Use of Paperclip RNA Structure to Inhibit Target Gene Expression

Abstract

Described herein is a “paperclip” structured dsRNA (pcRNA) that has two closed ends (FIG. 1), and was developed and tested for its ability to enter insect cells and induce RNAi. While conventional dsRNAs, with their two collinear RNA strands, enter insect cells by clathrin-mediated endocytosis, the pcRNAs can enter cells by a clathrin-independent mechanism. The new structured dsRNA can be used to deliver dsRNA to insects that either develop resistance through alterations to their conventional uptake mechanisms and to insects that are naturally refractory to dsRNAs, including lepidopteran pests.

Claims

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10. A method of reducing feeding damage to a plant from a biological pest of interest comprising: applying to the plant to be protected from the biological pest of interest an effective amount of a synthetic RNA molecule to at least a portion of the plant to be protected from the biological pest of interest, the synthetic RNA molecule folding to form a secondary structure following synthesis that comprises: a) a double stranded region comprising an RNAi sequence specific for the biological pest of interest, said RNAi sequence being at least 21 nucleotides; b) two single-stranded hairpin loops flanking either end of the double-stranded region; and c) a 5′ end and a 3′ end within the double stranded region that are closed but not covalently sealed, said synthetic RNA molecule reducing feeding damage to the plant to be protected by reducing feeding activity of the biological pest of interest following ingestion of said synthetic RNA molecule by the insect of interest.

11. The method according to claim 10 wherein the synthetic RNA molecule folds spontaneously.

12. The method according to claim 10 wherein in the folded RNA molecule, the 5′ end and the 3′ end are separated by a gap corresponding to the length of at least one phosphodiester bond.

13. The method according to claim 12 wherein the length of the double stranded RNA region is at least 23 nucleotides on a 5′ side of the gap.

14. The method according to claim 13 wherein the length of the double stranded region is at least 3 nucleotides on a 3′ side of the gap.

15. The method according to claim 10 wherein each hairpin loop independently comprises 3-15 nucleotides.

16. (canceled)

17. (canceled)

18. (canceled)

19. The method according to claim 10 wherein the synthetic RNA molecule is co-administered with a nuclease inhibitor.

20. A method of protecting a plant from feeding damage from a biological pest of interest comprising: applying to the plant to be protected from the biological pest of interest an effective amount of a synthetic RNA molecule to at least a portion of the plant to be protected from the biological pest of interest, the synthetic RNA molecule folding to form a secondary structure following synthesis that comprises: a) a double stranded region comprising an RNAi sequence specific for the biological pest of interest, said RNAi sequence being at least 21 nucleotides; b) two single-stranded hairpin loops flanking either end of the double-stranded region; and c) a 5′ end and a 3′ end within the double stranded region that are closed but not covalently sealed, said synthetic RNA molecule reducing feeding damage to the plant to be protected by reducing feeding activity of the biological pest of interest following ingestion of said synthetic RNA molecule by the insect of interest.

21. The method according to claim 20 wherein the synthetic RNA molecule folds spontaneously.

22. The method according to claim 20 wherein in the folded RNA molecule, the 5′ end and the 3′ end are separated by a gap corresponding to the length of at least one phosphodiester bond.

23. The method according to claim 22 wherein the length of the double stranded RNA region is at least 23 nucleotides on a 5′ side of the gap.

24. The method according to claim 23 wherein the length of the double stranded region is at least 3 nucleotides on a 3′ side of the gap.

25. The method according to claim 20 wherein each hairpin loop independently comprises 3-15 nucleotides.

26. (canceled)

27. (canceled)

28. (canceled)

29. The method according to claim 20 wherein the synthetic RNA molecule is co-administered with a nuclease inhibitor.

30. A method of killing a biological pest of interest comprising: applying to at least a portion of a food source of the biological pest of interest an effective amount of a synthetic RNA molecule, the synthetic RNA molecule folding to form a secondary structure following synthesis that comprises: a) a double stranded region comprising an RNAi sequence specific for the biological pest of interest, said RNAi sequence being at least 21 nucleotides; b) two single-stranded hairpin loops flanking either end of the double-stranded region; and c) a 5′ end and a 3′ end within the double stranded region that are closed but not covalently sealed, said synthetic RNA molecule killing the biological pest of interest following ingestion of said synthetic RNA molecule by the biological pest of interest.

31. The method according to claim 30 wherein the synthetic RNA molecule folds spontaneously.

32. The method according to claim 30 wherein in the folded RNA molecule, the 5′ end and the 3′ end are separated by a gap corresponding to the length of at least one phosphodiester bond.

33. The method according to claim 32 wherein the length of the double stranded RNA region is at least 23 nucleotides on a 5′ side of the gap.

34. The method according to claim 33 wherein the length of the double stranded region is at least 3 nucleotides on a 3′ side of the gap.

35. The method according to claim 30 wherein each hairpin loop independently comprises 3-15 nucleotides.

36. (canceled)

37. (canceled)

38. (canceled)

39. The method according to claim 30 wherein the synthetic RNA molecule is co-administered with a nuclease inhibitor.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0031] FIG. 1. Schematic drawing of an exemplary short paperclip RNAs (pcRNAs), with two closed ends, but not covalently sealed. The “missing” phosphodiester bond is highlighted with an asterisk. Image produced using RNAfold program, with base-pairing probabilities scored using the heat map. Nucleotides labelled red indicate high certainty of predicted secondary structure.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0032] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned hereunder are incorporated herein by reference.

[0033] As discussed herein, we have developed dsRNAs, both long and short, that are sealed on both ends to create what we are calling a “paperclip” dsRNA, otherwise known as a pcRNA. A previously described covalently closed dsRNA molecule proved ineffective at producing RNAi, as it failed to be processed by the enzyme Dicer [15], which is responsible for dicing exogenous dsRNAs into the effector 21 nt-long siRNAs that mediate RNAi. In contrast, the pcRNAs are not completely closed circles, but rather, they possess two free ends (3′ and 5′), a feature that facilitates efficient processing by Dicer and enables effective RNAi. Aside from this structural difference, unlike long linear dsRNAs, siRNAs, and hpRNAs, which enter insect cells by CME, the pcRNAs are capable of entering the cells by a clathrin-independent mechanism.

[0034] Although the molecular mechanism of this clathrin-independent uptake process has not been identified, the fact that the pcRNAs can enter insect cells by an alternative mechanism allows for the delivery of dsRNAs to insects that have either developed uptake resistance or they are naturally recalcitrant to uptake of double-stranded RNA. Furthermore, while not wishing to be bound to a particular theory or hypothesis, it is believed that the pcRNA may be entering the cell by caveolin-dependent endocytosis; lipid-raft-dependent endocytosis; or by an unidentified dsRNA transporter, perhaps distantly related to SID proteins.

[0035] As discussed herein, this “paperclip” structured dsRNA (pcRNA), shown schematically in FIG. 1 has two closed ends (FIG. 1), and was developed and tested for its ability to enter insect cells and induce RNAi. While conventional dsRNAs, with their two collinear RNA strands, enter insect cells by clathrin-mediated endocytosis, the pcRNAs can enter cells by a clathrin-independent mechanism, as discussed above. This alternative structured dsRNA can readily enter lepidopteran cells. The new structured dsRNA can therefore be used to deliver dsRNA to insects that either develop resistance through alterations to their conventional uptake mechanisms and to insects that are naturally refractory to dsRNAs, including lepidopteran pests.

[0036] This dsRNA has been developed as an alternative to conventional (linear) dsRNAs for insecticidal applications. Like other dsRNAs, it can be designed to be species-specific, thereby providing pest insect control without adversely affecting non-target species. Specifically, as discussed herein, the pcRNA includes an RNAi sequence of at least 21 nucleotides that is capable of interfering with expression of a target gene. As will be known by those of skill in the art, the target gene selected is typically a gene whose expression is critical for growth and/or development of the target organism.

[0037] Examples of suitable gene targets will be readily apparent to one of skill in the art and are within the scope of the invention. However, by way of example, suitable targets may include but are by no means limited to: a) genes involved in insect cuticle formation (for example, chitin synthase; chitinase); b) genes involved in control of metamorphosis (for example, ecdysone receptor; ultraspiracle; prothoracicotropic hormone; molting defective); c) genes involved in cellular respiration (for example cytochrome c heme lyase, superoxide dismutase; catalase); d) genes involved in DNA structure/organization (for example, dre4, ssrp, condensin); e) genes involved intracellular transport (for example, Snf7; Rab6; dynamin); and f) genes involved in neural function (for example, Fez2; Ace). Alternatively, the target gene may be a gene targets for producing sterile male insects. As will be apparent to one of skill in the art, these gene targets (for example, Tssk1, Vasa, Doublesex, Zpg, and gas8) may be used for sterile insect technique applications.

[0038] As discussed herein, pcRNAs enter insect cells by a clathrin-independent manner, unlike other conventional dsRNAs that use clathrin-mediated endocytosis as the main mode of cellular entry. pcRNAs can therefore provide an alternative dsRNA to overcome dsRNA uptake resistance, which has already been demonstrated in lab-selected insects.

[0039] Furthermore, as discussed herein, pcRNAs enter lepidopteran (moth) cells and induce RNAi, unlike conventional dsRNAs, which generally fail to induce RNAi in this large group of ecumenically-important pests. Hence, these new molecules can be used to control pests that are currently refractory to dsRNA.

[0040] Described herein is a synthetic RNA molecule that, following synthesis, folds to form a secondary structure comprising:

[0041] a) a double stranded region comprising an RNAi sequence of at least 21 nucleotides;

[0042] b) two single-stranded hairpin loops flanking either end of the double-stranded region; and

[0043] c) a 5′ end and a 3′ end within the double stranded region that are closed but not covalently sealed.

[0044] As will be appreciated by one of skill in the art, the synthetic RNA molecule is a single stranded RNA in which both ends fold over on itself to create a perfectly duplexed double-stranded RNA, but lacking a phosphodiester bond where the 5′ and 3′ ends meet.

[0045] As discussed herein, the RNAi sequence is a nucleotide sequence that is specific for a target gene of biological subject of interest.

[0046] In some embodiments, the biological subject of interest is a biological pest, for example, an insect.

[0047] As discussed herein, the synthetic RNA molecule of the invention is designed to fold such that the 5′ end and the 3′ end are proximal to one another and are separated by at least one phosphodiester bond. As will be apparent to one of skill in the art, if the “missing” phosphodiester bond that corresponds to the gap between the 5′ end and the 3′ end of the synthetic RNA molecule was present, the synthetic RNA molecule would be covalently closed.

[0048] As discussed herein, the folded RNA molecule is arranged to fold spontaneously such that the 5′ end and the 3′ end are separated by a gap corresponding to the length of at least one phosphodiester bond.

[0049] The fact that the 5′ end and the 3′ end are proximal but not covalently closed, that is, are separated by at least a missing phosphodiester bond is important as closed circular dsRNA are not effectively processed into siRNAs (the effector molecules of RNAi) by Dicer [15].

[0050] Furthermore, as discussed herein, the synthetic RNA molecule of the invention is designed such that the gap corresponding to at least the missing phosphodiester bond is positioned such that the double stranded region comprises at least 23 nt on the 5′ side of the gap. As will be apparent to one of skill in the art, these at least 23 nt of the double stranded RNA region correspond to the RNAi sequence. Furthermore, this arrangement allows for Dicer to generate a 21 bp siRNA, as discussed herein. The two extra nucleotides ensure that Dicer can cut dsRNA efficiently.

[0051] The other side or 3′ side of the gap or the “missing” phosphodiester bond is, as discussed herein, a minimum of 3 nt to ensure stability of the 3′ end. That is, at least 3 nt are required so that the paperclip structure is held securely as well as so that the secondary structure of the synthetic RNA molecule forms as desired when folding spontaneously.

[0052] As will be apparent to one of skill in the art, as used herein, “spontaneously” in regards the folding of the synthetic RNA molecule refers to the fact that it is not necessary to add any additional factors or agents known in the art to promote proper folding of the synthetic RNA molecule so that the desired RNA secondary structure forms.

[0053] As will be appreciated by one of skill in the art, the synthetic RNA molecule may be of any suitable length. As will be known by those of skill in the art, typically, dsRNAs up to 300 bp are effective at knocking down transcripts, anything larger is not usually found to be any more effective, and there is greater potential for synthesis errors, thereby reducing the overall effectiveness of the synthetic RNA molecule.

[0054] Furthermore, as discussed herein, while not wishing to be bound to a particular theory or hypothesis, it is believed that relatively smaller pcRNAs may be binding more efficiently to the endocytic protein machinery or that larger pcRNAs may not hold together as firmly, as the dsRNA regions may open and close in places, leaving them subject to refolding or possible endonuclease attack.

[0055] As discussed above, the length of the double stranded RNA region is at least 23 nucleotides on a 5′ side of the gap. That is, in the double stranded region of the synthetic RNA molecule when the synthetic RNA molecule is folded, there is at least a 23 nt double stranded region starting at the 5′ end of the synthetic RNA molecule.

[0056] Dicer will generate 21 nt short interfering RNAs (siRNAs) from any length of dsRNA >21 nt in length. Specifically, Dicer slides along a double-stranded RNA molecule and cuts at 21 nt intervals. Hence, a double stranded region comprising 42 nt would be cut into two siRNA molecules by Dicer, while a 100 bp dsRNA would produce 4 siRNAs. Once the siRNA molecules have been generated, the RNA interference machinery [e.g. RISC] in the cell is able to use the siRNA to impair expression of the target gene as discussed herein. Accordingly, while the RNAi sequence is at least 23 nts, it may be longer, for example, 44 nts, 65 nt, 86 nts, 107 nt, 128 nt, 149 nts, for generation of multiple, different siRNA molecules, as discussed herein. Furthermore, this 5′ end double stranded region which comprises the RNAi sequence may be any suitable length, bearing in mind synthesis fidelity and mis-folding concerns as will be apparent to those of skill in the art and as discussed herein.

[0057] As discussed above, the length of the double stranded region of the synthetic RNA molecule of the invention starting with the 3′ end of the synthetic RNA molecule is at least 3 nucleotides, that is, there is at least 3 nt of double stranded RNA on a 3′ side of the gap. As discussed above, this 3 nt sequence is the minimum sequence required from stability of the RNA molecule of the invention, specifically, so that the desired secondary structure forms spontaneously and stably. As will be appreciated by one of skill in the art, this 3′ end double stranded region may be of any suitable length, as greater lengths will increase stability of the synthetic RNA molecule when folded, bearing in mind synthesis fidelity and mis-folding concerns as will be apparent to those of skill in the art and as discussed herein.

[0058] As will be apparent to one of skill in the art on consultation with at least FIG. 1, the RNA sequences that complementarily bind together to form the double stranded region are each separated by a hairpin loop, wherein each hairpin loop independently comprises 3-15 nucleotides, 3-12 nucleotides, 6-15 nucleotides, or 6-12 nucleotides.

[0059] As will be appreciated by one of skill in the art, the hairpin loops can be variable in size. For example, loops as short a 3 nt may be used in some hairpins, but these short loops may not always fold over 100% of the time. Loops between 6 and 15 nt are often used for hairpins we tested loops of 6 nts, 9 nts, and 12 nts and observed that 9 bp worked consistently. Specifically, while all worked equally well, 9 nt loops gave more consistent knockdown. Ideally, the loop should be kept as small as possible to make it easy to synthesize. Furthermore, while not wishing to be bound to a particular theory or hypothesis, it is believed that loops longer than 15 nts, for example, longer than 12 nt, can potentially generate their own internal secondary structure, and were avoided.

[0060] According to another aspect of the invention, there is provided a method of reducing feeding damage to a plant from a biological pest of interest comprising:

[0061] applying to the plant to be protected from the biological pest of interest an effective amount of a synthetic RNA molecule to at least a portion of the plant to be protected from the biological pest of interest, the synthetic RNA molecule folding to form a secondary structure following synthesis that comprises:

[0062] a) a double stranded region comprising an RNAi sequence specific for the biological pest of interest, said RNAi sequence being at least 21 nucleotides;

[0063] b) two single-stranded hairpin loops flanking either end of the double-stranded region; and

[0064] c) a 5′ end and a 3′ end within the double stranded region that are closed but not covalently sealed,

[0065] said synthetic RNA molecule reducing feeding damage to the plant to be protected by reducing feeding activity of the biological pest of interest following ingestion of said synthetic RNA molecule by the insect of interest.

[0066] The synthetic RNA molecule may be co-administered with a nuclease inhibitor.

[0067] Alternatively, the synthetic RNA molecule of the invention may be co-administered with a second synthetic RNA molecule, for example, a synthetic RNA molecule of the invention comprising a different RNAi sequence than the first synthetic RNA molecule. Alternatively, the second synthetic RNA molecule may be a dsRNA molecule such as those known in the prior art for generating siRNA transcripts.

[0068] According to another aspect of the invention, there is provided a method of protecting a plant from feeding damage from a biological pest of interest comprising:

[0069] applying to the plant to be protected from the biological pest of interest an effective amount of a synthetic RNA molecule to at least a portion of the plant to be protected from the biological pest of interest, the synthetic RNA molecule folding to form a secondary structure following synthesis that comprises: [0070] a) a double stranded region comprising an RNAi sequence specific for the biological pest of interest, said RNAi sequence being at least 21 nucleotides; [0071] b) two single-stranded hairpin loops flanking either end of the double-stranded region; and [0072] c) a 5′ end and a 3′ end within the double stranded region that are closed but not covalently sealed,

[0073] said synthetic RNA molecule reducing feeding damage to the plant to be protected by reducing feeding activity of the biological pest of interest following ingestion of said synthetic RNA molecule by the insect of interest.

[0074] According to another aspect of the invention, there is provided a method of killing a biological pest of interest comprising:

[0075] applying to at least a portion of a food source of the biological pest of interest an effective amount of a synthetic RNA molecule, the synthetic RNA molecule folding to form a secondary structure following synthesis that comprises: [0076] a) a double stranded region comprising an RNAi sequence specific for the biological pest of interest, said RNAi sequence being at least 21 nucleotides; [0077] b) two single-stranded hairpin loops flanking either end of the double-stranded region; and [0078] c) a 5′ end and a 3′ end within the double stranded region that are closed but not covalently sealed,

[0079] said synthetic RNA molecule killing the biological pest of interest following ingestion of said synthetic RNA molecule by the biological pest of interest.

[0080] As will be appreciated by one of skill in the art, an “effective amount” as used herein refers to an amount that is sufficient to achieve the desired result, that is, to reduce feeding activity of the biological pest of interest or to reduce damage to a plant from a biological pest of interest compared to an untreated control plant of similar size, age and type or to a food source of the biological pest of interest to kill the biological pest of interest. As such, an “effective amount” may depend on several factors, such as the environmental conditions, weather conditions, and the number of the biological pest of interest that the plant or food source may encounter or is expected to encounter, which can be estimated using any of a variety of means known in the art.

[0081] As discussed herein, the synthetic RNA molecule of the invention may be applied to at least one leaf of the plant to be protected or to at least a portion of the food source. The synthetic RNA molecule may be applied to the plant to be protected or to the food source at a concentration of at least about 0.1 ng per mm.sup.2, or at least about 0.5 ng per mm.sup.2, that is, per mm.sup.2 of plant material being coated, for example, one or more leaves.

[0082] As discussed herein, the synthetic RNA molecule of the invention was applied onto the leaves in just water. However, it is of note that there are a variety of formulation additives known in the art that act for example as spreaders, stickers and penetrants when applied to plants or other food sources, for example, to leaves of plants. As one of skill in the art will appreciate, such formulation additives can be tested and optimized by one of routine skill in the art and are within the scope of the invention.

[0083] In some embodiments of the invention, the effective amount of the synthetic RNA molecule of the invention is co-administered with an effective amount of a nuclease inhibitor.

[0084] Examples of known nuclease inhibitors include but are by no means limited to strong protein denaturants, such as guanidinium isothiocyanate; anionic polymers such as polyvinylsulfonic acid and protein-based nuclease inhibitors, such as: GamS Nuclease Inhibitor; Superase protein-based RNase inhibitor; and Recombinant RNase Inhibitor.

[0085] The invention will now be further explained and/or elucidated by way of examples; however, the invention is not necessarily limited to or by the examples.

Example 1—PcRNAs can Induce RNAi when Delivered to Insect Cells

[0086] Two genes were targeted for RNAi-mediated knockdown in cultured Aedes aegypti mosquito cells: snf7 and kermit. To produce long linear dsRNAs, gene fragments of approximately 200 bp were PCR-amplified from mosquito cDNA. All PCR primers contained T7 sequences to facilitate RNA synthesis in in vitro transcription reactions, to produce dsRNA. A list of the dsRNAs used is provided in Table 1. For the other dsRNAs (siRNAs, long/short hpRNAs, and long/short pcRNA), DNA oligonucleotides, also with T7 linkers, were purchased from IDT, ligated by slow cooling (from 95oC down to 25° C.), and then used directly in in vitro transcription reactions.

[0087] Mosquito cells were transfected with the different lengths and structures of dsRNA using Lipofectamine-3000 transfection reagent, to evaluate their ability to knock down the targeted genes if delivered directly into the cells. The long dsRNAs targeting Snf7 and kermit were 204 and 213 bp, respectively, whereas the siRNA, short hpRNA, and short pcRNA each contained a contiguous 23 bp long dsRNA section. For the short dsRNAs, a 10× higher concentration of each was delivered to the cells, to provide approximately the same number of siRNA to the cells as the long dsRNA would generate from dicer processing. The values shown in Table 2 represent the means and standard errors of 10 replicate well treatments. No significant difference in % knockdown was observed for any of the different dsRNA treatments (ANOVA, p >0.7 for all comparisons).

[0088] As can be seen in Table 2, all of the dsRNAs, regardless of the length or shape, could mediate target transcript knockdown, as assessed qRT-PCR analyses. For the qRT-PCR analyses, two reference genes, actin and RpS7, were used to normalize the transcript levels in the different treatments. Using the transfection reagent to deliver the dsRNAs to the mosquito cells resulted in similar levels of knockdown of each of the two transcripts, regardless of their lengths or structure. This result indicates that the pcRNA is a suitable substrate for Dicer and the other RNAi machinery, for both the long and short pcRNA structures.

[0089] Two genes were similarly targeted for RNAi mediated knockdown in cultured Sf9 fall armyworm cells: actin and the putative orthologue of kermit. Using the same approach, knockdown of each gene was achieved using each of the different lengths and structures of dsRNA in this lepidopteran cell line, indicating that the RNAi machinery is functional and that the pcRNA could also be used as an effective substrate for dicer and the other RNAi machinery, provided the dsRNAs were delivered by a transfection reagent. The results are shown in Table 3, which demonstrates that all dsRNA molecules were equally effective (when doses were adjusted to accommodate different numbers of siRNA generated by the long dsRNAs). All values represent the means and standard errors from 10 replicate wells of cells. The extent of knockdown was similar for all treatments (ANOVA, p>0.5)

Example 2—Treatment of Mosquito Cells with Clathrin Inhibitors Prevents RNAi, Except when pcRNAs are Delivered

[0090] To examine DsRNA uptake in cultured Aedes aegypti cells, CCL125 cells were seeded to six well plates. Once cells and reached 80% confluence, culture media was withdrawn and cells were washed with PBS, and were then treated for 30 minutes to one of two clathrin inhibitors (0.2 μM bafilomycin-A1, 10 μM chlorpromazine), a micropinocytosis/phagocytosis inhibitor (10 μM cytochalsin-D), and a calveolae-dependent endocytosis inhibitor (2 mM methyl βcyclodextrin). The cells were washed with PBS and different lengths and structures of dsRNA targeting actin transcripts were added to the media. After two hours, the cells were washed to remove excess dsRNA, and 24 hours later the cells were harvested, RNA extracted, and the level of actin transcripts was evaluated by qRT-PCR. Table 4 shows the percent transcript knockdown of snf7 or kermit following exposure of the A. aegypti CCL-125 cells to different structured dsRNAs in the presence or absence of chlorpromazine. Values represent the means and standard errors of 5 replicate experiments consisting of 6 well each. Treatments demonstrating significantly impaired RNAi, relative to the “no inhibit control” are highlighted with an asterisk.

[0091] As can be seen in Table 4, in the absence of chlorpromazine, the long linear dsRNA, the long hpRNA, the long pcRNA, and the short pcRNA were equally effective at knocking down the targeted transcripts, whereas the siRNA and shRNA were less effective, but nevertheless still caused a knockdown. Only the clathrin inhibitors were observed to inhibit RNAi-mediated knockdown of actin, using long linear dsRNA, siRNA, and hpRNA. The other chemical inhibitors had no impact on the ability of the cells to take up double-stranded RNA and mediate RNAi. Interestingly, both the long and short pcRNAs were able to enter mosquito cells and induce RNAi even in the presence of the clathrin inhibitors. This finding indicates that CME is important for the uptake of long linear dsRNA, siRNAs, and hpRNAs but is not needed for uptake of pcRNA.

Example 3—PcRNA can Enter Spodoptera (Sf9) Cells Whereas Linear or Hairpin dsRNAs Cannot

[0092] DsRNA targeting actin transcripts were applied to Spodoptera frugiperda (fall armyworm) Sf9 cells, and after two hours, the cells were washed and 24 hours later, the cells were harvested, RNA extracted, and qRT-PCR was used to assess knockdown of actin transcripts. Table 5 shows the percent knockdown of actin transcripts in Sf9 cells treated with different dsRNAs. The values represent the means and averages of 5 replicates of 4 wells of cells.

[0093] As can be seen in Table 5, the linear or hairpin dsRNAs failed to induce knockdown in the lepidopteran cells, which is not unexpected, given that these cells are known to be refractory to exogenous dsRNA. In contrast, the long and the short pcRNAs were able to enter the cells, but interestingly, the short pcRNAs were able to mediate a three-fold stronger knockdown of the actin transcripts. These findings indicate that the pcRNA structure facilitates entry into these lepidopteran cells, and the short pcRNAs appear to either enter the cells more readily or are processed more efficiently than the longer pcRNAs.

Example 4—Treatment of Mosquito Larvae with the Clathrin Inhibitor Prevents RNAi, Unless pcRNAs are Applied

[0094] Aedes aegypti fourth instar larvae were treated with 2.5 μM chlorpromazine for two hours to inhibit clathrin, then rinsed in water for 15 minutes, and then soaked in small groups of five in different dsRNA lengths and structures targeting the snf7 gene's transcripts for three hours. The larvae were then transferred to water containing food, and after 30 hours, larvae were sacrificed, RNA extracted, and qRT-PCR was used to measure snf7 transcript levels. Table 6 shows the results of RNAi-mediated knockdown of snf7 by alternative dsRNA molecules in A. aegypti larvae treated with chlorpromazine. Value represent the mean and standard deviations of 5 replicate experiments of pools of 5 larvae. Treatments with significant knockdown, even in the presence of chlorpromazine, are highlighted with an asterisk.

[0095] As can be seen in Table 6, chlorpromazine treatment completely abolished transcript knockdown mediated by long dsRNA, siRNA, long and short and hpRNAs. Larvae treated with pcRNA demonstrated strong snf7 transcript knockdown, even when treated with chlorpromazine. These findings indicate that CME is the primary means of dsRNA entry into mosquito gut cells, but that pcRNA is able to enter the gut cells independently of CME mechanisms.

Example 5—Treatment of Spodoptera Larvae with pcRNA Induces RNAi, Whereas Other dsRNA Structures Fail

[0096] First instar Spodoptera frugiperda larvae were starved for 12 hours and then droplet fed different dsRNAs targeting the actin gene. The insects were transferred to an artificial diet for 24 hours, and were then sacrificed, RNA extracted, and qRT-PCR was used to assess actin transcript levels. Table 7 shows the results of the RNAi-mediated knockdown of actin transcripts by alternative dsRNA molecules in first instar S. frugiperda larvae. The values represent the means and standard errors for 15 individual insects.

[0097] As can be seen in Table 7, linear dsRNAs (long or short) and hairpin RNAs (long and short) failed to induce knockdown, but larvae that fed short pcRNAs showed significant 52% knockdown of actin transcripts. These findings indicate the pcRNAs can mediate RNAi in an dsRNA insensitive insect.

[0098] While the preferred embodiments of the invention have been described above, it will be recognized and understood that various modifications may be made therein, and the appended claims are intended to cover all such modifications which may fall within the spirit and scope of the invention.

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TABLE-US-00001 TABLE 1 List of dsRNAs used in this study. Double- stranded Gene DsRNA sequences length Aedes Snf7 Derived from Vectorbase Accession number: AAEL001698 Long-dsRNA AGCTGAGGGACATCGAGAATATGCTGACCAAAAAGCAGGAAT 204 bp SEQ ID No. 1 TCCTGGAAAAAAAGATCGAAGTTGAATTGGACACTGCGCGAAA GAACGGAACCAAAAACAAGAGAGCTGCACTTCAGGCACTCAA ACGAAAGAAGCGATATGAGAAACAACTGACTCAGATCGATGG CACTTTGTCCACGATTGAAATGCAACGGGAAGCCT SIRNA AGATTCAAATTGAGATTGAAGAC  23 bp SEQ ID No. 2 Long hpRNA AGCTGAGGGACATCGAGAATATGCTGACCAAAAAGCAGGAAT 106 bp SEQ ID No. 3 TCCTGGAAAAAAAGATCGAAGTTGAATTGGACACTGCGCGAAA GAACGGAACCAAAAACAAGAGAAAAAAAAACTCTTGTTTTTGG TTCCGTTCTTTCGCGCAGTGTCCAATTCAACTTCGATCTTTTTT TCCAGGAATTCCTGCTTTTTGGTCAGCATATTCTCGATGTCCCT CAGCT shRNA AGATTCAAATTGAGATTGAAGACAAAAAAAAAGTCTTCAATCTC  23 bp SEQ ID No. 4 AATTTGAATCT Long pcRNA AGCTGAGGGACATCGAGAATATGCTGACCAAAAAGCAGGAAT 106 bp SEQ ID No. 5 TCCTGGAAAAAAAGATCGAAGTTGAATTGGACACTGCGCGAAA GAACGGAACCAAAAACAAGAGAAAAAAAAACTCTTGTTTTTGG TTCCGTTCTTTCGCGCAGTGTCCAATTCAACTTCGATCTTTTTT TCCAGGAATTCCTGCTTTTTGGTCAGCATATTCTCGATGTCCCT CAGCTTCAGAAAAAAAAACAGA Short pcRNA AGATTCAAATTGAGATTGAAGACAAAAAAAAAGTCTTCAATCTC  28 bp SEQ ID No. 6 AATTTGAAT CTTATCTAAAAAAAAAAGATA Aedes kermit Derived from Vectorbase Accession number: AAEL012658 Long-dsRNA CTACGAGAATAACAACGGCTTTAACGGCAAGAATCTGGCAGG 213 bp SEQ ID No. 7 GAGCACAATGCCCACGGCACCGCACATCGATGACCTTAACGG GAGCCACGAGGCCAACGTGACCAAGCCACAGCTCGTGTTCAA CTGCCAGCTGGCCCACGGCAGTCCAACGGGCTTCATCACCGG GTTTGCCAGCGTCAAGGAGCTGTACCAGAAGATCGCCGAATG CTA SIRNA GATACGACACTTCATGGAATGTT  23 bp SEQ ID No. 8 Long hairpin CTACGAGAATAACAACGGCTTTAACGGCAAGAATCTGGCAGG 106 bp SEQ ID No. 9 GAGCACAATGCCCACGGCACCGCACATCGATGACCTTAACGG GAGCCACGAGGCCAACGTGACCAAAAAAAAAAATTGGTCACG TTGGCCTCGTGGCTCCCGTTAAGGTCATCGATGTGCGGTGCC GTGGGCATTGTGCTCCCTGCCAGATTCTTGCCGTTAAAGCCGT TGTTATTCTCGTAG shRNA GATACGACACTTCATGGAATGTTAAAAAAAAAAACATTCCATGA  23 bp SEQ ID No. 10 AGTGTCGT ATC Long pcRNA CTACGAGAATAACAACGGCTTTAACGGCAAGAATCTGGCAGG SEQ ID No. 11 GAGCACAATGCCCACGGCACCGCACATCGATGACCTTAACGG GAGCCACGAGGCCAACGTGACCAAAAAAAAAAATTGGTCACG TTGGCCTCGTGGCTCCCGTTAAGGTCATCGATGTGCGGTGCC GTGGGCATTGTGCTCCCTGCCAGATTCTTGCCGTTAAAGCCGT TGTTATTCTCGTAGAAAAGAAAAAAAAACTTTT pcRNA GATACGACACTTCATGGAATGTTAAAAAAAAAAACATTCCATGA  28 bp SEQ ID No. 12 AGTGTCGTA TCAGAATAAAAAAAAAATTCT Spodoptera actin Derived from NCBI accession: KY231202.1 Long dsRNA CGTTCGTGACATCAAGGAGAAGCTGTGCTATGTCGCCCTCGA 200 bp SEQ ID No. 13 CTTCGAGCAGGAGATGGCCACCGCTGCCGCCTCCACCTCCCT CGAGAAGTCCTACGAACTTCCCGACGGTCAGGTCATCACCAT CGGTAACGAGAGGTTCCGTTGCCCTGAAGCCCTCTTCCAGCC TTCCTTCTTGGGTATGGAATCTTGCGGTATCC SIRNA CGTTCGTGACATCAAGGAGAA  23 bp SEQ ID No. 14 Long hairpin CGTTCGTGACATCAAGGAGAAGCTGTGCTATGTCGCCCTCGA 108 bp SEQ ID No. 15 CTTCGAGCAGGAGATGGCCACCGCTGCCGCCTCCACCTCCCT CGAGAAGTCCTACGAACTTCCCGACAAAAAAAAAGTCGGGAA GTTCGTAGGACTTCTCGAGGGAGGTGGAGGCGGCAGCGGTG GCCATCTCCTGCTCGAAGTCGAGGGCGACATAGCACAGCTTC TCCTTGATGTCACGAACG Short hpRNA CGTTCGTGACATCAAGGAGAAAAAAAAAAATTCTCCTTGATGT  23 bp SEQ ID No. 16 CACGAACG Long pcRNA CGTTCGTGACATCAAGGAGAAGCTGTGCTATGTCGCCCTCGA 108 bp SEQ ID No. 17 CTTCGAGCAGGAGATGGCCACCGCTGCCGCCTCCACCTCCCT CGAGAAGTCCTACGAACTTCCCGACAAAAAAAAAGTCGGGAA GTTCGTAGGACTTCTCGAGGGAGGTGGAGGCGGCAGCGGTG GCCATCTCCTGCTCGAAGTCGAGGGCGACATAGCACAGCTTC TCCTTGATGTCACGAACGATTTCAAAAAAAAAGAAAT Short pcRNA CGTTCGTGACATCAAGGAGAAAAAAAAAAATTCTCCTTGATGT  23 bp SEQ ID No. 18 CACGAACGATTTCAAAAAAAAAGAAAT Derived from: Spodoptera genome_corn_v3.0_spodoptera_frugiperda_v3.0: scaffold kermit 3463 Long dsRNA AATCAACAGTTTGCTGGAATCGTTCATGGGCATCAACGACTCC 205 bp SEQ ID No. 19 GAGTTAGCATCTCAGATGTGGGATCTTGCCGAAGGAAAAGCA SIRNA AATTCTATGCAACTAGCTGAAGCCATCGATAATAGTGACTTACA SEQ ID No. 20 AGAATTCGGATTCACTGACGAGTTTATAATTGAATTATGGGGT GTTATTACTGACGCTAGATCTGGTAGACTCAGT ACGCTAGATCTGGTAGACTCAGT Long hairpin AATCAACAGTTTGCTGGAATCGTTCATGGGCATCAACGACTCC  23 bp SEQ ID No. 21 GAGTTAGCATCTCAGATGTGGGATCTTGCCGAAGGAAAAGCA AATTCTATGCAACTAGCTGAAGCCAAAAAAAAAGGCTTCAGCT 205 bp AGTTGCATAGAATTTGCTTTTCCTTCGGCAAGATCCCACATCT GAGATGCTAACTCGGAGTCGTTGATGCCCATGAACGATTCCA GCAAACTGTTGATT short hp ACGCTAGATCTGGTAGACTCAGTAAAAAACTGAGTCTACCAGA  23 bp SEQ ID No. 22 TCTAGCGGT Long pcRNA AATCAACAGTTTGCTGGAATCGTTCATGGGCATCAACGACTCC 205 bp SEQ ID No. 23 GAGTTAGCATCTCAGATGTGGGATCTTGCCGAAGGAAAAGCA AATTCTATGCAACTAGCTGAAGCCAAAAAAAAAGGCTTCAGCT AGTTGCATAGAATTTGCTTTTCCTTCGGCAAGATCCCACATCT GAGATGCTAACTCGGAGTCGTTGATGCCCATGAACGATTCCA GCAAACTGTTGATTTTCTAAAAAAAAAAGAA Short pcRNA ACGCTAGATCTGGTAGACTCAGTAAAAAACTGAGTCTACCAGA  23 bp SEQ ID No. 24 TCTAGCGTTTCTAAAAAAAAAAGAA

TABLE-US-00002 TABLE 2 Knockdown of two genes using different lengths and structured dsRNAs in Ae. aegypti CCL125 cells using Lipofectamine ™(3000) transfection reagent to deliver the dsRNAs to the cells. The long dsRNAs targeting Snf7 and kermit were 204 and 213 bp, respectively, whereas the siRNA, short hpRNA, and short pcRNA each contained a contiguous 23 bp long dsRNA section. Hence, the cells were treated with 10x higher doses of the short dsRNAs to provide the cells with equivalent numbers of Dicer-generated (in vivo) siRNAs. The values represent the means and standard errors of 10 replicate well treatments. No significant difference in % knockdown was observed for any of the different dsRNA treatments (ANOVA, p > .7 for all comparisons). Target Gene dsRNA (dose/well) % Knockdown Snf7 Long (20 pmol) 91 ± 3 siRNA (200 pmol) 88 ± 4 Long hpRNA (20 pmol) 92 ± 5 shRNA (200 pmol) 93 ± 2 Long pcRNA 93 ± 6 pcRNA (200 pmol) 94 ± 3 Kermit Long (20 pmol) 88 ± 5 siRNA (200 pmol) 86 ± 4 Long hpRNA 84 ± 5 shRNA (200 pmol) 89 ± 3 Long pcRNA 94 ± 4 pcRNA (200 pmol) 91 ± 4

TABLE-US-00003 TABLE 3 Knockdown of two genes in Spodoptera Sf9 cells when different dsRNAs are delivered by transfection reagents. All dsRNA molecules were equally effective (when doses were adjusted to accommodate different numbers of siRNA generated by the long dsRNAs). All values represent the means and standard errors from 10 replicate wells of cells. The extent of knockdown was similar for all treatments (ANOVA, p > 0.5) Target Gene dsRNA (dose/well) % Knockdown actin Long (20 pmol) 83 ± 5 siRNA (200 pmol) 80 ± 6 Long hpRNA (20 pmol) 86 ± 7 shRNA (200 pmol) 79 ± 5 Long pcRNA 83 ± 5 pcRNA (200 pmol) 78 ± 5 kermit Long (20 pmol) 78 ± 6 siRNA (200 pmol) 73 ± 5 Long hpRNA 74 ± 6 shRNA (200 pmol) 72 ± 6 Long pcRNA 74 ± 6 pcRNA (200 pmol) 77 ± 5

TABLE-US-00004 TABLE 4 Percent transcript knockdown of two target genes (snf7 or kermit) following exposure of A. aegypti CCL-125 cells to different structured dsRNAs in the presence or absence of chlorpromazine, an inhibitor of clathrin-mediated endocytosis. Values represent the means and standard errors of 5 replicate experiments consisting of 6 well each. Treatments demonstrating significantly impaired RNAi, relative to the “no inhibit control” are highlighted with an asterisk. DSRNA TREATMENT NO INHIBITOR +CPZ +CYTO-D +MBC SNF7 NO DSRNA  0.00 ± 2.35 — — — LONG-DSRNA 86.65 ± 1.25 8.22 ± 2.59* 84.45 ± 2.31 83.89 ± 2.43 SIRNA 25.14 ± 2.17 11.56 ± 2.11*  26.34 ± 2.42 24.98 ± 2.16 LONG HPRNA 88.37 ± 2.36 13.22 ± 3.24*  84.76 ± 2.07 86.94 ± 2.16 SHRNA 32.40 ± 2.23 12.98 ± 2.99*  33.03 ± 2.47 31.86 ± 2.78 LONG PCRNA 89.56 ± 2.09 85.43 ± 2.46  85.89 ± 2.19 87.56 ± 2.44 PCRNA 82.64 ± 2.34 74.58 ± 2.83  82.49 ± 2.66 81.77 ± 3.15 KERMIT NO DSRNA  0.00 ± 2.10 — — — LONG DSRNA 76.73 ± 1.68 9.27 ± 2.63* 75.53 ± 2.06 77.83 ± 2.23 SIRNA 38.64 ± 2.47 4.86 ± 1.54* 37.09 ± 3.28 39.53 ± 2.97 LONG HPRNA 79.44 ± 2.82 6.41 ± 2.03* 78.51 ± 1.98 80.46 ± 3.12 SHRNA 65.02 ± 1.52 9.96 ± 1.43* 67.22 ± 3.11 64.91 ± 2.47 LONG PCRNA 80.23 ± 3.38 74.21 ± 4.51  80.01 ± 3.26 79.88 ± 2.93 PCRNA 75.42 ± 1.85 66.90 ± 1.90  76.37 ± 2.44 74.28 ± 3.17

TABLE-US-00005 TABLE 5 Percent knockdown of actin transcripts in Sf9 cells treated with different dsRNAs. The values represent the means and averages of 5 replicates of 4 wells of cells. ACTIN SIGNIFICANT FROM KNOCKDOWN NEGATIVE CONTROL? DSRNA (%) (Y/N) NONE (NEG. 0.00 ± 4.22 — CONTROL) LONG DSRNA 4.25 ± 3.07 N SIRNA 0.12 ± 3.36 N LONG HPRNA 4.55 ± 3.72 N SHORT HPRNA 4.86 ± 3.60 N LONG PCRNA 17.36 ± 3.81  Y SHORT PCRNA 51.21 ± 4.63  Y

TABLE-US-00006 TABLE 6 RNAi-mediated knockdown of snf7 by alternative dsRNA molecules in A. aegypti larvae treated with chlorpromazine. Value represent the mean and standard deviations of 5 replicate experiments of pools of 5 larvae. Treatments with significant knockdown, even in the presence of chlorpromazine are highlighted with an asterisk. DSRNA TREATMENT −CPZ +CPZ NO DSRNA  0.00 ± 3.97 2.66 ± 3.01 LONG-DSRNA 59.42 ± 3.09 4.98 ± 4.10 SIRNA 17.12 ± 4.83 1.30 ± 3.02 LONG HPRNA 64.27 ± 4.12 3.44 ± 2.32 SHORT HPRNA 41.40 ± 5.24 5.00 ± 3.94 LONG PCRNA 64.94 ± 4.21 41.38 ± 5.46* PCRNA 45.82 ± 5.30 33.04 ± 4.66*

TABLE-US-00007 TABLE 7 RNAi-mediated knockdown of actin transcripts by alternative dsRNA molecules in first instar S. frugiperda larvae. The values represent the means and standard errors for 15 individual insects. SIGNIFICANTLY PERCENT DIFFERENT FROM ACTIN NEGATIVE CONTROL DSRNA TREATMENT KNOCKDOWN (YES/NO) NO DSRNA (NEG. 0.00 ± 5.62 — CONT.) LONG-DSRNA 4.01 ± 4.33 N SIRNA 2.34 ± 2.01 N LONG HPRNA 6.55 ± 4.83 N SHORT HPRNA 3.79 ± 4.04 N LONG PCRNA 24.36 ± 7.44  Y SHORT PCRNA 52.2. ± 8.23  Y