formula (II) is the anhydride. [00165] In another embodiment, the compound of Formula (V) is the anhydride.
[00166] In yet another embodiment, R1 is selected from:
[00167] In another embodiment, x is an integer from 80-1000.
[00168] In another embodiment, x is an integer from 50-900.
[00169] In another embodiment, a is an integer from 180-1000. [00170] In yet another embodiment, b is an integer from 80-1000.
[00171] In a further embodiment, c is an integer from 80-1000.
[00172] In an embodiment, R1 has a molecular weight between 5,000 and 10,000 Da.
[00173] In an embodiment, R1 has a molecular weight between 5,000 and 40,000 Da.
[00174] In an embodiment, R1 is polyethylene glycol (PEG).
[00175] In an embodiment, R1 is poloxamer 407, 338, 188, 184, 401, or any combination thereof.
[00176] In an embodiment, a is 101, b is 56, and c is 101.
[00177] In an embodiment, R1 is a HMW polymer with an HLB ranging from about 2 to about 30. In another embodiment, R1 is a HMW polymer with an HLB ranging from about 8 to about 27.
[00178] In an embodiment, steps (c) and (d) are present.
[00179] In an embodiment, steps (b) and (d) are performed sequentially.
[00180] In an embodiment, steps (b) and (d) are performed simultaneously.
[00181] In another embodiment, the MdsRNA comprises a sequence complementary to an expressed RNA in a target insect, fungus, weed or acari, as discussed in detail in this disclosure, such as but not limited to target (insect fungus, weed or acari), target sequences and target regions.
[00182] In another embodiment, R2 is selected from N-methyl anthranoyl (NMA), N- benzyl anthranoyl (NBA), dimethyl furoyl, -Tyr, -Trp, -Leu, octanoyl, lauroyl, linoleyl, oleyl, nicotinoyl or benzoyl.
[00183] Embodiments in other sections of the disclosure regarding the composition, methods of making the compositions, nucleotide sequences and uses for target insects, fungi, weeds or acari are intended to be covered herein this disclosure.
[00184] In some embodiments, the disclosed method of preparation is superior to prior methods in both cost and scalability. Prior methods of synthesis required water or another suitable polar protic solvent to reduce the need for excess solvent dilution. However, this led to significant degradation of the necessary polyalkyloxy polymer anhydrides. Prior methods sometimes employed as much as 200 equivalents of modifying groups per one bp of dsRNA. The addition of ionic solvent has provided the superior and unexpected benefit of reducing the amount of solvent used, reducing the degradation of starting material, and allowing efficient synthesis of the desired product. In some embodiments of the method, (b) has a ratio of less than 0.05 equivalents of the compound of Formula (IIA) (e.g., the polyalkyloxy polymer) per one bp of the dsRNA. In another embodiment of the method, (b) has a ratio of less than 0.1 equivalents of the compound of Formula (IIA). In particular embodiments of the method, (b) has a ratio of about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, or about 1.0 equivalents of the compound of Formula (IIA) per one bp of the dsRNA.
[00185] Without wishing to be held to any particular theory, the ionic solvent is not acting as a phase transfer catalyst. Rather, the ionic solvent is acting, unexpectedly, as a superior solvating agent. The ionic solvent also serves to help “unzip” the dsRNA by shielding the charges on the molecules. This use of such an ionic solvent is unreported in the prior art.
[00186] Thus, in an embodiment, the ionic solvent improves dsRNA dissociation by shielding the resulting charge on the single strand RNA.
Method of Preparation of Low-Molecular Weight Modifications by Ionic Solvation [00187] In another aspect, provided herein are methods of preparing a composition comprising a post-transcriptionally modified double strand RNA (MdsRNA) wherein the MdsRNA comprises a double strand RNA wherein no more than about 30% of all the nucleotides independently comprise Formula (VI): or an acceptable salt thereof, wherein:
B is a nucleobase;
R3 is selected from: amino acids, fatty acids, alkyl; substituted alkyl; alkenyl; substituted alkenyl; alkynyl; substituted alkynyl; aryl; substituted aryl; Cl -CIO alkyl, Cl- C10 alkenyl, or Cl -CIO alkynyl wherein alkyl and alkenyl can be linear, branched or cyclic; hydrogen; methyl; ethyl; propyl; isopropyl; butyl; isobutyl; tert-butyl; pentyl; hexyl; cyclohexyl; heptyl; octyl; nonyl; decyl; vinyl; allyl; ethynyl; benzyl; cinnamyl; COCM aryl; C6-C14 substituted aryl; heterocyclyl; C5-C14 heterocyclyl; phenyl; mono or disubstituted phenyl wherein the substituents are selected from Cl -CIO alkyl, Cl -CIO alkenyl, C1-C6 alkoxy, halogen, nitro, methylsulfonyl, and trifluoromethyl; 2-nitrophenyl;
4-nitrophenyl; 2;4-dinitrophenyl; 2-trifluoromethylphenyl; 4-trifluorom ethylphenyl; styryl; C8-C16 substituted styryl; 2-aminophenyl; mono or disubstituted 2-aminophenyl wherein the substituents are selected from Cl -CIO alkyl, Cl -CIO alkenyl, C1-C6 alkoxy, halogen, nitro, methylsulfonyl, and trifluoromethyl; N-alkyl-2-aminophenyl or N-aryl-2- aminophenyl wherein alkyl has the formula -CmFbm+i (wherein m is an integer less than or equal to 12) and aryl is an aromatic moiety; 2-amino-3-methyl-phenyl; 2-amino-
5-chlorophenyl; 2-methyl-5-chlorophenyl; N-methyl-2-aminophenyl; N-ethyl-2- aminophenyl; N-propyl-2-aminophenyl; N-butyl-2-aminophenyl; N-pentyl-2- aminophenyl; N-methyl-2-amino-4-nitrophenyl; 2-methyl-3-furyl; 2-methylnicotyl or N- trifluoromethyl-2-aminophenyl; silanyl; substituted silanyl; C1-C10 alkylsilanyl; C3-C12 trialkylsilanyl; C2-C12 alkoxyalkyl; C2-C12 alkoxy alkenyl; C2-C12 alkylthioalkyl; alkyl sulfonyl; Cl -CIO alkylsulfonyl; Cl -CIO haloalkyl; Cl -CIO haloalkenyl or Cl -CIO aminoalkyl; -(CH2CH2O)PCH3, -(CH2CH2O)PH, or -(CH2CH2O)PCOOR4 wherein p is an integer from 2 to 8 and R4 is H, alkyl, substituted alkyl, aryl, or substituted aryl;
-(CH2CH2O)SCOOH; -CH2CH2OH; -(CH2CH2O)4OH; -(CH2CH2O)6OH; -(CH2CH2O)SOH; -(CH2CH2O)sCOOMe; -(CH2CH2O)4OMe; -(CH2CH2O)6OMe; -(CH2CH2O)sOMe; -CH2OCH3; -CH2OCH2CH3; or -CH2OCH2CH2OCH3.; and the method comprising:
(a) contacting a compound of Formula (IV): (IV), with an activation agent to form a compound of Formula (IVA): (IVA), wherein X is a suitable leaving group; and
(b) contacting a compound of Formula (VIA): with a compound of Formula (IVA) to form a compound of Formula (VI).
[00188] In an embodiment, (a) and (b) are carried out in an anhydrous solvent.
[00189] In an embodiment, the anhydrous solvent is selected from DMSO or DCM.
[00190] In another embodiment, (a) and (b) are carried out without intervening purification.
[00191] In yet another embodiment, there is a purification step between (a) and (b).
[00192] In an embodiment, an ionic solvent is added after (a).
[00193] In an embodiment, the ionic solvent is selected from benzyltributyl ammonium chloride or benzyltrimethyl ammonium chloride.
[00194] In another embodiment, the activation agent is carbonyldiimidazole.
[00195] In yet another embodiment, the suitable leaving group is: wherein »AAA/' represents the covalent point of attachment to carbonyl of Formula (IVA). [00196] In a further embodiment, (b) has a ratio of between about two equivalents and about fifty equivalents of the compound of Formula (IVA) per nucleotide of the dsRNA. [00197] In another embodiment, (b) has a ratio of between about four equivalents and about twenty -five equivalents of the compound of Formula (IVA) per nucleotide of the dsRNA.
[00198] In another embodiment, the compound of Formula (IV) is the anhydride.
[00199] In another embodiment, R3 is selected from N-methyl anthranoyl (NMA), N- benzyl anthranoyl (NBA), dimethyl furoyl, -Tyr, -Trp, -Leu, octanoyl, lauroyl, linoleyl, oleyl, nicotinoyl or benzoyl.
[00200] In another embodiment, the MdsRNA comprises a sequence complementary to an expressed RNA in a target insect, fungus, weed or acari, as discussed in detail in this disclosure, such as but not limited to target (insect fungus, weed or acari), target sequences and target regions.
[00201] Embodiments in other sections of the disclosure regarding the composition, methods of making the compositions, nucleotide sequences and uses for target insects, fungi, weeds or acari are intended to be covered in this disclosure.
[00202] In some embodiments, the disclosed method of preparation is superior to prior methods in both cost and scalability. Prior methods of synthesis required water or another suitable polar protic solvent to reduce the need for excess solvent dilution. However, this led to significant degradation of the necessary anhydrides. Prior methods sometimes employed as much as 200 equivalents of modifying groups per one bp of dsRNA. The addition of ionic solvent has provided the superior and unexpected benefit of reducing the amount of solvent used, reducing the degradation of starting material, and allowing efficient synthesis of the desired product. In some embodiments of the method, (b) has a ratio of less than 0.05 equivalents of the compound of Formula (IVA) per one bp of the dsRNA. In another embodiment of the method, (b) has a ration of less than 0.1 equivalents of the compound of Formula (IVA). In particular embodiments of the method, (b) has a ratio of 0.2 equivalents of the compound of Formula (IVA) per one bp of the dsRNA.
[00203] Without wishing to be held to any particular theory, the ionic solvent is not acting as a phase transfer catalyst. Rather, the ionic solvent is acting, unexpectedly, as a superior solvating agent. The ionic solvent also serves to help “unzip” the dsRNA by shielding the charges on the molecules. This use of such an ionic solvent is unreported in the prior art.
[00204] Low molecular weight compositions have been described in U.S. patents 10,131,911, 10,640,769, and 11,174,480, the entire teachings of which are incorporated by reference.
Methods of Use
[00205] In another aspect of the disclosure, provided herein is a method of modifying the expression of a polynucleotide of interest in an insect comprising administering a composition of the disclosure.
[00206] In some embodiments, a target gene is selected such that inhibiting expression of the target gene kills, inhibits growth or appetite of, or slows reproduction of an animal, fungus, or weed. Inhibiting expression of the target gene can control, kill, inhibit growth or appetite of, or slow reproduction of the animal, fungus, or weed. In some embodiments, the insect, fungus, or plant is of agricultural significance. In some embodiments, an agriculturally significant animal, fungus, or plant is an insect, fungus, or weed. In an embodiment, the modified expression reduces the fertility rate of the target insect. In some embodiments, the described MdsRNAs can be used to control, kill, inhibit growth, appetite, or feeding of, or slow reproduction of an animal, fungus, or plant in an agricultural or urban setting. In some embodiments, a plant target gene is selected such that inhibiting expression of the gene in the plant increases plant growth, viability, quality, or yield.
[00207] In another embodiment, the MdsRNA comprises a sequence complementary to an expressed RNA in a target insect. For example, an efficacious sequence or target gene useful in the products and methods of the disclosure will inhibit the expression of the target gene, act on the mid gut of the insect and increase mortality or induce growth stunting, or stop instar development.
[00208] In an embodiment, the target insect is Diamondback moth (Plutella xylostella), Gypsy moth, Red imported fire ant (Solenopsis invicta), Fall armyworm, Colorado potato beetle, Canola flea beetle, Aedes aegypti, or Western corn root worm (Diabrotica virgifera virgifera). In another embodiment, the target insect is Pea aphid (Acyrthosiphon pisum), Soybean aphid, Piezodorus guildinii. In another embodiment the target is an acari, such as Verroa mite. In another embodiment the target is a weed, such as Palmer amaranth. In yet another embodiment, the target is a fungus such as Palmer amaranth, Fusarium graminearum (Gibberella zeae) or Botrytis. Accordingly, the MdsRNA used in the compositions of the disclosure will be a nucleotide sequence that can inhibit the expression of target genes or regions in these insects. For example, the MdsRNA comprises a sequence complementary to a target region in Diamondback moth, such as but not limited to AChE2, P450, CYP6BFlvl, DOUX, Cytokine receptor DOMELESS, Protein MESH transcript variant XI, Venom carboylesterase-6 and VPASE-E. In another embodiment, the MdsRNA comprises a sequence complementary to a target region in Fall armyworm, such as but not limited to P450, Cytokine receptor DOMELESS, VPASE, Dredd, Protein MESH transcript variant XI, P450 CYP9A58, P450 CYP321 A8, P450 CYP6B2-like.
[00209] In another embodiment, the MdsRNA comprises a sequence complementary to a target region in Western corn root worm, such as but not limited to SNF7. [00210] In an embodiment, the MdsRNA comprises a sequence selected from one of SEQ ID Nos. 1-269 and its perfect or imperfect reverse complementary strand. In a particular embodiment, the sequence is selected from one of SEQ ID Nos. 13 or 248-251 and its perfect or imperfect reverse complementary strand. In an embodiment, the target insect is a Lepidopteran and can be targeted using any one of SEQ ID Nos. 1-269 and its perfect or imperfect reverse complementary strand thereof for each sequence specified. In an embodiment, the Lepidopteran is targeted using any one of SEQ ID Nos. 13 or 248- 251.
[00211] In an embodiment, the target insect is a Lepidopteran. In an embodiment, the Lepidopteran is an army worm, com ear worm, cabbage butterfly, or cotton boll worm.
[00212] In another embodiment, the MdsRNA comprises a sequence complementary to the P450, CYP6FBlvl, MESH, AChE2, VPASE, DOMELESS, DOUX or Venom target region in a Lepidopteran.
[00213] In an embodiment, the treatment of at least about 30% control of the target insect. In an embodiment, the treatment of at least about 50% control of the target insect. In an embodiment, the treatment shows greater than about 50% control of the target insect. In an embodiment, the treatment shows between about 50% to about 90% control of the target insect. In an embodiment, the treatment shows between about 50% to about 75% control of the target insect. In an embodiment, the treatment shows between about 30% to about 50% control of the target insect.
[00214] The compositions and methods herein described are further illustrated in the following examples, which are provided by way of illustration and are not intended to be limiting. It will be appreciated that variations in proportions and alternatives in elements of the components shown will be apparent to those skilled in the art and are within the scope of embodiments of the present disclosure. Theoretical aspects are presented with the understanding that Applicant does not seek to be bound by the theory presented. All parts or amounts, unless otherwise specified, are by weight.
Agricultural/Agrochemical Compositions
[00215] In some embodiments, compositions containing the described MdsRNAs are described. In some embodiments, the MdsRNA-containing compositions are formulated for agricultural application (agrochemical compositions).
[00216] As used herein, an agrochemical composition comprises an effective amount of at least one MdsRNA and optionally one or more acceptable carriers or excipients. Carriers and excipients are substances other than the MdsRNA that have been appropriately evaluated for safety and are intentionally included in a composition. Excipients may act to a) aid in processing of the MdsRNA during manufacture, b) protect, support or enhance stability or bioavailability of the MdsRNA, c) assist in product identification, and/or d) enhance any other attribute of the overall safety, effectiveness, of delivery of the MdsRNA during storage or use. An acceptable carrier or excipient may or may not be an inert substance. As used herein, "effective amount," refers to that amount of a MdsRNA to produce the intended result.
[00217] Carrier and excipients include, but are not limited to, absorption enhancers, anti-adherents, anti-foaming agents, anti-oxidants, binders, buffering agents (pH regulating agents), chelating agents, coating agents, colors, delivery enhancers, dextran, dextrose, diluents, disintegrates, dispersants, emulsifiers, extenders, fillers, foam control agents, glidants, humectants, lubricants, oils, pigments, polymers, preservatives, saline, salts, solvents, sugars, surfactants, suspending agents, sustained release matrices, sweeteners, thickening agents, tonicity agents, vehicles, water-repelling agents, and wetting agents.
[00218] In some embodiments, an agrochemical composition comprises one or more adjuvants or surfactants. In some embodiments, the one or more adjuvant or surfactants are independently selected from anionic surfactants, cationic surfactants, amphoteric surfactants, nonionic surfactants, anti-condensates, thickeners, emulsifiers, spreaders, stickers, organosilanes, fatty esters and oils. In a particular embodiment, the one or more adjuvants or surfactants are optionally selected from non-ionic, organo silicone surfactants (e.g., KINETIC ® from the Helena company; nonionic organosilicone-based wetter/spreader/penetrant spray adjuvant), DYNE-AMIC ® (from the Helena company; blend of highly refined methylated seed oils in combination with specialized organosilicone-based nonionic surfactants) and SIL WET ® (from Momentive; nonionic surfactant).
[00219] In some embodiments, an agrochemical composition comprises one or more agents selected from an herbicide, fungicide, insecticide, acaricide and fertilizer.
[00220] The described MdsRNAs and compositions containing MdsRNAs can be processed in a number of different ways known to those skilled in the art to facilitate application of such material onto plants or into baits and for use in the field or in urban environments. The described MdsRNAs and compositions comprising MdsRNAs disclosed herein can be packaged or included in a kit, container, pack, or dispenser. [00221] In some embodiments, an agrochemical composition contains two or more different MdsRNAs. The MdsRNAs may have different antisense sequences complementary to the same target gene, different antisense sequences complementary to different traget genes in the same or different hosts, different or similar lengths, or different or similar post transcriptional modification.
[00222] In some embodiments, an agrochemical composition is an emulsifiable agricultural concentrate. In some embodiments, an emulsifiable agricultural concentrate further contains a least one agent that can be, but is not limited to, carrier, or organic solvent, surfactant, excipient, herbicide, fungicide, insecticide, fertilizer, or combinations thereof.
[00223] In some embodiments, an agrochemical composition contains one or more herbicides. Non-limiting examples of suitable herbicides include, but are not limited to, imidazolinone, acetochlor, acifluorfen, aclonifen, acrolein, AKH-7088, alachlor, alloxydim, ametryn, amidosulfuron, amitrole, ammonium sulfamate, anilofos, asulam, atrazine, azafenidin, azimsulfuron, BAS 620H, BAS 654 OOH, BAY FOE 5043, benazolin, benfluralin, benfuresate, bensulfuron-methyl, bensulide, bentazone, benzofenap, bifenox, bilanafos, bispyribac-sodium, bromacil, bromobutide, bromofenoxim, bromoxynil, butachlor, butamifos, butralin, butroxydim, butylate, cafenstrole, carbetamide, carfentrazone-ethyl, chlormethoxy fen, chloramben, chlorbromuron, chloridazon, chlorimuron-ethyl, chloroacetic acid, chlorotoluron, chlorpropham, chlorsulfuron, chlorthal-dimethyl, chlorthiamid, cinmethylin, cinosulfuron, clethodim, clodinafop- propargyl, clomazone, clomeprop, clopyralid, cloransulam-methyl, cyanazine, cycloate, cyclosulfamuron, cycloxydim, cyhalofop-butyl, 2,4-D, daimuron, dalapon, dazomet, 2,4DB, desmedipham, desmetryn, dicamba, dichlobenil, dichlorprop, dichlorprop-P, diclofop-methyl, difenzoquat metilsulfate, diflufenican, dimefuron, dimepiperate, dimethachlor, dimethametryn, dimethenamid, dimethipin, dimethylarsinic acid, dinitramine, dinocap, dinoterb, diphenamid, diquat dibromide, dithiopyr, diuron, DNOC, EPTC, esprocarb, ethalfluralin, ethametsulfuron-methyl, ethofumesate, ethoxysulfuron, etobenzanid, fenoxaprop-P-ethyl, fenuron, ferrous sulfate, flamprop-M, flazasulfuron, fluazifop-butyl, fluazifop-P -butyl, fluchloralin, flumetsulam, flumiclorac-pentyl, flumioxazin, fluometuron, fluoroglycofen-ethyl, flupoxam, flupropanate, flupyrsulfuron- methyl-sodium, flurenol, fluridone, flurochloridone, fluroxypyr, flurtamone, fluthiacet- methyl, fomesafen, fosamine, glufosinate-ammonium, glyphosate, glyphosinate, halosulfuron-methyl, haloxyfop, HC-252, hexazinone, imazamethabenz-methyl, imazamox, imazapyr, imazaquin, imazethapyr, imazosuluron, imidazilinone, indanofan, ioxynil, isoproturon, isouron, isoxaben, isoxaflutole, lactofen, lenacil, linuron, MCPA, MCPA-thioethyl, MCPB, mecoprop, mecoprop-P, mefenacet, metamitron, metazachlor, methabenzthiazuron, methylarsonic acid, methyldymron, methyl isothiocyanate, metobenzuron, metobromuron, metolachlor, metosulam, metoxuron, metribuzin, metsulfuron-methyl, molinate, monolinuron, naproanilide, napropamide, naptalam, neburon, nicosulfuron, nonanoic acid, norflurazon, oleic acid (fatty acids), orbencarb, oryzalin, oxadiargyl, oxadiazon, oxasulfuron, oxyfluorfen, paraquat dichloride, pebulate, pendimethalin, pentachlorophenol, pentanochlor, pentoxazone, petroleum oils, phenmedipham, picloram, piperophos, pretilachlor, primisulfuron-methyl, prodiamine, prometon, prometryn, propachlor, propanil, propaquizafop, propazine, propham, propisochlor, propyzamide, prosulfocarb, prosulfuron, pyraflufen-ethyl, pyrazolynate, pyrazosulfuron-ethyl, pyrazoxyfen, pyributicarb, pyridate, pyriminobac-methyl, pyrithiobac-sodium, quinclorac, quinmerac, quinoclamine, quizalofop, quizalofop-P, rimsulfuron, sethoxydim, siduron, simazine, simetryn, sodium chlorate, STS system (sulfonylurea), sulcotrione, sulfentrazone, sulfometuron-methyl, sulfosulfuron, sulfuric acid, tar oils, 2,3,6-TBA, TCA-sodium, tebutam, tebuthiuron, terbacil, terbumeton, terbuthylazine, terbutryn, thenylchlor, thiazopyr, thifensulfuron-methyl, thiobencarb, tiocarbazil, tralkoxydim, tri-allate, triasulfuron, triaziflam, tribenuron-methyl, triclopyr, trietazine, trifluralin, triflusulfuron-methyl, vernolate, and combinations thereof.
[00224] In some embodiments, an agrochemical composition contains one or more fungicides. Suitable fungicides include, but are not limited to, carbamate fungicides such as 3,3'-ethylenebis(tetrahydro-4,6-dimethyl-2H-l,3,5-thiadiazine-2-thione), zinc or manganese ethylenebis(dithiocarbamate), bis(dimethyldithiocarbamoyl)disulfide, zinc propylene-bis-(dithiocarbamate), bis(dimethyldithiocarbamoyl)ethylenediamine, nickel dimethyl-dithiocarbamate, methyl 1 -(butylcarbamoyl)-2-benzimidazolecarbamate, l,2-bis(3-methoxycarbonyl-2-thioureido)benzene, l-isopropylcarbamoyl-3-(3,5- dichlorophenyl)-hydantoin, potassium N-hydroxymethyl-N-methyldithiocarbamate, and 5-methyl-10-butoxycarbonylamino-10,l 1 -dehydrodibenzo (b,f)azepine; pyridine fungicides such as zinc bis(l-hydroxy-2(lH)pyridinethionate) and 2-pyridinethiol-l -oxide sodium salt; phosphorus fungicides such as O,O-diisopropyl S-benzylphosphorothioate and O-ethyl S,S-diphenyldithiophosphate; phthalimide fungicides such as N-(2,6-diethylphenyl)phthalimide and N-(2,6-diethylphenyl)-4-methylphthalimide; dicarboxyimide fungicides such as N-trichloromethylthio-4-cyclohexene-l,2- dicarboxyimide and N-tetrachloroethylthio-4-cyclohexene-l,2-dicarboxyimide; oxathine fungicides such as 5,6-dihydro-2-methyl-l,4-oxathine-3-carboxanilido-4,4-dioxide and 5,6-dihydro-2-methyl-l,4-oxathine-3-carboxanilide; naphthoquinone fungicides such as
2.3-dichloro-l,4-naphthoquinone, 2-oxy-3-chloro-l,4-naphthoquinone copper sulfate; pentachloronitrobenzene; 1,4-di chi oro-2, 5-dimethoxybenzene; 5-methyl-s- triazol(3,4-b)benzthiazole; 2-(thiocyanomethylthio)benzothiazole; 3-hydroxy-5- methylisooxazole; N-2,3-dichlorophenyltetrachlorophthalamic acid; 5-ethoxy-3- trichloromethyl-l-2,4-thiadiazole; 2,4-dichloro-6-(O-chloroanilino)-l,3,5-triazine;
2.3-dicyano-l,4-dithio-anthraquinone; copper 8-quinolinate, poly oxine; validamycin; cycloheximide; iron methanearsonate; diisopropyl-l,3-dithiolane-2-iridene malonate; 3-allyloxy-l,2-benzoisothiazol-l,l-dioxide; kasugamycin; Blasticidin S; 4,5,6,7-tetra- chlorophthalide; 3-(3,5-dichlorophenyl)-5-ethenyl-5-methyloxazolizine-2, 4-dione; N-(3,5- di chlorophenyl)- 1 ,2-dimethylcyclopropane- 1 ,2-dicarboxyimide; S-n-butyl-5 '-para-t- butylbenzyl-N-3-pyridyldithiocarbonylimidate; 4-chlorophenoxy-3, 3 -dimethyl- 1- (lH,l,3,4-triazol-l-yl)-2-butanone; methyl-D,L-N-(2,6-dimethylphenyl)-N-(2'- methoxyacetyl)alaninate; N-propyl-N-[2-(2,4,6-trichlorophenoxy)ethyl]phosphor- 1 - carboxamide; N-(3,5-dichlorophenyl)-succinimide; tetrachloroisophthalonitrile;
2-dimethylamino-4-methyl-5-n-butyl-6-hydroxypyrimidine; 2,6-dichloro-4-nitroaniline;
3-methyl-4-chlorobenzthiazol-2-one; l,2,5,6-tetrahydro-4H-pyrrolo[3,2,l-I,j]phosphor-2- one; 3'-isopropoxy-2-methyl-benzanilide; l-[2-(2,4-dichlorophenyl)-4-ethyl-l,3- dioxorane-2-ylmethyl]-lH,l,2,4-triaz ol; l,2-benzisothiazoline-3-one; basic copper chloride; basic copper sulfate; N'-dichlorofluoromethylthio-N,N-dimethyl-N- phenylsulfamide; ethyl-N-(3-dimethylamino-propyl)thiocarbamate hydrochloride; piomycin; S,S-6-methylquinoxaline-2,3-diyldithio-carbonate; complex of zinc and manneb; di-zinc bis(dimethyldithiocarbamate) ethylenebis (dithiocarbamate) and glyphosate; chlorothalonil-based fungicides, strobilurin-based fungicides such as azoxystrobin, pyraclostrobin, and trifloxystrobin; and triazole-based fungicide such as myclobutanil, propi conazole, tebuconazol, tetraconazole, and combinations thereof. [00225] In some embodiments, an agrochemical composition contains one or more insecticides. Suitable insecticides include, but are not limited to, phosphoric insecticides such as 0,0-diethyl O-(2-isopropyl-4-methyl-6-pyrimidinyl)phosphorothioate, 0,0-dimethyl S-2-[(ethylthio)ethyl]phosphorodithioate, 0,0-dimethyl O-(3-methyl-4- nitrophenyl)-thiophosphate, 0,0-dimethyl S-(N-methylcarbamoylmethyl)- phosphorodithioate, 0,0-dimethyl S-(N-methyl-N-formylcarbamoylmethyl) phosphoro- dithioate, O,O-dimethyl S-2-[(ethylthio)ethyl] phosphorodithioate, 0,0-diethyl S-2-[(ethylthio)ethyl] phosphorodithioate, 0,0-dimethyl- 1 -hydroxy-2, 2, 2- trichloroethylphophonate, O,O-diethyl-O-(5-phenyl-3-isooxazolyl)phosphorothioate, 0,0- dimethyl O-(2,5-dichloro-4-bromophenyl)phosphorothioate, 0,0-dimethyl O-(3-methyl-4- methylmercaptophenyl)-thiophosphate, O-ethyl O-p-cyanophenyl phenyl- phosphorothioate, O,O-dimethyl-S-(l,2-dicarboethoxyethyl)phosphorodithioate, 2-chloro- (2,4,5-trichlorophenyl)vinyldimethyl phosphate, 2-chloro-l-(2,4-dichlorophenyl)- vinyldimethyl phosphate, 0,0-dimethyl O-p-cyanophenyl phosphorothioate, 2,2-dichlorovinyl dimethyl phosphate, 0,0-diethyl 0-2,4-dichlorophenyl phosphorothioate, ethyl mercaptophenylacetate 0,0-dimethyl phosphoro-dithioate, S-[(6- chloro-2-oxo-3-benzooxazolinyl)methyl] 0,0-diethyl phosphorodithioate, 2-chloro-l- (2,4-dichlorophenyl)vinyl diethylphosphate, 0,0-diethyl O-(3-oxo-2-phenyl-2H- pyridazine-6-yl) phosphorothioate, 0,0-dimethyl S-(l-methyl-2-ethylsulfinyl)-ethyl phosphorothiolate, 0,0-dimethyl S-phthalimidomethyl phosphorodithioate, 0,0-diethyl S-(N-ethoxycarbonyl-N-methylcarbamoylmethyl)phosphorodithioate, 0,0-dimethyl S-[2- methoxy-l,3,4-thiadiazol-5-(4H)-I-(4)-methyl] dithiophosphate, 2-methoxy-4H- 1,3,2- benzooxaphosphorine 2-sulfide, 0,0-diethyl O-(3,5,6-trichloro-2-pyridyl)phosphorothiate, O-ethyl 0-2,4-dichlorophenyl thionobenzene phosphonate, S-[4,6-diamino-s-triazine-2-yl- methyl] 0,0-dimethyl phosphorodithioate, O-ethyl O-p-nitrophenyl phenyl phosphorothioate, O,S-dimethyl N-acetyl phosphoroamidothioate, 2-diethylamino-6- methylpyrimidine-4-yl-diethylphosphorothi onate, 2-diethylamino-6-methylpyrimidine-4- yl-dimethylphosphorothionate, 0,0-diethyl O-N-(methylsulfinyl) phenyl phosphorothioate, O-ethyl S-propyl 0-2,4-dichlorophenyl phosphorodithioate and cis-3- (dimethoxyphosphinoxy)N-methyl-cis-crotone amide; carbamate insecticides such as 1 -naphthyl N-methylcarbamate, S-methyl N-[methylcarbamoyloxy]thioacetoimidate, m-tolyl methylcarbamate, 3,4-xylyl methylcarbamate, 3,5-xylyl methylcarbamate, 2-sec- butylphenyl N-methylcarbamate, 2,3-dihydro-2,2-dimethyl-7-benzofuranylmethyl- carbamate, 2-isopropoxyphenyl N-methylcarbamate, l,3-bis(carbamoylthio)-2-(N,N- dimethylamino)propane hydrochloride and 2-diethylamino-6-methylpyrimidine-4-yl- dimethylcarbamate; and other insecticides such as N,N-dimethyl N'-(2-methyl-4- chlorophenyl)formamidine hydrochloride, nicotine sulfate, milbemycin, 6-methyl-2,3- quinoxalinedithiocyclic S,S-dithiocarbonate, 2,4-dinitro-6-sec-butylphenyl dimethylacrylate, l,l-bis(p-chlorophenyl) 2,2,2-trichloroethanol, 2-(p-tert-butylphenoxy)isopropyl- 2'-chloroethylsulfite, azoxybenzene, di-(p-chlorophenyl)-cyclopropyl carbinol, di[tri(2,2- dimethyl-2-phenylethyl)tin]oxide, l-(4-chlorophenyl)-3-(2,6-difluorobenzoyl) urea, S-tricyclohexyltin O,O-diisopropylphosphorodithioate, and combinations thereof.
[00226] In some embodiments, an agrochemical composition contains one or more fertilizers. A variety of fertilizers are suitable for inclusion in the compositions. The fertilizer can be a single nutrient fertilizer (N, P, or K), binary fertilizer (e.g., NP, NK, or PK), a NPK fertilizer, or a multinutrient fertilizer (e.g., may provide one or more of calcium, magnesium, sulfur, copper, iron, manganese, molybdenum, zinc, boron, silicon, cobalt, or vanadium). The fertilizer can be of natural origin or synthetic origin. The fertilizer can be liquid or solid, and may provide slow or controlled release.
[00227] In some embodiments, the MdsRNAs comprise less than 50% by weight of a composition. In some embodiments, the amount of MdsRNA in an agriculture composition is less than 5% by weight of the composition. In some embodiments, the MdsRNA is present in the composition in an amount less than about 1% by weight, less than about 0.9% by weight, less than about 0.8% by weight, less than about 0.7% by weight, less than about 0.6% by weight, less than about 0.5% by weight, less than about 0.4% by weight, less than about 0.3% by weight, less than about 0.2% by weight, less than about 0.1% by weight, less than about 0.05% by weight, less than about 0.01% by weight, or less than about 0.001% by weight of the composition.
[00228] In some embodiments, the agrochemical composition is formulated as a liquid. Liquid formulations can be prepared by mixing the MdsRNA and other agents in a liquid until dissolution of all the components is achieved in the weight percentages described below. The liquid can be an aqueous, ionic, or organic liquid. Suitable liquids include, but are not limited to, water, alcohols (e.g. methanol and ethanol), ketones (e.g. acetone, methyl ethyl ketone and cyclohexanone), aromatic hydrocarbons (e.g. benzene, toluene, xylene, ethylbenzene and methylnaphthalene), aliphatic hydrocarbons (e.g. hexane and kerosene), esters (e.g. ethyl acetate and butyl acetate), nitriles (e.g. acetonitrile and isobutyronitrile), ethers (e.g. dioxane and diisopropyl ether), acid amides (e.g. dimethylformamide and dimethylacetamide), and halogenated hydrocarbons (e.g. di chloroethane, trichloroethylene and carbon tetrachloride).
[00229] In some embodiments, the liquid formulation is an aqueous formulation. In some embodiments, an aqueous formulation contains only water, the MdsRNA and other agents. In some embodiments, additional compounds, solvents, or adjuvants are provided with the aqueous formulation. [00230] In some embodiments, the agrochemical composition is formulated as a powder or dust. The powder or dust can be granulated to be suitable for applying the powder or dust directly to a crop (i.e., by dusting the crop), or it can be granulated for eventual dissolution in a solvent such as water. In some embodiments, the composition is a lyophilisate. Typically, the MdsRNA and the other agents are lyophilized together. In some embodiments, one or more MdsRNAs and the other agents can be lyophilized separately.
[00231] A variety of suitable solid and gaseous carriers can be utilized in the compositions. Suitable solid carriers include, but are not limited to, fine powders or granules of clays (e.g. kaolin clay, diatomaceous earth, synthetic hydrated silicon dioxide, attapulgite clay, bentonite and acid clay), talcs, bulking agents, inorganic minerals (e.g., sericite, powdered quartz, powdered sulfur, activated carbon, calcium carbonate and hydrated silica), and salts for chemical fertilizers (e.g. ammonium sulfate, ammonium phosphate, ammonium nitrate, urea and ammonium chloride). Suitable gaseous carriers include, for example, butane gas, carbon dioxide, and fluorocarbon gas.
[00232] In some embodiments, an agrochemical composition includes a dispersant. Examples of dispersants include, but are not limited to, methyl cellulose, polyvinyl alcohol, sodium lignin sulfonates, polymeric alkyl naphthalene sulfonates, sodium naphthalene sulfonate, polymethylene bisnaphthalene sulfonate, neutralized polyoxyethylated derivatives, and ring-substituted alkyl phenol phosphates. Stabilizers may also be used to produce stable emulsions. Exemplary stabilizers include, but are not limited to magnesium, aluminum silicate, and xanthan gum.
[00233] In some embodiments, an agrochemical composition is formulated as a spray in the form of an aerosol. When formulated as an aerosol spray, the composition is generally charged in a container under pressure together with a propellant. Examples of suitable propellants include fluorotrichloromethane and dichlorodifluoromethane.
[00234] In some embodiments, an agrochemical composition includes a seed. In some embodiments, an agrochemical composition comprises an antifungal MdsRNA and a seed. In some embodiments, an agrochemical composition comprises a MdsRNA, a seed, and further comprises a fungicide.
[00235] In some embodiments, the amount of the MdsRNA in a fungicidal composition (agrochemical composition containing a fungicide) is less than about 5% by weight, less than about 1% by weight, less than about 0.9% by weight, less than about 0.8% by weight, less than about 0.7% by weight, less than about 0.6% by weight, less than about 0.5% by weight, less than about 0.4% by weight, less than about 0.3% by weight, less than about 0.2% by weight, less than about 0.1% by weight, less than about 0.05% by weight, less than about 0.01% by weight, or less than about 0.001% by weight of the fungicidal composition. The weight of the fungicidal composition does not include the weight of the seed.
[00236] In some embodiments, the fungicidal composition is present inside the seed coat, or internal to the seed. In some embodiments, the fungicidal composition is formed over the seed such that it covers the exterior of the seed, either fully or partially. Methods for coating a seed include those known in the art.
Methods for Controlling Agricultural Pests
[00237] In some embodiments, MdsRNAs or compositions containing MdsRNAs are used to control agricultural pests or treat agricultural pest infestation. The MdsRNAs can be administered to the pest, to an area occupied by the pest, or to a food source of the pest. [00238] In some embodiments, methods are provided for treating for or controlling pests. In some embodiments, the pest is an insect, fungus, acari or weed. The methods comprise applying a composition comprising one or more described MdsRNAs to an area to be treated. In some embodiments, the MdsRNA is present in the composition in an amount of less than 5% by weight. In some embodiments, the composition is applied directly to a surface. In some embodiments, the surface is a plant surface upon which the targeted insect or fungal pest feeds.
[00239] In some embodiments, the gene expression level and/or mRNA level of a target gene in a target host is reduced by at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, following application of MdsRNAs or MdsRNA-containing composition. In some embodiments, mortality of the agricultural pest in increased at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% following application of MdsRNAs or MdsRNA-containing composition.
[00240] As used herein, controlling a pest means to reduce crop damage or decreased yield caused by the pest, or to increase morbidity, inhibit growth or appetite or feeding of, or slow reproduction of the pest compared with the damage, decreased yield morbidity, growth, appetite, feeding, or reproduction as measured in the absence of treatment with MdsRNAs. Crop Application
[00241] In some embodiments, methods of reducing expression of a target gene in a target plant other than a weed are described. The methods comprise applying a composition containing one or more of the described MdsRNAs to the plant. In some embodiments, the plant is a crop plant. A crop plant is a plant that can be grown and harvested for profit or subsistence. A crop plant can be, but is not limited to, a food plant, horticultural plant, Horticulture plant, or industrial plant. In some embodiments, the plant is a cultivated plant. The plant can be in a laboratory, greenhouse, nursery, field, orchard or other agricultural setting, garden, or another natural or urban setting. In some embodiments, a target plant is a plant considered desirable in a particular situation or location.
Insect Infestation
[00242] In some embodiments, the animal is an insect. In some embodiments, the insect is a Coleopteran (such as a beetle). A Coleopteran can be, but are not limited to, bark beetle, elm leaf beetle, Asian longhorn beetle, death watch beetle, mountain pine beetle, coconut hispine beetle or the Colorado potato beetle. In some embodiments, the insect is a Lepidopteran (such as a butterfly or moth). A Lepidopteran can be, but is not limited to, army worm, corn ear worm, cabbage butterfly, or cotton boll worm. In some embodiments, the insect is a Hymenopteran (such as sawflies, wasps, bees, ants). A Hymenopteran can be, but is not limited to, fire ant, argentine ant, carpenter ant, leafcutter ant, army ant, wheat stem sawfly, larch sawfly, spruce sawfly, or bed bug. In some embodiments, the insect is a Dipteran (such as a fly). A Dipteran can be, but is limited to, fly, mosquito, gnat, or leafminer. In some embodiments, the insect is a Hemipteran (such as a true bug). A Hemipteran can be, but is not limited to, aphid, hopper, bug, whiteflies, mealybug, or flea. In some embodiments, the insect is a Western corn root worm.
[00243] In some embodiments, the insect is an insect having resistance to one or more conventional known insecticides. In some embodiments, the insect, such as a Red imported fire ant has the potential to have a negative impact on biodiversity (Wojcik et al. 2001 and/or resistance to insecticides (Zhang et al. 2016). In some embodiments, the insect, such as a mosquito, has the potential to impact human health as a vector for disease, such as, but not limited to, Malaria, Dengue, Zika and Chikungunya (Hemingway et al. 2004). In some embodiments, the insect, such as Asian citrus psyllid, is a vector of the citrus greening disease (Tiwari et al. 2011). [00244] Coleopteran, Lepidopteran, Hymenopteran, Dipteran, and Hemipteran insect pests are known to be susceptible to RNAi introduced either by direct injection or by feeding on plant matter treated with siRNA precursors. Field application of naked RNAs is generally impractical due to the sensitivity of RNA to environmental specific and nonspecific degradation (Baum 2016). Furthermore, RNA is highly susceptible to degradation during the course of feeding and in transit through the insect gut. For example, in general, the Lepidoptera seem to degrade RNA much more aggressively than the Coleoptera, which may account for their relatively poor susceptibility to RNAi mediated control methods. The stability of the described MdsRNAs serves to protect the MdsRNA from host nucleases before delivery to the RNAi pathway, and limits nonspecific environmental degradation. The described MdsRNAs are nevertheless sufficiently biodegradable to be considered environmentally safe.
[00245] A composition comprising one or more MdsRNAs can be applied to a plant prior to infection to prevent an insect infection. The composition may also be applied after the appearance of signs of infection to treat an insect infection. The composition can be applied by a variety of methods depending on the plant part to be treated. By way of example, the composition can be applied to a plant seed prior to planting to prevent insect infection of the seed. The composition can be applied to the soil at the time of planting or just before planting to prevent insect infestation of the newly planted seed (i.e., as a pre- emergent). In some embodiments, the composition can be applied to a plant after its germination or to the foliage of the plant after emergence to either treat or prevent insect infestation (i.e., as a post-emergent). In an exemplary embodiment, the application occurs during the stages of germination, seedling growth, vegetative growth, and reproductive growth. In some embodiments, application occurs during vegetative and reproductive growth stages.
[00246] Applying the composition to a pre-emergent seed may involve various seed coating techniques such as film coating, pelleting, encapsulation, drum coating, and fluidized bed coating. Applying to a post-emergent plant may involve spraying or crop dusting techniques.
[00247] An effective amount of the composition can be applied to a plant or seed by several methods generally known in the art. As will be appreciated by a skilled artisan, the amount of composition comprising an “an effective amount” can and will vary depending upon the plant and its stage of production, the fungal target, and environmental conditions. Generally speaking, for a typical application, the plant or its progeny is treated with an amount of the composition sufficient to provide a concentration of active ingredients from about 0.01 mg/kg to about 10% by weight. It is envisioned that the method may involve more than one application of the composition to the plant or its progeny. For example, the number of applications may range from about 1 to about 5 or more. The applications, as detailed herein, can be applied at the same or different stages of the plant’s life cycle.
Fungal Infection
[00248] In some embodiments, the MdsRNAs are used to treat or prevent fungal infection. In some embodiments, the fungus can be, but is not limited to, a Hypocrealesan, Venturia, Podosphaera, Erysiphe, Monolinia, Mycosphaerella, Uncinula; Basidiomycete, Hemileia, Rhizoctonia, Puccinia, Fungi imperfecti, Botrytis, Helminthosporium, Rhynchosporium, Fusarium, Septoria, Cercospora, Alternaria, Pyricularia, Pseudocercosporella, Oomycete fungi, Phytophthora, Peronospora, Bremia, Pythium, Plasmopara, Phakopsora Pachyrhizi, P. meibomiae, Scleropthora macrospora, Sclerophthora rayissiae, Sclerospora graminicola, Peronosclerospora sorghi, Peronosclerospora philippinensis, Peronosclerospora sacchari Peronosclerospora maydis, Physopella zeae, Cercospora zeae-maydis, Colletotrichum graminicola, Hypocreale, Gibberella zeae, Exserohilum turcicum, Kabatiellu zeae, Bipolaris maydis, Gibberella avenacea, Fusarium culmorum, Fusarium oxysporum, Fusarium sporotrichioides, or Fusarium graminearum. In some embodiments, treatment of Fusarium graminearum can reduce the production of mycotoxins, the risk of emergence of resistance to fungicides based on demethylation inhibitors (DMI), orcarcinogenicity concerns about conventional DMI like Tebuconazole.
[00249] In some embodiments, the described agrochemical compositions can be applied to a plant prior to infection to prevent a fungal infection. In some embodiments, the described agrochemical compositions can be applied to a plant after the appearance of signs of infection to treat a fungal infection. The composition can be applied by a variety of methods depending on the plant part to be treated. By way of example, the composition can be applied to a plant seed prior to planting to prevent fungal infection of the seed. The composition can be applied to the soil at the time of planting or just before planting to prevent microbial infestation of the newly planted seed (i.e., as a pre-emergent). In some embodiments, the composition can be applied to a plant after its germination or to the foliage of the plant after emergence to either treat or prevent microbial infestation (i.e., as a post emergent). In an exemplary embodiment, the application occurs during the stages of germination, seedling growth, vegetative growth, and reproductive growth. More typically, applications occur during vegetative and reproductive growth stages.
[00250] Applying the composition to a pre-emergent seed may involve various seed coating techniques such as film coating, pelleting, encapsulation, drum coating, and fluidized bed coating. Applying to a post-emergent plant may involve spraying or crop dusting techniques.
[00251] An effective amount of the composition can be applied to a plant or seed by several methods generally known in the art. As will be appreciated by a skilled artisan, the amount of composition comprising an “an effective amount” can and will vary depending upon the plant and its stage of production, the fungal target, and environmental conditions. Generally speaking, for a typical application, the plant or its progeny is treated with an amount of the composition sufficient to provide a concentration of active ingredients from about 0.01 mg/kg to about 5,000 mg/kg. It is envisioned that the method may involve more than one application of the composition to the plant or its progeny. For example, the number of applications may range from about 1 to about 5 or more. The applications, as detailed herein, can be made at the same or different stages of the plant’s life cycle.
Weeds
[00252] The compostions of the disclosure can be used to control, prevent, eliminate, slow the growth of weeds. A weed is a plant considered undesirable in a particular situation or location. A weed can be, but is not limited to, Palmer Amaranth, Common Lambsquarters, Horseweed, Morning Glory, Waterhemp, Nutsedge, Kochia, Common Ragweed, Giant Ragweed, or Nightshade.
EXAMPLES
Example 1. Preparation of High Molecular Weight Polyalkyloxy Polymer dsRNA in DMSO.
Activation of PEG
[00253] One equivalent PEG-acetic acid was dissolved in dimethyl sulfoxide (DMSO) at 50 mg/ml and to this solution was added 1.05 equivalent of carbonyldiimidazole (CDI). The reaction mixture was stirred for at least 2 hours, preferably overnight under argon. This solution was then added directly to dsRNA following the ionic solvation.
Reaction of dsRNA with activated PEG
[00254] Benzyl triethyl ammonium chloride (Benzyl-TriBA-Cl) was dissolved in DMSO at 200 mM. An aliquot of this solution was added to freeze-dried dsRNA in a reaction tube. The tube was warmed to 65°C in a shaker for 1 hr. Then the temperature was reduced to 55°C and catalytic amount of pyridine was added followed by a 200 pg/pL solution of DMAP. A 200 ug/uL solution of activated PEG was added in 1-3 portions.
The mixture was kept at 55 °C for 90 min and then was quenched with 500 mM citrate pH 4.5, and diluted to 40 mL with water. The resulting mixture containing the crude PEG- dsRNA was purified using tangential flow filtration, TFF, to yield MdsRNA with >90% purity.
[00255] The following MdsRNAs were prepared according to the procedure described above:
Table 1: Modified MdsRNA Compounds Synthesized by Procedure of Example 1. aInput is moles of PEG/mole of RNA nucleotide. bModifier is the -OC(O)R1 modification as defined in Formula I. cSequence is the sequence of nucelobases of the RNA strand backbone.
[00256] As can be seen by FIG. 4, the agarose gel (2%) analysis of the activated 10k PEG/ dsRNA reaction mixture (line 3) demonstrates the covalent modification of the 300 bp molecule with 1, 2, 3, 4 and so on chains of 10k PEG polymer. The original unmodified dsRNA molecule is all consumed during the reaction.
Example 2. Preparation of High Molecular Weight Polyalkyloxy Polymer dsRNA using DCM for the activation reaction.
[00257] One equivalent PEG-acetic acid was dissolved in dichloromethane (DCM) at 300 mg/ml and to this solution was added 1.05 equivalent of CDI. The reaction mixture was stirred for at least 2 hours, preferably overnight under argon. The reaction mixture was then diluted to 5 mg/ml with fresh DCM and extracted with 0.5 volumes of IN HCL followed by 0.5 volumes saturated NaCl. The aqueous layer was then washed with Ethyl acetate followed by DCM. The combined organic layers were dried over sodium sulfate and solvent was evaporated under vacuum to yield a viscous liquid. The viscous liquid was triturated with 2 volumes of acetonitrile (CAN) and stripped under a stream of argon to remover water and then dried under high vac and P2O5 for several hours. The resulting solid was reacted with dsRNA following the ionic solvation method. The following MdsRNAs were prepared according to this procedure:
Table 2: Modified MdsRNA Compounds Synthesized by Procedure of Example 2. aInput is moles of PEG/mole of RNA nucleotide. bModifier is the -OC(O)R1 modification as defined in Formula I. cSequence is the sequence of nucelobases of the RNA strand backbone. Example 3. Preparation of High Molecular Weight Polyalkyloxy Polymer dsRNA in DMSO.
Activation ofPolyalkyloxy Polymer PEG
[00258] The polyaloxy polymer was reacted with a linker to introduce a reactive COOH at one end of the polymer chain. One eq. of polyalkyloxy polymer was dissolved in 1,4- dioxane. DMAP (0.5 eq.), DIEA (3 eq.), and the corresponding anhydride (1.5. eq.) were added and the reaction mixture was stirred overnight at room temperature. Then the mixture was diluted with DCM and extracted with sodium bicarbonate, brine. The organic layer was dried over sodium sulfate, and concentrated at reduced pressure. The resulting gum was triturated with fresh DCM and the solid polyalkyloxy-COOH was collected via filtration.
[00259] One eq. of poyalkyloxy-COOH was dissolved in dimethyl sulfoxide (DMSO) at 50 mg/ml and to this solution was added 1.05 equivalent of carbonyldiimidazole (CDI). The reaction mixture was stirred for at least 2 hours, preferably overnight under argon. This solution was then added directly to dsRNA.
[00260] The preparation method described above was used to prepare the following activated polyalkyloxy polymers which were used to yield the desired MdsRNAs: Methoxy PEG acetic acid (MPEGA) IK, 2K, 5K, and 10K; Methoxy PEG acetic acid (MPEG A- Y) 40K, Y-shaped; Methoxy PEG succinic acid (MPEGS) 5K; Methoxy PEG glutamic acid (MPEGG) 5K; Methoxy PEG 3, 3 -methylglutamic acid (MPEGM) 5K; Carbamoyl PEG 5K (CPEG), Poloxalene succinic L64, 2,9K; Poloxalene succinic L68; Poloxalene succinic L121; Poloxalene succinic Fl 08, 14K; Poloxalene succinic Fl 27, 12.5K.
Reaction of dsRNA with activated Polyalkyloxy Polymer PEG
[00261] Benzyl-TriButyl Ammonium-Chloride was dissolved in DMSO at 200 mM. An aliquot of this solution was added to freeze-dried dsRNA in a reaction tube. The tube was warmed to 65°C in a shaker for 1 hr. Then the temperature was reduced to 55°C and catalytic amount of pyridine was added followed by a 200 pg/pL solution of DMAP. A 200 ug/uL solution of activated polyalkyloxy polymer was added in portions. The mixture was kept at 55 °C for 90 min and then was quenched with 500 mM citrate pH 4.5 and diluted to 40 mL with water. The resulting mixture containing the crude M-dsRNA was purified using tangential flow filtration (TFF), to yield MdsRNA with >90% purity. The MdsRNAs were characterized by gel electrophoresis with non-denaturing agarose gel and denaturing polyacrylamide gel.
[00262] The extent of modification (%Modification), i.e. the ratio of number of bases esterified with polymer to the total number of bases in the dsRNA was determined by a combination hydrolysis/HPLC-ELSD method. Hydrolysis of the MdsRNA samples was obtained upon heating at 99°C a mixture of an aqueous solution of the purified product and 0.5M NaOH solution. The resulting polyalkyloxy polymer and RNA nucleotides were quantified using a Shimadzu LC-2030C HPLC fitted with a Cl 8 100 A LC Column 250x4.6 mm and an ELSD. A calibration curve was built using dsRNA starting material and polyalkyloxy polymer as standards.
[00263] The insecticidal activity of the MdsRNAs was screened using a leaf disc assay. MdsRNAs targeting Diamondback moth (DBM) (P. xylostella) were tested on DBM larvae using cabbage leaf discs. The MdsRNA treatments were dissolved in water with an adjuvant and diluted to obtain the desired concentration. The desired treatment solution was sprayed on both sides of fresh cabbage leaf discs (3.5 cm diameter). Each treatment was applied with 3 repetitions and a water only control. The discs were placed on a wet paper towel in a container. P. xylostella eggs (8-12) were transferred to each treated disc and the containers were incubated at 26 °C and 72% relative humidity (RH). On days 2, 3, and 4 fresh treated discs were provided, and untreated discs were provided daily after that. On days 5 and 8 larvae mortality and stunting were recorded. The % efficacy and the std dev. were calculated for each treatment and used to rate the treatments on a scale of 1 to 3 as follows: % efficacy 0-30% Rate=l; % efficacy 30-50% Rate=2, % efficacy >50% Rate=3. Treatments with rating of 3 were advanced to further optimization studies.
[00264] MdsRNAs targeting Fall army worm (FAW) (S. frugiperda) were tested on
DBM larvae using corn leaf squares (9 cm2). One egg of FAW was placed on each corn leaf and the containers were incubated as described above. A minimum of 10 repetitions per treatment and a negative control were run. On days 4, 5, and 6 fresh treated leaf squares were provided, and untreated discs were provided daily after that. On days 8 and 11 larvae mortality and stunting were recorded. The % efficacy was calculated for each treatment and used to rate the treatments on a scale of 1 to 3 as follows: % efficacy 0-30% Rate=l; % efficacy 30-50% Rate=2, % efficacy >50% Rate=3. Treatments with rating of 3 were advanced to further optimization studies. [00265] The following MdsRNAs were prepared according to the procedures described above:
Table 3: Modified dsRNA Triggers Synthesized Using Procedure in Example 3.
^Estimated from moles of PEG/moles of RNA nucleotide in the reacting mixture. bModifier is the -OC(O)R1 modification as defined in Formula I. cSequence is the sequence of nucleobases of the RNA strand backbone.
Example 4. Preparation of High Molecular Weight Polyalkyloxy Polymer- NMIA dsRNA. [00266] MPEGA- dsAChE2 was prepared and purified according to the procedure described in Example 3 using Methoxy PEG acetic acid 10K at input 0.1. Benzyl-TriBA- C1 was dissolved in DMSO at 200 mM. An aliquot (3.4 mL) of this solution was added to freeze-dried MPEGA-dsAChE (34 mg) in a reaction tube. The tube was warmed to 65°C in a shaker for 1 hr. Then the temperature was reduced to 55°C and 110 pL pyridine are added followed by 500 pL of a 200 pg/pL solution of DMAP and 442 pL of a 200 pg/pL solution of NMIA. After 5 min, another 442 pL of NMIA solution were added (Input =10, moles of NMIA/mole of RNA nucleotide). The mixture was kept at 55°C for 90 min and then is quenched with 500 mM citrate, and diluted to 40 mL with water. The resulting mixture containing the crude PEG-NMA-dsAChE2 was purified using tangential flow filtration, TFF, to yield MdsRNA with >90% purity. The PEG-NMA-dsAChE2 was characterized by gel electrophoresis with non-denaturing agarose gel and denaturing polyacrylamide gel. %Modification by HPLC: PEG 1%, NMIA 37%.
Example 5. Preparation of High Molecular Weight Polyalkyloxy Polymer- FA dsRNAs. [00267] Following a similar procedure as described in Example 4. MPEGA-P450 5K (115) was reacted with activated lauryl (LAU), oleic (OLE) and linoleic (LIN) fatty acids to yield the MPEGA-FA-P450 materials described in Table 4.
Table 4: Modified dsRNA Triggers Synthesized Using Procedure in Example 4.
Example 6. Cabbage Disc Assay
[00268] The insecticidal activity of modified dsRNA materials were tested on DBM larvae using a cabbage leaf disc bioassay and DBM eggs collected from the field.
Cabbage leaves were collected at stage 4-5. Leaves were prepared by washing with tap water using a nozzle to remove insects, dust etc. The leaves were then wiped with paper towels. Leaf discs of 3.5 cm diameter were punched from the cabbage leaves using a metal cutter. An aqueous solution of the desired treatment solution was prepared at 500 ppm or 150 ppm concentrationand 60uL were sprayed on both sides of the disc. After the treatment was applied to both sides of the leaf disc, four 1 cm diameter discs were then punched from the treated 3.5 cm. The resulting treated 1 cm diameter discs were used in the assay. Three P. xylostella egg wassere placed on each treated disc placed in a petri dish. The plates were incubated at 26 °C and 72% room humidity (RH). After 48 hours only one neonate was transferred to a new (2nd) treated disc. A third treated disc was provided after 24 hours of the 2nd treated disc. The larvae were then provided with an untreated disc every day for up to 10 days. Mortality was recorded daily starting on the 2nd day after incubation. Mortality rate was calculated for each test during the duration of the experiment and cumulative mortality after 5 days and 7 or 9 days was calculated.
After 7 days of incubation the number of 2nd or 1st instar larva was added to the dead larva count. The efficacy of some PEG- P450 (115) examples on DBM larva is shown in FIG.
1. The efficacy of two PEG-dsRNA examples on field collected DBM eggs vs. efficacy of a leading commercial product is shown in FIG. 3 and Table 5.
Table 5: DBM Larva %Control (Calculated using Schneider-Orelli)*
DA-A: days after infestation, infestation. *Means followed by the same letter do not significantly differ (P=0.05, LSD)
Example 7. Preparation of NMA dsRNAs using ionic solvation method.
[00269] Benzyl-TriButyl Ammonium-Chloride is dissolved in DMSO at 200 mM. An aliquot of this solution is added to freeze-dried dsRNA in a reaction tube. The tube is warmed to 65°C in a shaker for 1 hr. Then the temperature is reduced to 55C and 110 uL pyridine are added followed by a 200 ug/uL solution of DMAP and two additions of a 200 ug/uL solution of N-methyl isatoic anhydride (NMIA). The mixture is kept at 55°C for 90 min and then is quenched with 500 mM citrate, and diluted with water. The resulting mixture containing the crude NMA-dsRNA is purified using tangential flow filtration, TFF, to yield MdsRNA with >90% purity. The following NMA-dsRNA were prepared according to the procedure described above: NMA-P450(l 15) (37% NMA), NMA- VPASE(7) (81% NMA), NMIA-AChE(l) (40%, 80% NMA), NMA-B1(4), NMA-TH(3). This procedure was also used to prepare dsRNA analogs for NBA, dimethyl furoyl, -Tyr, - Trp, -Leu, and octanoyl. This procedure can be used for other modifications such as, but not limited to, lauroyl, linoleyl, and the like.
Example 8. Persistence ofPEG-dsRNA in field cabbage plants
[00270] Longevity of an RNAi active ingredient in a field has always been of primary importance in development of advanced pesticides. RNA is known to be a very unstable molecule vulnerable to enzymatic degradation. Estimations of half-life for dsRNA in the field range between 0.5 and 0.7 days after foliar application on soybean plants (Bachman et al., 2020). The time decay of dsRNA sequences sprayed on cabbage leaves for native dsRNAs and 2’-0 modified dsRNAs was studied in a small plot field trial.
[00271] Cabbage leaves samples exposed to sprays were collected using a leaf puncher at these time points after spraying: 0 days (1 hr), 1, 2, 3, and 5 days. Per each data point, 12 leaf circles from 12 individual plants from each plot were collected and immediately frozen on dry ice. Then the bags should be stored in dry ice or in -80°C freezer until ready to be analyzed.
[00272] The dsRNA used in this trial is a dsSNF7 sequence that has been previously reported in field dissipation study (Bachman et al., 2020). QuantGene, a nucleic acid detection platform marketed by Invitrogen, was the preferred analytical method used to quantify of dsRNA sequences on the cabbage plant leaves after spray. The sequence and corresponding Quantigene probe sets were published previously (Armstrong et al., 2013). The sequence is not directed against Diamondback Moth (DBM) and used here only for analytical purposes. Naive (unmodified) dsSNF7, treatment C2, was used as a comparison.
[00273] Two modifying groups were used: N-methyl anthranoyl and PEGA. The dsRNAs modified with these two groups demonstrated exceptional biological activities against DBM in previous studies (see, for example, Example 3, Table 3). Treatment NS2 (PEGA-NMA-dsSNF7, MdsRNA 90, Table 3) showed a different profile from all others dsRNAs including NS5 (NMA-dsSNF7, NMA -100%), hinting at existence of a highly stabilized MdsRNA fraction which exhibited almost no decay in the tested timeframe (See FIG. 2).
SEQUENCES - dsRNA sequences for Lepidoptera.
[00274] SEQ ID NOs: 1-12 are provided above.
[00275] ID 1. Plutella xylostella V-type proton ATPase subunit E (LOC 105389010), mRNA. NCBI Reference Sequence. NM 001305532 1 . 51-150 GGCAGCAACCATG GCGC TCAGCGATGCAGA TGTCCAAAAACAGATCAAGCATATGATGGCCTTCA TCGAGCAAGAGGCAAATGAAAAGGCCGAAGAAATC (SEQ ID NO: 13).
[00276] ID 2. Plutella xylostella V-type proton ATPase subunit E (LOC 105389010), mRNA 151-250 GATGCTAAGGCTGAGGAGGAGTTCAACATCGAGAAGGGGCGTCTGG
TGCAGCAGCAGCGCCTCAAGATCATGGAGTACTACGAGAAGAAGGAGAAGCA GG (SEQ ID NO: 14).
[00277] ID 3. Plutella xylostella V-type proton ATPase subunit E (LOC 105389010), mRNA 251-350 TGGAACTCCAGAAGAAGATCCAATCCTCCAACATGCTGAACCAGGCCCGTCTG
AAGGTGCTGAAGGTGCGCGAGGACCACGTGGGCCACGTGTTGGACGA (SEQ ID NO: 15).
[00278] ID 4. Plutella xylostella V-type proton ATPase subunit E (LOC 105389010), mRNA 351-450 GACGCGCCGCCGCCTCGCCGAGGTGCCCAACGACCAGGGGCTCTACTCCGACC
TGGTGGTCAAGCTCATCGTGCAGGCGCTGTTCCAGCTGGTTGAGCCA (SEQ ID NO: 16).
[00279] ID 5. Plutella xylostella V-type proton ATPase subunit E (LOC 105389010), mRNA 451-550 ACCGTAACCCTCCGCGTGCGCGAGGCCGACAAGCCGCTGATCGACAGCCTGCT
CGAGCGCGCGCAGGCGCAGTACAAGGAGAAGATCAAGAAGGATGTGA (SEQ ID NO: 17).
[00280] ID 6. Plutella xylostella V-type proton ATPase subunit E (LOC 105389010), mRNA 551-650 CCTTGAAGGTGGACACGGAGCACTACCTGCCGGTGGGCACCTGCGGCGGGAT TGAGTTGGTCGCCGCTAGGGGCCGCATCAAGATCATCAACACCCTGGA (SEQ ID NO: 18).
[00281] ID 7. Plutella xylostella V-type proton ATPase subunit E (LOCI 05389010), mRNA 651-750
GTCGCGCATGGAGCTGATCGCGCAGCAGCTGCTGCCCGAGATCCGCACGGCG
CTGTTCGGACGGAACCCCAACCGCAAGTTCACCGACTAAACACCAACC (SEQ ID NO: 19).
[00282] ID 8. Plutella xylostella V-type proton ATPase subunit E (LOCI 05389010), mRNA 1501-1600
AGATTTGACTGGGCGCAGGGCGGTGTCACCCATACATCTTTCAGCTGAAAAAG
ACATTCCTCTTTCATGTTTCCTGTCCTGGCAATCAAATGTTTCGGCT (SEQ ID NO: 20).
[00283] ID 9. Plutella xylostella V-type proton ATPase subunit E (LOCI 05389010), mRNA 1601-1700 TGCTTTTAACAGTTCTATCGAAGAGCACCGTAGCTCTATAAATTACATAACGA
ATATAATGTTTAATCCCCATTATGGCACTGTTAAAATTCTTATGTAA (SEQ ID NO: 21).
[00284] ID 10. Plutella xylostella V-type proton ATPase subunit E (LOCI 05389010), mRNA 1401-1500 ATACTGTTTACTATCGTGGACTTCCTGGGAATTATTTGATGCTGTAAGGTTTAT
GGGTGACGGCAGCATCCCGCCTTATTCCCACTGAAATCGAAGTGAA (SEQ ID NO: 22).
[00285] ID 11. Plutella xylostella V-type proton ATPase subunit E (LOCI 05389010), mRNA 51-166
GGCAGCAACCATGGCGCTCAGCGATGCAGATGTCCAAAAACAGATCAAGCAT
ATGATGGCCTTCATCGAGCAAGAGGCAAATGAAAAGGCCGAAGAAATCGATG CTAAGGCTGAGG (SEQ ID NO: 23).
[00286] ID 12. Plutella xylostella V-type proton ATPase subunit E (LOCI 05389010), mRNA 1476-1649
TTATTCCCACTGAAATCGAAGTGAAAGATTTGACTGGGCGCAGGGCGGTGTCA
CCCATACATCTTTCAGCTGAAAAAGACATTCCTCTTTCATGTTTCCTGTCCTGG CAATCAAATGTTTCGGCTTGCTTTTAACAGTTCTATCGAAGAGCACCGTAGCTC TATAAATTACATA (SEQ ID NO: 24). [00287] ID 13. Plutella xylostella V-type proton ATPase subunit E (LOCI 05389010), mRNA sequence GGCAGCAACCATGGCGCTCAGCGATGCAGATGTCCAAAAACAGATCAAGCAT
ATGATGGCCTTCATCGAGCAAGAGGCAAATGAAAAGGCCGAAGAAATCGATG
CTAAGGCTGAGGTTATTCCCACTGAAATCGAAGTGAAAGATTTGACTGGGCGC
AGGGCGGTGTCACCCATACATCTTTCAGCTGAAAAAGACATTCCTCTTTCATG
TTTCCTGTCCTGGCAATCAAATGTTTCGGCTTGCTTTTAACAGTTCTATCGAAG AGCACCGTAGCTCTATAAATTACATA (SEQ ID NO: 25).
[00288] ID 14. XM_038113977. l|:71-220 PREDICTED: Plutella xylostella V-type proton ATPase catalytic subunit A (LOC105392322), transcript variant XI, mRNA GTGATAAGTTGTGATTCTCCGCCATTCTCGGTCTGTCTTTTCCATCTTTCTCGTC
CAAAAAACAAGGTAATTGTTCAAGATGAGTCACGGGTTGAAGAGGATTGCCG
ATGAGGACAATGAAACCCAGTTCGGTTATGTCTTCGCTGTGTC (SEQ ID NO: 26).
[00289] ID 15. NM_001305532.1|:51-200 Plutella xylostella V-type proton ATPase subunit E (LOCI 05389010), mRNA GGCAGCAACCATGGCGCTCAGCGATGCAGATGTCCAAAAACAGATCAAGCAT
ATGATGGCCTTCATCGAGCAAGAGGCAAATGAAAAGGCCGAAGAAATCGATG
CTAAGGCTGAGGAGGAGTTCAACATCGAGAAGGGGCGTCTGGTGCA (SEQ ID NO: 27).
[00290] ID 16. Plutella xylostella V-type proton ATPase Sequence Cover both submit A and E
GTGATAAGTTGTGATTCTCCGCCATTCTCGGTCTGTCTTTTCCATCTTTCTCGTC
CAAAAAACAAGGTAATTGTTCAAGATGAGTCACGGGTTGAAGAGGATTGCCG
ATGAGGACAATGAAACCCAGTTCGGTTATGTCTTCGCTGTGTCGGCAGCAACC
ATGGCGCTCAGCGATGCAGATGTCCAAAAACAGATCAAGCATATGATGGCCTT
CATCGAGCAAGAGGCAAATGAAAAGGCCGAAGAAATCGATGCTAAGGCTGAG GAGGAGTTCAACATCGAGAAGGGGCGTCTGGTGCA (SEQ ID NO: 28).
[00291] ID 17. 101-200 PREDICTED: Plutella xylostella venom carboxylesterase-6
(LOCI 05388350), mRNA
ACGGAGGGAGATCCTCCTTCTGTGACTATAGCGCAAGGGTCCGTTGTAGGTAC
TGCTGTGACCAGCTCTGGATTTACACACTATGAATTCCACGGGATAC (SEQ ID NO: 29). [00292] ID 18. 201-300 PREDICTED: Plutella xylostella venom carboxylesterase-6 (LOCI 05388350), mRNA CGTATGCTGACTCTACTTCTGGATCTCACAGGTTCAAGGCGCCACGACCAGCA
CCCACATTCACACAGACTTTTGTTGCTGATCGCAAAGGAATCAAATG (SEQ ID NO: 30).
[00293] ID 19. 301-400 PREDICTED: Plutella xylostella venom carboxylesterase-6 (LOCI 05388350), mRNA TGTGAAAGCTATAAAGGGGGGATACGAGGGCACCGAAGATTGCTTGGTGGCC
AACGTCTACACACCGGCCATTGATCCAGAAAAGAAATACCCAGTAATG (SEQ ID NO: 31).
[00294] ID 20. 401-500 PREDICTED: Plutella xylostella venom carboxylesterase-6 (LOCI 05388350), mRNA GTTTGGATTAAAGGGTCCGAGTTTGAGAAAACTAAGGGACCTGAACTATCTTT
TAGAAATCTTATTGAAAAAGAAGTAATAGTCGTGTCTCTAAACTTCA (SEQ ID NO: 32).
[00295] ID 21. 501-600 PREDICTED: Plutella xylostella venom carboxylesterase-6 (LOCI 05388350), mRNA GAGAGTCGATTCTCGGGTATCTTTGTCTTGGAACAGAAACTGCGCCTGGTAAC
GCTGGATTGAAAGATATAATTGCTGGACTTCAATGGGTGAAAGATAA (SEQ ID NO: 33).
[00296] ID 22. 601-700 PREDICTED: Plutella xylostella venom carboxylesterase-6 (LOCI 05388350), mRNA CATTGAACAGTTTGGTGGAGACCCTGAGAGTATAACCCTATTTGGGCATGGTT
CTGGAGCTGCGGCGGTAGATTTAGTCACACTCTCTCCAATGTCTAAG (SEQ ID NO: 34).
[00297] ID 23. 801-900 PREDICTED: Plutella xylostella venom carboxylesterase-6 (LOCI 05388350), mRNA TTGGGCATGAAATTACTGAGGAATTAGATATTGAAAAGCTTTCGGAAGTTTTT
ACTAGAACAAGTGTTGCCGCTCTAATGGCAGTTATAGATGAGTTGGA (SEQ ID NO: 35).
[00298] ID 24. 901-1000 PREDICTED: Plutella xylostella venom carboxylesterase-6 (LOCI 05388350), mRNA TTTAACTGATAACTCATTGGCTTTTGCTCCGTGTATCGAAAATGAGCATTTAGA TGATGAAAAGTTTCTAGAAAAATCACCTTTTAGTACGCTAACTGAA (SEQ ID NO: 36).
[00299] ID 25. 1001-1100 PREDICTED: Plutella xylostella venom carboxylesterase-6 (LOCI 05388350), mRNA GGAACTTACACTAAAATACCTATGATCTTCGGATTTGTTGAAAACGAAGGAAC
AATACGTTTTGATGAGGCACTAGAAGCTGATTGGCTAACAAAGATGG (SEQ ID NO: 37).
[00300] ID 25-2. 351-500 PREDICTED: Plutella xylostella venom carboxylesterase-6 (LOCI 05388350), mRNA CCAACGTCTACACACCGGCCATTGATCCAGAAAAGAAATACCCAGTAATGGTT
TGGATTAAAGGGTCCGAGTTTGAGAAAACTAAGGGACCTGAACTATCTTTTAG AAATCTTATTGAAAAAGAAGTAATAGTCGTGTCTCTAAACTTCA (SEQ ID NO:
38).
[00301] ID 25-3. 951-1100 PREDICTED: Plutella xylostella venom carboxylesterase-6 (LOCI 05388350), mRNA TAGATGATGAAAAGTTTCTAGAAAAATCACCTTTTAGTACGCTAACTGAAGGA
ACTTACACTAAAATACCTATGATCTTCGGATTTGTTGAAAACGAAGGAACAAT ACGTTTTGATGAGGCACTAGAAGCTGATTGGCTAACAAAGATGG (SEQ ID NO:
39).
[00302] ID 25-4. XM_011559245.2|:Plutella xylostella venom carboxylesterase-6 (LOCI 05388350), mRNA 351-500, 951-1100 CCAACGTCTACACACCGGCCATTGATCCAGAAAAGAAATACCCAGTAATGGTT
TGGATTAAAGGGTCCGAGTTTGAGAAAACTAAGGGACCTGAACTATCTTTTAG AAATCTTATTGAAAAAGAAGTAATAGTCGTGTCTCTAAACTTCA TAGATGATGAAAAGTTTCTAGAAAAATCACCTTTTAGTACGCTAACTGAAGGA ACTTACACTAAAATACCTATGATCTTCGGATTTGTTGAAAACGAAGGAACAAT ACGTTTTGATGAGGCACTAGAAGCTGATTGGCTAACAAAGATGG (SEQ ID NO:
40).
[00303] ID 26. 101-250 Plutella xylostella strain DBM1 Ac-S mitogen-activated protein kinase kinase kinase kinase 4 isoform XI (MAP4K4) mRNA, complete cds, alternatively spliced AATATAAAGTGCGTGATTTTACACATGTCGAATGTCATGAGTGAAAGGATCTT
TGTAGGTTTCTTTACATTGAAGTGAATTCTCGTGCTTTGTCTTCGTGTGTGATA ATACTCAAAATGGCGCATCAACTGGCTCCGTCTGTGAATTGCT (SEQ ID NO:
41).
[00304] ID 27. 901-1050 Plutella xylostella strain DBM1 Ac-S mitogen-activated protein kinase kinase kinase kinase 4 isoform XI (MAP4K4) mRNA, complete cds, alternatively spliced ATGGCCGAGAGTCAGCCGCCCCTGTGTGACCTTCACCCAATGAGAGCATTGTT
TCTTATACCAAGAAATCCTCCACCCCGTTTAAAGTCAAAGAAATGGGCAAAGA AATTTCATAGTTTTATTGAAACTGTTCTTGTGAAAGACTATCAC (SEQ ID NO:
42).
[00305] ID 28. 51-150 Plutella xylostella prophenoloxidase 1 mRNA, complete cds TTGCTTCATGCAGAAGGGCGACGACAAGACCGTCTTCCAGATCCCGGACAACT TCTACCCAGAAAAGTACAAGAAGGTGGGCAACCAGCTGGCCGACCGG (SEQ ID NO: 43).
[00306] ID 29. 301-400 Plutella xylostella prophenoloxidase 1 mRNA, complete cds GGAATGCGTGACGTGGAGGACCTGCAGTCCGTGTGTAGCTACTGCCAGCTCCG CATCAACCCCTACATGTTCAACTACTGCCTGTCGGTCGCCATGCTGC (SEQ ID NO: 44).
[00307] ID 30. 401-500 Plutella xylostella prophenoloxidase 1 mRNA, complete cds ACAGACCAGACACGAAGGGCCTGTCGCCGCCGACGCTGGCGGAGACGTTCCC CGACAAGTTCATGGACCCCAAGGTGTTCCGCCGCGCGCGGGAGACCTC (SEQ ID NO: 45).
[00308] ID 31. 501-600 Plutella xylostella prophenoloxidase 1 mRNA, complete cds CACCACCGCGCCTGCTGGGGACAGGATGCCAGTCCTAATCCCGGTCAACTACA CGGCCTCCGACGCTGAGCCAGAACAACGCATCGCGTACTTCCGCGAA (SEQ ID NO: 46).
[00309] ID 32. 601-700 Plutella xylostella prophenoloxidase 1 mRNA, complete cds GACATCGGCATCAACCTGCACCACTGGCACTGGCACCTGGTGTACCCCTTCGA GGCGGCCGACCGCGCCGTGGTGGACAAGGACAGGCGCGGCGAGCTGCGC (SEQ ID NO: 47).
[00310] ID 33. 601-700 Plutella xylostella prophenoloxidase 1 mRNA, complete cds TGTACTACATGCACCAGCAGATCATCGCCAGATACAACGCAGAGCGTCTGTGC AACAACCTGGGCTTCGTGACGCGCTACAACGACTTCCGCGGGCCCAT (SEQ ID NO: 48). [00311] ID 34. 801-900 Plutella xylostella prophenoloxidase 1 mRNA, complete cds CGCCGAGGGGTACTTCCCCAAGATGGACTCGCAGGTCGCCAGCAGGGCCTGG CCTCCTAGGTTCTCCGGCACCACGATCCGCGACCTGGACCGCCCCGTG (SEQ ID NO: 49).
[00312] ID 35. 901-1000 Plutella xylostella prophenol oxidase 1 mRNA, complete cds GACCAGATCCGCTCCGACGTGTCTGAGATGGAGACCTGGAGGGACCGCTTCAT CCAGGCCATCGACAGCGGCACTATTGTTCTGCCCAACGGCCGCACCC (SEQ ID NO: 50).
[00313] ID 36. 1001-1100 Plutella xylostella prophenoloxidase 1 mRNA, complete cds AGCGCCTCGACGAGGAGACCGGCATCGACGTGCTGGCCAACCTCATGGAGTC GTCCATCATCAGCCGCAACCGCGCCTACTACGGGGACCTGCACAACAT (SEQ ID NO: 51)
[00314] ID 37. 1101-1200 Plutella xylostella prophenoloxidase 1 mRNA, complete cds GGGGCATGTGTTCATCTCCTATGCGCACGACCCCGACCACCGGCACTTGGAAC AATTCGGCGTGATGGGAGACCCGGCCACGGCCATGAGGGACCCGATC (SEQ ID NO: 52).
[00315] ID 38. 1201-1300 Plutella xylostella prophenoloxidase 1 mRNA, complete cds TTCTACCGCTGGCACGCGTACGTCGACGACATCTTCCAGAGATACAAGGCCAC ACTACCAGCCTACACCAGGGAGAGGTTGGACTTCCCAGGCATCCGCG (ID SEQ ID NO: 53).
[00316] ID 40. 1301-1400 Plutella xylostella prophenoloxidase 1 mRNA, complete cds TCTCCTCCATCGCCATCTCGGGCCGCACTCCGAACCAGTTCTCGACGCAGTGG GAGCAGAGTTCAGTGAACCTGGCGCGCGGGCTGGACTTCATGCCGCG (SEQ ID NO: 54).
[00317] ID 41. 1401-1500 Plutella xylostella prophenoloxidase 1 mRNA, complete cds CGGCGCCGTGCTGGCGCGGTTCACGCATCTGCAGCATGACGAGTTTGAGTACA CCATCGAGTGCGACAACACAACCGGCCAAGCAGCCATGGGCACCGTC (SEQ ID NO: 55).
[00318] ID 42. 1501-1600 Plutella xylostella prophenol oxidase 1 mRNA, complete cds CGCATATTCCTCGCCCCGACCACCGACCAGGCCGGCAACGCACTCAACTTCGA GGAGCAGAGGCGACTCATGATCGAGCTGGACAAGTTTACTCAGGGCT (SEQ ID NO: 56)
[00319] ID 43. 1601-1700 Plutella xylostella prophenol oxidase 1 mRNA, complete cds TGCGCCCCGGCAGCAACACCATCCGGCGTCGCAGCATCGACTCCTCAGTTACC ATCCCCTACGAGCGAACATTCCGGGACGAGTCCCAACACCCCGGAGA (SEQ ID NO: 57)
[00320] ID 44. 1701-1800 Plutella xylostella prophenol oxidase 1 mRNA, complete cds CGCTGGCTCAGCTCAGTCAGCCGACTTCGACTTCTGCGGCTGCGGCTGGCCGC ACCACATGCTGATACCGAAGGGGACTCAGCAGGGATGGAACTGTGT (SEQ ID NO: 58)
[00321] ID 45. 1801-1900 Plutella xylostella prophenol oxidase 1 mRNA, complete cds CTCTTCTGCATGATTACCAACTGGAATGAAGATCGGGTGGAGCAAGACACAGT AGGAACCTGCAACGACGCAGCCTCCTACTGCGGTATCCGGGACCGCC (SEQ ID NO: 59)
[00322] ID 46. 1901-2000 Plutella xylostella prophenol oxidase 1 mRNA, complete cds GCTACCCGGACCGCAAGCCTATGGGATTCCCCTTCGATAGACCAGCGCCATCT ACCGGCAGTTTGGGAGACTTCTTGACCCCCAACATGACTGTGCAGAA (SEQ ID NO: 60)
[00323] ID 46-1. 101-200 Plutella xylostella prophenol oxidase 1 mRNA, complete cds TTACAGTGTTCGTGGTATTTTAAGCCCAAACGCTAATCCAAGATGGCGGACAA AAACAACCTGCTGCTGTTCTTCGACCGCCCCACGGAGCCCTGCTTCA (SEQ ID NO: 61)
[00324] ID 46-2. Plutella xylostella prophenol oxidase 1 mRNA, complete cds TCAGGTTTACTGATGCGGTCAGGCAGCGTCAGCAGCGGTAGAGTGTGGGAGG GAGACATGGTTCATACGTTACGAGAGTTCTAGCTAAACTTAAACACACAATGA AAGCTGACTGTAACTTTTGTAGATTTTGCCTTCAAAATGACAATTACTTAGGCA CTAATTAGGTTCTAATTTTGTATAATTCATTTTTAGCGACA (SEQ ID NO: 62) [00325] ID 46-3. Plutella xylostella prophenol oxidase 1 sequence mRNA, complete cds TTACAGTGTTCGTGGTATTTTAAGCCCAAACGCTAATCCAAGATGGCGGACAA AAACAACCTGCTGCTGTTCTTCGACCGCCCCACGGAGCCCTGCTTCATCAGGT TTACTGATGCGGTCAGGCAGCGTCAGCAGCGGTAGAGTGTGGGAGGGAGACA TGGTTCATACGTTACGAGAGTTCTAGCTAAACTTAAACACACAATGAAAGCTG ACTGTAACTTTTGTAGATTTTGCCTTCAAAATGACAATTACTTAGGCACTAATT AGGTTCTAATTTTGTATAATTCATTTTTAGCGACA (SEQ ID NO: 63)
[00326] ID 47. 151-250 Plutella xylostella glutathione synthetase (Gss), mRNA CGACCCACGGCTCCAAGTCCGTGCTGACCCCCAACCTCGACGTGCTGACCCGC TCAGGAGTGTCCCTCCACCGCTACTACACCCACGCTCTCTGCTCGCC (SEQ ID NO: 64) [00327] ID 48. 251-350 Plutella xylostella glutathione synthetase (Gss), mRNA CGCCCGTACCGCTGTGCTCACCGGCAAATACGCCCACACCGTCGGTATGCAGG GTATGCCTCTGTCCAACGCTGAGGAGCGTGGTATCCCCCTAGAGGAG (SEQ ID NO: 65)
[00328] ID 49. 351-450 Plutella xylostella glutathione synthetase (Gss), mRNA CGCCTGATCTCTCAGTACCTACAGGACGCTGGTTACAGGACCCAGATGGTCGG AAAGTGGCACGTCGGTCACGCCTTCTTCGAGCAGCTGCCCACTTACA (SEQ ID NO: 66)
[00329] ID 50. 451-550 Plutella xylostella glutathione synthetase (Gss), mRNA GAGGATTCGAGAACCACTTCGGTGTCCGCGGTGGATTCATCGACTACTACGAA TACAACGCTCAGGAGCA GCTTGACGGCAGGCCAGTCACTGGACTGTG (SEQ ID NO: 67)
[00330] ID 51. 551-650 Plutella xylostella glutathione synthetase (Gss), mRNA TCTGTTCGACGACCTGCAGCCCGACTGGACCACCGAGGGATACATCACCGACG TCTACACCGAGAAGTCCACCACCATCATTGAGAACCACAACGTCTCC (SEQ ID NO: 68)
[00331] ID 52. 651-750 Plutella xylostella glutathione synthetase (Gss), mRNA GAGCCCCTGTACCTGCTGCTGACCCACCACGCTCCCCACAACGGCAACGAAGA CGCTTCCCTGCAGGCCCCTCCTGAAGAGGTCCGCGCCCAGAGGCACG (SEQ ID NO: 69)
[00332] ID 53. 751-850 Plutella xylostella glutathione synthetase (GSS), mRNA TCGAGCTCCACCCCAGACGTATCTTCGCCGCTATGGTTAAGAAACTGGACGAC AGTATCGGAGAAATCGTCGCTACCCTCGAGAAGAAGGGCATGCTCGA (SEQ ID NO: 70)
[00333] ID 54. 851-950 Plutella xylostella glutathione synthetase (Gss), mRNA GAACACCATCATCACCTTCTCCACTGACAACGGTGCCCCCACCGTCGGTCTTG GCGCCAACTCTGGTTCCAACTACCCCCTGAGAGGAGTCAAGAAGTCC (SEQ ID NO: 71)
[00334] ID 54. 951-1050 Plutella xylostella glutathione synthetase (Gss), mRNA CCCTGGGAGGGAGGTATCCGTGGTAACGCCATGATCTGGGCCGGTCCCGAGGT CGCCCCCGGAAACGCGTGGCGTGGAAAGGTTTACGACGGCAACATGC (SEQ ID NO: 72)
[00335] ID 57. 1051-1150 Plutella xylostella glutathione synthetase (Gss), mRNA ACGCCGCTGACTGGGTCCCCACTCTGCTTGAGGCCATCGGTGAGAAGATCCCC GCCGGTCTGGACGGTATCCCCATGTGGAGCCACATCATCGAGAACAA (SEQ ID NO: 73)
[00336] ID 58. 1151-1250 Plutella xylostella glutathione synthetase (Gss), mRNA GCCTTCTCCCCGTACCGAGATCTTCGAGATCGACGACTACTTCAACCACTCCTC TGTCACCCTCGGCCGCCACAAGCTCGTCAAGGGAACCATCGACGAG (SEQ ID NO: 74)
[00337] ID 59. 1251-1350 Plutella xylostella glutathione synthetase (Gss), mRNA TCTCTCAGCAAGCACTACGGTGAAGACCTCCGTGGCATCATCGGAACTCCCCC AGACTACAAGCAGAAGCTGCGCGACAGCAAGGCATGGGAGTCTCTGG (SEQ ID NO: 75)
[00338] ID 60. 1351-1450 Plutella xylostella glutathione synthetase (Gss), mRNA AGACCATCGGCATCCCTCTGGACGCTGACGTCATGGCTGACCGCGATGAGGCT ATCGTCACTTGCGGAAATGTCGTCCCCAAGCCTTGCAGTCCTTCTGC (SEQ ID NO: 76)
[00339] ID 61. 1451-1550 Plutella xylostella glutathione synthetase (Gss), mRNA CGAGTCTTGGTGCCTGTACGACATCATCGAGGACCCTTGTGAGCTTCGTGACC TGTCTGAGGAGCTTCCTCAGCTGGCTCAGATCCTTCTGTACCGTCTG (SEQ ID NO: 77)
[00340] ID 62. 1551-1650 Plutella xylostella glutathione synthetase (Gss), mRNA GAGCAGGAAGAGGCCAAGATCATCCCCAGGGAGGGCCAGTACGTCGCTGACC CCAAGTCTGCCCCCAAGTACTTCAACTACACCTGGGACGCGTACCTGT (SEQ ID NO: 78)
[00341] ID 63. 1651-1750 Plutella xylostella glutathione synthetase (Gss), mRNA CCGTCGAACCCTACTCCGACTCCGAATAGACGAAGCTCAGCTCAAGCGGCGCA GTTCGCCGTGAAAGTTGTAAATGTTGATCCTGGCCTTAATTTCAGTA (SEQ ID NO: 79)
[00342] ID 63-2. Plutella xylostella glutathione synthetase (Gss), mRNA
GAGGATTCGAGAACCACTTCGGTGTCCGCGGTGGATTCATCGACTACTACGAA TACAACGCTCAGGAGCAGCTTGACGGCAGGCCAGTCACTGGACTGTGTCGAG CTCCACCCCAGACGTATCTTCGCCGCTATGGTTAAGAAACTGGACGACAGTAT CGGAGAAATCGTCGCTACCCTCGAGAAGAAGGGCATGCTCGACCGTCGAACC CTACTCCGACTCCGAATAGACGAAGCTCAGCTCAAGCGGCGCAGTTCGCCGTG
AAAGTTGTAAATGTTGATCCTGGCCTTAATTTCAGTA (SEQ ID NO: 80) [00343] ID 64. AY904342.1: 160-460 Plutella xylostella pheromone biosynthesis activating neuropeptide (PBAN) mRNA, complete cds
ATCCAGCGGGACGCCCGCGACCGCGCCTCAATGTGGTTCGGGCCGCGCCTCGG
GAAGCGAGCCATGCACCTCGCGCCCGACGGTGACGGACAAGCAGTATACAGG
ATGCTCGAAGCTGCAGACGCGCTCAAGTACTACTACGACCAGCTCCAGTATTA
TGGGGCTCAGGCAGACGATCCTGAGACTAAAGTGACAAAGAAGGTGATCTTC
ACCCCCAAGTTGGGCCGCAACGCTGATGAAGACCAGCAGCAGTCGGTGGACT
TCACGCCGAGACTAGGGC
GCCGCCGGCTCAAGGACTCGG (SEQ ID NO: 81)
[00344] ID 65. 151-250 PREDICTED: Plutella xylostella charged multivesicular body protein 4b (LOC105396929), mRNA
ACAACCATGAGTTTTCTGGGGAAAATATTCGGTGGTAAGAAGGAGGAGAAAG
GTCCATCAACACACGAAGCTATTCAAAAATTACGCGAGACCGAAGAAT (SEQ ID NO: 82)
[00345] ID 66. 251-350 PREDICTED: Plutella xylostella charged multivesicular body protein 4b (LOC105396929), mRNA
TGCTCATCAAAAAGCAGGACTTCCTGGAGAAGAAAATACAATTAGAAGTAGA
CACAGCCAGGAAACATGGCACTAAGAACAAAAGAGCGGCCATCGCTGC (SEQ ID NO: 83)
[00346] ID 67. 351-450 PREDICTED: Plutella xylostella charged multivesicular body protein 4b (LOC105396929), mRNA
ACTTAAACGCAAGAAGCGTTACGAGAAGCAGCTCACACAGATCGACGGCACG
CTCAGCCAGATAGAGATGCAGAGAGAAGCATTGGAGGGAGCCAACACT (SEQ ID NO: 84)
[00347] ID 68. 451-550 PREDICTED: Plutella xylostella charged multivesicular body protein 4b (LOC105396929), mRNA
AACACTCAAGTACTGAACACGATGCGAGAGGCCGCCGCGGCTATGAAGCTCG
CTCACAAGGATATTGACGTAGACAAAGTGCACGATATCATGGACGACA (SEQ ID NO: 85)
[00348] ID 69. 551-650 PREDICTED: Plutella xylostella charged multivesicular body protein 4b (LOC105396929), mRNA
TCGCTGAACAACATGATGTGGCTCGCGAGATCACGGATGCAATCAGCAACAA
TGTGGCCTTCCCGAGTGACATTGATGATGAGGAGCTGGAGAGAGAGTT (SEQ ID NO: 86) [00349] ID 70. 651-751 PREDICTED: Plutella xylostella charged multivesicular body protein 4b (LOC105396929), mRNA
GGATGAACTGGAACAGGAGGACCTGGACAAGGAGATGCTGGGCATCAACGTG
CCCACGGACCAGCTGCCCGACGTGCCGTCCGCCGAGCCCGCCGCGCCGC (SEQ ID NO: 87)
[00350] ID 71. 751-850 PREDICTED: Plutella xylostella charged multivesicular body protein 4b (LOC105396929), mRNA
CGCCCCGCCAAGGCCAAGCCTGCTAGCGAAGACGACGATGATCTGGCTAAAC
TGCAATCGTGGGCGACATAAATGTAAGTGTTGGAAGCGAAACCGAATA (SEQ ID NO: 88)
[00351] ID 72. 851-950 PREDICTED: Plutella xylostella charged multivesicular body protein 4b (LOC105396929), mRNA
CATATGTATAGTCTCCGTTACACATCCAACTACTTAGCTTATTCTAAGGCTGCG
TCCCTGTAGGCAAACAGTATTCTCGTTACTAGTCTATGGTAATTTG (SEQ ID
NO: 89)
[00352] ID 73. 951-1050 PREDICTED: Plutella xylostella charged multivesicular body protein 4b (LOC105396929), mRNA AGTAGAGTGAAAGAACAACTAGCTAATAAAAATAAGGAAGGCCCAAGACAG
AGTGTTGTGGCGCTCCTTGGGATTGGTCTATGTCCAACATTGGACGATA (SEQ ID NO: 90)
[00353] ID 74. 1051-1150 PREDICTED: Plutella xylostella charged multivesicular body protein 4b (LOC105396929), mRNA
ACAGCTTTATGACTTTAATTTATGAGGTACTGGCTATGGCAGATCTATTTACGG
GCGCATCGCTTTGCGCTTTTAGGAGTTTCCCTCGGGAATTCCGGGA (SEQ ID NO: 91)
[00354] ID 75. 201-350 PREDICTED: Plutella xylostella charged multivesicular body protein 4b (LOC105396929), mRNA AGGTCCATCAACACACGAAGCTATTCAAAAATTACGCGAGACCGAAGAATTG
CTCATCAAAAAGCAGGACTTCCTGGAGAAGAAAATACAATTAGAAGTAGACA
CAGCCAGGAAACATGGCACTAAGAACAAAAGAGCGGCCATCGCTGC (SEQ ID NO: 92)
[00355] ID 76. 826-975 PREDICTED: Plutella xylostella charged multivesicular body protein 4b (LOC105396929), mRNA TAAGTGTTGGAAGCGAAACCGAATACATATGTATAGTCTCCGTTACACATCCA ACTACTTAGCTTATTCTAAGGCTGCGTCCCTGTAGGCAAACAGTATTCTCGTTA CTAGTCTATGGTAATTTGAGTAGAGTGAAAGAACAACTAGCTA (SEQ ID NO:
93)
[00356] ID 77. 826-975 PREDICTED: Plutella xylostella charged multivesicular body protein 4b (LOC105396929), mRNA TAAGTGTTGGAAGCGAAACCGAATACATATGTATAGTCTCCGTTACACATCCA ACTACTTAGCTTATTCTAAGGCTGCGTCCCTGTAGGCAAACAGTATTCTCGTTA CTAGTCTATGGTAATTTGAGTAGAGTGAAAGAACAACTAGCTA (SEQ ID NO:
94)
[00357] ID 78. 201-350, 826-975 PREDICTED: Plutella xylostella charged multivesicular body protein 4b (LOC105396929), purposed sequence mRNA AGGTCCATCAACACACGAAGCTATTCAAAAATTACGCGAGACCGAAGAATTG CTCATCAAAAAGCAGGACTTCCTGGAGAAGAAAATACAATTAGAAGTAGACA CAGCCAGGAAACATGGCACTAAGAACAAAAGAGCGGCCATCGCTGCTAAGTG TTGGAAGCGAAACCGAATACATATGTATAGTCTCCGTTACACATCCAACTACT TAGCTTATTCTAAGGCTGCGTCCCTGTAGGCAAACAGTATTCTCGTTACTAGTC TATGGTAATTTGAGTAGAGTGAAAGAACAACTAGCTA (SEQ ID NO: 95)
[00358] ID 79. XM 038113977.1|: 101-200 PREDICTED: Plutella xylostella V-type proton ATPase catalytic subunit A (LOC105392322), transcript variant XI, mRNA GTCTGTCTTTTCCATCTTTCTCGTCCAAAAAACAAGGTAATTGTTCAAGATGAG TCACGGGTTGAAGAGGATTGCCGATGAGGACAATGAAACCCAGTTC (SEQ ID NO: 96)
[00359] ID 80. 201-300 PREDICTED: Plutella xylostella V-type proton ATPase catalytic subunit A (LOC105392322), transcript variant XI, mRNA GGTTATGTCTTCGCTGTGTCCGGTCCCGTGGTCACAGCGGAGAAGATGTCCGG CTCGGCCATGTACGAGCTGGTGCGCGTCGGCTACAACGAGCTGGTCG (SEQ ID NO: 97)
[00360] ID 81. XM 038113977.1|:301-400 PREDICTED: Plutella xylostella V-type proton ATPase catalytic subunit A (LOC105392322), transcript variant XI, mRNA GAGAGATCATCCGTCTGGAGGGGGACATGGCCACCATCCAGGTGTACGAAGA GACCTCAGGCGTAACCGTCGGCGACCCCGTGCTCCGCACCGGCAAGCC (SEQ ID NO: 98)
[00361] ID 82. XM 038113977.1|:401-500 PREDICTED: Plutella xylostella V-type proton ATPase catalytic subunit A (LOC105392322), transcript variant XI, mRNA TCTCTCCGTAGAACTGGGACCCGGCATCCTCGGCTCCATCTTCGACGGCATCC AGCGCCGCTGAAGGACATCAACGAGCTCACGCAGAGCATCTACATC (SEQ ID NO: 99)
[00362] ID 83. XM_038113977. l|:501-600 PREDICTED: Plutella xylostella V-type proton ATPase catalytic subunit A (LOC105392322), transcript variant XI, mRNA CCCAAGGGGGTGAACGTGCCCGCGCTGGCCCGCGACACCGAGTGGGAGTTCC ACCGCAGTACATCAAGGTCGGCACCCACATCACCGGCGGGGACTTAT (SEQ ID NO: 100)
[00363] ID 84. XM 038113977.1|:601-700 PREDICTED: Plutella xylostella V-type proton ATPase catalytic subunit A (LOC105392322), transcript variant XI, mRNA ACGGGATCGTGCACGAAAACACGCTGGTGAAGCACCGCATGCTGGTGCCGCC CAAGGCCAAGGGCACCGTCACCTACATCGCGCCCGAGGGGAACTACAA (SEQ ID NO: 101)
[00364] ID 85. XM_038113977.1|:1501-1600 PREDICTED: Plutella xylostella V-type proton ATPase catalytic subunit A (LOC105392322), transcript variant XI, mRNA CTTACAGTAAGTACATGCGCGCCCTGGACGACTTCTACGACAAGAACTACCCC GAGTTCGTGCCGCTCAGGACCAAGGTCAAGGAGATCCTGCAAGAGGA (SEQ ID NO: 102)
[00365] ID 86. XM_038113977.1|:1601-1700 PREDICTED: Plutella xylostella V-type proton ATPase catalytic subunit A (LOC105392322), transcript variant XI, mRNA GGAGGACCTGTCTGAAATCGTGCAGCTGGTCGGAAAGGCGTCCCTCGCCGAG ACCGACAAGATCACCCTCGAGGTGGCCAAGCTGCTGAAGGACGACTTC (SEQ ID NO: 103)
[00366] ID 87. XM_038113977.1|:1701-1800 PREDICTED: Plutella xylostella V-type proton ATPase catalytic subunit A (LOC105392322), transcript variant XI, mRNA TTGCAGCAGAACAGCTACTCGAACTACGACCGTTTCTGCCCGTTCTACAAGAC GACCGGCATGCTGAAGAACATCATCACGTTCTACGACATGTCGCGAC (SEQ ID NO: 104)
[00367] ID 88. XM_038113977.1|:1801-1900 PREDICTED: Plutella xylostella V-type proton ATPase catalytic subunit A (LOC105392322), transcript variant XI, mRNA ACGCTGTCGAGTCCACCGCGCAGTCCGACAACAAGGTGACGTGGAACACGAT CCGTGACGCCATGGGCCCCGTGCTCTACCAGCTGTCCAGCATGAAGTT (SEQ ID NO: 105) [00368] ID 89. XM_038113977.1|:1901-2000 PREDICTED: Plutella xylostella V-type proton ATPase catalytic subunit A (LOC105392322), transcript variant XI, mRNA CAAGGACCCCGTGAAAGATGGAGAAGCCAAGATCAAGGCTGACTTCGACCAG ATCGTCGAGGACATGGCCGCTGCCTTCCGTAACCTAGAGGACTAAGTT (SEQ ID NO: 106)
[00369] ID 90. XM 038113977.1|:2001-2100 PREDICTED: Plutella xylostella V-type proton ATPase catalytic subunit A (LOC105392322), transcript variant XI, mRNA ATCATGCGCAATTATACTCTTTATTCTTGAAGAGGATGTTTGGGTCGGACCTCT TGCCGCGCGGTGAAAAAAATAAAACTGTCAACTTAACCATAGAGCT (SEQ ID NO: 107)
[00370] ID 91. XM 038113977.1|:2101-2200 PREDICTED: Plutella xylostella V-type proton ATPase catalytic subunit A (LOC105392322), transcript variant XI, mRNA GACAGTGTCTATGGTTAGAAATAGCGCGAAAAGATACTGGCTACTGCTAAGAT AGGTTGATGTTTGAGGTCCGATCCAACATTGTTTTGTTAATAAATAT (SEQ ID NO: 108)
[00371] ID 92. XM_038113977.1|:2301-2400 PREDICTED: Plutella xylostella V-type proton ATPase catalytic subunit A (LOC105392322), transcript variant XI, mRNA AAAGTCGTGTTTCAAATCTTGTTGAATTTCATCTTAGACGTTGAATATAAGCCG TTGACGGTGTCAATGTATTTTTTATGTAACACGCGGTCACTAGATA (SEQ ID NO: 109)
[00372] ID 93. XM_038113977.11 :2401-2500 PREDICTED: Plutella xylostella V-type proton ATPase catalytic subunit A (LOC105392322), transcript variant XI, mRNA CTTTCTCGACAATATGATATATACATTTTGTAAATAAAGCCCCCTTTCCATTCA AGATTGTGTAATGTTATATAGAGGTACATGGTGCATCGGTCTAGTG (SEQ ID NO: 110)
[00373] ID 94. XM_038113977.11 :2601-2700 PREDICTED: Plutella xylostella V-type proton ATPase catalytic subunit A (LOC105392322), transcript variant XI, mRNA GCCCAATGATAATTGAATGATTGTTTTCGTCGGTTCATATACCCTGTTGCATTC CAGCTTATTTTTAGATAGTTAAAACAACAAATCGGTTATTTTTACT (SEQ ID NO: 111)
[00374] ID 95. XM_038113977.11 :71-220 PREDICTED: Plutella xylostella V-type proton ATPase catalytic subunit A (LOC105392322), transcript variant XI, mRNA GTGATAAGTTGTGATTCTCCGCCATTCTCGGTCTGTCTTTTCCATCTTTCTCGTC CAAAAAACAAGGTAATTGTTCAAGATGAGTCACGGGTTGAAGAGGATTGCCG ATGAGGACAATGAAACCCAGTTCGGTTATGTCTTCGCTGTGTC (SEQ ID NO:
112)
[00375] ID 96. XM_038113977.1|:1971-2120 PREDICTED: Plutella xylostella V-type proton ATPase catalytic subunit A (LOC105392322), transcript variant XI, mRNA
GCTGCCTTCCGTAACCTAGAGGACTAAGTTATCATGCGCAATTATACTCTTTAT TCTTGAAGAGGATGTTTGGGTCGGACCTCTTGCCGCGCGGTGAAAAAAATAAA ACTGTCAACTTAACCATAGAGCTGACAGTGTCTATGGTTAGAA (SEQ ID NO:
113)
[00376] ID 97. 300 BP Plutella xylostella V-type proton ATPase catalytic subunit A (LOC105392322), transcript variant XI, sequence mRNA
GTGATAAGTTGTGATTCTCCGCCATTCTCGGTCTGTCTTTTCCATCTTTCTCGTC CAAAAAACAAGGTAATTGTTCAAGATGAGTCACGGGTTGAAGAGGATTGCCG ATGAGGACAATGAAACCCAGTTCGGTTATGTCTTCGCTGTGTCGCTGCCTTCC GTAACCTAGAGGACTAAGTTATCATGCGCAATTATACTCTTTATTCTTGAAGA GGATGTTTGGGTCGGACCTCTTGCCGCGCGGTGAAAAAAATAAAACTGTCAAC TTAACCATAGAGCTGACAGTGTCTATGGTTAGAA (SEQ ID NO: 114)
[00377] ID 98. AY971374.1|:676-975 Plutella xylostella cytochrome P450 (CYP6BFlvl) mRNA, complete cds
ACCAATTGCAGGCGTGAAGAATGTCCTCAGATACGGCTACCCTTCCTTCTTCT ACAGCGTGGGATTGGAGCTCTATTCCAGCAAAATTTACCGTTTCTTCCGATCTG TTATACTTGACGTTATAAACAGTCGTAACGGCGCCAAATCTTCGAGGAATGAC ATGGTGGATCTTATTTCCGATTGGAAGAAGAACAAATACATAACGGGAGACA
GTATTGATAATGGCATAGACGGTGGAAACAAGAAGGTGCGTATCGAAGTCGA CGACGAACTTTTGGTGAGCCAATGTGTGCTGTTCTT (SEQ ID NO: 115) [00378] ID 99. 101-200 Plutella xylostella cytochrome P450 (CYP6BFlvl) mRNA, complete cds
AACTACTGGAAGAAACAGAACGTCAAGTACTTGACGCCGATCCCTTTCCTGGG GAACGTGGCTGATGTGATCTTCCAGAGGGACACCTTCGGAGCCGTGA (SEQ ID NO: 116)
[00379] ID 100. 201-300 Plutella xylostella cytochrome P450 (CYP6BFlvl) mRNA, complete cds
CGCAACGGATCTGCCAGCAGTTCCCCGATGAAGCTGTGGTCGGCATGTTCTAC TGCAGCAACCCTGCAGCCCTCGTACAGTGCCCTGACATGCTCAAGAC (SEQ ID NO: 117) [00380] ID 101. 301-400 Plutella xylostella cytochrome P450 (CYP6BFlvl) mRNA, complete cds AGTCATGGTCAAGGACTACGCCTACTGCTCCAGTAAGGAGGTCTCCGTCCACA
GCCACAAGGAACCCATGACCAAGAACATGTTCTTCACCTTCGGAGAC (SEQ ID NO: 118)
[00381] ID 102. AY971374.1|:401-500 Plutella xylostella cytochrome P450 (CYP6BFlvl) mRNA, complete cds
AAGTGGAAGCTCATCCGGCAGAACCTCACGCCGGTCTACACGTCCGCCAAAAT
GAAGAACATGTTTCCACTGGTACAGGATTGCTGCAGAATATTCCAGA (SEQ ID NO: 119)
[00382] ID 103. AY971374.1|:501-600 Plutella xylostella cytochrome P450 (CYP6BFlvl) mRNA, complete cds AGGTTCTCGATGATGAGATAGGAAAGGGCCGGGTGGTGGAAGTGAAGTCTTT
GATAGCTCGGTATACTATGGACTGTATAACTTCGTGTGCATTCGGCGT (SEQ ID NO: 120)
[00383] ID 104. AY971374.1|:601-700 Plutella xylostella cytochrome P450 (CYP6BFlvl) mRNA, complete cds CGACTCTGGCACGATGTCGAAGGGCGAGGAAGGGAACCCTTTCACAGAAACA
GGTCACCTTTTATTTGATGAAAGACCAATTGCAGGCGTGAAGAATGTC (SEQ ID NO: 121)
[00384] ID 105. 701-800 Plutella xylostella cytochrome P450 (CYP6BFlvl) mRNA, complete cds
CTCAGATACGGCTACCCTTCCTTCTTCTACAGCGTGGGATTGGAGCTCTATTCC
AGCAAAATTTACCGTTTCTTCCGATCTGTTATACTTGACGTTATAA (SEQ ID
NO: 122)
[00385] IDs 106. 801-900 Plutella xylostella cytochrome P450 (CYP6BFlvl) mRNA, complete cds ACAGTCGTAACGGCGCCAAATCTTCGAGGAATGACATGGTGGATCTTATTTCC
GATTGGAAGAAGAACAAATACATAACGGGAGACAGTATTGATAATGG (SEQ
ID NO: 123)
[00386] ID 107. 1201-1300 Plutella xylostella cytochrome P450 (CYP6BFlvl) mRNA, complete cds
CAAAGACTACACGCTACCGAATGGTGTGCATCTAAAGAAGGGGATGATGATA CATATTCCTGTTTATCATTTGCATCACAATCCGAAGTATTTCCCGGAG (SEQ ID NO: 124)
[00387] ID 108. 1301-1400 Plutella xylostella cytochrome P450 (CYP6BFlvl) mRNA, complete cds
CCCGAGGTGTTTCGTCCGGAGCGGTTTTCTGAAGAAGGACGGAAAAGTATTGT
CCCGTATACCTACTTGCCCTTTGGGGACGGGCCGAGGATGTGTATAG (SEQ ID NO: 125)
[00388] ID 109. AY971374.1|:1401-1500 Plutella xylostella cytochrome P450 (CYP6BFlvl) mRNA, complete cds
GCTACCGTTTCGCAAGGCTAGAGATCTTCTCCAGCCTAGCAGTTCTGTTGAAG AAATACCGAGTGGAGCTGGCCCCCCACATGCCGAGGAAGCTGCAGTT (SEQ ID NO: 126)
[00389] ID 109-2 AY971374.1|:700-849 Plutella xylostella cytochrome P450 (CYP6BFlvl) mRNA, complete cds CCTCAGATACGGCTACCCTTCCTTCTTCTACAGCGTGGGATTGGAGCTCTATTC
CAGCAAAATTTACCGTTTCTTCCGATCTGTTATACTTGACGTTATAAACAGTCG
TAACGGCGCCAAATCTTCGAGGAATGACATGGTGGATCTTAT (SEQ ID NO:
127)
[00390] ID 109-3 AY971374.1|:1151-1300 Plutella xylostella cytochrome P450 (CYP6BFlvl) mRNA, complete cds GAATCCCTCCGCATGTATCCTCCAGTCTCGGTGCTCATGAGAGAGATTTACAA
AGACTACACGCTACCGAATGGTGTGCATCTAAAGAAGGGGATGATGATACAT ATTCCTGTTTATCATTTGCATCACAATCCGAAGTATTTCCCGGAG (SEQ ID NO:
128)
[00391] ID 109-3. AY971374.1|:1151-1300 Plutella xylostella cytochrome P450 (CYP6BFlvl) purposed sequence mRNA, complete cds CTCAGATACGGCTACCCTTCCTTCTTCTACAGCGTGGGATTGGAGCTCTATTCC
AGCAAAATTTACCGTTTCTTCCGATCTGTTATACTTGACGTTATAAACAGTCGT
AACGGCGCCAAATCTTCGAGGAATGACATGGTGGATCTTATTGAATCCCTCCG
CATGTATCCTCCAGTCTCGGTGCTCATGAGAGAGATTTACAAAGACTACACGC
TACCGAATGGTGTGCATCTAAAGAAGGGGATGATGATACATATTCCTGTTTAT
CATTTGCATCACAATCCGAAGTATTTCCCGGAG (SEQ ID NO: 129) [00392] ID 109.4. AY971374.1|:650-707 Plutella xylostella cytochrome P450 (CYP6BFlvl) mRNA, complete cds ACAGGTCACCTTTTATTTGATGAAAGACCAATTGCAGGCGTGAAGAATGTCCT
CAGAT (SEQ ID NO: 130)
[00393] ID 109.5. AY971374.1|:765-822 Plutella xylostella cytochrome P450
(CYP6BFlvl) mRNA, complete cds
ACCGTTTCTTCCGATCTGTTATACTTGACGTTATAAACAGTCGTAACGGCGCCA
AATC (SEQ ID NO: 131)
[00394] ID 109.6 AY971374.1|: 823-880 Plutella xylostella cytochrome P450
(CYP6BFlvl) mRNA, complete cds
TTCGAGGAATGACATGGTGGATCTTATTTCCGATTGGAAGAAGAACAAATACA
TAACG (SEQ ID NO: 132)
[00395] ID 109.7 AY971374.1|: 1159-1216 Plutella xylostella cytochrome P450
(CYP6BFlvl) mRNA, complete cds
CCGCATGTATCCTCCAGTCTCGGTGCTCATGAGAGAGATTTACAAAGACTACA
CGCTA (SEQ ID NO: 133)
[00396] ID 109.8 AY971374.1|:1217-1274 Plutella xylostella cytochrome P450
(CYP6BFlvl) mRNA, complete cds
CCGAATGGTGTGCATCTAAAGAAGGGGATGATGATACATATTCCTGTTTATCA
TTTGC (SEQ ID NO: 134)
[00397] ID 109.9 AY971374.1|: 881-938 Plutella xylostella cytochrome P450
(CYP6BFlvl) mRNA, complete cds
GGAGACAGTATTGATAATGGCATAGACGGTGGAAACAAGAAGGTGCGTATCG
AAGTCG (SEQ ID NO: 135)
[00398] ID 109-10 939-996 Plutella xylostella cytochrome P450 (CYP6BFlvl) mRNA, complete cds
ACGACGAACTTTTGGTGAGCCAATGTGTGCTGTTCTTCCAAGCTGGCTTCCAG
CCAAG (SEQ ID NO: 136)
[00399] ID 109-11 1101-1158 Plutella xylostella cytochrome P450 (CYP6BFlvl) mRNA, complete cds
TGCAGACCGACTGCGTGACCGCCCTGCCTTTCCTCGCCCAGTGCATGGAGGAA
TCCCT (SEQ ID NO: 137)
[00400] ID 109-11 1159-1216 Plutella xylostella cytochrome P450 (CYP6BFlvl) mRNA, complete cds
CCGCATGTATCCTCCAGTCTCGGTGCTCATGAGAGAGATTTACAAAGACTACA
CGCTA (SEQ ID NO: 138) [00401] ID 109-12 1217-1274 Plutella xylostella cytochrome P450 (CYP6BFlvl) mRNA, complete cds
CCGAATGGTGTGCATCTAAAGAAGGGGATGATGATACATATTCCTGTTTATCA
TTTGC (SEQ ID NO: 139)
[00402] ID 109-13 1275-1333 Plutella xylostella cytochrome P450 (CYP6BFlvl) mRNA, complete cds
ATCACAATCCGAAGTATTTCCCGGAGCCCGAGGTGTTTCGTCCGGAGCGGTTT
TCTGAA (SEQ ID NO: 140)
[00403] ID 109-14 1334-1390 Plutella xylostella cytochrome P450 (CYP6BFlvl) mRNA, complete cds
GAAGGACGGAAAAGTATTGTCCCGTATACCTACTTGCCCTTTGGGGACGGGCC
GAGG (SEQ ID NO: 141)
[00404] ID Pl. 101-250 Plutella xylostella cytochrome P450 6kl-like
(LOC105392167), mRNA
CAGTTTATACTATCGAAATGATTTTAGCAATAATATTGTTATTAGTGATTATTA
TAATAACATTTTCGTTTCTACTGGGCTCCTACAATGAGTCTTACTGGGCAAAAC
GAAACGTTAAATACCATGGCGGGAAAAATGCCATAGCAACAT (SEQ ID NO:
142)
[00405] ID P2. 251-400 Plutella xylostella cytochrome P450 6kl-like
(LOC105392167), mRNA
TCTCAGAGTTTTTGTTCACTAGCCGTGGCATTTTCGATATATTCGGTAACATTT
ACAAGCTGTACCCTGAAGAACCTGCAGTGGCCACGCCGTCGCTGCTTCAACCG
GCTTTATTCGTCAAACATCCAGAAAACATACAACATGTGCTGA (SEQ ID NO:
143)
[00406] ID P3. 401-550 Plutella xylostella cytochrome P450 6kl-like
(LOC105392167), mRNA
CAGACAATTTTAAAAACTTTTACCACAGAGGTGTTGAAATCGCTAAGAAAGAT
AAACTAGCACAAAATGT (SEQ ID NO: 144)
[00407] Plutella xylostella, mRNA
ACCTTTCCTGAACGGCAGTCGGTGGAAACTTATGAGACAAAAAATGACGCCG
CTGTCACTAGTGCGAAGCTGAAGAACA (SEQ ID NO: 145)
[00408] ID P4. 551-700 Plutella xylostella cytochrome P450 6kl-like
(LOC105392167), mRNA
TGCACTACATCATAGACAGATGTGCCCAAGACTACATCGGTTATCTGAAAGAA CATGTCAACGATAAAAATGCTAATGCATTCGAAACATTATCTGTGTACAGCTG CTCATCGCTACTAGCCCCGATATTCGGAATCCACAGCGGACAGT (SEQ ID NO:
146)
[00409] ID P5. 701-850 Plutella xylostella cytochrome P450 6kl-like (LOC105392167), mRNA
CAACGGTAACTTCACCGCTTCTAAATATGGCGAGAAATGCCACGAAACCGACT TTGAAAGCAAATTTAAAATTCATTTTGAACTCTTTGTCGCCGAAAGTCTTCCAA ATGCTGGGACTTAGTTTCTTTGGCGAATATGAGGAACAGTTCA (SEQ ID NO:
147)
[00410] ID P6. 851-1000 Plutella xylostella cytochrome P450 6kl-like (LOC105392167), mRNA
TCGGCGCGATCAGTCAAGTGATAAGACAACGTAAGGAAGAGAATGTGAAAAA GCACGATTTTGCTGACATTGCTGTGAGTTTGCAGAATGCTGGTACGATGAAAG ACGAATCCAGCGGCTGTGAAATAGAGCCTACGGATGAAGTTCTAG (SEQ ID NO: 148)
[00411] ID P7. 751-1050 Plutella xylostella cytochrome P450 6kl-like (LOC105392167), mRNA
ACTTTGAAAGCAAATTTAAAATTCATTTTGAACTCTTTGTCGCCGAAAGTCTTC CAAATGCTGGGACTTAGTTTCTTTGGCGAATATGAGGAACAGTTCATCGGCGC GATCAGTCAAGTGATAAGACAACGTAAGGAAGAGAATGTGAAAAAGCACGAT TTTGCTGACATTGCTGTGAGTTTGCAGAATGCTGGTACGATGAAAGACGAATC
CAGCGGCTGTGAAATAGAGCCTACGGATGAAGTTCTAGCTGCACAAGCGTTCT TCTTCCTTATAGCAGGAGTAGATCCAGTAACAATG (SEQ ID NO: 149)
[00412] ID P8. 450-751 Plutella xylostella cytochrome P450 6kl-like (LOC105392167), mRNA
AGATAAACTAGCACAAAATGTACCTTTCCTGAACGGCAGTCGGTGGAAACTTA TGAGACAAAAAATGACGCCGCTGTTCACTAGTGCGAAGCTGAAGAACATGCA CTACATCATAGACAGATGTGCCCAAGACTACATCGGTTATCTGAAAGAACATG TCAACGATAAAAATGCTAATGCATTCGAAACATTATCTGTGTACAGCTGCTCA
TCGCTACTAGCCCCGATATTCGGAATCCACAGCGGACAGTCAACGGTAACTTC ACCGCTTCTAAATATGGCGAGAAATGCCACGAAACCGA (SEQ ID NO: 150) [00413] ID 110. KC789751.1|:72-1432 Spodoptera frugiperda cytochrome P450 CY321A8 mRNA, complete cds
TGAGAGCTACTGGAAGAAGCGTGGCGTAAAGTTCTACAGTAAAAACAAAGTG ATTGACCTTACTGGGATTATTTCATAACACAGCGTGCGTTGTTCGAAGTCTTAA CAGACCTGTACAAAAAATATAGACATGAACCAGCTATTGGTATTGGTCAAGTC CTAACACCTGCGCTCTTTGTCATCGATCCGAAGAATGTTCAGCAAGTTTTATCA AGTGACTTCCAATCTTTTAACCACAGAGGCATTGAAAGTATTGAAGGGGATCA ACTGACCGATAATATTCTCATGATGAATGGTCCAAGATGGAAGCTGATGCGAC AGAATATGACTCCGTTGTTTACGGCAAACAAATTGAAGAACATGTATTACATC ATGGACAAAAGTGCTCAAGACTTTGCGAACTATTTGAAAAACAACCCCAAGA CGCGTGATGGTAACTTATTTGAAACTCTTATGATGTTCTGTAATGCTGCAGTTT GTGGAGCTATCTTCGGCATTGGGTCAGAGTCAATCTTTGATTCACCTTTCCTCA AGCTTGCCAAAAACATATCGCAATCCAATTTTAAGATGAGAATGAAATTCGTT ATTTATAGTCTCAGTCCAAAGTTGTACAAACTGTTGAGATTACAAGCTTTCAA CGAGATCGAAGACTTCTTCATTGGTTCAATAAGTCAAGTGATGAAATCAAGAG
AACAAGAAAATGTAAAGAGACACGACTTTGCTGAAATTGCTGTGGCTATACA GAAAAATGGTATTATGAAAGACCGTACAACAGGTTGCGAGATAGAGCCTACT
ACCGGCATTTTGTCTGCACAGGCATTTTTCTTCTTTAGTGCTGGTGTAGAGCCT TGTGCTAATGCAATCTTTTCAACATTATTTCTTTTAAGCAGTCACCCAGAAATA
CTAGAAAGAGTTCACCAAGAAATTGACGAACATTTTGAAAAGCATAGCAACA
ACATAAATTACGATGTTATATGTGAAATGAAGTACACAGACAAGGTGCTGAGT GAAGCGATGAGAATGTTACCTCCAATTGGTCACTTGACAAGACAATGTGTTCA
AAATACTGTCCTGCCTGTTGGTAATATCCCAGTAGAAAAGGGGACAAAAATGT TCACTCCAATTTATGCTATTCATCACGACCCAGAACTATATCCAGACCCAGAA
GTGTTCGACCCGGAGCGATTTGCCAATGATAGAAAACCGAATGATAACATTTA
CATGCCATTTGGAATGGGCAACAGGGCATGCATAGGGGAGAGGTATGCCAAA
TTACAAGTACAAGCCGGTTTAGTTCACGTCTTGCGAAACTTCACTGTTAAACC
GCAGAAACATGTAAAAGTAACATTTGCCCAGGAT (SEQ ID NO: 151)
[00414] ID 111. 301-400 Spodoptera frugiperda cytochrome P450 CY321A8 mRNA, complete cds
TTTAACCACAGAGGCATTGAAAGTATTGAAGGGGATCAACTGACCGATAATAT
TCTCATGATGAATGGTCCAAGATGGAAGCTGATGCGACAGAATATGAAAGCTT
TCAACGAGATCGAAGACTTCTTCATTGGTTCAATAAGTCAAGTGATGAAATCA
AGAGAACAAGAAAATGTAAAGAGACACGACTTTGCTGAAATTCCAATTGGTC ACTTGACAAGACAATGTGTTCAAAATACTGTCCTGCCTGTTGGTAATATCCCA
GTAGAAAAGGGGACAAAAATGTTCACTCCAATTTAT (SEQ ID NO: 152) [00415] ID 112. 701-800 Spodoptera frugiperda cytochrome P450 CY321 A8 mRNA, complete cds AAGCTTTCAACGAGATCGAAGACTTCTTCATTGGTTCAATAAGTCAAGTGATG
AAATCAAGAGAACAAGAAAATGTAAAGAGACACGACTTTGCTGAAAT (SEQ
ID NO: 153)
[00416] ID 113. 1101-1200 Spodoptera frugiperda cytochrome P450 CY321 A8 mRNA, complete cds
TCCAATTGGTCACTTGACAAGACAATGTGTTCAAAATACTGTCCTGCCTGTTG GTAATATCCCAGTAGAAAAGGGGACAAAAATGTTCACTCCAATTTAT (SEQ ID NO: 154)
[00417] ID 114. MN480661.1|:55-1500 Spodoptera frugiperda cytochrome P450 CYP6AE44 mRNA, complete cds CTCGTATCAAAACGAAAATTCAGATACTGGGAACAGAAGAAAGTGCCACACC
TACCACCAAAACCAATTCTGGGAAATTTCTCCGAATACATTCTCCAGCAGAAA
TTCTATGGACGTGTTGAACAGGAGATCTGCAACAAGTTTCCTGAAGAACCATA
CGTTGGCTCTTACTTGGGCACGGAACCGACCCTCATCATACAAGATCCCGAAT
ACATCAAGACCGTCATGACCAAGGACTACTATTTCTTCAGTGGCCGTGAAGTC
TCTGCATACTGTGAAAAGGAACCGTTAACTCAGAACCTATTCTTCACTTATGG
CGATAAGTGGAAGGTACTGCGTCAGAACCTTACGCCTTTGTTCTCATCCGCAA
AGATGAAGAACATGTTCCATTTGATCGAGAAATGTGCCCGTATCTTCGAGAAC
ATGGTCGACCAGGAAGTACAGAAAAGTAAAGACATTGAAGTGCGAGCCCTGA
CGGGAAAATTCACCATGGATGCCATTGGAAATTTGCTTTTGGAGTTGAGACCC
AGACGATGGTAAAGACTGACAATAATCCATTCACAAAGTTGGCGATGTTATCT TCGATACTGCCAGAATGAGAGCATTAAAGGGTGTGCTAAGAAGTATTTGGCCA GCCATGTTTTATAGTTTTGGAGGTAAAGCAGTCCCTGCTGATGTGATAGCTTTT TCTTCAATTTGATGACGAGTATCTTCAAAGGACGCAACTATAATCTGATCAAG GAACGACTTTGTGGACCTCCTATTGAAGTTCCATAACAACAAAACAGTGACTG GGGATAGTATGAGAAACTTGAAGGGTGATTCAGGAAAGAAAGTCAGTTTAGA AGTGGACGATGAGTTTTTGATTGCACAATGTTTCCTGTTCTTTGCTGCTGGGTA TGAGACGTCGGCGACTACGTTGAGTTACACTTTATATGAGTTGGCAAAGAATC CTGAAGCTCAAGAGTTAGCAATTCAAGACGTGGACAACTACTTGCGTCGCAAT GACAATGTGTTGAAGTACGAGTGTGTGACCGAGTTGCCTTATGTGGAAGCTTG TGTTGATGAGGCTCTTCGTCTATACCCAGTGCTAGGAGTAATTACTCGGGAGG TTATTGAGGATTATACATTCCCGACGGGATTGAAATTGGAGAAAGGACTTCGC GTGCATTTACCGGTGTACCACATGCACACAACCCTAATTACTTCCCGGAACCA
GAGCAATATCGTCCGGAGCGGTTCTTGGGCGACGAGAAGAAAAATATTAAGC
CGTATACATACTTCCCATTTGGCGAAGGACCTCGACTATGTATCGGAATGAGG
TTCGCAAAGATGCAGATTACGGCTGGAATTATAACATTACTGAAGAAATATCG
CGTAGAACTTGCGCCGGGTATGAGCGAGACAATACAGTTCGAACCTCGATCTG
TAATCACCGCC (SEQ ID NO: 155)
[00418] ID 115. MN480661.1|:701-1001 Spodoptera frugiperda cytochrome P450
CYP6AE44 mRNA, complete cds
ATAGTTTTGGAGGTAAAGCAGTCCCTGCTGATGTTGATAGCTTTTTCTTCAATT
TGATGACGAGTATCTTCAAAGGACGCAACTATAATCTGACATCAAGGAACGA
CTTTGTGGACCTCCTATTGAAGTTCCATAACAACAAAACAGTGACTGGGGATA
GTATGAGAAATTGAAGGGTGATTCAGGAAAGAAAGTCAGTTTAGAAGTGGAC
GATGAGTTTTTGATTGCACAATGTTTCCTGTTCTTTGCTGCTGGGTATGAGACG
TCGGCGACTACGTTGAGTTACACTTTATATGAGTT (SEQ ID NO: 156)
[00419] ID 116. Spodoptera frugiperda cytochrome P450 6B2-like (LOCI 18273915), mRNA NCBI Reference Sequence: XM 035591116.1
TATAAATGAT TCATAAGTGT TCGGACCGCG TATTTGGCCA GTCGCAACCA
TGGCGGCCTT ATATTTCCTC GCCGCAGTGC TAGTGTTAGT GTACGCGTTA
TATTATTACT TCACAAGGAC ATTCAACTAC TGGAAGAGTA GAAATGTGCG
AGGACCAAAA CCAGTTGCAT TATTTGGAAA CATTAAGGAC GCAGCTCTTC
GCAAAGAAAA TTATGGCGTC GTAATGCAAA ATATATACAA TGCATATCCA
AATGAAAAAG TGGTCGGCAT ATTCAGGATG ACTTCGCCTT GTCTCCTTAT
TCGAGACCTG GACATTATCA AACATATCAT GATCAAAGAC TTCGAAGCCT
TCAGTGATCG TGGAGTGGAA TTCAGCAAAG AAGGATTGGG ACAAAACTTA
TTCCACGCGG ACGGAGATAC ATGGACTGCC TTGAGGAACA GATTCACTCC
CATTTTCACA ACAGGTAAAT TGAAGAACAT GTTTTACCTA ATAAATGAGG
GAGGCGATTC ATTTGTAGAG TACATCCGTA CAGAATGCCA AAAGAAGGAA
GAATTTGATA TTCAGCCTCT CCTCCAGACG TATACTTTGT CTACGATCTC
CGCCTGTGCA TTCGGAATTA GCTATGACAG TCTTGATGTT AAAATGGATA
CTCTGAAACT TGTGGATAAA ATATTTTCTT CACCAAGTTT TGCAGTTGAA
TTGGATATGA TGTATCCCGG TCTCCTGAAA TCTCTAAACC TTTCTTTATT
CCCTACCGCC ATAAAAAAGT TCTTTGATAA TCTAGTGAAT AATGTTATAG
AGCAAAGAAA TGGTAAACCA TCGGGTCGAA ATGATTTCAT GGATCTTATT TTGGCGCTCC GTGAAATGGG AGAGGTCACA AACTCAAAAT ATGACTCTGC
AAAGCCAGTT GAAATAACAC CTGGTGTGAT AGCAGCGCAA GCTTTTGTGT
TTTATGCGGC TGGTTATGAA ACCAGTGCTA CCACTATGAC GTACATGCTT
TACCAACTAG CAATGAATCC AGACATCCAA AAGAAGTTGA CTGAAGAAAT
TGACGAATCT CTCAAAGCAA ATAATGGACA AGTTACATAC GAGAGCATTA
AGGAAATGAA GTATTTGAAC AAAGTGTTTG ATGAAACTCT ACGAATGTAC
TCGATTGTAG AACCTCTGCA GAGGAAAGCT GTAAGAGATT ACAAAGTGCC
CGGTACTGAC TTGACGATAG AAAAGAACAC AATTGTGCTG GTATCTCCGA
GAGGTATCCA CTACGACGAG AAATATTACG ACAACCCTGA ACAGTTCAAC
CCTGACAGAT TTGACGCGGA GGAGGTGGGC AAGCGACATC CGTGCGCTTA
CATGCCGTTT GGAATTGGAC AGAGAAACTG CATCGGAATG AGGTTCGGCA
GACTTCAATC CCAACTGTGC ATAACCAAGT TGCTGTCTAA GTTCCAAGTG
GAGCCATCGA GGAATACTGC AAGGAAGCTG GAAGTGGAAC CTTGTCGCTT
TATCATCGGA CCCAAAGGAG GGATACGTCT GAATATTGTT CCAAGAAAGC
TGAAGGCTTA ACACATTAAA CGCCATGGGG GGCAAATGTG ACCGGCGTTA
GCAGAGGATT TGCCTTCAAC AGATTTTTGT TGGCAGTCAA TGTATAACGA
CTTTTAAAAC TGTTTATAAA TCCATCCATC TACCAGGTAA ACTTAGGTAT
ACCAATGTGT TATTTTATTT TTTTCATTGA AAATTCGACT TTGTATGTAA
ATAATACGGT TGAAATCTAA TACAGAAAAC TATCAGATTG CAGTAGCTTA
ACCTGCTGCT CATTGTATAT AGAATATCGT TTTAAATTTG ATTCAAAATA
ATAATTGTAT GTGGCCAGTT TCACCAGTCA TGTTATTATG GAGTGCGCGG
ACTTAGTGGG CTACTCCTTA GGTGGCGAAC AGGGGTGAGG GGGGGGGAGG
CAATTCGCCT ACGCATATGA GAATAGACAA TGATGATTGA TGACCAATCT
TGAGATATCG GGAATATGAA TGGTTCAATT ATTGAAACAA AAGACAATTG
AACGTATAGA ACTTTTACGC ACGCAACCAA CTAGTTCCGC GCGCACTATT
ATTGAGATAT TATAAGACTC ATAAAGGGAA GAAACCATCA ACAAACATCG
ATTACAAATC CATTCATTTG CAAATTGTAT ATAATTACTC TTAGCACATT
AATCATGCAT TTAGTTTATA AGTAAGCTAT ATTTAATTAA ATTATTTAAA
AACTA (SEQ ID NO: 157)
[00420] ID 117. 101-300 PREDICTED: Spodoptera frugiperda cytochrome P450 6B2- like (LOCI 18273915), mRNA
TATTATTACTTCACAAGGACATTCAACTACTGGAAGAGTAGAAATGTGCGAGG
ACCAAAACCAGTTGCATTATTTGGAAACATTAAGGACGCAGCTCTTCGCAAAG AAAATTATGGCGTCGTAATGCAAAATATATACAATGCATATCCAAATGAAAA AGTGGTCGGCATATTCAGGATGACTTCGCCTTGTCTCCTTAT (SEQ ID NO: 158) [00421] ID 118. 1101-1200 PREDICTED: Spodoptera frugiperda cytochrome P450 6B2-like (LOCI 18273915), mRNA AGGAAATGAAGTATTTGAACAAAGTGTTTGATGAAACTCTACGAATGTACTCG
ATTGTAGAACCTCTGCAGAGGAAAGCTGTAAGAGATTACAAAGTGCC (SEQ ID NO: 159)
[00422] ID 119. Spodoptera frugiperda cytochrome P450 CYP9A58 mRNA, complete cds GenBank- MN480666 11: 101-250, 1101-1250
TCAAGCCTATCCCATTGCTGGGCAATATGGGCACTGTATTGCTTCGAAGACAA CACTTAGCATATAGTCTTATTGATTTGTATAATGCCTTCCCTGAAGAAAAATTT GTAGGAAGGTTTGAGTTCATGAACGAGGCAGTGCTGATCAGAGCAAGTTCGA CTTCAACTCGATACAGAGCATGAAGTATATGGATAATGTGGTGTCAGATTATT ACGACGATGGCCTGTAGCTGTAGCTACTGACAGAATTTGTGAAAAGGACTACA
ACATGGGTAAACCAAATGAAAGGCTGAGAAGGA (SEQ ID NO: 160) [00423] ID 120. 101-200 PREDICTED: Spodoptera frugiperda cytochrome P450 dislike (LOCI 18273915), mRNA
TATTATTACTTCACAAGGACATTCAACTACTGGAAGAGTAGAAATGTGCGAGG ACCAAAACCAGTTGCATTATTTGGAAACATTAAGGACGCAGCTCTTC (SEQ ID NO: 161)
[00424] ID 121. 201-300 PREDICTED: Spodoptera frugiperda cytochrome P450 6B2- like (LOCI 18273915), mRNA
GCAAAGAAAATTATGGCGTCGTAATGCAAAATATATACAATGCATATCCAAAT GAAAAAGTGGTCGGCATATTCAGGATGACTTCGCCTTGTCTCCTTAT (SEQ ID NO: 162)
[00425] ID 122. 101-300 PREDICTED: Spodoptera frugiperda cytochrome P450 6B2- like (LOCI 18273915), mRNA
TATTATTACTTCACAAGGACATTCAACTACTGGAAGAGTAGAAATGTGCGAGG ACCAAAACCAGTTGCATTATTTGGAAACATTAAGGACGCAGCTCTTCGCAAAG AAAATTATGGCGTCGTAATGCAAAATATATACAATGCATATCCAAATGAAAA AGTGGTCGGCATATTCAGGATGACTTCGCCTTGTCTCCTTAT (SEQ ID NO: 163) [00426] ID 123. 301-400 PREDICTED: Spodoptera frugiperda cytochrome P450 6B2- like (LOCI 18273915), mRNA
TCGAGACCTGGACATTATCAAACATATCATGATCAAAGACTTCGAAGCCTTCA GTGATCGTGGAGTGGAATTCAGCAAAGAAGGATTGGGACAAAACTTA (SEQ ID NO: 164)
[00427] ID 125. 1201-1300 PREDICTED: Spodoptera frugiperda cytochrome P450 6B2-like (LOCI 18273915), mRNA
CGGTACTGACTTGACGATAGAAAAGAACACAATTGTGCTGGTATCTCCGAGAG GTATCCACTACGACGAGAAATATTACGACAACCCTGAACAGTTCAA (SEQ ID NO: 165)
[00428] ID 126. 1301-1400 PREDICTED: Spodoptera frugiperda cytochrome P450 6B2-like (LOCI 18273915), mRNA
CCTGACAGATTTGACGCGGAGGAGGTGGGCAAGCGACATCCGTGCGCTTACA TGCCGTTTGGAATTGGACAGAGAAACTGCATCGGAATGAGGTTCGGCA (SEQ ID NO: 166)
[00429] ID 127. 101-300 PREDICTED: Spodoptera frugiperda cytochrome P450 6B2- like (LOCI 18273915), mRNA
TATTATTACTTCACAAGGACATTCAACTACTGGAAGAGTAGAAATGTGCGAGG ACCAAAACCAGTTGCATTATTTGGAAACATTAAGGACGCAGCTCTTCGCAAAG AAAATTATGGCGTCGTAATGCAAAATATATACAATGCATATCCAAATGAAAA AGTGGTCGGCATATTCAGGATGACTTCGCCTTGTCTCCTTAT (SEQ ID NO: 167) [00430] ID 129. PREDICTED: Spodoptera frugiperda cytochrome P450 6B2-like (LOCI 18273915), mRNA
TATTATTACTTCACAAGGACATTCAACTACTGGAAGAGTAGAAATGTGCGAGG ACCAAAACCAGTTGCATTATTTGGAAACATTAAGGACGCAGCTCTTCGCAAAG AAAATTATGGCGTCGTAATGCAAAATATATACAATGCATATCCAAATGAAAA AGTGGTCGGCATATTCAGGATGACTTCGCCTTGTCTCCTTATAGGAAATGAAG
TATTTGAACAAAGTGTTTGATGAAACTCTACGAATGTACTCGATTGTAGAACC TCTGCAGAGGAAAGCTGTAAGAGATTACAAAGTGCC (SEQ ID NO: 168) [00431] ID 130. 201-300 Spodoptera frugiperda cytochrome P450 CY321A8 mRNA, complete cds
ACATGAACCAGCTATTGGTATTGGTCAAGTCCTAACACCTGCGCTCTTTGTCAT CGATCCGAAGAATGTTCAGCAAGTTTTATCAAGTGACTTCCAATCT (SEQ ID NO: 169)
[00432] ID 133. 801-900 Spodoptera frugiperda cytochrome P450 CY321A8 mRNA, complete cds
TGCTGTGGCTATACAGAAAAATGGTATTATGAAAGACCGTACAACAGGTTGCG AGATAGAGCCTACTACCGGCATTTTGTCTGCACAGGCATTTTTCTTC (SEQ ID NO: 170)
[00433] ID 135. 1201-1300 Spodoptera frugiperda cytochrome P450 CY321A8 mRNA, complete cds
GCTATTCATCACGACCCAGAACTATATCCAGACCCAGAAGTGTTCGACCCGGA GCGATTTGCCAATGATAGAAAACCGAATGATAACATTTACATGCCAT (SEQ ID NO: 171)
[00434] ID. 1749-2049 Plutella xylostella strain DBM1 Ac-S ABC transporter subfamily H member 1 (ABCH1) mRNA, complete cds Sequence ID: KP260785.1 GACTACAACCCGAAGGTGGGCGACATCCCGATCGACTTCAAGGAGCCCATCT
ACGGCGACACCAACCCCTCCTTCACTGACTTCGTCGCTCCCGGTGTTATTCTCA
CTATCGTGTTCTTCCTGGCGGTGGCGCTGACGTCGTCGGCGCTGATCGTGGAG CGCATGGAGGGGCTGCTGGACCGCTCGTGGGTGGCCGGCGTGTCCCCCGGGG AGATCCTGTTCTCGCACGTCGTCACGCAGTTCGTCGTCATGTGCGGCCAGACC GCGCTCGTGCTCGTGTTCATGATACTGGTGTTCGGCG (SEQ ID NO: 172)
[00435] ID 137. |MN480666.1|:101-250 Spodoptera frugiperda cytochrome P450 CYP9A58 mRNA, complete cds
TCAAGCCTATCCCATTGCTGGGCAATATGGGCACTGTATTGCTTCGAAGACAA CACTTAGCATATAGTCTTATTGATTTGTATAATGCCTTCCCTGAAGAAAAATTT GTAGGAGGTTTGAGTTCATGAACGAGGCAGTGCTGATCAGAG (SEQ ID NO:
173)
[00436] ID 138. 1101-1250 Spodoptera frugiperda cytochrome P450 CYP9A58 mRNA, complete cds
CAAGTTCGACTTCAACTCGATACAGAGCATGAAGTATATGGATAATGTGGTGT CAGAATTATTACGACGATGGCCTGTAGCTGTAGCTACTGACAGAATTTGTGAA AAGGACTCAACATGGGTAAACCAAATGCAAAGGCTGAGAAGGA (SEQ ID NO:
174)
[00437] ID 139. 151-300 PREDICTED: Spodoptera frugiperda ABC transporter G family member 20-like (LOCI 18270582), transcript variant XI, mRNA
GCGCCAAAATTTTGTGACAGTGACTTTTGTGATTATCTGTGACACTTTATTTTT TCTATTTTTACTTCCCTTCGGATTGTTTGAGAGATGGCGCATCCTACAGAGTTG CGCTCCGAGAGAGTGAAACAAATGTTTAGTTGGACTACGGG (SEQ ID NO: 175) [00438] ID 140. XM_03558621 l.l|:2801-3000 PREDICTED: Spodoptera frugiperda
ABC transporter G family member 20-like (LOCI 18270582), transcript variant XI, mRNA
TTATTTTGACAATTGTTTATATTTGATGCGTAATCTGAACAAAAACATAGTGGA
TTACATTTGTAAATGTTTTGGTACGATTGTAAGCTCACGGTAGGAAGGAATTA GAAGTTCAGGACCACGCCTTATAGTCAGTAAATACTTCTTCATTTAAACGGTG AAGGGCGACGAGTCAGTTTGTTGTACACTCTCGAAACTAT (SEQ ID NO: 176) [00439] ID 141. 201 -300 PREDICTED : Spodoptera frugiperda PB AN-type neuropeptides (LOCI 18281022), mRNA
ATTCTTCAAACTCTTGGAAGCGGCAGACGCATTGAAATATTACTACGATCGCT TACCTTACGAGATGCAAGCGGACGAACCTGAAACCAGAGTTACCAAA (SEQ ID NO: 177)
[00440] ID 142. 251-350 PREDICTED: Spodoptera frugiperda PBAN-type neuropeptides (LOCI 18281022), mRNA
GCTTACCTTACGAGATGCAAGCGGACGAACCTGAAACCAGAGTTACCAAAAA AGTGATATTTACTCCTAAATTGGGAAGGAGTTTGGCTTATGATGATAA (SEQ ID NO: 178)
[00441] ID 143. 351-450 PREDICTED: Spodoptera frugiperda PBAN-type neuropeptides (LOCI 18281022), mRNA
AGTCTTTGAGAATGTTGAGTTCACACCACGGTTGGGAAGACGGTTGGCTGATG ATATGCCCGCGACGCCGGCTGATCAGGAACTGTATAGACCAGACCCG (SEQ ID NO: 179)
[00442] ID 144. 451-550 PREDICTED: Spodoptera frugiperda PBAN-type neuropeptides (LOCI 18281022), mRNA
GATCAGATCGACAGCAGGACGAAGTACTTCTCGCCTAGGCTCGGCAGGACCA TGATTTTTCACCACGACTCGGCAGGGAATTGTCCTATGAAATGTTAC (SEQ ID NO: 180)
[00443] ID 145. 551-650 PREDICTED: Spodoptera frugiperda PBAN-type neuropeptides (LOCI 18281022), mRNA
CAAGCAAACTAAGAATGGTGAGGAGTGCCAACAGAACGCAATCGACATAAAC TTGACATGAGCCCGCCATCAAACATAACCTCAAAATGAGACAAACTAA (SEQ ID NO: 181)
[00444] ID 146. 551-700 PREDICTED: Spodoptera frugiperda PBAN-type neuropeptides (LOCI 18281022), mRNA
CAAGCAAACTAAGAATGGTGAGGAGTGCCAACAGAACGCAATCGACATAAAC TTGACATGAGCCCGCCATCAAACATAACCTCAAAATGAGACAAACTAATGTAC TGTACAATGTATAAATAAATCGCTATTTTGGACGAAATAATGTAC (SEQ ID NO:
182)
[00445] ID 147. XM_035601452.1|:201-400 PREDICTED: Spodoptera frugiperda PBAN-type neuropeptides (LOCI 18281022), mRNA
ATTCTTCAAACTCTTGGAAGCGGCAGACGCATTGAAATATTACTACGATCGCT TACCTTACGAGATGCAAGCGGACGAACCTGAAACCAGAGTTACCAAAAAAGT GATATTTACTCCTAAATTGGGAAGGAGTTTGGCTTATGATGATAAAGTCTTTG AGAATGTTGAGTTCACACCACGGTTGGGAAGACGGTTGGCTG (SEQ ID NO:
183)
[00446] ID 149. 151-300 PREDICTED: Spodoptera frugiperda charged multivesicular body protein 4b-like (LOCI 18279222), mRNA AGTTTTCTGGGGAAGTTATTCGGTGGTAAGAAGGAAGAAAAGGGCCCGACAA
CACATGAAGCTATTCAGAAATTACGCGAGACAGAGGAGCTCTTGCTGAAAAA ACAAGAGTTTCTAGAGAAGAAAATCGATATAGAAATACAGACTGCT (SEQ ID NO: 184)
[00447] ID 150. 801-950 PREDICTED: Spodoptera frugiperda charged multivesicular body protein 4b-like (LOCI 18279222), mRNA
GTCTTGGGCCACATAATGTATGCACCTCAAGTTGTAATTTTGTGATATATTATA GGTATTGTATAGCCATCCGCTTTGTTACGCACTCTTGTGGGCACAGCACGTCG ATGTATACTGGTTTTGTATTGAAAATATGTAGCTTGATATGCT (SEQ ID NO: 185)
[00448] ID 151. 151-300, and 801-950 Suggested Spodoptera frugiperda charged multivesicular body protein 4b-like (LOCI 18279222), mRNA
AGTTTTCTGGGGAAGTTATTCGGTGGTAAGAAGGAAGAAAAGGGCCCGACAA CACATGAAGCTATTCAGAAATTACGCGAGACAGAGGAGCTCTTGCTGAAAAA ACAAGAGTTTCTAGAGAAGAAAATCGATATAGAAATACAGACTGCTGTCTTG GGCCACATAATGTATGCACCTCAAGTTGTAATTTTGTGATATATTATAGGTATT GTATAGCCATCCGCTTTGTTACGCACTCTTGTGGGCACAGCACGTCGATGTAT ACTGGTTTTGTATTGAAAATATGTAGCTTGATATGCT (SEQ ID NO: 186) [00449] ID 152. 301-450 and 1851-2000 Spodoptera frugiperda cytokine receptor mRNA, complete cds
TTGTATATTGAACATTTGGACAAACAGGTATACACATTTTACTGTAAGAATAA TGTGACAAACAAACCTTGCACCACTAGAGTGCTAGTGGACGACTCTCCCTCCA ATGTTACTGACTTTAGCTGCATATCAAAGAATCTTGATGAACTGAAACATAAC TGATCTTAAACCTTATACAACATATCTTTTTACCCTCGCCTTAAATACTACTTA
TGGGTTAAAGACAATTGAAAATGCTTCCACTGGAGTGACAACTATCGAGGATA
CTCCCACAAGTCCAAGGAATGTACGCATTACAGA (SEQ ID NO: 187) [00450] ID 153. 301-450 Spodoptera frugiperda cytokine receptor mRNA, complete cds
TTGTATATTGAACATTTGGACAAACAGGTATACACATTTTACTGTAAGAATAA
TGTGACAAACAAACCTTGCACCACTAGAGTGCTAGTGGACGACTCTCCCTCCA
ATGTTACTGACTTTAGCTGCATATCAAAGAATCTTGATGAACTG (SEQ ID NO:
188)
[00451] ID 154. 1851-2000 Spodoptera frugiperda cytokine receptor mRNA, complete cds
AAACATAACTGATCTTAAACCTTATACAACATATCTTTTTACCCTCGCCTTAAA
TACTACTTATGGGTTAAAGACAATTGAAAATGCTTCCACTGGAGTGACAACTA
TCGAGGATACTCCCACAAGTCCAAGGAATGTACGCATTACAGA (SEQ ID NO:
189)
[00452] ID 155. 201-300 Spodoptera frugiperda cytokine receptor mRNA, complete cds
GATCTTTTGCGTGGCTGAAAACTATACTGCTGAGAACTTGGAGTTTTACCACG
GTCGAAAATTTATAGAATCAGAAAAAGTGAATGCAACCACCAGGAGG (SEQ
ID NO: 190)
[00453] ID 156. 301-400 Spodoptera frugiperda cytokine receptor mRNA, complete cds
TTGTATATTGAACATTTGGACAAACAGGTATACACATTTTACTGTAAGAATAA
TGTGACAAACAAACCTTGCACCACTAGAGTGCTAGTGGACGACTCTC (SEQ ID NO: 191)
[00454] ID 157. 401-500 Spodoptera frugiperda cytokine receptor mRNA, complete cds
CCTCCAATGTTACTGACTTTAGCTGCATATCAAAGAATCTTGATGAACTGAAC
TGTACTTGGACTTCTCCAGAGATTTACAGCATAACTACATACCGGTT (SEQ ID NO: 192)
[00455] ID 158. 101-200 Spodoptera frugiperda Dredd mRNA, complete cds AAATAATCATTTAAAACGAGAAAATGTTTTCTGTAGACTCAGTTACCACAACA CCACAAGGAATGGAAGTGGAAAATGTTATTGGAAACAGTGATATTAT (SEQ ID
NO: 193) [00456] ID 159. 201-300 Spodoptera frugiperda Dredd mRNA, complete cds GAATACTGATATGATGTCAGAAATTGAAAAAGAATTACAAGACAATCCCAGC GACTTGATATCCCTAGTGTTTCTTCTTTATGAAGTACCGGACACAGCA (SEQ ID NO: 194)
[00457] ID 160. 301-400 Spodoptera frugiperda Dredd mRNA, complete cds CCACAACGTTTAATTGTTTTTCAACGCGTTTCCAACGATGCCTGTAACTCTATT AATCTTAACATGCTACACGAGTGGTTTCGATCTACTAAGCACAACC (SEQ ID NO: 195)
[00458] ID 161. 401-500 Spodoptera frugiperda Dredd mRNA, complete cds CCAACTGGAAACATCAATTTGTGGAAGCTCTCCTTATCTGTCAACTGTACAGT ATTGTCAGAAAACTTGGATTGAATGTCCCTACAGCACGCAAGTACTA (SEQ ID NO: 196)
[00459] ID 162. 801-900 Spodoptera frugiperda Dredd mRNA, complete cds GATGAATATGTTGCAACACTCATTGAATGATGAGCACAAGCCAAGTGCCTCAG CTGCTACCAGTACTCCTATGATGAAACATATGAAAGTGGATGAAACA (SEQ ID NO: 197)
[00460] ID 163. 901-1000 Spodoptera frugiperda Dredd mRNA, complete cds AATCAAGGGAAAGCTGATGACAATTATTACAATATGGATTTTAATGATGTGTT TGAAATGCTTGGAGAGTTACAGTTGGATGAAAGAATGCAAGAGTCTC (SEQ ID NO: 198)
[00461] ID 164. 1001-1100 Spodoptera frugiperda Dredd mRNA, complete cds TGAAGTCTGATAGGAAGCTACTGGACAATGATAGCTATGAAATCAAGAGTAA CAAAAGGGTTGGAGTTTGTGTCATCATAAATCAGGAAACATTTTATCC (SEQ ID NO: 199)
[00462] ID 165. 1401-1500 Spodoptera frugiperda Dredd mRNA, complete cds TGATGCCAAAAAAATACACAACATGCCTAAACTTTTGATAGTTCAAGCATGCC AAGTTGATGAAAATACTCCCCAGATTGTAGTGGCTGACAGCCCAAGA (SEQ ID NO: 200)
[00463] ID 166. 1451-1550 Spodoptera frugiperda Dredd mRNA, complete cds GCCAAGTTGATGAAAATACTCCCCAGATTGTAGTGGCTGACAGCCCAAGAGAT TACAATTTAAGGAAATCTAACTTCCTTGTTTACTATGCCACTGCACC (SEQ ID NO: 201)
[00464] ID 167. 1501-1600 Spodoptera frugiperda Dredd mRNA, complete cds GATTACAATTTAAGGAAATCTAACTTCCTTGTTTACTATGCCACTGCACCTGAA CTAGAAGCTTACAGAAATGAAAAAAGAGGATCGATATTCATTCAGG (SEQ ID NO: 202)
[00465] ID 168. 1601-1700 Spodoptera frugiperda Dredd mRNA, complete cds TGCTATGTAGAACAATTAGAAAGTATGCTAATACTGAACATGTCTATGATATT TTTACTAAGGTCAATAATAATGTGAATTTCATTTGCCAAAAGGTGGG (SEQ ID NO: 203)
[00466] ID 169. Spodoptera frugiperda Dredd mRNA, complete cds GAATACTGATATGATGTCAGAAATTGAAAAAGAATTACAAGACAATCCCAGC GACTTGATATCCCTAGTGTTTCTTCTTTATGAAGTACCGGACACAGCATGAAGT CTGATAGGAAGCTACTGGACAATGATAGCTATGAAATCAAGAGTAACAAAAG GGTTGGAGTTTGTGTCATCATAAATCAGGAAACATTTTATCCTGATGCCAAAA AAATACACAACATGCCTAAACTTTTGATAGTTCAAGCATGCCAAGTTGATGAA AATACTCCCCAGATTGTAGTGGCTGACAGCCCAAGA (SEQ ID NO: 204) [00467] ID 170. Spodoptera frugiperda Dredd mRNA, complete cds ACACCACAAGGAATGGAAGTGGAAAATGTTATTGGAAACAGTGATATTATGA ATACTGATATGATGTCAGAAATTGAAAAAGAATTACAAGACAATCCCAGCGA CTTGATATCCCTAGTGTTTCTTCTTTATGAAGTACCGGACACAGCACCACAACG TTTAATTGTTTTTCAACGCGTTTCCAACGATGCCTGTAACTCTATTAATCTTAA CATGCTACACGAGTGGTTTCGATCTACTAAGCACAACCCCAACTGGAAACATC AATTTGTGGAAGCTCTCCTTATCTGTCAACTGTAC (SEQ ID NO: 205)
[00468] ID 171. 200-300 PREDICTED: Spodoptera frugiperda dual oxidase-like (LOCI 18269141), transcript variant XI, mRNA ATGTTGGTCTCAACGTTACAAGTTGTGATACCGGTGTTCTGTGATCCAGAAGT GTACTACGAGAAACAGCGGTATGATGGCTGGTTCAACAACAGAGCTTA (SEQ ID NO: 206)
[00469] ID 172. 300-400 PREDICTED: Spodoptera frugiperda dual oxidase-like (LOCI 18269141), transcript variant XI, mRNA ACCCAGACTGGGGATCCGTTGGCAGTCGTCTCACACGCAAGACTCCAGCATCA TACGCTGATGGGGTCTACATGATGGCAGGCGTCGATAGACCCGGCGCC (SEQ ID NO: 207)
[00470] ID 173. 400-500 PREDICTED: Spodoptera frugiperda dual oxidase-like (LOCI 18269141), transcript variant XI, mRNA CAGAACATTATCCAAACTCTTCATGAGAGGGCAAGATGGACTGCCTTCTTTAG CCAACAGGACAGCATTATTGGCGTTCTTTGGCCAGGTCGTAACCGGCG (SEQ
ID NO: 208)
[00471] ID 174. 500-600 PREDICTED: Spodoptera frugiperda dual oxidase-like
(LOCI 18269141), transcript variant XI, mRNA
[00472] GAGATTGTAATGGCATCCGAGTCGGGCTGTCCTATCGAGCACCATC
GCATACCAGTGGACAAATGTGACCACATGTATGACCCAGAATGTAATGGAGC
CAA (SEQ ID NO: 209)
[00473] ID 175. PREDICTED: Spodoptera frugiperda dual oxidase-like
(LOCI 18269141), transcript variant XI, mRNA 4100-4200
TCACATCAAAGCACAAGGTCCCTGGACGTGGAAACTCAGGAATTACTTCGATC
CCTGCAACTTTAATACTGAAGATCATCCTAAAATCAGGATCCAAGGTC (SEQ
ID NO: 210)
[00474] ID 176. 600-700 PREDICTED: Spodoptera frugiperda dual oxidase-like
(LOCI 18269141), transcript variant XI, mRNA
AGTACATGCCGTTTCTTAGAGCAGCATATGATCGGAACACGGGGCAGAGTCCC
AACAGTCCTAGAGAACAGATAAACCAAATGACTTCGTGGATCGACGGC (SEQ ID NO: 211)
[00475] ID 177. 2600-2700 PREDICTED: Spodoptera frugiperda dual oxidase-like
(LOCI 18269141), transcript variant XI, mRNA
CTTGGAATGAAAGCAGATGCTGTATTTGTGAAGAAAATGTTCAACATTGTCGA
CAAAGACGGAGATGGCAGGATCTCTTTCCAGGAATTTCTGGACACGGT (SEQ ID NO: 212)
[00476] ID 178. 2700-2800 PREDICTED: Spodoptera frugiperda dual oxidase-like
(LOCI 18269141), transcript variant XI, mRNA
TAGTCCTATTTTCTCGAGGAGCAACAGAAGACAAACTTCGCATTATCTTCGAC
ATGTGTGACGATGATCGTAACGGTGTGATCGACAAGGGAGAGCTCAGT (SEQ ID NO: 213)
[00477] ID 179. 3800-3900 PREDICTED: Spodoptera frugiperda dual oxidase-like
(LOCI 18269141), transcript variant XI, mRNA
CTACGAACTAAGTATCTAGCACTGGATGTTTTGGAAACAGAAATGCTACCATC
GGATGTGATTAAAATAAAGTTCTATAGACCACCAAATCTAAAATATTT (SEQ
ID NO: 214)
[00478] ID 180. 3900-4000 PREDICTED: Spodoptera frugiperda dual oxidase-like
(LOCI 18269141), transcript variant XI, mRNA TATCAGGTCAATGGGTACGGTTATCTTGTACGGCGTTCAAGAAAGAAGAGTTC CACTCGTTCACATTAACCTCAGCTCCTCACGAGAACTTCTTATCGTGT (SEQ ID NO: 215)
[00479] ID 181. Spodoptera frugiperda dual oxidase-like (LOCI 18269141), transcript variant XI, mRNA
ATGTTGGTCTCAACGTTACAAGTTGTGATACCGGTGTTCTGTGATCCAGAAGT GTACTACGAGAAACAGCGGTATGATGGCTGGTTCAACAACAGAGCTTACTAC GAACTAAGTATCTAGCACTGGATGTTTTGGAAACAGAAATGCTACCATCGGAT GTGATTAAAATAAAGTTCTATAGACCACCAAATCTAAAATATTTCTACGAACT
AAGTATCTAGCACTGGATGTTTTGGAAACAGAAATGCTACCATCGGATGTGAT TAAAATAAAGTTCTATAGACCACCAAATCTAAAATATTT (SEQ ID NO: 216) [00480] ID 181. Spodoptera frugiperda dual oxidase-like (LOCI 18269141), transcript variant XI, mRNA
CTACGAACTAAGTATCTAGCACTGGATGTTTTGGAAACAGAAATGCTACCATC GGATGTGATTAAAATAAAGTTCTATAGACCACCAAATCTAAAATATTTTATCA GGTCAATGGGTACGGTTATCTTGTACGGCGTTCAAGAAAGAAGAGTTCCACTC GTTCACATTAACCTCAGCTCCTCACGAGAACTTCTTATCGTGTTCACATCAAAG CACAAGGTCCCTGGACGTGGAAACTCAGGAATTACTTCGATCCCTGCAACTTT AATACTGAAGATCATCCTAAAATCAGGATCCAAGGTC (SEQ ID NO: 217) [00481] ID 182. Spodoptera frugiperda dual oxidase-like (LOCI 18269141), transcript variant XI, mRNA
TAAAATGGCGGCCCCGGATACGGTAGGGAGTCGAATATGGCTCCTAGTAATGT TGGTCTCAACGTTACAAGTTGTGATACCGGTGTTCTGTGATCCAGAAGTGTAC TACGAGAAACAGCGGTATGATGGCTGGTTCAACAACAGAGCTTAAGCCAACA GGACAGCATTATTGGCGTTCTTTGGCCAGGTCGTAACCGGCGAGATTGTAATG GCATCCGAGTCGGGCTGTCCTATCGAGCACCATCGCATACCAGTGGACAAATG TGACCACATGTATGACCCAGAATGTAATGGAGCCAA (SEQ ID NO: 218) [00482] ID 183. 101-200 PREDICTED: Spodoptera frugiperda glutathione S- transf erase 1-like (LOCI 18261931), mRNA
CATGCGAGAAGAGGCTTTGATGGGTGTGAAACTATATGCTGCAGACTTCAGCC CACCGGTGCGTTCCAGCATGATGGCCCTTGATATCTTCAATGTACCA (SEQ ID NO: 219)
[00483] ID 184. 201-300 PREDICTED: Spodoptera frugiperda glutathione S- transf erase 1-like (LOCI 18261931), mRNA TTTGAGAAGATAACTCTCAACTTACTGAAACTGGAACACCGCACTCCAGAATA TTTGGAAAAAAATCCGATTCACTCGATTCCAGTATTGGAAGATGGAG (SEQ ID NO: 220)
[00484] ID 185. 301-400 PREDICTED: Spodoptera frugiperda glutathione S- transf erase 1-like (LOCI 18261931), mRNA
ATTTAATATTACATGATAGCCACGCAATAATGGCCTACCTCGCTGACACCTAC
GGAAAGGATGAGTCTTGGTATCCTAAGGACGTGAAAAAACGAGCTTT (SEQ ID NO: 221)
[00485] ID 186. 401-500 PREDICTED: Spodoptera frugiperda glutathione S- transf erase 1-like (LOCI 18261931), mRNA
AGTTAACCAAAAACTGTTTTTCACCACAGGTGTAATTTTCCCTAGACTACGAA
TCATTACATACTATCTTCTAGAAAAAGGAAGAAAAGCTATAGAACAA (SEQ ID NO: 222)
[00486] ID 187. 501-600 PREDICTED: Spodoptera frugiperda glutathione S- transf erase 1-like (LOCI 18261931), mRNA
GAATGGCTAGACAACATTGAAGAAGCCATTGGTTTTGTGGAGCAGTTCTTGTC
TCGGACAAAATTCATCGCCTTGGACCATGTCACCATTGCCGATATTG (SEQ ID NO: 223)
[00487] ID 188. 601-700 PREDICTED: Spodoptera frugiperda glutathione S- transf erase 1-like (LOCI 18261931), mRNA
CAGTGTTAAGTCACCTGTCCTCTGTGGAACCTATTCTACCCATAGATCCGAAA
AAGTACCCAAAGACGGTTGCCTGGTTAGAAATTATGAAAGCGACGCC (SEQ ID NO: 224)
[00488] ID 189. 651-750 PREDICTED: Spodoptera frugiperda glutathione S- transf erase 1-like (LOCI 18261931), mRNA
AAAAAGTACCCAAAGACGGTTGCCTGGTTAGAAATTATGAAAGCGACGCCAT
ATTGCAAGAAATATAACGAAGAAGGTGTTAAATCTCTGACTGCTATTA (SEQ ID NO: 225)
[00489] ID 190. Spodoptera frugiperda glutathione S-transferase 1-like
(LOCI 18261931), mRNA
TTTGAGAAGATAACTCTCAACTTACTGAAACTGGAACACCGCACTCCAGAATA
TTTGGAAAAAAATCCGATTCACTCGATTCCAGTATTGGAAGATGGAGAGTTAA CCAAAAACTGTTTTTCACCACAGGTGTAATTTTCCCTAGACTACGAATCATTAC ATACTATCTTCTAGAAAAAGGAAGAAAAGCTATAGAACAAAAAAAGTACCCA AAGACGGTTGCCTGGTTAGAAATTATGAAAGCGACGCCATATTGCAAGAAAT ATAACGAAGAAGGTGTTAAATCTCTGACTGCTATTA (SEQ ID NO: 226) [00490] ID 191. 201-500 PREDICTED: Spodoptera frugiperda glutathione S- transf erase 1-like (LOCI 18261931), mRNA TTTGAGAAGATAACTCTCAACTTACTGAAACTGGAACACCGCACTCCAGAATA TTTGGAAAAAAATCCGATTCACTCGATTCCAGTATTGGAAGATGGAGATTTAA TATTACATGATAGCCACGCAATAATGGCCTACCTCGCTGACACCTACGGAAAG GATGAGTCTTGGTATCCTAAGGACGTGAAAAAACGAGCTTTAGTTAACCAAAA ACTGTTTTTCACCACAGGTGTAATTTTCCCTAGACTACGAATCATTACATACTA TCTTCTAGAAAAAGGAAGAAAAGCTATAGAACAA (SEQ ID NO: 227) [00491] ID 192. 101-200 PREDICTED: Spodoptera frugiperda protein mesh (LOCI 18271033), transcript variant XI, mRNA ACGGGCACACAATGTTCCAACACTTTAGTCGACCAGTACATTAGCGAGCCACC TGTTGTCGTGAAAATTGATAAAAAATAATGTGATGGACATTTTAATA (SEQ ID NO: 228)
[00492] ID 193. 201-300 PREDICTED: Spodoptera frugiperda protein mesh (LOCI 18271033), transcript variant XI, mRNA TAAGTGTGAAAATGGGTGTTAAGGTTTTAGCTTTAATAGCACTCTTAGTTGTTA GTGTACTCGGACAAGACGTTACTGTCAGTGACAACGATGTAACAAA (SEQ ID NO: 229)
[00493] ID 194. 301-400 PREDICTED: Spodoptera frugiperda protein mesh (LOCI 18271033), transcript variant XI, mRNA GGAAGTTGTGGTGGACAATTTAGCTAGTGAGGACCCGATAGTATTAGACACA GTTACAGAAGCCACAAAAGATGAAGTTGCTGAAGATGCGAACCCAGTG (SEQ ID NO: 230)
[00494] ID 195. 401-500 PREDICTED: Spodoptera frugiperda protein mesh (LOCI 18271033), transcript variant XI, mRNA GAAATACTAAGTCCAACGGATGATCTGCAAGTAAGAAGCGGGAAATATCAGC TCAATGATGGGCTCGTGGGTGAAGAGCCGATGCCACTAGATGCTGTTA (SEQ ID NO: 231)
[00495] ID 196. 1901-2000 PREDICTED: Spodoptera frugiperda protein mesh (LOCI 18271033), transcript variant XI, mRNA AAGGAGATTACCATCAGACCCCAGCTTGAATACATAGATATTATCGAAATGGG CGTGGCTAACACTGGAGAATATGTGATCAATCCCCAAAACTTTAGGA (SEQ ID NO: 232)
[00496] ID 197. 2001-2100 PREDICTED: Spodoptera frugiperda protein mesh
(LOCI 18271033), transcript variant XI, mRNA
ACCGGGATAACTTCATGCACAATGATATGCAGTTCGGTTTCCTCCAGATTAAT
TTAACTACGCCTGAAGTCTTCAAAGGAGTCTCTATCTCGCCTGTACT (SEQ ID NO: 233)
[00497] ID 198. 201-350 PREDICTED: Spodoptera frugiperda protein mesh
(LOCI 18271033), transcript variant XI, mRNA
TAAGTGTGAAAATGGGTGTTAAGGTTTTAGCTTTAATAGCACTCTTAGTTGTTA
GTGTACTCGGACAAGACGTTACTGTCAGTGACAACGATGTAACAAAGGAAGT
TGTGGTGGACAATTTAGCTAGTGAGGACCCGATAGTATTAGACA (SEQ ID NO:
234)
[00498] ID 199. 1901-2050 PREDICTED: Spodoptera frugiperda protein mesh
(LOCI 18271033), transcript variant XI, mRNA
AAGGAGATTACCATCAGACCCCAGCTTGAATACATAGATATTATCGAAATGGG
CGTGGCTAACACTGGAGAATATGTGATCAATCCCCAAAACTTTAGGAACCGGG
ATAACTTCATGCACAATGATATGCAGTTCGGTTTCCTCCAGATT (SEQ ID NO:
235)
[00499] ID 200. 201-350, 1901-1050 PREDICTED: Spodoptera frugiperda protein mesh (LOCI 18271033), transcript variant XI, mRNA
TAAGTGTGAAAATGGGTGTTAAGGTTTTAGCTTTAATAGCACTCTTAGTTGTTA
GTGTACTCGGACAAGACGTTACTGTCAGTGACAACGATGTAACAAAGGAAGT
TGTGGTGGACAATTTAGCTAGTGAGGACCCGATAGTATTAGACAAAGGAGATT
ACCATCAGACCCCAGCTTGAATACATAGATATTATCGAAATGGGCGTGGCTAA
CACTGGAGAATATGTGATCAATCCCCAAAACTTTAGGAACCGGGATAACTTCA
TGCACAATGATATGCAGTTCGGTTTCCTCCAGATT (SEQ ID NO: 236)
[00500] ID 202. MT544380.1|: 101-400 Spodoptera frugiperda clone Sf_17445 protein mesh mRNA, partial cds
TGGACAATTTAGCTAGTGAGGACCCAATAGTATTAGACACAGTTACAGAAGCC
ACAAAAGATGAAGTTGCTGAAGATGCGAACCCAGTGGAAATACTAAGTCCTA CGGATGATCTGCAAGTAAGAAGTGGGAAATATCAGCTCAATGATGGGCTCGT GGGCGAAGAGCCGATACCACTAGATGCTGTTAATTTCGACTCGAATAATGATG CCGGAGAGAGTGAGAAGCAGTTGCTCTCTCCTGGCACAACTCAAGTCACGAA CAACGAGTATGCTCATATCGATGGCCGAGTTTTACCGG (SEQ ID NO: 237) [00501] ID 203. 301-400 PREDICTED: Spodoptera frugiperda uncharacterized LOCI 18263801 (LOCI 18263801), transcript variant XI, mRNA AAGTCGGACGTTATACAAAGAACGATGAGTGTGCTGAGGGGTTTTTAGTGAG AAATTGTGAAAAATAGAAAGAGTGAAGATGTCGGTAAAAGCGAGCGTT (SEQ ID NO: 238)
[00502] ID 204. 401-500 PREDICTED: Spodoptera frugiperda uncharacterized LOCI 18263801 (LOCI 18263801), transcript variant XI, mRNA GGTTAAACAAAAAGGGTAAAAATGGCCTTCAAAGGATTCTGTGGCGAAGTGA TCGGGTTTTTCCTGGCTGTGGGTTTTTGCATCATATGTCCGGAATATG (SEQ ID NO: 239)
[00503] ID 205. 701-800 PREDICTED: Spodoptera frugiperda uncharacterized LOCI 18263801 (LOCI 18263801), transcript variant XI, mRNA AACTGCCGAGTCAAGAATTTAGGAAATAAAACGTTAAACATGCAAGTATCGT GGGTACGGCATAGAGACATCCATCTGCTGACAGTCGGCCGGTACACAT (SEQ ID NO: 240)
[00504] ID 206. 801-900 PREDICTED: Spodoptera frugiperda uncharacterized LOCI 18263801 (LOCI 18263801), transcript variant XI, mRNA ACACGAGCGATCAAAGGTTTAGAGCTATTCACTTACCGCACTCCGAGGACTGG ACTTTACAGATCAAGTATCCGCAACACAGGGATTCGGGAATTTATGA (SEQ ID NO: 241)
[00505] ID 207. 1351-1450 PREDICTED: Spodoptera frugiperda uncharacterized LOCI 18263801 (LOCI 18263801), transcript variant XI, mRNA GTTGGTTGCCTGCCTGTTCATCGCACTCTCTTGACACATACGGATCTTTAACTA AATGTAAATACAAGGATTTTATCAACATCAGCTGCTTATATTGACA (SEQ ID NO: 242)
[00506] ID 211. Spodoptera frugiperda uncharacterized LOC 118263801 (LOCI 18263801), transcript variant XI, mRNA AAGTCGGACGTTATACAAAGAACGATGAGTGTGCTGAGGGGTTTTTAGTGAG AAATTGTGAAAAATAGAAAGAGTGAAGATGTCGGTAAAAGCGAGCGTTGGTT AAACAAAAAGGGTAAAAATGGCCTTCAAAGGATTCTGTGGCGAAGTGATCGG GTTTTTCCTGGCTGTGGGTTTTTGCATCATATGTCCGGAATATG (SEQ ID NO: 243) [00507] ID 212. 401-500 Spodoptera frugiperda V-ATPase subunit A mRNA, complete cds AATTCAACCCCTTGAATGTTAAGGTCGGCTCCCACATCACCGGAGGAGACTTG
TACGGTATCGTACACGAGAACACATTGGTTAAGCACAAGATGTTGAT (SEQ ID NO: 244)
[00508] ID 213. 1301-1400 Spodoptera frugiperda V - ATPase subunit A mRNA, complete cds
TGGACAAGAAGCTCGCGCAGCGCAAGCACTTCCCCGCCATCAACTGGCTCATC
TCCTACAGCAAGTACATGCGAGCGCTGGATGACTTCTATGAGAAGAA (SEQ ID NO: 245)
[00509] ID 214. 1501-1600 Spodoptera frugiperda V-ATPase subunit A mRNA, complete cds
GCCGAGACTGACAAAATCACCCTTGAGGTTGCCAAGCTGCTCAAAGACGACTT
CTTGCAACAAAACAGCTACTCGTCATACGATCGTTTCTGTCCGTTCT (SEQ ID NO: 246)
[00510] ID 218. 100-159, 1-60, Spodoptera frugiperda V-type proton ATPase catalytic subunit A (LOCI 18267501), transcript variant XI, X2, X3, and X4
AATTCAACCCCTTGAATGTTAAGGTCGGCTCCCACATCACCGGAGGAGACTTG TACGGTATCGTACACGAGAACACATTGGTTAAGCACAAGATGTTGATTGGACA AGAAGCTCGCGCAGCGCAAGCACTTCCCCGCCATCAACTGGCTCATCTCCTAC AGCAAGTACATGCGAGCGCTGGATGACTTCTATGAGAAGAAGCCGAGACTGA
CAAAATCACCCTTGAGGTTGCCAAGCTGCTCAAAGACGACTTCTTGCAACAAA
ACAGCTACTCGTCATACGATCGTTTCTGTCCGTTCT (SEQ ID NO: 247)
[00511] ID 219. XM_035591116.1|:100-1600 PREDICTED: Spodoptera frugiperda cytochrome P450 6B2-like (LOCI 18273915), mRNA
ATATTATTACTTCACAAGGACATTCAACTACTGGAAGAGTAGAAATGTGCGAG
GACCAAAACCAGTTGCATTATTTGGAAACATTAAGGACGCAGCTCTTCGCAAA
GAAAATTATGGCGTCGTAATGCAAAATATATACAATGCATATCCAAATGAAA
AAGTGGTCGGCATATTCAGGATGACTTCGCCTTGTCTCCTTATTCGAGACCTGG
ACATTATCAAACATATCATGATCAAAGACTTCGAAGCCTTCAGTGATCGTGGA
GTGGAATTCAGCAAAGAAGGATTGGGACAAAACTTATTCCACGCGGACGGAG
ATACATGGACTGCCTTGAGGAACAGATTCACTCCCATTTTCACAACAGGTAAA TTGAAGAACATGTTTTACCTAATAAATGAGGGAGGCGATTCATTTGTAGAGTA CATCCGTACAGAATGCCAAAAGAAGGAAGAATTTGATATTCAGCCTCTCCTCC AGACGTATACTTTGTCTACGATCTCCGCCTGTGCATTCGGAATTAGCTATGACA GTCTTGATGTTAAAATGGATACTCTGAAACTTGTGGATAAAATATTTTCTTCAC CAAGTTTTGCAGTTGAATTGGATATGATGTATCCCGGTCTCCTGAAATCTCTAA ACCTTTCTTTATTCCCTACCGCCATAAAAAAGTTCTTTGATAATCTAGTGAATA ATGTTATAGAGCAAAGAAATGGTAAACCATCGGGTCGAAATGATTTCATGGAT CTTATTTTGGCGCTCCGTGAAATGGGAGAGGTCACAAACTCAAAATATGACTC TGCAAAGCCAGTTGAAATAACACCTGGTGTGATAGCAGCGCAAGCTTTTGTGT
TTTATGCGGCTGGTTATGAAACCAGTGCTACCACTATGACGTACATGCTTTACC
AACTAGCAATGAATCCAGACATCCAAAAGAAGTTGACTGAAGAAATTGACGA
ATCTCTCAAAGCAAATAATGGACAAGTTACATACGAGAGCATTAAGGAAATG
AAGTATTTGAACAAAGTGTTTGATGAAACTCTACGAATGTACTCGATTGTAGA
ACCTCTGCAGAGGAAAGCTGTAAGAGATTACAAAGTGCCCGGTACTGACTTG
ACGATAGAAAAGAACACAATTGTGCTGGTATCTCCGAGAGGTATCCACTACG
ACGAGAAATATTACGACAACCCTGAACAGTTCAACCCTGACAGATTTGACGCG
GAGGAGGTGGGCAAGCGACATCCGTGCGCTTACATGCCGTTTGGAATTGGAC
AGAGAAACTGCATCGGAATGAGGTTCGGCAGACTTCAATCCCAACTGTGCATA
ACCAAGTTGCTGTCTAAGTTCCAAGTGGAGCCATCGAGGAATACTGCAAGGA
AGCTGGAAGTGGAACCTTGTCGCTTTATCATCGGACCCAAAGGAGGGATACGT
CTGAATATTGTTCCAAGAAAGCTGAAGGCTTAACACATTAAACGCCATGGGGG
GCAAATGTGACCGGCGTTA (SEQ ID NO: 248)
[00512] ID 220. 301-400 Spodoptera frugiperda cytochrome P450 CY321A8 mRNA, complete cds
TTTAACCACAGAGGCATTGAAAGTATTGAAGGGGATCAACTGACCGATAATAT
TCTCATGATGAATGGTCCAAGATGGAAGCTGATGCGACAGAATATGA (SEQ ID NO: 249)
[00513] ID 221. 701-800 Spodoptera frugiperda cytochrome P450 CY321A8 mRNA, complete cdsAAGCTTTCAACGAGATCGAAGACTTCTTCATTGGTTCAATAAGTCAAGTGA
TGAAATCAAGAGAACAAGAAAATGTAAAGAGACACGACTTTGCTGAAAT (SEQ ID NO: 250)
[00514] ID 222. 1101-1200 Spodoptera frugiperda cytochrome P450 CY321 A8 mRNA, complete cds
TCCAATTGGTCACTTGACAAGACAATGTGTTCAAAATACTGTCCTGCCTGTTG GTAATATCCCAGTAGAAAAGGGGACAAAAATGTTCACTCCAATTTAT (SEQ ID NO: 251)
[00515] ID 223. 501-600 PREDICTED: Spodoptera frugiperda uncharacterized LOCI 18263801 (LOCI 18263801), transcript variant XI, mRNA
TATCATCAGCACAGCGCGAACTCGGCAAGGGTTCGCCGACTAACGCCTCCGCC ACGGCATCGCCGGCCGGTTCTGTGGACAACGCGATCCGGTCCGGAGC (SEQ ID NO: 252)
[00516] ID. 61-360 Plutella xylostella prophenol oxidase 1 mRNA, complete cds GenBank: KT006134.1 cagaagggcg acgacaagac cgtcttccag atcccggaca acttctaccc agaaaagtac aagaaggtgg gcaaccagct ggccgaccgg ttcggcacgg acgcgggccg catggtgccg gtgcgcaaca tcgcgctgcc ggacctcagc ctgccgcagc agctgccgta ccacaaccag ttctcgctgt ttgtgccgaa acacaggcgg atggctgcta agctgattga tatatttatg ggaatgcgtg acgtggagga cctgcagtcc gtgtgtagct actgccagct ccgcatcaac (SEQ ID NO: 253)
[00517] ID. BI-P450 Plutella xylostella cytochrome P450 (CYP6BFlvl) mRNA, GenBank: AY971374.1 nt#s 471-520, 556-605, 629-678, 752-802, 856-905, 1317-1366. TGGTACAGGATTGCTGCAGAATATTCCAGAAGGTTCTCGATGATGAGATAAGC TCGGTATACTATGGACTGTATAACTTCGTGTGCATTCGGCGTCGACTGAAGGG AACCCTTTCACAGAAACAGGTCACCTTTTATTTGATGAAAGACCTCCAGCAAA ATTTACCGTTTCTTCCGATCTGTTATACTTGACGTTATAAATTGGAAGAAGAAC AAATACATAACGGGAGACAGTATTGATAATGGCATAGCGGAGCGGTTTTCTG
AAGAAGGACGGAAAAGTATTGTCCCGTATACCTAC (SEQ ID NO: 254) [00518] ID. 201-500 Spodoptera frugiperda cytochrome P450 CYP6AE44 mRNA, complete cds Sequence ID: MN480661.1
TGAAGAACCATACGTTGGCTCTTACTTGGGCACGGAACCGACCCTCATCATAC AAGATCCCGAATACATCAAGACCGTCATGACCAAGGACTACTATTTCTTCAGT GGCCGTGAAGTCTCTGCATACTGTGAAAAGGAACCGTTAACTCAGAACCTATT CTTCACTTATGGCGATAAGTGGAAGGTACTGCGTCAGAACCTTACGCCTTTGT TCTCATCCGCAAAGATGAAGAACATGTTCCATTTGATCGAGAAATGTGCCCGT ATCTTCGAGAACATGGTCGACCAGGAAGTACAGAA (SEQ ID NO: 255) [00519] ID. 1601-1789 Plutella xylostella strain DBM1 Ac-S ABC transporter subfamily H member 1 (ABCH1) mRNA, complete cds Sequence ID: KP260785.1 tttggctgacactgcggacgacgagaccatcgagtcgtcagaagtgcaggtgtggctcgacatgtccaaccagcagatcggcct catgctcaaccgagacatacagttctcatacagggacttcgctaagaacttgctgtcgacttgcgactacaacccgaaggtgggcg acatcccgatcgacttcaa (SEQ ID NO: 256) [00520] ID. 332-632 Plutella xylostella juvenile hormone epoxide hydrolase mRNA, complete cds GenBank: JX297814.2 tagattttcggatgagatggtaaaagacttgttatatcgcatcaaccatcgccgtaaaataagaccctcactgcaagggtcaggaaa ccactatgggccaaactctgcgctggtgaaacaagtattggaccactggagtcaaaaatacgatttcaaagaacgtgccaagaaa ctaaaccggcatcctcaatacataaccaatgttaacggcctggacatccactacatacatgttcagccttcagggaataaacgcgtt gtgccaatcctgctcatccacagcgtagaatccaacagtctg (SEQ ID NO: 257)
[00521] ID. M_011553525.1|:161-460 PREDICTED: Plutella xylostella protein meshlike (LOCI 05383478), mRNA ACTTATTAGTGTAAGTGTAAAAGGGCAAGAGGACGCTATAATTAATGAGGAC AGTTTAGTGAATGACGTCACGAGTGTAGTGAGTAATGACTTGGAGGCTCCTGT AGTTGCTGAAGCGAAAGAGGTGGGAGTGGAAGGGTTTGTGGAGGACCCTGCT CCTGTGGAAGTGATAGCCGATTCTGAAGCCGTGGCGGTGAGGAGTGGGCGGT ACCAGGCGCTCAATGATGCCCTGCAGGAGGGGCCGGTGGACCTCCAGGCAGT GGACCTGGACGAGCAGCTCGGGAGGCAGCTGTTGAACCC (SEQ ID NO: 258) [00522] XM_011553525.11 :3300-3599 PREDICTED: Plutella xylostella protein meshlike (LOCI 05383478), mRNA CGGTCCTTTGACGTATCCAGGGCCACCGAACTCTGCCAAGATTCCTACCAGTG CCGATATGACTACGGAATGACCCTCAACAGAGATATGGCTGAGTTCACCAAG AACTATTTGTCTTCTATCACAAACATCAAGGAGAAAAACGCAAGGAGAGTCAT CAGTTGTGGCATCTTGGAGACACCGCGGTTTGGACGGAAGAGTAACTTCTTCT TTACTCCCGGAACTACGGTGAACTTTGAGTGCAATCAAAACTTTATCCTGATT GGTGACAAGCGCCGAGCTTGCGAGTCCGACGGCCGG (SEQ ID NO: 259)
[00523] ID 25-2. 351-500 PREDICTED: Plutella xylostella venom carboxylesterase-6 (LOCI 05388350), mRNA CCAACGTCTACACACCGGCCATTGATCCAGAAAAGAAATACCCAGTAATGGTT TGGATTAAAGGGTCCGAGTTTGAGAAAACTAAGGGACCTGAACTATCTTTTAG AAATCTTATTGAAAAAGAAGTAATAGTCGTGTCTCTAAACTTCATAGATGATG AAAAGTTTCTAGAAAAATCACCTTTTAGTACGCTAACTGAAGGAACTTACACT AAAATACCTATGATCTTCGGATTTGTTGAAAACGAAGGAACAATACGTTTTGA TGAGGCACTAGAAGCTGATTGGCTAACAAAGATGG (SEQ ID NO: 260).
[00524] ID. 4 -303 Spodoptera frugiperda V-ATPase subunit E mRNA, complete cds [00525] Sequence ID : MT707618.1 GCGCTCAGCGATGCAGATGTTCAAAAACAGATCAAGCACATGATGGCCTTCAT CGAGCAAGAAGCCAATGAAAAGGCTGAGGAAATCGATGCAAAGGCCGAGGA GGAGTTCAACATCGAAAAGGGGCGTCTGGTCCAGCAGCAGAGGTTGAAGATC ATGGAGTACTATGAGAAGAAGGAAAAGCAGGTCGAACTCCAGAAAAAGATCC AATCCTCAAACATGCTGAACCAGGCTCGTCTCAGGGTGCTCAAAGTACGTGAA GACCATGTACGCAACCTGTTGGACGAGGCCCGCAAGCGC (SEQ ID NO: 261) [00526] ID. Plutella xylostella mRNA for vacuolar ATP synthethase subunit E, GenBank: AB189032.1 191-240, 505-554, 683-812, 875-924, 1091-1140, 1172-1222 GTCTGGTGCAGCAGCAGCGCCTCAAGATCATGGAGTACTACGAGAAGAAGGA GCGCGCGCAGGCGCAGTACAAGGAGAAGATCAAGAAGGATGTGACCTTGCCC CCCGCGCAAATATTCAACTTTCTTATTATTATTATGTAGAACTAAAAATGCGTA CAACATTATTAACATGTATGAAAGAATCCGTATTAAGTCAGACTTAGCTGTAG CTTTTAGGAGACACAGTTAAATTGAAATGTTATACCGATAGCGTAAGCATTTT ATTGATATAATTCTGGATTGTTGCCATAACAATTAT (SEQ ID NO: 262) [00527] ID. 301-400, 401-500, 1351-1450 PREDICTED: Spodoptera frugiperda uncharacterized LOCI 18263801 (LOCI 18263801), transcript variant XI, X2, and X3, mRNA
AAGTCGGACGTTATACAAAGAACGATGAGTGTGCTGAGGGGTTTTTAGTGAG AAATTGTGAAAAATAGAAAGAGTGAAGATGTCGGTAAAAGCGAGCGTTGGTT AAACAAAAAGGGTAAAAATGGCCTTCAAAGGATTCTGTGGCGAAGTGATCGG GTTTTTCCTGGCTGTGGGTTTTTGCATCATATGTCCGGAATATGGTTGGTTGCC TGCCTGTTCATCGCACTCTCTTGACACATACGGATCTTTAACTAAATGTAAATA CAAGGATTTTATCAACATCAGCTGCTTATATTGACA (SEQ ID NO: 263) [00528] ID. 252-491 Diabrotica virgifera virgifera charged multivesicular body protein 4b (LOCI 14337301), mRNA Sequence ID: XM_028287710.1 gcaaagaaaaatgcgtcgaaaaataaaagagttgcactccaagccctcaaaaagaagaaacgattggaaaagacccaactaca aatagatggaacccttacaactattgaaatgcagagggaagccctcgaaggagctagcacaaatactgctgtattagattctatgaa aaatgctgcagatgcccttaagaaagctcataagaatttgaatgtagatgatgttcacgatatcatggat (SEQ ID NO: 264)
[00529] ID. 87-386 PREDICTED: Plutella xylostella cytokine receptor-like (LOCI 05380229), mRNA Sequence ID: XM_011549746.3 Domeless: CTTCAAATTATTGTAGTGATGATCATACGGAAGCCGATTTGATAGAATTTTAA TTCACATACGATACTAGCCTATATAACAGTCGATTTGTGCAGTCGGTCAGTGA GTGTTGTTTAGTGAGTCGTGCAACTCGTACCCTGCATGACTTTGGACTCCGATG CGCCATTTGGCCTCAGCTATAAAAACAAGGAATGTGTTTATCGTCTCTCCCGA TTTCCCAAACGACTTTAGCTTGCAGTGATGGTGTGATAAGTGAACTTTAGTGA
TATTCCCCGACATGGCAATTTGTCAGAGCACTCG (SEQ ID NO: 265) [00530] ID. 77-376 PREDICTED: Plutella xylostella caspase-8 (LOC105390324), mRNA Sequence ID: XM 011561609.3 Dredd:
ATAGGTAGTTAGCTATTTTATTTAGTATTCGGGTGTTACGATAATGTTGCAACC
TGACGCTCTCAAAGGTCATGAGAATGAGGAGTTGAACAGTGTTCACAAAAAC
ATACATAATATCACTGTTAATGTGATAGCTGAGGTACAAAGGGACTTGACTCC ATACGATATCACTTCGCTGGTGTTTCTTCTATACGATATCCCGGAAACGGCGCT TCAGCGGCTTACTCTGCTGCAGAGAGTGAGCCGGGACATCAGTGGCAAGGAT
CTGAACTTGCTCTATGACTGGGCGGTGTACGCTCA (SEQ ID NO: 266) [00531] ID. 96-395 Plutella xylostella glutathione synthetase (Gss), mRNA Sequence ID: NM_001309054.2 GSS1:
ACCAAGCCCCACGTCATCATGATCATGGCTGACGACATGGGATGGGACGACA
CCTCGACCCACGGCTCCAAGTCCGTGCTGACCCCCAACCTCGACGTGCTGACC
CGCTCAGGAGTGTCCCTCCACCGCTACTACACCCACGCTCTCTGCTCGCCCGCC CGTACCGCTGTGCTCACCGGCAAATACGCCCACACCGTCGGTATGCAGGGTAT GCCTCTGTCCAACGCTGAGGAGCGTGGTATCCCCCTAGAGGAGCGCCTGATCT
CTCAGTACCTACAGGACGCTGGTTACAGGACCCAG (SEQ ID NO: 267) [00532] ID. PREDICTED: Plutella xylostella dual oxidase (LOC105389437), mRNA Sequence ID: XM_048622382.1 Duox
CCCCAGCTCCTGCGCCCGCGCACGAATCACTTCCGCGATCGAATACCCGCTTT
CGCTCCGTAAACAGTCCGTGTGCAAATCATATTTTCCGTTTTCTCATCAACATT
GCAAAACGCTAAATTGCACTTCTACACCGGCGGATGTGTTCGGTGTCAACAAA TGTGGTGTTTCTTGGCGCTCGTTGGAATAATGTCTAATTGGAGTTGAGATTGAG ACTATACACGATGGCGCGATGGTCCTCCAGCGAAGTGTGGACACTCGTAGCGC
TCTGTGCCCTCGTCACATCCTGCCTGTCAGACC (SEQ ID NO: 268) [00533] ID. MH899215.1|:461-761 Plutella xylostella chitinase 5 mRNA, complete cds
ACCTGGACTGGGAGTACCCTGGAGCAGCCGACCGCGGCGGCTCGTTCTCCGAC
AAGGACAAGTTCCTGTACCTCGTCCAGGAGTTGAGACGAGCTTTCCTGAGGGT TGGGAAGGGCTGGGAGCTGACCGCTGCGGTACCGCTGGCCAACTTCCGACTG ATGGAGGGGTACCATGTGCCTGAGTTGTGCCAGGAGCTGGACGCGATCCACGT
GATGGCGTACGACCTGCGCGGCAACTGGGCCGGGTTCGCCGACGTGCACTCCC CGCTGTACTCCCGCCCGCACGACCAGTACGCATACTC (SEQ ID NO: 269) REFERENCES
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[00534] The teachings of all patents, published applications and references cited herein are incorporated by reference in their entirety.

Claims

What is claimed is:
1. A composition comprising a post-transcriptionally chemically modified double strand RNA (MdsRNA) wherein the MdsRNA comprises a double strand RNA wherein no more than about 30% of all the nucleotides independently comprise Formula (I): or an acceptable salt thereof, wherein:
B is a nucleobase;
R1 is selected from: wherein y is an integer from 1-8, x is an integer from 12-1000, a is an integer from 12- 1000, b is an integer from 12-1000, and c is an integer from 12-1000; and optionally wherein at least 2% of all the nucleotides independently comprise LMW nucleotides of Formula (III):
or an acceptable salt thereof, wherein:
B is a nucleobase;
R2 is selected from C1-C25 alkyl, C1-C25 alkenyl, C1-C25 alkynyl, C5-C12 aryl or C5-C12 heteroaryl, wherein R2 is optionally substituted with one or more substituents selected from halo, C1-12 alkyl, C1-C12 aminoalkyl, or Ci-C 12 alkoxy.
The composition of claim 1, wherein R1 is selected from:
The composition of claim 1 or claim 2, wherein x is an integer from 80-1000.
The composition of any one of claims 1-3, wherein a is an integer from 80-1000. The composition of any one of claims 1-4, wherein b is an integer from 80-1000. The composition of any one of claims 1-5, wherein c is an integer from 80-1000. The composition of any one of claims 1-6, wherein R1 has a molecular weight between 5,000 and 40,000 Da. The composition of any one of claims 1-7, wherein the MdsRNA comprises a sequence complementary to an expressed RNA in a target insect. The composition of any one of claims 1-8, wherein the MdsRNA comprises a sequence complementary to a target region in Diamondback moth AChE2, P450, DOMELESS, DOUX, MESH, P450 CYP6BFlvl, Venom or VPASE; Western corn root worm SNF7; Fall armyworm P450, CYP9A58, Cytokine receptor DOMELESS, Dredd, VPASE and Protein MESH. A method of preparing a composition comprising a post-transcriptionally chemically modified double strand RNA (MdsRNA) of claim 1; and the method comprising: (a) contacting a compound of Formula (II): with an activation agent to form a compound of Formula (IIA):
(IIA), wherein X is a suitable leaving group;
(b) contacting a compound of Formula (IA): with a compound of Formula (IIA) to form a compound of Formula (I);
(c) optionally contacting a compound of Formula (II):
(IV), with an activation agent to form a compound of Formula (IVA):
O
XAR2
(IVA), wherein X is a suitable leaving group;
(d) optionally contacting a compound of Formula (IA): (IA) with a compound of Formula (IVA) to form a compound of Formula (III). The method of claim 10, wherein (a), (b), (c), or (d) are carried out in a substantially anhydrous solvent. The method of claim 11, wherein the anhydrous solvent is selected from DMSO or DCM. The method of any one of claims 10-12, wherein (a), (b), (c), and (d) are carried out without intervening purification. The method of any one of claims 10-12, wherein there is a purification step between (a), (b), (c), and (d). The method of any one of claims 10-14, wherein an ionic solvent is added after (a) or (c). The method of claim 15, wherein the ionic solvent is selected from benzyltributyl ammonium chloride or benzyltrimethyl ammonium chloride. The method of any one of claims 10-16, wherein the activation agent is carbonyldiimidazole. The method of any one of claims 10-17, wherein the suitable leaving group is:
N The method of any one of claims 10-18, wherein (b) has a ratio of less than ten equivalents of the compound of Formula (IIA) per nucleotides of the dsRNA and (d) has a ratio of between two and fifty equivalents of the compound of Formula (VA) per nucleotide of the dsRNA. The method of any one of claims 10-19, wherein (b) has a ratio of less than two equivalents of the compound of Formula (IIA) per nucleotide of the dsRNA and (d) has a ratio of between four and twenty -five equivalents of the compound of Formula (VA) per nucleotide of the dsRNA. The method of any one of claims 10-20, wherein R1 is , The method of any one of claims 10-21, wherein x is an integer from 80-1000.
125 The method of any one of claims 10-22, wherein a is an integer from 80-1000. The method of any one of claims 10-23, wherein b is an integer from 80-1000. The method of any one of claims 10-24, wherein c is an integer from 80-1000. The method of any one of claims 10-25, wherein R1 has a molecular weight between 5,000 and 40,000 Da. The method of any one of claims 10-26, wherein the MdsRNA comprises a sequence complementary to an expressed RNA in a target insect. The method of any one of claims 10-27, wherein the MdsRNA comprises a sequence complementary to a target region in Diamondback moth AChE2, P450, DOMELESS, DOUX, MESH, P450 CYP6BFlvl, Venom or VPASE; Western corn root worm SNF7; Fall armyworm P450, CYP9A58, Cytokine receptor DOMELESS, Dredd, VPASE and Protein MESH. A method of preparing a composition comprising a post-transcriptionally chemically modified double strand RNA (MdsRNA) wherein the MdsRNA comprises a double strand RNA wherein at least about 2% of the nucleotides independently comprise Formula (VI): or an acceptable salt thereof, wherein:
B is a nucleobase;
R3 is selected from: amino acids, fatty acids, alkyl; substituted alkyl; alkenyl; substituted alkenyl; alkynyl; substituted alkynyl; aryl; substituted aryl; Cl -CIO alkyl, Cl -CIO alkenyl, or Cl -CIO alkynyl wherein alkyl and alkenyl can be linear, branched or cyclic; hydrogen; methyl; ethyl; propyl; isopropyl; butyl; isobutyl; tert-butyl; pentyl; hexyl; cyclohexyl; heptyl; octyl; nonyl; decyl; vinyl; allyl; ethynyl; benzyl; cinnamyl; C6-C14 aryl; C6-C14 substituted aryl; heterocyclyl; C5-C14 heterocyclyl; phenyl; mono or disubstituted phenyl wherein the substituents are selected from Cl -CIO alkyl, Cl -CIO alkenyl, C1-C6 alkoxy,
126 halogen, nitro, methylsulfonyl, and trifluoromethyl; 2-nitrophenyl; 4-nitrophenyl; 2;4-dinitrophenyl; 2-trifluorom ethylphenyl; 4-triflouromethylphenyl; styryl; C8- C16 substituted styryl; 2-aminophenyl; mono or di substituted 2-aminophenyl wherein the substituents are selected from Cl -CIO alkyl, Cl -CIO alkenyl, C1-C6 alkoxy, halogen, nitro, methylsulfonyl, and trifluoromethyl; N-alkyl-2- aminophenyl or N-aryl-2-aminophenyl wherein alkyl has the formula -Cmffcm+i (wherein m is an integer less than or equal to 12) and aryl is an aromatic moiety; 2- amino-3-methyl-phenyl; 2-amino-5-chlorophenyl; 2-methyl-5-chlorophenyl; N-methyla-2-minophenyl; N-ethyl-2-aminophenyl; N-propyl-2-aminophenyl; N- butyl2-aminophenyl; N-pentyl-2-aminophenyl; N-methyl-2-amino-4-nitrophenyl; 2-methyl-3-furyl; 2-methylnicotyl or N-trifluoromethyl-2-aminophenyl; silanyl; substituted silanyl; C1-C10 alkylsilanyl; C3-C12 trialkyl silanyl; C2-C12 alkoxyalkyl; C2-C12 alkoxy alkenyl; C2-C12 alkylthioalkyl; alkyl sulfonyl; C1-C10 alkyl sulfonyl; Cl -CIO haloalkyl; Cl -CIO haloalkenyl or Cl -CIO aminoalkyl; - (CH2CH2O)PCH3, -(CH2CH2O)PH, or -(CH2CH2O)PCOOR4 wherein p is an integer from 2 to 8 and R4 is H, alkyl, substituted alkyl, aryl, or substituted aryl; -(CH2CH2O)SCOOH; -CH2CH2OH; -(CH2CH2O)4OH; -(CH2CH2O)6OH;
-(CH2CH2O)8OH; -(CH2CH2O)8COOMe; -(CH2CH2O)4OMe; -(CH2CH2O)6OMe; -(CH2CH2O)8OMe; -CH2OCH3; -CH2OCH2CH3; or -CH2OCH2CH2OCH3.; and the method comprising:
(a) contacting a compound of Formula (IV): (IV), with an activation agent to form a compound of Formula (IVA):
O XAR= (IVA), wherein X is a suitable leaving group; and
(b) contacting a compound of Formula (VIA):
127 with a compound of Formula (IVA) to form a compound of Formula (VI). The method of claim 29, wherein (a) or (b) are carried out in an anhydrous solvent. The method of claim 30, wherein the anhydrous solvent is selected from DMSO or DCM. The method of any one of claims 29-31, wherein (a) and (b) are carried out without intervening purification. The method of any one of claims 29-31, wherein there is a purification step between (a) and (b). The method of any one of claims 29-33, wherein an ionic solvent is added after (a). The method of claim 34, wherein the ionic solvent is selected from benzyltributyl ammonium chloride or benzyltrimethyl ammonium chloride. The method of any one of claims 29-35, wherein the activation agent is carbonyldiimidazole. The method of any one of claims 29-36, wherein the suitable leaving group is: The method of any one of claims 29-37, wherein (b) has a ratio of between two and fifty equivalents of the compound of Formula (IVA) per nucleotide of the dsRNA. The method of any one of claims 29-38, wherein (b) has a ratio of between four and twenty -five equivalents of the compound of Formula (IVA) per nucleotide of the dsRNA. The method of any one of claims 29-39, wherein R3 is N-methyl anthranoyl (NMA), N-benzyl anthranoyl (NBA), dimethyl furoyl, -Tyr, -Trp, -Leu, octanoyl, lauroyl, linoleyl, oleoyl, nicotinoyl or benzoyl.
128 The method of any one of claims 29-40, wherein the MdsRNA comprises a sequence complementary to an expressed RNA in a target insect. The method of any one of claims 29-41, wherein the MdsRNA comprises a sequence complementary to a target region in Diamondback moth AChE2, P450, DOMELESS, DOUX, MESH, P450 CYP6BFlvl, Venom or VPASE; Western corn root worm SNF7; Fall armyworm P450, Cytokine receptor DOMELESS, Dredd, VPASE and Protein MESH. A method of modifying the expression of a polynucleotide of interest in any of the following: an insect, an acari, a fungus or a weed comprising administering a composition of any one of claims 1-9. The method of claim 43, wherein the expression is for a target region in Diamondback moth AChE2, P450, DOMELESS, DOUX, MESH, P450 CYP6BFlvl, Venom or VPASE; Western com root worm SNF7; Fall armyworm P450, VPASE, Cytokine receptor DOMELESS, Dredd, and Protein MESH. The method of claim 44, wherein the target region is in P450 CYP6BFlvl, in MESH transcript variant XI or Venom carboylesterase-6. The method of claim 44, wherein the expression is for a target region in Fall armyworm P450 CYP9A58, P450 CYP321A8, P450 CYP6B2-like, Cytokine receptor DOMELESS, Dredd, and Protein MESH transcript variant XI . The method of claim 43, wherein the expression is for target region in Western corn root worm SNF7. The method of claims 43-47, wherein the modified expression increase mortality or induce growth stunting, or stop instar development or reduces the fertility of the target insect. The method of any one of claims 43-48, wherein the modified expression reduces fertility of Lepidopteran insects. The method of claim 49, wherein the Lepidopteran insect is Diamondback moth, Gypsy moth, or Fall armyworm. The method of any one of claims 43-48, wherein the expression increase mortality or induce growth stunting, or stop instar development or reduces the fertility of Coleopteran insects. The method of claim 51, wherein the Coleopteran insect is Colorado potato beetle, Canola flea beetle or Western corn root worm. The method of claim 43, wherein the weed is Palmer Amaranth.
129
54. The method of claim 43, wherein the fungus is Fusarium Graminearum or Botrytis.
55. The method of claim 43, wherein the acari is Verroa mite.
56. The composition of any one of claims 1-9, wherein at least about 2% to about 50% of all the nucleotides independently comprise LMW nucleotides of Formula (III).
57. The composition of any one of claims 1-9 and 56, wherein R2 is N-methyl anthranoyl (NMA), N-benzyl anthranoyl (NBA), dimethyl furoyl, -Tyr, -Trp, -Leu, octanoyl, lauroyl, linoleyl, oleoyl, nicotinoyl or benzoyl.
58. The method of any one of claims 10-55, wherein at least about 2% to about 50% of all the nucleotides independently comprise LMW nucleotides of Formula (III).
130
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