Asymmetric RNA molecules

Abstract

The present invention relates to asymmetric RNA molecules, precursors thereof, and their use in gene silencing.

Description

[0001] ASYMMETRIC RNA MOLECULES

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to asymmetric RNA molecules, precursors thereof, and their use in gene silencing.

[0004] BACKGROUND OF THE INVENTION

[0005] RNA silencing is an evolutionarily conserved gene silencing mechanism in eukaryotes that is induced by double-stranded RNA (dsRNA) which may be of a form designated hairpin structured RNA (hpRNA). In the basic RNA silencing pathway, dsRNA is processed by Dicer proteins into short, 20-25 nucleotide (nt) small RNA duplexes, of which one strand is bound to Argonaute (AGO) proteins to form an RNA- induced silencing complex (RISC). This silencing complex uses the small RNA as a guide to find and bind to complementary single-stranded RNA, where the AGO protein cleaves the RNA resulting in its degradation, or translation of the target RNA is reduced without target RNA cleavage.

[0006] In plants, multiple RNA silencing pathways exist, including microRNA (miRNA), trans-acting small interfering RNA (tasiRNA), repeat-associated siRNA (rasiRNA) and exogenic (virus and transgene) siRNA (exosiRNA) pathways. miRNAs are 20-24-nt small RNAs processed in the nucleus by Dicer-like 1 (DCL1) from short stem -loop precursor RNAs that are transcribed by RNA polymerase II from MIR genes. tasiRNAs are phased siRNAs of primarily 2 Int in size derived from DCL4 processing of long dsRNA synthesised by RNA-dependent RNA polymerase 6 (RDR6) from miRNA- cleaved TAS RNA fragment. The 24-nt rasiRNAs are produced by DCL3, and the precursor dsRNA is generated by the combined function of plant-specific DNA- dependent RNA polymerase IV (PolIV) and RDR2 from repetitive DNA in the genome. The exosiRNA pathway overlaps with the tasiRNA and rasiRNA pathways and both DCL4 and DCL3 are involved in exosiRNA processing. In addition to DCL1, DCL3 and DCL4, the model plant Arabidopsis thaliana and other higher plants encodes DCL2 or equivalent, which generates 22-nt siRNAs including 22-nt exosiRNAs, and plays a key role in systemic and transitive gene silencing in plants. All of these plant small RNAs are methylated at the 2’ -hydroxyl group of the 3’ terminal nucleotide by HUA Enhancer 1 (HEN1), and this 3’ terminal 2’-O-methylation is thought to stabilise the small RNAs in plant cells. miRNAs, tasiRNAs and exosiRNAs are functionally similar to small RNAs in animal cells which are involved in posttranscriptional gene silencing or sequencespecific degradation of RNA in animals. The rasiRNAs, however, are unique to plants and function to direct de novo cytosine methylation at the cognate DNA, a transcriptional gene silencing mechanism known as RNA-directed DNA methylation (RdDM).

[0007] RNA silencing induced by dsRNA has been extensively exploited to reduce gene activity in various eukaryotic systems, and a number of gene silencing technologies have been developed. Different organisms are often amenable to different gene silencing approaches. For instance, long dsRNA (at least 100 basepairs in length) is less suited to inducing RNA silencing in mammalian cells due to dsRNA-induced interferon responses, and so shorter dsRNAs (less than 30 basepairs) are generally used in mammalian cells, whereas in plants, hairpin RNA (hpRNA) with a long dsRNA stem is highly effective. In plants, the different RNA silencing pathways have led to different gene silencing technologies, such as artificial miRNA, artificial tasiRNA and virus- induced gene silencing technologies. However, successful applications of RNA silencing in plants have so far been achieved primarily by using long hpRNA transgenes. A hpRNA transgene construct typically consists of an inverted repeat made up of fully complementary sense and antisense sequences of a target gene sequence (which when transcribed form the dsRNA stem of hpRNA) separated by a spacer sequence (forming the loop of hpRNA), which is inserted between a promoter and a transcription terminator for expression in plant cells. The spacer sequence functions to stabilise the inverted- repeat DNA in bacteria during construct preparation. The dsRNA stem of the resulting hpRNA transcript is processed by DCL proteins into siRNAs that direct target gene silencing. hpRNA transgenes have been widely used to knock down gene expression, modify metabolic pathways and enhance disease and pest resistance in plants for crop improvement, and many successful applications of the technology in crop improvement have now been reported (Guo et al., 2016; Kim et al., 2019).

[0008] WO2019 / 051563 discloses RNA molecules having double-stranded structures and their use in gene silencing, including a double hairpin structure. W02020 / 024019 discloses double-stranded RNA structures having non-canonical basepairs in the doublestranded RNA region and their use in gene silencing. WO2021 / 022325 discloses doublestranded RNA molecules for use in modulating flowering in plants.

[0009] Whilst dsRNA induced gene silencing has proven to be a valuable tool in altering the phenotype of an organism, there is a need for alternate, preferably improved, dsRNA molecules which can be used for RNA interference (RNAi).

[0010] SUMMARY OF THE INVENTION

[0011] The present inventors have identified double -stranded product RNA molecules, and precursor RNA molecules encoding the double-stranded product RNA molecules, with desirable characteristics. These are especially useful for down-regulating gene expression or reducing the amount or activity of one or more target RNA molecules in a sequence-specific manner in a eukaryotic cell. In particular, the precursor RNA molecules and therefore also the double-stranded product RNA molecules have an asymmetric design feature that provides one or more bulged ribonucleotides in the precursor and product RNA molecules, preferably also having a ledRNA structure or comprising multiple non-canonical basepairs, preferably G:U basepairs, in a doublestranded region of the RNA molecules. The precursor RNA molecules may be applied topically to a eukaryotic cell, tissue, organ or organism, or be ingested by an organism such as an insect pest, or be expressed from a polynucleotide that encodes the precursor RNA molecules. The precursor RNA molecules thereby provide improved means to control pests and pathogens such as insect pests, nematodes and fungal and viral pathogens, or to reduce the incidence of, or treat, a disease in a eukaryotic organism.

[0012] Examples of the precursor RNA molecules and product RNA molecules of the following aspects and embodiments are illustrated schematically in Figure 53 herein and the reader is encouraged to view Figure 53 in concert with the description of the following aspects and embodiments.

[0013] In a first aspect, the present invention provides asymmetric precursor RNA molecules which are processed to produce siRNA molecules consisting of 21nt sense RNA sequences hybridised to 22nt antisense RNA sequences. Therefore, the present invention provides a precursor RNA molecule (A) comprising at least one doublestranded RNA region (B), wherein:

[0014] (i) the double-stranded RNA region (B) comprises:

[0015] (a) a first RNA strand (D) of at least 23 contiguous ribonucleotides which comprises a first RNA sequence (E) of at least 21 contiguous ribonucleotides, and

[0016] (b) a second RNA strand (F) of at least 24 contiguous ribonucleotides which comprises a second RNA sequence (G) of at least 22 contiguous ribonucleotides, wherein the first RNA strand (D) and second RNA strand (F) are covalently linked by a linking RNA sequence (L), wherein the first RNA sequence (E) is hybridised to the second RNA sequence (G) by basepairing between at least 20 of the at least 21 contiguous ribonucleotides of the first RNA sequence (E) and at least 20 of the at least 22 contiguous ribonucleotides of the second RNA sequence (G), forming at least a part (C) of the double-stranded RNA region (B), wherein 1 or 2 ribonucleotides of the at least 22 ribonucleotides of (G) are nonbasepaired, and 0 or 1, respectively, ribonucleotides of the at least 21 ribonucleotides of (E) are non-basepaired in the part (C) of the double-stranded RNA region (B), forming one or two or three bulges in the part (C) of the double-stranded RNA region (B), wherein each of the one or two or three bulges is immediately flanked by ribonucleotides which are basepaired in the part (C) of the double-stranded RNA region (B); and

[0017] (ii) the precursor RNA molecule (A) is capable of being cleaved in a eukaryotic cell by one or more ribonucleases (RNases) to produce one or more double-stranded product RNA molecule(s) (P) consisting of a sense RNA sequence (H) of 21 contiguous ribonucleotides from the first RNA sequence (E) and an antisense RNA sequence (J) of 22 contiguous ribonucleotides from the second RNA strand (F), including at least 20 contiguous ribonucleotides from (G), wherein the one or more double-stranded product RNA molecule(s) (P) comprise the one or two or three bulges, wherein 18 or 19 of ribonucleotides 1 to 19 of the sense RNA sequence (H) each basepair with a ribonucleotide of ribonucleotides 1 to 20 of antisense RNA sequence (J) in the one or more double-stranded product RNA molecule(s) (P), wherein ribonucleotides 20 and 21 of the sense RNA sequence (H) form a 2- ribonucleotide unpaired 3' overhang and ribonucleotides 21 and 22 of the antisense RNA sequence (J) form a 2-ribonucleotide unpaired 3' overhang in the one or more doublestranded product RNA molecule(s) (P), wherein ribonucleotides 1 and 2 of the sense RNA sequence (H) basepair with ribonucleotides 19 and 20, respectively, of the antisense RNA sequence (J) in the one or more double-stranded product RNA molecule(s) (P), and wherein ribonucleotides 18 and 19 of the sense RNA sequence (H) basepair with ribonucleotides 1 and 2, respectively, of the antisense RNA sequence (J) in the one or more double-stranded product RNA molecule(s) (P). Exemplary RNA molecules of this aspect are illustrated schematically in Figure 53, panels A to H.

[0018] In an embodiment of the first aspect, one or both of ribonucleotides 1 and 2 of the at least 23 contiguous ribonucleotides of the first RNA strand (D) are not basepaired with one or both, respectively, of ribonucleotides 23 and 24 of the at least 24 contiguous ribonucleotides of the second RNA strand (F). Exemplary RNA molecules of this embodiment are illustrated schematically in Figure 53, panels F to H.

[0019] In an embodiment of the first aspect, the first RNA sequence (E) is hybridised to the second RNA sequence (G) by basepairing between at least 20 of the at least 21 contiguous ribonucleotides of the first RNA sequence (E) and at least 20 of the at least 22 contiguous ribonucleotides of the second RNA sequence (G), wherein 2 ribonucleotides of the at least 22 contiguous ribonucleotides of (G) are nonbasepaired and 1 ribonucleotide of the at least 21 contiguous ribonucleotides of the first RNA sequence (E) is non-basepaired in the part (C) of the double -stranded RNA region (B), the non-basepaired ribonucleotides forming two bulges in the part (C) of the doublestranded RNA region (B), wherein each bulge is immediately flanked by ribonucleotides which are basepaired in the part (C) of the double-stranded RNA region (B), wherein the one or more double-stranded product RNA molecule(s) (P) comprise the bulges, wherein one bulge is formed by a mismatched ribonucleotide pair between the first RNA sequence (E) and the second RNA sequence (G) and the second bulge is a singleribonucleotide bulge, and wherein all but one of ribonucleotides 1 to 19 of the sense RNA sequence (H) each basepair with a ribonucleotide of ribonucleotides 1 to 20 of antisense RNA sequence (J) in the one or more double-stranded product RNA molecule(s) (P). Exemplary RNA molecules of this embodiment are illustrated schematically in Figure 53, panels A and F.

[0020] In an embodiment of the first aspect, the first RNA sequence (E) is hybridised to the second RNA sequence (G) by basepairing between at least 20 of the at least 21 contiguous ribonucleotides of the first RNA sequence (E) and at least 20 of the at least 22 contiguous ribonucleotides of the second RNA sequence (G), wherein 2 contiguous ribonucleotides of the at least 22 ribonucleotides of (G) are non- basepaired and 1 ribonucleotide of the at least 21 ribonucleotides of (E) is non-basepaired in the part (C) of the double -stranded RNA region (B), the non-basepaired ribonucleotides forming a bulge in the part (C) of the double-stranded RNA region (B), wherein the bulge is immediately flanked by ribonucleotides which are basepaired in the part (C) of the double-stranded RNA region (B), wherein the one or more double-stranded product RNA molecule(s) (P) comprise the bulge, and wherein all but one of ribonucleotides 1 to 19 of the sense RNA sequence (H) each basepair with a ribonucleotide of ribonucleotides 1 to 20 of antisense RNA sequence (J) in the one or more double-stranded product RNA molecule(s). Exemplary RNA molecules of this embodiment are illustrated schematically in Figure 53, panel B.

[0021] In an embodiment of the first aspect, the first RNA sequence (E) is hybridised to the second RNA sequence (G) by basepairing between at least 20 of the at least 21 contiguous ribonucleotides of the first RNA sequence (E) and at least 20 of the at least 22 contiguous ribonucleotides of the second RNA sequence (G), wherein 2 ribonucleotides of the at least 22 ribonucleotides of (G) are non-basepaired and 1 ribonucleotide of the at least 21 ribonucleotides of (E) is non-basepaired in the part (C) of the double-stranded RNA region (B), the non-basepaired ribonucleotides forming three bulges in the part (C) of the double-stranded RNA region (B), wherein each bulge is immediately flanked by ribonucleotides which are basepaired in the part (C) of the double-stranded RNA region (B), wherein each bulge is a single-ribonucleotide bulge, wherein the one or more double-stranded product RNA molecule(s) (P) comprise the bulges, wherein all but one of ribonucleotides 1 to 19 of the sense RNA sequence (H) each basepair with a ribonucleotide of ribonucleotides 1 to 20 of antisense RNA sequence (J) in the one or more double -stranded product RNA molecule(s). Exemplary RNA molecules of this embodiment are illustrated schematically in Figure 53, panels C and G.

[0022] In an embodiment of the first aspect, the first RNA sequence (E) is hybridised to the second RNA sequence (G) by basepairing between at least 21 of the at least 21 contiguous ribonucleotides of the first RNA sequence (E) and at least 21 of the at least 22 contiguous ribonucleotides of the second RNA sequence (G), wherein 1 ribonucleotide of the at least 22 ribonucleotides of (G) is non-basepaired and all of the at least 21 ribonucleotides of (E) are basepaired in the part (C) of the doublestranded RNA region (B), the 1 non-basepaired ribonucleotide forming a single nucleotide bulge in the part (C) of the double-stranded RNA region (B), wherein the bulge is immediately flanked by ribonucleotides which are basepaired in the part (C) of the double-stranded RNA region (B), wherein the one or more double-stranded product RNA molecule(s) (P) comprise the bulge, and wherein ribonucleotides 1 to 19 of the sense RNA sequence (H) each basepair with a ribonucleotide of ribonucleotides 1 to 20 of antisense RNA sequence (J) in the one or more double-stranded product RNA molecule(s) (P). Exemplary RNA molecules of this embodiment are illustrated schematically in Figure 53, panels D, E and H.

[0023] In an embodiment of the first aspect, the double-stranded RNA region (B) comprises:

[0024] (a) a first RNA strand (D) which comprises a first RNA sequence (E) of at least 23 contiguous ribonucleotides, and

[0025] (b) a second RNA strand (F) which comprises a second RNA sequence (G) of at least 24 contiguous ribonucleotides, wherein the first RNA sequence (E) is hybridised to the second RNA sequence (G) by basepairing between at least 22 of the at least 23 contiguous ribonucleotides of the first RNA sequence (E) and at least 22 of the at least 24 contiguous ribonucleotides of the second RNA sequence (G), forming at least a part (C) of the double-stranded RNA region (B), wherein 1 or 2 ribonucleotides of the at least 24 ribonucleotides of (G) are non-basepaired and 0 or 1, respectively, ribonucleotides of the at least 23 ribonucleotides of (E) are non- basepaired in the part (C) of the double -stranded RNA region (B), forming one or two or three bulges in the part (C) of the double-stranded RNA region (B). Exemplary RNA molecules of this embodiment are illustrated schematically in Figure 53, panels A to E.

[0026] Thus, in an embodiment the present invention provides a precursor RNA molecule

[0027] (A) comprising at least one double-stranded RNA region (B), wherein:

[0028] (i) the double-stranded RNA region (B) comprises:

[0029] (a) a first RNA strand (D) which comprises a first RNA sequence (E) of at least 23 contiguous ribonucleotides, and

[0030] (b) a second RNA strand (F) which comprises a second RNA sequence (G) of at least 24 contiguous ribonucleotides, wherein the first RNA strand (D) and second RNA strand (F) are covalently linked by a linking RNA sequence (L), wherein the first RNA sequence (E) is hybridised to the second RNA sequence (G) by basepairing between at least 22 of the at least 23 contiguous ribonucleotides of the first RNA sequence (E) and at least 22 of the at least 24 contiguous ribonucleotides of the second RNA sequence (G), forming at least a part (C) of the double-stranded RNA region

[0031] (B), wherein 1 or 2 ribonucleotides of the at least 24 ribonucleotides of (G) are non- basepaired, and 0 or 1, respectively, ribonucleotides of the at least 23 ribonucleotides of (E) are non-basepaired, in the part (C) of the double -stranded RNA region (B) forming one or two or three bulges in the part (C) of the double-stranded RNA region (B), wherein each of the one or two or three bulges is immediately flanked by ribonucleotides which are basepaired in the part (C) of the double-stranded RNA region (B); and

[0032] (ii) the precursor RNA molecule (A) is capable of being cleaved in a eukaryotic cell by one or more ribonucleases (RNases) to produce one or more double-stranded product RNA molecule(s) (P) consisting of a sense RNA sequence (H) of 21 contiguous ribonucleotides from the first RNA sequence (E) and an antisense RNA sequence (J) of 22 contiguous ribonucleotides from the second RNA sequence (G), wherein the one or more double-stranded product RNA molecule(s) (P) comprise the one or two or three bulges, wherein 18 or 19 of ribonucleotides 1 to 19 of the sense RNA sequence (H) each basepair with a ribonucleotide of ribonucleotides 1 to 20 of antisense RNA sequence (J) in the one or more double-stranded product RNA molecule(s) (P), wherein ribonucleotides 20 and 21 of the sense RNA sequence (H) form a 2- ribonucleotide unpaired 3' overhang and ribonucleotides 21 and 22 of the antisense RNA sequence (J) form a 2-ribonucleotide unpaired 3' overhang in the one or more doublestranded product RNA molecule(s) (P), wherein ribonucleotides 1 and 2 of the sense RNA sequence (H) basepair with ribonucleotides 19 and 20, respectively, of the antisense RNA sequence (J) in the one or more double-stranded product RNA molecule(s) (P), and wherein ribonucleotides 18 and 19 of the sense RNA sequence (H) basepair with ribonucleotides 1 and 2, respectively, of the antisense RNA sequence (J) in the one or more double-stranded product RNA molecule(s) (P). Exemplary RNA molecules of this embodiment are illustrated schematically in Figure 53, panels A to E.

[0033] In an embodiment of the first aspect, the first RNA sequence (E) is hybridised to the second RNA sequence (G) by basepairing between at least 22 of the at least 23 contiguous ribonucleotides of the first RNA sequence (E) and at least 22 of the at least 24 contiguous ribonucleotides of the second RNA sequence (G), wherein 2 ribonucleotides of the at least 24 ribonucleotides of (G) are non-basepaired and 1 ribonucleotide of the at least 23 ribonucleotides of (E) is non-basepaired in the part (C) of the double-stranded RNA region (B), the non-basepaired ribonucleotides forming two bulges in the part (C) of the double-stranded RNA region (B), wherein each bulge is immediately flanked by ribonucleotides which are basepaired in the part (C) of the double-stranded RNA region (B), wherein the one or more double-stranded product RNA molecule(s) (P) comprise the bulges, wherein one bulge is formed by a mismatched ribonucleotide pair between the first RNA sequence (E) and the second RNA sequence (G) and the second bulge is a singleribonucleotide bulge, and wherein all but one of ribonucleotides 1 to 19 of the sense RNA sequence (H) each basepair with a ribonucleotide of ribonucleotides 1 to 20 of antisense RNA sequence (J) in the one or more double-stranded product RNA molecule(s) (P). Exemplary RNA molecules of this embodiment are illustrated schematically in Figure 53, panel A. In an embodiment of the first aspect, the first RNA sequence (E) is hybridised to the second RNA sequence (G) by basepairing between at least 22 of the at least 23 contiguous ribonucleotides of the first RNA sequence (E) and at least 22 of the at least 24 contiguous ribonucleotides of the second RNA sequence (G), wherein 2 contiguous ribonucleotides of the at least 24 ribonucleotides of (G) are nonbasepaired and 1 ribonucleotide of the at least 23 ribonucleotides of (E) is non-basepaired in the part (C) of the double -stranded RNA region (B), the non-basepaired ribonucleotides forming a bulge in the part (C) of the double-stranded RNA region (B), wherein the bulge is immediately flanked by ribonucleotides which are basepaired in the part (C) of the double-stranded RNA region (B), wherein the one or more double-stranded product RNA molecule(s) (P) comprise the bulge, and wherein all but one of ribonucleotides 1 to 19 of the sense RNA sequence (H) each basepair with a ribonucleotide of ribonucleotides 1 to 20 of antisense RNA sequence (J) in the one or more double-stranded product RNA molecule(s). Exemplary RNA molecules of this embodiment are illustrated schematically in Figure 53, panel B.

[0034] In an embodiment of the first aspect, the first RNA sequence (E) is hybridised to the second RNA sequence (G) by basepairing between at least 22 of the at least 23 contiguous ribonucleotides of the first RNA sequence (E) and at least 22 of the at least 24 contiguous ribonucleotides of the second RNA sequence (G), wherein 2 ribonucleotides of the at least 24 ribonucleotides of (G) are non-basepaired and 1 ribonucleotide of the at least 23 ribonucleotides of (E) is non-basepaired in the part (C) of the double-stranded RNA region (B), the non-basepaired ribonucleotides forming three bulges in the part (C) of the double-stranded RNA region (B), wherein each bulge is immediately flanked by ribonucleotides which are basepaired in the part (C) of the double-stranded RNA region (B), wherein each bulge is a single-ribonucleotide bulge, wherein the one or more double-stranded product RNA molecule(s) (P) comprise the bulges, wherein all but one of ribonucleotides 1 to 19 of the sense RNA sequence (H) each basepair with a ribonucleotide of ribonucleotides 1 to 20 of antisense RNA sequence (J) in the one or more double -stranded product RNA molecule(s). Exemplary RNA molecules of this embodiment are illustrated schematically in Figure 53, panel C.

[0035] In an embodiment of the first aspect, the first RNA sequence (E) is hybridised to the second RNA sequence (G) by basepairing between at least 23 of the at least 23 contiguous ribonucleotides of the first RNA sequence (E) and at least 23 of the at least 24 contiguous ribonucleotides of the second RNA sequence (G), wherein 1 ribonucleotide of the at least 24 ribonucleotides of (G) is non-basepaired and all of the at least 23 ribonucleotides of (E) are basepaired in the part (C) of the doublestranded RNA region (B), the 1 non-basepaired ribonucleotide forming a single nucleotide bulge in the part (C) of the double-stranded RNA region (B), wherein the bulge is immediately flanked by ribonucleotides which are basepaired in the part (C) of the double-stranded RNA region (B), wherein the one or more double-stranded product RNA molecule(s) (P) comprise the bulge, and wherein ribonucleotides 1 to 19 of the sense RNA sequence (H) each basepair with a ribonucleotide of ribonucleotides 1 to 20 of antisense RNA sequence (J) in the one or more double-stranded product RNA molecule(s) (P). Exemplary RNA molecules of this embodiment are illustrated schematically in Figure 53, panels D and E.

[0036] The design principles for the asymmetric precursor RNA molecules of the first aspect can be applied in an extended fashion to longer double -stranded regions. For example, the non-basepaired ribonucleotides in the second RNA sequence (G) (antisense sequence) may be arranged in a periodic fashion to provide a population of product RNA molecules (P) having multiple, non-overlapping antisense RNA sequences (J) of 22nt. Such precursor RNA molecules are particularly useful for reducing expression of a target RNA molecule in a plant cell, fungal cell or nematode cell. They are also useful in other invertebrate animal cells such as an arthropod cell or insect cell, or in a non-mammalian vertebrate animal cell. They may be produced in a plant cell to reduce an insect target RNA molecule or a fungal pathogen or nematode target RNA molecule, or applied topically to a plant or insect to reduce a target RNA molecule. For example, in an embodiment of the first aspect, the first RNA sequence (E) comprises at least 44 contiguous ribonucleotides and the second RNA sequence (G) comprises at least 46 contiguous ribonucleotides, wherein the first RNA sequence (E) is hybridised to the second RNA sequence (G) by basepairing between at least 42, preferably at least 43 or at least 44, of the at least 44 contiguous ribonucleotides of the first RNA sequence (E) and at least 42, preferably at least 43 or at least 44, of the at least 46 contiguous ribonucleotides of the second RNA sequence (G), wherein 2, 3 or 4 ribonucleotides of the at least 46 contiguous ribonucleotides of (G) are non-basepaired and 0, 1 or 2, respectively, ribonucleotides of the at least 44 contiguous ribonucleotides of (E) are non-basepaired in the part (C) of the doublestranded RNA region (B), forming bulges in the part (C) of the double-stranded RNA region (B), wherein each of the bulges is immediately flanked by ribonucleotides which are basepaired in the part (C) of the double-stranded RNA region (B), wherein the precursor RNA molecule is capable of being cleaved in a eukaryotic cell by one or more ribonucleases (RNases) to produce multiple, different doublestranded product RNA molecules (P) consisting of a sense RNA sequence (H) of 21 contiguous ribonucleotides and an antisense RNA sequence (J) of 22 contiguous ribonucleotides, wherein the multiple, different double-stranded product RNA molecule(s) (P) each comprise at least one of the bulges, and wherein at least some of the multiple, different double -stranded product RNA molecules (P) have non-overlapping antisense RNA sequences (J), preferably adjacent non-overlapping antisense RNA sequences (J).

[0037] In another embodiment of the first aspect, the first RNA strand (D) comprises a first RNA sequence (E) of at least 44 contiguous ribonucleotides and the second RNA strand (F) comprises a second RNA sequence (G) of at least 46 contiguous ribonucleotides, wherein the first RNA sequence (E) is hybridised to the second RNA sequence (G) by basepairing between all 44 contiguous ribonucleotides of the first RNA sequence (E) and 44 of the 46 contiguous ribonucleotides of the second RNA sequence (G), forming a part (C) of the double-stranded RNA region (B), wherein 2 ribonucleotides of the 46 contiguous ribonucleotides of (G) are nonbasepaired in the part (C) of the double -stranded RNA region (B), forming single ribonucleotide bulges in the part (C) of the double-stranded RNA region (B), wherein each of the bulges is immediately flanked by ribonucleotides which are basepaired in the part (C) of the double-stranded RNA region (B), wherein the precursor RNA molecule is capable of being cleaved in a eukaryotic cell by one or more ribonucleases (RNases) to produce multiple, different doublestranded product RNA molecules (P) consisting of a sense RNA sequence (H) of 21 contiguous ribonucleotides and an antisense RNA sequence (J) of 22 contiguous ribonucleotides, wherein the multiple, different double-stranded product RNA molecule(s) (P) each comprise at least one of the bulges, and wherein at least some of the multiple, different double -stranded product RNA molecules (P) have non-overlapping antisense RNA sequences (J), preferably adjacent non-overlapping antisense RNA sequences (J). In this and the previous embodiments, the single ribonucleotide bulges are preferably spaced apart by 16-26 contiguous basepairs, more preferably by 17-24 or 18-23 contiguous basepairs, even more preferably by 19-22 contiguous basepairs, most preferably by 20 or 21 contiguous basepairs. In specific examples, the single ribonucleotide bulges are spaced apart by 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 contiguous basepairs. In an example, all of these basepairs are canonical basepairs. Alternatively, 1, 2, 3, 4, 5 or 6 of the basepairs between the bulges are G:U basepairs. The precursor RNA molecule may comprise two or more such double-stranded RNA regions, for example comprise an ledRNA structure. In these embodiments, the eukaryotic cell in which the precursor RNA molecule (A) is cleaved is preferably a plant cell, or a fungal cell, or a nematode cell, or an arthropod cell such as an insect cell, arachnid, or decapod, or the target RNA molecule is preferably in a plant cell, or a fungal cell, or a nematode cell, or an arthropod cell such as an insect, arachnid, or decapod cell. The precursor RNA molecule may be produced in a cell-free system or in a microbial cell such as a bacterial cell or yeast cell, and such cells applied topically to the plant or insect or are ingested by the insect.

[0038] In a different embodiment with longer dsRNA regions of at least 44 basepairs, the precursor RNA molecule lacks the linking RNA sequence (L) and the first RNA strand

[0039] (D) and the second RNA strand (F) hybridise to form the double -stranded RNA region (B). Such precursor RNA molecule may be readily produced in a cell -free system, for example in vitro. In an embodiment, the first RNA strand (D) comprises a first RNA sequence (E) of at least 44 contiguous ribonucleotides and the second RNA strand (F) comprises a second RNA sequence (G) of at least 46 contiguous ribonucleotides, wherein the first RNA sequence (E) is hybridised to the second RNA sequence (G) by basepairing between all 44 contiguous ribonucleotides of the first RNA sequence

[0040] (E) and 44 of the 46 contiguous ribonucleotides of the second RNA sequence (G), forming a part (C) of the double-stranded RNA region (B), wherein 2 ribonucleotides of the 46 contiguous ribonucleotides of (G) are nonbasepaired in the part (C) of the double -stranded RNA region (B), forming single ribonucleotide bulges in the part (C) of the double-stranded RNA region (B), wherein each of the bulges is immediately flanked by ribonucleotides which are basepaired in the part (C) of the double-stranded RNA region (B), wherein the precursor RNA molecule is capable of being cleaved in a eukaryotic cell by one or more ribonucleases (RNases) to produce multiple, different doublestranded product RNA molecules (P) consisting of a sense RNA sequence (H) of 21 contiguous ribonucleotides and an antisense RNA sequence (J) of 22 contiguous ribonucleotides, wherein the multiple, different double-stranded product RNA molecule(s) (P) each comprise at least one of the bulges, and wherein at least some of the multiple, different double -stranded product RNA molecules (P) have non-overlapping antisense RNA sequences (J), preferably adjacent non-overlapping antisense RNA sequences (J). In this embodiment, the single ribonucleotide bulges are preferably spaced apart by 16-26 contiguous basepairs, more preferably by 17-24 or 18-23 contiguous basepairs, even more preferably by 19-22 contiguous basepairs, most preferably by 20 or 21 contiguous basepairs. In specific examples, the single ribonucleotide bulges are spaced apart by 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 contiguous basepairs. In a preferred example, all of these basepairs are canonical basepairs. Alternatively, 1, 2, 3, 4, 5 or 6 of the basepairs between the bulges are G:U basepairs.

[0041] In the previous three embodiments, the first RNA sequence (E) and the second RNA sequence (G) may extend to longer than 44 and 46 ribonucleotides, respectively. In another embodiment of the first aspect, for even longer dsRNA region(s), the first RNA sequence (E) comprises at least 65 contiguous ribonucleotides and the second RNA sequence (G) comprises at least 68 contiguous ribonucleotides, and wherein the first RNA sequence (E) is hybridised to the second RNA sequence (G) by basepairing between at least 62, preferably at least 63, at least 64 or at least 65, of the at least 65 contiguous ribonucleotides of the first RNA sequence (E) and at least 62, preferably at least 63, at least 64 or at least 65, of the at least 68 contiguous ribonucleotides of the second RNA sequence (G).

[0042] In another embodiment of the first aspect, the first RNA strand (D) comprises a first RNA sequence (E) of at least 65 contiguous ribonucleotides and the second RNA strand (F) comprises a second RNA sequence (G) of at least 68 contiguous ribonucleotides, wherein the first RNA sequence (E) is hybridised to the second RNA sequence (G) by basepairing between all 65 contiguous ribonucleotides of the first RNA sequence (E) and 65 of the 68 contiguous ribonucleotides of the second RNA sequence (G), forming a part (C) of the double-stranded RNA region (B), wherein 3 ribonucleotides of the 68 contiguous ribonucleotides of (G) are nonbasepaired in the part (C) of the double -stranded RNA region (B), forming single ribonucleotide bulges in the part (C) of the double-stranded RNA region (B), wherein each of the bulges is immediately flanked by ribonucleotides which are basepaired in the part (C) of the double-stranded RNA region (B), wherein the precursor RNA molecule is capable of being cleaved in a eukaryotic cell by one or more ribonucleases (RNases) to produce multiple, different doublestranded product RNA molecules (P) consisting of a sense RNA sequence (H) of 21 contiguous ribonucleotides and an antisense RNA sequence (J) of 22 contiguous ribonucleotides, wherein the multiple, different double-stranded product RNA molecule(s) (P) each comprise at least one of the bulges, and wherein at least some of the multiple, different double -stranded product RNA molecules (P) have non-overlapping antisense RNA sequences (J), preferably adjacent non-overlapping antisense RNA sequences (J). In this and the previous embodiments, the single ribonucleotide bulges are preferably spaced apart periodically, for example by 16-26 contiguous basepairs, more preferably by 17-24 or 18-23 contiguous basepairs, even more preferably by 19-22 contiguous basepairs, most preferably by 20 or 21 contiguous basepairs. In specific examples, the single ribonucleotide bulges are spaced apart by 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 contiguous basepairs. In an example, all of these basepairs are canonical basepairs. Alternatively, 1, 2, 3, 4, 5 or 6 of the basepairs between the bulges are G:U basepairs. The precursor RNA molecule may comprise two or more such double-stranded RNA regions, for example comprise an ledRNA structure.

[0043] In a different embodiment with longer dsRNA regions of at least 65 basepairs, the precursor RNA molecule lacks the linking RNA sequence (L) and the first RNA strand

[0044] (D) and the second RNA strand (F) hybridise to form the double -stranded RNA region (B). Such precursor RNA molecule may be readily produced in a cell -free system, for example in vitro. In an embodiment, the first RNA strand (D) comprises a first RNA sequence (E) of at least 65 contiguous ribonucleotides and the second RNA strand (F) comprises a second RNA sequence (G) of at least 68 contiguous ribonucleotides, wherein the first RNA sequence (E) is hybridised to the second RNA sequence (G) by basepairing between all 65 contiguous ribonucleotides of the first RNA sequence

[0045] (E) and 65 of the 68 contiguous ribonucleotides of the second RNA sequence (G), forming a part (C) of the double-stranded RNA region (B), wherein 3 ribonucleotides of the 68 contiguous ribonucleotides of (G) are nonbasepaired in the part (C) of the double -stranded RNA region (B), forming single ribonucleotide bulges in the part (C) of the double-stranded RNA region (B), wherein each of the bulges is immediately flanked by ribonucleotides which are basepaired in the part (C) of the double-stranded RNA region (B), wherein the precursor RNA molecule is capable of being cleaved in a eukaryotic cell by one or more ribonucleases (RNases) to produce multiple, different doublestranded product RNA molecules (P) consisting of a sense RNA sequence (H) of 21 contiguous ribonucleotides and an antisense RNA sequence (J) of 22 contiguous ribonucleotides, wherein the multiple, different double-stranded product RNA molecule(s) (P) each comprise at least one of the bulges, and wherein at least some of the multiple, different double -stranded product RNA molecules (P) have non-overlapping antisense RNA sequences (J), preferably adjacent non-overlapping antisense RNA sequences (J). In this embodiment, the single ribonucleotide bulges are preferably spaced apart by 16-26 contiguous basepairs, more preferably by 17-24 or 18-23 contiguous basepairs, even more preferably by 19-22 contiguous basepairs, most preferably by 20 or 21 contiguous basepairs. In specific examples, the single ribonucleotide bulges are spaced apart by 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 contiguous basepairs. In a preferred example, all of these basepairs are canonical basepairs. Alternatively, 1, 2, 3, 4, 5 or 6 of the basepairs between the bulges are G:U basepairs.

[0046] In the previous three embodiments, the first RNA sequence (E) and the second RNA sequence (G) may extend to longer than 65 and 68 ribonucleotides, respectively. In a further embodiment of the first aspect, the first RNA sequence (E) comprises at least 86 contiguous ribonucleotides and the second RNA sequence (G) comprises at least 90 contiguous ribonucleotides, and wherein the first RNA sequence (E) is hybridised to the second RNA sequence (G) by basepairing between at least 82, preferably at least 83, at least 84, at least 85 or at least 86, of the at least 86 contiguous ribonucleotides of the first RNA sequence (E) and at least 82, preferably at least 83, at least 84, at least 85 or at least 86, of the at least 90 contiguous ribonucleotides of the second RNA sequence (G).

[0047] In another embodiment of the first aspect, the first RNA strand (D) comprises a first RNA sequence (E) of at least 86 contiguous ribonucleotides and the second RNA strand (F) comprises a second RNA sequence (G) of at least 90 contiguous ribonucleotides, wherein the first RNA sequence (E) is hybridised to the second RNA sequence (G) by basepairing between all 86 contiguous ribonucleotides of the first RNA sequence (E) and 86 of the 90 contiguous ribonucleotides of the second RNA sequence (G), forming a part (C) of the double-stranded RNA region (B), wherein 4 ribonucleotides of the 90 contiguous ribonucleotides of (G) are nonbasepaired in the part (C) of the double -stranded RNA region (B), forming single ribonucleotide bulges in the part (C) of the double-stranded RNA region (B), wherein each of the bulges is immediately flanked by ribonucleotides which are basepaired in the part (C) of the double-stranded RNA region (B), wherein the precursor RNA molecule is capable of being cleaved in a eukaryotic cell by one or more ribonucleases (RNases) to produce multiple, different doublestranded product RNA molecules (P) consisting of a sense RNA sequence (H) of 21 contiguous ribonucleotides and an antisense RNA sequence (J) of 22 contiguous ribonucleotides, wherein the multiple, different double-stranded product RNA molecule(s) (P) each comprise at least one of the bulges, and wherein at least some of the multiple, different double -stranded product RNA molecules (P) have non-overlapping antisense RNA sequences (J), preferably adjacent non-overlapping antisense RNA sequences (J). In this and the previous embodiments, the single ribonucleotide bulges are preferably spaced apart periodically, for example by 16-26 contiguous basepairs, more preferably by 17-24 or 18-23 contiguous basepairs, even more preferably by 19-22 contiguous basepairs, most preferably by 20 or 21 contiguous basepairs or, on average, by about 21 basepairs. In specific examples, the single ribonucleotide bulges are spaced apart, independently, by 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 contiguous basepairs. In an example, all of these basepairs are canonical basepairs. Alternatively, 1, 2, 3, 4, 5 or 6 of the basepairs between the bulges are G:U basepairs. The precursor RNA molecule may comprise two or more such doublestranded RNA regions, for example comprise a ledRNA structure.

[0048] In a different embodiment with longer dsRNA regions of at least 86 basepairs, the precursor RNA molecule lacks the linking RNA sequence (L) and the first RNA strand

[0049] (D) and the second RNA strand (F) hybridise to form the double -stranded RNA region (B). Such precursor RNA molecule may be readily produced in a cell -free system, for example in vitro. In an embodiment, the first RNA strand (D) comprises a first RNA sequence (E) of at least 86 contiguous ribonucleotides and the second RNA strand (F) comprises a second RNA sequence (G) of at least 90 contiguous ribonucleotides, wherein the first RNA sequence (E) is hybridised to the second RNA sequence (G) by basepairing between all 86 contiguous ribonucleotides of the first RNA sequence

[0050] (E) and 86 of the 90 contiguous ribonucleotides of the second RNA sequence (G), forming a part (C) of the double-stranded RNA region (B), wherein 4 ribonucleotides of the 86 contiguous ribonucleotides of (G) are nonbasepaired in the part (C) of the double -stranded RNA region (B), forming single ribonucleotide bulges in the part (C) of the double-stranded RNA region (B), wherein each of the bulges is immediately flanked by ribonucleotides which are basepaired in the part (C) of the double-stranded RNA region (B), wherein the precursor RNA molecule is capable of being cleaved in a eukaryotic cell by one or more ribonucleases (RNases) to produce multiple, different doublestranded product RNA molecules (P) consisting of a sense RNA sequence (H) of 21 contiguous ribonucleotides and an antisense RNA sequence (J) of 22 contiguous ribonucleotides, wherein the multiple, different double-stranded product RNA molecule(s) (P) each comprise at least one of the bulges, and wherein at least some of the multiple, different double -stranded product RNA molecules (P) have non-overlapping antisense RNA sequences (J), preferably adjacent non-overlapping antisense RNA sequences (J). In this embodiment, the single ribonucleotide bulges are preferably spaced apart by 16-26 contiguous basepairs, more preferably by 17-24 or 18-23 contiguous basepairs, even more preferably by 19-22 contiguous basepairs, most preferably by 20 or 21 contiguous basepairs, or, on average, about 21 basepairs. In specific examples, the single ribonucleotide bulges are spaced apart, independently, by 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 contiguous basepairs. In a preferred example, all of these basepairs are canonical basepairs. Alternatively, 1, 2, 3, 4, 5 or 6 of the basepairs between the bulges are G:U basepairs.

[0051] In the previous three embodiments, the first RNA sequence (E) and the second RNA sequence (G) may extend to longer than 86 and 90 ribonucleotides, respectively. In a further embodiment of the first aspect, the first RNA sequence (E) comprises at least 107 contiguous ribonucleotides and the second RNA sequence (G) comprises at least 112 contiguous ribonucleotides, and wherein the first RNA sequence (E) is hybridised to the second RNA sequence (G) by basepairing between at least 102, preferably at least 103, at least 104, at least 105, at least 106 or at least 107, of the at least 107 contiguous ribonucleotides of the first RNA sequence (E) and at least 102, preferably at least 103, at least 104, at least 105, at least 106 or at least 107, of the at least 112 contiguous ribonucleotides of the second RNA sequence (G). In this embodiment, at least 5 ribonucleotides of the second RNA sequence, up to a maximum of 10 ribonucleotides, are non-basepaired and form bulges, preferably single ribonucleotide bulges.

[0052] In another embodiment of the first aspect, the first RNA strand (D) comprises a first RNA sequence (E) of at least 107 contiguous ribonucleotides and the second RNA strand (F) comprises a second RNA sequence (G) of at least 112 contiguous ribonucleotides, wherein the first RNA sequence (E) is hybridised to the second RNA sequence (G) by basepairing between all 107 contiguous ribonucleotides of the first RNA sequence (E) and 107 of the 112 contiguous ribonucleotides of the second RNA sequence (G), forming a part (C) of the double-stranded RNA region (B), wherein 5 ribonucleotides of the 112 contiguous ribonucleotides of (G) are nonbasepaired in the part (C) of the double -stranded RNA region (B), forming single ribonucleotide bulges in the part (C) of the double-stranded RNA region (B), wherein each of the bulges is immediately flanked by ribonucleotides which are basepaired in the part (C) of the double-stranded RNA region (B), wherein the precursor RNA molecule is capable of being cleaved in a eukaryotic cell by one or more ribonucleases (RNases) to produce multiple, different doublestranded product RNA molecules (P) consisting of a sense RNA sequence (H) of 21 contiguous ribonucleotides and an antisense RNA sequence (J) of 22 contiguous ribonucleotides, wherein the multiple, different double-stranded product RNA molecule(s) (P) each comprise at least one of the bulges, and wherein at least some of the multiple, different double -stranded product RNA molecules (P) have non-overlapping antisense RNA sequences (J), preferably adjacent non-overlapping antisense RNA sequences (J). In this and the previous embodiments, the single ribonucleotide bulges are preferably spaced apart periodically, for example by 16-26 contiguous basepairs, more preferably by 17-24 or 18-23 contiguous basepairs, even more preferably by 19-22 contiguous basepairs, most preferably by 20 or 21 contiguous basepairs or, on average, by about 21 basepairs. In specific examples, the single ribonucleotide bulges are spaced apart, independently, by 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 contiguous basepairs. In an example, all of these basepairs are canonical basepairs. Alternatively, 1, 2, 3, 4, 5 or 6 of the basepairs between the bulges are G:U basepairs. The precursor RNA molecule may comprise two or more such doublestranded RNA regions, for example comprise an ledRNA structure.

[0053] In a different embodiment with longer dsRNA regions of at least 107 basepairs, the precursor RNA molecule lacks the linking RNA sequence (L) and the first RNA strand (D) and the second RNA strand (F) hybridise to form the double-stranded RNA region (B). Such precursor RNA molecule may be readily produced in a cell-free system, for example in vitro. In an embodiment, the first RNA strand (D) comprises a first RNA sequence (E) of at least 107 contiguous ribonucleotides and the second RNA strand (F) comprises a second RNA sequence (G) of at least 112 contiguous ribonucleotides, wherein the first RNA sequence (E) is hybridised to the second RNA sequence (G) by basepairing between all 107 contiguous ribonucleotides of the first RNA sequence (E) and 107 of the 112 contiguous ribonucleotides of the second RNA sequence (G), forming a part (C) of the double-stranded RNA region (B), wherein 5 ribonucleotides of the 112 contiguous ribonucleotides of (G) are nonbasepaired in the part (C) of the double -stranded RNA region (B), forming single ribonucleotide bulges in the part (C) of the double-stranded RNA region (B), wherein each of the bulges is immediately flanked by ribonucleotides which are basepaired in the part (C) of the double-stranded RNA region (B), wherein the precursor RNA molecule is capable of being cleaved in a eukaryotic cell by one or more ribonucleases (RNases) to produce multiple, different doublestranded product RNA molecules (P) consisting of a sense RNA sequence (H) of 21 contiguous ribonucleotides and an antisense RNA sequence (J) of 22 contiguous ribonucleotides, wherein the multiple, different double-stranded product RNA molecule(s) (P) each comprise at least one of the bulges, and wherein at least some of the multiple, different double -stranded product RNA molecules (P) have non-overlapping antisense RNA sequences (J), preferably adjacent non-overlapping antisense RNA sequences (J). In this embodiment, the single ribonucleotide bulges are preferably spaced apart by 16-26 contiguous basepairs, more preferably by 17-24 or 18-23 contiguous basepairs, even more preferably by 19-22 contiguous basepairs, most preferably by 20 or 21 contiguous basepairs, or, on average, about 21 basepairs. In specific examples, the single ribonucleotide bulges are spaced apart, independently, by 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 contiguous basepairs. In a preferred example, all of these basepairs are canonical basepairs. Alternatively, 1, 2, 3, 4, 5 or 6 of the basepairs between the bulges are G:U basepairs

[0054] In each of the above embodiments, the second RNA sequence (G) is longer than the first RNA sequence (E) because of the non-basepaired ribonucleotides that bulge out in (G). Preferably, the length of the first RNA sequence (E) is 94%-97% or 94%-96% of the length of the second RNA sequence (G). These features may also apply across the full length of the double-stranded region (B), where the part (C) of the double-stranded region is the full length of (B). As the skilled person would appreciate, further embodiments include a first RNA sequence (E) hybridised to a second RNA sequence (G) having longer contiguous ribonucleotides following the same principles described in the above embodiments.

[0055] In an embodiment, the first RNA sequence (E) and the second RNA sequence (G) both comprise at least 100 contiguous ribonucleotides, or at least 150, or at least 200, or at least 250, or at least 300 contiguous ribonucleotides, preferably to a maximum of 1000 contiguous ribonucleotides, more preferably to a maximum of 800 contiguous ribonucleotides, or even more preferably to a maximum of 600 contiguous ribonucleotides. For example, the first RNA sequence (E) and the second RNA sequence (G) both comprise contiguous ribonucleotides in the range 100-1000, 100-800, or 100- 600 contiguous ribonucleotides, or in the range 150-1000, 150-800, or 150-600 contiguous ribonucleotides. In preferred embodiments, the length of the sense RNA sequence of the dsRNA region is 94%-97% or 94%-96% the length of the antisense sequence. These features are applicable to hairpin RNAs and to dsRNAs formed by annealing of two RNA strands, i.e. without a joining loop sequence. Each of these features may also be applied to a second dsRNA region in the precursor RNA molecule, for example in a ledRNA molecule. In these embodiments, the eukaryotic cell in which the precursor RNA molecule (A) is cleaved is preferably a plant cell, a fungal cell, a nematode cell, or an arthropod cell such as an insect, arachnid, or decapod cell, or the target RNA molecule is preferably in a plant cell, a fungal cell, a nematode cell, or an arthropod cell such as an insect, arachnid, or decapod cell.

[0056] Each of the embodiments of the first aspect may have the following feature:

[0057] (i) ribonucleotides 1, 2 and 3 of the sense RNA sequence (H) basepair with ribonucleotides 18, 19 and 20, respectively, of the antisense RNA sequence (J) in at least some of the one or more double-stranded product RNA molecule(s) (P), or

[0058] (ii) ribonucleotides 17, 18 and 19 of the sense RNA sequence (H) basepair with ribonucleotides 1, 2 and 3, respectively, of the antisense RNA sequence (J) in at least some of the one or more double-stranded product RNA molecule(s) (P), or preferably both (i) and (ii). Exemplary RNA molecules of this embodiment are illustrated schematically in Figure 53, panels A and H, which all show product RNA molecules with at least three contiguous basepairs at both ends of the double-stranded region of the product RNA molecules. In further features of these embodiments, the product RNA molecules may have at least four, at least five or at least six contiguous basepairs at one or both ends of the double-stranded molecule before any bulge. In these embodiments, any of those basepairs may be G:U basepairs, preferably one or more of ribonucleotides 1, 2 and 3 of the antisense sequence (J) are involved in G:U basepairs, more preferably one or both of ribonucleotides 1 and 2 of the antisense sequence (J) are involved in G:U basepairs, even more preferably ribonucleotide 1 of the antisense sequence (J) is involved in G:U basepairs. Most preferably, one or more of ribonucleotides 1, 2 and 3 of the antisense sequence (J) are U ribonucleotides, or one or both of ribonucleotides 1 and 2 are U ribonucleotides, or ribonucleotide 1 is a U ribonucleotide, involved in a G:U basepair.

[0059] Each of the features of the embodiments of the first aspect can be applied to longer double-stranded regions to provide essentially either multimers of the product RNA molecules (P) or combinations of different designs of product RNA molecules (P). In an embodiment of the first aspect, the precursor RNA molecule is capable of being cleaved in a eukaryotic cell by one or more ribonucleases (RNases) to produce multiple, different double-stranded product RNA molecules (P) which each consist of a sense RNA sequence (H) of 21 contiguous ribonucleotides from the first RNA sequence (E) and an antisense RNA sequence (J) of 22 contiguous ribonucleotides from the second RNA sequence (G), wherein at least some of the multiple, different double-stranded product RNA molecules (P) have overlapping antisense RNA sequences (J). In a further embodiment, at least some of the multiple, different double-stranded product RNA molecules (P) have non-overlapping antisense RNA sequences (J), where the population of double-stranded product RNA molecules (P) produced from the precursor RNA molecule includes some overlapping and some non-overlapping antisense RNA sequences (J). Preferably, there are more non-overlapping antisense RNA sequences (J), as readily occurs with longer (>42 basepairs) double-stranded regions (B) where siRNA molecules each consisting of 21nt sense RNA sequences hybridised to 22nt antisense RNA sequences are phased along the length of (B).

[0060] In an embodiment of the first aspect, the precursor RNA molecule is capable of being cleaved in a eukaryotic cell by one or more ribonucleases (RNases) to produce multiple, different double-stranded product RNA molecules (P) which each consist of a sense RNA sequence (H) of 21 contiguous ribonucleotides from a first RNA sequence (E) and an antisense RNA sequence (J) of 22 contiguous ribonucleotides from a second RNA sequence (G), wherein at least some of the multiple, different double-stranded product RNA molecules (P) have non-overlapping antisense RNA sequences (J), preferably adjacent non-overlapping antisense RNA sequences (J). This readily occurs with longer double-stranded regions (B) where the siRNA molecules each consisting of 21nt sense RNA sequences hybridised to 22nt antisense RNA sequences are repeated along the length of (B), where phased cleavage by Dicer can occur. In an embodiment of the first aspect, the precursor RNA molecule is capable of being cleaved in a eukaryotic cell by one or more ribonucleases (RNases) to produce multiple, different double-stranded product RNA molecules (P) which comprise doublestranded RNA product molecules as defined in two or more of the above embodiments.

[0061] Each of the embodiments of the first aspect may have the following feature: at least some of the one or more double -stranded product RNA molecule(s) (P) comprise one or two or three non-basepaired ribonucleotide(s) selected from the group consisting of ribonucleotides 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 and 17 of the antisense RNA sequence (J).

[0062] Each of the embodiments of the first aspect may have the following feature: at least some of the one or more double -stranded product RNA molecule(s) (P) comprise one or two non-basepaired ribonucleotide(s), preferably one non-basepaired ribonucleotide, selected from the group consisting of ribonucleotides 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 and 17 of the antisense RNA sequence (J).

[0063] In a second aspect, the present invention provides asymmetric precursor RNA molecules which are processed to produce siRNA molecules consisting of 21nt sense RNA sequences hybridised to 23nt antisense RNA sequences. Therefore, in this aspect the present invention provides a precursor RNA molecule (A) comprising at least one double-stranded RNA region (B), wherein:

[0064] (i) the double-stranded RNA region (B) comprises:

[0065] (a) a first RNA strand (D) of at least 23 contiguous ribonucleotides which comprises a first RNA sequence (E) of at least 21 contiguous ribonucleotides, and

[0066] (b) a second RNA strand (F) of at least 25 contiguous ribonucleotides which comprises a second RNA sequence (G) of at least 23 contiguous ribonucleotides, wherein the first RNA strand (D) and second RNA strand (F) are covalently linked by a linking RNA sequence (L), wherein the first RNA sequence (E) is hybridised to the second RNA sequence (G) by basepairing between at least 20 of the at least 21 contiguous ribonucleotides of the first RNA sequence (E) and at least 20 of the at least 23 contiguous ribonucleotides of the second RNA sequence (G), forming at least a part (C) of the double-stranded RNA region (B), wherein 2 or 3 ribonucleotides of the at least 23 ribonucleotides of (G) are non-basepaired and 0 or 1, respectively, ribonucleotides of the at least 21 ribonucleotides of (E) are non- basepaired in the part (C) of the double-stranded RNA region (B), forming one, two, three or four bulges in the part (C) of the double-stranded RNA region (B), wherein each of the one, two, three or four bulges is immediately flanked by ribonucleotides which are basepaired in the part (C) of the double-stranded RNA region (B); and

[0067] (ii) the precursor RNA molecule (A) is capable of being cleaved in a eukaryotic cell by one or more ribonucleases (RNases) to produce one or more double-stranded product RNA molecule(s) (P) consisting of a sense RNA sequence (H) of 21 contiguous ribonucleotides from the first RNA sequence (E) and an antisense RNA sequence (J) of 23 contiguous ribonucleotides from the second RNA strand (F), including at least 21 contiguous ribonucleotides from (G), wherein the one or more double-stranded product RNA molecule(s) (P) comprise the one, two, three or four bulges, wherein 18 or 19 of ribonucleotides 1 to 19 of the sense RNA sequence (H) each basepair with a ribonucleotide of ribonucleotides 1 to 21 of antisense RNA sequence (J) in the one or more double-stranded product RNA molecule(s) (P), wherein ribonucleotides 20 and 21 of the sense RNA sequence (H) form a 2- ribonucleotide unpaired 3' overhang and ribonucleotides 22 and 23 of the antisense RNA sequence (J) form a 2-ribonucleotide unpaired 3' overhang in the one or more doublestranded product RNA molecule(s) (P), wherein ribonucleotides 1 and 2 of the sense RNA sequence (H) basepair with ribonucleotides 20 and 21, respectively, of the antisense RNA sequence (J) in the one or more double-stranded product RNA molecule(s) (P), and wherein ribonucleotides 18 and 19 of the sense RNA sequence (H) basepair with ribonucleotides 1 and 2, respectively, of the antisense RNA sequence (J) in the one or more double-stranded product RNA molecule(s) (P). Exemplary RNA molecules of this aspect are illustrated schematically in Figure 53, panels I to Q.

[0068] In an embodiment of the second aspect, one or both of ribonucleotides 1 and 2 of the at least 23 contiguous ribonucleotides of the first RNA strand (D) are not basepaired with one or both, respectively, of ribonucleotides 24 and 25 of the at least 25 contiguous ribonucleotides of the second RNA strand (F). Exemplary RNA molecules of this embodiment are illustrated schematically in Figure 53, panels O to Q.

[0069] In an embodiment of the second aspect, the first RNA sequence (E) is hybridised to the second RNA sequence (G) by basepairing between at least 20 of the at least 21 contiguous ribonucleotides of the first RNA sequence (E) and at least 20 of the at least 23 contiguous ribonucleotides of the second RNA sequence (G), wherein 3 ribonucleotides, 2 of which are contiguous, of the at least 23 ribonucleotides of (G) are non-basepaired and 1 ribonucleotide of the at least 21 ribonucleotides of (E) is non-basepaired in the part (C) of the double-stranded RNA region (B), the non- basepaired ribonucleotides forming two bulges in the part (C) of the double-stranded RNA region (B), wherein each bulge is immediately flanked by ribonucleotides which are basepaired in the part (C) of the double-stranded RNA region (B), wherein one bulge is formed by a mismatched ribonucleotide pair between the first RNA sequence (E) and the second RNA sequence (G) and the second bulge is a diribonucleotide bulge, wherein the one or more double-stranded product RNA molecule(s) (P) comprise the bulges, and wherein all but one of ribonucleotides 1 to 19 of the sense RNA sequence (H) each basepair with a ribonucleotide of ribonucleotides 1 to 21 of antisense RNA sequence (J) in the one or more double-stranded product RNA molecule(s) (P). Exemplary RNA molecules of this embodiment are illustrated schematically in Figure 53, panel I.

[0070] In an embodiment of the second aspect, the first RNA sequence (E) is hybridised to the second RNA sequence (G) by basepairing between at least 20 of the at least 21 contiguous ribonucleotides of the first RNA sequence (E) and at least 20 of the at least 23 contiguous ribonucleotides of the second RNA sequence (G), wherein 3 ribonucleotides of the at least 23 ribonucleotides of (G) are non-basepaired, and 1 ribonucleotide of the at least 21 ribonucleotides of (E) is non-basepaired in the part (C) of the double-stranded RNA region (B), the non-basepaired ribonucleotides forming four single-ribonucleotide bulges in the part (C) of the double-stranded RNA region (B), wherein each bulge is immediately flanked by ribonucleotides which are basepaired in the part (C) of the double-stranded RNA region (B), wherein the one or more double-stranded product RNA molecule(s) (P) comprise the bulges, and wherein all but one of ribonucleotides 1 to 19 of the sense RNA sequence (H) each basepair with a ribonucleotide of ribonucleotides 1 to 21 of antisense RNA sequence (J) in the one or more double-stranded product RNA molecule(s) (P). Exemplary RNA molecules of this embodiment are illustrated schematically in Figure 53, panel J.

[0071] In an embodiment of the second aspect, the first RNA sequence (E) is hybridised to the second RNA sequence (G) by basepairing between at least 21 of the at least 21 contiguous ribonucleotides of the first RNA sequence (E) and at least 21 of the at least 23 contiguous ribonucleotides of the second RNA sequence (G), wherein 2 contiguous ribonucleotides of the at least 23 ribonucleotides of (G) are nonbasepaired and all of the at least 21 ribonucleotides of (E) are basepaired in the part (C) of the double-stranded RNA region (B), the 2 non-basepaired ribonucleotides forming a di-ribonucleotide bulge, or two single -ribonucleotide bulges, in the part (C) of the double-stranded RNA region (B), wherein the bulge is immediately flanked by ribonucleotides of the second RNA sequence (G) which are basepaired to ribonucleotides of the first RNA sequence (E), wherein the one or more double-stranded product RNA molecule(s) (P) comprise the bulge, and wherein ribonucleotides 1 to 19 of the sense RNA sequence (H) each basepair with a ribonucleotide of ribonucleotides 1 to 21 of antisense RNA sequence (J) in the one or more double-stranded product RNA molecule(s) (P). Exemplary RNA molecules of this embodiment are illustrated schematically in Figure 53, panels K, N and P.

[0072] In an embodiment of the second aspect, the first RNA sequence (E) is hybridised to the second RNA sequence (G) by basepairing between at least 21 of the at least 21 contiguous ribonucleotides of the first RNA sequence (E) and at least 21 of the at least 23 contiguous ribonucleotides of the second RNA sequence (G), wherein 2 ribonucleotides of the at least 23 ribonucleotides of (G) are non-basepaired and all of the at least 21 ribonucleotides of (E) are basepaired in the part (C) of the double - stranded RNA region (B), the 2 non-basepaired ribonucleotide forming two singleribonucleotide bulges in the part (C) of the double-stranded RNA region (B), wherein each bulge is immediately flanked by ribonucleotides of the second RNA sequence (G) which are basepaired to ribonucleotides of the first RNA sequence (E), wherein the one or more double-stranded product RNA molecule(s) (P) comprise the two bulges, and wherein ribonucleotides 1 to 19 of the sense RNA sequence (H) each basepair with a ribonucleotide of ribonucleotides 1 to 21 of antisense RNA sequence (J) in the one or more double-stranded product RNA molecule(s) (P). Exemplary RNA molecules of this embodiment are illustrated schematically in Figure 53, panels L, M and Q.

[0073] In an embodiment of the second aspect, the first RNA sequence (E) is hybridised to the second RNA sequence (G) by basepairing between at least 20 of the at least 21 contiguous ribonucleotides of the first RNA sequence (E) and at least 20 of the at least 23 contiguous ribonucleotides of the second RNA sequence (G), wherein 3 contiguous ribonucleotides of the at least 23 ribonucleotides of (G) are non- basepaired, and 1 ribonucleotide of the at least 21 ribonucleotides of (E) is non- basepaired in the part (C) of the double-stranded RNA region (B), the non-basepaired ribonucleotides forming a bulge in the part (C) of the double -stranded RNA region (B), wherein the bulge is immediately flanked by ribonucleotides which are basepaired in the part (C) of the double-stranded RNA region (B), wherein the one or more double-stranded product RNA molecule(s) (P) comprise the bulge, and wherein all but one of ribonucleotides 1 to 19 of the sense RNA sequence (H) each basepair with a ribonucleotide of ribonucleotides 1 to 21 of antisense RNA sequence (J) in the one or more double-stranded product RNA molecule(s) (P).

[0074] In an embodiment of the second aspect, the first RNA sequence (E) is hybridised to the second RNA sequence (G) by basepairing between at least 20 of the at least 21 contiguous ribonucleotides of the first RNA sequence (E) and at least 20 of the at least 23 contiguous ribonucleotides of the second RNA sequence (G), wherein 3 ribonucleotides of the at least 23 ribonucleotides of (G) are non-basepaired, and 1 ribonucleotide of the at least 21 ribonucleotides of (E) is non-basepaired in the part (C) of the double-stranded RNA region (B), the non-basepaired ribonucleotides forming three bulges in the part (C) of the double-stranded RNA region (B), wherein each bulge is immediately flanked by ribonucleotides which are basepaired in the part (C) of the double-stranded RNA region (B), wherein one bulge is formed by a mismatched ribonucleotide pair between the first RNA sequence (E) and the second RNA sequence (G) and the other two bulges are singleribonucleotide bulges, wherein the one or more double-stranded product RNA molecule(s) (P) comprise the bulges, and wherein all but one of ribonucleotides 1 to 19 of the sense RNA sequence (H) each basepair with a ribonucleotide of ribonucleotides 1 to 21 of antisense RNA sequence (J) in the one or more double-stranded product RNA molecule(s) (P).

[0075] In an embodiment of the second aspect, the first RNA sequence (E) is hybridised to the second RNA sequence (G) by basepairing between at least 20 of the at least 21 contiguous ribonucleotides of the first RNA sequence (E) and at least 20 of the at least 23 contiguous ribonucleotides of the second RNA sequence (G), wherein 3 ribonucleotides of the at least 23 ribonucleotides of (G) are non-basepaired, and 1 ribonucleotide of the at least 21 ribonucleotides of (E) is non-basepaired in the part (C) of the double-stranded RNA region (B), the non-basepaired ribonucleotides forming two bulges in the part (C) of the double-stranded RNA region (B), wherein each bulge is immediately flanked by ribonucleotides which are basepaired in the part (C) of the double-stranded RNA region (B), wherein one bulge is a single-ribonucleotide bulge, and the other non-basepaired ribonucleotides form the other bulge, wherein the one or more double-stranded product RNA molecule(s) (P) comprise the bulges, and wherein all but one of ribonucleotides 1 to 19 of the sense RNA sequence (H) each basepair with a ribonucleotide of ribonucleotides 1 to 21 of antisense RNA sequence (J) in the one or more double-stranded product RNA molecule(s) (P). Exemplary RNA molecules of this embodiment are illustrated schematically in Figure 53, panel O.

[0076] In an embodiment of the second aspect, the double-stranded RNA region (B) comprises:

[0077] (a) a first RNA strand (D) which comprises a first RNA sequence (E) of at least 23 contiguous ribonucleotides, and

[0078] (b) a second RNA strand (F) which comprises a second RNA sequence (G) of at least 25 contiguous ribonucleotides, wherein the first RNA strand (D) and second RNA strand (F) are covalently linked by a linking RNA sequence (L), wherein the first RNA sequence (E) is hybridised to the second RNA sequence (G) by basepairing between at least 22 of the at least 23 contiguous ribonucleotides of the first RNA sequence (E) and at least 22 of the at least 25 contiguous ribonucleotides of the second RNA sequence (G), forming at least a part (C) of the double-stranded RNA region (B), wherein 2 or 3 ribonucleotides of the at least 25 ribonucleotides of (G) are non-basepaired and 0 or 1, respectively, ribonucleotides of the at least 23 ribonucleotides of (E) are non- basepaired in the part (C) of the double-stranded RNA region (B), forming one, two, three or four bulges in the part (C) of the double-stranded RNA region (B), wherein each of the bulges is immediately flanked by ribonucleotides which are basepaired in the part (C) of the double-stranded RNA region (B). Exemplary RNA molecules of this embodiment are illustrated schematically in Figure 53, panels I to Q.

[0079] Thus, in an embodiment the present invention provides a precursor RNA molecule (A) comprising at least one double-stranded RNA region (B), wherein:

[0080] (i) the double-stranded RNA region (B) comprises:

[0081] (a) a first RNA strand (D) which comprises a first RNA sequence (E) of at least 23 contiguous ribonucleotides, and (b) a second RNA strand (F) which comprises a second RNA sequence (G) of at least 25 contiguous ribonucleotides, wherein the first RNA strand (D) and second RNA strand (F) are covalently linked by a linking RNA sequence (L), wherein the first RNA sequence (E) is hybridised to the second RNA sequence (G) by basepairing between at least 22 of the at least 23 contiguous ribonucleotides of the first RNA sequence (E) and at least 22 of the at least 25 contiguous ribonucleotides of the second RNA sequence (G), forming at least a part (C) of the double-stranded RNA region (B), wherein 2 or 3 ribonucleotides of the at least 25 ribonucleotides of (G) are non-basepaired and 0 or 1, respectively, ribonucleotides of the at least 23 ribonucleotides of (E) are non- basepaired in the part (C) of the double-stranded RNA region (B), forming one, two, three or four bulges in the part (C) of the double-stranded RNA region (B), wherein each of the bulges is immediately flanked by ribonucleotides which are basepaired in the part (C) of the double-stranded RNA region (B); and

[0082] (ii) the precursor RNA molecule (A) is capable of being cleaved in a eukaryotic cell by one or more ribonucleases (RNases) to produce one or more double-stranded product RNA molecule(s) (P) consisting of a sense RNA sequence (H) of 21 contiguous ribonucleotides from the first RNA sequence (E) and an antisense RNA sequence (J) of 23 contiguous ribonucleotides from the second RNA sequence (G), wherein the one or more double-stranded product RNA molecule(s) (P) comprise the one, two, three or four bulges, wherein 18 or 19 of ribonucleotides 1 to 19 of the sense RNA sequence (H) each basepair with a ribonucleotide of ribonucleotides 1 to 21 of antisense RNA sequence (J) in the one or more double-stranded product RNA molecule(s) (P), wherein ribonucleotides 20 and 21 of the sense RNA sequence (H) form a 2- ribonucleotide unpaired 3' overhang and ribonucleotides 22 and 23 of the antisense RNA sequence (J) form a 2-ribonucleotide unpaired 3' overhang in the one or more doublestranded product RNA molecule(s) (P), wherein ribonucleotides 1 and 2 of the sense RNA sequence (H) basepair with ribonucleotides 20 and 21, respectively, of the antisense RNA sequence (J) in the one or more double-stranded product RNA molecule(s) (P), and wherein ribonucleotides 18 and 19 of the sense RNA sequence (H) basepair with ribonucleotides 1 and 2, respectively, of the antisense RNA sequence (J) in the one or more double-stranded product RNA molecule(s) (P). In an embodiment of the second aspect, the first RNA sequence (E) is hybridised to the second RNA sequence (G) by basepairing between at least 22 of the at least 23 contiguous ribonucleotides of the first RNA sequence (E) and at least 22 of the at least 25 contiguous ribonucleotides of the second RNA sequence (G), wherein 3 ribonucleotides, 2 of which are contiguous, of the at least 25 ribonucleotides of (G) are non-basepaired, and 1 ribonucleotide of the at least 23 ribonucleotides of (E) is non-basepaired in the part (C) of the double-stranded RNA region (B), the non- basepaired ribonucleotides forming two bulges in the part (C) of the double-stranded RNA region (B), wherein each bulge is immediately flanked by ribonucleotides which are basepaired in the part (C) of the double-stranded RNA region (B), wherein one bulge is formed by a mismatched ribonucleotide pair between the first RNA sequence (E) and the second RNA sequence (G) and the second bulge is a diribonucleotide bulge, wherein the one or more double-stranded product RNA molecule(s) (P) comprise the bulges, and wherein all but one of ribonucleotides 1 to 19 of the sense RNA sequence (H) each basepair with a ribonucleotide of ribonucleotides 1 to 21 of antisense RNA sequence (J) in the one or more double-stranded product RNA molecule(s) (P).

[0083] In an embodiment of the second aspect, the first RNA sequence (E) is hybridised to the second RNA sequence (G) by basepairing between at least 22 of the at least 23 contiguous ribonucleotides of the first RNA sequence (E) and at least 22 of the at least 25 contiguous ribonucleotides of the second RNA sequence (G), wherein 3 ribonucleotides of the at least 25 ribonucleotides of (G) are non-basepaired, and 1 ribonucleotide of the at least 23 ribonucleotides of (E) is non-basepaired in the part (C) of the double-stranded RNA region (B), the non-basepaired ribonucleotides forming four single-ribonucleotide bulges in the part (C) of the double-stranded RNA region (B), wherein each bulge is immediately flanked by ribonucleotides which are basepaired in the part (C) of the double-stranded RNA region (B), wherein the one or more double-stranded product RNA molecule(s) (P) comprise the bulges, and wherein all but one of ribonucleotides 1 to 19 of the sense RNA sequence (H) each basepair with a ribonucleotide of ribonucleotides 1 to 21 of antisense RNA sequence (J) in the one or more double-stranded product RNA molecule(s) (P). Exemplary RNA molecules of this embodiment are illustrated schematically in Figure 53, panel J. In an embodiment of the second aspect, the first RNA sequence (E) is hybridised to the second RNA sequence (G) by basepairing between at least 23 of the at least 23 contiguous ribonucleotides of the first RNA sequence (E) and at least 23 of the at least 25 contiguous ribonucleotides of the second RNA sequence (G), wherein 2 contiguous ribonucleotides of the at least 25 ribonucleotides of (G) are nonbasepaired and all of the at least 23 ribonucleotides of (E) are basepaired in the part (C) of the double-stranded RNA region (B), the 2 non-basepaired ribonucleotides forming a di-ribonucleotide bulge in the part (C) of the double-stranded RNA region (B), wherein the bulge is immediately flanked by ribonucleotides of the second RNA sequence (G) which are basepaired to ribonucleotides of the first RNA sequence (E), wherein the one or more double-stranded product RNA molecule(s) (P) comprise the bulge, and wherein ribonucleotides 1 to 19 of the sense RNA sequence (H) each basepair with a ribonucleotide of ribonucleotides 1 to 21 of antisense RNA sequence (J) in the one or more double-stranded product RNA molecule(s) (P). Exemplary RNA molecules of this embodiment are illustrated schematically in Figure 53, panels K and P.

[0084] In an embodiment of the second aspect, the first RNA sequence (E) is hybridised to the second RNA sequence (G) by basepairing between at least 23 of the at least 23 contiguous ribonucleotides of the first RNA sequence (E) and at least 23 of the at least 25 contiguous ribonucleotides of the second RNA sequence (G), wherein 2 ribonucleotides of the at least 25 ribonucleotides of (G) are non-basepaired and all of the at least 23 ribonucleotides of (E) are basepaired in the part (C) of the doublestranded RNA region (B), the 2 non-basepaired ribonucleotide forming two singleribonucleotide bulges in the part (C) of the double-stranded RNA region (B), wherein each bulge is immediately flanked by ribonucleotides of the second RNA sequence (G) which are basepaired to ribonucleotides of the first RNA sequence (E), wherein the one or more double-stranded product RNA molecule(s) (P) comprise the two bulges, and wherein ribonucleotides 1 to 19 of the sense RNA sequence (H) each basepair with a ribonucleotide of ribonucleotides 1 to 21 of antisense RNA sequence (J) in the one or more double-stranded product RNA molecule(s) (P). Exemplary RNA molecules of this embodiment are illustrated schematically in Figure 53, panels L, M and Q.

[0085] In an embodiment of the second aspect, the first RNA sequence (E) is hybridised to the second RNA sequence (G) by basepairing between at least 22 of the at least 23 contiguous ribonucleotides of the first RNA sequence (E) and at least 22 of the at least 25 contiguous ribonucleotides of the second RNA sequence (G), wherein 3 contiguous ribonucleotides of the at least 25 ribonucleotides of (G) are nonbasepaired, and 1 ribonucleotide of the at least 23 ribonucleotides of (E) is nonbasepaired in the part (C) of the double-stranded RNA region (B), the non-basepaired ribonucleotides forming a bulge in the part (C) of the double -stranded RNA region (B), wherein the bulge is immediately flanked by ribonucleotides which are basepaired in the part (C) of the double-stranded RNA region (B), wherein the one or more double-stranded product RNA molecule(s) (P) comprise the bulge, and wherein all but one of ribonucleotides 1 to 19 of the sense RNA sequence (H) each basepair with a ribonucleotide of ribonucleotides 1 to 21 of antisense RNA sequence (J) in the one or more double-stranded product RNA molecule(s) (P).

[0086] In an embodiment of the second aspect, the first RNA sequence (E) is hybridised to the second RNA sequence (G) by basepairing between at least 22 of the at least 23 contiguous ribonucleotides of the first RNA sequence (E) and at least 22 of the at least 25 contiguous ribonucleotides of the second RNA sequence (G), wherein 3 ribonucleotides of the at least 25 ribonucleotides of (G) are non-basepaired, and 1 ribonucleotide of the at least 23 ribonucleotides of (E) is non-basepaired in the part (C) of the double-stranded RNA region (B), the non-basepaired ribonucleotides forming three bulges in the part (C) of the double-stranded RNA region (B), wherein each bulge is immediately flanked by ribonucleotides which are basepaired in the part (C) of the double-stranded RNA region (B), wherein one bulge is formed by a mismatched ribonucleotide pair between the first RNA sequence (E) and the second RNA sequence (G) and the other two bulges are singleribonucleotide bulges, wherein the one or more double-stranded product RNA molecule(s) (P) comprise the bulges, and wherein all but one of ribonucleotides 1 to 19 of the sense RNA sequence (H) each basepair with a ribonucleotide of ribonucleotides 1 to 21 of antisense RNA sequence (J) in the one or more double-stranded product RNA molecule(s) (P).

[0087] In an embodiment of the second aspect, the first RNA sequence (E) is hybridised to the second RNA sequence (G) by basepairing between at least 22 of the at least 23 contiguous ribonucleotides of the first RNA sequence (E) and at least 22 of the at least 25 contiguous ribonucleotides of the second RNA sequence (G), wherein 3 ribonucleotides of the at least 25 ribonucleotides of (G) are non-basepaired, and 1 ribonucleotide of the at least 23 ribonucleotides of (E) is non-basepaired in the part (C) of the double-stranded RNA region (B), the non-basepaired ribonucleotides forming two bulges in the part (C) of the double-stranded RNA region (B), wherein each bulge is immediately flanked by ribonucleotides which are basepaired in the part (C) of the double-stranded RNA region (B), wherein one bulge is a single-ribonucleotide bulge, and the other non-basepaired ribonucleotides form the other bulge, wherein the one or more double-stranded product RNA molecule(s) (P) comprise the bulges, and wherein all but one of ribonucleotides 1 to 19 of the sense RNA sequence (H) each basepair with a ribonucleotide of ribonucleotides 1 to 21 of antisense RNA sequence (J) in the one or more double-stranded product RNA molecule(s) (P).

[0088] Each of the embodiments of the second aspect may have the following feature:

[0089] (i) ribonucleotides 1, 2 and 3 of the sense RNA sequence (H) basepair with ribonucleotides 19, 20 and 21, respectively, of the antisense RNA sequence (J) in at least some of the one or more double-stranded product RNA molecule(s) (P), or

[0090] (ii) ribonucleotides 17, 18 and 19 of the sense RNA sequence (H) basepair with ribonucleotides 1, 2 and 3, respectively, of the antisense RNA sequence (J) in at least some of the one or more double-stranded product RNA molecule(s) (P), or preferably both (i) and (ii). Exemplary RNA molecules of this embodiment are illustrated schematically in Figure 53, panels I to Q, which all show product RNA molecules with at least three contiguous basepairs at both ends of the double-stranded region of the product RNA molecules. In further features of these embodiments, the product RNA molecules may have at least four, at least five or at least six contiguous basepairs at one or both ends of the double-stranded molecule before any bulge. In these embodiments, any of those basepairs may be G:U basepairs, preferably one or more of ribonucleotides 1, 2 and 3 of the antisense sequence (J) are involved in G:U basepairs, more preferably one or both of ribonucleotides 1 and 2 of the antisense sequence (J) are involved in G:U basepairs, even more preferably ribonucleotide 1 of the antisense sequence (J) is involved in G:U basepairs. Most preferably, one or more of ribonucleotides 1, 2 and 3 of the antisense sequence (J) are U ribonucleotides, or one or both of ribonucleotides 1 and 2 are U ribonucleotides, or ribonucleotide 1 is a U ribonucleotide, involved in G:U basepairs.

[0091] The design principles for the asymmetric precursor RNA molecules of the second aspect can be applied in an extended fashion to longer double -stranded regions. For example, the non-basepaired ribonucleotides in the second RNA sequence (G) (antisense sequence) may be arranged in a periodic fashion to provide a population of product RNA molecules (P) having multiple, non-overlapping antisense RNA sequences (J) of 23nt. Such precursor RNA molecules are particularly useful for reducing expression of a target RNA molecule in a plant cell, fungal cell or nematode cell. They are also useful in other invertebrate animal cells such as an arthropod cell or insect cell, or in a non-mammalian vertebrate animal cell. They may be produced in a plant cell to reduce an insect target RNA molecule or a fungal pathogen or nematode target RNA molecule, or applied topically to a plant or insect to reduce a target RNA molecule. For example, in an embodiment of the second aspect, the first RNA sequence (E) comprises at least 44 contiguous ribonucleotides and the second RNA sequence (G) comprises at least 48 contiguous ribonucleotides, wherein the first RNA sequence (E) is hybridised to the second RNA sequence (G) by basepairing between at least 42, preferably at least 43 or at least 44, of the at least 44 contiguous ribonucleotides of the first RNA sequence (E) and at least 42, preferably at least 43 or at least 44, of the at least 48 contiguous ribonucleotides of the second RNA sequence (G), wherein 4, 5 or 6 ribonucleotides of the at least 48 contiguous ribonucleotides of (G) are non-basepaired, and 0, 1 or 2, respectively, ribonucleotides of the at least 44 contiguous ribonucleotides of (E) are non-basepaired in the part (C) of the doublestranded RNA region (B), forming bulges in the part (C) of the double-stranded RNA region (B), wherein each of the bulges is immediately flanked by ribonucleotides which are basepaired in the part (C) of the double-stranded RNA region (B), wherein the precursor RNA molecule is capable of being cleaved in a eukaryotic cell by one or more ribonucleases (RNases) to produce multiple, different doublestranded product RNA molecules (P) consisting of a sense RNA sequence (H) of 21 contiguous ribonucleotides and an antisense RNA sequence (J) of 23 contiguous ribonucleotides, wherein the multiple, different double-stranded product RNA molecule(s) (P) each comprise at least one of the bulges, and wherein at least some of the multiple, different double -stranded product RNA molecules (P) comprise non-overlapping antisense RNA sequences (J), preferably adjacent non-overlapping antisense RNA sequences (J).

[0092] In another embodiment of the second aspect, the first RNA strand (D) comprises a first RNA sequence (E) of at least 44 contiguous ribonucleotides and the second RNA strand (F) comprises a second RNA sequence (G) of at least 48 contiguous ribonucleotides, wherein the first RNA sequence (E) is hybridised to the second RNA sequence (G) by basepairing between all 44 contiguous ribonucleotides of the first RNA sequence (E) and 44 of the 48 contiguous ribonucleotides of the second RNA sequence (G), forming a part (C) of the double-stranded RNA region (B), wherein 4 ribonucleotides of the 48 contiguous ribonucleotides of (G) are nonbasepaired in the part (C) of the double-stranded RNA region (B), forming 2, 3 or 4 bulges in the part (C) of the double-stranded RNA region (B), wherein each of the bulges is immediately flanked by ribonucleotides which are basepaired in the part (C) of the double-stranded RNA region (B), wherein the precursor RNA molecule is capable of being cleaved in a eukaryotic cell by one or more ribonucleases (RNases) to produce multiple, different doublestranded product RNA molecules (P) consisting of a sense RNA sequence (H) of 21 contiguous ribonucleotides and an antisense RNA sequence (J) of 23 contiguous ribonucleotides, wherein the multiple, different double-stranded product RNA molecule(s) (P) each comprise at least one of the bulges, and wherein at least some of the multiple, different double -stranded product RNA molecules (P) have non-overlapping antisense RNA sequences (J), preferably adjacent non-overlapping antisense RNA sequences (J). In this and the previous embodiments, the bulges are preferably spaced apart by 6-16 contiguous basepairs, more preferably by 7-15 or 8-14 contiguous basepairs, even more preferably by 9-13 contiguous basepairs, most preferably by 10, 11 or 12 contiguous basepairs. In specific examples, the bulges are spaced apart by 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 contiguous basepairs. In an embodiment, all of these basepairs are canonical basepairs. Alternatively, 1, 2, 3, 4 or 5 of the basepairs between the bulges are G:U basepairs. The precursor RNA molecule may comprise two or more such double-stranded RNA regions, for example comprise an ledRNA structure.

[0093] In a different embodiment with longer dsRNA regions of at least 44 basepairs, the precursor RNA molecule lacks the linking RNA sequence (L) and the first RNA strand (D) and the second RNA strand (F) hybridise to form the double -stranded RNA region (B). Such precursor RNA molecule may be readily produced in a cell -free system, for example in vitro. In an embodiment, the first RNA strand (D) comprises a first RNA sequence (E) of at least 44 contiguous ribonucleotides and the second RNA strand (F) comprises a second RNA sequence (G) of at least 48 contiguous ribonucleotides, wherein the first RNA sequence (E) is hybridised to the second RNA sequence (G) by basepairing between all 44 contiguous ribonucleotides of the first RNA sequence (E) and 44 of the 48 contiguous ribonucleotides of the second RNA sequence (G), forming a part (C) of the double-stranded RNA region (B), wherein 4 ribonucleotides of the 48 contiguous ribonucleotides of (G) are nonbasepaired in the part (C) of the double -stranded RNA region (B), forming 2, 3 or 4 bulges in the part (C) of the double-stranded RNA region (B), wherein each of the bulges is immediately flanked by ribonucleotides which are basepaired in the part (C) of the double-stranded RNA region (B), wherein the precursor RNA molecule is capable of being cleaved in a eukaryotic cell by one or more ribonucleases (RNases) to produce multiple, different doublestranded product RNA molecules (P) consisting of a sense RNA sequence (H) of 21 contiguous ribonucleotides and an antisense RNA sequence (J) of 23 contiguous ribonucleotides, wherein the multiple, different double-stranded product RNA molecule(s) (P) each comprise at least one of the bulges, and wherein at least some of the multiple, different double -stranded product RNA molecules (P) have non-overlapping antisense RNA sequences (J), preferably adjacent non-overlapping antisense RNA sequences (J). In this embodiment, the bulges are preferably spaced apart by 6-16 contiguous basepairs, more preferably by 7-15 or 8-14 contiguous basepairs, even more preferably by 9-13 contiguous basepairs, most preferably by 10, 11 or 12 contiguous basepairs. In specific examples, the bulges are spaced apart by 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 contiguous basepairs. In a preferred example, all of these basepairs are canonical basepairs. Alternatively, 1, 2, 3, 4 or 5 of the basepairs between the bulges are G:U basepairs.

[0094] For example, in an embodiment, the first RNA sequence (E) comprises at least 65 contiguous ribonucleotides and the second RNA sequence (G) comprises at least 71 contiguous ribonucleotides, and wherein the first RNA sequence (E) is hybridised to the second RNA sequence (G) by basepairing between at least 62, preferably at least 63, at least 64 or at least 65, of the at least 65 contiguous ribonucleotides of the first RNA sequence (E) and at least 62, preferably at least 63, at least 64 or at least 65, of the at least 71 contiguous ribonucleotides of the second RNA sequence (G).

[0095] In another embodiment of the second aspect, the first RNA strand (D) comprises a first RNA sequence (E) of at least 65 contiguous ribonucleotides and the second RNA strand (F) comprises a second RNA sequence (G) of at least 71 contiguous ribonucleotides, wherein the first RNA sequence (E) is hybridised to the second RNA sequence (G) by basepairing between all 65 contiguous ribonucleotides of the first RNA sequence (E) and 65 of the 71 contiguous ribonucleotides of the second RNA sequence (G), forming a part (C) of the double-stranded RNA region (B), wherein 6 ribonucleotides of the 71 contiguous ribonucleotides of (G) are nonbasepaired in the part (C) of the double-stranded RNA region (B), forming 3, 4, 5 or 6 bulges in the part (C) of the double-stranded RNA region (B), wherein each of the bulges is immediately flanked by ribonucleotides which are basepaired in the part (C) of the double-stranded RNA region (B), wherein the precursor RNA molecule is capable of being cleaved in a eukaryotic cell by one or more ribonucleases (RNases) to produce multiple, different doublestranded product RNA molecules (P) consisting of a sense RNA sequence (H) of 21 contiguous ribonucleotides and an antisense RNA sequence (J) of 23 contiguous ribonucleotides, wherein the multiple, different double-stranded product RNA molecule(s) (P) each comprise at least one of the bulges, and wherein at least some of the multiple, different double -stranded product RNA molecules (P) have non-overlapping antisense RNA sequences (J), preferably adjacent non-overlapping antisense RNA sequences (J). In this and the previous embodiments, the bulges are preferably spaced apart periodically, for example by 6-16 contiguous basepairs, more preferably by 7-15 or 8-14 contiguous basepairs, even more preferably by 9-13 contiguous basepairs, most preferably by 10, 11 or 12 contiguous basepairs. In specific examples, the bulges are spaced apart by 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 contiguous basepairs. In an example, all of these basepairs are canonical basepairs. Alternatively, 1, 2, 3, 4 or 5 of the basepairs between the bulges are G:U basepairs. The precursor RNA molecule may comprise two or more such double-stranded RNA regions, for example comprise an ledRNA structure.

[0096] In a different embodiment with longer dsRNA regions of at least 65 basepairs, the precursor RNA molecule lacks the linking RNA sequence (L) and the first RNA strand

[0097] (D) and the second RNA strand (F) hybridise to form the double -stranded RNA region (B). Such precursor RNA molecule may be readily produced in a cell-free system, for example in vitro. In an embodiment, the first RNA strand (D) comprises a first RNA sequence (E) of at least 65 contiguous ribonucleotides and the second RNA strand (F) comprises a second RNA sequence (G) of at least 71 contiguous ribonucleotides, wherein the first RNA sequence (E) is hybridised to the second RNA sequence (G) by basepairing between all 65 contiguous ribonucleotides of the first RNA sequence

[0098] (E) and 65 of the 71 contiguous ribonucleotides of the second RNA sequence (G), forming a part (C) of the double-stranded RNA region (B), wherein 6 ribonucleotides of the 71 contiguous ribonucleotides of (G) are nonbasepaired in the part (C) of the double-stranded RNA region (B), forming 3, 4, 5 or 6 bulges in the part (C) of the double-stranded RNA region (B), wherein each of the bulges is immediately flanked by ribonucleotides which are basepaired in the part (C) of the double-stranded RNA region (B), wherein the precursor RNA molecule is capable of being cleaved in a eukaryotic cell by one or more ribonucleases (RNases) to produce multiple, different doublestranded product RNA molecules (P) consisting of a sense RNA sequence (H) of 21 contiguous ribonucleotides and an antisense RNA sequence (J) of 23 contiguous ribonucleotides, wherein the multiple, different double-stranded product RNA molecule(s) (P) each comprise at least one of the bulges, and wherein at least some of the multiple, different double -stranded product RNA molecules (P) have non-overlapping antisense RNA sequences (J), preferably adjacent non-overlapping antisense RNA sequences (J). In this embodiment, the bulges are preferably spaced apart by 6-16 contiguous basepairs, more preferably by 7-15 or 8-14 contiguous basepairs, even more preferably by 9-13 contiguous basepairs, most preferably by 10, 11 or 12 contiguous basepairs. In specific examples, the bulges are spaced apart by 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 contiguous basepairs. In a preferred example, all of these basepairs are canonical basepairs. Alternatively, 1, 2, 3, 4 or 5 of the basepairs between the bulges are G:U basepairs.

[0099] In the previous three embodiments, the first RNA sequence (E) and the second RNA sequence (G) may extend to longer than 65 and 71 ribonucleotides, respectively. In a further embodiment, the first RNA sequence (E) comprises at least 86 contiguous ribonucleotides and the second RNA sequence (G) comprises at least 94 contiguous ribonucleotides, and wherein the first RNA sequence (E) is hybridised to the second RNA sequence (G) by basepairing between at least 82, preferably at least 83, at least 84, at least 85 or at least 86, of the at least 86 contiguous ribonucleotides of the first RNA sequence (E) and at least 82, preferably at least 83, at least 84, at least 85 or at least 86, of the at least 94 contiguous ribonucleotides of the second RNA sequence (G).

[0100] In another embodiment of the second aspect, the first RNA strand (D) comprises a first RNA sequence (E) of at least 86 contiguous ribonucleotides and the second RNA strand (F) comprises a second RNA sequence (G) of at least 94 contiguous ribonucleotides, wherein the first RNA sequence (E) is hybridised to the second RNA sequence (G) by basepairing between all 86 contiguous ribonucleotides of the first RNA sequence (E) and 86 of the 94 contiguous ribonucleotides of the second RNA sequence (G), forming a part (C) of the double-stranded RNA region (B), wherein 8 ribonucleotides of the 94 contiguous ribonucleotides of (G) are nonbasepaired in the part (C) of the double-stranded RNA region (B), forming 4-8 bulges in the part (C) of the double-stranded RNA region (B), wherein each of the bulges is immediately flanked by ribonucleotides which are basepaired in the part (C) of the double-stranded RNA region (B), wherein the precursor RNA molecule is capable of being cleaved in a eukaryotic cell by one or more ribonucleases (RNases) to produce multiple, different doublestranded product RNA molecules (P) consisting of a sense RNA sequence (H) of 21 contiguous ribonucleotides and an antisense RNA sequence (J) of 23 contiguous ribonucleotides, wherein the multiple, different double-stranded product RNA molecule(s) (P) each comprise at least one of the bulges, and wherein at least some of the multiple, different double -stranded product RNA molecules (P) have non-overlapping antisense RNA sequences (J), preferably adjacent non-overlapping antisense RNA sequences (J). In this and the previous embodiments, the bulges are preferably spaced apart periodically, for example by 6-16 contiguous basepairs, more preferably by 7-15 or 8-14 contiguous basepairs, even more preferably by 9-13 contiguous basepairs, most preferably by 10, 11 or 12 contiguous basepairs. In specific examples, the bulges are spaced apart by 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 contiguous basepairs. In a preferred example, all of these basepairs are canonical basepairs. Alternatively, 1, 2, 3, 4 or 5 of the basepairs between the bulges are G:U basepairs. The precursor RNA molecule may comprise two or more such double-stranded RNA regions, for example comprise a ledRNA structure.

[0101] In a different embodiment with longer dsRNA regions of at least 86 basepairs, the precursor RNA molecule lacks the linking RNA sequence (L) and the first RNA strand

[0102] (D) and the second RNA strand (F) hybridise to form the double -stranded RNA region (B). Such precursor RNA molecule may be readily produced in a cell-free system, for example in vitro. In an embodiment, the first RNA strand (D) comprises a first RNA sequence (E) of at least 86 contiguous ribonucleotides and the second RNA strand (F) comprises a second RNA sequence (G) of at least 94 contiguous ribonucleotides, wherein the first RNA sequence (E) is hybridised to the second RNA sequence (G) by basepairing between all 86 contiguous ribonucleotides of the first RNA sequence

[0103] (E) and 86 of the 94 contiguous ribonucleotides of the second RNA sequence (G), forming a part (C) of the double-stranded RNA region (B), wherein 8 ribonucleotides of the 94 contiguous ribonucleotides of (G) are nonbasepaired in the part (C) of the double-stranded RNA region (B), forming 4-8 bulges in the part (C) of the double-stranded RNA region (B), wherein each of the bulges is immediately flanked by ribonucleotides which are basepaired in the part (C) of the double-stranded RNA region (B), wherein the precursor RNA molecule is capable of being cleaved in a eukaryotic cell by one or more ribonucleases (RNases) to produce multiple, different doublestranded product RNA molecules (P) consisting of a sense RNA sequence (H) of 21 contiguous ribonucleotides and an antisense RNA sequence (J) of 23 contiguous ribonucleotides, wherein the multiple, different double-stranded product RNA molecule(s) (P) each comprise at least one of the bulges, and wherein at least some of the multiple, different double -stranded product RNA molecules (P) have non-overlapping antisense RNA sequences (J), preferably adjacent non-overlapping antisense RNA sequences (J). In this embodiment, the bulges are preferably spaced apart by 6-16 contiguous basepairs, more preferably by 7-15 or 8-14 contiguous basepairs, even more preferably by 9-13 contiguous basepairs, most preferably by 10, 11 or 12 contiguous basepairs. In specific examples, the bulges are spaced apart by 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 contiguous basepairs. In a preferred example, all of these basepairs are canonical basepairs. Alternatively, 1, 2, 3, 4 or 5 of the basepairs between the bulges are G:U basepairs. In a preferred example, all of these basepairs are canonical basepairs. Alternatively, 1, 2, 3, 4 or 5 of the basepairs between the bulges are G:U basepairs.

[0104] In the previous three embodiments, the first RNA sequence (E) and the second RNA sequence (G) may extend to longer than 86 and 94 ribonucleotides, respectively. In a further embodiment of the second aspect, the first RNA sequence (E) comprises at least 107 contiguous ribonucleotides and the second RNA sequence (G) comprises at least 117 contiguous ribonucleotides, and wherein the first RNA sequence (E) is hybridised to the second RNA sequence (G) by basepairing between at least 102, preferably at least 103, at least 104, at least 105, at least 106 or at least 107, of the at least 107 contiguous ribonucleotides of the first RNA sequence (E) and at least 102, preferably at least 103, at least 104, at least 105, at least 106 or at least 107, of the at least 117 contiguous ribonucleotides of the second RNA sequence (G). In this embodiment, at least 10 ribonucleotides of the second RNA sequence, up to a maximum of 15 ribonucleotides, are non-basepaired and form bulges, preferably at least some are single ribonucleotide bulges. In another embodiment of the second aspect, the first RNA strand (D) comprises a first RNA sequence (E) of at least 107 contiguous ribonucleotides and the second RNA strand (F) comprises a second RNA sequence (G) of at least 117 contiguous ribonucleotides, wherein the first RNA sequence (E) is hybridised to the second RNA sequence (G) by basepairing between all 107 contiguous ribonucleotides of the first RNA sequence (E) and 107 of the 117 contiguous ribonucleotides of the second RNA sequence (G), forming a part (C) of the double-stranded RNA region (B), wherein 10 ribonucleotides of the 117 contiguous ribonucleotides of (G) are nonbasepaired in the part (C) of the double-stranded RNA region (B), forming 5-10 bulges in the part (C) of the double-stranded RNA region (B), wherein each of the bulges is immediately flanked by ribonucleotides which are basepaired in the part (C) of the double-stranded RNA region (B), wherein the precursor RNA molecule is capable of being cleaved in a eukaryotic cell by one or more ribonucleases (RNases) to produce multiple, different doublestranded product RNA molecules (P) consisting of a sense RNA sequence (H) of 21 contiguous ribonucleotides and an antisense RNA sequence (J) of 23 contiguous ribonucleotides, wherein the multiple, different double-stranded product RNA molecule(s) (P) each comprise at least one of the bulges, and wherein at least some of the multiple, different double -stranded product RNA molecules (P) have non-overlapping antisense RNA sequences (J), preferably adjacent non-overlapping antisense RNA sequences (J). In this and the previous embodiments, the bulges are preferably spaced apart periodically, for example by 6-16 contiguous basepairs, more preferably by 7-15 or 8-14 contiguous basepairs, even more preferably by 9-13 contiguous basepairs, most preferably by 10, 11 or 12 contiguous basepairs. In specific examples, the bulges are spaced apart by 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 contiguous basepairs. In an example, all of these basepairs are canonical basepairs. Alternatively, 1, 2, 3, 4 or 5 of the basepairs between the bulges are G:U basepairs. The precursor RNA molecule may comprise two or more such double-stranded RNA regions, for example comprise a ledRNA structure.

[0105] In a different embodiment with longer dsRNA regions of at least 107 basepairs, the precursor RNA molecule lacks the linking RNA sequence (L) and the first RNA strand (D) and the second RNA strand (F) hybridise to form the double-stranded RNA region (B). Such precursor RNA molecule may be readily produced in a cell-free system, for example in vitro. In an embodiment, the first RNA strand (D) comprises a first RNA sequence (E) of at least 107 contiguous ribonucleotides and the second RNA strand (F) comprises a second RNA sequence (G) of at least 117 contiguous ribonucleotides, wherein the first RNA sequence (E) is hybridised to the second RNA sequence (G) by basepairing between all 107 contiguous ribonucleotides of the first RNA sequence (E) and 107 of the 117 contiguous ribonucleotides of the second RNA sequence (G), forming a part (C) of the double-stranded RNA region (B), wherein 10 ribonucleotides of the 117 contiguous ribonucleotides of (G) are nonbasepaired in the part (C) of the double-stranded RNA region (B), forming bulges in the part (C) of the double-stranded RNA region (B), wherein each of the bulges is immediately flanked by ribonucleotides which are basepaired in the part (C) of the double-stranded RNA region (B), wherein the precursor RNA molecule is capable of being cleaved in a eukaryotic cell by one or more ribonucleases (RNases) to produce multiple, different doublestranded product RNA molecules (P) consisting of a sense RNA sequence (H) of 21 contiguous ribonucleotides and an antisense RNA sequence (J) of 23 contiguous ribonucleotides, wherein the multiple, different double-stranded product RNA molecule(s) (P) each comprise at least one of the bulges, and wherein at least some of the multiple, different double -stranded product RNA molecules (P) have non-overlapping antisense RNA sequences (J), preferably adjacent non-overlapping antisense RNA sequences (J). In this embodiment, the bulges are preferably spaced apart by 6-16 contiguous basepairs, more preferably by 7-15 or 8-14 contiguous basepairs, even more preferably by 9-13 contiguous basepairs, most preferably by 10, 11 or 12 contiguous basepairs. In specific examples, the bulges are spaced apart by 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 contiguous basepairs. In a preferred example, all of these basepairs are canonical basepairs. Alternatively, 1, 2, 3, 4 or 5 of the basepairs between the bulges are G:U basepairs.

[0106] In each of the above embodiments of the second aspect, the second RNA sequence (G) is longer than the first RNA sequence (E) because of the non-basepaired ribonucleotides that bulge out in (G). Preferably, the length of the first RNA sequence (E) is 90%-94% or 90%-93% or 91%-94% or 91%— 93% of the length of the second RNA sequence (G). These features may also apply across the full length of the doublestranded region (B), where the part (C) of the double -stranded region is the full length of (B). As the skilled person would appreciate, further embodiments include a first RNA sequence (E) and a second RNA sequence (G) having longer contiguous ribonucleotides following the same principles in the above embodiments.

[0107] In an embodiment of the first or second aspect, or the molecules lacking a loop, the first RNA sequence (E) and the second RNA sequence (G) both comprise at least 100 contiguous ribonucleotides, or at least 110 or 120, or at least 150, or at least 200, or at least 250, or at least 300 contiguous ribonucleotides, preferably to a maximum of 1000 ribonucleotides, more preferably to a maximum of 800 contiguous ribonucleotides, or even more preferably to a maximum of 600 contiguous ribonucleotides. For example, the first RNA sequence (E) and the second RNA sequence (G) both comprise contiguous ribonucleotides in the range 100-1000, 100-800, or 100-600 ribonucleotides, or in the range 150-1000, 150-800, or 150-600 ribonucleotides. In preferred embodiments, the length of the sense RNA sequence of the dsRNA region is 90%-97% or 90%-96% the length of the antisense sequence, for example 90%-94% or 90%-93% or 91%-94% or 91%— 93% compared to the antisense sequence. These features are applicable to hairpin RNAs and to dsRNAs formed by annealing of two RNA strands i.e. without a joining loop sequence. Each of these features may also be applied to a second dsRNA region in the precursor RNA molecule, for example in a ledRNA molecule. In these embodiments, the eukaryotic cell in which the precursor RNA molecule (A) is cleaved is preferably a plant cell, a fungal cell, a nematode cell, or an arthropod cell such as an insect cell, or the target RNA molecule is preferably in a plant cell, a fungal cell, a nematode cell, or an arthropod cell such as an insect cell. Such precursor RNA molecules are particularly useful for reducing expression of a target RNA molecule in a plant cell, fungal cell or nematode cell. They are also useful in other invertebrate animal cells such as an arthropod cell, for example an insect cell, or in a non-mammalian vertebrate animal cell. They may be produced in a plant cell to reduce an insect target RNA molecule or a fungal pathogen or nematode target RNA molecule, or applied topically to a plant or insect to reduce a target RNA molecule.

[0108] Each of the embodiments of the second aspect, or the molecules lacking a loop, may have the following feature: the precursor RNA molecule is capable of being cleaved in a eukaryotic cell by one or more ribonucleases (RNases) to produce multiple, different double-stranded product RNA molecules (P) which each consist of a sense RNA sequence (H) of 21 contiguous ribonucleotides from the first RNA sequence (E) and an antisense RNA sequence (J) of 23 contiguous ribonucleotides from the second RNA sequence (G), wherein at least some of the multiple, different double-stranded product RNA molecules (P) have overlapping antisense RNA sequences (J). In a further embodiment, at least some of the multiple, different double-stranded product RNA molecules (P) have non-overlapping antisense RNA sequences (J), where the population of double-stranded product RNA molecules (P) produced from the precursor RNA molecule includes some overlapping and some non-overlapping antisense RNA sequences (J). Preferably, there are more non-overlapping antisense RNA sequences (J), as readily occurs with longer (>42 basepairs) double-stranded regions (B) where the siRNA molecules each consisting of 21nt sense RNA sequences hybridised to 23nt antisense RNA sequences are phased along the length of (B).

[0109] Each of the embodiments of the second aspect, or the molecules lacking a loop, may have the following feature: the precursor RNA molecule is capable of being cleaved in a eukaryotic cell by one or more ribonucleases (RNases) to produce multiple, different double-stranded product RNA molecules (P) which each consist of a sense RNA sequence (H) of 21 contiguous ribonucleotides from a first RNA sequence (E) and an antisense RNA sequence (J) of 23 contiguous ribonucleotides from a second RNA sequence (G), wherein at least some of the multiple, different double-stranded product RNA molecules (P) have non-overlapping antisense RNA sequences (J), preferably adjacent non-overlapping antisense RNA sequences (J).

[0110] In an embodiment of the second aspect, or the molecules lacking a loop, the molecule is capable of being cleaved in a eukaryotic cell by one or more ribonucleases (RNases) to produce multiple, different double-stranded product RNA molecules (P) which comprise double-stranded RNA product molecules as defined in two or more embodiments of the second aspect.

[0111] In a third aspect, the present invention provides asymmetric precursor RNA molecules which are processed to produce siRNA molecules consisting of 21nt sense RNA sequences hybridised to 24nt antisense RNA sequences. Therefore, in this aspect the present invention provides a precursor RNA molecule (A) comprising at least one double-stranded RNA region (B), wherein:

[0112] (i) the double-stranded RNA region (B) comprises:

[0113] (a) a first RNA strand (D) of at least 23 contiguous ribonucleotides which comprises a first RNA sequence (E) of at least 21 contiguous ribonucleotides, and

[0114] (b) a second RNA strand (F) of at least 26 contiguous ribonucleotides which comprises a second RNA sequence (G) of at least 24 contiguous ribonucleotides, wherein the first RNA strand (D) and second RNA strand (F) are covalently linked by a linking RNA sequence (L), wherein the first RNA sequence (E) is hybridised to the second RNA sequence (G) by basepairing between at least 20 of the at least 21 contiguous ribonucleotides of the first RNA sequence (E) and at least 20 of the at least 24 contiguous ribonucleotides of the second RNA sequence (G), forming at least a part (C) of the double-stranded RNA region (B), wherein 3 or 4 ribonucleotides of the at least 24 ribonucleotides of (G) are non-basepaired and 0 or 1, respectively, ribonucleotides of the at least 21 ribonucleotides of (E) are non- basepaired in the part (C) of the double-stranded RNA region (B), forming one, two, three, four or five bulges in the part (C) of the double-stranded RNA region (B), wherein each of the one, two, three, four or five bulges is immediately flanked by ribonucleotides which are basepaired in the part (C) of the double-stranded RNA region (B); and

[0115] (ii) the precursor RNA molecule (A) is capable of being cleaved in a eukaryotic cell by one or more ribonucleases (RNases) to produce one or more double-stranded product RNA molecule(s) (P) consisting of a sense RNA sequence (H) of 21 contiguous ribonucleotides from the first RNA sequence (E) and an antisense RNA sequence (J) of 24 contiguous ribonucleotides from the second RNA strand (F), including at least 22 contiguous ribonucleotides from (G), wherein the one or more double-stranded product RNA molecule(s) (P) comprise the one, two, three, four or five bulges, wherein 18 or 19 of ribonucleotides 1 to 19 of the sense RNA sequence (H) each basepair with a ribonucleotide of ribonucleotides 1 to 22 of antisense RNA sequence (J) in the one or more double-stranded product RNA molecule(s) (P), wherein ribonucleotides 20 and 21 of the sense RNA sequence (H) form a 2- ribonucleotide unpaired 3' overhang and ribonucleotides 23 and 24 of the antisense RNA sequence (J) form a 2-ribonucleotide unpaired 3' overhang in the one or more doublestranded product RNA molecule(s) (P), wherein ribonucleotides 1 and 2 of the sense RNA sequence (H) basepair with ribonucleotides 21 and 22, respectively, of the antisense RNA sequence (J) in the one or more double-stranded product RNA molecule(s) (P), and wherein ribonucleotides 18 and 19 of the sense RNA sequence (H) basepair with ribonucleotides 1 and 2, respectively, of the antisense RNA sequence (J) in the one or more double-stranded product RNA molecule(s) (P). Exemplary RNA molecules of this embodiment are illustrated schematically in Figure 53, panels Rto Z.

[0116] In an embodiment of the third aspect, one or both of ribonucleotides 1 and 2 of the at least 23 contiguous ribonucleotides of the first RNA strand (D) are not basepaired with one or both, respectively, of ribonucleotides 25 and 26 of the at least 26 contiguous ribonucleotides of the second RNA strand (F).

[0117] In an embodiment of the third aspect, the first RNA sequence (E) is hybridised to the second RNA sequence (G) by basepairing between at least 20 of the at least 21 contiguous ribonucleotides of the first RNA sequence (E) and at least 20 of the at least 24 contiguous ribonucleotides of the second RNA sequence (G), wherein 4 ribonucleotides, 3 of which are contiguous, of the at least 24 ribonucleotides of (G) are non-basepaired and 1 ribonucleotide of the at least 21 ribonucleotides of (E) is non-basepaired in the part (C) of the double-stranded RNA region (B), the non- basepaired ribonucleotides forming two bulges in the part (C) of the double-stranded RNA region (B), wherein each bulge is immediately flanked by ribonucleotides which are basepaired in the part (C) of the double-stranded RNA region (B), wherein one bulge is formed by a mismatched ribonucleotide pair between the first RNA sequence (E) and the second RNA sequence (G) and the second bulge is a triribonucleotide bulge, wherein the one or more double-stranded product RNA molecule(s) (P) comprise the bulges, and wherein all but one of ribonucleotides 1 to 19 of the sense RNA sequence (H) each basepair with a ribonucleotide of ribonucleotides 1 to 22 of antisense RNA sequence (J) in the one or more double-stranded product RNA molecule(s) (P). Exemplary RNA molecules of this embodiment are illustrated schematically in Figure 53, panel R.

[0118] In an embodiment of the third aspect, the first RNA sequence (E) is hybridised to the second RNA sequence (G) by basepairing between at least 20 of the at least 21 contiguous ribonucleotides of the first RNA sequence (E) and at least 20 of the at least 24 contiguous ribonucleotides of the second RNA sequence (G), wherein 4 ribonucleotides of the at least 24 ribonucleotides of (G) are non-basepaired and 1 ribonucleotide of the at least 21 ribonucleotides of (E) is non-basepaired in the part (C) of the double-stranded RNA region (B), the non-basepaired ribonucleotides forming five single -ribonucleotide bulges in the part (C) of the double -stranded RNA region (B), wherein each bulge is immediately flanked by ribonucleotides which are basepaired in the part (C) of the double-stranded RNA region (B), wherein the one or more double-stranded product RNA molecule(s) (P) comprise the bulges, and wherein all but one of ribonucleotides 1 to 19 of the sense RNA sequence (H) each basepair with a ribonucleotide of ribonucleotides 1 to 22 of antisense RNA sequence (J) in the one or more double-stranded product RNA molecule(s) (P). Exemplary RNA molecules of this embodiment are illustrated schematically in Figure 53, panel S.

[0119] In an embodiment of the third aspect, the first RNA sequence (E) is hybridised to the second RNA sequence (G) by basepairing between at least 21 of the at least 21 contiguous ribonucleotides of the first RNA sequence (E) and at least 21 of the at least 24 contiguous ribonucleotides of the second RNA sequence (G), wherein 3 ribonucleotides of the at least 24 ribonucleotides of (G) are non-basepaired and all of the at least 21 ribonucleotides of (E) are basepaired in the part (C) of the double - stranded RNA region (B), the 3 non-basepaired ribonucleotides forming three singleribonucleotide bulges in the part (C) of the double-stranded RNA region (B), wherein each bulge is immediately flanked by ribonucleotides of the second RNA sequence (G) which are basepaired to ribonucleotides of the first RNA sequence (E), wherein the one or more double-stranded product RNA molecule(s) (P) comprise the bulges, and wherein ribonucleotides 1 to 19 of the sense RNA sequence (H) each basepair with a ribonucleotide of ribonucleotides 1 to 22 of antisense RNA sequence (J) in the one or more double-stranded product RNA molecule(s) (P). Exemplary RNA molecules of this embodiment are illustrated schematically in Figure 53, panels T and Z.

[0120] In an embodiment of the third aspect, the first RNA sequence (E) is hybridised to the second RNA sequence (G) by basepairing between at least 21 of the at least 21 contiguous ribonucleotides of the first RNA sequence (E) and at least 21 of the at least 24 contiguous ribonucleotides of the second RNA sequence (G), wherein 3 ribonucleotides of the at least 24 ribonucleotides of (G) are non-basepaired and all of the at least 21 ribonucleotides of (E) are basepaired in the part (C) of the double - stranded RNA region (B), the non-basepaired ribonucleotides forming two bulges in the part (C) of the double-stranded RNA region (B), wherein each bulge is immediately flanked by ribonucleotides of the second RNA sequence (G) which are basepaired to ribonucleotides of the first RNA sequence (E), wherein one bulge is a single-ribonucleotide bulge and the other bulge is a diribonucleotide bulge, wherein the one or more double-stranded product RNA molecule(s) (P) comprise the bulges, and wherein ribonucleotides 1 to 19 of the sense RNA sequence (H) each basepair with a ribonucleotide of ribonucleotides 1 to 22 of antisense RNA sequence (J) in the one or more double-stranded product RNA molecule(s) (P). Exemplary RNA molecules of this embodiment are illustrated schematically in Figure 53, panels U and Y. In an embodiment of the third aspect, the first RNA sequence (E) is hybridised to the second RNA sequence (G) by basepairing between at least 21 of the at least 21 contiguous ribonucleotides of the first RNA sequence (E) and at least 21 of the at least 24 contiguous ribonucleotides of the second RNA sequence (G), wherein 3 contiguous ribonucleotides of the at least 24 ribonucleotides of (G) are nonbasepaired and all of the at least 21 ribonucleotides of (E) are basepaired in the part (C) of the double-stranded RNA region (B), the 3 non-basepaired ribonucleotides forming a tri-ribonucleotide bulge in the part (C) of the double-stranded RNA region (B), wherein the bulge is immediately flanked by ribonucleotides of the second RNA sequence (G) which are basepaired to ribonucleotides of the first RNA sequence (E), wherein the one or more double-stranded product RNA molecule(s) (P) comprise the bulge, and wherein ribonucleotides 1 to 19 of the sense RNA sequence (H) each basepair with a ribonucleotide of ribonucleotides 1 to 22 of antisense RNA sequence (J) in the one or more double-stranded product RNA molecule(s) (P). Exemplary RNA molecules of this embodiment are illustrated schematically in Figure 53, panels V and X.

[0121] In an embodiment of the third aspect, the first RNA sequence (E) is hybridised to the second RNA sequence (G) by basepairing between at least 20 of the at least 21 contiguous ribonucleotides of the first RNA sequence (E) and at least 20 of the at least 24 contiguous ribonucleotides of the second RNA sequence (G), wherein 4 contiguous ribonucleotides of the at least 24 ribonucleotides of (G) are non- basepaired and 1 ribonucleotide of the at least 21 ribonucleotides of (E) is non-basepaired in the part (C) of the double -stranded RNA region (B), the non-basepaired ribonucleotides forming a bulge in the part (C) of the double-stranded RNA region (B), wherein the bulge is immediately flanked by ribonucleotides which are basepaired in the part (C) of the double-stranded RNA region (B), wherein the one or more double-stranded product RNA molecule(s) (P) comprise the bulge, and wherein all but one of ribonucleotides 1 to 19 of the sense RNA sequence (H) each basepair with a ribonucleotide of ribonucleotides 1 to 22 of antisense RNA sequence (J) in the one or more double-stranded product RNA molecule(s) (P).

[0122] In an embodiment of the third aspect, the first RNA sequence (E) is hybridised to the second RNA sequence (G) by basepairing between at least 20 of the at least 21 contiguous ribonucleotides of the first RNA sequence (E) and at least 20 of the at least 24 contiguous ribonucleotides of the second RNA sequence (G), wherein 4 ribonucleotides of the at least 24 ribonucleotides of (G) are non-basepaired and 1 ribonucleotide of the at least 21 ribonucleotides of (E) is non-basepaired in the part (C) of the double-stranded RNA region (B), the non-basepaired ribonucleotides forming four bulges in the part (C) of the double-stranded RNA region (B), wherein each bulge is immediately flanked by ribonucleotides which are basepaired in the part (C) of the double-stranded RNA region (B), wherein one bulge is formed by a mismatched ribonucleotide pair between the first RNA sequence (E) and the second RNA sequence (G), and other three bulges are single nucleotide bulges, wherein the one or more double-stranded product RNA molecule(s) (P) comprise the bulges, and wherein all but one of ribonucleotides 1 to 19 of the sense RNA sequence (H) each basepair with a ribonucleotide of ribonucleotides 1 to 22 of antisense RNA sequence (J) in the one or more double-stranded product RNA molecule(s) (P).

[0123] In an embodiment of the third aspect, the first RNA sequence (E) is hybridised to the second RNA sequence (G) by basepairing between at least 20 of the at least 21 contiguous ribonucleotides of the first RNA sequence (E) and at least 20 of the at least 24 contiguous ribonucleotides of the second RNA sequence (G), wherein 4 ribonucleotides, 2 of which are contiguous, of the at least 24 ribonucleotides of (G) are non-basepaired and 1 ribonucleotide of the at least 21 ribonucleotides of (E) is non-basepaired in the part (C) of the double-stranded RNA region (B), the non- basepaired ribonucleotides forming three bulges in the part (C) of the double-stranded RNA region (B), wherein each bulge is immediately flanked by ribonucleotides which are basepaired in the part (C) of the double-stranded RNA region (B), wherein two of the bulges are single nucleotide bulges, and the other non-basepaired ribonucleotides form the other bulge, wherein the one or more double-stranded product RNA molecule(s) (P) comprise the bulges, and wherein all but one of ribonucleotides 1 to 19 of the sense RNA sequence (H) each basepair with a ribonucleotide of ribonucleotides 1 to 22 of antisense RNA sequence (J) in the one or more double-stranded product RNA molecule(s) (P).

[0124] In an embodiment of the third aspect, the first RNA sequence (E) is hybridised to the second RNA sequence (G) by basepairing between at least 20 of the at least 21 contiguous ribonucleotides of the first RNA sequence (E) and at least 20 of the at least 24 contiguous ribonucleotides of the second RNA sequence (G), wherein 4 ribonucleotides, 2 of which are contiguous, of the at least 24 ribonucleotides of (G) are non-basepaired and 1 ribonucleotide of the at least 21 ribonucleotides of (E) is non-basepaired in the part (C) of the double-stranded RNA region (B), the non- basepaired ribonucleotides forming two bulges in the part (C) of the double-stranded RNA region (B), wherein each bulge is immediately flanked by ribonucleotides which are basepaired in the part (C) of the double-stranded RNA region (B), wherein one bulge is a di-ribonucleotide bulge and the other non-basepaired ribonucleotides form the other bulge, wherein the one or more double-stranded product RNA molecule(s) (P) comprise the bulges, and wherein all but one of ribonucleotides 1 to 19 of the sense RNA sequence (H) each basepair with a ribonucleotide of ribonucleotides 1 to 22 of antisense RNA sequence (J) in the one or more double-stranded product RNA molecule(s) (P).

[0125] In an embodiment of the third aspect, the double-stranded RNA region (B) comprises:

[0126] (a) a first RNA strand (D) which comprises a first RNA sequence (E) of at least 23 contiguous ribonucleotides, and

[0127] (b) a second RNA strand (F) which comprises a second RNA sequence (G) of at least 26 contiguous ribonucleotides, wherein the first RNA strand (D) and second RNA strand (F) are covalently linked by a linking RNA sequence (L), wherein the first RNA sequence (E) is hybridised to the second RNA sequence (G) by basepairing between at least 22 of the at least 23 contiguous ribonucleotides of the first RNA sequence (E) and at least 22 of the at least 26 contiguous ribonucleotides of the second RNA sequence (G), forming at least a part (C) of the double-stranded RNA region (B), wherein 3 or 4 ribonucleotides of the at least 26 ribonucleotides of (G) are non-basepaired and 0 or 1, respectively, ribonucleotides of the at least 23 ribonucleotides of (E) are non- basepaired in the part (C) of the double-stranded RNA region (B), forming one, two, three, four or five bulges in the part (C) of the double-stranded RNA region (B), wherein each of the bulges is immediately flanked by ribonucleotides which are basepaired in the part (C) of the double -stranded RNA region (B). Exemplary RNA molecules of this embodiment are illustrated schematically in Figure 53, panels Rto V.

[0128] Thus, in an embodiment the present invention provides a precursor RNA molecule (A) comprising at least one double-stranded RNA region (B), wherein: (i) the double-stranded RNA region (B) comprises:

[0129] (a) a first RNA strand (D) which comprises a first RNA sequence (E) of at least 23 contiguous ribonucleotides, and

[0130] (b) a second RNA strand (F) which comprises a second RNA sequence (G) of at least 26 contiguous ribonucleotides, wherein the first RNA strand (D) and second RNA strand (F) are covalently linked by a linking RNA sequence (L), wherein the first RNA sequence (E) is hybridised to the second RNA sequence (G) by basepairing between at least 22 of the at least 23 contiguous ribonucleotides of the first RNA sequence (E) and at least 22 of the at least 26 contiguous ribonucleotides of the second RNA sequence (G), forming at least a part (C) of the double-stranded RNA region (B), wherein 3 or 4 ribonucleotides of the at least 26 ribonucleotides of (G) are non-basepaired and 0 or 1, respectively, ribonucleotides of the at least 23 ribonucleotides of (E) are non- basepaired in the part (C) of the double-stranded RNA region (B), forming one, two, three, four or five bulges in the part (C) of the double-stranded RNA region (B), wherein each of the bulges is immediately flanked by ribonucleotides which are basepaired in the part (C) of the double-stranded RNA region (B); and

[0131] (ii) the precursor RNA molecule (A) is capable of being cleaved in a eukaryotic cell by one or more ribonucleases (RNases) to produce one or more double-stranded product RNA molecule(s) (P) consisting of a sense RNA sequence (H) of 21 contiguous ribonucleotides from the first RNA sequence (E) and an antisense RNA sequence (J) of 24 contiguous ribonucleotides from the second RNA sequence (G), wherein the one or more double-stranded product RNA molecule(s) (P) comprise the one, two, three, four or five bulges, wherein 18 or 19 of ribonucleotides 1 to 19 of the sense RNA sequence (H) each basepair with a ribonucleotide of ribonucleotides 1 to 22 of antisense RNA sequence (J) in the one or more double-stranded product RNA molecule(s) (P), wherein ribonucleotides 20 and 21 of the sense RNA sequence (H) form a 2- ribonucleotide unpaired 3' overhang and ribonucleotides 23 and 24 of the antisense RNA sequence (J) form a 2-ribonucleotide unpaired 3' overhang in the one or more doublestranded product RNA molecule(s) (P), wherein ribonucleotides 1 and 2 of the sense RNA sequence (H) basepair with ribonucleotides 21 and 22, respectively, of the antisense RNA sequence (J) in the one or more double-stranded product RNA molecule(s) (P), and wherein ribonucleotides 18 and 19 of the sense RNA sequence (H) basepair with ribonucleotides 1 and 2, respectively, of the antisense RNA sequence (J) in the one or more double-stranded product RNA molecule(s) (P). Exemplary RNA molecules of this embodiment are illustrated schematically in Figure 53, panels Rto V.

[0132] In an embodiment of the third aspect, the first RNA sequence (E) is hybridised to the second RNA sequence (G) by basepairing between at least 22 of the at least 23 contiguous ribonucleotides of the first RNA sequence (E) and at least 22 of the at least 26 contiguous ribonucleotides of the second RNA sequence (G), wherein 4 ribonucleotides, 3 of which are contiguous, of the at least 26 ribonucleotides of (G) are non-basepaired and 1 ribonucleotide of the at least 23 ribonucleotides of (E) is non-basepaired in the part (C) of the double-stranded RNA region (B), the non- basepaired ribonucleotides forming two bulges in the part (C) of the double-stranded RNA region (B), wherein each bulge is immediately flanked by ribonucleotides which are basepaired in the part (C) of the double-stranded RNA region (B), wherein one bulge is formed by a mismatched ribonucleotide pair between the first RNA sequence (E) and the second RNA sequence (G) and the second bulge is a triribonucleotide bulge, wherein the one or more double-stranded product RNA molecule(s) (P) comprise the bulges, and wherein all but one of ribonucleotides 1 to 19 of the sense RNA sequence (H) each basepair with a ribonucleotide of ribonucleotides 1 to 22 of antisense RNA sequence (J) in the one or more double-stranded product RNA molecule(s) (P). Exemplary RNA molecules of this embodiment are illustrated schematically in Figure 53, panel R.

[0133] In an embodiment of the third aspect, the first RNA sequence (E) is hybridised to the second RNA sequence (G) by basepairing between at least 22 of the at least 23 contiguous ribonucleotides of the first RNA sequence (E) and at least 22 of the at least 26 contiguous ribonucleotides of the second RNA sequence (G), wherein 4 ribonucleotides of the at least 26 ribonucleotides of (G) are non-basepaired and 1 ribonucleotide of the at least 23 ribonucleotides of (E) is non-basepaired in the part (C) of the double-stranded RNA region (B), the non-basepaired ribonucleotides forming five single -ribonucleotide bulges in the part (C) of the double -stranded RNA region (B), wherein each bulge is immediately flanked by ribonucleotides which are basepaired in the part (C) of the double-stranded RNA region (B), wherein the one or more double-stranded product RNA molecule(s) (P) comprise the bulges, and wherein all but one of ribonucleotides 1 to 19 of the sense RNA sequence (H) each basepair with a ribonucleotide of ribonucleotides 1 to 22 of antisense RNA sequence (J) in the one or more double-stranded product RNA molecule(s) (P). Exemplary RNA molecules of this embodiment are illustrated schematically in Figure 53, panel S.

[0134] In an embodiment of the third aspect, the first RNA sequence (E) is hybridised to the second RNA sequence (G) by basepairing between at least 23 of the at least 23 contiguous ribonucleotides of the first RNA sequence (E) and at least 23 of the at least 26 contiguous ribonucleotides of the second RNA sequence (G), wherein 3 ribonucleotides of the at least 26 ribonucleotides of (G) are non-basepaired and all of the at least 23 ribonucleotides of (E) are basepaired in the part (C) of the doublestranded RNA region (B), the 3 non-basepaired ribonucleotides forming three singleribonucleotide bulges in the part (C) of the double-stranded RNA region (B), wherein each bulge is immediately flanked by ribonucleotides of the second RNA sequence (G) which are basepaired to ribonucleotides of the first RNA sequence (E), wherein the one or more double-stranded product RNA molecule(s) (P) comprise the bulges, and wherein ribonucleotides 1 to 19 of the sense RNA sequence (H) each basepair with a ribonucleotide of ribonucleotides 1 to 22 of antisense RNA sequence (J) in the one or more double-stranded product RNA molecule(s) (P). Exemplary RNA molecules of this embodiment are illustrated schematically in Figure 53, panels T and Z.

[0135] In an embodiment of the third aspect, the first RNA sequence (E) is hybridised to the second RNA sequence (G) by basepairing between at least 23 of the at least 23 contiguous ribonucleotides of the first RNA sequence (E) and at least 23 of the at least 26 contiguous ribonucleotides of the second RNA sequence (G), wherein 3 ribonucleotides of the at least 26 ribonucleotides of (G) are non-basepaired and all of the at least 23 ribonucleotides of (E) are basepaired in the part (C) of the doublestranded RNA region (B), the non-basepaired ribonucleotides forming two bulges in the part (C) of the double-stranded RNA region (B), wherein each bulge is immediately flanked by ribonucleotides of the second RNA sequence (G) which are basepaired to ribonucleotides of the first RNA sequence (E), wherein one bulge is a single-ribonucleotide bulge, and the other bulge is a diribonucleotide bulge, wherein the one or more double-stranded product RNA molecule(s) (P) comprise the bulges, and wherein ribonucleotides 1 to 19 of the sense RNA sequence (H) each basepair with a ribonucleotide of ribonucleotides 1 to 22 of antisense RNA sequence (J) in the one or more double-stranded product RNA molecule(s) (P). Exemplary RNA molecules of this embodiment are illustrated schematically in Figure 53, panels U and Y.

[0136] In an embodiment of the third aspect, the first RNA sequence (E) is hybridised to the second RNA sequence (G) by basepairing between at least 23 of the at least 23 contiguous ribonucleotides of the first RNA sequence (E) and at least 23 of the at least 26 contiguous ribonucleotides of the second RNA sequence (G), wherein 3 contiguous ribonucleotides of the at least 26 ribonucleotides of (G) are nonbasepaired and all of the at least 23 ribonucleotides of (E) are basepaired in the part (C) of the double-stranded RNA region (B), the 3 non-basepaired ribonucleotides forming a tri-ribonucleotide bulge in the part (C) of the double-stranded RNA region (B), wherein the bulge is immediately flanked by ribonucleotides of the second RNA sequence (G) which are basepaired to ribonucleotides of the first RNA sequence (E), wherein the one or more double-stranded product RNA molecule(s) (P) comprise the bulge, and wherein ribonucleotides 1 to 19 of the sense RNA sequence (H) each basepair with a ribonucleotide of ribonucleotides 1 to 22 of antisense RNA sequence (J) in the one or more double-stranded product RNA molecule(s) (P). Exemplary RNA molecules of this embodiment are illustrated schematically in Figure 53, panels V and X.

[0137] In an embodiment of the third aspect, the first RNA sequence (E) is hybridised to the second RNA sequence (G) by basepairing between at least 22 of the at least 23 contiguous ribonucleotides of the first RNA sequence (E) and at least 22 of the at least 26 contiguous ribonucleotides of the second RNA sequence (G), wherein 4 contiguous ribonucleotides of the at least 26 ribonucleotides of (G) are non- basepaired and 1 ribonucleotide of the at least 23 ribonucleotides of (E) is non-basepaired in the part (C) of the double -stranded RNA region (B), the non-basepaired ribonucleotides forming a bulge in the part (C) of the double-stranded RNA region (B), wherein the bulge is immediately flanked by ribonucleotides which are basepaired in the part (C) of the double-stranded RNA region (B), wherein the one or more double-stranded product RNA molecule(s) (P) comprise the bulge, and wherein all but one of ribonucleotides 1 to 19 of the sense RNA sequence (H) each basepair with a ribonucleotide of ribonucleotides 1 to 22 of antisense RNA sequence (J) in the one or more double-stranded product RNA molecule(s) (P).

[0138] In an embodiment of the third aspect, the first RNA sequence (E) is hybridised to the second RNA sequence (G) by basepairing between at least 22 of the at least 23 contiguous ribonucleotides of the first RNA sequence (E) and at least 22 of the at least 26 contiguous ribonucleotides of the second RNA sequence (G), wherein 4 ribonucleotides of the at least 26 ribonucleotides of (G) are non-basepaired and 1 ribonucleotide of the at least 23 ribonucleotides of (E) is non-basepaired in the part (C) of the double-stranded RNA region (B), the non-basepaired ribonucleotides forming four bulges in the part (C) of the double-stranded RNA region (B), wherein each bulge is immediately flanked by ribonucleotides which are basepaired in the part (C) of the double-stranded RNA region (B), wherein one bulge is formed by a mismatched ribonucleotide pair between the first RNA sequence (E) and the second RNA sequence (G), and other three bulges are single nucleotide bulges, wherein the one or more double-stranded product RNA molecule(s) (P) comprise the bulges, and wherein all but one of ribonucleotides 1 to 19 of the sense RNA sequence (H) each basepair with a ribonucleotide of ribonucleotides 1 to 22 of antisense RNA sequence (J) in the one or more double-stranded product RNA molecule(s) (P).

[0139] In an embodiment of the third aspect, the first RNA sequence (E) is hybridised to the second RNA sequence (G) by basepairing between at least 22 of the at least 23 contiguous ribonucleotides of the first RNA sequence (E) and at least 22 of the at least 26 contiguous ribonucleotides of the second RNA sequence (G), wherein 4 ribonucleotides, 2 of which are contiguous, of the at least 26 ribonucleotides of (G) are non-basepaired and 1 ribonucleotide of the at least 23 ribonucleotides of (E) is non-basepaired in the part (C) of the double-stranded RNA region (B), the non- basepaired ribonucleotides forming three bulges in the part (C) of the double-stranded RNA region (B), wherein each bulge is immediately flanked by ribonucleotides which are basepaired in the part (C) of the double-stranded RNA region (B), wherein two of the bulges are single nucleotide bulges and the other non-basepaired ribonucleotides form the other bulge, wherein the one or more double-stranded product RNA molecule(s) (P) comprise the bulges, and wherein all but one of ribonucleotides 1 to 19 of the sense RNA sequence (H) each basepair with a ribonucleotide of ribonucleotides 1 to 22 of antisense RNA sequence (J) in the one or more double-stranded product RNA molecule(s) (P).

[0140] In an embodiment of the third aspect, the first RNA sequence (E) is hybridised to the second RNA sequence (G) by basepairing between at least 22 of the at least 23 contiguous ribonucleotides of the first RNA sequence (E) and at least 22 of the at least 26 contiguous ribonucleotides of the second RNA sequence (G), wherein 4 ribonucleotides, 2 of which are contiguous, of the at least 26 ribonucleotides of (G) are non-basepaired and 1 ribonucleotide of the at least 23 ribonucleotides of (E) is non-basepaired in the part (C) of the double-stranded RNA region (B), the non- basepaired ribonucleotides forming two bulges in the part (C) of the double-stranded RNA region (B), wherein each bulge is immediately flanked by ribonucleotides which are basepaired in the part (C) of the double-stranded RNA region (B), wherein one bulge is a di-nucleotide bulge and the other non-basepaired ribonucleotides form the other bulge, wherein the one or more double-stranded product RNA molecule(s) (P) comprise the bulges, and wherein all but one of ribonucleotides 1 to 19 of the sense RNA sequence (H) each basepair with a ribonucleotide of ribonucleotides 1 to 22 of antisense RNA sequence (J) in the one or more double-stranded product RNA molecule(s) (P).

[0141] Each of the embodiments of the third aspect may have the following feature:

[0142] (i) ribonucleotides 1, 2 and 3 of the sense RNA sequence (H) basepair with ribonucleotides 20, 21 and 22, respectively, of the antisense RNA sequence (J) in at least some of the one or more double-stranded product RNA molecule(s) (P), or

[0143] (ii) ribonucleotides 17, 18 and 19 of the sense RNA sequence (H) basepair with ribonucleotides 1, 2 and 3, respectively, of the antisense RNA sequence (J) in at least some of the one or more double-stranded product RNA molecule(s) (P), or preferably both (i) and (ii). Exemplary RNA molecules of this embodiment are illustrated schematically in Figure 53, panels Rto Z, which all show product RNA molecules with at least three contiguous basepairs at both ends of the product RNA molecules. In further features of these embodiments, the product RNA molecules may have at least four, at least five or at least six contiguous basepairs at one or both ends of the double-stranded molecule before any bulge. In these embodiments, any of those basepairs may be G:U basepairs, preferably one or more of ribonucleotides 1, 2 and 3 of the antisense sequence (J) are involved in G:U basepairs, more preferably one or both of ribonucleotides 1 and 2 of the antisense sequence (J) are involved in G:U basepairs, even more preferably ribonucleotide 1 of the antisense sequence (J) is involved in G:U basepairs. Most preferably, one or more of ribonucleotides 1, 2 and 3 of the antisense sequence (J) are U ribonucleotides, or one or both of ribonucleotides 1 and 2 are U ribonucleotides, or ribonucleotide 1 is a U ribonucleotide, involved in G:U basepairs. The design principles for the asymmetric precursor RNA molecules of the third aspect can be applied in an extended fashion to longer double -stranded regions. For example, the non-basepaired ribonucleotides in the second RNA sequence (G) (antisense sequence) may be arranged in a periodic fashion to provide a population of product RNA molecules (P) having multiple, non-overlapping antisense RNA sequences (J) of 24nt. Such precursor RNA molecules are particularly useful for reducing expression of a target RNA molecule in a plant cell, fungal cell or nematode cell. They are also useful in other invertebrate animal cells such as an arthropod cell or insect cell, or in a non-mammalian vertebrate animal cell. They may be produced in a plant cell to reduce an insect target RNA molecule or a fungal pathogen or nematode target RNA molecule, or applied topically to a plant or insect to reduce a target RNA molecule. For example, in an embodiment of the third aspect, the first RNA sequence (E) comprises at least 44 contiguous ribonucleotides and the second RNA sequence (G) comprises at least 50 contiguous ribonucleotides, wherein the first RNA sequence (E) is hybridised to the second RNA sequence (G) by basepairing between at least 42, preferably at least 43 or at least 44, of the at least 44 contiguous ribonucleotides of the first RNA sequence (E) and at least 42, preferably at least 43 or at least 44, of the at least 50 contiguous ribonucleotides of the second RNA sequence (G), wherein 6, 7 or 8 ribonucleotides of the at least 50 contiguous ribonucleotides of (G) are non-basepaired, and 0, 1 or 2, respectively, ribonucleotides of the at least 44 contiguous ribonucleotides of (E) are non-basepaired in the part (C) of the doublestranded RNA region (B), forming bulges in the part (C) of the double-stranded RNA region (B), wherein each of the bulges is immediately flanked by ribonucleotides which are basepaired in the part (C) of the double-stranded RNA region (B), wherein the precursor RNA molecule is capable of being cleaved in a eukaryotic cell by one or more ribonucleases (RNases) to produce multiple, different doublestranded product RNA molecules (P) consisting of a sense RNA sequence (H) of 21 contiguous ribonucleotides and an antisense RNA sequence (J) of 24 contiguous ribonucleotides, wherein the multiple, different double-stranded product RNA molecule(s) (P) each comprise at least one of the bulges, and wherein at least some of the multiple, different double -stranded product RNA molecules (P) comprise non-overlapping antisense RNA sequences (J), preferably adjacent non-overlapping antisense RNA sequences (J). In another embodiment of the third aspect, the first RNA strand (D) comprises a first RNA sequence (E) of at least 44 contiguous ribonucleotides and the second RNA strand (F) comprises a second RNA sequence (G) of at least 50 contiguous ribonucleotides, wherein the first RNA sequence (E) is hybridised to the second RNA sequence (G) by basepairing between all 44 contiguous ribonucleotides of the first RNA sequence (E) and 44 of the 50 contiguous ribonucleotides of the second RNA sequence (G), forming a part (C) of the double-stranded RNA region (B), wherein 6 ribonucleotides of the 50 contiguous ribonucleotides of (G) are nonbasepaired in the part (C) of the double-stranded RNA region (B), forming 2-6 bulges in the part (C) of the double-stranded RNA region (B), wherein each of the bulges is immediately flanked by ribonucleotides which are basepaired in the part (C) of the double-stranded RNA region (B), wherein the precursor RNA molecule is capable of being cleaved in a eukaryotic cell by one or more ribonucleases (RNases) to produce multiple, different doublestranded product RNA molecules (P) consisting of a sense RNA sequence (H) of 21 contiguous ribonucleotides and an antisense RNA sequence (J) of 24 contiguous ribonucleotides, wherein the multiple, different double-stranded product RNA molecule(s) (P) each comprise at least one of the bulges, and wherein at least some of the multiple, different double -stranded product RNA molecules (P) have non-overlapping antisense RNA sequences (J), preferably adjacent non-overlapping antisense RNA sequences (J). In this and the previous embodiments, the bulges are preferably spaced apart by 6-23 contiguous basepairs, more preferably by 7-22 or 8-21 contiguous basepairs, even more preferably by 9-20 contiguous basepairs, most preferably by 7, 8, 9, 10, 11 or 12 contiguous basepairs, or 7-12 or 7-11 or 7-10 contiguous basepairs.. In specific examples, the bulges are spaced apart by 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 contiguous basepairs. In an embodiment, all of these basepairs are canonical basepairs. Alternatively, 1, 2, 3, 4, 5 or 6 of the basepairs between the bulges are G:U basepairs. The precursor RNA molecule may comprise two or more such double-stranded RNA regions, for example comprise a ledRNA structure.

[0144] In a different embodiment with longer dsRNA regions of at least 44 basepairs, the precursor RNA molecule lacks the linking RNA sequence (L) and the first RNA strand (D) and the second RNA strand (F) hybridise to form the double -stranded RNA region (B). Such precursor RNA molecule may be readily produced in a cell-free system, for example in vitro. In an embodiment, the first RNA strand (D) comprises a first RNA sequence (E) of at least 44 contiguous ribonucleotides and the second RNA strand (F) comprises a second RNA sequence (G) of at least 50 contiguous ribonucleotides, wherein the first RNA sequence (E) is hybridised to the second RNA sequence (G) by basepairing between all 44 contiguous ribonucleotides of the first RNA sequence (E) and 44 of the 50 contiguous ribonucleotides of the second RNA sequence (G), forming a part (C) of the double-stranded RNA region (B), wherein 6 ribonucleotides of the 50 contiguous ribonucleotides of (G) are nonbasepaired in the part (C) of the double-stranded RNA region (B), forming 2-6 bulges in the part (C) of the double-stranded RNA region (B), wherein each of the bulges is immediately flanked by ribonucleotides which are basepaired in the part (C) of the double-stranded RNA region (B), wherein the precursor RNA molecule is capable of being cleaved in a eukaryotic cell by one or more ribonucleases (RNases) to produce multiple, different doublestranded product RNA molecules (P) consisting of a sense RNA sequence (H) of 21 contiguous ribonucleotides and an antisense RNA sequence (J) of 24 contiguous ribonucleotides, wherein the multiple, different double-stranded product RNA molecule(s) (P) each comprise at least one of the bulges, and wherein at least some of the multiple, different double -stranded product RNA molecules (P) have non-overlapping antisense RNA sequences (J), preferably adjacent non-overlapping antisense RNA sequences (J). In this embodiment, the bulges are preferably spaced apart by 6-23 contiguous basepairs, more preferably by 7-22 or 8-21 contiguous basepairs, even more preferably by 9-20 contiguous basepairs, most preferably by 7, 8, 9, 10, 11 or 12 contiguous basepairs, or 7-12 or 7-11 or 7-10 contiguous basepairs. In specific examples, the bulges are spaced apart by 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 contiguous basepairs. In a preferred example, all of these basepairs are canonical basepairs. Alternatively, 1, 2, 3, 4, 5 or 6 of the basepairs between the bulges are G:U basepairs.

[0145] In an embodiment of the third aspect, the first RNA sequence (E) comprises at least 65 contiguous ribonucleotides and the second RNA sequence (G) comprises at least 74 contiguous ribonucleotides, wherein the first RNA sequence (E) is hybridised to the second RNA sequence (G) by basepairing between at least 62, preferably at least 63 or at least 64 or at least 65, of the at least 65 contiguous ribonucleotides of the first RNA sequence (E) and at least 62, preferably at least 63 or at least 64 or at least 65, of the at least 74 contiguous ribonucleotides of the second RNA sequence (G), wherein 9-12 ribonucleotides of the at least 74 contiguous ribonucleotides of (G) are non-basepaired, and 0-3 ribonucleotides of the at least 44 contiguous ribonucleotides of (E) are non-basepaired in the part (C) of the double -stranded RNA region (B), forming bulges in the part (C) of the double-stranded RNA region (B), wherein each of the bulges is immediately flanked by ribonucleotides which are basepaired in the part (C) of the double-stranded RNA region (B), wherein the precursor RNA molecule is capable of being cleaved in a eukaryotic cell by one or more ribonucleases (RNases) to produce multiple, different doublestranded product RNA molecules (P) consisting of a sense RNA sequence (H) of 21 contiguous ribonucleotides and an antisense RNA sequence (J) of 24 contiguous ribonucleotides, wherein the multiple, different double-stranded product RNA molecule(s) (P) each comprise at least one of the bulges, and wherein at least some of the multiple, different double -stranded product RNA molecules (P) comprise non-overlapping antisense RNA sequences (J), preferably adjacent non-overlapping antisense RNA sequences (J).

[0146] In another embodiment of the third aspect, the first RNA strand (D) comprises a first RNA sequence (E) of at least 65 contiguous ribonucleotides and the second RNA strand (F) comprises a second RNA sequence (G) of at least 74 contiguous ribonucleotides, wherein the first RNA sequence (E) is hybridised to the second RNA sequence (G) by basepairing between all 65 contiguous ribonucleotides of the first RNA sequence (E) and 65 of the 74 contiguous ribonucleotides of the second RNA sequence (G), forming a part (C) of the double-stranded RNA region (B), wherein 9 ribonucleotides of the 74 contiguous ribonucleotides of (G) are non- basepaired in the part (C) of the double-stranded RNA region (B), forming 3-9 bulges in the part (C) of the double-stranded RNA region (B), wherein each of the bulges is immediately flanked by ribonucleotides which are basepaired in the part (C) of the double-stranded RNA region (B), wherein the precursor RNA molecule is capable of being cleaved in a eukaryotic cell by one or more ribonucleases (RNases) to produce multiple, different doublestranded product RNA molecules (P) consisting of a sense RNA sequence (H) of 21 contiguous ribonucleotides and an antisense RNA sequence (J) of 24 contiguous ribonucleotides, wherein the multiple, different double-stranded product RNA molecule(s) (P) each comprise at least one of the bulges, and wherein at least some of the multiple, different double -stranded product RNA molecules (P) have non-overlapping antisense RNA sequences (J), preferably adjacent non-overlapping antisense RNA sequences (J). In this and the previous embodiments, the bulges are preferably spaced apart by 6-23 contiguous basepairs, more preferably by 7-22 or 8-21 contiguous basepairs, even more preferably by 9-20 contiguous basepairs, most preferably by 7, 8, 9, 10, 11 or 12 contiguous basepairs, or 7-12 or 7-11 or 7-10 contiguous basepairs. In specific examples, the bulges are spaced apart by 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 contiguous basepairs. In an embodiment, all of these basepairs are canonical basepairs. Alternatively, 1, 2, 3, 4, 5 or 6 of the basepairs between the bulges are G:U basepairs. The precursor RNA molecule may comprise two or more such double-stranded RNA regions, for example comprise an ledRNA structure.

[0147] In a different embodiment with longer dsRNA regions of at least 65 basepairs, the precursor RNA molecule lacks the linking RNA sequence (L) and the first RNA strand

[0148] (D) and the second RNA strand (F) hybridise to form the double -stranded RNA region (B). Such precursor RNA molecule may be readily produced in a cell -free system, for example in vitro. In an embodiment, the first RNA strand (D) comprises a first RNA sequence (E) of at least 65 contiguous ribonucleotides and the second RNA strand (F) comprises a second RNA sequence (G) of at least 74 contiguous ribonucleotides, wherein the first RNA sequence (E) is hybridised to the second RNA sequence (G) by basepairing between all 65 contiguous ribonucleotides of the first RNA sequence

[0149] (E) and 65 of the 74 contiguous ribonucleotides of the second RNA sequence (G), forming a part (C) of the double-stranded RNA region (B), wherein 9 ribonucleotides of the 74 contiguous ribonucleotides of (G) are nonbasepaired in the part (C) of the double-stranded RNA region (B), forming 3-9 bulges in the part (C) of the double-stranded RNA region (B), wherein each of the bulges is immediately flanked by ribonucleotides which are basepaired in the part (C) of the double-stranded RNA region (B), wherein the precursor RNA molecule is capable of being cleaved in a eukaryotic cell by one or more ribonucleases (RNases) to produce multiple, different doublestranded product RNA molecules (P) consisting of a sense RNA sequence (H) of 21 contiguous ribonucleotides and an antisense RNA sequence (J) of 24 contiguous ribonucleotides, wherein the multiple, different double-stranded product RNA molecule(s) (P) each comprise at least one of the bulges, and wherein at least some of the multiple, different double -stranded product RNA molecules (P) have non-overlapping antisense RNA sequences (J), preferably adjacent non-overlapping antisense RNA sequences (J). In this embodiment, the bulges are preferably spaced apart by 6-23 contiguous basepairs, more preferably by 7-22 or 8-21 contiguous basepairs, even more preferably by 9-20 contiguous basepairs, most preferably by 7, 8, 9, 10, 11 or 12 contiguous basepairs, or 7-12 or 7-11 or 7-10 contiguous basepairs. In specific examples, the bulges are spaced apart by 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 contiguous basepairs. In a preferred example, all of these basepairs are canonical basepairs. Alternatively, 1, 2, 3, 4, 5 or 6 of the basepairs between the bulges are G:U basepairs.

[0150] In the previous three embodiments, the first RNA sequence (E) and the second RNA sequence (G) may extend to longer than 65 and 74 ribonucleotides, respectively. In a further embodiment, the first RNA sequence (E) comprises at least 86 contiguous ribonucleotides and the second RNA sequence (G) comprises at least 98 contiguous ribonucleotides, and wherein the first RNA sequence (E) is hybridised to the second RNA sequence (G) by basepairing between at least 82, preferably at least 83, at least 84, at least 85 or at least 86, of the at least 86 contiguous ribonucleotides of the first RNA sequence (E) and at least 82, preferably at least 83, at least 84, at least 85 or at least 86, of the at least 98 contiguous ribonucleotides of the second RNA sequence (G).

[0151] In another embodiment of the third aspect, the first RNA strand (D) comprises a first RNA sequence (E) of at least 86 contiguous ribonucleotides and the second RNA strand (F) comprises a second RNA sequence (G) of at least 98 contiguous ribonucleotides, wherein the first RNA sequence (E) is hybridised to the second RNA sequence (G) by basepairing between all 86 contiguous ribonucleotides of the first RNA sequence (E) and 86 of the 98 contiguous ribonucleotides of the second RNA sequence (G), forming a part (C) of the double-stranded RNA region (B), wherein 12 ribonucleotides of the 98 contiguous ribonucleotides of (G) are nonbasepaired in the part (C) of the double-stranded RNA region (B), forming 4-12 bulges in the part (C) of the double-stranded RNA region (B), wherein each of the bulges is immediately flanked by ribonucleotides which are basepaired in the part (C) of the double-stranded RNA region (B), wherein the precursor RNA molecule is capable of being cleaved in a eukaryotic cell by one or more ribonucleases (RNases) to produce multiple, different doublestranded product RNA molecules (P) consisting of a sense RNA sequence (H) of 21 contiguous ribonucleotides and an antisense RNA sequence (J) of 24 contiguous ribonucleotides, wherein the multiple, different double-stranded product RNA molecule(s) (P) each comprise at least one of the bulges, and wherein at least some of the multiple, different double -stranded product RNA molecules (P) have non-overlapping antisense RNA sequences (J), preferably adjacent non-overlapping antisense RNA sequences (J). In this and the previous embodiments, the bulges are preferably spaced apart periodically, for example by 6-23 contiguous basepairs, more preferably by 7—2 or 8-21 contiguous basepairs, even more preferably by 9-20 contiguous basepairs, most preferably by 7, 8, 9, 10, 11 or 12 contiguous basepairs, or 7-12 or 7-11 or 7-10 contiguous basepairs. In specific examples, the bulges are spaced apart by 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 contiguous basepairs. In a preferred example, all of these basepairs are canonical basepairs. Alternatively, 1, 2, 3, 4, 5 or 6 of the basepairs between the bulges are G:U basepairs. The precursor RNA molecule may comprise two or more such double-stranded RNA regions, for example comprise a ledRNA structure.

[0152] In a different embodiment with longer dsRNA regions of at least 86 basepairs, the precursor RNA molecule lacks the linking RNA sequence (L) and the first RNA strand

[0153] (D) and the second RNA strand (F) hybridise to form the double -stranded RNA region (B). Such precursor RNA molecule may be readily produced in a cell -free system, for example in vitro. In an embodiment, the first RNA strand (D) comprises a first RNA sequence (E) of at least 86 contiguous ribonucleotides and the second RNA strand (F) comprises a second RNA sequence (G) of at least 98 contiguous ribonucleotides, wherein the first RNA sequence (E) is hybridised to the second RNA sequence (G) by basepairing between all 86 contiguous ribonucleotides of the first RNA sequence

[0154] (E) and 86 of the 98 contiguous ribonucleotides of the second RNA sequence (G), forming a part (C) of the double-stranded RNA region (B), wherein 12 ribonucleotides of the 98 contiguous ribonucleotides of (G) are nonbasepaired in the part (C) of the double-stranded RNA region (B), forming 4-12 bulges in the part (C) of the double-stranded RNA region (B), wherein each of the bulges is immediately flanked by ribonucleotides which are basepaired in the part (C) of the double-stranded RNA region (B), wherein the precursor RNA molecule is capable of being cleaved in a eukaryotic cell by one or more ribonucleases (RNases) to produce multiple, different doublestranded product RNA molecules (P) consisting of a sense RNA sequence (H) of 21 contiguous ribonucleotides and an antisense RNA sequence (J) of 24 contiguous ribonucleotides, wherein the multiple, different double-stranded product RNA molecule(s) (P) each comprise at least one of the bulges, and wherein at least some of the multiple, different double -stranded product RNA molecules (P) have non-overlapping antisense RNA sequences (J), preferably adjacent non-overlapping antisense RNA sequences (J). In this embodiment, the bulges are preferably spaced apart by 66-23 contiguous basepairs, more preferably by 7-22 or 8-21 contiguous basepairs, even more preferably by 9-20 contiguous basepairs, most preferably by 7, 8, 9, 10, 11 or 12 contiguous basepairs, or 7-12 or 7-11 or 7-10 contiguous basepairs. In specific examples, the bulges are spaced apart by 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 contiguous basepairs. In a preferred example, all of these basepairs are canonical basepairs. Alternatively, 1, 2, 3, 4, 5 or 6 of the basepairs between the bulges are G:U basepairs. In a preferred example, all of these basepairs are canonical basepairs. Alternatively, 1, 2, 3, 4 or 5 of the basepairs between the bulges are G:U basepairs.

[0155] In each of these embodiments of the third aspect, or molecules lacking a loop, the first RNA sequence (E) and the second RNA sequence (G) may both comprise at least 100 contiguous ribonucleotides, or at least 110 or at least 120 contiguous ribonucleotides, or at least 150, or at least 200, or at least 250, or at least 300 contiguous ribonucleotides, preferably to a maximum of 1000 contiguous ribonucleotides, more preferably to a maximum of 800 contiguous ribonucleotides, or even more preferable to a maximum of 600 contiguous ribonucleotides. For example, the first RNA sequence (E) and the second RNA sequence (G) may both comprise contiguous ribonucleotides in the range 100- 1000, 100-800, 100-600 contiguous ribonucleotides, or in the range 150-1000, 150— 800, 150-600 contiguous ribonucleotides. In preferred embodiments, the length of the sense RNA sequence of the dsRNA region is 87%-97% or 87%-96% the length of the antisense sequence, for example 87%— 91% or 87%-90% compared to the antisense sequence. These features are applicable to hairpin RNAs and to dsRNAs formed by annealing of two RNA strands i.e. without a joining loop sequence. Each of these features may also be applied to a second dsRNA region in the precursor RNA molecule, for example in a ledRNA molecule. In these embodiments, the eukaryotic cell in which the precursor RNA molecule (A) is cleaved is preferably a plant cell, a fungal cell, a nematode cell, or an arthropod cell such as an insect, arachnid, or decapod cell, or the target RNA molecule is preferably in a plant cell, a fungal cell, a nematode cell, or an arthropod cell such as an insect, arachnid, or decapod cell.

[0156] Each of the embodiments of the third aspect, or the molecules lacking a loop, may have the following feature: the precursor RNA molecule is capable of being cleaved in a eukaryotic cell by one or more ribonucleases (RNases) to produce multiple, different double-stranded product RNA molecules (P) which each consist of a sense RNA sequence (H) of 21 contiguous ribonucleotides from the first RNA sequence (E) and an antisense RNA sequence (J) of 24 contiguous ribonucleotides from the second RNA sequence (G), wherein at least some of the multiple, different double-stranded product RNA molecules (P) have overlapping antisense RNA sequences (J). In a further embodiment, at least some of the multiple, different double-stranded product RNA molecules (P) have non-overlapping antisense RNA sequences (J), where the population of double-stranded product RNA molecules (P) produced from the precursor RNA molecule includes some overlapping and some non-overlapping antisense RNA sequences (J). Preferably, there are more product RNA molecules (P) comprising nonoverlapping antisense RNA sequences (J), as readily occurs with longer (>42 basepairs) double-stranded regions (B) where siRNA molecules each consisting of 21nt sense RNA sequences hybridised to 24nt antisense RNA sequence are phased along the length of (B).

[0157] Each of the embodiments of the third aspect may have the following feature: the precursor RNA molecule is capable of being cleaved in a eukaryotic cell by one or more ribonucleases (RNases) to produce multiple, different double-stranded product RNA molecules (P) which each consist of a sense RNA sequence (H) of 21 contiguous ribonucleotides from a first RNA sequence (E) and an antisense RNA sequence (J) of 24 contiguous ribonucleotides from a second RNA sequence (G), wherein at least some of the multiple, different double-stranded product RNA molecules (P) have nonoverlapping antisense RNA sequences (J), preferably adjacent non-overlapping antisense RNA sequences (J).

[0158] In an embodiment of the third aspect, the molecule is capable of being cleaved in a eukaryotic cell by one or more ribonucleases (RNases) to produce multiple, different double-stranded product RNA molecules (P) which comprise double-stranded RNA product molecules as defined in two or more embodiments of the third aspect.

[0159] The present invention also provides symmetric and asymmetric precursor RNA molecules comprising G:U basepairs in a double-stranded RNA region targeting one or more RNA molecules in an insect cell or fungal cell. Therefore, in a fourth aspect the present invention provides a precursor RNA molecule comprising at least one doublestranded RNA region, wherein:

[0160] (i) the double-stranded RNA region comprises: (a) a first RNA strand of at least 46 contiguous ribonucleotides which comprises a first RNA sequence of at least 44 contiguous ribonucleotides, and

[0161] (b) a second RNA strand of at least 46 contiguous ribonucleotides which comprises a second RNA sequence of at least 44 contiguous ribonucleotides, wherein the first RNA sequence hybridises to the second RNA sequence by basepairing between the at least 44 contiguous ribonucleotides of the first RNA sequence and the at least 44 contiguous ribonucleotides of the second RNA sequence, forming at least a part of the double-stranded RNA region, wherein between 10% and 35% of the ribonucleotides of the first RNA sequence and the second RNA sequence, in total, are basepaired in G:U basepairs, wherein the second RNA sequence is at least 80% identical to a sequence of at least 44 contiguous ribonucleotides which is fully complementary to a first region of a target RNA molecule in an insect cell; and

[0162] (ii) the precursor RNA molecule is capable of being cleaved in an insect cell by one or more Dicers to produce double-stranded product RNA molecules, each independently consisting of a sense RNA sequence of 21 contiguous ribonucleotides from the first RNA sequence and an antisense RNA sequence of 21 contiguous ribonucleotides from the second RNA sequence, wherein ribonucleotides 1 to 19 of the sense RNA sequence basepair with ribonucleotides 1 to 19 of the antisense RNA sequence in each of the double-stranded product RNA molecules, wherein the 19 basepairs between the sense RNA sequence and the antisense RNA sequence in each of the double-stranded product RNA molecules comprise, independently, 2, 3, 4, 5, or 6 G:U basepairs, wherein ribonucleotides 20 and 21 of the sense RNA sequence in each of the double-stranded product RNA molecules form a 2-ribonucleotide unpaired 3' overhang, and wherein ribonucleotides 20 and 21 of the antisense RNA sequence in each of the double-stranded product RNA molecules form a 2-ribonucleotide unpaired 3' overhang, and wherein either (c) at least some of the antisense RNA sequences in the doublestranded product RNA molecules are capable of reducing the expression and / or activity of the target RNA molecule in the insect cell, or

[0163] (d) the antisense RNA sequences in the double-stranded product RNA molecules are each, independently, at least 80% identical in sequence to a corresponding region of the complement of the target RNA molecule, or (e) both (c) and (d).

[0164] In a preferred embodiment, the insect cell is a Lepidopteran insect cell, for example of the genus Helicoverpci or Spodoptera, for example, of the species Helicoverpci cirmigerci or Spodoptera frugiperda, and / or the second RNA sequence is at least 90% identical, preferably 100% identical, to the sequence of at least 44 contiguous ribonucleotides which is fully complementary to the first region of the target RNA molecule in an insect cell.

[0165] In a fifth aspect the present invention provides a precursor RNA molecule comprising at least one double-stranded RNA region, wherein:

[0166] (i) the double-stranded RNA region comprises:

[0167] (a) a first RNA strand of at least 46 contiguous ribonucleotides which comprises a first RNA sequence of at least 44 contiguous ribonucleotides, and

[0168] (b) a second RNA strand of at least 46 contiguous ribonucleotides which comprises a second RNA sequence of at least 44 contiguous ribonucleotides, wherein the first RNA sequence hybridises to the second RNA sequence by basepairing between the at least 44 contiguous ribonucleotides of the first RNA sequence and the at least 44 contiguous ribonucleotides of the second RNA sequence, forming at least a part of the double-stranded RNA region, wherein between 10% and 35% of the ribonucleotides of the first RNA sequence and the second RNA sequence, in total, are basepaired in G:U basepairs, wherein the second RNA sequence is at least 80% identical to a sequence of at least 44 contiguous ribonucleotides which is fully complementary to a first region of a target RNA molecule in a fungal cell; and

[0169] (ii) the precursor RNA molecule is capable of being cleaved in the fungal cell by one or more Dicers to produce double-stranded product RNA molecules, each independently consisting of a sense RNA sequence of 21 contiguous ribonucleotides from the first RNA sequence and an antisense RNA sequence of 21 contiguous ribonucleotides from the second RNA sequence, wherein ribonucleotides 1 to 19 of the sense RNA sequence basepair with ribonucleotides 1 to 19 of the antisense RNA sequence in each of the double-stranded product RNA molecules, wherein the 19 basepairs between the sense RNA sequence and the antisense RNA sequence in each of the double-stranded product RNA molecules comprise, independently, 2, 3, 4, 5, or 6 G:U basepairs, wherein ribonucleotides 20 and 21 of the sense RNA sequence in each of the double-stranded product RNA molecules form a 2-ribonucleotide unpaired 3' overhang, and wherein ribonucleotides 20 and 21 of the antisense RNA sequence in each of the double-stranded product RNA molecules form a 2-ribonucleotide unpaired 3' overhang, and wherein either (c) at least some of the antisense RNA sequences in the doublestranded product RNA molecules are capable of reducing the expression and / or activity of the target RNA molecule in the fungal cell, or

[0170] (d) the antisense RNA sequences in the double-stranded product RNA molecules are each, independently, at least 80% identical in sequence to a corresponding region of the complement of the target RNA molecule, or

[0171] (e) both (c) and (d).

[0172] In a preferred embodiment, the fungal cell is a plant pathogenic fungal cell, for example of the genus Fusarium or Verticillium, and / or the second RNA sequence is at least 90% identical, preferably 100% identical, to the sequence of at least 44 contiguous ribonucleotides which is fully complementary to the first region of the target RNA molecule in a fungal cell.

[0173] In each embodiment of the fourth and fifth aspects, the features of the first RNA sequence (E) and the second RNA sequence (G) that apply to the precursor RNA molecules of the first, second and third aspects may also apply to the precursor RNA molecules of the fourth and fifth aspects, in particular the length features.

[0174] In an embodiment of the asymmetric precursor RNA molecule comprising G:U basepairs, the precursor RNA molecule comprises at least one double -stranded RNA region, wherein:

[0175] (i) the double-stranded RNA region comprises:

[0176] (a) a first RNA strand of at least 46 contiguous ribonucleotides which comprises a first RNA sequence of at least 44 contiguous ribonucleotides, and

[0177] (b) a second RNA strand of at least 48 contiguous ribonucleotides which comprises a second RNA sequence of at least 46 contiguous ribonucleotides, wherein the first RNA sequence hybridises to the second RNA sequence by basepairing between at least 42 ribonucleotides of the at least 44 contiguous ribonucleotides of the first RNA sequence and at least 42 ribonucleotides of the at least 46 contiguous ribonucleotides of the second RNA sequence, forming at least a part of the double-stranded RNA region, wherein 2, 3, or 4 ribonucleotides of the at least 46 contiguous ribonucleotides of the second RNA sequence are non-basepaired and 0, 1, or 2, respectively, ribonucleotides of the at least 44 contiguous ribonucleotides of the first RNA sequence are non- basepaired in the part of the double-stranded RNA region, forming bulges in the part of the double-stranded RNA region, wherein each of the bulges is immediately flanked by ribonucleotides which are basepaired in the part of the double-stranded RNA region, wherein between 10% and 35% of the ribonucleotides of the first RNA sequence and the second RNA sequence, in total, are basepaired in G:U basepairs, wherein the second RNA sequence is at least 80% identical to a sequence of at least 46 contiguous ribonucleotides which is fully complementary to a first region of a target RNA molecule in an insect cell or a fungal cell; and

[0178] (ii) the precursor RNA molecule is capable of being cleaved in the insect cell or fungal cell by one or more Dicers to produce double-stranded product RNA molecules, each independently consisting of a sense RNA sequence of 21 contiguous ribonucleotides from the first RNA sequence and an antisense RNA sequence of 22 contiguous ribonucleotides from the second RNA sequence, wherein the antisense RNA sequence of the double-stranded product RNA molecules each comprise at least one of the bulges, wherein the basepairs between the sense RNA sequence and the antisense RNA sequence in each of the double-stranded product RNA molecules comprise, independently, 2, 3, 4, 5, or 6 G:U basepairs, wherein ribonucleotides 20 and 21 of the sense RNA sequence in each of the double-stranded product RNA molecules form a 2-ribonucleotide unpaired 3' overhang, wherein ribonucleotides 21 and 22 of the antisense RNA sequence in each of the double-stranded product RNA molecules form a 2-ribonucleotide unpaired 3' overhang, and wherein either (c) at least some of the antisense RNA sequences in the doublestranded product RNA molecules are capable of reducing the expression and / or activity of the target RNA molecule in the insect cell or fungal cell, or

[0179] (d) the antisense RNA sequences in the double-stranded product RNA molecules are each, independently, at least 80% identical in sequence to a corresponding region of the complement of the target RNA molecule, or

[0180] (e) both (c) and (d). In another embodiment of the asymmetric precursor RNA molecule comprising G:U basepairs, the precursor RNA molecule comprises at least one double -stranded RNA region, wherein:

[0181] (i) the double-stranded RNA region comprises:

[0182] (a) a first RNA strand of at least 46 contiguous ribonucleotides which comprises a first RNA sequence of at least 44 contiguous ribonucleotides, and

[0183] (b) a second RNA strand of at least 50 contiguous ribonucleotides which comprises a second RNA sequence of at least 48 contiguous ribonucleotides, wherein the first RNA sequence hybridises to the second RNA sequence by basepairing between at least 42 ribonucleotides of the at least 44 contiguous ribonucleotides of the first RNA sequence and at least 42 ribonucleotides of the at least 48 contiguous ribonucleotides of the second RNA sequence, forming at least a part of the double-stranded RNA region, wherein 4, 5 or 6 ribonucleotides of the at least 48 contiguous ribonucleotides of the second RNA sequence are non-basepaired and 0, 1 or 2, respectively, ribonucleotides of the at least 44 contiguous ribonucleotides of the first RNA sequence are non- basepaired in the part of the double-stranded RNA region, forming bulges in the part of the double-stranded RNA region, wherein each of the bulges is immediately flanked by ribonucleotides which are basepaired in the part of the double-stranded RNA region, wherein between 10% and 35% of the ribonucleotides of the first RNA sequence and the second RNA sequence, in total, are basepaired in G:U basepairs, wherein the second RNA sequence is at least 80% identical to a sequence of at least 48 contiguous ribonucleotides which is fully complementary to a first region of a target RNA molecule in an insect cell or a fungal cell; and

[0184] (ii) the precursor RNA molecule is capable of being cleaved in the insect cell or fungal cell by one or more Dicers to produce double-stranded product RNA molecules, each independently consisting of a sense RNA sequence of 21 contiguous ribonucleotides from the first RNA sequence and an antisense RNA sequence of 23 contiguous ribonucleotides from the second RNA sequence, wherein the antisense RNA sequence of the double-stranded product RNA molecules each comprise at least one of the bulges, wherein the basepairs between the sense RNA sequence and the antisense RNA sequence in each of the double-stranded product RNA molecules comprise, independently, 2, 3, 4, 5, or 6 G:U basepairs, wherein ribonucleotides 20 and 21 of the sense RNA sequence in each of the double-stranded product RNA molecules form a 2-ribonucleotide unpaired 3' overhang, wherein ribonucleotides 22 and 23 of the antisense RNA sequence in each of the double-stranded product RNA molecules form a 2-ribonucleotide unpaired 3' overhang, and wherein either (c) at least some of the antisense RNA sequences in the doublestranded product RNA molecules are capable of reducing the expression and / or activity of the target RNA molecule in the insect cell or fungal cell, or

[0185] (d) the antisense RNA sequences in the double-stranded product RNA molecules are each, independently, at least 80% identical in sequence to a corresponding region of the complement of the target RNA molecule, or

[0186] (e) both (c) and (d).

[0187] In a further embodiment of the asymmetric precursor RNA molecule comprising G:U basepairs, the precursor RNA molecule comprises at least one double -stranded RNA region, wherein:

[0188] (i) the double-stranded RNA region comprises:

[0189] (a) a first RNA strand of at least 46 contiguous ribonucleotides which comprises a first RNA sequence of at least 44 contiguous ribonucleotides, and

[0190] (b) a second RNA strand of at least 52 contiguous ribonucleotides which comprises a second RNA sequence of at least 50 contiguous ribonucleotides, wherein the first RNA sequence hybridises to the second RNA sequence by basepairing between at least 42 ribonucleotides of the at least 44 contiguous ribonucleotides of the first RNA sequence and at least 42 ribonucleotides of the at least 50 contiguous ribonucleotides of the second RNA sequence, forming at least a part of the double-stranded RNA region, wherein 6, 7, or 8 ribonucleotides of the at least 50 contiguous ribonucleotides of the second RNA sequence are non-basepaired and 0, 1, or 2, respectively, ribonucleotides of the at least 44 contiguous ribonucleotides of the first RNA sequence are non- basepaired in the part of the double-stranded RNA region, forming bulges in the part of the double-stranded RNA region, wherein each of the bulges is immediately flanked by ribonucleotides which are basepaired in the part of the double-stranded RNA region, wherein between 10% and 35% of the ribonucleotides of the first RNA sequence and the second RNA sequence, in total, are basepaired in G:U basepairs, wherein the second RNA sequence is at least 80% identical to a sequence of at least 50 contiguous ribonucleotides which is fully complementary to a first region of a target RNA molecule in an insect cell or a fungal cell; and

[0191] (ii) the precursor RNA molecule is capable of being cleaved in the insect cell or fungal cell by one or more Dicers to produce double-stranded product RNA molecules, each independently consisting of a sense RNA sequence of 21 contiguous ribonucleotides from the first RNA sequence and an antisense RNA sequence of 24 contiguous ribonucleotides from the second RNA sequence, wherein the antisense RNA sequence of the double-stranded product RNA molecules each comprise at least one of the bulges, wherein the basepairs between the sense RNA sequence and the antisense RNA sequence in each of the double-stranded product RNA molecules comprise, independently, 2, 3, 4, 5, or 6 G:U basepairs, wherein ribonucleotides 20 and 21 of the sense RNA sequence in each of the double-stranded product RNA molecules form a 2-ribonucleotide unpaired 3' overhang, wherein ribonucleotides 23 and 24 of the antisense RNA sequence in each of the double-stranded product RNA molecules form a 2-ribonucleotide unpaired 3' overhang, and wherein either (c) at least some of the antisense RNA sequences in the doublestranded product RNA molecules are capable of reducing the expression and / or activity of the target RNA molecule in the insect cell or fungal cell, or

[0192] (d) the antisense RNA sequences in the double-stranded product RNA molecules are each, independently, at least 80% identical in sequence to a corresponding region of the complement of the target RNA molecule, or

[0193] (e) both (c) and (d).

[0194] It is understood in the context of the embodiments of the fourth or fifth aspects or the asymmetric precursor RNA molecules, that at least some, but not necessarily all, of the double-stranded product RNA molecules produced from the precursor RNA molecule have the features recited in part (ii). For example, sense or antisense RNAs of lengths other than 21 ribonucleotides, or 22-, 23- or 24-mers as the case may be, may be produced as well as those of the specified lengths such as 21 ribonucleotides.

[0195] In an embodiment of the first, second, third, fourth, or fifth aspects, or the embodiments of the asymmetric precursor RNA molecule, the precursor RNA molecule comprises a linking RNA sequence, wherein the linking RNA sequence links either the 3 ' end of the first RNA strand to the 5 ' end of the second RNA strand, or the 5 ' end of the first RNA strand to the 3' end of the second RNA strand. In an alternative embodiment, the precursor RNA molecule lacks a linking RNA sequence, i.e. the first RNA strand and the second RNA strands are not covalently linked by a linking RNA sequence. In an embodiment of the first, second, third, fourth, or fifth aspects, or the embodiments of the asymmetric precursor RNA molecule, the precursor RNA molecule comprises a second double-stranded RNA region and a second linking RNA sequence and forms a ledRNA structure.

[0196] In an embodiment of the first, second, third, fourth, or fifth aspects, or the embodiments of the asymmetric precursor RNA molecule, one or more or all of the following may apply:

[0197] (i) the linking RNA sequence comprises a sequence of at least 50 contiguous ribonucleotides which is at least 90% identical to a second region of the target RNA molecule in the eukaryotic cell such as a plant cell, nematode cell, insect cell or fungal cell, and / or the linking RNA sequence comprises a sequence of at least 50 contiguous ribonucleotides which is at least 90% identical to the complement of a second region of the target RNA molecule in the eukaryotic cell such as a plant cell, nematode cell, insect cell or fungal cell,

[0198] (ii) the precursor RNA molecule further comprises a sequence of at least 50 contiguous ribonucleotides which is at least 90% identical to a second region of the target RNA molecule in the eukaryotic cell such as a plant cell, nematode cell, insect cell or fungal cell, covalently linked to the 5' or 3' end of the first RNA strand or to the 5' or 3' end of the second RNA strand, and / or a sequence of at least 50 contiguous ribonucleotides which is at least 90% identical to the complement of a second region of the target RNA molecule in the eukaryotic cell such as a plant cell, nematode cell, insect cell or fungal cell, covalently linked to the 5' or 3' end of the first RNA strand or to the 5' or 3' end of the second RNA strand,

[0199] (iii) the linking RNA sequence comprises a single-stranded RNA sequence of at least 44 contiguous ribonucleotides which is identical to a region of either the first RNA sequence or the second RNA sequence,

[0200] (iv) the precursor RNA molecule comprises the linking RNA sequence and is encoded by a polynucleotide which lacks an intron in its region encoding the linking RNA sequence,

[0201] (v) the first RNA sequence and second RNA sequence are identical in length across the full length of the double -stranded RNA region,

[0202] (vi) in the embodiments of the asymmetric precursor RNA molecule, the first RNA sequence and second RNA sequence are different in length across the full length of the double-stranded RNA region, either the first RNA sequence is longer than the second RNA sequence or, preferably, the first RNA sequence is shorter than the second RNA sequence,

[0203] (vii) the first RNA sequence varies from the first region of the target RNA molecule only by the substitution of at least some A ribonucleotides with G ribonucleotides, or at least some C ribonucleotides with U ribonucleotides, or a combination of at least some A ribonucleotides with G ribonucleotides and at least some C ribonucleotides with U ribonucleotides, or wherein some but not all of the A ribonucleotides are substituted with G ribonucleotides, or some but not all C ribonucleotides are substituted with U ribonucleotides, or a combination of some but not all A ribonucleotides with G ribonucleotides and some but not all C ribonucleotides with U ribonucleotides, and optionally wherein the second RNA sequence is identical in sequence to the complement of the first region of the target RNA molecule,

[0204] (viii) the second RNA sequence varies from the complement of the first region of the target RNA molecule only by the substitution of at least some A ribonucleotides with G ribonucleotides, or at least some C ribonucleotides with U ribonucleotides, or a combination of at least some A ribonucleotides with G ribonucleotides and at least some C ribonucleotides with U ribonucleotides, or wherein some but not all of the A ribonucleotides are substituted with G ribonucleotides, or some but not all C ribonucleotides are substituted with U ribonucleotides, or a combination of some but not all A ribonucleotides with G ribonucleotides and some but not all C ribonucleotides with U ribonucleotides, and optionally wherein the first RNA sequence is identical in sequence to the first region of the target RNA molecule,

[0205] (ix) at least some of the double -stranded product RNA molecules have nonoverlapping antisense RNA sequences, preferably adjacent non-overlapping antisense RNA sequences, relative to the target RNA molecule,

[0206] (x) the sense RNA sequence of each of the double-stranded product RNA molecules varies from their corresponding sequence in the first region of the target RNA molecule only by the substitution of at least some A ribonucleotides with G ribonucleotides, or at least some C ribonucleotides with U ribonucleotides, or a combination of at least some A ribonucleotides with G ribonucleotides and at least some C ribonucleotides with U ribonucleotides, or wherein some but not all of the A ribonucleotides are substituted with G ribonucleotides, or some but not all C ribonucleotides are substituted with U ribonucleotides, or a combination of some but not all A ribonucleotides with G ribonucleotides and some but not all C ribonucleotides with U ribonucleotides,

[0207] (xi) the antisense RNA sequence of each of the double-stranded product RNA molecules varies from a corresponding sequence in the complement of the first region of the target RNA molecule only by the substitution of at least some A ribonucleotides with G ribonucleotides, or at least some C ribonucleotides with U ribonucleotides, or a combination of at least some A ribonucleotides with G ribonucleotides and at least some C ribonucleotides with U ribonucleotides, or wherein some but not all of the A ribonucleotides are substituted with G ribonucleotides, or some but not all C ribonucleotides are substituted with U ribonucleotides, or a combination of some but not all A ribonucleotides with G ribonucleotides and some but not all C ribonucleotides with U ribonucleotides,

[0208] (xii) the percentage of G ribonucleotides in the first RNA sequence is between 26- 40% of the total number of ribonucleotides in the first RNA sequence, and / or the percentage of G ribonucleotides in the second RNA sequence is between 26-40% of the total number of ribonucleotides in the second RNA sequence,

[0209] (xiii) the percentage of U ribonucleotides in the first RNA sequence is between 26- 40% of the total number of ribonucleotides in the first RNA sequence, and / or the percentage of U ribonucleotides in the second RNA sequence is between 26-40% of the total number of ribonucleotides in the second RNA sequence,

[0210] (xiv) the double-stranded RNA region has at most 18 contiguous canonical basepairs,

[0211] (xv) the precursor RNA molecule is capable of being made enzymatically by transcription in vitro or in a cell, or both.

[0212] In embodiments, combinations of the above features (i) to (xv) apply. Preferred combinations include (i) and (ii), (i) and (iv), (i) and any one of (v) to (xv), (ii) and (iii), (ii) and (iv), (ii) and any one of (v) to (xv), (iii) and (iv), (iii) and any one of (v) to (xv), (iv) and any one of (v) to (xv), (v) and (vii), (v) and (viii), (v) and (x), (v) and (xi), (v) and (xii), (v) and (xiii), (v) and (xiv), (vi) and (vii), (vi) and (viii), (vi) and (x), (vi) and (xi), (vi) and (xii), (vi) and (xiii), (vi) and (xiv), (vii) and (viii), (vii) and (x), (vii) and

[0213] (xi), (vii) and (xii), (vii) and (xiii), (vii) and (xiv), (viii) and (x), (viii) and (xi), (viii) and

[0214] (xii), (viii) and (xiii), (viii) and (xiv), (x) and (xi), (x) and (xii), (x) and (xiii), (x) and (xiv), (xi) and (xii), (xi) and (xiii), (xi) and (xiv), (xii) and (xiii), (xii) and (xiv).

[0215] In an embodiment of the first, second, third, fourth, or fifth aspects, or the embodiments of the asymmetric precursor RNA molecule, the precursor RNA molecule comprises a linking RNA sequence which comprises a sequence of at least 50 contiguous ribonucleotides which is at least 90% identical, preferably at least 95% or 100% identical, to a second region of the target RNA molecule. The second region of the target RNA molecule is preferably 3' of the first region of the target RNA molecule. In analogous fashion, the linking RNA sequence comprises a sequence of at least 50 contiguous ribonucleotides which is at least 90% identical, preferably at least 95% or 100% identical, to the complement of the second region of the target RNA molecule.

[0216] In an embodiment of the first, second, third, fourth, or fifth aspects, or the embodiments of the asymmetric precursor RNA molecule, the precursor RNA molecule further comprises a sequence of at least 50 contiguous ribonucleotides which is at least 90% identical, preferably at least 95% or 100% identical, to a second region of the target RNA molecule in the eukaryotic cell such as a plant cell, nematode cell, insect cell or fungal cell, covalently linked to the 5' or 3' end of the first RNA strand or to the 5' or 3' end of the second RNA strand, and / or a sequence of at least 50 contiguous ribonucleotides which is at least 90% identical, preferably at least 95% or 100% identical, to the complement of a second region of the target RNA molecule, covalently linked to the 5' or 3' end of the first RNA strand or to the 5' or 3' end of the second RNA strand.

[0217] In an embodiment of the first, second, third, fourth, or fifth aspects, or the embodiments of the asymmetric precursor RNA molecule, the linking RNA sequence comprises a single-stranded RNA sequence of at least 44, or at least 50, or at least 100, contiguous ribonucleotides which is identical to a region of either the first RNA sequence or the second RNA sequence of the double-stranded RNA region. Such a second copy of the RNA sequence in the precursor RNA molecule, in a single -stranded form, is considered to increase the inhibitory activity of the precursor RNA molecule, for example by increasing production of secondary siRNAs.

[0218] In an embodiment of the first, second, third, fourth, or fifth aspects, or the embodiments of the asymmetric precursor RNA molecule, the precursor RNA molecule comprises a linking RNA sequence and is encoded by a polynucleotide which lacks an intron in its region encoding the linking RNA sequence. That is, the initial transcript from the polynucleotide lacks an intron sequence, instead has a linking RNA sequence without an intron.

[0219] In an embodiment of the fourth or fifth aspects as applied to a symmetric precursor RNA molecule, also to the embodiments of the asymmetric precursor RNA molecule of the first, second, third, fourth, or fifth aspects, the first RNA sequence and second RNA sequence are identical in length across the full length of the double-stranded RNA region. That is, if there are non-basepaired ribonucleotides in the first and second RNA sequences, the number of non-basepaired ribonucleotides in the first RNA sequence is equal to the number of non-basepaired ribonucleotides in the second RNA sequence. This can be achieved through insertions and / or deletions in one or both sequences.

[0220] In embodiments of the asymmetric precursor RNA molecules of the first, second, third, fourth, or fifth aspects, or the embodiments of the asymmetric precursor RNA molecule, the first RNA sequence and second RNA sequence are different in length across the full length of the double-stranded RNA region, either the first RNA sequence is longer than the second RNA sequence or, preferably, the first RNA sequence is shorter than the second RNA sequence. In an embodiment, the different lengths are due at least in part, preferably entirely, to non-basepaired ribonucleotides in the second RNA sequence that bulge out from the double-stranded RNA region. In an embodiment, the sense sequence has a length which is between 87% and 97%, or 87% and 96%, or 91% and 97%, or 91% and 96%, or more preferably between 94% and 97% or 94% and 96% of the length of the antisense sequence, or the length of the sense sequence is about 21 / 22, 21 / 23, or 21 / 24 of the length of the antisense sequence, calculated as a fraction.

[0221] In an embodiment of the first, second, third, fourth, or fifth aspects, or the embodiments of the asymmetric precursor RNA molecule, the first RNA sequence and / or the second RNA sequence varies from the first region of the target RNA molecule or its complement, respectively, only by the substitution of at least some A ribonucleotides with G ribonucleotides, preferably 40-60% of the A ribonucleotides, or at least some C ribonucleotides with U ribonucleotides, preferably 40-60% of the C ribonucleotides, or a combination of at least some A ribonucleotides, preferably 40-60% of the A ribonucleotides, with G ribonucleotides and at least some C ribonucleotides, preferably 40-60% of the C ribonucleotides, with U ribonucleotides. In an embodiment, some but not all of the A ribonucleotides are substituted with G ribonucleotides, or some but not all C ribonucleotides are substituted with U ribonucleotides, or a combination of some but not all A ribonucleotides with G ribonucleotides and some but not all C ribonucleotides with U ribonucleotides. In a preferred embodiment, the second RNA sequence is identical in sequence to the complement of the first region of the target RNA molecule, i.e. has no substitutions. These features are also applicable to double-stranded product RNA molecules produced from the precursor RNA molecule.

[0222] In an embodiment of the first, second, third, fourth, or fifth aspects, or the embodiments of the asymmetric precursor RNA molecule, the ribonucleotide substitutions result in G:U basepairs in the double-stranded RNA region, for example between 10% and 35%, between 10% and 30%, or between 10% and 25% of the ribonucleotides in the double -stranded RNA region, in total, and / or the part of the double-stranded RNA region, in total, form a G:U basepair. In an embodiment, between 15% and 35%, between 15% and 30%, or between 15% and 25% of the ribonucleotides in the double-stranded RNA region, in total, and / or the part of the double-stranded RNA region, in total, form a G:U basepair. In an embodiment, between 17% and 35%, between 17% and 30%, or between 17% and 25% of the ribonucleotides in the double-stranded RNA region, in total, and / or the part of the double-stranded RNA region, in total, form a G:U basepair. In an embodiment, about 12%, about 15%, about 20%, about 25%, or about 30% of the ribonucleotides in the double-stranded RNA region, in total, and / or the part of the double -stranded RNA region, in total, form a G:U basepair.

[0223] In an embodiment of the first, second, third, fourth, or fifth aspects, or the embodiments of the asymmetric precursor RNA molecule, the percentage of G ribonucleotides in the first RNA sequence is increased by A to G substitutions. For example, between 26-40% of the total number of ribonucleotides in the first RNA sequence are G ribonucleotides, preferably 28-40%, 30-40%, 32-40%, 34-40%, or 26- 38%, 26-36%, or 26-34% of the total number of ribonucleotides in the first RNA sequence are G ribonucleotides. In an embodiment, the percentage of G ribonucleotides in the second RNA sequence is increased by A to G substitutions, for example, between 26-40% of the total number of ribonucleotides in the second RNA sequence are G ribonucleotides, preferably 28-40%, 30-40%, 32-40%, 34-40%, or 26-38%, 26-36%, or 26-34% of the total number of ribonucleotides in the first RNA sequence are G ribonucleotides. In this context, substitutions are relative to the sequence of the region of the target RNA molecule or its complement. In an embodiment, either the first or second RNA sequence, or both, lack A to G substitutions relative to the region of the target RNA molecule or its complement, respectively.

[0224] In an embodiment of the first, second, third, fourth, or fifth aspects, or the embodiments of the asymmetric precursor RNA molecule, the percentage of U ribonucleotides in the first RNA sequence is increased by C to U substitutions. For example, between 26-40% of the total number of ribonucleotides in the first RNA sequence are U ribonucleotides, preferably 28-40%, 30-40%, 32-40%, 34-40%, or 26- 38%, 26-36% or 26-34% of the total number of ribonucleotides in the first RNA sequence are U ribonucleotides. In an embodiment, the percentage of U ribonucleotides in the second RNA sequence is increased by C to U substitutions, for example, between 26-40% of the total number of ribonucleotides in the second RNA sequence are G ribonucleotides, preferably 28-40%, 30-40%, 32-40%, 34-40%, or 26-38%, 26-36% or 26-34% of the total number of ribonucleotides in the first RNA sequence are G ribonucleotides. In this context, substitutions are relative to the sequence of the region of the target RNA molecule or its complement. In an embodiment, either the first or second RNA sequence, or both, lack C to U substitutions relative to the region of the target RNA molecule or its complement, respectively.

[0225] In an embodiment of the first, second, third, fourth, or fifth aspects, or the embodiments of the asymmetric precursor RNA molecule, the double -stranded RNA region has at most 18 contiguous canonical basepairs, preferably at most 17, at most 16, at most 15, at most 14, at most 13, at most 12, at most 11, at most 10, at most 9, at most 8, or at most 7 contiguous canonical basepairs. That is, these numbers represent the maximum number of contiguous canonical basepairs for the longest subregion of contiguous canonical basepairing in the dsRNA region. The calculation of the number of contiguous canonical basepairs ignores the presence of any non-basepaired ribonucleotides in the double-stranded region. Reducing the number of contiguous canonical basepairs in any subregion can be achieved by regular spacing of A to G and C to U substitutions in either or both sequences.

[0226] In an embodiment of the fourth or fifth aspects, or the embodiments of the asymmetric precursor RNA molecule of the first, second, third, fourth, or fifth aspects, or the embodiments of the asymmetric precursor RNA molecule, at least some of the antisense RNA sequences in the double-stranded product RNA molecules produced from the precursor RNA molecule basepair along the full length of the antisense RNA sequences to the region of the target RNA molecule, preferably basepair along the full length by canonical basepairs.

[0227] In an embodiment of the fourth or fifth aspects, or the embodiments of the asymmetric precursor RNA molecule of the first, second, third, fourth, or fifth aspects, at least some of the antisense RNA sequences in the double-stranded product RNA molecules produced from the precursor RNA molecule are capable of reducing the expression and / or activity of the target RNA molecule in the eukaryotic cell such as a plant cell, nematode cell, insect cell or fungal cell. In an embodiment, the antisense RNA sequences in the double -stranded product RNA molecules produced from the precursor RNA molecule are capable of reducing the expression and / or activity of multiple, different target RNA molecules in the eukaryotic cell such as a plant cell, nematode cell, insect cell or fungal cell, wherein the different target RNA molecules are unrelated in sequence.

[0228] In an embodiment, the reduction in expression and / or activity of the target RNA molecule(s) in the insect cell results in death of larvae of the insect, or the mortality rate is increased relative to the use of antisense RNA sequences produced from a corresponding precursor RNA molecule with only canonical basepairing. For example, 87.5-100% of the insect larvae that ingest the precursor RNA molecule and / or the antisense RNA sequences are killed. In an embodiment, the reduction in expression and / or activity of the target RNA molecule(s) in the fungal cell results in decreased symptoms and / or increased resistance to fungal infection relative to the use of antisense RNA sequences produced from a corresponding precursor RNA molecule with only canonical basepairing.

[0229] In an embodiment of the ledRNA molecules of the first, second, third, fourth, or fifth aspects, or the embodiments of the asymmetric precursor RNA molecule, either the sense RNA sequences of the two double-stranded RNA regions are contiguous relative to the target RNA molecule, or the antisense RNA sequences of the two double-stranded RNA regions are contiguous relative to the complement of the target RNA molecule. In an embodiment, the two double-stranded RNA regions are capable of being cleaved by a Dicer to produce double-stranded product RNA molecules which comprise antisense RNA sequences which hybridise to one region of a target RNA molecule or to different, non-contiguous regions of the target RNA molecule. In an embodiment, wherein the two double-stranded RNA regions are capable of being cleaved by a Dicer to produce doublestranded product RNA molecules which comprise antisense RNA sequences which hybridise to regions of different target RNA molecules, or to corresponding regions in a family of target RNA molecules. In an embodiment, the second double-stranded region lacks non-basepaired ribonucleotide bulges.

[0230] In an embodiment of the ledRNA molecules of the first, second, third, fourth, or fifth aspects, or the embodiments of the asymmetric precursor RNA molecule, the second double-stranded region comprises a third RNA sequence of at least 23 contiguous ribonucleotides and a fourth RNA sequence of at least 26 contiguous ribonucleotides, wherein the third RNA sequence hybridises to the fourth RNA sequence by basepairing between at least 20 ribonucleotides of the at least 23 contiguous ribonucleotides of the third RNA sequence and at least 20 ribonucleotides of the at least 26 contiguous ribonucleotides of the fourth RNA sequence, forming at least a part of the second doublestranded RNA region, wherein 1, 2, 3, 4, 5, or 6 ribonucleotides of the at least 26 contiguous ribonucleotides of the fourth RNA sequence are non-basepaired and 0, 1, 2, or 3 ribonucleotides of the at least 23 contiguous ribonucleotides of the first RNA sequence are non-basepaired in the part of the double -stranded RNA region, forming bulges in part of the second double-stranded RNA region, wherein each of the bulges is immediately flanked by ribonucleotides which are basepaired in the part of the second double-stranded RNA region, wherein the fourth RNA sequence is at least 80% identical to a sequence of at least 26 contiguous ribonucleotides which is fully complementary to a region of a target RNA molecule in eukaryotic cell such as a plant cell, nematode cell, insect cell or fungal cell.

[0231] In an embodiment, (i) the first RNA sequence and / or the third RNA sequence differs from a corresponding wild-type RNA sequence in the target RNA molecule by deletion of one or more ribonucleotides from the corresponding wild-type RNA sequence to make the first or third RNA sequence, and / or (ii) the second RNA sequence and / or the fourth RNA sequence differs from a fully complementary sequence to the corresponding wild-type RNA sequence in the target RNA molecule by insertion of one or more ribonucleotides into the fully complementary sequence to make the second or fourth RNA sequence, preferably (i). In an embodiment, the deletion of ribonucleotides from the corresponding wild-type RNA sequence occurs at one or more or all of the ribonucleotide positions corresponding to the non-basepaired ribonucleotides in the second or fourth RNA sequence. In an embodiment, the precursor RNA molecule is capable of being cleaved in eukaryotic cell such as a plant cell, nematode cell, insect cell or fungal cell by one or more Dicers to produce any of the double-stranded product RNA molecules defined in any of the first, second, third, fourth, or fifth aspects.

[0232] In an embodiment of the first, second, third, fourth, or fifth aspects, or the embodiments of the asymmetric precursor RNA molecule, the precursor RNA molecule has a single double-stranded RNA region and, optionally, the part of the double-stranded RNA region extends to encompass the whole double-stranded RNA region.

[0233] In an embodiment of the first, second, third, fourth, or fifth aspects, or the embodiments of the asymmetric precursor RNA molecule, all of the ribonucleotides of the antisense RNA sequences are capable of basepairing to ribonucleotides in the region of the target RNA molecule.

[0234] In an embodiment of the first, second, third, fourth, or fifth aspects, or the embodiments of the asymmetric precursor RNA molecule, the precursor RNA molecule is produced in a plant cell or a microbial cell such as a yeast cell by transcription of an exogenous polynucleotide.

[0235] Each of the embodiments of the first, second and third aspects may have the following feature: the antisense RNA sequence (J) from at least one of the product RNA molecules (P) produced from the precursor RNA molecule is capable of hybridising to a region (R) of a target RNA molecule in a eukaryotic cell through at least all of ribonucleotides 2 to 8 of the antisense RNA sequence (J) basepairing with ribonucleotides within the region (R) of the target RNA molecule. The hybridisation may be through at least all of ribonucleotides 2 to 10 or 2 to 11 of the antisense RNA sequence (J).

[0236] In an embodiment, including for the immediately preceding paragraph, the eukaryotic cell is a vertebrate animal cell such as a mammalian animal cell, or a nonmammalian vertebrate animal cell, where reduction of activity of the target RNA molecule may be primarily through an inhibition of translation of the target RNA molecule. Where basepairing to the target RNA molecule occurs through a longer area than ribonucleotides 2 to 11, the reduction of activity may be through inhibition of translation and / or cleavage of the target RNA molecule.

[0237] Preferably, most of the antisense RNA sequences (J) from the product RNA molecules (P) produced from the precursor RNA molecule are capable of hybridising to a region (R), or more than one region (R), of the target RNA molecule. More preferably, all of the antisense RNA sequences (J) from the product RNA molecules (P) produced from the precursor RNA molecule are capable of hybridising to a region (R), or more than one region, of the target RNA molecule.

[0238] In each of the embodiments of the first, second, third, fourth, or fifth aspects, or the embodiments of the asymmetric precursor RNA molecule, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%, of the ribonucleotides of one or more or all of the antisense RNA sequences (J) are capable of basepairing to ribonucleotides of a region (R) of a target RNA molecule in a eukaryotic cell. Preferably, multiple, non-overlapping antisense RNA sequences (J) are capable of basepairing to ribonucleotides of the region (R) of the target RNA molecule or to multiple regions (R) of the target molecule, or to each of multiple target RNA molecules with any of these minimum percentages.

[0239] In an embodiment, all of the ribonucleotides of the antisense RNA sequence (J) are capable of basepairing to ribonucleotides of a region (R) of a target RNA molecule in a eukaryotic cell. Preferably, this feature occurs for each of multiple, non-overlapping antisense RNA sequences (J) that basepair to ribonucleotides of the region (R) of the target RNA molecule or to multiple regions (R) of the target molecule, or to multiple target RNA molecules. This can readily be achieved through the use of longer antisense sequences, at least 50nt or at least lOOnt in length, in the precursor RNA molecule that are fully complementary to the target RNA molecule. Preferably, all of the ribonucleotides of multiple, different antisense RNA sequences (J) are capable of basepairing to ribonucleotides across a length of a region (R) of a target RNA molecule of at least 150, or at least 200, or at least 250, or at least 300 ribonucleotides, preferably to a maximum of 1000 ribonucleotides, more preferably to a maximum of 800 ribonucleotides, or even more preferably to a maximum of 600 ribonucleotides. For example, preferably all of the ribonucleotides of multiple, different antisense RNA sequences (J) are capable of basepairing to ribonucleotides across a length of a region (R) of a target RNA molecule in the range 100-1000, 100-800, or 100-600 ribonucleotides, or in the range 150-1000, 150-800, or 150-600 ribonucleotides of the target RNA molecule.

[0240] Each of the embodiments of the first, second, third, fourth, or fifth aspects, or the embodiments of the asymmetric precursor RNA molecule may have the following feature: the basepairing to ribonucleotides of the region (R) of the target RNA molecule comprises one or more G:U basepairs, preferably 2, 3, 4 or 5 G:U basepairs.

[0241] Each of the embodiments of the first, second, third, fourth, or fifth aspects, or the embodiments of the asymmetric precursor RNA molecule may have the following feature: the basepairing to ribonucleotides of the region (R) of the target RNA molecule comprises only canonical basepairs.

[0242] Each of the embodiments of the first, second, third, fourth, or fifth aspects, or the embodiments of the asymmetric precursor RNA molecule may have one or more of the following features, where applicable: the region (R) of the target RNA molecule has a length of 22-30, 23-30, 23-33, 24-30, 30-50, 34-200, 50-100, 100-600, or 100-1000 ribonucleotides, and / or the length of the antisense sequence of the dsRNA region is 22- 30, 23-30, 23-33, 24-30, 30-50, 34-200, 50-100, 100-600, or 100-1000 ribonucleotides, and / or the sense sequence of the dsRNA region of the precursor RNA molecule within those ranges is shorter than the corresponding antisense sequence, preferably the sense sequence is shorter than the corresponding antisense sequence entirely because of the presence of non-basepaired ribonucleotides in the antisense sequence that bulge from the dsRNA region or the product RNA molecule(s) (P), more preferably the sense sequence has a length which is between 87%-97%, or 87%-96%, or 91%-97%, or 91%-96%, or more preferably 94%-97% or 94%-96% of the length of the antisense sequence, or the length of the sense sequence is about 21 / 22, 21 / 23 or 21 / 24 of the length of the antisense sequence, calculated as a fraction. Preferably, the antisense sequence in the precursor RNA molecule is fully complementary to the target RNA molecule along at least that length.

[0243] Each of the embodiments of the first, second, third, fourth, or fifth aspects, or the embodiments of the asymmetric precursor RNA molecule may have the following feature: the precursor RNA molecule comprises two or more different, double-stranded RNA regions (B), wherein each double -stranded RNA region (B) is independently defined herein. Examples of this embodiment are the ledRNA structures shown schematically in Figure 1.

[0244] In an embodiment, the two or more different, double-stranded RNA regions (B) are contiguous with regard to the sequence of the target RNA molecule.

[0245] In an alternative embodiment, the two different, double-stranded RNA regions (B) are linked covalently through one or two linking RNA sequence(s). In this context, when two linking RNA sequences are present, one links the 3' end of one strand of the first of the double-stranded regions to the 5' end of one strand of the second doublestranded region, and the other linking RNA sequence links the 3' end of the other strand of the second double -stranded region to the 5' end of the other strand of the first doublestranded region, effectively forming a longer double-stranded region in the precursor RNA molecule.

[0246] In an embodiment, one or both of the linkers comprise or consist of ribonucleotides that are non-basepaired, preferably that form one or more bulges or loops in the precursor RNA molecule.

[0247] Each of the embodiments of the first, second, third, fourth, or fifth aspects, or the embodiments of the asymmetric precursor RNA molecule may have the following feature, where applicable: the two or more different, double-stranded RNA regions (B) are capable of being cleaved by one or more RNases to produce product RNA molecules (P) which comprise antisense RNA sequences (J) which hybridise to one region (R) of a target RNA molecule or to different, non-contiguous regions (R) of the target RNA molecule.

[0248] Each of the embodiments of the first, second, third, fourth, or fifth aspects, or the embodiments of the asymmetric precursor RNA molecule may have the following feature, where applicable: the two or more different, double-stranded RNA regions (B) are capable of being cleaved by one or more RNases to produce product RNA molecules (P) which comprise antisense RNA sequences (J) which hybridise to regions (R) of different target RNA molecules, or to corresponding regions (R) in a family of target RNA molecules.

[0249] Each of the embodiments of the first, second, third, fourth, or fifth aspects, or the embodiments of the asymmetric precursor RNA molecule, may have the following feature: the target RNA molecule encodes one or more protein(s). Alternatively, the target RNA molecule does not encode a protein, for example the target RNA is a miRNA.

[0250] Each of the embodiments of the first, second and third aspects, or the embodiments of the asymmetric precursor RNA molecule, may have the following feature: the eukaryotic cell is a plant cell, an animal cell, or a fungal cell, preferably a plant cell, an arthropod cell such as an insect, arachnid, or decapod cell, a nematode cell, or a fungal cell. Preferred insect cells are from the orders Lepidoptera, Coleoptera, Diptera and Hemiptera. Other preferred arthropods include those in the Order Arachnida such as spiders and ticks, or Decapoda such as prawns. In an embodiment, the target RNA molecule is an RNA molecule of a viral pathogen of the eukaryotic cell or organism such as the plant, insect, or the decapod. In embodiments of the first, second, and third aspects where the double-stranded region (B) comprises between 20 and 30 basepairs, where the basepairs are all canonical basepairs, or where the basepairs include one or more G:U basepairs, the eukaryotic cell may be a vertebrate animal cell such as a mammalian cell, a human cell, or a non-human mammalian cell, or a non-mammalian vertebrate animal cell. In embodiments of the first, second and third aspects where the double-stranded region (B) comprises between 31 and 50 basepairs and where the eukaryotic cell is a vertebrate animal cell, it is preferred that between 10% and 40%, preferably between 16% and 30% or between 16% and 25%, of the basepairs are G:U basepairs. More preferred, in the context of these cells, is that the precursor RNA molecule comprises a ledRNA structure in addition to having the G:U basepairs. In an embodiment, the vertebrate animal cell is a mammalian cell, a human cell, a non-human mammalian cell or a non-mammalian vertebrate animal cell. In an embodiment, the nonmammalian vertebrate animal cell is a bird cell or a fish cell. The vertebrate animal cell, mammalian cell, human cell or non-human cell may be a cell in culture or in vitro. The cell may be used in a screening assay to identify suitable target RNA molecules.

[0251] In an embodiment, applicable to each of the embodiments of the first, second, third, fourth, or fifth aspects, or the embodiments of the asymmetric precursor RNA molecule, the target RNA molecule, or multiple different target RNA molecules, is in a eukaryotic cell which is a plant cell, an animal cell or a fungal cell, preferably a plant cell, an arthropod cell, a nematode cell or a fungal cell. The animal cell may be an arthropod cell such as an insect, arachnid, or decapod cell. Any of these cells may be cells in cell culture.

[0252] In an embodiment, applicable to each of the embodiments of the first, second, third, fourth, or fifth aspects, or the embodiments of the asymmetric precursor RNA molecule, the precursor RNA molecule is present in the eukaryotic cell and / or which is produced in the eukaryotic cell, optionally is cleaved by one or more RNases in the eukaryotic cell to produce the one or more double-stranded product RNA molecule(s) (P). Furthermore, the target RNA molecule may be in the same cell. Alternatively, the target RNA molecule is not in the same cell, or has not yet entered the cell e.g. the target RNA molecule is from a viral pathogen which may or may not enter the cell. For example, the precursor RNA molecule may be produced in a microbial cell such as a bacterial cell or a yeast cell, for example Saccharomyces cerevisiae, and applied, with or without extraction of the RNA from the microbial cell, to the cells or organism comprising the target RNA molecule. In an embodiment, the microbial cell is ingested by the target organism and the precursor RNA molecule and / or the siRNA products from the precursor RNA molecule are released from the microbial cell.

[0253] In an embodiment, applicable to each of the embodiments of the first, second, third, fourth, or fifth aspects, or the embodiments of the asymmetric precursor RNA molecule, the one or more double-stranded region(s) (B) comprise bulges which are evenly spaced apart along most or all of each double-stranded region (B). For example, the bulges for the A22 modification are spaced apart on average about one ribonucleotide bulge in the second RNA sequence (G) about every 22nd ribonucleotide, applicable to longer sequences (G). Analogously, the bulges for the A23 or A24 modifications are spaced apart on average about two or three bulging antisense ribonucleotides, respectively, in the second RNA sequence (G) about every 23 or 24 ribonucleotides. In this context, “about” means + / - 10%, preferably + / - 5%.

[0254] In an embodiment, applicable to each of the embodiments of the first, second, third, fourth, or fifth aspects, or the embodiments of the asymmetric precursor RNA molecule, each double -stranded region (B) comprises a bulge which is closer to the linking RNA sequence (L) than any other bulge in the double-stranded region (B), wherein said closer bulge and the linking RNA sequence (L) are separated by at least four or more contiguous intervening basepairs, preferably at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10 contiguous intervening basepairs, more preferably by 5-15, 5-14, 5-13, 5-12, or 5-11 contiguous intervening basepairs.

[0255] In an embodiment, applicable to each of the embodiments of the first, second, third, fourth, or fifth aspects, or the embodiments of the asymmetric precursor RNA molecule, the precursor RNA molecule comprises a terminal basepair at a junction of the double-stranded region (B) and the linking RNA sequence (L), wherein the terminal basepair comprises a U ribonucleotide as the last 3' ribonucleotide in the first RNA sequence (E) or the first 5' ribonucleotide in the second RNA sequence (G), preferably the first 5' ribonucleotide in the second RNA sequence (G) is a U ribonucleotide.

[0256] In an embodiment, the terminal basepair at the junction of the double-stranded region (B) and the linking RNA sequence (L) is an A:U basepair or a G:U basepair, preferably a G:U basepair, more preferably the U is in the second RNA sequence (G).

[0257] In an embodiment, applicable to each of the embodiments of the first, second, third, fourth, or fifth aspects, or the embodiments of the asymmetric precursor RNA molecule, the precursor RNA molecule has a single linking RNA sequence (L), thereby forming a hairpin RNA (hpRNA) structure. In an embodiment, the linking RNA sequence joins the 3' end of the first RNA strand (D) and the 5' end of the second RNA strand (F). Alternately, the linking RNA sequence joins the 3' end of the second RNA strand (F) and the 5' end of the first RNA strand (D). Presenting this alternate structure visually, the loop (L) would appear at the left-hand side of the schematic diagrams in Figure 53, and the order of transcription is second RNA strand (F)-loop (L)-first RNA strand (D).

[0258] Alternatively, the invention provides precursor RNA molecules (A) which are the same as the precursor RNA molecules (A) of the first, second, third, fourth, or fifth aspects, or the embodiments of the asymmetric precursor RNA molecule, except that they lack a linking RNA sequence (L). That is, the precursor RNA molecule is comprised of two RNA strands that anneal together. In an embodiment, the precursor RNA molecules (A) lacking the (L) comprise at least 44 basepairs, preferably at least 50 basepairs, more preferably at least 100 basepairs of the second RNA strand (F) is at least 100 ribonucleotides long. All of the features of the embodiments of the first, second and third aspects are applicable to these precursor RNA molecules (A) lacking the (L), singly or in combinations.

[0259] In an embodiment, applicable to each of the embodiments of the first, second, third, fourth, or fifth aspects, or the embodiments of the asymmetric precursor RNA molecule, the precursor RNA molecule has two double-stranded regions (B) and two linking RNA sequences (L) forming a ledRNA structure (also known as a dumbbell structure). All of the features of the embodiments of the ledRNA structures as described herein are applicable here, either singly or in combination.

[0260] In an embodiment, applicable to each of the embodiments of the hairpin RNAs or ledRNAs of the first, second, third, fourth, or fifth aspects, or the embodiments of the asymmetric precursor RNA molecule, the precursor RNA molecule comprises a terminal basepair at a junction of the double-stranded region (B) and the linking RNA sequence (L), wherein the terminal basepair is the first basepair of part (C) of the double-stranded RNA region.

[0261] In an embodiment, applicable to each of the embodiments of the hairpin RNAs or ledRNAs of the first, second, third, fourth, or fifth aspects, or the embodiments of the asymmetric precursor RNA molecule, the precursor RNA molecule comprises at least one linking RNA sequence (L) which is between 4 and 2000 ribonucleotides in length, preferably between 4 and 1000 ribonucleotides in length, more preferably between 4 and 200 ribonucleotides or 4 and 50 ribonucleotides in length and most preferably between 4 and 20 nucleotides in length, preferably wherein all of the linking RNA sequences in the precursor RNA molecule have the aforesaid length.

[0262] In an embodiment, applicable to each of the hairpin RNAs or ledRNAs of the embodiments of the first, second, third, fourth, or fifth aspects, or the embodiments of the asymmetric precursor RNA molecule as encoded by a polynucleotide, at least one linking RNA sequence (L) comprises an intron, preferably wherein all of the linking RNA sequences in the precursor RNA molecule comprise an intron. In an alternative embodiment, the precursor RNA molecule as encoded by a polynucleotide lacks an intron.

[0263] In embodiments of the first, second, third, fourth, or fifth aspects, or the embodiments of the asymmetric precursor RNA molecule, the loop sequence of a hairpin precursor RNA molecule, or either or both loops of a precursor ledRNA molecule, comprises a second copy of a sequence (seed sequence) from the dsRNA region, comprising either part or the whole of the sense or antisense sequence from the dsRNA region. For example, the precursor RNA molecule comprises a second sense or antisense sequence as the seed sequence which comprises at least 100 ribonucleotides from within the dsRNA region (seed region), incorporated into the loop sequence or elsewhere in the precursor RNA molecule. The loop sequence may be chimeric in comprising a seed sequence as well as other sequences related to the target RNA molecule or its complement. In an embodiment, the seed sequence is inserted into a region of the RNA molecule other than the loop, for example to the 5' or 3' end of the precursor RNA molecule. In an embodiment, the duplex regions of the first and second components of the precursor ledRNA molecule targets different target RNA molecules and the first and second loop sequences correspond to regions from the different target transcripts.

[0264] In an embodiment, applicable to each of the embodiments of the first, second, third, fourth, or fifth aspects, or the embodiments of the asymmetric precursor RNA molecule, including the ledRNA structures, the double -stranded region(s) (B) in the precursor RNA molecule comprises bulges only as defined herein.

[0265] In an embodiment, applicable to each of the embodiments of the first, second, third, fourth, or fifth aspects, or the embodiments of the asymmetric precursor RNA molecule, including the precursor hairpin RNA and ledRNA molecules, the precursor RNA molecule further comprises a double-stranded region which comprises at least 23 contiguous basepairs and which lacks bulges. In an embodiment, between 5% and 40% of the at least 23 contiguous basepairs are G:U basepairs. Alternatively, the at least 23 contiguous basepairs are all canonical basepairs. The precursor RNA molecules (A) of these embodiments thereby provide a mixture of symmetrical and asymmetrical product RNA molecules (P). In an embodiment, when cleaved by a Dicer, the precursor RNA molecule provides more asymmetrical than symmetrical product RNA molecules (P).

[0266] In an embodiment, the double-stranded region which lacks bulges has a length of 30-200 contiguous basepairs, preferably at least 100 basepairs, more preferably 100- 200, 100-300, 100-400, 100-500 or 100-600 basepairs in length.

[0267] In an embodiment, applicable to each of the embodiments of the first, second, third, fourth, or fifth aspects, or the embodiments of the asymmetric precursor RNA molecule, including the ledRNA structures, the one or more RNase(s) is a Type III ribonuclease, preferably a Dicer or Dicer-like (DCL) protein, more preferably a DCL2 and / or DCL4 protein or homologue. In an embodiment, the eukaryotic cell in which the precursor RNA is produced is a plant cell which is wild-type (unmodified) in gene(s) encoding DCL2 and / or DCL4 protein.

[0268] In an embodiment, applicable to each of the embodiments of the first, second, third, fourth, or fifth aspects, or the embodiments of the asymmetric precursor RNA molecule, including the precursor hairpin RNA and ledRNA molecules, one, two, three, four, five, or six, of the basepairs in at least some of the one or more double-stranded product RNA molecule(s) (P) are non-canonical basepairs, preferably G:U basepairs. Preferably, most of the double-stranded product RNA molecule(s) (P) produced from the precursor RNA molecule, independently comprise one, two, three, four, five, or six, G:U basepairs, for example at least 60%, at least 70%, at least 80%, or at least 90% of the product RNA molecules produced from the precursor RNA molecule comprise that number of G:U basepairs.

[0269] In an embodiment, applicable to each of the embodiments of the first, second, third, fourth, or fifth aspects, or the embodiments of the asymmetric precursor RNA molecule, including the ledRNA structures, on average every one in four to every one in six ribonucleotides in the dsRNA region (B) and / or the part (C) of the double-stranded RNA region and / or at least some of the product RNA molecules (P) form a non-canonical basepair or are not basepaired, preferably form a G:U basepair, and / or wherein between 5% and 40% of the ribonucleotides in the dsRNA region (B), in total, and / or the part (C) of the double-stranded RNA region, in total, and / or at least some of the product RNA molecules (P) form a non-canonical basepair or are not basepaired, preferably form a G:U basepair. Preferably, most of the double-stranded product RNA molecules (P) produced from the precursor RNA molecule, independently have on average about one- in-four to one-in-six basepairs that are G:U basepairs. Preferably, at least 60%, at least 70%, at least 80%, or at least 90% of the canonically-basepaired ribonucleotides in the dsRNA region, in total, are in subregions of 4-6 canonical basepairs. In an embodiment, more of the G:U basepairs of the population of the double-stranded product RNA molecules (P) produced from the precursor RNA molecule have the G ribonucleotide in a sense RNA sequence (H) and the U in an antisense RNA sequence (J).

[0270] In an embodiment, between 10% and 40%, between 10% and 35%, between 10% and 30%, or between 10% and 25% of the ribonucleotides in the dsRNA region (B), in total, and / or the part (C) of the double-stranded RNA region, in total, and / or in at least some of the product RNA molecules (P) form a G:U basepair. In an embodiment, between 15% and 40%, between 15% and 35%, between 15% and 30%, or between 15% and 25% of the ribonucleotides in the dsRNA region (B), in total, and / or the part (C) of the double-stranded RNA region, in total, and / or in at least some of the product RNA molecules (P) form a G:U basepair. In an embodiment, between 17% and 40%, between 17% and 35%, between 17% and 30%, or between 17% and 25% of the ribonucleotides in the dsRNA region (B), in total, and / or the part (C) of the double-stranded RNA region, in total, and / or in at least some of the product RNA molecules (P) form a G:U basepair. In an embodiment, about 10%, about 15%, about 20%, about 25%, about 30% or about 35% of the ribonucleotides in the dsRNA region (B), in total, and / or the part (C) of the double-stranded RNA region, in total, and / or in at least some of the product RNA molecules (P) form a G:U basepair.

[0271] In an embodiment, applicable to each of the embodiments of the first, second, third, fourth, or fifth aspects, or the embodiments of the asymmetric precursor RNA molecule, including the precursor hairpin RNA and ledRNA molecules, the at least one double-stranded RNA region (B), preferably all of the double-stranded RNA regions (B), in the precursor RNA molecule lacks 8 contiguous canonical basepairs. This can readily be achieved by the introduction of suitably distributed G:U basepairs in addition to bulges in the double -stranded RNA regions.

[0272] In an embodiment, applicable to each of the embodiments of the first, second, third, fourth, or fifth aspects, or the embodiments of the asymmetric precursor RNA molecule, including the precursor hairpin RNA and ledRNA molecules, the precursor RNA molecule, following cleavage by the one or more RNAses, produces at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, or at least 100 different product RNA molecules (P). In an embodiment, the precursor RNA molecule, following cleavage by the one or more RNAses, produces at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30, at least 40, up to about 50 non-overlapping different product RNA molecules (P). In an embodiment, applicable to each of the embodiments of the first, second, third, fourth, or fifth aspects, or the embodiments of the asymmetric precursor RNA molecule, including the precursor hairpin RNA and ledRNA molecules, the first RNA strand (D) and / or the second RNA strand (F), preferably both, comprise at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1,000, or 100 to 1,000, or 50 to 1000 nucleotides, or 50 to 500, or 100 to 500 ribonucleotides. In a preferred embodiment, the first RNA strand (D) is shorter than the second RNA strand (F).

[0273] In an embodiment, applicable to each of the embodiments of the first, second, third, fourth, or fifth aspects, or the embodiments of the asymmetric precursor RNA molecule, including the precursor hairpin RNA and ledRNA molecules, the precursor RNA molecule has a single double-stranded RNA region (B) and a single part (C) which extends to encompass the whole double -stranded RNA region (B), i.e. the single part (C) corresponds to the entire double -stranded RNA region (B).

[0274] In an embodiment, both RNA strands (D) and (F) of the double-stranded region (B) are 23 to 33 ribonucleotides in length, and the double-stranded region (B) comprises at least 18 basepairs, preferably at least 19 or at least 20 basepairs. This is preferred where the eukaryotic cell is a vertebrate animal cell, particularly a mammalian cell. In this embodiment, preferably one, two, three, four, five, or six of the basepairs in at least some of the one or more double-stranded product RNA molecule(s) (P) produced from the precursor RNA molecule are non-canonical basepairs, preferably G:U basepairs.

[0275] In an embodiment, applicable to each of the embodiments of the first, second and third aspects, including the ledRNA molecules,

[0276] (i) RNA strands (D) and (F) of the double -stranded region (B) are each 34 to 200 ribonucleotides in length,

[0277] (ii) at least 80%, preferably at least 90%, of the ribonucleotides in the RNA strands (D) and (F), in total, are basepaired, thereby forming the double -stranded region (B) of the precursor RNA molecule, and

[0278] (iii) between 5% and 40% of the ribonucleotides in the dsRNA region (B), in total, are basepaired in a non-canonical basepair or are not basepaired, preferably basepaired in a G:U basepair. In this context, preferably between 10% and 40%, between 10% and 35%, between 10% and 30%, or between 10% and 25% of the ribonucleotides in the dsRNA region (B), in total, and / or the part (C) of the double-stranded RNA region, in total, and / or in at least some of the product RNA molecules (P) form a G:U basepair. More preferably, between 15% and 40%, between 15% and 35%, between 15% and 30%, or between 15% and 25% of the ribonucleotides in the dsRNA region (B), in total, and / or the part (C) of the double-stranded RNA region, in total, and / or in at least some of the product RNA molecules (P) form a G:U basepair. Even more preferably, between 17% and 40%, between 17% and 35%, between 17% and 30%, or between 17% and 25% of the ribonucleotides in the dsRNA region (B), in total, and / or the part (C) of the doublestranded RNA region, in total, and / or in at least some of the product RNA molecules (P) form a G:U basepair. In an embodiment, about 10%, about 15%, about 20%, about 25%, about 30% or about 35% of the ribonucleotides in the dsRNA region (B), in total, and / or the part (C) of the double-stranded RNA region, in total, and / or in at least some of the product RNA molecules (P) form a G:U basepair. Such molecules are particularly useful in vertebrate animal cells such as mammalian animal cells.

[0279] In an embodiment, applicable to each of the embodiments of the first, second, third, fourth, or fifth aspects, or the embodiments of the asymmetric precursor RNA molecule, including the ledRNA molecules, the antisense RNA sequence (J) from at least one of the product RNA molecules (P) produced from the precursor RNA molecule, preferably multiple, different product RNA molecules, are capable of hybridising to a region (R) of a target RNA molecule in a eukaryotic cell through all of at least ribonucleotides 2 to 8 of the antisense RNA sequence (J) basepairing with ribonucleotides within the region (R) of the target RNA molecule, preferably all of at least ribonucleotides 2-10 or 2-11 of the antisense RNA sequence (J).

[0280] In an embodiment, at least 50%, at least 75%, at least 90%, or at least 95%, of the ribonucleotides of the antisense RNA sequence (J) are capable of basepairing to ribonucleotides within the region (R) of the target RNA molecule in the eukaryotic cell.

[0281] In an embodiment, all of the ribonucleotides of the antisense RNA sequence (J), preferably all of the ribonucleotides of multiple, different antisense RNA sequences (J), are capable of basepairing to ribonucleotides in a region (R) of a target RNA molecule in the eukaryotic cell. In a preferred embodiment, all of the ribonucleotides of the antisense RNA sequence (J), preferably all of the ribonucleotides of multiple, different antisense RNA sequences (J), are capable of basepairing by canonical basepairs to ribonucleotides in the region (R) of the target RNA molecule in the eukaryotic cell, particularly for a plant cell, a fungal cell, a nematode cell, or an arthropod cell such as an insect cell.

[0282] In an embodiment, applicable to each of the embodiments of the first, second and third aspects, or the embodiments of the asymmetric precursor RNA molecule, including the precursor hairpin RNA and ledRNA molecules, the eukaryotic cell comprising the target RNA molecule is a vertebrate animal cell, preferably a mammalian cell, more preferably a human cell, or a non-mammalian vertebrate animal cell such as a bird cell or fish cell. The vertebrate animal cell may be of a companion animal or a livestock animal.

[0283] In an embodiment, applicable to each of the embodiments of the first, second, third, fourth, or fifth aspects, or the embodiments of the asymmetric precursor RNA molecule, including the precursor hairpin RNA and ledRNA molecules,

[0284] (i) the first RNA sequence (E) differs from a corresponding wild-type RNA sequence in the target RNA molecule by deletion of one or more ribonucleotides from the corresponding wild-type RNA sequence to make the first RNA sequence, preferably by deletion of multiple ribonucleotides in the A22, A23 or A24 pattern, and / or

[0285] (ii) the second RNA sequence (G) differs from a fully complementary sequence to the corresponding wild-type RNA sequence in the target RNA molecule by insertion of one or more ribonucleotides into the fully complementary sequence to make the second RNA sequence, preferably (i). In a preferred embodiment, the second RNA strand (F), along its full length, is identical in sequence to the complement of the wild-type RNA sequence in the target RNA molecule, particularly for a target RNA molecule in a plant, arthropod such as insect, arachnid, or decapod, nematode or fungal cell.

[0286] In an embodiment, applicable to each of the embodiments of the first, second, third, fourth, or fifth aspects, or the embodiments of the asymmetric precursor RNA molecule, including the precursor hairpin RNA and ledRNA molecules, the deletion of ribonucleotides from the corresponding wild-type RNA sequence occurs at one or more or all of the ribonucleotide positions corresponding to the non-basepaired ribonucleotides in the second RNA sequence (G) as defined herein.

[0287] In an embodiment, applicable to each of the embodiments of the first, second, third, fourth, or fifth aspects, including the precursor hairpin RNA and ledRNA molecules, the second RNA sequence (G) is identical in length to the fully complementary sequence to the corresponding wild-type RNA sequence in the target RNA molecule. That is, the second RNA sequence (G) does not have insertions or deletions of ribonucleotides relative to the complement of the region of the target RNA molecule.

[0288] In an embodiment, applicable to each of the embodiments of the first, second, third, fourth, or fifth aspects, or the embodiments of the asymmetric precursor RNA molecule, including the precursor hairpin RNA and ledRNA molecules,

[0289] (i) the first RNA sequence (E) differs from the corresponding wild-type RNA sequence in the target RNA molecule by substitution of one or more A or C ribonucleotides in the corresponding wild-type RNA sequence to G or U ribonucleotides, respectively, thereby resulting in the first RNA sequence, wherein the G or U ribonucleotides are involved in G:U basepairs with ribonucleotides of the second RNA sequence (G), and / or

[0290] (ii) the second RNA sequence (G) differs from the fully complementary sequence by substitution of one or more A or C ribonucleotides in the fully complementary sequence to G or U ribonucleotides, thereby resulting in the second RNA sequence, wherein the G or U ribonucleotides are involved in G:U basepairs with ribonucleotides of the first RNA sequence (E), preferably (i).

[0291] In a further aspect, the present invention provides a precursor RNA molecule comprising; i) a first double-stranded RNA region (B) of a first aspect of the invention, and a second double-stranded RNA region (B) of a second aspect of the invention, ii) a first double-stranded RNA region (B) of a first aspect of the invention, and a second double-stranded RNA region (B) of a third aspect of the invention, iii) a first double -stranded RNA region (B) of a second aspect of the invention, and a second double-stranded RNA region (B) of a third aspect of the invention, iv) a first double-stranded RNA region (B) of a first aspect of the invention, a second double-stranded RNA region (B) of a second aspect of the invention, and a third double-stranded RNA region (B) of a third aspect of the invention, v) a first double-stranded RNA region (B) of a first aspect of the invention, and a second double-stranded RNA region (B) of a first aspect of the invention, vi) a first double-stranded RNA region (B) of a second aspect of the invention, and a second double-stranded RNA region (B) of a second aspect of the invention, vii) a first double-stranded RNA region (B) of a third aspect of the invention, and a second double -stranded RNA region (B) of a third aspect of the invention, or viii) any combination of i) to vii). All combinations of the embodiments of the first, second and third aspects are contemplated here.

[0292] In an embodiment, the precursor RNA molecule comprises two, three, four, five, 10, 15, 20 or more double-stranded RNA regions of a first aspect of the invention, two, three, four, five, 10, 15, 20 or more double-stranded RNA regions of a second aspect of the invention, two, three, four, five, 10, 15, 20 or more double-stranded RNA regions of a third aspect of the invention, or any combination thereof.

[0293] Each of the embodiments of the precursor RNA molecule of the first, second, third, fourth and fifth aspects as described above, and the precursor RNA molecule lacking a linking RNA sequence, are useful for reducing expression and / or activity of a target RNA molecule in a eukaryotic cell, preferably a plant cell, fungal cell or nematode cell, or an arthropod cell such as an insect cell or a decapod cell. They are also useful in reducing expression and / or activity of a viral target RNA molecule, such as for a plant virus, including the specific plant viruses mentioned herein. They are also useful for reducing expression and / or activity of a target RNA molecule in other invertebrate animal cells such as an arthropod cell or insect cell, nematode, or in a non-mammalian vertebrate animal cell. Single or combinations of precursor RNA molecules are also useful in reducing expression and / or activity of multiple target RNA molecules, for example produced from multiple genes. The precursor RNA molecules are useful by way of the processing that produces the product RNA molecules (P), wherein the antisense sequences (J) function with an Argonaute protein in a RISC, the mechanism well known in the art. They may also be useful through enhanced production of secondary antisense sRNA molecules relative to the corresponding conventional RNA molecule. The precursor RNA molecule may be produced in a plant cell to reduce an insect target RNA molecule upon ingestion, or a fungal pathogen or nematode target RNA molecule, or applied topically to a plant or insect to reduce a target RNA molecule.

[0294] In an aspect, the present invention provides a double-stranded RNA molecule (P) consisting of a sense RNA sequence (H) of 21 contiguous ribonucleotides and an antisense RNA sequence (I) of 22 contiguous ribonucleotides and one or two or three bulges, wherein 18 or 19 of ribonucleotides 1 to 19 of the sense RNA sequence (H) each basepair with a ribonucleotide of ribonucleotides 1 to 20 of the antisense RNA sequence (I), wherein each of the one or two or three bulges is immediately flanked by ribonucleotides which are basepaired in the double-stranded RNA molecule, wherein ribonucleotides 20 and 21 of the sense RNA sequence form a 2-ribonucleotide unpaired 3' overhang and ribonucleotides 21 and 22 of the antisense RNA sequence form a 2-ribonucleotide unpaired 3' overhang, wherein ribonucleotides 1 and 2 of the sense RNA sequence basepair with ribonucleotides 19 and 20, respectively, of the antisense RNA sequence, and wherein ribonucleotides 18 and 19 of the sense RNA sequence basepair with ribonucleotides 1 and 2, respectively, of the antisense RNA sequence.

[0295] In an embodiment,

[0296] (i) ribonucleotides 1, 2 and 3 of the sense RNA sequence (H) basepair with ribonucleotides 18, 19 and 20, respectively, of the antisense RNA sequence (I), or

[0297] (ii) ribonucleotides 17, 18 and 19 of the sense RNA sequence (H) basepair with ribonucleotides 1, 2 and 3, respectively, of the antisense RNA sequence (I), or preferably both (i) and (ii). In further features of these embodiments, the product RNA molecules may have at least four, at least five or at least six contiguous basepairs at one or both ends of the double-stranded molecule before any bulge. In these embodiments, any of those basepairs may be G:U basepairs, preferably one or more of ribonucleotides 1, 2 and 3 of the antisense sequence (J) are involved in G:U basepairs, more preferably one or both of ribonucleotides 1 and 2 of the antisense sequence (J) are involved in G:U basepairs, even more preferably ribonucleotide 1 of the antisense sequence (J) is involved in G:U basepairs. Most preferably, one or more of ribonucleotides 1, 2 and 3 of the antisense sequence (J) are U ribonucleotides, or one or both of ribonucleotides 1 and 2 are U ribonucleotides, or ribonucleotide 1 is a U ribonucleotide.

[0298] In an aspect, the present invention provides a double-stranded RNA molecule (P) consisting of a sense RNA sequence (H) of 21 contiguous ribonucleotides and an antisense RNA sequence (J) of 23 contiguous ribonucleotides and one or two or three or four bulges, wherein 18 or 19 of ribonucleotides 1 to 19 of the sense RNA sequence (H) each basepair with a ribonucleotide of ribonucleotides 1 to 21 of the antisense RNA sequence (J), wherein each of the one or two or three or four bulges is immediately flanked by ribonucleotides which are basepaired in the double -stranded RNA molecule, wherein ribonucleotides 20 and 21 of the sense RNA sequence form a 2-ribonucleotide unpaired 3' overhang and ribonucleotides 22 and 23 of the antisense RNA sequence form a 2-ribonucleotide unpaired 3' overhang, wherein ribonucleotides 1 and 2 of the sense RNA sequence basepair with ribonucleotides 20 and 21, respectively, of the antisense RNA sequence, and wherein ribonucleotides 18 and 19 of the sense RNA sequence basepair with ribonucleotides 1 and 2, respectively, of the antisense RNA sequence.

[0299] In an embodiment,

[0300] (i) ribonucleotides 1, 2 and 3 of the sense RNA sequence (H) basepair with ribonucleotides 19, 20 and 21, respectively, of the antisense RNA sequence (J), or

[0301] (ii) ribonucleotides 17, 18 and 19 of the sense RNA sequence (H) basepair with ribonucleotides 1, 2 and 3, respectively, of the antisense RNA sequence (J), or preferably both (i) and (ii). In further features of these embodiments, the product RNA molecules may have at least four, at least five or at least six contiguous basepairs at one or both ends of the double-stranded molecule before any bulge. In these embodiments, any of those basepairs may be G:U basepairs, preferably one or more of ribonucleotides 1, 2 and 3 of the antisense sequence (J) are involved in G:U basepairs, more preferably one or both of ribonucleotides 1 and 2 of the antisense sequence (J) are involved in G:U basepairs, even more preferably ribonucleotide 1 of the antisense sequence (J) is involved in G:U basepairs. Most preferably, one or more of ribonucleotides 1, 2 and 3 of the antisense sequence (J) are U ribonucleotides, or one or both of ribonucleotides 1 and 2 are U ribonucleotides, or ribonucleotide 1 is a U ribonucleotide.

[0302] In an aspect, the present invention provides a double-stranded RNA molecule (P) consisting of a sense RNA sequence (H) of 21 contiguous ribonucleotides and an antisense RNA sequence (J) of 24 contiguous ribonucleotides and one or two or three or four or five bulges, wherein 18 or 19 of ribonucleotides 1 to 19 of the sense RNA sequence each basepair with a ribonucleotide of ribonucleotides 1 to 22 of the antisense RNA sequence (J), wherein each of the one or two or three or four or five bulges is immediately flanked by ribonucleotides which are basepaired in the double -stranded RNA molecule, wherein ribonucleotides 20 and 21 of the sense RNA sequence form a 2-ribonucleotide unpaired 3' overhang and ribonucleotides 23 and 24 of the antisense RNA sequence form a 2-ribonucleotide unpaired 3' overhang, wherein ribonucleotides 1 and 2 of the sense RNA sequence basepair with ribonucleotides 21 and 22, respectively, of the antisense RNA sequence, and wherein ribonucleotides 18 and 19 of the sense RNA sequence basepair with ribonucleotides 1 and 2, respectively, of the antisense RNA sequence.

[0303] In an embodiment,

[0304] (i) ribonucleotides 1, 2 and 3 of the sense RNA sequence (H) basepair with ribonucleotides 20, 21 and 22, respectively, of the antisense RNA sequence (J), or

[0305] (ii) ribonucleotides 17, 18 and 19 of the sense RNA sequence (H) basepair with ribonucleotides 1, 2 and 3, respectively, of the antisense RNA sequence (J), or preferably both (i) and (ii). In further features of these embodiments, the product RNA molecules may have at least four, at least five or at least six contiguous basepairs at one or both ends of the double-stranded molecule before any bulge. In these embodiments, any of those basepairs may be G:U basepairs, preferably one or more of ribonucleotides 1, 2 and 3 of the antisense sequence (J) are involved in G:U basepairs, more preferably one or both of ribonucleotides 1 and 2 of the antisense sequence (J) are involved in G:U basepairs, even more preferably ribonucleotide 1 of the antisense sequence (J) is involved in G:U basepairs. Most preferably, one or more of ribonucleotides 1, 2 and 3 of the antisense sequence (J) are U ribonucleotides, or one or both of ribonucleotides 1 and 2 are U ribonucleotides, or ribonucleotide 1 is a U ribonucleotide.

[0306] In an embodiment, applicable to each of these aspects, the basepairing between ribonucleotides of the sense RNA sequence (H) and ribonucleotides of the antisense RNA sequence (J) comprises one, two, three, four, five or six G:U basepairs. Preferably, all of the G:U basepairs have the G ribonucleotide in the sense RNA sequence (H) and the U ribonucleotide in the antisense RNA sequence (J).

[0307] In an embodiment, applicable to each of these aspects, the basepairing between ribonucleotides of the sense RNA sequence (H) and ribonucleotides of the antisense RNA sequence (J) lacks G:U basepairs, preferably lacks non-canonical basepairs.

[0308] In an embodiment, applicable to all of the above aspects, ribonucleotide 1 of the antisense RNA sequence (J) in at least some of the product RNA molecules (P) is a U ribonucleotide. In an embodiment, ribonucleotide 1 of the antisense RNA sequence (J) in at least some of the product RNA molecules (P) is a U ribonucleotide which is basepaired to a G ribonucleotide in the sense RNA sequence (H).

[0309] In an embodiment, applicable to all of the above aspects, all of at least ribonucleotides 2-8, or 2-10 or 2-11, of the antisense RNA sequence (J) basepair to ribonucleotides in a region (R) of a target RNA molecule in a eukaryotic cell, preferably all of the ribonucleotides of the antisense RNA sequence basepair to ribonucleotides in the region (R) of the target RNA molecule. In a preferred embodiment, all of the ribonucleotides of the antisense RNA sequence (J) basepair to ribonucleotides in the region (R) of the target RNA molecule by canonical basepairs.

[0310] In an embodiment of the fourth or fifth aspects or the embodiments of the asymmetric precursor RNA molecule of the first, second, third, fourth or fifth aspects, the double-stranded RNA molecule consists of a sense RNA sequence of 21 contiguous ribonucleotides and an antisense RNA sequence of 22 contiguous ribonucleotides and one or two or three bulges, wherein 18 or 19 of ribonucleotides 1 to 19 of the sense RNA sequence each basepair with a ribonucleotide of ribonucleotides 1 to 20 of the antisense RNA sequence, wherein the basepairs between the sense RNA sequence and the antisense RNA sequence in each of the double-stranded product RNA molecules comprise, independently, 2, 3, 4, 5, or 6 G:U basepairs, wherein each of the one or two or three bulges is immediately flanked by ribonucleotides which are basepaired in the double -stranded RNA molecule, wherein ribonucleotides 20 and 21 of the sense RNA sequence form a 2- ribonucleotide unpaired 3' overhang and ribonucleotides 21 and 22 of the antisense RNA sequence form a 2-ribonucleotide unpaired 3' overhang, wherein ribonucleotides 1 and 2 of the sense RNA sequence basepair with ribonucleotides 19 and 20, respectively, of the antisense RNA sequence, and wherein ribonucleotides 18 and 19 of the sense RNA sequence basepair with ribonucleotides 1 and 2, respectively, of the antisense RNA sequence. In an embodiment, (i) ribonucleotides 1, 2, and 3 of the sense RNA sequence basepair with ribonucleotides 18, 19 and 20, respectively, of the antisense RNA sequence, or (ii) ribonucleotides 17, 18, and 19 of the sense RNA sequence basepair with ribonucleotides 1, 2, and 3, respectively, of the antisense RNA sequence, or preferably both (i) and (ii).

[0311] In an embodiment of the fourth or fifth aspects or the embodiments of the asymmetric precursor RNA molecule of the first, second, third, fourth, or fifth aspects, the double-stranded RNA molecule consists of a sense RNA sequence of 21 contiguous ribonucleotides and an antisense RNA sequence of 23 contiguous ribonucleotides and one or two or three or four bulges, wherein 18 or 19 of ribonucleotides 1 to 19 of the sense RNA sequence each basepair with a ribonucleotide of ribonucleotides 1 to 21 of the antisense RNA sequence, wherein the basepairs between the sense RNA sequence and the antisense RNA sequence in each of the double-stranded product RNA molecules comprise, independently, 2, 3, 4, 5, or 6 G:U basepairs, wherein each of the one or two or three or four bulges is immediately flanked by ribonucleotides which are basepaired in the double -stranded RNA molecule, wherein ribonucleotides 20 and 21 of the sense RNA sequence form a 2- ribonucleotide unpaired 3' overhang and ribonucleotides 22 and 23 of the antisense RNA sequence form a 2-ribonucleotide unpaired 3' overhang, wherein ribonucleotides 1 and 2 of the sense RNA sequence basepair with ribonucleotides 20 and 21, respectively, of the antisense RNA sequence, and wherein ribonucleotides 18 and 19 of the sense RNA sequence basepair with ribonucleotides 1 and 2, respectively, of the antisense RNA sequence.

[0312] In an embodiment, (i) ribonucleotides 1, 2, and 3 of the sense RNA sequence basepair with ribonucleotides 19, 20, and 21, respectively, of the antisense RNA sequence, or (ii) ribonucleotides 17, 18, and 19 of the sense RNA sequence basepair with ribonucleotides 1, 2 and 3, respectively, of the antisense RNA sequence, or preferably both (i) and (ii).

[0313] In an embodiment of the fourth or fifth aspects or the embodiments of the asymmetric precursor RNA molecule of the first, second, third, fourth, or fifth aspects, the double-stranded RNA molecule consists of a sense RNA sequence of 21 contiguous ribonucleotides and an antisense RNA sequence of 24 contiguous ribonucleotides and one or two or three or four or five bulges, wherein 18 or 19 of ribonucleotides 1 to 19 of the sense RNA sequence each basepair with a ribonucleotide of ribonucleotides 1 to 22 of the antisense RNA sequence, wherein the basepairs between the sense RNA sequence and the antisense RNA sequence in each of the double-stranded product RNA molecules comprise, independently, 2, 3, 4, 5, or 6 G:U basepairs, wherein each of the one or two or three or four or five bulges is immediately flanked by ribonucleotides which are basepaired in the double-stranded RNA molecule, wherein ribonucleotides 20 and 21 of the sense RNA sequence form a 2- ribonucleotide unpaired 3' overhang and ribonucleotides 23 and 24 of the antisense RNA sequence form a 2-ribonucleotide unpaired 3' overhang, wherein ribonucleotides 1 and 2 of the sense RNA sequence basepair with ribonucleotides 21 and 22, respectively, of the antisense RNA sequence, and wherein ribonucleotides 18 and 19 of the sense RNA sequence basepair with ribonucleotides 1 and 2, respectively, of the antisense RNA sequence.

[0314] In an embodiment, (i) ribonucleotides 1, 2 and 3 of the sense RNA sequence basepair with ribonucleotides 20, 21, and 22, respectively, of the antisense RNA sequence, or (ii) ribonucleotides 17, 18, and 19 of the sense RNA sequence basepair with ribonucleotides 1, 2, and 3, respectively, of the antisense RNA sequence, or preferably both (i) and (ii).

[0315] In an embodiment, ribonucleotide 1 of the antisense RNA sequence is a U ribonucleotide. In an embodiment, ribonucleotide 1 of the antisense RNA sequence is a U ribonucleotide which is basepaired to a G ribonucleotide in the sense RNA sequence.

[0316] In an embodiment, the double-stranded RNA molecule (P) has one or more of any of the features defined herein.

[0317] As exemplified herein, it is possible to change the numbers of siRNA molecules in a eukaryotic cell comprising an antisense RNA sequence (J) of 22, 23 or 24 ribonucleotides relative to 21 ribonucleotides by the A22, A23 or A24 modifications in a precursor RNA molecule, relative to a corresponding conventional precursor RNA molecule without the modifications, especially along the full length of a double-stranded region (B) comprising a second RNA strand (F) of at least 100 ribonucleotides in length. Therefore, in an aspect, the present invention provides a population of multiple, different double-stranded RNA molecules (P) of the invention. In another aspect, the present invention provides a population of multiple, different double-stranded RNA molecules comprising double-stranded RNA molecules of the invention, or any combination thereof.

[0318] In an embodiment, the population of multiple, different double-stranded RNA molecules (P) comprises at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, or at least 100 different product RNA molecules (P) which each independently comprise:

[0319] (i) an antisense RNA sequence (J) of 22 contiguous ribonucleotides and one or two or three bulges,

[0320] (ii) an antisense RNA sequence (J) of 23 contiguous ribonucleotides and one or two or three or four bulges,

[0321] (iii) an antisense RNA sequence (J) of 24 contiguous ribonucleotides and one or two or three or four or five bulges, or

[0322] (iv) any combination of (i), (ii) and (iii).

[0323] In an embodiment, the population of multiple, different double -stranded RNA molecules (P) comprises (i) or (ii) above.

[0324] In an embodiment,

[0325] (i) more antisense RNA sequences (J) in the population consist of 22 ribonucleotides than consist of 21 ribonucleotides,

[0326] (ii) more antisense RNA sequences (J) in the population consist of 23 ribonucleotides than consist of 21 ribonucleotides,

[0327] (iii) more antisense RNA sequences (J) in the population consist of 24 ribonucleotides than consist of 21 ribonucleotides, or

[0328] (iv) any combination of (i), (ii) and (iii).

[0329] In an embodiment of the first, second, third, fourth or fifth aspects or the embodiments of the asymmetric precursor RNA molecule, the present invention provides a population of multiple, different double-stranded RNA molecules of the invention. In an embodiment, the population of multiple, different double-stranded RNA molecules comprises at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, or at least 100 different product RNA molecules which each independently comprise:

[0330] (i) an antisense RNA sequence of 22 contiguous ribonucleotides and one or two or three bulges,

[0331] (ii) an antisense RNA sequence of 23 contiguous ribonucleotides and one or two or three or four bulges,

[0332] (iii) an antisense RNA sequence of 24 contiguous ribonucleotides and one or two or three or four or five bulges, or

[0333] (iv) any combination of (i), (ii) and (iii).

[0334] In an embodiment, the population of multiple, different double -stranded RNA molecules comprise the following features: (i) more antisense RNA sequences in the population consist of 22 ribonucleotides than consist of 21 ribonucleotides,

[0335] (ii) more antisense RNA sequences in the population consist of 23 ribonucleotides than consist of 21 ribonucleotides,

[0336] (iii) more antisense RNA sequences in the population consist of 24 ribonucleotides than consist of 21 ribonucleotides, or

[0337] (iv) any combination of (i), (ii) and (iii).

[0338] In an embodiment of the first, second, third, fourth or fifth aspects or the embodiments of the asymmetric precursor RNA molecule, the invention provides a method of increasing the amount of small interfering RNAs (sRNAs) of 22, 23 or 24 ribonucleotides in length in an insect cell or insect, or a fungal cell or fungus, or of increasing the ratio of the amount of sRNAs of 22, 23 or 24 ribonucleotides relative to the amount of sRNAs of 21 ribonucleotides in length, the method comprising delivering to the eukaryotic cell such as a plant cell, nematode cell, insect cell or fungal cell, insect, or fungus one or more or all of the precursor RNA molecule, the double-stranded RNA molecule, the population of multiple, different double-stranded RNA molecules, the polynucleotide, the vector, the cell, the non-human organism or part thereof, the extract, and the composition of the invention.

[0339] In an aspect, the present invention provides a eukaryotic cell comprising the double-stranded RNA molecule (P) of the invention.

[0340] In an aspect, the present invention provides a eukaryotic cell comprising the population of multiple, different double-stranded RNA molecules (P) of the invention.

[0341] In an embodiment of the first, second, third, fourth or fifth aspects or the embodiments of the asymmetric precursor RNA molecule, the present invention provides a eukaryotic cell such as a plant cell, nematode cell, insect cell or fungal cell, comprising one or more or all of the precursor RNA molecules, the double-stranded RNA molecules, and the population of multiple, different double-stranded RNA molecules of the invention, wherein the eukaryotic cell is preferably a plant cell, insect cell, fungal cell such as a yeast cell.

[0342] In an embodiment, the eukaryotic cell comprises one or both of the precursor RNA molecule, and the population of multiple, different double-stranded RNA molecules of the invention.

[0343] In an aspect, the present invention provides a double-stranded RNA molecule (P) obtainable by, or obtained from, the cleavage of the precursor RNA molecule of the invention, wherein the double-stranded RNA molecule has one or more of any of the features defined herein. In an aspect, the present invention provides a double-stranded RNA molecule (P) obtainable by, or obtained from, the cleavage of the precursor RNA molecule of the invention.

[0344] In an aspect, the present invention provides a method of identifying a doublestranded RNA molecule (P), or a precursor RNA molecule (A), for reducing the amount and / or activity of a target RNA molecule of interest, the method comprising: i) producing a precursor RNA molecule (A) and / or a double-stranded RNA molecule of the invention, or a population of multiple, different precursor RNA molecules (A) and / or multiple, different double-stranded RNA molecules of the invention, ii) determining the ability of the precursor RNA molecule or double-stranded RNA molecule, or members of the population of multiple, different precursor RNA molecules or multiple, different double-stranded RNA molecules, to reduce the amount and / or activity of a target RNA molecule of interest. The RNA molecules of the invention can thereby by used in screening assays to identify a suitable precursor RNA molecule that down-regulates a known target RNA molecule, or to identify a target RNA molecule associated with a suitable phenotype or function in the eukaryotic cell.

[0345] In an embodiment, step i) comprises expressing a precursor RNA molecule (A) of the invention in a eukaryotic cell, wherein the precursor RNA molecule is cleaved in the eukaryotic cell by one or more ribonucleases (RNases) to produce the doublestranded RNA molecule or the population of multiple, different double-stranded RNA molecules.

[0346] In an embodiment, the method further comprises designing the double-stranded RNA molecule based on a region (R) of the ribonucleotide sequence of the target RNA molecule of interest, preferably using the features defined herein.

[0347] In an embodiment, the method further comprises a step of producing more of the double-stranded RNA molecule or the precursor RNA molecule after step ii), for example in commercial quantities of for a kit comprising the double-stranded RNA molecule or the precursor RNA molecule.

[0348] In an embodiment for the precursor RNA molecules of the fourth or fifth aspects or the embodiments of the asymmetric precursor RNA molecule, the invention provides a method of identifying a double-stranded RNA molecule, or a precursor RNA molecule, for reducing the amount and / or activity of a target RNA molecule of interest in an insect cell or a fungal cell, the method comprising: i) producing a precursor RNA molecule, and / or a double-stranded RNA molecule, or a population of multiple, different precursor RNA molecules of the invention, and ii) determining the ability of the precursor RNA molecule or double-stranded RNA molecule, or members of the population of multiple, different precursor RNA molecules, or the population of multiple, different double-stranded RNA molecules, to reduce the amount and / or activity of the target RNA molecule of interest in the insect or fungal cell.

[0349] In an embodiment, step i) comprises introducing a precursor RNA molecule of the invention into: (i) an insect cell, preferably by ingestion, soaking, dusting, spraying or injection of an insect comprising the insect cell; and / or (ii) a fungal cell, preferably by topical application such as soaking, dusting, spraying or applying a composition comprising the precursor RNA molecule to the fungal cell; wherein the precursor RNA molecule is cleaved in the insect cell or fungal cell by a Dicer to produce the doublestranded RNA molecule or the population of multiple, different double-stranded RNA molecules.

[0350] In an embodiment, step i) comprises introducing a precursor RNA molecule of the invention into a plant cell or microbial cell such as a bacterial cell or yeast cell, and step (ii) comprises delivering the plant cell or microbial cell of step (i) to the insect cell or the fungal cell, preferably by ingestion of the plant cell or microbial cell, wherein the precursor RNA molecule is cleaved in the insect cell or fungal cell by a Dicer to produce the double-stranded RNA molecule or the population of multiple, different doublestranded RNA molecules. In an embodiment, the method further comprises a step of producing more of the double-stranded RNA molecule or the precursor RNA molecule after step ii).

[0351] Also provided is a double-stranded RNA molecule (P) or a precursor RNA molecule (A) identified or produced using a method of the invention.

[0352] In an aspect, the present invention provides an isolated and / or exogenous polynucleotide encoding the precursor RNA molecule of the invention.

[0353] In an embodiment, the polynucleotide is a DNA construct such as a chimeric DNA construct. Alternatively, the polynucleotide is a RNA construct, for example a RNA construct based on a viral RNA genome.

[0354] In an embodiment, the polynucleotide is operably linked to a promoter capable of directing expression of the precursor RNA molecule in a host cell, preferably a eukaryotic cell such as a plant cell, and optionally a polyadenylation region / transcription terminator or a transcription termination sequence. In an embodiment, the promoter is heterologous to the target RNA molecule. In an embodiment, the promoter is as an RNA polymerase III promoter, an RNA polymerase II promoter, or a promoter which functions in an in vitro transcription reaction.

[0355] In an aspect, the present invention provides a vector comprising a polynucleotide of the invention.

[0356] In an embodiment, the vector is a viral vector, such as a DNA viral vector or an RNA viral vector or a plasmid.

[0357] In an aspect, the present invention provides a host cell comprising one or more or all of the precursor RNA molecule of the invention, the double-stranded RNA molecule of the invention, the population of multiple, different double-stranded RNA molecules of the invention, the polynucleotide of the invention, or the vector of the invention.

[0358] In an embodiment, the host cell comprises a population of two, three, four, five, 10, 15, 20, 50, 100 or more different, double-stranded RNA molecules of the invention.

[0359] In an embodiment, the host cell is a eukaryotic cell. In an embodiment, the host cell is a micro-organism, for example a bacterial cell such as an E. coli cell, or a yeast cell such as, for example, Saccharomyces cerevisiae. In an embodiment, the microorganism has been modified to reduce catabolism of double-stranded RNA molecules or to enhance their accumulation. In an embodiment, the host cell is a non-human host cell, or a cell in cell culture or in vitro, or a cell in a non-human organism.

[0360] In a further aspect, the present invention provides eukaryotic cell comprising one or more or all of a precursor RNA molecule of the invention, a polynucleotide encoding the precursor RNA molecule, the double-stranded RNA molecule (P) of the invention, and the population of multiple, different double-stranded RNA molecules (P) of the invention, preferably wherein the cell is a non-human cell or a eukaryotic cell in vitro.

[0361] In an embodiment, the host cell is a plant cell, a fungal cell, or an animal cell, preferably a plant cell, an arthropod cell such as an insect, arachnid, or decapod cell, a nematode cell or a fungal cell. In an embodiment, the plant cell is wild-type for a gene encoding a DCL4 protein and / or a gene encoding a DCL2 protein. In an embodiment, the plant cell is other than a Nicotiana benthamiana or Nicotiana tabcicum cell. In an embodiment, the host cell is a fungal cell or an animal cell which is wild-type for Dicer protein(s).

[0362] In an embodiment, applicable to all of the embodiments of the above aspects, the double-stranded RNA molecule and / or the precursor RNA molecule and / or the population of multiple, different double-stranded RNA molecules do not naturally occur in the cell. In an embodiment, applicable to all of the embodiments of the above aspects, the host cell is dead and / or incapable of reproduction. For example, the dead or inactivated host cell is a microbial cell. Alternatively, the dead or inactivated cell is a plant cell, or of an insect pest.

[0363] In an aspect, the present invention provides a non-human organism, or a part thereof, comprising a cell of the invention.

[0364] In an embodiment, the non-human organism or part thereof is a transgenic non- human organism or part thereof, being transgenic for a polynucleotide of the invention, preferably a transgenic plant or part thereof.

[0365] In an embodiment, the polynucleotide is stably integrated into the genome of the organism or part thereof, preferably into the nuclear genome of the organism or part thereof, for example the non-human eukaryotic organism such as a plant or yeast. Alternatively, the polynucleotide is not integrated into the genome of the organism or part thereof, but is expressed in the organism or part thereof, for example transiently.

[0366] In an embodiment, the plant is transgenic for the polynucleotide which is stably integrated into the genome of the plant, wherein the polynucleotide encodes a precursor RNA molecule of the first, second, third, fourth or fifth aspects, or an embodiment of the asymmetric RNA molecules, wherein the precursor RNA molecule and / or at least some of the antisense RNA sequences in the double -stranded product RNA molecules produced from the precursor RNA molecules are capable of reducing the expression and / or activity of a target RNA molecule in the insect, such as for example a Lepidopteran insect, when ingested or in a fungal pathogen. In an embodiment, the transgenic plant has increased resistance to the insect or to the fungal pathogen relative to a corresponding plant lacking the polynucleotide. For example, at least some of the insect larvae are killed after ingesting some of the transgenic plant cells expressing the precursor RNA molecules. In an embodiment, the transgenic plant is a cotton plant and the insect is of the genus Helicoverpa, or a maize or sorghum or rice plant and the insect is of the genus Spodoptera. In an embodiment, the fungal pathogen is of the genus Fusarium or Verticillium.

[0367] In an embodiment of the fourth or fifth aspects or the embodiments of the asymmetric precursor RNA molecule, the invention provides a non-human organism, or a part thereof, preferably a plant or part thereof, or an insect or part thereof, or a fungus or part thereof, comprising one or more or all of the precursor RNA molecule, the doublestranded RNA molecule, the population of multiple, different double-stranded RNA molecules, the polynucleotide, the vector, or the host cell of the invention. In an embodiment, the non-human organism or part thereof is a transgenic plant or part thereof, being transgenic for a polynucleotide of the invention, preferably wherein the polynucleotide is stably integrated into the genome of the plant or part thereof.

[0368] In an aspect, the present invention provides a method of producing the host cell of the invention, the method comprising introducing into a cell one or more or all of the precursor RNA molecule of the invention, the double-stranded RNA molecule of the invention, the population of multiple, different double-stranded RNA molecules of the invention, the polynucleotide of the invention, or the vector of the invention. In a preferred embodiment, if the host cell is an animal cell, the step of introducing said molecules occurs ex vivo. For example, the introducing step occurs in vitro. The introducing step may be followed by a step of culturing or propagating the molecules into which the molecules were introduced, which may comprise a step of selecting a transformed cell and / or identifying a cell or progeny cell comprising the molecule(s). Progeny cells may be assayed to identify cells having a suitable phenotype.

[0369] In an aspect, the present invention provides a method of producing a non-human organism of the invention, the method comprising introducing one or more or all of the precursor RNA molecule of the invention, the double-stranded RNA molecule of the invention, the population of multiple, different double-stranded RNA molecules of the invention, the polynucleotide of the invention, or the vector of the invention, into a cell and generating the non-human organism from the cell. In an embodiment, where the non- human organism is a plant, the step of generating the non-human organism from the cell comprises regenerating a transgenic plant from the cell. The step of introducing a polynucleotide of the invention may be followed by a step of selecting or identifying a cell or progeny cell comprising the polynucleotide.

[0370] In an embodiment, the polynucleotide of the invention is stably integrated into the genome of the cell or the organism, preferably into the nuclear genome of the organism or part thereof, preferably a plant.

[0371] In an aspect, the present invention provides a method of producing the precursor RNA molecule of the invention, the double-stranded RNA molecule of the invention, and / or the population of multiple, different double-stranded RNA molecules of the invention, the method comprising expressing the polynucleotide of the invention in a host cell or cell-free expression system. In an embodiment, the host cell is a eukaryotic cell, preferably a plant cell or a yeast cell. In an embodiment, the host cell is a cell that has been modified to reduce dsRNA degradation, for example by removal of a Type III ribonuclease.

[0372] In an embodiment, the method further comprises extracting and / or at least partially purifying some of the precursor RNA molecule of the invention or double- stranded RNA molecule of the invention or the population of multiple, different doublestranded RNA molecules of the invention. In an embodiment, the method does not comprise a step of extracting the RNA molecules from the host cell. In an embodiment, the host cell is inactivated or killed after the precursor RNA is produced, for example by heat treatment.

[0373] In an aspect, the present invention provides an extract of a cell of the invention, wherein the extract comprises one or more or all of the precursor RNA molecule of the invention, the double-stranded RNA molecule of the invention, the population of multiple, different double-stranded RNA molecules of the invention, the polynucleotide of the invention, or the vector of the invention. In an embodiment, the extract comprises the precursor RNA molecule of the invention but not the double-stranded RNA molecules of the invention or the population of multiple, different double -stranded RNA molecules of the invention. In an embodiment, the extract of the cell is purified to remove one or more impurities and / or concentrate the RNA molecules.

[0374] In an aspect, the present invention provides a composition comprising one or more or all of the precursor RNA molecule of the invention, the double-stranded RNA molecule of the invention, the population of double-stranded RNA molecules of the invention, the polynucleotide of the invention, the vector of the invention, the cell of the invention, the non-human organism or part thereof of the invention, or the extract of the invention. In an embodiment, the composition comprises the precursor RNA molecule of the invention but not the double-stranded RNA molecules of the invention or the population of multiple, different double-stranded RNA molecules of the invention. Alternatively, the composition comprises the double-stranded RNA molecules of the invention or the population of multiple, different double-stranded RNA molecules of the invention but not the precursor RNA molecule. In an embodiment, the extract of the cell is purified to remove one or more impurities and / or concentrate the RNA molecules.

[0375] In an embodiment, the extract or the composition comprises a population of two, three, four, five, 10, 15, 20, 50, or 100 or more double-stranded RNA molecules of the invention.

[0376] In an embodiment, the composition is a pharmaceutical composition.

[0377] In an embodiment, the composition is suitable for application to plants growing in a field, such as by spraying or dusting onto plants. For example, the composition comprises a surfactant.

[0378] In an embodiment, the composition further comprises at least one compound which enhances the stability, or entry into a eukaryotic cell or both, of the precursor RNA molecule, the double -stranded RNA molecule, population of double-stranded RNA molecules, the polynucleotide, the vector, the host cell, the non-human organism or part thereof, or the extract. An example of such a compound is a transfection promoting agent such as, for example, a detergent.

[0379] In an aspect, the present invention provides a method for increasing the number of double-stranded RNA molecules of the invention in a eukaryotic cell or organism, comprising expressing in the cell or organism a polynucleotide of the invention or a vector of the invention, or contacting the cell or organism with the double-stranded RNA molecule of the invention.

[0380] In an aspect, the invention provides a method for increasing the total number of sRNA molecules produced in a eukaryotic cell or organism, preferably 22-mers, 23-mers and / or 24-mers, preferably in a plant, fungus, nematode, or an arthropod such as an insect, arachnid, or decapod, the method comprising delivering to the cell or organism one or more or all of the precursor RNA molecule of the invention, the double-stranded RNA molecule of the invention, the population of double-stranded RNA molecules of the invention, the polynucleotide of the invention, the vector of the invention, the cell of the invention, the non-human organism or part thereof of the invention, the extract of the invention, or the composition of the invention. In an embodiment, the total number sRNA molecules in the cell or organism is assayed. Alternatively, the total number sRNA molecules in the cell or organism is not assayed directly, but a suitable phenotype is observed. Preferably, the number of antisense sRNAs that hybridise to a target RNA molecule of interest is increased. In a related aspect, the invention provides a method for modifying the ratio of sRNA molecules of the invention in a eukaryotic cell or organism, relative to the total number of 21-mer sRNAs, preferably increasing the ratio of 22-mers, 23-mers and / or 24-mers relative to the total number of 21-mer sRNAs, more preferably with regard to a specific target RNA molecule, especially in a plant, fungus or nematode, or an arthropod such as an insect, arachnid, or decapod.

[0381] In an embodiment for the precursor RNA molecules of the fourth or fifth aspects or the embodiments of the asymmetric precursor RNA molecule, the invention provides a method for increasing the number of double-stranded RNA molecules of the invention in an insect or an insect cell, or a fungal cell or fungus, comprising expressing in the insect or an insect cell, or fungal cell or fungus a polynucleotide or a vector of the invention, or contacting the insect or an insect cell, or fungal cell or fungus with the double-stranded RNA molecule or the population of double-stranded RNA molecules of the invention.

[0382] In an aspect, the invention provides a method for identifying a phenotype or function associated with a target RNA molecule in a eukaryotic cell or organism, the method comprising (i) delivering to the cell or organism, one or more or all of: the precursor RNA molecule of the invention, the double-stranded RNA molecule of the invention, the population of multiple, different double-stranded RNA molecules of the invention, the polynucleotide of the invention, the vector of the invention, the extract of the invention, or the composition of the invention, and (ii) observing the cell or organism, or a progeny cell or organism thereof, for the phenotype or function, or assaying the cell or organism, or a progeny cell or organism thereof, for a molecule associated with the phenotype or function, thereby identifying the phenotype or function associated with a target RNA.

[0383] In an embodiment for the precursor RNA molecules of the fourth or fifth aspects or the embodiments of the asymmetric precursor RNA molecule, the invention provides a method for identifying a function or phenotype associated with a target RNA molecule in an insect or insect cell, or a fungal cell or fungus, the method comprising (i) delivering to the insect or insect cell, or fungal cell or fungus, one or more or all of: the precursor RNA molecule, the double-stranded RNA molecule, the population of multiple, different double-stranded RNA molecules, the polynucleotide, the vector, the cell, the non-human organism or part thereof, the extract, and the composition of the invention, and (ii) determining a function or phenotype of the insect or insect cell, or fungal cell or organism, or a progeny insect or fungal cell or progeny insect or progeny fungus thereof, or assaying the insect or insect cell, or fungal cell or fungus, or a progeny insect or insect cell, or a progeny fungal cell or fungus thereof, for a molecule associated with the function or phenotype, thereby identifying the function or phenotype associated with a target RNA.

[0384] In an aspect, the invention provides a method for identifying a region of a target RNA molecule in a eukaryotic cell or organism that is susceptible to down-regulation by RNAi, the method comprising (i) delivering to the cell or organism one or more or all of: multiple precursor RNA molecules of the invention, multiple double-stranded RNA molecules of the invention, populations of multiple, different double-stranded RNA molecules of the invention, polynucleotides of the invention, vectors of the invention, extracts of the invention, or compositions of the invention, wherein the multiple precursor RNA molecules, double-stranded RNA molecules or populations of multiple double-stranded RNA molecules target different regions of the target RNA molecule, and (ii) assaying the cell or organism, or a progeny cell or organism thereof, for one or more of: the amount of target RNA molecule, the amount of protein encoded by the target RNA molecule, and / or for a phenotype or function associated with the target RNA molecule, and (iii) selecting a region of the target RNA molecule based on assay results from step (ii), thereby identifying the region.

[0385] In an embodiment for the precursor RNA molecules of the fourth or fifth aspects or the embodiments of the asymmetric precursor RNA molecule, the invention provides a method for identifying a region of a target RNA molecule in an insect cell or insect, or a fungal cell or fungus that is susceptible to down-regulation by RNAi, the method comprising (i) delivering to the cell or organism one or more or all of: multiple precursor RNA molecules, multiple double -stranded RNA molecules, populations of multiple, different double-stranded RNA molecules, polynucleotides, vectors, extracts, or compositions of the invention, wherein the multiple precursor RNA molecules, doublestranded RNA molecules or populations of multiple double-stranded RNA molecules target different regions of the target RNA molecule, and (ii) assaying the insect cell or insect, or fungal cell or fungus, or a progeny insect cell or insect, or a progeny fungal cell or fungus thereof, for one or more of: the amount of target RNA molecule, the amount of protein encoded by the target RNA molecule, and / or for a function or phenotype associated with the target RNA molecule, and (iii) selecting a region of the target RNA molecule based on assay results from step (ii), thereby identifying the region.

[0386] In an aspect, the invention provides a method for identifying a RNA molecule that is capable of having an effect on a pest or pathogen of a eukaryotic cell or organism, the method comprising (i) delivering to the eukaryotic cell or organism, one or more or all of the precursor RNA molecule of the invention, the double-stranded RNA molecule of the invention, the population of different double-stranded RNA molecules of the invention, the polynucleotide of the invention, the vector of the invention, the extract of the invention, or the composition of the invention, (ii) contacting the cell or organism of step (i), or a progeny cell or organism thereof, with the pest or pathogen, (iii) determining whether or not the precursor RNA molecule, double-stranded RNA molecule or population of different double-stranded RNA molecules has an effect on the pest or pathogen, and optionally (iv) if the precursor RNA molecule, double-stranded RNA molecule or population of different double-stranded RNA molecules has a desirable effect on the pest or pathogen, selecting an RNA molecule based on results from step (iii), thereby identifying the RNA molecule.

[0387] In an embodiment for the precursor RNA molecules of the fourth or fifth aspects or the embodiments of the asymmetric precursor RNA molecule, the invention provides a method for identifying an RNA molecule that is capable of having an effect on an insect pest, or a fungal pathogen, the method comprising (i) delivering to the insect pest or fungal pathogen, one or more or all of the precursor RNA molecule, the double-stranded I l l

[0388] RNA molecule, the population of multiple, different double-stranded RNA molecules, the polynucleotide, the vector, the cell, the non-human organism or part thereof, the extract, and the composition of the invention, and (ii) determining whether or not the precursor RNA molecule, double-stranded RNA molecule or population of different double-stranded RNA molecules has an effect on the insect pest or fungal pathogen, and optionally (iii) if the precursor RNA molecule, double -stranded RNA molecule or population of different double-stranded RNA molecules has a desirable effect on the insect pest or fungal pathogen, selecting an RNA molecule based on results from step (ii), thereby identifying the RNA molecule.

[0389] In an aspect, the invention provides a method for identifying a RNA molecule that is capable of having an effect on a pest or pathogen of a eukaryotic cell or organism, the method comprising (i) delivering to the pest or pathogen, one or more or all of the precursor RNA molecule of the invention, the double-stranded RNA molecule of the invention, the population of multiple, different double-stranded RNA molecules of the invention, the extract of the invention, or the composition of the invention, (ii) testing the pest or pathogen for an effect of the precursor RNA molecule, double-stranded RNA molecule or population of double-stranded RNA molecules, and optionally (iii) selecting a RNA molecule based on results from step (ii), thereby identifying the RNA molecule.

[0390] In an aspect, the present invention provides a method for reducing or downregulating the level and / or activity of a target RNA molecule in a eukaryotic cell or organism, the method comprising delivering to the cell or organism one or more or all of the precursor RNA molecule of the invention, the double-stranded RNA molecule of the invention, the population of double-stranded RNA molecules of the invention, the polynucleotide of the invention, the vector of the invention, the cell of the invention, the non-human organism or part thereof of the invention, the extract of the invention, or the composition of the invention. In an embodiment, the cell or organism is not an animal cell or organism, preferably the cell or organism is a plant or fungal cell or organism. In an embodiment, the cell is a nematode cell such as a plant-pathogenic nematode (PPN) cell, or a nematode, or PPN.

[0391] In an embodiment, the method reduces or down-regulates at least two, at least three, at least four, at least five, or at least six different target RNA molecules in a eukaryotic cell using either a single precursor RNA molecule, which may have chimeric target sequences in its dsRNA regio...

Claims

CLAIMS1. A precursor RNA molecule (A) comprising at least one double -stranded RNA region (B), wherein:(i) the double-stranded RNA region (B) comprises:(a) a first RNA strand (D) of at least 23 contiguous ribonucleotides which comprises a first RNA sequence (E) of at least 21 contiguous ribonucleotides, and(b) a second RNA strand (F) of at least 24 contiguous ribonucleotides which comprises a second RNA sequence (G) of at least 22 contiguous ribonucleotides, wherein the first RNA strand (D) and second RNA strand (F) are covalently linked by a linking RNA sequence (L), wherein the first RNA sequence (E) is hybridised to the second RNA sequence (G) by basepairing between at least 20 of the at least 21 contiguous ribonucleotides of the first RNA sequence (E) and at least 20 of the at least 22 contiguous ribonucleotides of the second RNA sequence (G), forming at least a part (C) of the double-stranded RNA region (B), wherein 1 or 2 ribonucleotides of the at least 22 ribonucleotides of (G) are nonbasepaired, and 0 or 1, respectively, ribonucleotides of the at least 21 ribonucleotides of (E) are non-basepaired in the part (C) of the double-stranded RNA region (B), forming one or two or three bulges in the part (C) of the double-stranded RNA region (B), wherein each of the one or two or three bulges is immediately flanked by ribonucleotides which are basepaired in the part (C) of the double-stranded RNA region (B); and(ii) the precursor RNA molecule (A) is capable of being cleaved in a eukaryotic cell by one or more ribonucleases (RNases) to produce one or more double-stranded product RNA molecule(s) (P) consisting of a sense RNA sequence (H) of 21 contiguous ribonucleotides from the first RNA sequence (E) and an antisense RNA sequence (J) of 22 contiguous ribonucleotides from the second RNA strand (F), including at least 20 contiguous ribonucleotides from (G), wherein the one or more double-stranded product RNA molecule(s) (P) comprise the one or two or three bulges, wherein 18 or 19 of ribonucleotides 1 to 19 of the sense RNA sequence (H) each basepair with a ribonucleotide of ribonucleotides 1 to 20 of antisense RNA sequence (J) in the one or more double-stranded product RNA molecule(s) (P),wherein ribonucleotides 20 and 21 of the sense RNA sequence (H) form a 2- ribonucleotide unpaired 3' overhang and ribonucleotides 21 and 22 of the antisense RNA sequence (J) form a 2-ribonucleotide unpaired 3' overhang in the one or more doublestranded product RNA molecule(s) (P), wherein ribonucleotides 1 and 2 of the sense RNA sequence (H) basepair with ribonucleotides 19 and 20, respectively, of the antisense RNA sequence (J) in the one or more double-stranded product RNA molecule(s) (P), and wherein ribonucleotides 18 and 19 of the sense RNA sequence (H) basepair with ribonucleotides 1 and 2, respectively, of the antisense RNA sequence (J) in the one or more double -stranded product RNA molecule(s) (P).

2. The precursor RNA molecule of claim 1, wherein the first RNA sequence (E) is hybridised to the second RNA sequence (G) by basepairing between at least 21 of the at least 21 contiguous ribonucleotides of the first RNA sequence (E) and at least 21 of the at least 22 contiguous ribonucleotides of the second RNA sequence (G), wherein 1 ribonucleotide of the at least 22 ribonucleotides of (G) is non-basepaired and all of the at least 21 ribonucleotides of (E) are basepaired in the part (C) of the doublestranded RNA region (B), the 1 non-basepaired ribonucleotide forming a single nucleotide bulge in the part (C) of the double-stranded RNA region (B), wherein the bulge is immediately flanked by ribonucleotides which are basepaired in the part (C) of the double-stranded RNA region (B), wherein the one or more double-stranded product RNA molecule(s) (P) comprise the bulge, and wherein ribonucleotides 1 to 19 of the sense RNA sequence (H) each basepair with a ribonucleotide of ribonucleotides 1 to 20 of antisense RNA sequence (J) in the one or more double-stranded product RNA molecule(s) (P).

3. The precursor RNA molecule of claim 1 or claim 2, wherein the first RNA sequence (E) comprises at least 44 contiguous ribonucleotides and the second RNA sequence (G) comprises at least 46 contiguous ribonucleotides, wherein the first RNA sequence (E) is hybridised to the second RNA sequence (G) by basepairing between at least 42 of the at least 44 contiguous ribonucleotides of the first RNA sequence (E) and at least 42 of the at least 46 contiguous ribonucleotides of the second RNA sequence (G),wherein 2, 3 or 4 ribonucleotides of the at least 46 contiguous ribonucleotides of (G) are non-basepaired and 0, 1 or 2, respectively, ribonucleotides of the at least 44 contiguous ribonucleotides of (E) are non-basepaired in the part (C) of the doublestranded RNA region (B), forming bulges in the part (C) of the double-stranded RNA region (B), wherein each of the bulges is immediately flanked by ribonucleotides which are basepaired in the part (C) of the double-stranded RNA region (B), wherein the precursor RNA molecule is capable of being cleaved in a eukaryotic cell by one or more ribonucleases (RNases) to produce multiple, different doublestranded product RNA molecules (P) consisting of a sense RNA sequence (H) of 21 contiguous ribonucleotides and an antisense RNA sequence (J) of 22 contiguous ribonucleotides, wherein the multiple, different double-stranded product RNA molecule(s) (P) each comprise at least one of the bulges, and wherein at least some of the multiple, different double -stranded product RNA molecules (P) have non-overlapping antisense RNA sequences (J), preferably adjacent non-overlapping antisense RNA sequences (J).

4. The precursor RNA molecule according to any one of claims 1 to 3, wherein(i) ribonucleotides 1, 2 and 3 of the sense RNA sequence (H) basepair with ribonucleotides 18, 19 and 20, respectively, of the antisense RNA sequence (J) in at least some of the one or more double-stranded product RNA molecule(s) (P), or(ii) ribonucleotides 17, 18 and 19 of the sense RNA sequence (H) basepair with ribonucleotides 1, 2 and 3, respectively, of the antisense RNA sequence (J) in at least some of the one or more double-stranded product RNA molecule(s) (P), or preferably both (i) and (ii).

5. The precursor RNA molecule according to any one of claims 1 to 4, wherein the molecule is capable of being cleaved in a eukaryotic cell by one or more ribonucleases (RNases) to produce multiple, different double-stranded product RNA molecules (P) which each consist of a sense RNA sequence (H) of 21 contiguous ribonucleotides from the first RNA sequence (E) and an antisense RNA sequence (J) of 22 contiguous ribonucleotides from the second RNA sequence (G), wherein at least some of the multiple, different double -stranded product RNA molecules (P) have overlapping antisense RNA sequences (J), or non-overlapping antisense RNA sequences (J), preferably adjacent non-overlapping antisense RNA sequences (J).

6. A precursor RNA molecule (A) comprising at least one double-stranded RNA region (B), wherein:(i) the double-stranded RNA region (B) comprises:(a) a first RNA strand (D) of at least 23 contiguous ribonucleotides which comprises a first RNA sequence (E) of at least 21 contiguous ribonucleotides, and(b) a second RNA strand (F) of at least 25 contiguous ribonucleotides which comprises a second RNA sequence (G) of at least 23 contiguous ribonucleotides, wherein the first RNA strand (D) and second RNA strand (F) are covalently linked by a linking RNA sequence (L), wherein the first RNA sequence (E) is hybridised to the second RNA sequence (G) by basepairing between at least 20 of the at least 21 contiguous ribonucleotides of the first RNA sequence (E) and at least 20 of the at least 23 contiguous ribonucleotides of the second RNA sequence (G), forming at least a part (C) of the double-stranded RNA region (B), wherein 2 or 3 ribonucleotides of the at least 23 ribonucleotides of (G) are non-basepaired and 0 or 1, respectively, ribonucleotides of the at least 21 ribonucleotides of (E) are non- basepaired in the part (C) of the double-stranded RNA region (B), forming one, two, three or four bulges in the part (C) of the double-stranded RNA region (B), wherein each of the one, two, three or four bulges is immediately flanked by ribonucleotides which are basepaired in the part (C) of the double-stranded RNA region (B); and(ii) the precursor RNA molecule (A) is capable of being cleaved in a eukaryotic cell by one or more ribonucleases (RNases) to produce one or more double-stranded product RNA molecule(s) (P) consisting of a sense RNA sequence (H) of 21 contiguous ribonucleotides from the first RNA sequence (E) and an antisense RNA sequence (J) of 23 contiguous ribonucleotides from the second RNA strand (F), including at least 21 contiguous ribonucleotides from (G), wherein the one or more double-stranded product RNA molecule(s) (P) comprise the one, two, three or four bulges, wherein 18 or 19 of ribonucleotides 1 to 19 of the sense RNA sequence (H) each basepair with a ribonucleotide of ribonucleotides 1 to 21 of antisense RNA sequence (J) in the one or more double-stranded product RNA molecule(s) (P),wherein ribonucleotides 20 and 21 of the sense RNA sequence (H) form a 2- ribonucleotide unpaired 3' overhang and ribonucleotides 22 and 23 of the antisense RNA sequence (J) form a 2-ribonucleotide unpaired 3' overhang in the one or more doublestranded product RNA molecule(s) (P), wherein ribonucleotides 1 and 2 of the sense RNA sequence (H) basepair with ribonucleotides 20 and 21, respectively, of the antisense RNA sequence (J) in the one or more double-stranded product RNA molecule(s) (P), and wherein ribonucleotides 18 and 19 of the sense RNA sequence (H) basepair with ribonucleotides 1 and 2, respectively, of the antisense RNA sequence (J) in the one or more double -stranded product RNA molecule(s) (P).

7. The precursor RNA molecule of claim 6, wherein the first RNA sequence (E) is hybridised to the second RNA sequence (G) by basepairing between at least 21 of the at least 21 contiguous ribonucleotides of the first RNA sequence (E) and at least 21 of the at least 23 contiguous ribonucleotides of the second RNA sequence (G), wherein 2 contiguous ribonucleotides of the at least 23 ribonucleotides of (G) are nonbasepaired and all of the at least 21 ribonucleotides of (E) are basepaired in the part (C) of the double-stranded RNA region (B), the 2 non-basepaired ribonucleotides forming a di -ribonucleotide bulge or two single-ribonucleotide bulges in the part (C) of the doublestranded RNA region (B), wherein the bulge is immediately flanked by ribonucleotides of the second RNA sequence (G) which are basepaired to ribonucleotides of the first RNA sequence (E), wherein the one or more double-stranded product RNA molecule(s) (P) comprise the bulge, and wherein ribonucleotides 1 to 19 of the sense RNA sequence (H) each basepair with a ribonucleotide of ribonucleotides 1 to 21 of antisense RNA sequence (J) in the one or more double-stranded product RNA molecule(s) (P).

8. The precursor RNA molecule of claim 6 or claim 7, wherein the first RNA sequence (E) comprises at least 44 contiguous ribonucleotides and the second RNA sequence (G) comprises at least 48 contiguous ribonucleotides, wherein the first RNA sequence (E) is hybridised to the second RNA sequence (G) by basepairing between at least 42 of the at least 44 contiguous ribonucleotides of the first RNA sequence (E) and at least 42 of the at least 48 contiguous ribonucleotides of the second RNA sequence (G),wherein 4, 5 or 6 ribonucleotides of the at least 48 contiguous ribonucleotides of (G) are non-basepaired, and 0, 1 or 2, respectively, ribonucleotides of the at least 44 contiguous ribonucleotides of (E) are non-basepaired in the part (C) of the doublestranded RNA region (B), forming bulges in the part (C) of the double-stranded RNA region (B), wherein each of the bulges is immediately flanked by ribonucleotides which are basepaired in the part (C) of the double-stranded RNA region (B), wherein the precursor RNA molecule is capable of being cleaved in a eukaryotic cell by one or more ribonucleases (RNases) to produce multiple, different doublestranded product RNA molecules (P) consisting of a sense RNA sequence (H) of 21 contiguous ribonucleotides and an antisense RNA sequence (J) of 23 contiguous ribonucleotides, wherein the multiple, different double-stranded product RNA molecule(s) (P) each comprise at least one of the bulges, and wherein at least some of the multiple, different double -stranded product RNA molecules (P) comprise non-overlapping antisense RNA sequences (J), preferably adjacent non-overlapping antisense RNA sequences (J).

9. The precursor RNA molecule according to any one of claims 6 to 8, wherein the molecule is capable of being cleaved in a eukaryotic cell by one or more ribonucleases (RNases) to produce multiple, different double-stranded product RNA molecules (P) which each consist of a sense RNA sequence (H) of 21 contiguous ribonucleotides from the first RNA sequence (E) and an antisense RNA sequence (J) of 23 contiguous ribonucleotides from the second RNA sequence (G), wherein at least some of the multiple, different double -stranded product RNA molecules (P) have overlapping antisense RNA sequences (J), or non-overlapping antisense RNA sequences (J), preferably adjacent non-overlapping antisense RNA sequences (J).

10. A precursor RNA molecule (A) comprising at least one double-stranded RNA region (B), wherein:(i) the double-stranded RNA region (B) comprises:(a) a first RNA strand (D) of at least 23 contiguous ribonucleotides which comprises a first RNA sequence (E) of at least 21 contiguous ribonucleotides, and(b) a second RNA strand (F) of at least 26 contiguous ribonucleotides which comprises a second RNA sequence (G) of at least 24 contiguous ribonucleotides, wherein the first RNA strand (D) and second RNA strand (F) are covalently linked by a linking RNA sequence (L), wherein the first RNA sequence (E) is hybridised to the second RNA sequence (G) by basepairing between at least 20 of the at least 21 contiguous ribonucleotides of the first RNA sequence (E) and at least 20 of the at least 24 contiguous ribonucleotides of the second RNA sequence (G), forming at least a part (C) of the double-stranded RNA region (B), wherein 3 or 4 ribonucleotides of the at least 24 ribonucleotides of (G) are non-basepaired and 0 or 1, respectively, ribonucleotides of the at least 21 ribonucleotides of (E) are non- basepaired in the part (C) of the double-stranded RNA region (B), forming one, two, three, four or five bulges in the part (C) of the double-stranded RNA region (B), wherein each of the one, two, three, four or five bulges is immediately flanked by ribonucleotides which are basepaired in the part (C) of the double-stranded RNA region (B); and(ii) the precursor RNA molecule (A) is capable of being cleaved in a eukaryotic cell by one or more ribonucleases (RNases) to produce one or more double-stranded product RNA molecule(s) (P) consisting of a sense RNA sequence (H) of 21 contiguous ribonucleotides from the first RNA sequence (E) and an antisense RNA sequence (J) of 24 contiguous ribonucleotides from the second RNA strand (F), including at least 22 contiguous ribonucleotides from (G), wherein the one or more double-stranded product RNA molecule(s) (P) comprise the one, two, three, four or five bulges, wherein 18 or 19 of ribonucleotides 1 to 19 of the sense RNA sequence (H) each basepair with a ribonucleotide of ribonucleotides 1 to 22 of antisense RNA sequence (J) in the one or more double-stranded product RNA molecule(s) (P), wherein ribonucleotides 20 and 21 of the sense RNA sequence (H) form a 2- ribonucleotide unpaired 3' overhang and ribonucleotides 23 and 24 of the antisense RNA sequence (J) form a 2-ribonucleotide unpaired 3' overhang in the one or more doublestranded product RNA molecule(s) (P), wherein ribonucleotides 1 and 2 of the sense RNA sequence (H) basepair with ribonucleotides 21 and 22, respectively, of the antisense RNA sequence (J) in the one or more double-stranded product RNA molecule(s) (P), andwherein ribonucleotides 18 and 19 of the sense RNA sequence (H) basepair with ribonucleotides 1 and 2, respectively, of the antisense RNA sequence (J) in the one or more double -stranded product RNA molecule(s) (P).

11. The precursor RNA molecule of claim 10, wherein the first RNA sequence (E) comprises at least 44 contiguous ribonucleotides and the second RNA sequence (G) comprises at least 50 contiguous ribonucleotides, wherein the first RNA sequence (E) is hybridised to the second RNA sequence (G) by basepairing between at least 42 of the at least 44 contiguous ribonucleotides of the first RNA sequence (E) and at least 42 of the at least 50 contiguous ribonucleotides of the second RNA sequence (G), wherein 6, 7 or 8 ribonucleotides of the at least 50 contiguous ribonucleotides of (G) are non-basepaired, and 0, 1 or 2, respectively, ribonucleotides of the at least 44 contiguous ribonucleotides of (E) are non-basepaired in the part (C) of the doublestranded RNA region (B), forming bulges in the part (C) of the double-stranded RNA region (B), wherein each of the bulges is immediately flanked by ribonucleotides which are basepaired in the part (C) of the double-stranded RNA region (B), wherein the precursor RNA molecule is capable of being cleaved in a eukaryotic cell by one or more ribonucleases (RNases) to produce multiple, different doublestranded product RNA molecules (P) consisting of a sense RNA sequence (H) of 21 contiguous ribonucleotides and an antisense RNA sequence (J) of 24 contiguous ribonucleotides, wherein the multiple, different double-stranded product RNA molecule(s) (P) each comprise at least one of the bulges, and wherein at least some of the multiple, different double -stranded product RNA molecules (P) comprise non-overlapping antisense RNA sequences (J), preferably adjacent non-overlapping antisense RNA sequences (J).

12. The precursor RNA molecule of claim 10 or claim 11, wherein the molecule is capable of being cleaved in a eukaryotic cell by one or more ribonucleases (RNases) to produce multiple, different double-stranded product RNA molecules (P) which each consist of a sense RNA sequence (H) of 21 contiguous ribonucleotides from the first RNA sequence (E) and an antisense RNA sequence (J) of 24 contiguous ribonucleotides from the second RNA sequence (G), wherein at least some of the multiple, different double-stranded product RNA molecules (P) have overlapping antisense RNA sequences(J), or non-overlapping antisense RNA sequences (J), preferably adjacent nonoverlapping antisense RNA sequences (J).

13. The precursor RNA molecule according to any one of claims 1 to 12, wherein one or more or all of the following apply:(a) the antisense RNA sequence (J) from at least one of the product RNA molecules (P) produced from the precursor RNA molecule is capable of hybridising to a region (R) of a target RNA molecule in a eukaryotic cell through at least ribonucleotides 2 to 8 of the antisense RNA sequence (J) basepairing with ribonucleotides within the region (R) of the target RNA molecule,(b) at least 50%, at least 75%, at least 90%, or at least 95%, of the ribonucleotides of the antisense RNA sequence (J) are capable of basepairing to ribonucleotides of a region (R) of a target RNA molecule in a eukaryotic cell,(c) all of the ribonucleotides of the antisense RNA sequence (J) are capable of basepairing to ribonucleotides of a region (R) of a target RNA molecule in a eukaryotic cell,(d) the basepairing to ribonucleotides of the region (R) of the target RNA molecule comprises one or more G:U basepairs, preferably 2, 3, 4 or 5 G:U basepairs,(e) the basepairing to ribonucleotides of the region (R) of the target RNA molecule comprises only canonical basepairs,(f) the region (R) of the target RNA molecule has a length of 22-30, 23-30, 23- 33, 24-30, 30-50, 34-200, 50-100, 100-600, or 100-1000 ribonucleotides, and / or the length of the antisense sequence of the dsRNA region is 22-30, 23-30, 23-33, 24-30, 30-50, 34-200, 50-100, 100-600, or 100-1000 ribonucleotides, and / or wherein the sense sequence of the dsRNA region of the precursor RNA molecule is shorter than the corresponding antisense sequence, preferably wherein the sense sequence is shorter than the corresponding antisense sequence entirely because of the presence of non-basepaired ribonucleotides in the antisense sequence that bulge from the dsRNA region or the product RNA molecule(s) (P), more preferably wherein the sense sequence has a length which is between 87%-97%, or 87%-96%, or 91%-97%, or 91%-96%, or more preferably 94%-97% or 94%-96% of the length of the antisense sequence, or the length of the sense sequence is about 21 / 22, 21 / 23 or 21 / 24 of the length of the antisense sequence, calculated as a fraction, and(g) the precursor RNA molecule comprises two or more different, doublestranded RNA regions (B), wherein each double-stranded RNA region (B) is independently defined in any one of claims 1 to 12.

14. The precursor RNA molecule of claim 13, wherein the two or more different, double-stranded RNA regions (B) are capable of being cleaved by one or more RNases to produce product RNA molecules (P) which comprise antisense ribonucleotide sequences (J) which hybridise to one region (R) of a target RNA molecule or to different, non-contiguous regions (R) of the target RNA molecule, or to regions (R) of different target RNA molecules, or to corresponding regions (R) in a family of target RNA molecules.

15. The precursor RNA molecule according to any one of claims 1 to 14, wherein the eukaryotic cell is a plant cell, an animal cell or a fungal cell, preferably a plant cell, an arthropod cell, a nematode cell or a fungal cell, and / or the target RNA molecule is in a eukaryotic cell which is a plant cell, an animal cell or a fungal cell, preferably a plant cell, an arthropod cell, a nematode cell or a fungal cell.

16. The precursor RNA molecule according to any one of claims 1 to 15, wherein the one or more double-stranded region(s) (B) comprise bulges which are evenly spaced apart along most or all of each double-stranded region (B), and / or the precursor RNA molecule has a single linking RNA sequence (L), thereby forming a hairpin RNA (hpRNA) structure, or the precursor RNA molecule comprises two double -stranded regions (B) and two linking RNA sequences (L) forming a ledRNA structure.

17. The precursor RNA molecule according to any one of claims 1 to 16 which further comprises a double-stranded region which comprises at least 23 contiguous basepairs and which lacks bulges, preferably wherein the double -stranded region which lacks bulges has a length of 30-200 contiguous basepairs.

18. The precursor molecule according to any one of claims 1 to 17, wherein(i) the first RNA sequence (E) differs from a corresponding wild-type RNA sequence in the target RNA molecule by deletion of one or more ribonucleotides from the corresponding wild-type RNA sequence to make the first RNA sequence, and / or(ii) the second RNA sequence (G) differs from a fully complementary sequence to the corresponding wild-type RNA sequence in the target RNA molecule by insertion of one or more ribonucleotides into the fully complementary sequence to make the second RNA sequence, preferably (i), optionally wherein the deletion of ribonucleotides from the corresponding wild-type RNA sequence occurs at one or more or all of theribonucleotide positions corresponding to the non-basepaired ribonucleotides in the second RNA sequence (G) as defined in any one of claims 1 to 17.

19. The precursor molecule according to any one of claims 1 to 18, wherein(i) the first RNA sequence (E) differs from the corresponding wild-type RNA sequence in the target RNA molecule by substitution of one or more A or C ribonucleotides in the corresponding wild-type RNA sequence to G or U ribonucleotides, respectively, thereby making the first RNA sequence, wherein the G or U ribonucleotides are involved in G:U basepairs with ribonucleotides of the second RNA sequence (G), and / or(ii) the second RNA sequence (G) differs from the fully complementary sequence by substitution of one or more C ribonucleotides in the fully complementary sequence to U ribonucleotides, thereby making the second RNA sequence, wherein the U ribonucleotides are involved in G:U basepairs with ribonucleotides of the first RNA sequence (E), preferably (i).

20. A double-stranded RNA molecule (P) consisting of a sense RNA sequence (H) of 21 contiguous ribonucleotides and an antisense RNA sequence (J) of 22 contiguous ribonucleotides and one or two or three bulges, wherein 18 or 19 of ribonucleotides 1 to 19 of the sense RNA sequence (H) each basepair with a ribonucleotide of ribonucleotides 1 to 20 of the antisense RNA sequence (J), wherein each of the one or two or three bulges is immediately flanked by ribonucleotides which are basepaired in the double-stranded RNA molecule, wherein ribonucleotides 20 and 21 of the sense RNA sequence form a 2-ribonucleotide unpaired 3' overhang and ribonucleotides 21 and 22 of the antisense RNA sequence form a 2-ribonucleotide unpaired 3' overhang, wherein ribonucleotides 1 and 2 of the sense RNA sequence basepair with ribonucleotides 19 and 20, respectively, of the antisense RNA sequence, and wherein ribonucleotides 18 and 19 of the sense RNA sequence basepair with ribonucleotides 1 and 2, respectively, of the antisense RNA sequence.

21. A double-stranded RNA molecule (P) consisting of a sense RNA sequence (H) of 21 contiguous ribonucleotides and an antisense RNA sequence (J) of 23 contiguous ribonucleotides and one or two or three or four bulges, wherein 18 or 19 of ribonucleotides 1 to 19 of the sense RNA sequence (H) each basepair with a ribonucleotide of ribonucleotides 1 to 21 of the antisense RNA sequence (J),wherein each of the one or two or three or four bulges is immediately flanked by ribonucleotides which are basepaired in the double -stranded RNA molecule, wherein ribonucleotides 20 and 21 of the sense RNA sequence form a 2-ribonucleotide unpaired 3' overhang and ribonucleotides 22 and 23 of the antisense RNA sequence form a 2-ribonucleotide unpaired 3' overhang, wherein ribonucleotides 1 and 2 of the sense RNA sequence basepair with ribonucleotides 20 and 21, respectively, of the antisense RNA sequence, and wherein ribonucleotides 18 and 19 of the sense RNA sequence basepair with ribonucleotides 1 and 2, respectively, of the antisense RNA sequence.

22. A double-stranded RNA molecule (P) consisting of a sense RNA sequence (H) of 21 contiguous ribonucleotides and an antisense RNA sequence (J) of 24 contiguous ribonucleotides and one or two or three or four or five bulges, wherein 18 or 19 of ribonucleotides 1 to 19 of the sense RNA sequence each basepair with a ribonucleotide of ribonucleotides 1 to 22 of the antisense RNA sequence (J), wherein each of the one or two or three or four or five bulges is immediately flanked by ribonucleotides which are basepaired in the double -stranded RNA molecule, wherein ribonucleotides 20 and 21 of the sense RNA sequence form a 2-ribonucleotide unpaired 3' overhang and ribonucleotides 23 and 24 of the antisense RNA sequence form a 2-ribonucleotide unpaired 3' overhang, wherein ribonucleotides 1 and 2 of the sense RNA sequence basepair with ribonucleotides 21 and 22, respectively, of the antisense RNA sequence, and wherein ribonucleotides 18 and 19 of the sense RNA sequence basepair with ribonucleotides 1 and 2, respectively, of the antisense RNA sequence.

23. A population of multiple, different double-stranded RNA molecules (P) comprising double-stranded RNA molecules according to any one of claims 20 to 22 or any combination thereof.

24. The population of multiple, different double -stranded RNA molecules (P) of claim 23, wherein(i) more antisense RNA sequences (J) in the population consist of 22 ribonucleotides than consist of 21 ribonucleotides,(ii) more antisense RNA sequences (J) in the population consist of 23 ribonucleotides than consist of 21 ribonucleotides,(iii) more antisense RNA sequences (J) in the population consist of 24 ribonucleotides than consist of 21 ribonucleotides, or(iv) any combination of (i), (ii) and (iii), preferably (i) and (ii).

25. A eukaryotic cell comprising one or more or all of a precursor RNA molecule according to any one of claims 1 to 19, a polynucleotide encoding the precursor RNA molecule, the double-stranded RNA molecule (P) according to any one of claims 20 to 22, and the population of multiple, different double-stranded RNA molecules (P) of claim 23 or claim 24, preferably wherein the cell is a non-human cell or a eukaryotic cell in vitro.

26. A double-stranded RNA molecule (P) obtainable by, or obtained from, the cleavage of the precursor RNA molecule according to any of claims 1 to 19.

27. A method of identifying a double-stranded RNA molecule (P), or a precursor RNA molecule (A), for reducing the amount and / or activity of a target RNA molecule of interest, the method comprising i) producing a precursor RNA molecule according to any one of claims 1 to 19, and / or a double-stranded RNA molecule according to any one of claims 20 to 22, or a population of multiple, different precursor RNA molecules according to any one of claims 1 to 19, and / or multiple, different double-stranded RNA molecules of claim 23 or claim 24, ii) determining the ability of the precursor RNA molecule or double-stranded RNA molecule, or members of the population of multiple, different precursor RNA molecules, or the population of multiple, different double-stranded RNA molecules, to reduce the amount and / or activity of a target RNA molecule of interest, optionally wherein step i) comprises expressing a precursor RNA molecule (A) according to any one of claims 1 to 19 in a eukaryotic cell, wherein the precursor RNA molecule is cleaved in the eukaryotic cell by one or more ribonucleases (RNases) to produce the doublestranded RNA molecule or the population of multiple, different double-stranded RNA molecules.

28. An isolated and / or exogenous polynucleotide, or a vector comprising the polynucleotide, encoding the precursor RNA molecule according to any one of claims 1 to 19, optionally wherein the polynucleotide is operably linked to a promoter capable of directing expression of the precursor RNA molecule in a host cell, preferably aeukaryotic cell, and optionally a polyadenylation region / transcription terminator or a transcription termination sequence.

29. A host cell comprising one or more or all of the precursor RNA molecule of any one of claims 1 to 19, the double -stranded RNA molecule according to any one of claims 20 to 22, the population of multiple, different double-stranded RNA molecules of claim 23 or claim 24, the polynucleotide or the vector of claim 28, preferably a eukaryotic cell, more preferably a plant cell, a fungal cell, or an animal cell, an arthropod cell such as an insect cell, or a nematode cell.

30. A non-human organism, or a part thereof, comprising a cell of claim 25 or claim 29, preferably a transgenic non-human organism or part thereof, being transgenic for a polynucleotide of claim 28, and / or wherein the polynucleotide is stably integrated into the genome of the organism or part thereof.

31. A method of producing the cell of claim 25 or 29, the method comprising introducing into a cell one or more or all of the precursor RNA molecule of any one of claims 1 to 19, the double-stranded RNA molecule according to any one of claims 20 to 22, the population of multiple, different double-stranded RNA molecules of claim 23 or claim 24, and the polynucleotide or the vector of claim 28.

32. A method of producing a non-human organism of claim 30, the method comprising introducing one or more or all of the precursor RNA molecule according to of any one of claims 1 to 19, the double-stranded RNA molecule according to any one of claims 20 to 22, the population of multiple, different double-stranded RNA molecules of claim 23 or claim 24, and the polynucleotide or the vector of claim 28, into a cell and generating the non-human organism from the cell.

33. A method of producing the precursor RNA molecule of any one of claims 1 to 19, the double-stranded RNA molecule according to any one of claims 20 to 22, and / or the population of multiple, different double-stranded RNA molecules of claim 23 or claim 24, the method comprising expressing the polynucleotide or vector of claim 28 in a host cell or cell-free expression system.

34. An extract of a cell of claim 25 or 29, wherein the extract comprises one or more or all of the precursor RNA molecule of any one of claims 1 to 19, the double-strandedRNA molecule according to any one of claims 20 to 22, the population of multiple, different double-stranded RNA molecules of claim 23 or claim 24, and the polynucleotide or the vector of claim 28.

35. A composition comprising one or more or all of the precursor RNA molecule of any one of claims 1 to 19, the double-stranded RNA molecule according to any one of claims 20 to 22, the population of multiple, different double-stranded RNA molecules of claim 23 or claim 24, the polynucleotide or the vector of claim 28, the cell of claim 25 or 29, the non-human organism or part thereof of claim 30, and the extract of claim 34.

36. A method for increasing the number of double-stranded RNA molecules according to any one of claims 20 to 22 in a eukaryotic cell or organism, comprising expressing in the cell or organism a polynucleotide or a vector of claim 28, or contacting the cell or organism with the precursor RNA molecule according to any one of claims 1 to 19, or the double-stranded RNA molecule according to any one of claims 20 to 22.

37. A method for reducing or down-regulating the level and / or activity of a target RNA molecule in a eukaryotic cell or organism, the method comprising delivering to the cell or organism one or more or all of the precursor RNA molecule according to any one of claims 1 to 19, the double-stranded RNA molecule according to any one of claims 20 to 22, the population of multiple, different double-stranded RNA molecules of claim 23 or claim 24, the polynucleotide or the vector of claim 28, the extract of claim 34, and the composition of claim 35.

38. The method of claim 37, wherein one or more or all of the precursor RNA molecule, the double-stranded RNA molecule, the population of double-stranded RNA molecules, the polynucleotide, the vector, the extract, or the composition, are contacted with the cell or organism, preferably a plant cell, plant, nematode cell, nematode, fungus, insect cell or insect, by topical application to the cell or organism such as by spraying, dusting or injection, or provided in a feed for the organism.

39. A method for identifying a phenotype or function associated with a target RNA molecule in a eukaryotic cell or organism, the method comprising (i) delivering to the cell or organism, one or more or all of: the precursor RNA molecule according to any one of claims 1 to 19, the double -stranded RNA molecule according to any one of claims 20 to 22, the population of multiple, different double-stranded RNA molecules of claim23 or claim 24, the polynucleotide or vector of claim 28, the extract of claim 34, or the composition of claim 35, and (ii) observing the cell or organism, or a progeny cell or organism thereof, for the phenotype or function, or assaying the cell or organism, or a progeny cell or organism thereof, for a molecule associated with the phenotype or function, thereby identifying the phenotype or function associated with a target RNA.

40. A method for identifying a region of a target RNA molecule in a eukaryotic cell or organism that is susceptible to down-regulation by RNAi, the method comprising (i) delivering to the cell or organism one or more or all of: multiple precursor RNA molecules according to any one of claims 1 to 19, multiple double-stranded RNA molecules according to any one of claims 20 to 22, populations of multiple, different double-stranded RNA molecules of claim 23 or claim 24, polynucleotides or vectors of claim 28, extracts of claim 34, and compositions of claim 35, wherein the multiple precursor RNA molecules, double-stranded RNA molecules or populations of multiple double-stranded RNA molecules target different regions of the target RNA molecule, and (ii) assaying the cell or organism, or a progeny cell or organism thereof, for one or more of: the amount of target RNA molecule, the amount of protein encoded by the target RNA molecule, and / or for a phenotype or function associated with the target RNA molecule, and (iii) selecting a region of the target RNA molecule based on assay results from step (ii), thereby identifying the region.

41. A method for identifying an RNA molecule that is capable of having an effect on a pest or pathogen of a eukaryotic cell or organism, the method comprising (i) delivering to the eukaryotic cell or organism, one or more or all of the precursor RNA molecule according to any one of claims 1 to 19, the double -stranded RNA molecule according to any one of claims 20 to 22, the population of different double -stranded RNA molecules of claim 23 or claim 24, the polynucleotide or vector of claim 28, the extract of claim 34, and the composition of claim 35, (ii) contacting the cell or organism of step (i), or a progeny cell or organism thereof, with the pest or pathogen, (iii) determining whether or not the precursor RNA molecule, double -stranded RNA molecule or population of different double-stranded RNA molecules has an effect on the pest or pathogen, and optionally (iv) if the precursor RNA molecule, double -stranded RNA molecule or population of different double-stranded RNA molecules has a desirable effect on the pest or pathogen, selecting an RNA molecule based on results from step (iii), thereby identifying the RNA molecule.

42. A method for identifying an RNA molecule that is capable of having an effect on a pest or pathogen of a eukaryotic cell or organism, the method comprising (i) delivering to the pest or pathogen, one or more or all of the precursor RNA molecule according to any one of claims 1 to 19, the double-stranded RNA molecule according to any one of claims 20 to 22, the population of multiple, different double-stranded RNA molecules of claim 23 or claim 24, the extract of claim 34, and the composition of claim 35, (ii) testing the pest or pathogen for an effect of the precursor RNA molecule, double-stranded RNA molecule or population of double-stranded RNA molecules, and optionally (iii) selecting an RNA molecule based on results from step (ii), thereby identifying the RNA molecule.

43. A method for reducing or down-regulating the level and / or activity of a target RNA molecule in an eukaryotic organism, the method comprising orally or parenterally delivering to the organism one or more or all of the precursor RNA molecule according to any one of claims 1 to 19, the double -stranded RNA molecule according to any one of claims 20 to 22, the population of double-stranded RNA molecules of claim 23 or claim 24, the polynucleotide or vector of claim 28, the cell of claim 25 or claim 29, the nonhuman organism or part thereof of claim 30, the extract of claim 34, and the composition of claim 35.

44. A method of reducing or preventing damage caused by a pest or pathogen to a non-human organism, or to a eukaryotic cell in vitro, the method comprising delivering to the pest or pathogen or cell, or contacting the pest or pathogen or cell with, one or more or all of the precursor RNA molecule according to any one of claims 1 to 19, the double-stranded RNA molecule according to any one of claims 20 to 22, the population of double-stranded RNA molecules of claim 23 or claim 24, the cell of claim 25 or claim 29, the non-human organism or part thereof of claim 30, the extract of claim 34, and the composition of claim 35.

45. A method of controlling a non-human eukaryotic organism, the method comprising delivering to the non-human organism one or more or all of the precursor RNA molecule according to any one of claims 1 to 19, the double-stranded RNA molecule according to any one of claims 20 to 22, the population of double-stranded RNA molecules of claim 23 or claim 24, the polynucleotide or vector of claim 28, the cell of claim 25 or claim 29, the non-human organism or part thereof of claim 30, the extract of claim 34, and the composition of claim 35, wherein the precursor RNA molecule, double-stranded RNA molecule or population of double-stranded RNAmolecules has a deleterious effect on the non-human organism, preferably wherein the non-human organism is an arthropod such as an insect, or a nematode, or a plant.

46. A method of increasing the amount of small interfering RNAs (sRNAs) of 22, 23 or 24 ribonucleotides in length in a eukaryotic cell or organism, or of increasing the ratio of the amount of sRNAs of 22, 23 or 24 ribonucleotides relative to the amount of sRNAs of 21 ribonucleotides in length, the method comprising delivering to the cell or organism one or more or all of the precursor RNA molecule according to any one of claims 1 to 19, the double-stranded RNA molecule according to any one of claims 20 to 22, the population of double -stranded RNA molecules of claim 23 or claim 24, the cell of claim 25 or claim 29, the non-human organism or part thereof of claim 30, the extract of claim 34, and the composition of claim 35.

47. A method of treating a disease in an organism, the method comprising administering to the subject one or more or all of the precursor RNA molecule according to any one of claims 1 to 19, the double -stranded RNA molecule according to any one of claims 20 to 22, the population of double-stranded RNA molecules of claim 23 or claim 24, the polynucleotide or vector of claim 28, the cell of claim 25 or claim 29, the non- human organism or part thereof of claim 30, the extract of claim 34, and the composition of claim 35, preferably wherein one or more or all of the precursor RNA molecule, the double-stranded RNA molecule, the population of double-stranded RNA molecules, the polynucleotide, the vector, the cell, the non-human organism or part thereof, the extract, or the composition, are administered topically, orally or parenterally, such as injected, optionally wherein the organism is a vertebrate or a plant.

48. The method according to any one of claims 31 to 33, or 36 to 47, wherein the sense sequence of the dsRNA region of the precursor RNA molecule, or the product RNA molecule(s) (P), is shorter than the antisense sequence, preferably wherein the sense sequence is shorter than the antisense sequence entirely because of the presence of nonbasepaired ribonucleotides in the antisense sequence that bulge from the dsRNA region or the product RNA molecule(s) (P), more preferably wherein the sense sequence has a length which is between 87% and 97%, or 87% and 96%, or 91% and 97%, or 91% and 96%, or more preferably between 94% and 97% or 94% and 96% of the length of the antisense sequence, or the length of the sense sequence is about 21 / 22, 21 / 23 or 21 / 24 of the length of the antisense sequence, calculated as a fraction.

49. One or more or all of the precursor RNA molecule according to any one of claims 1 to 19, the double-stranded RNA molecule according to any one of claims 20 to 22, the population of double-stranded RNA molecules of claim 23 or claim 24, the polynucleotide or vector of claim 28, the cell of claim 25 or claim 29, the non-human organism or part thereof of claim 30, the extract of claim 34, and the composition of claim 35, for use in treating a disease in a subject, wherein the double-stranded RNA molecule has a beneficial effect on at least one symptom of the disease.

50. Use of one or more or all of the precursor RNA molecule according to any one of claims 1 to 19, the double-stranded RNA molecule according to any one of claims 20 to 22, the population of double-stranded RNA molecules of claim 23 or claim 24, the polynucleotide or vector of claim 28, the cell of claim 25 or claim 29, the non-human organism or part thereof of claim 30, the extract of claim 34, and the composition of claim 35 in the manufacture of a medicament for treating a disease.

51. A kit comprising one or more or all of the precursor RNA molecule according to any one of claims 1 to 19, the double-stranded RNA molecule according to any one of claims 20 to 22, the population of double-stranded RNA molecules of claim 23 or claim 24, the polynucleotide or vector of claim 28, the cell of claim 25 or claim 29, the non- human organism or part thereof of claim 30, the extract of claim 134, and the composition of claim 35.

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