Double stranded RNA molecules with a to g substitutions

Abstract

The present invention relates to double-stranded RNA molecules having A to G substitutions, precursor RNA molecules thereof, and their use in modifying cells such as for gene silencing.

Description

[0001] DOUBLE STRANDED RNA MOLECULES WITH A TO G SUBSTITUTIONS

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to double- stranded RNA molecules, precursors thereof, and their use to modify cells or 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 21nt 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 precursor RNA molecules comprising one or more double-stranded regions which produce double- stranded product RNA molecules, and the double-stranded product RNA molecules produced from the precursor RNA molecules, with desirable characteristics. The precursor RNA molecules are useful for increasing the number of such product RNA molecules in a eukaryotic cell, such as for example a plant cell. They are also useful for down-regulating gene expression or reducing the amount or activity of one or more target RNA molecules in a sequencespecific manner in a eukaryotic cell, such as for example a plant cell. In embodiments, 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 G:U basepairs, in a double- stranded 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] The present invention also provides such precursor RNA molecules which comprise a double-stranded RNA (dsRNA) region having a relatively high guanine (G) ribonucleotide content in at least one strand of the dsRNA region, referred to as the first RNA strand or sense strand, and where G:U basepairing constitutes 10-40% of the total basepairing in the dsRNA region. These molecules have an increased G ribonucleotide content relative to a corresponding control RNA molecule which has only canonical basepairing in the dsRNA region, i.e. lacking the G:U basepairing, and are readily designed by substituting at least some, preferably all, of the adenine (A) ribonucleotides in the first RNA sequence (sense sequence) of the dsRNA region to guanine (G) ribonucleotides. That is, at least some, preferably all, A:U basepairs in the control RNA molecule which have the A ribonucleotides in the first RNA sequence are replaced with G:U basepairs in the precursor RNA molecules of the invention. In plant cells, such precursor RNA molecules are processed differently by Dicers to yield increased levels of 23nt and / or 24nt antisense small RNA molecules (sRNA), particularly 24nt antisense sRNAs, relative to the control RNA molecule which is processed to produced predominantly 21nt sRNAs. This therefore provides a means and method for generating more 23nt and / or 24nt sRNAs in the cell, and thereby a means for gene silencing.

[0013] Therefore, in a first aspect, the present invention provides a precursor RNA molecule comprising at least one double- stranded RNA region, wherein the doublestranded RNA region comprises (a) a first RNA strand which comprises a first RNA sequence of at least 24 contiguous ribonucleotides, and

[0014] (b) a second RNA strand which comprises a second RNA sequence of at least 24 contiguous ribonucleotides, wherein the first RNA sequence hybridises to the second RNA sequence by basepairing between the at least 24 contiguous ribonucleotides of the first RNA sequence and the at least 24 contiguous ribonucleotides of the second RNA sequence, forming the double-stranded RNA region, wherein the first RNA sequence along the full length of the double-stranded RNA region comprises a guanine (G) ribonucleotide content such that 36-55%, preferably 40.1-55%, of the ribonucleotides of the first RNA sequence are G ribonucleotides, and wherein between 10% and 40%, preferably between 15% and 40%, or more preferably between 20% and 40%, of the ribonucleotides of the first RNA sequence and the second RNA sequence, in total, are basepaired in G:U basepairs. It is not necessary for all of the at least 24 contiguous ribonucleotides of the first RNA sequence and the at least 24 contiguous ribonucleotides of the second RNA sequence to basepair, but it is preferred they do i.e. full basepairing along the dsRNA region.

[0015] In an embodiment, the G ribonucleotide content of the first RNA sequence is 36- 55% and between 10% and 40% of the ribonucleotides of the first RNA sequence and the second RNA sequence, in total, are basepaired in G:U basepairs.

[0016] In an embodiment, the G ribonucleotide content of the first RNA sequence is 36- 55% and between 15% and 40% of the ribonucleotides of the first RNA sequence and the second RNA sequence, in total, are basepaired in G:U basepairs.

[0017] In an embodiment, the G ribonucleotide content of the first RNA sequence is 36- 55% and between 20% and 40% of the ribonucleotides of the first RNA sequence and the second RNA sequence, in total, are basepaired in G:U basepairs.

[0018] In an embodiment, the G ribonucleotide content of the first RNA sequence is 40.1- 55% and between 10% and 40% of the ribonucleotides of the first RNA sequence and the second RNA sequence, in total, are basepaired in G:U basepairs.

[0019] In an embodiment, the G ribonucleotide content of the first RNA sequence is 40.1- 55% and between 15% and 40% of the ribonucleotides of the first RNA sequence and the second RNA sequence, in total, are basepaired in G:U basepairs.

[0020] In an embodiment, the G ribonucleotide content of the first RNA sequence is 40.1- 55% and between 20% and 40% of the ribonucleotides of the first RNA sequence and the second RNA sequence, in total, are basepaired in G:U basepairs. Other G ribonucleotide contents and percentages of G:U basepairs are as described herein. In embodiments of this aspect, the first RNA sequence along the full length of the double-stranded RNA region comprises an adenine (A) ribonucleotide content such that less than 10%, preferably less than 8%, more preferably less than 6%, even more preferably less than 4% or less than 2%, most preferably zero of the ribonucleotides of the first RNA sequence are A ribonucleotides. Alternatively, or in addition, the second RNA sequence is at least 80% identical to a sequence of at least 24 contiguous ribonucleotides which is fully complementary to a first region of a target RNA molecule in a eukaryotic cell such as for example an insect cell or a fungal cell, preferably a plant cell.

[0021] In an embodiment, the A ribonucleotide content of the first RNA sequence along the full length of the double-stranded RNA region is less than 10%.

[0022] In an embodiment, the A ribonucleotide content of the first RNA sequence along the full length of the double-stranded RNA region is less than 8%.

[0023] In an embodiment, the A ribonucleotide content of the first RNA sequence along the full length of the double-stranded RNA region is less than 6%.

[0024] In an embodiment, the A ribonucleotide content of the first RNA sequence along the full length of the double-stranded RNA region is less than 4%.

[0025] In an embodiment, the A ribonucleotide content of the first RNA sequence along the full length of the double-stranded RNA region is less than 2%.

[0026] In an embodiment, the A ribonucleotide content of the first RNA sequence along the full length of the double- stranded RNA region is zero. Each of the above A ribonucleotide contents may be combined with the G ribonucleotide content and the percentage of G:U basepairs in the first RNA sequence and the second RNA sequence, in total, as described herein.

[0027] In a preferred embodiment, the A ribonucleotide content of the first RNA sequence is zero, the G ribonucleotide content of the first RNA sequence is 36-55%, and between 10% and 40% of the ribonucleotides of the first RNA sequence and the second RNA sequence, in total, are basepaired in G:U basepairs.

[0028] In a preferred embodiment, the A ribonucleotide content of the first RNA sequence is zero, the G ribonucleotide content of the first RNA sequence is 36-55%, and between 15% and 40% of the ribonucleotides of the first RNA sequence and the second RNA sequence, in total, are basepaired in G:U basepairs.

[0029] In a preferred embodiment, the A ribonucleotide content of the first RNA sequence is zero, the G ribonucleotide content of the first RNA sequence is 36-55%, and between 20% and 40% of the ribonucleotides of the first RNA sequence and the second RNA sequence, in total, are basepaired in G:U basepairs.

[0030] In a preferred embodiment, the A ribonucleotide content of the first RNA sequence is zero, the G ribonucleotide content of the first RNA sequence is 40.1-55%, and between 10% and 40% of the ribonucleotides of the first RNA sequence and the second RNA sequence, in total, are basepaired in G:U basepairs.

[0031] In a preferred embodiment, the A ribonucleotide content of the first RNA sequence is zero, the G ribonucleotide content of the first RNA sequence is 40.1-55%, and between 15% and 40% of the ribonucleotides of the first RNA sequence and the second RNA sequence, in total, are basepaired in G:U basepairs.

[0032] In a preferred embodiment, the A ribonucleotide content of the first RNA sequence is zero, the G ribonucleotide content of the first RNA sequence is 40.1-55%, and between 20% and 40% of the ribonucleotides of the first RNA sequence and the second RNA sequence, in total, are basepaired in G:U basepairs.

[0033] In each of the above embodiments, the second RNA sequence may be at least 80% identical to a sequence of at least 24, preferably at least 48, contiguous ribonucleotides which is fully complementary to a first region of a target RNA molecule in a eukaryotic cell such as for example an insect cell or a fungal cell, preferably a plant cell.

[0034] In an embodiment, the second RNA sequence is at least 85% identical to a sequence of at least 24, preferably at least 48, contiguous ribonucleotides which is fully complementary to a first region of a target RNA molecule in a eukaryotic cell such as for example an insect cell or a fungal cell, preferably a plant cell.

[0035] In an embodiment, the second RNA sequence is at least 90% identical to a sequence of at least 24, preferably at least 48, contiguous ribonucleotides which is fully complementary to a first region of a target RNA molecule in a eukaryotic cell such as for example an insect cell or a fungal cell, preferably a plant cell.

[0036] In an embodiment, the second RNA sequence is at least 95% identical to a sequence of at least 24, preferably at least 48, contiguous ribonucleotides which is fully complementary to a first region of a target RNA molecule in a eukaryotic cell such as for example an insect cell or a fungal cell, preferably a plant cell.

[0037] In an embodiment, the second RNA sequence is 100% identical to a sequence of at least 24, preferably at least 48, contiguous ribonucleotides which is fully complementary to a first region of a target RNA molecule in a eukaryotic cell such as for example an insect cell or a fungal cell, preferably a plant cell.

[0038] In an embodiment, the precursor RNA molecule is cleaved in a eukaryotic cell, preferably a plant cell, to produce double-stranded product RNA molecules each independently consisting of a sense RNA sequence of 24 contiguous ribonucleotides from the first RNA sequence and an antisense RNA sequence of 24 contiguous ribonucleotides from the second RNA sequence, or an increased amount of such product RNA molecules relative to a corresponding control RNA molecule having only canonical basepairing, wherein ribonucleotides 1 to 22 of the sense RNA sequence basepair with ribonucleotides 1 to 22 of the antisense RNA sequence in each of the double-stranded product RNA molecules, wherein the basepairs in each of the double- stranded product RNA molecules comprise, independently, 1, 2, 3, 4, 5, 6, 7, 8 or 9 G:U basepairs, preferably 2-9 G:U basepairs, wherein ribonucleotides 23 and 24 of the sense RNA sequence in each of the double-stranded product RNA molecules form a 2 -ribonucleotide unpaired 3' overhang, and 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. Such double-stranded product RNA molecules are referred to as 24 / 24-mers. This feature may be combined with each of the previously described embodiments, specifically the A ribonucleotide content and the G ribonucleotide content of the first RNA sequence and the percentage of G:U basepairs in the first and second RNA sequences, in total.

[0039] In an embodiment, the precursor RNA molecule is cleaved in a eukaryotic cell, preferably a plant cell, to also produce double-stranded product RNA molecules each independently consisting of a sense RNA sequence of 23 contiguous ribonucleotides from the first RNA sequence and an antisense RNA sequence of 23 contiguous ribonucleotides from the second RNA sequence, or an increased amount of such product RNA molecules relative to a corresponding control RNA molecule having only canonical basepairing, wherein ribonucleotides 1 to 21 of the sense RNA sequence basepair with ribonucleotides 1 to 21 of the antisense RNA sequence in each of the double-stranded product RNA molecules, wherein the basepairs in each of the double- stranded product RNA molecules comprise, independently, 1, 2, 3, 4, 5, 6, 7, 8 or 9 G:U basepairs, preferably 2-9 G:U basepairs, wherein ribonucleotides 22 and 23 of the sense RNA sequence in each of the double-stranded product RNA molecules form a 2 -ribonucleotide unpaired 3' overhang, and 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. Such double- stranded product RNA molecules are referred to as 23 / 23-mers.

[0040] In an embodiment, at least some of the 24 / 24-mer and / or 23 / 23-mer doublestranded product RNA molecules produced from the precursor RNA molecule of the invention have only 1 G:U basepair.

[0041] In an embodiment, at least some of the 24 / 24-mer and / or 23 / 23-mer doublestranded product RNA molecules produced from the precursor RNA molecule of the invention have exactly 2 G:U basepairs.

[0042] In an embodiment, at least some of the 24 / 24-mer and / or 23 / 23-mer doublestranded product RNA molecules produced from the precursor RNA molecule of the invention have exactly 3 G:U basepairs.

[0043] In an embodiment, at least some of the 24 / 24-mer and / or 23 / 23-mer doublestranded product RNA molecules produced from the precursor RNA molecule of the invention have exactly 4 G:U basepairs.

[0044] In an embodiment, at least some of the 24 / 24-mer and / or 23 / 23-mer doublestranded product RNA molecules produced from the precursor RNA molecule of the invention have exactly 5 G:U basepairs.

[0045] In an embodiment, at least some of the 24 / 24-mer and / or 23 / 23-mer doublestranded product RNA molecules produced from the precursor RNA molecule of the invention have exactly 6 G:U basepairs.

[0046] In an embodiment, at least some of the 24 / 24-mer and / or 23 / 23-mer doublestranded product RNA molecules produced from the precursor RNA molecule of the invention have exactly 7 G:U basepairs.

[0047] In an embodiment, at least some of the 24 / 24-mer and / or 23 / 23-mer doublestranded product RNA molecules produced from the precursor RNA molecule of the invention have exactly 8 G:U basepairs.

[0048] In an embodiment of this and the previous embodiments, at least some of the double-stranded product RNA molecules may comprise an adenine (A) ribonucleotide content of the sense RNA sequence, or of the sense and antisense RNA sequences in total, such that less than 5, preferably less than 4, more preferably less than 3, even more preferably less than 2 or less than 1, most preferably zero of the ribonucleotides are A ribonucleotides. This feature may be combined with each of the previously described embodiments, specifically the A ribonucleotide content and the G ribonucleotide content of the first RNA sequence and the percentage of G:U basepairs in the first and second RNA sequences, in total. In an embodiment, which may be combined with the previous embodiments, the double-stranded product RNA molecules comprise an adenine (A) ribonucleotide content of the sense RNA sequence, or of the sense and antisense RNA sequences in total, such that not more than 5 of the ribonucleotides are A ribonucleotides.

[0049] In an embodiment, which may be combined with the previous embodiments, the double-stranded product RNA molecules comprise an adenine (A) ribonucleotide content of the sense RNA sequence, or of the sense and antisense RNA sequences in total, such that not more than 4 of the ribonucleotides are A ribonucleotides.

[0050] In an embodiment, which may be combined with the previous embodiments, the double-stranded product RNA molecules comprise an adenine (A) ribonucleotide content of the sense RNA sequence, or of the sense and antisense RNA sequences in total, such that not more than 3 of the ribonucleotides are A ribonucleotides.

[0051] In an embodiment, which may be combined with the previous embodiments, the double-stranded product RNA molecules comprise an adenine (A) ribonucleotide content of the sense RNA sequence, or of the sense and antisense RNA sequences in total, such that not more than 2 of the ribonucleotides are A ribonucleotides.

[0052] In an embodiment, which may be combined with the previous embodiments, the double-stranded product RNA molecules comprise an adenine (A) ribonucleotide content of the sense RNA sequence, or of the sense and antisense RNA sequences in total, such that not more than of the ribonucleotides are A ribonucleotides.

[0053] In an embodiment, which may be combined with the previous embodiments, at least some of the double- stranded product RNA molecules comprise an adenine (A) ribonucleotide content of the sense RNA sequence, or of the sense and antisense RNA sequences in total, such that none of the ribonucleotides are A ribonucleotides.

[0054] In an embodiment, which may be combined with the previous embodiments, at least some of the double- stranded product RNA molecules comprise an adenine (A) ribonucleotide content such that none of the ribonucleotides of the sense RNA sequence are A ribonucleotides.

[0055] In an embodiment, the precursor RNA molecule comprises a longer dsRNA region whereby multiple, non-overlapping siRNAs of 21, 23 or 24 ribonucleotides in length may be produced from the precursor RNA molecule. Therefore, in an embodiment, the double- stranded RNA region comprises

[0056] (a) a first RNA strand which comprises a first RNA sequence of at least 42, at least 46 or at least 48 contiguous ribonucleotides, and

[0057] (b) a second RNA strand comprises a second RNA sequence of at least 42, at least 46 or at least 48 contiguous ribonucleotides, wherein the first RNA sequence hybridises to the second RNA sequence by basepairing between the at least 42, at least 46 or at least 48 contiguous ribonucleotides of the first RNA sequence and the at least 42, at least 46 or at least 48 contiguous ribonucleotides of the second RNA sequence, respectively, forming the double- stranded RNA region, optionally wherein the double-stranded RNA region comprises a guanine (G) ribonucleotide content such that 36-55%, preferably 40.1-55%, of the ribonucleotides of the first RNA sequence are G ribonucleotides, and optionally wherein between 10% and 40%, preferably between 15% and 40%, or more preferably between 20% and 40%, of the ribonucleotides of the first RNA sequence and the second RNA sequence, in total, are basepaired in G:U basepairs. In an embodiment, the first RNA sequence along the full length of the double- stranded RNA region comprises an adenine (A) ribonucleotide content such that less than 10%, preferably less than 8%, more preferably less than 6%, even more preferably less than 4% or less than 2%, most preferably zero of the ribonucleotides of the first RNA sequence are A ribonucleotides. Alternatively, or in addition, 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 the eukaryotic cell such as for example an insect cell or a fungal cell, preferably a plant cell.

[0058] In embodiments that comprise longer double-stranded RNA regions, the first RNA sequence comprises at least 72, at least 96 or at least 120 contiguous ribonucleotides and the second RNA sequence comprises at least the same length, providing extended basepairing in the dsRNA region. Preferably, the maximum length of the first and second RNA sequences is 1000, 800 or preferably 600 ribonucleotides, but any length of sequences that can be synthesized can be accommodated, for example if multiple gene transcripts are targeted by the second RNA sequence.

[0059] In these embodiments where the precursor RNA molecule comprises a longer dsRNA region, the length feature may be combined with the preferred A ribonucleotide content, the G ribonucleotide content and the percentage of G:U basepairs as described in the above embodiments. In a preferred embodiment, the length of the first RNA sequence is at least 24, at least 48, at least 72, at least 96 or at least 120 contiguous ribonucleotides, the A ribonucleotide content of the first RNA sequence is zero, the G ribonucleotide content of the first RNA sequence is 36-55%, and between 10% and 40% of the ribonucleotides of the first RNA sequence and the second RNA sequence, in total, are basepaired in G:U basepairs. In an embodiment, the length of the first RNA sequence is at least 24, at least 48, at least 72, at least 96 or at least 120 contiguous ribonucleotides, the A ribonucleotide content of the first RNA sequence is zero, the G ribonucleotide content of the first RNA sequence is 36-55%, and between 15% and 40% of the ribonucleotides of the first RNA sequence and the second RNA sequence, in total, are basepaired in G:U basepairs.

[0060] In an embodiment, the length of the first RNA sequence is at least 24, at least 48, at least 72, at least 96 or at least 120 contiguous ribonucleotides, the A ribonucleotide content of the first RNA sequence is zero, the G ribonucleotide content of the first RNA sequence is 36-55%, and between 20% and 40% of the ribonucleotides of the first RNA sequence and the second RNA sequence, in total, are basepaired in G:U basepairs.

[0061] In an embodiment, the length of the first RNA sequence is at least 24, at least 48, at least 72, at least 96 or at least 120 contiguous ribonucleotides, the A ribonucleotide content of the first RNA sequence is zero, the G ribonucleotide content of the first RNA sequence is 40.1-55%, and between 10% and 40% of the ribonucleotides of the first RNA sequence and the second RNA sequence, in total, are basepaired in G:U basepairs.

[0062] In an embodiment, the length of the first RNA sequence is at least 24, at least 48, at least 72, at least 96 or at least 120 contiguous ribonucleotides, the A ribonucleotide content of the first RNA sequence is zero, the G ribonucleotide content of the first RNA sequence is 40.1-55%, and between 15% and 40% of the ribonucleotides of the first RNA sequence and the second RNA sequence, in total, are basepaired in G:U basepairs.

[0063] In an embodiment, the length of the first RNA sequence is at least 24, at least 48, at least 72, at least 96 or at least 120 contiguous ribonucleotides, the A ribonucleotide content of the first RNA sequence is zero, the G ribonucleotide content of the first RNA sequence is 40.1-55%, and between 20% and 40% of the ribonucleotides of the first RNA sequence and the second RNA sequence, in total, are basepaired in G:U basepairs.

[0064] One consequence of the A to G substitutions relative to the corresponding control RNA molecule is that the adenine (A) ribonucleotide content of at least the first RNA sequence, or in both the first and second RNA sequences, along the full length of the dsRNA region, is reduced. In embodiments, the precursor RNA molecule comprises an A ribonucleotide content in the first RNA sequence along the full length of the dsRNA region, wherein the A ribonucleotide content is less than 10% of the total number of ribonucleotides in the first RNA sequence, preferably less than 8%, more preferably less than 6%, even more preferably less than 4% or less than 2% or most preferably zero, and / or the A ribonucleotide content of the sense and antisense RNA sequences, in total, of the product RNA molecules is not more than 1, not more than 2, not more than 3, not more than 4, not more than 5, not more than 6, not more than 7, not more than 8, or not more than 9 A ribonucleotides. In this context, the percentage for the A ribonucleotide content is determined along the full length of the dsRNA region, bounded by the first and last basepairs. In an embodiment, the first and second RNA sequences each comprise at least 21 or at least 24 contiguous ribonucleotides and the A ribonucleotide content of the first RNA sequence is less than 10%, preferably less than 8%, more preferably less than 6%, even more preferably less than 4% or less than 2% or most preferably zero. In an embodiment, the first and second RNA sequences each comprise at least 48 contiguous ribonucleotides and the A ribonucleotide content of the first RNA sequence is less than 10%, preferably less than 8%, more preferably less than 6%, even more preferably less than 4% or less than 2% or most preferably zero. In an embodiment, the first and second RNA sequences each comprise at least 72 contiguous ribonucleotides and the A ribonucleotide content of the first RNA sequence is less than 10%, preferably less than 8%, more preferably less than 6%, even more preferably less than 4% or less than 2% or most preferably zero. In an embodiment, the first and second RNA sequences each comprise at least 96 contiguous ribonucleotides and the A ribonucleotide content of the first RNA sequence is less than 10%, preferably less than 8%, more preferably less than 6%, even more preferably less than 4% or less than 2% or most preferably zero. In an embodiment, the first and second RNA sequences each comprise at least 120 contiguous ribonucleotides and the A ribonucleotide content of the first RNA sequence is less than 10%, preferably less than 8%, more preferably less than 6%, even more preferably less than 4% or less than 2% or most preferably zero. In an embodiment, the first and second RNA sequences each comprise at least 21 or at least 24 contiguous ribonucleotides and the A ribonucleotide content of the first RNA sequence is only 1, or only 2, preferably zero, A ribonucleotides. In an embodiment, the first and second RNA sequences each comprise at least 48 contiguous ribonucleotides and the A ribonucleotide content of the first RNA sequence is not more than 1, not more than 2, not more than 3, not more than 4, or preferably zero A ribonucleotides. In an embodiment, the first and second RNA sequences each comprise at least 72 contiguous ribonucleotides and the A ribonucleotide content of the first RNA sequence is not more than 1, not more than 2, not more than 3, not more than 4, not more than 5, not more than 6, not more than 7, or preferably zero A ribonucleotides. In an embodiment, the first and second RNA sequences each comprise at least 96 contiguous ribonucleotides and the A ribonucleotide content of the first RNA sequence is not more than 1, not more than 2, not more than 3, not more than 4, not more than 5, not more than 6, not more than 7, not more than 8, not more than 9 or preferably zero A ribonucleotides. In an embodiment, the first and second RNA sequences each comprise at least 120 contiguous ribonucleotides and the A ribonucleotide content of the first RNA sequence is not more than 1, not more than 2, not more than 3, not more than 4, not more than 5, not more than 6, not more than 7, not more than 8, not more than 9, not more than 10, or not more than 11, or preferably zero, A ribonucleotides.

[0065] In a preferred embodiment, the dsRNA region does not comprise non-basepaired ribonucleotides i.e. all ribonucleotides are basepaired in either canonical or G:U basepairs, and, optionally, the minimum lengths of the first and second RNA sequences the A ribonucleotide content and / or G ribonucleotide content of the first RNA sequence are as described in the preceding embodiments. Alternatively, the dsRNA region may comprise non-basepaired ribonucleotides in both the first RNA sequence and the second RNA sequence. Non-basepaired nucleotides may be opposite each other in the dsRNA region, e.g. a mismatched ribonucleotide pair, or not opposite a non-basepaired ribonucleotide i.e. forming a bulge in the dsRNA region. These features of mismatched ribonucleotide pairs or bulged ribonucleotides may also apply to the above embodiments, provided the first and second RNA sequences are equal in length, or alternatively if the first RNA sequence is shorter than the second RNA sequence due to bulged ribonucleotides. In embodiments, less than 20%, less than 18%, less than 16%, less than 14%, less than 12%, less than 10%, less than 8%, less than 6%, less than 4%, or less than 2%, but in each case at least two ribonucleotides of the total number of ribonucleotides of the dsRNA region are non-basepaired. In embodiments, the first and second RNA sequences are both at least 48 ribonucleotides in length, and less than 20%, less than 18%, less than 16%, less than 14%, less than 12%, less than 10%, less than 8%, less than 6%, less than 4%, or less than 2%, but in each case at least two ribonucleotides, of the ribonucleotides of the dsRNA region are non-basepaired. In each of these embodiments, it is preferred that the G content of the first RNA sequence is between 40.1% and 55%, or the adenine (A) content of the first RNA sequence is less than 10%, or both. In each of these embodiments, it is preferred that the G content of the first RNA sequence is between 40.1% and 55%, and the adenine (A) content of the first RNA sequence is less than 8%. In each of these embodiments, it is preferred that the G content of the first RNA sequence is between 40.1% and 55%, and the adenine (A) content of the first RNA sequence is less than 6%. In each of these embodiments, it is preferred that the G content of the first RNA sequence is between 40.1% and 55%, and the adenine (A) content of the first RNA sequence is less than 4%. In each of these embodiments, it is preferred that the G content of the first RNA sequence is between 40.1% and 55%, and the adenine (A) content of the first RNA sequence is less than 2%. In each of these embodiments, it is preferred that the G content of the first RNA sequence is between 40.1% and 55%, and the adenine (A) content of the first RNA sequence is zero. In this context, these numbers are determined along the full length of the dsRNA region, bounded by the first and last basepairs.

[0066] The present inventors have also generated precursor RNA molecules which comprise a double- stranded RNA (dsRNA) region having an A ribonucleotide content which is low or even zero, optionally where G:U basepairing constitutes 10-40% of the total basepairing in the dsRNA region, optionally with a relatively high guanine (G) ribonucleotide content in at least one strand of the dsRNA region, referred to as the first RNA sequence or sense strand. These molecules have a decreased A ribonucleotide content and an increased G ribonucleotide content relative to a corresponding control RNA molecule which has only canonical basepairing in the dsRNA region, i.e. the corresponding control RNA lacking the G:U basepairing, and are readily designed by substituting at least some, preferably all, of the adenine (A) ribonucleotides in the first RNA sequence of the corresponding control RNA molecule, or both the first and second RNA sequences, to guanine (G) ribonucleotides. That is, at least some, preferably all, A:U basepairs in the control RNA molecule are replaced with G:U basepairs to generate the precursor RNA molecules of the invention. Thus, a hairpin RNA molecule or ledRNA molecule have a reduced A ribonucleotide content or preferably lacking A ribonucleotides in its dsRNA region or both dsRNA regions is contemplated. In an embodiment, the second (antisense) RNA sequence does not have such A to G substitutions, only the first RNA sequence has the substitutions.

[0067] Therefore, in a second, related aspect for precursor RNA molecules, that overlaps with the precursor RNA molecules of the first aspect, the present invention provides a precursor RNA molecule comprising at least one double-stranded RNA region, wherein the double- stranded RNA region comprises

[0068] (a) a first RNA strand which comprises a first RNA sequence of at least 21 contiguous ribonucleotides, preferably at least 24 contiguous ribonucleotides, and

[0069] (b) a second RNA strand which comprises a second RNA sequence of at least 21 contiguous ribonucleotides, preferably at least 24 contiguous ribonucleotides, wherein the first RNA strand and second RNA strand are covalently linked by a linking RNA sequence, wherein the first RNA sequence hybridises to the second RNA sequence by basepairing between the at least 21 or at least 24 contiguous ribonucleotides of the first RNA sequence and the at least 21 or at least 24 contiguous ribonucleotides of the second RNA sequence, forming the double- stranded RNA region, wherein the first RNA sequence along the full length of the double-stranded RNA region comprises an adenine (A) ribonucleotide content such that less than 10%, preferably less than 8%, more preferably less than 6%, even more preferably less than 4% or less than 2%, most preferably zero of the ribonucleotides of the first RNA sequence are A ribonucleotides, optionally wherein the double-stranded RNA region comprises a guanine (G) ribonucleotide content such that 36-55%, preferably 40.1-55%, of the ribonucleotides of the first RNA sequence are G ribonucleotides, and optionally wherein between 10% and 40%, preferably between 15% and 40%, or more preferably between 20% and 40%, of the ribonucleotides of the first RNA sequence and the second RNA sequence, in total, are basepaired in G:U basepairs. In an embodiment, the precursor RNA molecules comprise both the aforesaid G ribonucleotide content and the percentage of G:U basepairs. In an embodiment, the precursor RNA molecules comprise both the aforesaid A ribonucleotide content and the minimum length of the dsRNA region.

[0070] In an embodiment of the second aspect, the A ribonucleotide content of the first RNA sequence along the full length of the double- stranded RNA region is less than 10%.

[0071] In an embodiment of the second aspect, the A ribonucleotide content of the first RNA sequence along the full length of the double- stranded RNA region is less than 8%.

[0072] In an embodiment of the second aspect, the A ribonucleotide content of the first RNA sequence along the full length of the double- stranded RNA region is less than 6%.

[0073] In an embodiment of the second aspect, the A ribonucleotide content of the first RNA sequence along the full length of the double- stranded RNA region is less than 4%.

[0074] In an embodiment of the second aspect, the A ribonucleotide content of the first RNA sequence along the full length of the double- stranded RNA region is less than 2%.

[0075] In an embodiment of the second aspect, the A ribonucleotide content of the first RNA sequence along the full length of the double- stranded RNA region is zero.

[0076] In an embodiment, the A ribonucleotide content of the first and second RNA sequences, in total, is less than 20%, preferably less than 16%, more preferably less than

[0077] 12%, even more preferably less than 8% or 4%, most preferably zero.

[0078] In an embodiment, the A ribonucleotide content of the first and second RNA sequences, in total, is less than 20%.

[0079] In an embodiment, the A ribonucleotide content of the first and second RNA sequences, in total, is less than 16%.

[0080] In an embodiment, the A ribonucleotide content of the first and second RNA sequences, in total, is less than 12%.

[0081] In an embodiment, the A ribonucleotide content of the first and second RNA sequences, in total, is less than 8%. In an embodiment, the A ribonucleotide content of the first and second RNA sequences, in total, is less than 4%.

[0082] In an embodiment, the A ribonucleotide content of the first and second RNA sequences, in total, is zero.

[0083] Additionally, the second RNA sequence may be at least 80% identical to a sequence of at least 21, or at least 24, at least 48, at least 72, at least 96 or at least 120 contiguous ribonucleotides which is fully complementary to a first region of a target RNA molecule in a eukaryotic cell such as for example an insect cell or a fungal cell, preferably a plant cell. Any and all combinations of these features are contemplated for the precursor RNA molecule of the invention.

[0084] In an embodiment, the second RNA sequence is at least 80% identical to a sequence of at least 21, or at least 24, at least 48, at least 72, at least 96 or at least 120 contiguous ribonucleotides which is fully complementary to a first region of a target RNA molecule in a eukaryotic cell such as for example an insect cell or a fungal cell, preferably a plant cell.

[0085] In an embodiment, the second RNA sequence is at least 85% identical to a sequence of at least 21, or at least 24, at least 48, at least 72, at least 96 or at least 120 contiguous ribonucleotides which is fully complementary to a first region of a target RNA molecule in a eukaryotic cell such as for example an insect cell or a fungal cell, preferably a plant cell.

[0086] In an embodiment, the second RNA sequence is at least 90% identical to a sequence of at least 21, or at least 24, at least 48, at least 72, at least 96 or at least 120 contiguous ribonucleotides which is fully complementary to a first region of a target RNA molecule in a eukaryotic cell such as for example an insect cell or a fungal cell, preferably a plant cell.

[0087] In an embodiment, the second RNA sequence is at least 95% identical to a sequence of at least 21, or at least 24, at least 48, at least 72, at least 96 or at least 120 contiguous ribonucleotides which is fully complementary to a first region of a target RNA molecule in a eukaryotic cell such as for example an insect cell or a fungal cell, preferably a plant cell.

[0088] In an embodiment, the second RNA sequence is at least 100% identical to a sequence of at least 21, or at least 24, at least 48, at least 72, at least 96 or at least 120 contiguous ribonucleotides which is fully complementary to a first region of a target RNA molecule in a eukaryotic cell such as for example an insect cell or a fungal cell, preferably a plant cell. The recited G ribonucleotide content and / or low A ribonucleotide content of the precursor RNA molecule of the first or second aspect and all their embodiments, with the aforesaid G:U basepairing, can also be combined with the A22 or A23 or A24 modification in the dsRNA region as described herein, providing for non-basepaired ribonucleotides in the second RNA sequence which form bulges, thereby producing an asymmetric precursor RNA molecule. Such asymmetric precursor RNA molecules are processed by a Dicer to produce at least some asymmetric double- stranded product RNA molecules, having a sense RNA sequence which is sorter than the antisense RNA sequence. In such embodiments, the first RNA sequence of the precursor RNA molecule, is preferably shorter than the second RNA sequence by virtue of the bulged ribonucleotides in the second RNA sequence. For example, the asymmetric precursor RNA molecules of the invention may combine the A22 modification with the low A ribonucleotide content, the high G ribonucleotide content and G:U basepairs as described above. Therefore, in a third aspect, the present invention provides an asymmetric precursor RNA molecule comprising at least one double-stranded RNA region, wherein:

[0089] (i) the double- stranded RNA region comprises:

[0090] (a) a first RNA strand which comprises a first RNA sequence of at least 23 contiguous ribonucleotides, and

[0091] (b) a second RNA strand which comprises a second RNA sequence of at least 24 contiguous ribonucleotides, respectively, wherein the first RNA sequence hybridises to the second RNA sequence by basepairing between at least 22 of the at least 23 contiguous ribonucleotides of the first RNA sequence and at least 22 of the at least 24 ribonucleotides of the second RNA sequence, forming the double- stranded RNA region, wherein 1 or 2 ribonucleotides of the 24 ribonucleotides of the second RNA sequence are not basepaired to ribonucleotides of the first RNA sequence, and 0 or 1 ribonucleotides, respectively, of the first RNA sequence are not basepaired to ribonucleotides of the second RNA sequence, wherein the non-basepaired ribonucleotides form 1, 2 or 3 bulges in the doublestranded RNA region, wherein the first RNA sequence along the full length of the double-stranded RNA region comprises a guanine (G) ribonucleotide content such that 36-55%, preferably 40.1-55%, of the ribonucleotides of the first RNA sequence are G ribonucleotides, and wherein between 10% and 40%, preferably between 10% and 35%, or more preferably between 20% and 40%, of the ribonucleotides of the first RNA sequence and the second RNA sequence, in total, are basepaired in G:U basepairs. In an embodiment, the second RNA sequence is at least 80% identical to a sequence of at least 24 contiguous ribonucleotides which is fully complementary to a first region of a target RNA molecule in a eukaryotic cell such as for example an insect cell or a fungal cell, preferably a plant cell.

[0092] In an embodiment of this aspect, the first RNA sequence comprises at least 46 contiguous ribonucleotides, and the second RNA sequence comprises at least 48 contiguous ribonucleotides, wherein the first RNA sequence hybridises to the second RNA sequence by basepairing between at least 44 of the at least 46 contiguous ribonucleotides of the first RNA sequence and at least 44 of the at least 48 ribonucleotides of the second RNA sequence, forming the double- stranded RNA region, wherein 2, 3 or 4 ribonucleotides of the 48 ribonucleotides of the second RNA sequence are not basepaired to ribonucleotides of the first RNA sequence, and 0, 1 or 2 ribonucleotides, respectively, of the first RNA sequence are not basepaired to ribonucleotides of the second RNA sequence, and wherein the non-basepaired ribonucleotides form bulges in the double-stranded RNA region. In an embodiment, the first RNA sequence hybridises to the second RNA sequence by basepairing between the at least 46 contiguous ribonucleotides of the first RNA sequence and at least 46 of the at least 48 ribonucleotides of the second RNA sequence, forming the double- stranded RNA region, wherein 2 ribonucleotides of the 48 ribonucleotides of the second RNA sequence are not basepaired to ribonucleotides of the first RNA sequence, and 0 ribonucleotides of the first RNA sequence are not basepaired to ribonucleotides of the second RNA sequence, and wherein the non-basepaired ribonucleotides form one or two bulges in the doublestranded RNA region. In embodiments, the dsRNA region is extended in the same manner, whereby the second RNA sequence comprises at least 72, at least 96, or at least 120 ribonucleotides, with 1 bulge per 24 ribonucleotides when hybridised to the first RNA sequence.

[0093] In the previous two embodiments, the precursor RNA molecule may be cleaved in a eukaryotic cell, preferably a plant cell, to produce double- stranded product RNA molecules each independently consisting of a sense RNA sequence of 23 contiguous ribonucleotides from the first RNA sequence and an antisense RNA sequence of 24 contiguous ribonucleotides from the second RNA sequence, or an increased amount of such product RNA molecules relative to a corresponding control RNA molecule having only canonical basepairing, wherein at least 20 of ribonucleotides 1 to 21 of each sense RNA sequence basepair with at least 20 ribonucleotides from ribonucleotides 1 to 22 of the antisense RNA sequence, independently, in each of the double-stranded product RNA molecules, wherein the basepairs in each of the double- stranded product RNA molecules comprise, independently, 1, 2, 3, 4, 5, 6, 7, or 8 G:U basepairs, preferably 2-8 G:U basepairs, wherein 1 or 2 ribonucleotides of ribonucleotides 3-20 of the second RNA sequence are not basepaired to ribonucleotides of the first RNA sequence, and 0 or 1 ribonucleotides, respectively, of ribonucleotides 3-19 of the first RNA sequence are not basepaired to ribonucleotides of the second RNA sequence, wherein the non -basepaired ribonucleotides form 1, 2 or 3 bulges in the doublestranded product RNA molecules, wherein ribonucleotides 22 and 23 of the sense RNA sequence in each of the double-stranded product RNA molecules form a 2 -ribonucleotide unpaired 3' overhang, and 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.

[0094] The asymmetric precursor RNA molecules of the invention may also combine the A23 modification with the high G ribonucleotide content and G:U basepairs as described above. In a fourth aspect, the present invention provides an asymmetric precursor RNA molecule comprising at least one double-stranded RNA region, wherein:

[0095] (i) the double- stranded RNA region comprises:

[0096] (a) a first RNA strand which comprises a first RNA sequence of at least 22 contiguous ribonucleotides, and

[0097] (b) a second RNA strand which comprises a second RNA sequence of at least 24 contiguous ribonucleotides, respectively, wherein the first RNA sequence hybridises to the second RNA sequence by basepairing between at least 21 of the at least 22 contiguous ribonucleotides of the first RNA sequence and at least 21 of the at least 24 ribonucleotides of the second RNA sequence, forming the double- stranded RNA region, wherein 2 or 3 ribonucleotides of the 24 ribonucleotides of the second RNA sequence are not basepaired to ribonucleotides of the first RNA sequence, and 0 or 1 ribonucleotides, respectively, of the first RNA sequence are not basepaired to ribonucleotides of the second RNA sequence, wherein the non-basepaired ribonucleotides form bulges in the double-stranded RNA region, wherein the first RNA sequence along the full length of the double-stranded RNA region comprises a guanine (G) ribonucleotide content such that 36-55%, preferably 40.1-55%, of the ribonucleotides of the first RNA sequence are G ribonucleotides, and wherein between 10% and 40%, preferably between 10% and 35%, or more preferably between 20% and 40%, of the ribonucleotides of the first RNA sequence and the second RNA sequence, in total, are basepaired in G:U basepairs.

[0098] In an embodiment, the first RNA sequence hybridises to the second RNA sequence by basepairing between the at least 22 contiguous ribonucleotides of the first RNA sequence and at least 22 of the at least 24 ribonucleotides of the second RNA sequence, forming the double- stranded RNA region, wherein 2 ribonucleotides of the 24 ribonucleotides of the second RNA sequence are not basepaired to ribonucleotides of the first RNA sequence, and all of the at least 22 ribonucleotides of the first RNA sequence are basepaired to ribonucleotides of the second RNA sequence, wherein the non-basepaired ribonucleotides form bulges in the double-stranded RNA region.

[0099] In an embodiment of this aspect, the first RNA sequence comprises at least 44 contiguous ribonucleotides, and the second RNA sequence comprises at least 48 contiguous ribonucleotides, wherein the first RNA sequence hybridises to the second RNA sequence by basepairing between at least 42 of the at least 44 contiguous ribonucleotides of the first RNA sequence and at least 42 of the at least 48 ribonucleotides of the second RNA sequence, forming the double- stranded RNA region, wherein 4, 5 or 6 ribonucleotides of the 48 ribonucleotides of the second RNA sequence are not basepaired to ribonucleotides of the first RNA sequence, and 0, 1 or 2 ribonucleotides, respectively, of the first RNA sequence are not basepaired to ribonucleotides of the second RNA sequence, and wherein the non-basepaired ribonucleotides form bulges in the double-stranded RNA region.

[0100] In an embodiment, the precursor RNA molecule is cleaved in a eukaryotic cell, preferably a plant cell, to produce double-stranded product RNA molecules each independently consisting of a sense RNA sequence of 22 contiguous ribonucleotides from the first RNA sequence and an antisense RNA sequence of 24 contiguous ribonucleotides from the second RNA sequence, or an increased amount of such product RNA molecules relative to a corresponding control RNA molecule having only canonical basepairing, wherein at least 19 of ribonucleotides 1 to 20 of the sense RNA sequence basepair with at least 19 ribonucleotides from ribonucleotides 1 to 22 of the antisense RNA sequence, independently, in each of the double- stranded product RNA molecules, wherein the basepairs in each of the double- stranded product RNA molecules comprise, independently, 1, 2, 3, 4, 5, 6, 7, or 8 G:U basepairs, preferably 2-8 G:U basepairs, wherein 2 or 3 ribonucleotides of ribonucleotides 3-20 of the second RNA sequence are not basepaired to ribonucleotides of the first RNA sequence, and 0 or 1 ribonucleotides, respectively, of ribonucleotides 3-18 of the first RNA sequence are not basepaired to ribonucleotides of the second RNA sequence, wherein the non -basepaired ribonucleotides form 1, 2 or 3 bulges in the doublestranded product RNA molecules, wherein ribonucleotides 21 and 22 of the sense RNA sequence in each of the double-stranded product RNA molecules form a 2 -ribonucleotide unpaired 3' overhang, and 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.

[0101] In an embodiment, the precursor RNA molecule is cleaved in a eukaryotic cell, preferably a plant cell, to also 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, or an increased amount of such product RNA molecules relative to a corresponding control RNA molecule having only canonical basepairing, wherein at least 18 ribonucleotides from ribonucleotides 1 to 19 of the sense RNA sequence basepair with at least 18 ribonucleotides from ribonucleotides 1 to 21 of the antisense RNA sequence in each of the double- stranded product RNA molecules, wherein the basepairs in each of the double- stranded product RNA molecules comprise, independently, 1, 2, 3, 4, 5, 6, 7, or 8 G:U basepairs, preferably 2-8 G:U basepairs, wherein 2 or 3 ribonucleotides of ribonucleotides 3-19 of the second RNA sequence are not basepaired to ribonucleotides of the first RNA sequence, and 0 or 1 ribonucleotides, respectively, of ribonucleotides 3-17 of the first RNA sequence are not basepaired to ribonucleotides of the second RNA sequence, wherein the non-basepaired ribonucleotides form bulges in the double-stranded product RNA molecules, 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 22 and 23 of the antisense RNA sequence in each of the double-stranded product RNA molecules form a 2-ribonucleotide unpaired 3' overhang.

[0102] The asymmetric precursor RNA molecules of the invention may also combine the A24 modification with the high G ribonucleotide content and G:U basepairs as described above. Therefore, in a fifth aspect, the present invention provides an asymmetric precursor RNA molecule comprising at least one double-stranded RNA region, wherein:

[0103] (i) the double- stranded RNA region comprises:

[0104] (a) a first RNA strand which comprises a first RNA sequence of at least 21 contiguous ribonucleotides, and

[0105] (b) a second RNA strand which comprises a second RNA sequence of at least 24 contiguous ribonucleotides, respectively, wherein the first RNA sequence hybridises to the second RNA sequence by basepairing between at least 20 of the at least 21 contiguous ribonucleotides of the first RNA sequence and at least 20 of the at least 24 ribonucleotides of the second RNA sequence, forming the double- stranded RNA region, wherein 3 or 4 ribonucleotides of the 24 ribonucleotides of the second RNA sequence are not basepaired to ribonucleotides of the first RNA sequence, and 0 or 1 ribonucleotides, respectively, of the first RNA sequence are not basepaired to ribonucleotides of the second RNA sequence, wherein the non-basepaired ribonucleotides form bulges in the double-stranded RNA region, wherein the first RNA sequence along the full length of the double-stranded RNA region comprises a guanine (G) ribonucleotide content such that 36-55%, preferably 40.1-55%, of the ribonucleotides of the first RNA sequence are G ribonucleotides, and wherein between 10% and 40%, preferably between 10% and 35%, or more preferably between 20% and 40%, of the ribonucleotides of the first RNA sequence and the second RNA sequence, in total, are basepaired in G:U basepairs.

[0106] In an embodiment of this aspect, the first RNA sequence hybridises to the second RNA sequence by basepairing between the at least 21 contiguous ribonucleotides of the first RNA sequence and at least 21 of the at least 24 ribonucleotides of the second RNA sequence, forming the double- stranded RNA region, wherein 3 ribonucleotides of the 24 ribonucleotides of the second RNA sequence are not basepaired to ribonucleotides of the first RNA sequence, and all of the ribonucleotides of the first RNA sequence are basepaired to ribonucleotides of the second RNA sequence, wherein the non-basepaired ribonucleotides form bulges in the double-stranded RNA region.

[0107] In an embodiment of this aspect, the first RNA sequence comprises at least 42 contiguous ribonucleotides, and the second RNA sequence comprises at least 48 contiguous ribonucleotides, wherein the first RNA sequence hybridises to the second RNA sequence by basepairing between at least 40 of the at least 42 contiguous ribonucleotides of the first RNA sequence and at least 40 of the at least 48 ribonucleotides of the second RNA sequence, forming the double- stranded RNA region, wherein 6, 7 or 8 ribonucleotides of the 48 ribonucleotides of the second RNA sequence are not basepaired to ribonucleotides of the first RNA sequence, and 0, 1 or 2 ribonucleotides, respectively, of the first RNA sequence are not basepaired to ribonucleotides of the second RNA sequence, and wherein the non-basepaired ribonucleotides form bulges in the double-stranded RNA region.

[0108] In an embodiment, the precursor RNA molecule is cleaved in a eukaryotic cell, preferably a plant cell, 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, or an increased amount of such product RNA molecules relative to a corresponding control RNA molecule having only canonical basepairing, wherein at least 18 of ribonucleotides 1 to 19 of the sense RNA sequence basepair with at least 18 ribonucleotides from ribonucleotides 1 to 22 of the antisense RNA sequence, independently, in each of the double- stranded product RNA molecules, wherein the basepairs in each of the double- stranded product RNA molecules comprise, independently, 1, 2, 3, 4, 5, 6, 7, or 8 G:U basepairs, preferably 2-8 G:U basepairs, wherein 3 or 4 ribonucleotides of ribonucleotides 3-20 of the second RNA sequence are not basepaired to ribonucleotides of the first RNA sequence, and 0 or 1 ribonucleotides, respectively, of ribonucleotides 3-17 of the first RNA sequence are not basepaired to ribonucleotides of the second RNA sequence, wherein the non-basepaired ribonucleotides form bulges in the double-stranded product RNA molecules, 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 23 and 24 of the antisense RNA sequence in each of the double-stranded product RNA molecules form a 2-ribonucleotide unpaired 3' overhang.

[0109] In an embodiment, the precursor RNA molecule is cleaved in a eukaryotic cell, preferably a plant cell, to also produce double -stranded product RNA molecules each independently consisting of a sense RNA sequence of 20 contiguous ribonucleotides from the first RNA sequence and an antisense RNA sequence of 23 contiguous ribonucleotides from the second RNA sequence, or an increased amount of such product RNA molecules relative to a corresponding control RNA molecule having only canonical basepairing, wherein at least 17 ribonucleotides from ribonucleotides 1 to 18 of the sense RNA sequence basepair with at least 17 ribonucleotides from ribonucleotides 1 to 21 of the antisense RNA sequence in each of the double- stranded product RNA molecules, wherein the basepairs in each of the double- stranded product RNA molecules comprise, independently, 1, 2, 3, 4, 5, 6, 7, or 8 G:U basepairs, preferably 2-8 G:U basepairs, wherein 3 or 4 ribonucleotides of ribonucleotides 3-19 of the second RNA sequence are not basepaired to ribonucleotides of the first RNA sequence, and 0 or 1 ribonucleotides, respectively, of ribonucleotides 3-16 of the first RNA sequence are not basepaired to ribonucleotides of the second RNA sequence, wherein the non-basepaired ribonucleotides form bulges in the double-stranded product RNA molecules, wherein ribonucleotides 19 and 20 of the sense RNA sequence in each of the double-stranded product RNA molecules form a 2-ribonucleotide unpaired 3' overhang, and 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.

[0110] In an embodiment of the above aspects, the second RNA sequence is at least 80% identical to a sequence of at least 23 or at least 24 contiguous ribonucleotides, as the context determines, which is fully complementary to a first region of a target RNA molecule in a eukaryotic cell such as for example an insect cell or a fungal cell, preferably a plant cell.

[0111] Additionally in the above embodiments of the asymmetric RNA molecules of the invention, the first RNA sequence along the full length of the double-stranded RNA region may comprise an adenine (A) ribonucleotide content such that less than 10%, preferably less than 8%, more preferably less than 6%, even more preferably less than 4% or less than 2%, most preferably zero of the ribonucleotides of the first RNA sequence are A ribonucleotides. In an embodiment, the asymmetric precursor RNA molecule comprises both the aforesaid A ribonucleotide content and the minimum length of the dsRNA region as described above. Alternatively, or in addition, the second RNA sequence is at least 80% identical to a sequence of at least 48, at least 72, at least 86 or at least 120 contiguous ribonucleotides which is fully complementary to a first region of a target RNA molecule in a eukaryotic cell such as for example an insect cell or a fungal cell, preferably a plant cell.

[0112] The G ribonucleotide content in the double- stranded RNA region of the precursor RNA molecules of the above embodiments is increased relative to a corresponding conventional RNA molecule (control RNA molecule) having the same second RNA sequence and a first RNA sequence which is fully canonically-basepaired to the second RNA sequence. That is, the increase in G ribonucleotide content is at least in part because of A to G substitutions in at least the first RNA sequence relative to the first RNA sequence of the control RNA molecule. In an embodiment, the second RNA sequence also comprises A to G substitutions, relative to the complement of the first region of the target RNA molecule, in addition to the A to G substitutions in the first RNA sequence relative to the control RNA molecule. In an embodiment, all of the A ribonucleotides in the first RNA sequence of the control RNA molecule are replaced with G ribonucleotides to make the precursor RNA molecule of the invention, thereby providing G:U basepairs in the dsRNA region formed by hybridisation of the first and second RNA sequences, and resulting in the increase in the content of G ribonucleotides to the desired level. That is, the G:U basepairs formed in the precursor RNA molecule of the invention that have a G in the first RNA sequence and a U in the second RNA sequence are at positions occupied by A:U basepairs in the control RNA molecule. The dsRNA region of the precursor RNA molecule may also have G:U basepairs that result from A to G or C to T substitutions in the second RNA sequence, relative to the control RNA molecule. In an embodiment, some of the G:U basepairs, preferably less than 50% of the total number, have the U ribonucleotide in the first RNA sequence and the G ribonucleotide in the second RNA sequence.

[0113] In each case, it is understood that the defined ranges are with regard to the full length of the first RNA sequence, or the full length of the first and second RNA sequences, i.e. to the full extent of the dsRNA region, as the context determines.

[0114] In an embodiment, at least 20, preferably at least 21 or 22, more preferably at least 23 or most preferably all 24 ribonucleotides of the 24nt antisense RNA sequences in the double-stranded product RNA molecules produced in a plant cell are each, independently, identical in sequence to the complement of 24 contiguous ribonucleotides of a target RNA molecule. In this embodiment, the increase in the number of doublestranded product RNA molecules consisting of a sense RNA sequence of 24 contiguous ribonucleotides from the first RNA sequence and an antisense RNA sequence of 24 contiguous ribonucleotides from the second RNA sequence (24 / 24-mers) from the precursor RNA molecule is relative to the number of 24 / 24-mers from the corresponding control RNA molecule. Likewise, the increase in the number of double- stranded product RNA molecules consisting of a sense RNA sequence of 23 contiguous ribonucleotides from the first RNA sequence and an antisense RNA sequence of 24 contiguous ribonucleotides from the second RNA sequence (23 / 24-mers) from the precursor RNA molecule is relative to the number of 23 / 24-mers from the corresponding control RNA molecule. Again, the increase in the number of double-stranded product RNA molecules consisting of a sense RNA sequence of 22 contiguous ribonucleotides from the first RNA sequence and an antisense RNA sequence of 24 contiguous ribonucleotides from the second RNA sequence (22 / 24-mers) from the precursor RNA molecule is relative to the number of 22 / 24-mers from the corresponding control RNA molecule. Further, the increase in the number of double-stranded product RNA molecules 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 (21 / 24-mers) from the precursor RNA molecule is relative to the number of 21 / 24-mers from the corresponding control RNA molecule.

[0115] The analogous comparisons apply to the double- stranded product RNA molecules comprising an antisense RNA sequence of 23 contiguous ribonucleotides from the second RNA sequence. In an embodiment, more of the double- stranded product RNA molecules consisting of a sense RNA sequence of 23 contiguous ribonucleotides from the first RNA sequence and an antisense RNA sequence of 23 contiguous ribonucleotides from the second RNA sequence (23 / 23-mers) are produced from the precursor RNA molecule than from the corresponding control RNA molecule. In an embodiment, both the 23 / 23-mer and 24 / 24-mer product RNA molecules are produced from the precursor RNA molecule, preferably both at an increased level compared to those produced from the corresponding control RNA molecule, and preferably more 24 / 24-mers than 23 / 23- mers.

[0116] In an embodiment, more of the double- stranded product RNA molecules consisting of a sense RNA sequence of 22 contiguous ribonucleotides from the first RNA sequence and an antisense RNA sequence of 23 contiguous ribonucleotides from the second RNA sequence (22 / 23 -mers) are produced from the precursor RNA molecule than from a corresponding control RNA molecule having the same second RNA sequence and completely canonical basepairing in the dsRNA region. In an embodiment, both the 22 / 23-mer and 23 / 24-mer product RNA molecules are produced, preferably both at an increased level compared to those produced from the control RNA molecule, and preferably more 23 / 24-mers than 22 / 23-mers.

[0117] The increased number of specific siRNA product molecules such as the 24 / 24- mers or 23 / 23-mers from the symmetrical precursor RNA molecules, or the 23 / 24-mers, 22 / 23-mers, 21 / 23-mers or 22 / 24-mers etc from the asymmetric precursor RNA molecules relative to the control RNA molecule in plant cells is thought to be due to the altered susceptibility to cleavage by DCL-3 and DCL-4 by virtue of the altered ribonucleotide composition, in particular the A to G substitutions in the first RNA sequence or the first and second RNA sequences. This may merely be due to the low level of A ribonucleotides, the presence of A ribonucleotides assisting the activity of DCL4 and DCL2 in the plant cells. That is, DCL-4 activity which produces mainly 21 / 21- mers from a symmetric control RNA molecule or 21 / 22-mers from the asymmetric RNA precursor molecule is reduced through the nucleotide compositions of the dsRNA region, and DCL-3 activity which produces mainly 24 / 24-mers or 23 / 24-mers, respectively, is relatively increased compared to the activities on the control RNA.

[0118] In an embodiment, the increase in the number of double-stranded product RNA molecules consisting of a sense RNA sequence of 22 contiguous ribonucleotides from the first RNA sequence and an antisense RNA sequence of 24 contiguous ribonucleotides from the second RNA sequence (22 / 24-mers) from the precursor RNA molecule is relative to the number of 22 / 24-mers from a corresponding control RNA molecule having the A23 modification but lacking the A to G substitutions in the first RNA sequence.

[0119] In an embodiment, at least 20, preferably at least 21, more preferably at least 22 or most preferably all 23 ribonucleotides of the 23nt antisense RNA sequences in the double-stranded product RNA molecules are each, independently, identical in sequence to the complement of 23 contiguous ribonucleotides of the target RNA molecule. In an embodiment, more of those double- stranded product RNA molecules 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 (21 / 23-mers) are produced from the precursor RNA molecule than from a control RNA molecule having the same second RNA sequence and completely canonical basepairing in the dsRNA region. In an embodiment, both the 21 / 23-mer and 22 / 24-mer product RNA molecules are produced, preferably both at an increased level compared to those produced from the control RNA molecule, and preferably more 22 / 24-mers than 21 / 23-mers.

[0120] The G ribonucleotide content of the first RNA sequence, or first and second RNA sequences, can be readily adjusted to a desired level through the use of A to G substitutions, optionally also with C to T substitutions to provide more G:U basepairs. In an embodiment (i), the G ribonucleotide content of the first RNA sequence of the precursor RNA molecule of the invention is in the range 37-55%, 38-55%, 39-55%, 40.1- 55%, 41-55%, 36-54%, 37-54%, 38-54%, 39-54%, 40.1-54%, 41-54%, 36-53%, 37- 53%, 38-53%, 39-53% or 40.1-53%, in each case inclusive of the endpoints of the range, or about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49% or about 50%. In an embodiment (ii), the A ribonucleotide content of the first RNA sequence is less than 10%. In an embodiment (iii), the A ribonucleotide content of the first RNA sequence is less than 8%. In an embodiment (iv), the A ribonucleotide content of the first RNA sequence is less than 6%. In an embodiment (v), the A ribonucleotide content of the first RNA sequence is less than 4%. In an embodiment (vi), the A ribonucleotide content of the first RNA sequence is less than 2%. In a preferred embodiment (vii), the A ribonucleotide content of the first RNA sequence is zero. It is understood that the defined ranges are with regard to the full length of the first RNA sequence. Each of the combinations of ranges (i) and (ii), (i) and (iii), (i) and (iv), (i) and (v), (i) and (vi), and (i) and (vii) are contemplated.

[0121] In an embodiment (viii), the G ribonucleotide content of the first and second RNA sequences, in total, of the precursor RNA molecule is in the range 30-55%, 31-55%, 32- 55%, 33-55%, 34-55%, 35-55%, 30-52%, 31-52%, 32-52%, 33-52%, 34-52%, 35-52%, 30-50%, 31-50%, 32-50%, 33-50%, 34-50% or 35-50%, in each case inclusive of the endpoints of the range, or about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49% or about 50%. In this context, “about” means + / - 10% of the recited value. In an embodiment (ix), the A ribonucleotide content of the first and second RNA sequences, in total, is less than 20%. In an embodiment (x), the A ribonucleotide content of the first and second RNA sequences, in total, is less than 16%. In an embodiment (xi), the A ribonucleotide content of the first and second RNA sequences, in total, is less than 12%. In an embodiment (xii), the A ribonucleotide content of the first and second RNA sequences, in total, is less than 8%. In an embodiment (xiii), the A ribonucleotide content of the first and second RNA sequences, in total, is less than 4%. It is understood that the defined ranges are with regard to the full length of the first and second RNA sequences, i.e. to the full extent of the dsRNA region. Each of the combinations (viii) and (ix), (viii) and (x), (viii) and (xi), (viii) and (xii), and (viii) and (xiii), are contemplated, as are the combinations (i) and (viii), (ii) and (ix), (iii) and (x), (iv) and (xi), (v) and (xii) and (vi) and (xiii), (vii) and (viii), (vii) and (xi), (vii) and (xii). Each of the ranges (i) to (vi) may be combined with each of the ranges (viii) to (xiii).

[0122] The design principles for the asymmetric precursor RNA molecules can be applied in an extended fashion to longer double -stranded regions. For example, the nonbasepaired ribonucleotides in the second RNA sequence may be arranged in a periodic fashion to provide a population of product RNA molecules having multiple, nonoverlapping antisense RNA sequences of 24nt. Such precursor RNA molecules are particularly useful for reducing expression of a target RNA molecule in a plant cell, insect 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, the first RNA sequence comprises at least 48 contiguous ribonucleotides and the second RNA sequence comprises at least 50 contiguous ribonucleotides.

[0123] In the above embodiments, the ribonucleotide bulges, preferably singleribonucleotide bulges, may be spaced apart by 16-26 contiguous basepairs, preferably by 17-24 or 18-23 contiguous basepairs, 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 embodiment, 1, 2, 3, 4, 5 or 6 of the basepairs between the bulges are G:U basepairs.

[0124] The precursor RNA molecule may comprise two or more such double-stranded RNA regions, for example comprise a ledRNA structure. In these embodiments, the eukaryotic cell in which the precursor RNA molecule 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.

[0125] In a different embodiment with longer dsRNA regions of at least 48 basepairs, the precursor RNA molecule lacks the linking RNA sequence, or alternatively comprises the linking RNA sequence, and the first RNA sequence and the second RNA sequence hybridise to form the double-stranded RNA region. Such precursor RNA molecules may be readily produced in a cell-free system, for example in vitro. In a preferred embodiment of the molecule lacking the linking RNA sequence, the first RNA sequence comprises at least 48 contiguous ribonucleotides and the second RNA sequence at least 50 contiguous ribonucleotides, to aid hybridisation of the two sequences.

[0126] In an embodiment, the precursor RNA molecule is cleaved in a plant cell by one or more ribonucleases (RNases), preferably DCL3, to produce multiple, different doublestranded product RNA molecules consisting of a sense RNA sequence of 21, 22, 23 or 24 contiguous ribonucleotides and an antisense RNA sequence of 23 or 24 contiguous ribonucleotides.

[0127] The first RNA sequence and the second RNA sequence may extend to longer than 48 and 50 ribonucleotides, respectively. In another embodiment, for even longer dsRNA region(s), the first RNA sequence comprises at least 69 contiguous ribonucleotides and the second RNA sequence comprises at least 72 contiguous ribonucleotides, wherein the first RNA sequence hybridises to the second RNA sequence by basepairing between at least 66, preferably at least 67, at least 68 or at least 69, of the at least 69 contiguous ribonucleotides of the first RNA sequence and at least 66, preferably at least 67, at least 668 or at least 69, respectively, of the at least 72 contiguous ribonucleotides of the second RNA sequence.

[0128] In the above embodiments for the asymmetric precursor RNA molecules, the second RNA sequence may be longer than the first RNA sequence because of the nonbasepaired ribonucleotides that bulge out in the second RNA sequence. In a preferred embodiment using the A22 modification, the length of the first RNA sequence is 94%- 97% or 94%-96% of the length of the second RNA sequence. These features apply across the full length of the double- stranded region, where the part of the double- stranded region is the full length of the double- stranded region.

[0129] As the skilled person would appreciate, further embodiments include a first RNA sequence hybridised to a second RNA sequence having longer contiguous ribonucleotides following the same principles described in the above embodiments.

[0130] In an embodiment, the first RNA sequence and the second RNA sequence 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 and the second RNA sequence 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 such sense RNA sequences of the dsRNA region is 94%-97% or 94%-96% the length of the antisense sequence. These features are applicable to both 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 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.

[0131] In embodiments of the precursor RNA molecules, the double- stranded product RNA molecules produced therefrom may have the following feature:

[0132] (i) ribonucleotides 1, 2 and 3 of the sense RNA sequence basepair with ribonucleotides 20, 21 and 22, respectively, of the 24nt antisense RNA sequences, or ribonucleotides 19, 20 and 21 of the 23nt antisense RNA sequences, in at least some of the one or more double- stranded product RNA molecule(s), or

[0133] (ii) ribonucleotides 20, 21 and 22 of the 24nt sense RNA sequences, or ribonucleotides 19, 20 and 21 of the 23nt sense RNA sequences, basepair with ribonucleotides 1, 2 and 3, respectively, of the antisense RNA sequence in at least some of the one or more double-stranded product RNA molecule(s), 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. 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 are involved in G:U basepairs, more preferably one or both of ribonucleotides 1 and 2 of the antisense sequence are involved in G:U basepairs, even more preferably ribonucleotide 1 of the antisense sequence is involved in G:U basepairs. Most preferably, one or more of ribonucleotides 1, 2 and 3 of the antisense sequence 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.

[0134] Each of the features of the embodiments of these aspects can be applied to longer double-stranded regions to provide essentially either multimers of the product RNA molecules or combinations of different designs of product RNA molecules. In an embodiment, the asymmetric precursor RNA molecule is cleaved in a eukaryotic cell by one or more ribonucleases (RNases) to produce multiple, different double- stranded product RNA molecules which each consist of a sense RNA sequence of 23 contiguous ribonucleotides from the first RNA sequence and an antisense RNA sequence of 24 contiguous ribonucleotides from the second RNA sequence, wherein at least some of the multiple, different double- stranded product RNA molecules have overlapping antisense RNA sequences. In a further embodiment, at least some of the multiple, different doublestranded product RNA molecules have non-overlapping antisense RNA sequences, where the population of double- stranded product RNA molecules produced from the precursor RNA molecule includes some overlapping and some non-overlapping antisense RNA sequences. Preferably, there are more non-overlapping antisense RNA sequences, as readily occurs with longer (>48 basepairs) double- stranded regions, where phased cleavage by Dicer can occur.

[0135] Each of the embodiments may have the following feature: at least some of the one or more double- stranded product RNA molecule(s) comprise one or two or three nonbasepaired ribonucleotide(s) selected from the group consisting of ribonucleotides 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 and 19 of the antisense RNA sequence.

[0136] Each of the embodiments may have the following feature: at least some of the one or more double- stranded product RNA molecule(s) 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, 17, 18 and 19 of the antisense RNA sequence.

[0137] In another aspect, the present invention provides asymmetric precursor RNA molecules which are processed to produce siRNA molecules consisting of 22nt sense RNA sequences hybridised to 24nt antisense RNA sequences. Therefore, in this aspect the present invention provides a precursor RNA molecule comprising at least one doublestranded RNA region, wherein:

[0138] (i) the double- stranded RNA region comprises:

[0139] (a) a first RNA strand which comprises a first RNA sequence of at least 22 contiguous ribonucleotides, and a second RNA strand which comprises a second RNA sequence of at least 24 contiguous ribonucleotides, wherein the first RNA strand and second RNA strand are covalently linked by a linking RNA sequence, wherein the first RNA sequence hybridises to the second RNA sequence by basepairing between at least 21 of the at least 22 contiguous ribonucleotides of the first RNA sequence and at least 21 of the at least 24 contiguous ribonucleotides of the second RNA sequence, forming the double- stranded RNA region, wherein the double- stranded RNA region comprises a guanine (G) ribonucleotide content such that 36-55%, preferably 40.1-55%, of the ribonucleotides of the first RNA sequence are G ribonucleotides, wherein between 10% and 40%, preferably between 15% and 40%, or more preferably between 20% and 40%, of the ribonucleotides of the first RNA sequence and the second RNA sequence, in total, are basepaired in G:U basepairs, wherein 2 or 3 ribonucleotides of the at least 24 ribonucleotides of the second RNA sequence are non-basepaired and 0 or 1, respectively, ribonucleotides of the at least 22 ribonucleotides of the first RNA sequence are non-basepaired, forming one, two, three or four bulges in the part of the double-stranded RNA region, wherein each of the one, two, three or four bulges is immediately flanked by ribonucleotides which are basepaired in the double- stranded RNA region. In an embodiment, the precursor RNA molecule is cleaved in a eukaryotic cell by one or more ribonucleases (RNases) to produce one or more double-stranded product RNA molecule(s) consisting of a sense RNA sequence of 22 contiguous ribonucleotides from the first RNA sequence and an antisense RNA sequence of 24 contiguous ribonucleotides from the second RNA strand, including at least 22 contiguous ribonucleotides from the second RNA sequence, wherein the one or more double- stranded product RNA molecule(s) comprise the one, two, three or four bulges, wherein 19 or 20 of ribonucleotides 1 to 20 of the sense RNA sequence each basepair with a ribonucleotide of ribonucleotides 1 to 22 of antisense RNA sequence in the one or more double- stranded product RNA molecule(s), wherein ribonucleotides 21 and 22 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 in the one or more doublestranded product RNA molecule(s), wherein ribonucleotides 1 and 2 of the sense RNA sequence basepair with ribonucleotides 21 and 22, respectively, of the antisense RNA sequence in the one or more double-stranded product RNA molecule(s), and wherein ribonucleotides 19 and 20 of the sense RNA sequence basepair with ribonucleotides 1 and 2, respectively, of the antisense RNA sequence in the one or more double-stranded product RNA molecule(s). In an embodiment, the first RNA sequence hybridises to the second RNA sequence by basepairing between at least 21 of the at least 22 contiguous ribonucleotides of the first RNA sequence and at least 21 of the at least 24 contiguous ribonucleotides of the second RNA sequence, wherein the double- stranded RNA region comprises a guanine (G) ribonucleotide content such that 36-55%, preferably 40.1-55%, of the ribonucleotides of the first RNA sequence are G ribonucleotides, wherein between 10% and 40%, preferably between 15% and 40%, or more preferably between 20% and 40%, of the ribonucleotides of the first RNA sequence and the second RNA sequence, in total, are basepaired in G:U basepairs, wherein 3 ribonucleotides, 2 of which are contiguous, of the at least 24 ribonucleotides of the second RNA sequence are non-basepaired and 1 ribonucleotide of the at least 22 ribonucleotides of the first RNA sequence is non-basepaired, the non- basepaired ribonucleotides forming two bulges in the double- stranded RNA region, wherein each bulge is immediately flanked by ribonucleotides which are basepaired in the part of the double- stranded RNA region, wherein one bulge is formed by a mismatched ribonucleotide pair between the first RNA sequence and the second RNA sequence and the second bulge is a diribonucleotide bulge, wherein the one or more double- stranded product RNA molecule(s) comprise the bulges, and wherein all but one of ribonucleotides 1 to 20 of the sense RNA sequence each basepair with a ribonucleotide of ribonucleotides 1 to 22 of the antisense RNA sequence in the one or more double-stranded product RNA molecule(s).

[0140] In an embodiment, the first RNA sequence hybridises to the second RNA sequence by basepairing between at least 21 of the at least 22 contiguous ribonucleotides of the first RNA sequence and at least 21 of the at least 24 contiguous ribonucleotides of the second RNA sequence, wherein 3 ribonucleotides of the at least 24 ribonucleotides of the second rNA sequence are non-basepaired, and 1 ribonucleotide of the at least 22 ribonucleotides of the first RNA sequence is non-basepaired, the non-basepaired ribonucleotides forming four single-ribonucleotide bulges in the double-stranded RNA region, wherein the double- stranded RNA region comprises a guanine (G) ribonucleotide content such that 36-55%, preferably 40.1-55%, of the ribonucleotides of the first RNA sequence are G ribonucleotides, wherein between 10% and 40%, preferably between 15% and 40%, or more preferably between 20% and 40%, of the ribonucleotides of the first RNA sequence and the second RNA sequence, in total, are basepaired in G:U basepairs, wherein each bulge is immediately flanked by ribonucleotides which are basepaired in the double- stranded RNA region, wherein the one or more double- stranded product RNA molecule(s) comprise the bulges, and wherein all but one of ribonucleotides 1 to 20 of the sense RNA sequence each basepair with a ribonucleotide of ribonucleotides 1 to 22 of antisense RNA sequence in the one or more double- stranded product RNA molecule(s).

[0141] In an embodiment, the first RNA sequence hybridises to the second RNA sequence by basepairing between at least 22 of the at least 22 contiguous ribonucleotides of the first RNA sequence and at least 22 of the at least 24 contiguous ribonucleotides of the second RNA sequence, wherein 2 contiguous ribonucleotides of the at least 24 ribonucleotides of the second RNA sequence are non-basepaired and all of the at least 22 ribonucleotides of the first RNA sequence are basepaired in the double- stranded RNA region, the 2 non- basepaired ribonucleotides forming a di-ribonucleotide bulge, or two singleribonucleotide bulges, in the double-stranded RNA region, wherein the double- stranded RNA region comprises a guanine (G) ribonucleotide content such that 36-55%, preferably 40.1-55%, of the ribonucleotides of the first RNA sequence are G ribonucleotides, wherein between 10% and 40%, preferably between 15% and 40%, or more preferably between 20% and 40%, of the ribonucleotides of the first RNA sequence and the second RNA sequence, in total, are basepaired in G:U basepairs, wherein each bulge is immediately flanked by ribonucleotides of the second RNA sequence which are basepaired to ribonucleotides of the first RNA sequence, wherein the one or more double-stranded product RNA molecule(s) comprise at least one bulge, and wherein ribonucleotides 1 to 20 of the sense RNA sequence each basepair with a ribonucleotide of ribonucleotides 1 to 22 of antisense RNA sequence in the one or more double-stranded product RNA molecule(s).

[0142] In an embodiment, the first RNA sequence hybridises to the second RNA sequence by basepairing between at least 22 of the at least 22 contiguous ribonucleotides of the first RNA sequence and at least 22 of the at least 24 contiguous ribonucleotides of the second RNA sequence, wherein 2 ribonucleotides of the at least 24 ribonucleotides of the second RNA sequence are non-basepaired and all of the at least 22 ribonucleotides of the first RNA sequence are basepaired in the double- stranded RNA region, the 2 non-basepaired ribonucleotide forming two single-ribonucleotide bulges in the double-stranded RNA region, wherein the double- stranded RNA region comprises a guanine (G) ribonucleotide content such that 36-55%, preferably 40.1-55%, of the ribonucleotides of the first RNA sequence are G ribonucleotides, wherein between 10% and 40%, preferably between 15% and 40%, or more preferably between 20% and 40%, of the ribonucleotides of the first RNA sequence and the second RNA sequence, in total, are basepaired in G:U basepairs, wherein each bulge is immediately flanked by ribonucleotides of the second RNA sequence which are basepaired to ribonucleotides of the first RNA sequence, wherein the one or more double- stranded product RNA molecule(s) comprise the two bulges, and wherein ribonucleotides 1 to 20 of the sense RNA sequence each basepair with a ribonucleotide of ribonucleotides 1 to 22 of antisense RNA sequence in the one or more double-stranded product RNA molecule(s).

[0143] In an embodiment, the first RNA sequence hybridises to the second RNA sequence by basepairing between at least 21 of the at least 22 contiguous ribonucleotides of the first RNA sequence and at least 21 of the at least 24 contiguous ribonucleotides of the second RNA sequence, wherein 3 contiguous ribonucleotides of the at least 24 ribonucleotides of the second RNA sequence are non-basepaired, and 1 ribonucleotide of the at least 22 ribonucleotides of the first RNA sequence is non-basepaired, the non-basepaired ribonucleotides forming a bulge in the double- stranded RNA region, wherein the double- stranded RNA region comprises a guanine (G) ribonucleotide content such that 36-55%, preferably 40.1-55%, of the ribonucleotides of the first RNA sequence are G ribonucleotides, wherein between 10% and 40%, preferably between 15% and 40%, or more preferably between 20% and 40%, of the ribonucleotides of the first RNA sequence and the second RNA sequence, in total, are basepaired in G:U basepairs, wherein the bulge is immediately flanked by ribonucleotides which are basepaired in the double-stranded RNA region, wherein the one or more double- stranded product RNA molecule(s) comprise the bulge, and wherein all but one of ribonucleotides 1 to 20 of the sense RNA sequence each basepair with a ribonucleotide of ribonucleotides 1 to 22 of antisense RNA sequence in the one or more double- stranded product RNA molecule(s).

[0144] In an embodiment, the first RNA sequence hybridises to the second RNA sequence by basepairing between at least 21 of the at least 22 contiguous ribonucleotides of the first RNA sequence and at least 21 of the at least 24 contiguous ribonucleotides of the second RNA sequence, wherein the double- stranded RNA region comprises a guanine (G) ribonucleotide content such that 36-55%, preferably 40.1-55%, of the ribonucleotides of the first RNA sequence are G ribonucleotides, wherein between 10% and 40%, preferably between 15% and 40%, or more preferably between 20% and 40%, of the ribonucleotides of the first RNA sequence and the second RNA sequence, in total, are basepaired in G:U basepairs, wherein 3 ribonucleotides of the at least 24 ribonucleotides of the second RNA sequence are non-basepaired, and 1 ribonucleotide of the at least 22 ribonucleotides of the first RNA sequence is non-basepaired, the non-basepaired ribonucleotides forming three bulges in the double- stranded RNA region, wherein each bulge is immediately flanked by ribonucleotides which are basepaired in the double- stranded RNA region, wherein one bulge is formed by a mismatched ribonucleotide pair between the first RNA sequence and the second RNA sequence and the other two bulges are singleribonucleotide bulges, wherein the one or more double- stranded product RNA molecule(s) comprise the bulges, and wherein all but one of ribonucleotides 1 to 20 of the sense RNA sequence each basepair with a ribonucleotide of ribonucleotides 1 to 22 of antisense RNA sequence in the one or more double- stranded product RNA molecule(s).

[0145] In an embodiment, the first RNA sequence hybridises to the second RNA sequence by basepairing between at least 21 of the at least 22 contiguous ribonucleotides of the first RNA sequence and at least 21 of the at least 24 contiguous ribonucleotides of the second RNA sequence, wherein 3 ribonucleotides of the at least 23 ribonucleotides of the second RNA sequence are non-basepaired, and 1 ribonucleotide of the at least 21 ribonucleotides of the first RNA sequence is non-basepaired, the non-basepaired ribonucleotides forming two bulges in the double- stranded RNA region, wherein the double- stranded RNA region comprises a guanine (G) ribonucleotide content such that 36-55%, preferably 40.1-55%, of the ribonucleotides of the first RNA sequence are G ribonucleotides, wherein between 10% and 40%, preferably between 15% and 40%, or more preferably between 20% and 40%, of the ribonucleotides of the first RNA sequence and the second RNA sequence, in total, are basepaired in G:U basepairs, wherein each bulge is immediately flanked by ribonucleotides which are basepaired in the double- stranded RNA region, 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) comprise the bulges, and wherein all but one of ribonucleotides 1 to 20 of the sense RNA sequence each basepair with a ribonucleotide of ribonucleotides 1 to 22 of antisense RNA sequence in the one or more double- stranded product RNA molecule(s).

[0146] Each of the above embodiments may have the following feature:

[0147] (i) ribonucleotides 1, 2 and 3 of the sense RNA sequence basepair with ribonucleotides 18, 19 and 20, respectively, of the antisense RNA sequence in at least some of the one or more double-stranded product RNA molecule(s), or

[0148] (ii) ribonucleotides 18, 19 and 20 of the sense RNA sequence basepair with ribonucleotides 1, 2 and 3, respectively, of the antisense RNA sequence in at least some of the one or more double- stranded product RNA molecule(s), 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 are involved in G:U basepairs, more preferably one or both of ribonucleotides 1 and 2 of the antisense sequence are involved in G:U basepairs, even more preferably ribonucleotide 1 of the antisense sequence is involved in a G:U basepair. Most preferably, one or more of ribonucleotides 1, 2 and 3 of the antisense sequence 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.

[0149] The design principles for the asymmetric precursor RNA molecules can be applied in an extended fashion to longer double -stranded regions. For example, the non- basepaired ribonucleotides in the second RNA sequence (antisense sequence) may be arranged in a periodic fashion to provide a population of product RNA molecules having multiple, non-overlapping antisense RNA sequences of 23nt. Such precursor RNA molecules are particularly useful for reducing expression of a target RNA molecule in a plant cell, insect 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, the first RNA sequence comprises at least 46 contiguous ribonucleotides and the second RNA sequence comprises at least 50 contiguous ribonucleotides, wherein the first RNA sequence hybridises to the second RNA sequence by basepairing between at least 44, preferably at least 45 or at least 46, of the at least 46 contiguous ribonucleotides of the first RNA sequence and at least 44, preferably at least 45 or at least 46, of the at least 50 contiguous ribonucleotides of the second RNA sequence, wherein the double- stranded RNA region comprises a guanine (G) ribonucleotide content such that 36-55%, preferably 40.1-55%, of the ribonucleotides of the first RNA sequence are G ribonucleotides, wherein between 10% and 40%, preferably between 15% and 40%, or more preferably between 20% and 40%, of the ribonucleotides of the first RNA sequence and the second RNA sequence, in total, are basepaired in G:U basepairs, wherein 4, 5 or 6 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 46 contiguous ribonucleotides of the first RNA sequence are non- basepaired, forming bulges in the double- stranded RNA region, wherein each of the bulges is immediately flanked by ribonucleotides which are basepaired in the double- stranded RNA region, wherein the precursor RNA molecule is cleaved in a eukaryotic cell by one or more ribonucleases (RNases) to produce multiple, different double- stranded product RNA molecules consisting of a sense RNA sequence of 22 contiguous ribonucleotides and an antisense RNA sequence of 24 contiguous ribonucleotides, wherein the multiple, different double-stranded product RNA molecule(s) each comprise at least one of the bulges, and wherein at least some of the multiple, different double-stranded product RNA molecules comprise non-overlapping antisense RNA sequences, preferably adjacent nonoverlapping antisense RNA sequences. The precursor RNA molecule may or may not comprise the linking RNA sequence. 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, 16 or 17 contiguous basepairs. In an embodiment, 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.

[0150] In an embodiment, the first RNA sequence comprises at least 68 contiguous ribonucleotides and the second RNA sequence comprises at least 74 contiguous ribonucleotides, and wherein the first RNA sequence hybridises to the second RNA sequence by basepairing between at least 65, preferably at least 66, at least 67 or at least 68, of the at least 68 contiguous ribonucleotides of the first RNA sequence and at least 65, preferably at least 66, at least 67 or at least 68, of the at least 74 contiguous ribonucleotides of the second RNA sequence.

[0151] In the previous embodiments, the first RNA sequence and the second RNA sequence may extend to longer than 68 and 74 ribonucleotides, respectively. In a further embodiment, the first RNA sequence comprises at least 90 contiguous ribonucleotides and the second RNA sequence comprises at least 98 contiguous ribonucleotides, and wherein the first RNA sequence hybridises to the second RNA sequence by basepairing between at least 86, preferably at least 87, at least 88, at least 89 or at least 90, of the at least 90 contiguous ribonucleotides of the first RNA sequence and at least 86, preferably at least 87, at least 88, at least 89 or at least 90, of the at least 98 contiguous ribonucleotides of the second RNA sequence. The precursor RNA molecule may or may not comprise the linking RNA sequence.

[0152] In the previous embodiments, the first RNA sequence and the second RNA sequence may extend to longer than 90 and 98 ribonucleotides, respectively. In a further embodiment, the first RNA sequence comprises at least 107 contiguous ribonucleotides and the second RNA sequence comprises at least 117 contiguous ribonucleotides, and wherein the first RNA sequence hybridises to the second RNA sequence 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 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. 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.

[0153] In the above embodiments, the second RNA sequence is longer than the first RNA sequence because of the non-basepaired ribonucleotides that bulge out in the second RNA sequence. Preferably, the length of the first RNA sequence is 90%-94% or 90%- 93% or 91%-94% or 91%— 93% of the length of the second RNA sequence. These features apply across the full length of the double- stranded region, where the part of the double-stranded region is the full length of the double-stranded region.

[0154] As the skilled person would appreciate, further embodiments include a first RNA sequence and a second RNA sequence having longer contiguous ribonucleotides following the same principles in the above embodiments.

[0155] Each of the embodiments may have the following feature: the precursor RNA molecule is cleaved in a eukaryotic cell by one or more ribonucleases (RNases) to produce multiple, different double-stranded product RNA molecules which each consist of a sense RNA sequence of 22 contiguous ribonucleotides from the first RNA sequence and an antisense RNA sequence of 24 contiguous ribonucleotides from the second RNA sequence, wherein at least some of the multiple, different double- stranded product RNA molecules have overlapping antisense RNA sequences. In a further embodiment, at least some of the multiple, different double-stranded product RNA molecules have nonoverlapping antisense RNA sequences, where the population of double-stranded product RNA molecules produced from the precursor RNA molecule includes some overlapping and some non-overlapping antisense RNA sequences. Preferably, there are more nonoverlapping antisense RNA sequences, as readily occurs with longer (>42 basepairs) double-stranded regions where the siRNA molecules each consisting of 22nt sense RNA sequences hybridised to 24nt antisense RNA sequences are phased along the length of the double- stranded RNA region.

[0156] In another 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 comprising at least one doublestranded RNA region, wherein:

[0157] (i) the double- stranded RNA region comprises:

[0158] (a) a first RNA strand which comprises a first RNA sequence of at least 21 contiguous ribonucleotides, and a second RNA strand which comprises a second RNA sequence of at least 24 contiguous ribonucleotides, wherein the first RNA strand and second RNA strand are covalently linked by a linking RNA sequence, wherein the first RNA sequence hybridises to the second RNA sequence by basepairing between at least 20 of the at least 21 contiguous ribonucleotides of the first RNA sequence and at least 20 of the at least 24 contiguous ribonucleotides of the second RNA sequence, forming the double- stranded RNA region, wherein the double- stranded RNA region comprises a guanine (G) ribonucleotide content such that 36-55%, preferably 40.1-55%, of the ribonucleotides of the first RNA sequence are G ribonucleotides, wherein between 10% and 40%, preferably between 15% and 40%, or more preferably between 20% and 40%, of the ribonucleotides of the first RNA sequence and the second RNA sequence, in total, are basepaired in G:U basepairs, wherein 3 or 4 ribonucleotides of the at least 24 ribonucleotides of the second RNA sequence are non-basepaired and 0 or 1, respectively, ribonucleotides of the at least 21 ribonucleotides of the first RNA sequence are non-basepaired, forming one, two, three, four or five bulges in the double-stranded RNA region, wherein each of the one, two, three, four or five bulges is immediately flanked by ribonucleotides which are basepaired in the double- stranded RNA region. In an embodiment, the precursor RNA molecule is cleaved in a eukaryotic cell by one or more ribonucleases (RNases) to produce one or more double-stranded product RNA molecule(s) 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 strand, including at least 22 contiguous ribonucleotides from the second RNA sequence, wherein the one or more double- stranded product RNA molecule(s) comprise the one, two, three, 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 antisense RNA sequence in the one or more double- stranded product RNA molecule(s), 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 in the one or more doublestranded product RNA molecule(s), wherein ribonucleotides 1 and 2 of the sense RNA sequence basepair with ribonucleotides 21 and 22, respectively, of the antisense RNA sequence in the one or more double-stranded product RNA molecule(s), and wherein ribonucleotides 18 and 19 of the sense RNA sequence basepair with ribonucleotides 1 and 2, respectively, of the antisense RNA sequence in the one or more double-stranded product RNA molecule(s).

[0159] In an embodiment, the first RNA sequence hybridises to the second RNA sequence by basepairing between at least 23 of the at least 23 contiguous ribonucleotides of the first RNA sequence and at least 23 of the at least 26 contiguous ribonucleotides of the second RNA sequence, wherein 3 ribonucleotides of the at least 26 ribonucleotides of the second RNA sequence are non-basepaired and all of the at least 23 ribonucleotides of the first RNA sequence are basepaired in the double- stranded RNA region, the non-basepaired ribonucleotides forming two or three bulges in the double-stranded RNA region, wherein the double- stranded RNA region comprises a guanine (G) ribonucleotide content such that 36-55%, preferably 40.1-55%, of the ribonucleotides of the first RNA sequence are G ribonucleotides, wherein between 10% and 40%, preferably between 15% and 40%, or more preferably between 20% and 40%, of the ribonucleotides of the first RNA sequence and the second RNA sequence, in total, are basepaired in G:U basepairs, wherein each bulge is immediately flanked by ribonucleotides of the second RNA sequence which are basepaired to ribonucleotides of the first RNA sequence, wherein one bulge is a single-ribonucleotide bulge, and the other bulge is a diribonucleotide bulge, or all three bulges are single-ribonucleotide bulges, wherein the one or more double- stranded product RNA molecule(s) comprise the bulges, and wherein ribonucleotides 1 to 19 of the sense RNA sequence each basepair with a ribonucleotide of ribonucleotides 1 to 22 of the antisense RNA sequence in the one or more double-stranded product RNA molecule(s).

[0160] Each of the embodiments may have the following feature:

[0161] (i) ribonucleotides 1, 2 and 3 of the sense RNA sequence basepair with ribonucleotides 20, 21 and 22, respectively, of the antisense RNA sequence in at least some of the one or more double-stranded product RNA molecule(s), or

[0162] (ii) ribonucleotides 17, 18 and 19 of the sense RNA sequence basepair with ribonucleotides 1, 2 and 3, respectively, of the antisense RNA sequence in at least some of the one or more double- stranded product RNA molecule(s), 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 are involved in G:U basepairs, more preferably one or both of ribonucleotides 1 and 2 of the antisense sequence are involved in G:U basepairs, even more preferably ribonucleotide 1 of the antisense sequence is involved in a G:U basepair. Most preferably, one or more of ribonucleotides 1, 2 and 3 of the antisense sequence 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.

[0163] The design principles for the asymmetric precursor RNA molecules can be applied in an extended fashion to longer double-stranded regions. In an embodiment, the first RNA sequence comprises at least 44 contiguous ribonucleotides and the second RNA sequence comprises at least 50 contiguous ribonucleotides, wherein the first RNA sequence hybridises to the second RNA sequence 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 and at least 42, preferably at least 43 or at least 44, of the at least 50 contiguous ribonucleotides of the second RNA sequence, wherein the double- stranded RNA region comprises a guanine (G) ribonucleotide content such that 36-55%, preferably 40.1-55%, of the ribonucleotides of the first RNA sequence are G ribonucleotides, wherein between 10% and 40%, preferably between 15% and 40%, or more preferably between 20% and 40%, of the ribonucleotides of the first RNA sequence and the second RNA sequence, in total, are basepaired in G:U basepairs, 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, forming bulges in the double- stranded RNA region, wherein each of the bulges is immediately flanked by ribonucleotides which are basepaired in the double- stranded RNA region. In an embodiment, the precursor RNA molecule is cleaved in a eukaryotic cell by one or more ribonucleases (RNases) to produce multiple, different double-stranded product RNA molecules consisting of a sense RNA sequence of 21 contiguous ribonucleotides and an antisense RNA sequence of 24 contiguous ribonucleotides, wherein the multiple, different double-stranded product RNA molecule(s) each comprise at least one of the bulges, and wherein at least some of the multiple, different double-stranded product RNA molecules comprise non-overlapping antisense RNA sequences, preferably adjacent nonoverlapping antisense RNA sequences.

[0164] In these 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, 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. The precursor RNA molecule may or may not comprise a linking RNA sequence.

[0165] In an embodiment, the first RNA sequence comprises at least 65 contiguous ribonucleotides and the second RNA sequence comprises at least 74 contiguous ribonucleotides, wherein the first RNA sequence hybridises to the second RNA sequence 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 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, wherein the double- stranded RNA region comprises a guanine (G) ribonucleotide content such that 36-55%, preferably 40.1-55%, of the ribonucleotides of the first RNA sequence are G ribonucleotides, wherein between 10% and 40%, preferably between 15% and 40%, or more preferably between 20% and 40%, of the ribonucleotides of the first RNA sequence and the second RNA sequence, in total, are basepaired in G:U basepairs, wherein 9-12 ribonucleotides of the at least 74 contiguous ribonucleotides of the second RNA sequence are non-basepaired, and 0-3 ribonucleotides of the at least 65 contiguous ribonucleotides of the first RNA sequence are non-basepaired, forming bulges in the double-stranded RNA region, wherein each of the bulges is immediately flanked by ribonucleotides which are basepaired in the double- stranded RNA region, wherein the precursor RNA molecule is cleaved in a eukaryotic cell by one or more ribonucleases (RNases) to produce multiple, different double- stranded product RNA molecules consisting of a sense RNA sequence of 21 contiguous ribonucleotides and an antisense RNA sequence of 24 contiguous ribonucleotides, wherein the multiple, different double-stranded product RNA molecule(s) each comprise at least one of the bulges, and wherein at least some of the multiple, different double-stranded product RNA molecules comprise non-overlapping antisense RNA sequences, preferably adjacent nonoverlapping antisense RNA sequences.

[0166] In another embodiment, the first RNA strand comprises a first RNA sequence of at least 65 contiguous ribonucleotides and the second RNA strand comprises a second RNA sequence of at least 74 contiguous ribonucleotides, wherein the first RNA sequence hybridises to the second RNA sequence by basepairing between all 65 contiguous ribonucleotides of the first RNA sequence and 65 of the 74 contiguous ribonucleotides of the second RNA sequence, forming the doublestranded RNA region, wherein the double- stranded RNA region comprises a guanine (G) ribonucleotide content such that 36-55%, preferably 40.1-55%, of the ribonucleotides of the first RNA sequence are G ribonucleotides, wherein between 10% and 40%, preferably between 15% and 40%, or more preferably between 20% and 40%, of the ribonucleotides of the first RNA sequence and the second RNA sequence, in total, are basepaired in G:U basepairs, wherein 9 ribonucleotides of the 74 contiguous ribonucleotides of the second RNA sequence are non-basepaired, forming 3-9 bulges in the double- stranded RNA region, wherein each of the bulges is immediately flanked by ribonucleotides which are basepaired in the double-stranded RNA region. In an embodiment, the precursor RNA molecule is cleaved in a eukaryotic cell by one or more ribonucleases (RNases) to produce multiple, different double- stranded product RNA molecules each consisting of a sense RNA sequence of 21 contiguous ribonucleotides and an antisense RNA sequence of 24 contiguous ribonucleotides, wherein the multiple, different double-stranded product RNA molecule(s) each comprise at least one of the bulges, and wherein at least some of the multiple, different double-stranded product RNA molecules have non-overlapping antisense RNA sequences, preferably adjacent nonoverlapping antisense RNA sequences. 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, 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.

[0167] The embodiments may have the following feature: the precursor RNA molecule is cleaved in a eukaryotic cell by one or more ribonucleases (RNases) to produce multiple, different double-stranded product RNA molecules which each consist 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 at least some of the multiple, different double- stranded product RNA molecules have overlapping antisense RNA sequences. In a further embodiment, at least some of the multiple, different double-stranded product RNA molecules have nonoverlapping antisense RNA sequences, where the population of double-stranded product RNA molecules produced from the precursor RNA molecule includes some overlapping and some non-overlapping antisense RNA sequences. Preferably, there are more product RNA molecules comprising non-overlapping antisense RNA sequences, as readily occurs with longer (>48 basepairs) double-stranded regions where siRNA molecules each consisting of a 21 nt sense RNA sequence hybridised to a 24nt antisense RNA sequence are phased along the length of the double- stranded RNA region.

[0168] In an embodiment, the precursor RNA molecule is cleaved in a eukaryotic cell by one or more ribonucleases (RNases) to produce multiple, different double- stranded product RNA molecules which comprise double- stranded RNA product molecules as defined in two or more of the above embodiments.

[0169] The symmetric and asymmetric precursor RNA molecules comprising the G ribonucleotide content and the G:U basepairs in a double-stranded RNA region may be targeted to one or more target RNA molecules in an insect cell, fungal cell, or preferably a plant cell.

[0170] In a preferred embodiment, the insect cell is a Lepidopteran insect cell, for example of the genus Helicoverpa or Spodoptera, for example, of the species Helicoverpa armigera or Spodoptera frugiperda, and / or the second RNA sequence is at least 90% identical, preferably 100% identical, to the sequence which is fully complementary to the first region of the target RNA molecule in an insect cell.

[0171] In an 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 which is fully complementary to the first region of the target RNA molecule in a fungal cell. In an embodiment, (i) 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, fungal cell, or preferably the plant cell, and / or (ii) 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. It is understood in the context of the above embodiments 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 23 or 24 ribonucleotides.

[0172] In an embodiment, 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, the precursor RNA molecule comprises a second double-stranded RNA region and a second linking RNA sequence and forms a ledRNA structure.

[0173] In embodiments, one or more or all of the following may apply:

[0174] (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,

[0175] (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,

[0176] (iii) the linking RNA sequence comprises a single-stranded RNA sequence of at least 48 contiguous ribonucleotides which is identical to a region of either the first RNA sequence or the second RNA sequence,

[0177] (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,

[0178] (v) the first RNA sequence and second RNA sequence are identical in length across the full length of the double-stranded RNA region,

[0179] (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,

[0180] (vii) the first RNA sequence varies from the first region of the target RNA molecule only by the substitution of at least some, preferably all, A ribonucleotides with G 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,

[0181] (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, preferably all, of the 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, (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,

[0182] (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, preferably all, of the A ribonucleotides with G 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,

[0183] (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, preferably all, of the 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,

[0184] (xii) the percentage of G ribonucleotides in the second RNA sequence is between 26- 40% or between 40.1-55% of the total number of ribonucleotides in the second RNA sequence,

[0185] (xiii) the percentage of U ribonucleotides in the first RNA sequence is between 26- 40% or between 40.1-55% 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% or between 40.1-55% of the total number of ribonucleotides in the second RNA sequence,

[0186] (xiv) the double- stranded RNA region has at most 12, 11, 10, 9, 8, or 7 contiguous canonical basepairs,

[0187] (xv) the precursor RNA molecule is capable of being made enzymatically by transcription in vitro or in a cell, or both. 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

[0188] (xi), (vii) and (xii), (vii) and (xiii), (vii) and (xiv), (viii) and (x), (viii) and (xi), (viii) and

[0189] (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).

[0190] In an embodiment, 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.

[0191] In an embodiment, 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.

[0192] In an embodiment, the linking RNA sequence comprises a single-stranded RNA sequence of at least 48, 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.

[0193] In an embodiment, 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.

[0194] In an embodiment, 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.

[0195] In embodiments, 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 first (sense) RNA 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 second (antisense) RNA sequence, or the length of the first RNA sequence is about 21 / 22, 21 / 23, or 21 / 24 of the length of the antisense sequence, calculated as a fraction. These features are applicable to both hairpin RNAs and to dsRNAs formed by annealing of two RNA strands i.e. without a joining loop sequence.

[0196] In an embodiment, 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, preferably all of the A ribonucleotides are substituted with G. 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.

[0197] In an embodiment, 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 G:U basepairs. In an embodiment, between 15% and 40%, between 20% and 40%, or between 25% and 40% of the ribonucleotides in the double- stranded RNA region, in total, and / or the part of the double- stranded RNA region, in total, form G:U basepairs. 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 G:U basepairs. 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 G:U basepairs.

[0198] 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% or between 40.1-55% 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%, more preferably 41-55%, 42-55%, 43-55%, 44-55%, 46-55% or about 50%, 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, the second RNA sequence lacks A to G substitutions relative to the region of the target RNA molecule or its complement, respectively.

[0199] In an embodiment, the percentage of U ribonucleotides in the first RNA sequence is increased by C to U substitutions, additional to the A to G 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%, more preferably 41-55%, 42-55%, 43-55%, 44-55%, 46-55% or about 50%, 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 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 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.

[0200] In an embodiment, the double-stranded RNA region has at most 12 contiguous canonical basepairs, preferably 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 doublestranded 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.

[0201] In an embodiment, at least some of the antisense RNA sequences in the doublestranded 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.

[0202] In an embodiment, at least some of the antisense RNA sequences in the doublestranded 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.

[0203] 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.

[0204] In an embodiment of the ledRNA molecules, 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 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 cleaved by a Dicer to produce double- stranded 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.

[0205] In an embodiment of the ledRNA molecules, the second double- stranded region comprises a third RNA sequence of at least 24 contiguous ribonucleotides and a fourth RNA sequence of at least 24 contiguous ribonucleotides, wherein the third RNA sequence hybridises to the fourth RNA sequence by basepairing between at least 23 ribonucleotides of the at least 24 contiguous ribonucleotides of the third RNA sequence and at least 23 ribonucleotides of the at least 24 contiguous ribonucleotides of the fourth RNA sequence, forming the second double- stranded RNA region, wherein the second double- stranded RNA region comprises a guanine (G) ribonucleotide content such that 36-55%, preferably 40.1-55%, of the ribonucleotides of the first RNA sequence are G ribonucleotides, wherein between 10% and 40%, preferably between 15% and 40%, or more preferably between 20% and 40%, of the ribonucleotides of the third RNA sequence and the fourth RNA sequence, in total, are basepaired in G:U basepairs.

[0206] In an embodiment, the fourth RNA sequence is at least 80% identical to a sequence of at least 24 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.

[0207] In an embodiment of the ledRNA molecule, (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 wildtype 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 non-basepaired ribonucleotides in the second or fourth RNA sequence. In an embodiment, the precursor RNA molecule is cleaved in a 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 above.

[0208] In an embodiment, the precursor RNA molecule has a single double- stranded RNA region.

[0209] In an embodiment of the ledRNA molecule, all of the ribonucleotides of the second and fourth (antisense) RNA sequences are capable of basepairing to ribonucleotides in the region of the target RNA molecule.

[0210] In an embodiment, 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.

[0211] Each of the embodiments may have the following feature: the antisense RNA sequence from at least one of the product RNA molecules produced from the precursor RNA molecule is capable of hybridising to a region of a target RNA molecule in a eukaryotic cell through at least all of ribonucleotides 2 to 8 of the antisense RNA sequence basepairing with ribonucleotides within the region 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.

[0212] 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.

[0213] Preferably, most of the antisense RNA sequences from the product RNA molecules produced from the precursor RNA molecule are capable of hybridising to a region, or more than one region, of the target RNA molecule. More preferably, all of the antisense RNA sequences from the product RNA molecules produced from the precursor RNA molecule are capable of hybridising to a region, or more than one region, of the target RNA molecule.

[0214] In each of the embodiments, 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 are capable of basepairing to ribonucleotides of a region of a target RNA molecule in a eukaryotic cell. Preferably, multiple, nonoverlapping antisense RNA sequences are capable of basepairing to ribonucleotides of the region of the target RNA molecule or to multiple regions of the target molecule, or to each of multiple target RNA molecules with any of these minimum percentages. In an embodiment, all of the ribonucleotides of the antisense RNA sequence are capable of basepairing to ribonucleotides of a region of a target RNA molecule in a eukaryotic cell. Preferably, this feature occurs for each of multiple, non -overlapping antisense RNA sequences that basepair to ribonucleotides of the region of the target RNA molecule or to multiple regions of the target molecule, or to multiple target RNA molecules. This can readily be achieved through the use of longer second (antisense) RNA 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 are capable of basepairing to ribonucleotides across a length of a region 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 are capable of basepairing to ribonucleotides across a length of a region 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.

[0215] Each of the embodiments may have the following feature: the basepairing to ribonucleotides of the region of the target RNA molecule comprises only canonical basepairs.

[0216] Each of the embodiments may have one or more of the following features, where applicable: the region 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), 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. Each of the embodiments may have the following feature: the precursor RNA molecule comprises two or more different, double-stranded RNA regions, wherein each double-stranded RNA region is independently defined herein. Examples of this embodiment are the ledRNA structures shown schematically in Figure 1.

[0217] In an embodiment, the two or more different, double-stranded RNA regions are contiguous with regard to the sequence of the target RNA molecule.

[0218] In an alternative embodiment, the two different, double- stranded RNA regions 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 double- stranded 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.

[0219] 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.

[0220] Each of the embodiments may have the following feature, where applicable: the two or more different, double-stranded RNA regions are cleaved by one or more RNases, preferably a DCL3 RNase, to produce 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.

[0221] Each of the embodiments may have the following feature, where applicable: the two or more different, double- stranded RNA regions are cleaved by one or more RNases to produce 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.

[0222] Each of the embodiments 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.

[0223] Each of the embodiments 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 where the double- stranded region comprises between 20 and 30 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 where the double-stranded region comprises between 31 and 50 basepairs and where the eukaryotic cell is a vertebrate animal cell, it is preferred that between 15% 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 non-mammalian 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.

[0224] In an embodiment, 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.

[0225] In an embodiment, 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). 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.

[0226] In an embodiment, the one or more double- stranded region(s) comprise bulges which are evenly spaced apart along most or all of each double-stranded region. For example, the bulges for the A22 modification are spaced apart on average about one ribonucleotide bulge in the second RNA sequence about every 22nd ribonucleotide, applicable to longer sequences. 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 about every 23 or 24 ribonucleotides, respectively. In this context, “about” means + / - 10%, preferably + / - 5%.

[0227] In an embodiment, each double-stranded region comprises a bulge which is closer to the linking RNA sequence than any other bulge in the double-stranded region, wherein said closer bulge and the linking RNA sequence 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.

[0228] In an embodiment, the precursor RNA molecule comprises a terminal basepair at a junction of the double- stranded region and the linking RNA sequence, wherein the terminal basepair comprises a U ribonucleotide as the last 3' ribonucleotide in the first RNA sequence or the first 5' ribonucleotide in the second RNA sequence, preferably the first 5' ribonucleotide in the second RNA sequence is a U ribonucleotide.

[0229] In an embodiment, the terminal basepair at the junction of the double- stranded region and the linking RNA sequence 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.

[0230] In an embodiment, the precursor RNA molecule has a single linking RNA sequence, thereby forming a hairpin RNA (hpRNA) structure. In an embodiment, the linking RNA sequence joins the 3' end of the first RNA strand and the 5' end of the second RNA strand. Alternately, the linking RNA sequence joins the 3' end of the second RNA strand and the 5' end of the first RNA strand.

[0231] In an embodiment, the precursor RNA molecule has two double-stranded regions and two linking RNA sequences, forming a ledRNA structure (also known as a dumbbell structure). All of the features of the embodiments of the dsRNA structures as described herein are applicable to the ledRNA structures, either singly or in combination.

[0232] In an embodiment, applicable to each of the embodiments of the hairpin RNAs or ledRNAs, the precursor RNA molecule comprises a terminal basepair at a junction of the double-stranded region and the linking RNA sequence, wherein the terminal basepair is the first basepair of the double- stranded RNA region.

[0233] In an embodiment, applicable to each of the embodiments of the hairpin RNAs or ledRNAs, the precursor RNA molecule comprises at least one linking RNA sequence 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.

[0234] In an embodiment, applicable to each of the hairpin RNAs or ledRNAs, at least one linking RNA sequence 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 is encoded by a polynucleotide that lacks an intron.

[0235] In embodiments, 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.

[0236] In an embodiment, the double-stranded region(s) in the precursor RNA molecule comprises bulges only as defined herein.

[0237] In an embodiment, the precursor RNA molecule further comprises a doublestranded region which comprises at least 24 contiguous basepairs and which lacks bulges. In an embodiment, between 10% and 40% of the at least 24 contiguous basepairs are G:U basepairs. The precursor RNA molecules of these embodiments may thereby provide a mixture of symmetrical and asymmetrical product RNA molecules. In an embodiment, when cleaved by a Dicer, the precursor RNA molecule provides more asymmetrical than symmetrical product RNA molecules.

[0238] 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.

[0239] In an embodiment, the one or more RNase(s) is a Type III ribonuclease, preferably a Dicer or Dicer-like (DCL) protein, more preferably a DCL3 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, DCL3 and / or DCL4 proteins.

[0240] In an embodiment, on average every one in four to every one in six ribonucleotides in the dsRNA region and / or at least some of the product RNA molecules form G:U basepairs, and / or wherein between 10% and 40% of the ribonucleotides in the dsRNA region, in total, and / or the double- stranded RNA region, in total, and / or at least some of the product RNA molecules form G:U basepairs. Preferably, most of the doublestranded product RNA molecules 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 produced from the precursor RNA molecule have the G ribonucleotide in a sense RNA sequence and the U in an antisense RNA sequence.

[0241] In an embodiment, all of the double- stranded RNA regions, in the precursor RNA molecule lack 8 contiguous canonical basepairs. This can readily be achieved by the introduction of suitably distributed G:U basepairs.

[0242] 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 15, at least 20, at least 30, at least 40, at least 50, or at least 100 different product RNA molecules. 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 nonoverlapping different product RNA molecules.

[0243] In an embodiment, the first RNA sequence and / or the second RNA sequence, 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 contiguous ribonucleotides. In a preferred embodiment, the first RNA sequence is shorter than the second RNA sequence.

[0244] In an embodiment, both RNA strands of the double-stranded region are 24 to 33 ribonucleotides in length, and the double-stranded region comprises at least 19 basepairs, preferably at least 20 or at least 21 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) produced from the precursor RNA molecule are G:U basepairs.

[0245] In an embodiment,

[0246] (i) the first and second RNA strands of the double-stranded region are each 34 to 200 ribonucleotides in length, preferably 34 to 50 ribonucleotides in length,

[0247] (ii) at least 80%, preferably at least 90%, of the ribonucleotides in the RNA strand, in total, are basepaired, thereby forming the double-stranded region of the precursor RNA molecule. Such molecules are particularly useful in vertebrate animal cells such as mammalian animal cells.

[0248] In an embodiment, at least 50%, at least 75%, at least 90%, or at least 95%, of the ribonucleotides of the antisense RNA sequence are capable of basepairing to ribonucleotides within the region of the target RNA molecule in the eukaryotic cell.

[0249] In an embodiment, 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.

[0250] Each of the embodiments of the precursor RNA molecule as described above, including 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, wherein the antisense sequences 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.

[0251] In another aspect, the present invention provides a double-stranded product RNA molecule produced from the precursor RNA molecule of the invention. In an embodiment (i), the double- stranded product RNA molecule consists of a sense RNA sequence of 24 contiguous ribonucleotides and an antisense RNA sequence of 24 contiguous ribonucleotides, wherein ribonucleotides 1 to 22 of the sense RNA sequence each basepair with a ribonucleotide of ribonucleotides 1 to 22 of the antisense RNA sequence, wherein ribonucleotides 23 and 24 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 the sense RNA sequence comprises a guanine (G) ribonucleotide content such that 36-55%, preferably 40.1-55%, of the ribonucleotides of the sense RNA sequence are G ribonucleotides, and wherein between 10% and 40%, preferably between 15% and 40%, or more preferably between 20% and 40%, of the ribonucleotides of the sense RNA sequence and the antisense RNA sequence, in total, are basepaired in G:U basepairs.

[0252] In an embodiment (ii), the double-stranded product RNA molecule consists of a sense RNA sequence of 23 contiguous ribonucleotides and an antisense RNA sequence of 23 contiguous ribonucleotides, wherein ribonucleotides 1 to 21 of the sense RNA sequence each basepair with a ribonucleotide of ribonucleotides 1 to 21 of the antisense RNA sequence, wherein ribonucleotides 22 and 23 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 the sense RNA sequence comprises a guanine (G) ribonucleotide content such that 36-55%, preferably 40.1-55%, of the ribonucleotides of the sense RNA sequence are G ribonucleotides, and wherein between 10% and 40%, preferably between 15% and 40%, or more preferably between 20% and 40%, of the ribonucleotides of the sense RNA sequence and the antisense RNA sequence, in total, are basepaired in G:U basepairs.

[0253] In an embodiment (iii), the double- stranded product RNA molecule consists of a sense RNA sequence of 23 contiguous ribonucleotides and an antisense RNA sequence of 24 contiguous ribonucleotides, wherein ribonucleotides 1 to 21 of the sense RNA sequence each basepair with a ribonucleotide of ribonucleotides 1 to 22 of the antisense RNA sequence, wherein one of ribonucleotides 3 to 20 of the antisense RNA sequence is nonbasepaired, forming a bulge in the double- stranded RNA molecule, wherein ribonucleotides 22 and 23 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 the sense RNA sequence comprises a guanine (G) ribonucleotide content such that 36-55%, preferably 40.1-55%, of the ribonucleotides of the sense RNA sequence are G ribonucleotides, and wherein between 10% and 40%, preferably between 15% and 40%, or more preferably between 20% and 40%, of the ribonucleotides of the sense RNA sequence and the antisense RNA sequence, in total, are basepaired in G:U basepairs.

[0254] In an embodiment (iv), the double-stranded product RNA molecule consists of a sense RNA sequence of 23 contiguous ribonucleotides and an antisense RNA sequence of 24 contiguous ribonucleotides, wherein 20 ribonucleotides of ribonucleotides 1 to 21 of the sense RNA sequence each basepair with a ribonucleotide of ribonucleotides 1 to 22 of the antisense RNA sequence, wherein one ribonucleotide of ribonucleotides 3 to 19 of the sense RNA sequence and two ribonucleotides of ribonucleotides 3 to 20 of the antisense RNA sequence are non-basepaired, forming one or more bulges in the double- stranded RNA molecule, wherein ribonucleotides 22 and 23 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 the sense RNA sequence comprises a guanine (G) ribonucleotide content such that 36-55%, preferably 40.1-55%, of the ribonucleotides of the sense RNA sequence are G ribonucleotides, and wherein between 10% and 40%, preferably between 15% and 40%, or more preferably between 20% and 40%, of the ribonucleotides of the sense RNA sequence and the antisense RNA sequence, in total, are basepaired in G:U basepairs.

[0255] In an embodiment (v), the double- stranded product RNA molecule consists of a sense RNA sequence of 22 contiguous ribonucleotides and an antisense RNA sequence of 24 contiguous ribonucleotides, wherein ribonucleotides 1 to 20 of the sense RNA sequence each basepair with a ribonucleotide of ribonucleotides 1 to 22 of the antisense RNA sequence, wherein two of ribonucleotides 3 to 20 of the antisense RNA sequence are nonbasepaired, forming one or two bulges in the double-stranded RNA molecule, wherein ribonucleotides 21 and 22 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 the sense RNA sequence comprises a guanine (G) ribonucleotide content such that 36-55%, preferably 40.1-55%, of the ribonucleotides of the sense RNA sequence are G ribonucleotides, and wherein between 10% and 40%, preferably between 15% and 40%, or more preferably between 20% and 40%, of the ribonucleotides of the sense RNA sequence and the antisense RNA sequence, in total, are basepaired in G:U basepairs.

[0256] In an embodiment (vi), the double-stranded product RNA molecule consists of a sense RNA sequence of 22 contiguous ribonucleotides and an antisense RNA sequence of 24 contiguous ribonucleotides, wherein 19 ribonucleotides of ribonucleotides 1 to 20 of the sense RNA sequence each basepair with a ribonucleotide of ribonucleotides 1 to 22 of the antisense RNA sequence, wherein one ribonucleotide of ribonucleotides 3 to 17 of the sense RNA sequence and three ribonucleotides of ribonucleotides 3 to 20 of the antisense RNA sequence are non-basepaired, forming one or more bulges in the double- stranded RNA molecule, wherein ribonucleotides 21 and 22 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 the sense RNA sequence comprises a guanine (G) ribonucleotide content such that 36-55%, preferably 40.1-55%, of the ribonucleotides of the sense RNA sequence are G ribonucleotides, and wherein between 10% and 40%, preferably between 15% and 40%, or more preferably between 20% and 40%, of the ribonucleotides of the sense RNA sequence and the antisense RNA sequence, in total, are basepaired in G:U basepairs.

[0257] In an embodiment (vii), the double- stranded product RNA molecule consists of a sense RNA sequence of 21 contiguous ribonucleotides and an antisense RNA sequence of 24 contiguous ribonucleotides, wherein 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 three ribonucleotides of ribonucleotides 3 to 18 of the antisense RNA sequence are non-basepaired, forming one, two or three bulges 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 the sense RNA sequence comprises a guanine (G) ribonucleotide content such that 36-55%, preferably 40.1-55%, of the ribonucleotides of the sense RNA sequence are G ribonucleotides, and wherein between 10% and 40%, preferably between 15% and 40%, or more preferably between 20% and 40%, of the ribonucleotides of the sense RNA sequence and the antisense RNA sequence, in total, are basepaired in G:U basepairs.

[0258] In an embodiment (viii), the double- stranded product RNA molecule consists of a sense RNA sequence of 21 contiguous ribonucleotides and an antisense RNA sequence of 24 contiguous ribonucleotides, wherein 18 ribonucleotides 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 one ribonucleotide of ribonucleotides 3 to 17 of the sense RNA sequence and four ribonucleotides of ribonucleotides 3 to 20 of the antisense RNA sequence are non-basepaired, forming one or more bulges 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 the sense RNA sequence comprises a guanine (G) ribonucleotide content such that 36-55%, preferably 40.1-55%, of the ribonucleotides of the sense RNA sequence are G ribonucleotides, and wherein between 10% and 40%, preferably between 15% and 40%, or more preferably between 20% and 40%, of the ribonucleotides of the sense RNA sequence and the antisense RNA sequence, in total, are basepaired in G:U basepairs.

[0259] In each of embodiments (i) to (viii) of the double- stranded product RNA molecules described above, specific combinations of A ribonucleotide content, G ribonucleotide content and the number of G:U basepairs are applicable. Therefore, in an embodiment, the G ribonucleotide content of the sense RNA sequence of at least some of, preferably most of, the double-stranded product RNA molecules is between 8-13 G ribonucleotides, inclusive, and between 10% and 40% of the ribonucleotides of the first RNA sequence and the second RNA sequence, in total, are basepaired in G:U basepairs.

[0260] In an embodiment, the G ribonucleotide content of the sense RNA sequence of at least some of, preferably most of, the double-stranded product RNA molecules is 8, optionally with an A ribonucleotide content of not more than 2, not more than 1, preferably zero.

[0261] In an embodiment, the G ribonucleotide content of the sense RNA sequence of at least some of, preferably most of, the double-stranded product RNA molecules is 9, optionally with an A ribonucleotide content of not more than 2, not more than 1, preferably zero.

[0262] In an embodiment, the G ribonucleotide content of the sense RNA sequence of at least some of, preferably most of, the double- stranded product RNA molecules is 10, optionally with an A ribonucleotide content of not more than 2, not more than 1, preferably zero.

[0263] In an embodiment, the G ribonucleotide content of the sense RNA sequence of at least some of, preferably most of, the double- stranded product RNA molecules is 11, optionally with an A ribonucleotide content of not more than 2, not more than 1, preferably zero.

[0264] In an embodiment, the G ribonucleotide content of the sense RNA sequence of at least some of, preferably most of, the double- stranded product RNA molecules is 12, optionally with an A ribonucleotide content of not more than 2, not more than 1, preferably zero.

[0265] In an embodiment, the G ribonucleotide content of the sense RNA sequence of at least some of, preferably most of, the double- stranded product RNA molecules is 13, optionally with an A ribonucleotide content of not more than 2, not more than 1, preferably zero.

[0266] In an embodiment, the G ribonucleotide content of the sense RNA sequence of at least some of, preferably most of, the double- stranded product RNA molecules is at least

[0267] 9, optionally with an A ribonucleotide content of not more than 2, not more than 1, preferably zero.

[0268] In an embodiment, the G ribonucleotide content of the sense RNA sequence of at least some of, preferably most of, the double- stranded product RNA molecules is at least

[0269] 10, optionally with an A ribonucleotide content of not more than 2, not more than 1, preferably zero.

[0270] In an embodiment, the G ribonucleotide content of the sense RNA sequence of at least some of, preferably most of, the double- stranded product RNA molecules is at least 11, optionally with an A ribonucleotide content of not more than 2, not more than 1, preferably zero.

[0271] In an embodiment, the G ribonucleotide content of the sense RNA sequence of at least some of, preferably most of, the double- stranded product RNA molecules is at least

[0272] 12, optionally with an A ribonucleotide content of not more than 2, not more than 1, preferably zero. The above numbers are determined in the context, the full length of the sense RNA sequence of the double- stranded product RNA molecules, in particular for the 23- and 24-mers.

[0273] In preferred embodiments of those described above, at least 60%, at least 70%, at least 80%, at least 90% or essentially all of the double- stranded product RNA molecules produced from the precursor RNA molecule have the recited G ribonucleotide and A ribonucleotide contents.

[0274] In an embodiment that can be applied to the above embodiments of the doublestranded product RNA molecules,

[0275] (i) ribonucleotides 1, 2 and 3 of the sense RNA sequence basepair with ribonucleotides 20, 21 and 22, respectively, of the antisense RNA sequence of 24 ribonucleotides, or

[0276] (ii) ribonucleotides 19, 20 and 21 of the sense RNA sequence of 23 ribonucleotides basepair with ribonucleotides 1, 2 and 3, respectively, of the antisense RNA sequence, or preferably both (i) and (ii).

[0277] In an embodiment that can be applied to the above embodiments of the doublestranded product RNA molecules,

[0278] (i) ribonucleotides 1, 2 and 3 of the sense RNA sequence basepair with ribonucleotides 19, 20 and 21, respectively, of the antisense RNA sequence of 23 ribonucleotides, or

[0279] (ii) ribonucleotides 18, 19 and 20 of the sense RNA sequence of 22 ribonucleotides basepair with ribonucleotides 1, 2 and 3, respectively, of the antisense RNA sequence, or preferably both (i) and (ii).

[0280] In an embodiment that can be applied to the above embodiments of the doublestranded product RNA molecules,

[0281] (i) ribonucleotides 1, 2 and 3 of the sense RNA sequence basepair with ribonucleotides 20, 21 and 22, respectively, of the antisense RNA sequence of 24 ribonucleotides, or

[0282] (ii) ribonucleotides 17, 18 and 19 of the sense RNA sequence of 21 ribonucleotides basepair with ribonucleotides 1, 2 and 3, respectively, of the antisense RNA sequence, 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 are involved in G:U basepairs, more preferably one or both of ribonucleotides 1 and 2 of the antisense sequence are involved in G:U basepairs, even more preferably ribonucleotide 1 of the antisense sequence is involved in a G:U basepair. Most preferably, one or more of ribonucleotides 1, 2 and 3 of the antisense sequence are U ribonucleotides, or one or both of ribonucleotides 1 and 2 are U ribonucleotides, or ribonucleotide 1 is a U ribonucleotide.

[0283] The basepairing between ribonucleotides of the sense RNA sequence and ribonucleotides of the antisense RNA sequence in the double- stranded product RNA molecules may comprise three, four, five, six, seven or eight G:U basepairs. In a preferred embodiment, all of the G:U basepairs have the G ribonucleotide in the sense RNA sequence and the U ribonucleotide in the antisense RNA sequence.

[0284] In an embodiment, ribonucleotide 1 of the antisense RNA sequence in at least some of the product RNA molecules is a U ribonucleotide. In an embodiment, ribonucleotide 1 of the antisense RNA sequence in at least some of the product RNA molecules is a U ribonucleotide which is basepaired to a G ribonucleotide in the sense RNA sequence.

[0285] In an embodiment, all of at least ribonucleotides 2-8, or 2-10 or 2-11, of the antisense RNA sequence basepair to ribonucleotides in a region 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 of the target RNA molecule. In a preferred embodiment, all of the ribonucleotides of the antisense RNA sequence basepair to ribonucleotides in the region of the target RNA molecule by canonical basepairs.

[0286] As exemplified herein, it is possible to change the numbers of siRNA molecules in a eukaryotic cell, for example siRNA molecules comprising an antisense RNA sequence of 23 and / or 24 ribonucleotides relative to antisense RNA sequences of 21 ribonucleotides, by the high guanine (G) ribonucleotide content and / or low adenine (A) ribonucleotide content in at least the sense RNA sequence of a precursor RNA molecule, and the G:U basepair content, relative to a corresponding conventional precursor RNA molecule without the modifications, especially along the full length of a double- stranded region comprising a second RNA strand of at least 100 ribonucleotides in length. The modifications may be combined with any of the A22, A23 or A24 modifications in the precursor RNA molecule. Therefore, in an aspect, the present invention provides a population of multiple, different double- stranded product RNA molecules of the invention, produced from one or more precursor RNA molecules of the invention. The population of multiple, different double-stranded product RNA molecules may comprise double-stranded product RNA molecules according to embodiments (i) to (viii) described above, or any combination thereof, wherein the different, double-stranded product RNA molecules comprise different lengths of the sense RNA sequence, antisense RNA sequence, or both, or are produced from a combination of a symmetrical precursor RNA molecule and an asymmetric precursor RNA molecule.

[0287] The population may comprise two or more of the double-stranded product RNA molecules described in embodiments (i) to (viii) above. In an embodiment, the population comprises double-stranded product RNA molecules described in at least embodiments (i) and (ii). In an embodiment, the population comprises double- stranded product RNA molecules described in at least embodiments (i) and (iii). In an embodiment, the population comprises double- stranded product RNA molecules described in at least embodiments (i) and (iv). In an embodiment, the population comprises double-stranded product RNA molecules described in at least embodiments (i) and (v). In an embodiment, the population comprises double-stranded product RNA molecules described in at least embodiments (i) and (vi). In an embodiment, the population comprises double-stranded product RNA molecules described in at least embodiments (i) and (vii). In an embodiment, the population comprises double- stranded product RNA molecules described in at least embodiments (i) and (viii). In an embodiment, the population comprises double-stranded product RNA molecules described in at least embodiments (iii) and (iv). In an embodiment, the population comprises double-stranded product RNA molecules described in at least embodiments (iii) and (v). In an embodiment, the population comprises double- stranded product RNA molecules described in at least embodiments (iii) and (vi). In an embodiment, the population comprises double- stranded product RNA molecules described in at least embodiments (iii) and (vii). In an embodiment, the population comprises double- stranded product RNA molecules described in at least embodiments (iiii) and (viii). In an embodiment, the population comprises double-stranded product RNA molecules described in at least embodiments (v) and (vi). In an embodiment, the population comprises double- stranded product RNA molecules described in at least embodiments (v) and (vii). In an embodiment, the population comprises double- stranded product RNA molecules described in at least embodiments (v) and (viii).

[0288] In an embodiment, the population of multiple, different double-stranded product 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, each produced from one or more precursor RNA molecules of the invention.

[0289] In an embodiment,

[0290] (i) more antisense RNA sequences in the population consist of 24 ribonucleotides than consist of 21 ribonucleotides, and / or

[0291] (ii) more antisense RNA sequences in the population consist of 23 ribonucleotides than consist of 21 ribonucleotides.

[0292] In an embodiment, the present invention provides a double-stranded RNA product molecule obtainable by, or obtained from, the cleavage by Dicer, preferably DCL3, of a precursor RNA molecule of the invention, wherein the double- stranded product RNA molecule has one or more of any of the features defined herein.

[0293] In an aspect, the invention provides an isolated and / or exogenous polynucleotide, or a vector comprising the polynucleotide, encoding the precursor RNA molecule of the invention, and / or that produces a double-stranded product RNA molecule of the invention or a population of double-stranded product RNA molecules of the invention, optionally wherein 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, and optionally a polyadenylation region / transcription terminator or a transcription termination sequence.

[0294] 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.

[0295] In an embodiment, the polynucleotide is operably linked to a promoter capable of directing expression of the precursor RNA molecule in a host cell or in a cell-free expression system, preferably a eukaryotic cell such as for example a plant cell, and optionally a polyadenylation region / transcription terminator or a transcription termination sequence. It is understood that the promoter is heterologous to the target RNA molecule.

[0296] 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, for example a T7 RNA polymerase promoter, well known in the art.

[0297] In an aspect, the present invention provides a vector comprising a polynucleotide of the invention, for example comprising a polynucleotide operably linked to a promoter.

[0298] In an embodiment, the vector is a viral vector, such as a DNA viral vector or an RNA viral vector or a plasmid. In an aspect, the present invention provides a eukaryotic cell comprising a precursor RNA molecule of the invention, a double-stranded product RNA molecule of the invention and / or a polynucleotide of the invention, preferably two of these or all three.

[0299] In an aspect, the present invention provides a method of identifying a doublestranded product RNA molecule, or a precursor RNA molecule, for reducing the amount and / or activity of a target RNA molecule of interest in a eukaryotic cell, the method comprising i) producing a precursor RNA molecule of the invention, and / or a double-stranded product RNA molecule of the invention, or a population of multiple, different precursor RNA molecules of the invention, or a population of multiple, different double- stranded product RNA molecules ii) determining the ability of the precursor RNA molecule or double-stranded product RNA molecule, or members of a population of multiple, different precursor RNA molecules, or the population of multiple, different double- stranded product RNA molecules, to reduce the amount and / or activity of the target RNA molecule of interest, optionally wherein step i) comprises producing the precursor RNA molecule in the eukaryotic cell, wherein the precursor RNA molecule is cleaved in the eukaryotic cell by one or more ribonucleases (RNases) to produce the double- stranded product RNA molecule or the population of multiple, different double- stranded product RNA molecules.

[0300] In an embodiment, step i) comprises expressing a precursor RNA molecule of the invention in the eukaryotic cell comprising the target RNA molecule, or in a eukaryotic cell that is different to the eukaryotic cell comprising the target RNA molecule or in vitro, wherein the precursor RNA molecule is cleaved in the eukaryotic cell by one or more ribonucleases (RNases) to produce the double-stranded product RNA molecule or the population of multiple, different double- stranded product RNA molecules.

[0301] In an embodiment, the method further comprises designing the precursor RNA molecule based on a region of the ribonucleotide sequence of the target RNA molecule of interest, preferably using the features defined herein.

[0302] In an embodiment, the method further comprises a step of producing more of the double-stranded product RNA molecules or the precursor RNA molecule after step ii), for example in commercial quantities for a kit comprising the double- stranded product RNA molecule and / or the precursor RNA molecule.

[0303] In an embodiment for the precursor RNA molecules, the invention provides a method of identifying a double-stranded RNA product molecule, or a precursor RNA molecule, for reducing the amount and / or activity of a target RNA molecule of interest in a plant cell, an insect cell or a fungal cell, the method comprising: i) producing a precursor RNA molecule, and / or a double- stranded product RNA molecule, or a population of multiple, different precursor RNA molecules of the invention or a population of double-stranded product RNA molecules of the invention, and ii) determining the ability of the precursor RNA molecule or double-stranded product RNA molecule, or members of the population of multiple, different precursor RNA molecules, or the population of multiple, different double- stranded product RNA molecules, to reduce the amount and / or activity of the target RNA molecule of interest in the plant, insect or fungal cell.

[0304] In an embodiment, step i) comprises introducing a precursor RNA molecule of the invention into: (a) a plant cell by contacting the plant cell with the precursor RNA molecule or a polynucleotide expressing the precursor RNA molecule, (b) an insect cell, preferably by ingestion, soaking, dusting, spraying or injection of an insect comprising the insect cell; and / or (c) 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 plant cell, insect cell or fungal cell by a Dicer to produce the double- stranded product RNA molecule or the population of multiple, different double-stranded product RNA molecules.

[0305] 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 by the insect 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 product RNA molecule or the population of multiple, different double- stranded product RNA molecules. In an embodiment, the method further comprises a step, after step ii), of producing more of the double- stranded product RNA molecule or the precursor RNA molecule.

[0306] Also provided is a double- stranded product RNA molecule or a precursor RNA molecule identified or produced using a method of the invention.

[0307] In an aspect, the invention provides a host cell, preferably a eukaryotic cell, comprising one or more or all of a precursor RNA molecule of the invention, a polynucleotide encoding the precursor RNA molecule, a double-stranded product RNA molecule of the invention, and / or a population of multiple, different double- stranded product RNA molecules of the invention. In an embodiment, the cell is a micro-organism, a non-human cell or a eukaryotic cell in vitro.

[0308] In an embodiment, the cell, preferably a eukaryotic cell, comprises a population of multiple, different double-stranded product RNA molecules of the invention. In an embodiment, the eukaryotic cell such as a plant cell, nematode cell, insect cell or fungal cell, comprises one or more or all of the precursor RNA molecules, the double- stranded product RNA molecules, and the population of multiple, different double- stranded product RNA molecules of the invention, wherein the eukaryotic cell may be a yeast cell. In an embodiment, the eukaryotic cell comprises one or more precursor RNA molecule and a population of multiple, different double- stranded product RNA molecules of the invention.

[0309] In an embodiment, the host cell comprises a population of two, three, four, five, 10, 15, 20, 50, 100 or more different, double-stranded product RNA molecules of the invention.

[0310] 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.

[0311] 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, a DCL3 protein, and / or a gene encoding a DCL2 protein. In an embodiment, the plant cell is other than a Nicotiana benthamiana and / or Nicotiana tabacum cell. In an embodiment, the host cell is a fungal cell or an animal cell which is wild-type for Dicer protein(s).

[0312] In an embodiment, applicable to all of the embodiments of the host cell or eukaryotic cell, the 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.

[0313] In an aspect, the present invention provides a non-human organism, or a part thereof, comprising one or more or all of a precursor RNA molecule of the invention, a polynucleotide encoding the precursor RNA molecule, the double- stranded product RNA molecule of the invention, and / or the population of multiple, different double- stranded product RNA molecules of the invention, a cell of the invention, preferably a transgenic non-human organism or part thereof, being transgenic for a polynucleotide of the invention, and / or wherein the polynucleotide is stably integrated into the genome of the organism or part thereof.

[0314] 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, or a transgenic fungus such as, for example, a yeast cell.

[0315] In an embodiment, the polynucleotide is stably integrated into the genome of the organism or part thereof, preferably into the nuclear genome or plastid 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.

[0316] 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 invention, 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 plant, or in an insect, such as for example a Lepidopteran insect, when ingested, or in a fungal pathogen when contacted with 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. 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, or a soyabean plant, and the insect is of the genus Spodoptera. In an embodiment, the fungal pathogen is of the genus Fusarium or Verticillium.

[0317] 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 product RNA molecule of the invention, the population of multiple, different double- stranded product 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.

[0318] 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 product RNA molecule of the invention, the population of multiple, different double- stranded product 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.

[0319] In an embodiment of the method, 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.

[0320] In an aspect, the present invention provides a method of producing the precursor RNA molecule of the invention, the double-stranded product RNA molecule of the invention, and / or the population of multiple, different double-stranded product 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. In an embodiment, the host cell is a cell that does not comprise the target RNA molecule, or a cell of a different species than the cell that comprises the target RNA molecule. For example, the host cell is a yeast cell or a plant cell and the cell that comprises the target RNA molecule is an insect cell or a fungal cell.

[0321] In an embodiment, the method further comprises extracting and / or at least partially purifying some of the precursor RNA molecule of the invention or doublestranded product RNA molecule of the invention or the population of multiple, different double-stranded product 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. 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 product RNA molecule of the invention, the population of multiple, different double- stranded product 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 doublestranded product RNA molecules of the invention or the population of multiple, different double-stranded product 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.

[0322] In an aspect, the invention provides a method of increasing the amount of doublestranded product RNA molecules of the invention in a eukaryotic cell or organism, or increasing the amount of a population of double- stranded product RNA molecules of the invention in the cell or organism, comprising expressing in the cell or organism a polynucleotide or a vector of the invention, or contacting the cell or organism with the precursor RNA molecule of the invention, or the double-stranded product RNA molecule of the invention. The increase is relative to the use of the corresponding control RNA molecule, expressed in the same manner.

[0323] In an embodiment, the method increases the amount of small interfering RNAs (sRNAs) of 23 and / or 24 ribonucleotides in length in a plant or plant cell, an insect cell or insect, or a fungal cell or fungus, or increases the ratio of the amount of antisense sRNAs of 23 and / or 24 ribonucleotides relative to the amount of antisense 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, plant, insect, or fungus one or more or all of the precursor RNA molecule, the double-stranded product RNA molecule, the population of multiple, different double- stranded product RNA molecules, the polynucleotide, the vector, the cell, the non-human organism or part thereof, the extract, and the composition of the invention.

[0324] 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.

[0325] 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 product RNA molecule of the invention, the population of double- stranded product 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 product RNA molecules of the invention or the population of multiple, different double- stranded product RNA molecules of the invention. Alternatively, the composition comprises the doublestranded product RNA molecules of the invention or the population of multiple, different double-stranded product 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.

[0326] In an embodiment, the extract or the composition comprises a population of at least two, at least three, at least four, at least five, at least 10, at least 15, at least 20, at least 50, or at least 100 or more double- stranded product RNA molecules of the invention.

[0327] In an embodiment, the composition is a pharmaceutical composition, for example formulated with a carrier as nano-particles.

[0328] 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.

[0329] 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 or a lipid formulation.

[0330] 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, preferably a plant, insect or fungus, the method comprising (i) delivering to the cell or organism, one or more or all of: the precursor RNA molecule of the invention, the doublestranded product RNA molecule of the invention, the population of multiple, different double-stranded product 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. 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, preferably a plant, insect or fungus, 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.

[0331] 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, preferably a plant, insect, nematode or fungus, 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 product RNA molecule of the invention, the population of double- stranded product 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.

[0332] 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 regions, or a combination of precursor RNA molecules and / or the population of double-stranded product RNA molecules of the invention. The different target RNA molecules may be produced by differential splicing from a single gene, or be produced from a gene family. Alternatively, the different target RNA molecules may be unrelated in sequence.

[0333] In an embodiment, one or more or all of the precursor RNA molecule, the doublestranded product RNA molecule, the population of double- stranded product 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. In an embodiment, the composition comprises at least two, at least three, at least four, at least five, or at least six different precursor RNA molecules, each targeting a different target RNA molecule. The different target RNA molecules may be unrelated in sequence.

[0334] In an embodiment, the method for reducing or down-regulating the level and / or activity of a target RNA molecule in an eukaryotic organism comprises orally or parenterally delivering to the organism one or more or all of the precursor RNA molecule of the invention, the double-stranded product RNA molecule of the invention, the population of double- stranded product 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, at least two, at least three, at least four, at least five, or at least six different precursor RNA molecules, each targeting a different target RNA molecule, are delivered. The different target RNA molecules may be unrelated in sequence.

[0335] 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, preferably an insect, nematode or fungus, 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 product RNA molecule of the invention, the population of different double- stranded product 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 product RNA molecule or population of different double-stranded product RNA molecules has an effect on the pest or pathogen, and optionally (iv) if the precursor RNA molecule, double-stranded product RNA molecule or population of different double-stranded product 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.

[0336] 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, preferably an insect, nematode or fungus, 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 product RNA molecule of the invention, the population of multiple, different double-stranded product 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 product RNA molecule or population of double- stranded product RNA molecules, and optionally (iii) selecting a RNA molecule based on results from step (ii), thereby identifying the RNA molecule.

[0337] In an aspect, the present invention provides 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 of the invention, the double-stranded product RNA molecule of the invention, the population of double- stranded product 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 RNA molecule targets a target RNA molecule which is involved in feeding, growth, development, perception, movement, reproduction, hormone function, or survival of the pest or pathogen.

[0338] In an aspect, the present invention provides 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 of the invention, the double-stranded product RNA molecule of the invention, the population of double- stranded product 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, wherein the precursor RNA molecule, double- stranded product RNA molecule or population of double-stranded product RNA molecules has a deleterious effect on the non-human organism. In an embodiment, the organism is a plant and the double- stranded product RNA molecule or population of double- stranded product RNA molecules reduces the amount and / or activity of a target RNA molecule in the plant, where the target RNA molecule normally functions for the growth, development or reproduction of the plant. In an embodiment, the target RNA molecule functions for photosynthesis or amino acid metabolism, such as for example, EPSP synthase.

[0339] In an embodiment, the non-human organism is an arthropod, preferably an insect, a nematode, or a plant. In an embodiment, the non-human organism is a plant, and the insect or nematode eats the plant or a portion thereof such as a vegetative plant part or feeds on a plant part e.g. roots.

[0340] In an embodiment, the method does not comprise delivering the defined substance to an animal such as, for example, a human or another mammal. In an embodiment, the method does not comprise delivering the defined substance to a human.

[0341] In an aspect, the present invention provides 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 of the invention, the double-stranded product RNA molecule of the invention, the population of double-stranded product 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 organism is not an animal, preferably the organism is a plant or fungus.

[0342] In an embodiment, one or more or all of the precursor RNA molecule, the doublestranded product RNA molecule, the population of double- stranded product 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.

[0343] In an embodiment, the organism is a vertebrate animal, or alternatively a nonvertebrate animal. In an embodiment, the organism is a mammal. In an embodiment, the organism is a human. In an embodiment, the organism is a plant.

[0344] In an embodiment, the precursor RNA molecule(s) are produced in a eukaryotic cell or organism, or in a eukaryotic host cell, or by in vitro transcription, wherein the eukaryotic cell is preferably a plant cell, microbial cell such as a fungal cell, preferably a yeast cell. In an embodiment, the first RNA sequence of the double- stranded RNA region of the precursor RNA molecule, or the sense RNA sequence of the product RNA molecules, is shorter than the second RNA sequence or the antisense RNA sequence, respectively, preferably wherein the first RNA sequence is shorter than the second RNA sequence entirely because of the presence of non-basepaired ribonucleotides in the second RNA sequence that bulge from the double- stranded RNA region or the product RNA molecules, more preferably wherein the first RNA 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 second RNA sequence, or the length of the sense RNA sequence is about 21 / 22, 21 / 23 or 21 / 24 of the length of the antisense RNA sequence in the product RNA molecule, calculated as a fraction.

[0345] In an aspect, the present invention provides a one or more or all of the precursor RNA molecule of the invention, the double-stranded product RNA molecule of the invention, the population of double-stranded product 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, for use in treating a disease in a subject, the doublestranded product RNA molecule or population has a beneficial effect on at least one symptom of the disease. The subject may be a human or other mammalian animal, or a non-vertebrate animal.

[0346] In an aspect, the present invention provides the use of one or more or all of the precursor RNA molecule of the invention, the double-stranded product RNA molecule of the invention, the population of double-stranded product 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 the manufacture of a medicament for treating a disease, for example a disease caused by a fungal pathogen or transmitted by an insect pest.

[0347] In an aspect, the present invention provides a kit comprising one or more or all of the precursor RNA molecule of the invention, the double- stranded product RNA molecule of the invention, the population of double- stranded product 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. The kit may comprise instructions for its use.

[0348] Any embodiment herein shall be taken to apply mutatis mutandis to any other embodiment unless specifically stated otherwise.

[0349] The present invention is not to be limited in scope by the specific embodiments described herein, which are intended for the purpose of exemplification only. Functionally-equivalent products, compositions and methods are clearly within the scope of the invention, as described herein.

[0350] Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, compositions of matter, groups of steps or group of compositions of matter.

[0351] The invention is hereinafter described by way of the following non-limiting Examples and with reference to the accompanying figures.

[0352] BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

[0353] Figure 1: Synthesis of ledRNA constructs. Schematic designs of two ledRNA molecules: (A) This ledRNA molecule comprises a central sense sequence which can be considered to be two adjacent sense sequences, covalently linked without an intervening spacer sequence and having identity to the target RNA, an antisense sequence which is complementary to the sense sequence and which is divided into two regions, a 5' region and a 3' region, which flank the central sense sequence, and two loops that separate the sense from the antisense sequences. (B) This ledRNA molecule comprises a central antisense sequence which can be considered to be two adjacent antisense sequences, covalently linked without an intervening spacer sequence and having identity to the complement of a target RNA, a sense sequence which is complementary to the antisense sequence and which is divided into two regions, a 5' sense region and a 3' sense region, which flank the central antisense sequence, and two loops that separate the sense and antisense sequences. The RNA molecule produced by transcription, for example by in vitro transcription from a promoter such as a T7 or SP6 promoter, self-anneals by basepairing between the complementary sense and antisense sequences to form a double - stranded region with a loop at each end and having a “nick” in either the antisense or sense sequence. Additional sequences may be linked to the 5' and / or 3' ends as 5'- or 3'- extensions.

[0354] Figure 2: Alignment for the sense sequence of the target region of GUS mRNA (GUS WT; SEQ ID NO: 2) and the modified sense sequence (GUS GU; SEQ ID NO: 3), showing the positions of the C to T substitutions. Asterisks indicate nucleotides that were not changed.

[0355] Figure 3: GUS activity in plants transformed with constructs encoding modified hairpin RNAs for reducing expression of a GUS target gene. The control PPGH11 and PPGH24 plants without the hairpin constructs gave about 4000 MUG units.

[0356] Figure 4: Autoradiogram of a Northern blot of RNAs from six transgenic tobacco plants transformed with a 35S-hpGUS[G:U] construct and six tobacco plants transformed with a corresponding 35S-hpGUS[Con] construct. A. Upper panel shows larger RNAs hybridising to a 200-nucleotide GUS sense probe. Lower panel shows the staining of RNA in the gel prior to blotting, as a loading control. B. Upper panel shows small antisense RNAs hybridising to the sense GUS probe in the same plants as in A. The sample was lost for lane 4 of the hpGUS[G:U] plants resulting in no hybridising signal for that lane. Lower panel shows the same blot probed with an antisense probe to mirl68, as a loading control. The lane labelled M was loaded with size markers for 24 nucleotides (nt) and 21 nt.

[0357] Figure 5: Upper panel: Autoradiogram of Northern blot of small RNAs (21-24 nucleotides) from independently transformed plants expressing a 35S-hpGUS[G:U] construct (lanes 1-10) or a 35S-hpGUS[Con] construct (lanes 11-19). The blot was first probed with a sense GUS probe and then re-probed with U6 RNA probe as a loading control. Lower panels: Frequency distribution of sRNAs of 18-25 nucleotides length, calculated from RNA seq analysis (reads per million sequences). The upper part of each panel shows the frequency of sense sRNAs and the lower part antisense sRNAs. The panels labelled hpGUS[G:U]-12 and -13 show data from hpGUS[G:U] plant numbers 12 and 13 from the Northern blot, and the panel labelled hpGUS[Con]-3 shows data from hpGUS[Con] plant number 3.

[0358] Figure 6: Schematic representation of stem-loop structures of transcripts expressed from GUS hpRNA constructs. The transcripts have complementary sense and antisense sequences which basepair to form GUS sequence- specific dsRNA stems, with the indicated lengths in basepairs (bp) for the stems, and the number of nucleotides (nt) in the loops.

[0359] Figure 7: Northern blot analysis showing RNA from expression of the two short hpRNA transgenes GUShp93-l and GUShp93-2 in stably transformed A. thaliana plants. RNA samples were either treated (+) or not treated (-) with RNase I. The RNA blot was hybridised with a loop-specific antisense RNA probe.

[0360] Figure 8: (A) Schematic map of the genetic construct to express hpPBAN[Con] and hpPBAN[G:U] RNA molecules in plant cells. p35S: 35S promoter of CaMV; pT7: T7 RNA polymerase promoter; OCS-T: transcription terminator / polyadenylation region of the Agrobacterium ocs gene. (B) Alignment of the sense DNA sequences for hpPBAN[Con] (top row, PBAN-WT) and hpPBAN[G:U]. Asterisks show identity of nucleotides, absence of asterisks show the C to T substitutions.

[0361] Figure 9: Northern blot hybridisation to detect hpPBAN RNA transcripts in transgenic N. tabacum plants using an antisense loop sequence (upper panel) or a dsRNA stem sequence (middle panel) as probe. The lower panel shows the stained RNA on the gel before transfer to the Northern blot, as a control for loading. The bands corresponding to the stem-loop molecules (SL) and the processed loop (Loop) are arrowed.

[0362] Figure 10: Northern blot hybridisation to detect sRNA molecules from the dsRNA region of hpPBAN transcripts in transgenic N. tabacum plants to express hpPBAN[Con] or hpPBAN[G:U]. The size markers in the first lane were 21-mer and 24-mer in length.

[0363] Figure 11: Upper panel: Northern blot hybridisation to detect hpPBAN[Con] or hpPBAN[G:U] RNA molecules in independent transgenic A. thaliana plants (lanes hpPBAN[Con] and hpPBAN[G:U]), one N. benthamiana hpPBAN[Con] plant and one hpPBAN[G:U] plant (lanes Nb), one N. tabacum hpPBAN[Con] plant and one hpPBAN[G:U] plant (lanes W38), and S. cerevisiae strain HF7C (yeast) transformed with the construct to express hpPBAN[Con] in one lane or hpPBAN[G:U] RNA in the next lane, using an antisense loop sequence as probe. The A. thaliana plants were T2 plants homozygous for the transgene whereas the N. benthamiana and N. tabacum plants were TO plants. The two lanes at the extreme right contained in vitro produced RNA transcripts to show the position of the band for the full-length transcripts — the position of the main band (arrow) was evident at a shorter exposure time. Lower panel: the gel stained for RNA before blotting to the membrane, to show the RNA loading. The leftmost lane in the lower panel shows the size markers (RNA ladder).

[0364] Figure 12: Northern blot hybridisation detection of hpPBAN[Con] and hpPBAN[G:U] derived RNAs in total leaf RNA (Total RNA), loaded at 10 pg per lane, or from nuclear RNA loaded at 1.5 pg per lane (Nuclear RNA). Lanes labelled 7, 12 and 13 were from three N. benthamiana plants independently transformed with the hpPBAN[Con] construct, and lanes labelled 6, 10 and 12 were from three N. benthamiana plants independently transformed with the hpPBAN[G:U] construct. Lanes marked WT were loaded with RNA from a control, non-transgenic plant. The lower panel shows the gel stained for RNA as a loading control, with prominent bands for rRNA. Figure 13: Growth test of cotton bollworm larvae on detached leaf pieces from transgenic tobacco plants transformed with the hpPBAN[Con] or hpPBAN[G:U] constructs, compared to leaf pieces from untransformed tobacco plants. The lower panel shows the sizes of the resultant larvae.

[0365] Figure 14: Alignment of the sense DNA sequences for hpAChE[Con] (top row; SEQ ID NO: 17) and hpAChE[G:U] (SEQ ID NO: 18). Vertical dashes show identity of nucleotides, gaps show the C to T substitutions.

[0366] Figure 15: Alignment of the sense DNA sequences for hpHal[Con] (top row; SEQ ID NO: 23) and hpHal[G:U] (SEQ ID NO: 24). Vertical dashes show identity of nucleotides, gaps show the 73 C to T substitutions.

[0367] Figure 16: Alignment of the sense DNA sequences for hpHa2[Con] (top row; SEQ ID NO: 29) and hpHa2[G:U] (SEQ ID NO: 30). Vertical dashes show identity of nucleotides, gaps show the 73 C to T substitutions.

[0368] Figure 17: Alignment of the sense DNA sequences for hpHa3[Con] (top row; SEQ ID NO: 35) and hpHa3[G:U] (SEQ ID NO: 36). Vertical dashes show identity of nucleotides, gaps show the 75 C to T substitutions.

[0369] Figure 18: Alignment of the sense DNA sequences for hpHa4[Con] (top row; SEQ ID NO: 41) and hpHa4[G:U] (SEQ ID NO: 42). Vertical dashes show identity of nucleotides, gaps show the 77 C to T substitutions.

[0370] Figure 19: Alignment of the sense DNA sequences for hpHa5[Con] (top row; SEQ ID NO: 47) and hpHa5[G:U] (SEQ ID NO: 48). Vertical dashes show identity of nucleotides, gaps show the 75 C to T substitutions.

[0371] Figure 20: Alignment of the sense DNA sequences for hpHa6[Con] (top row; SEQ ID NO: 53) and hpHa6[G:U] (SEQ ID NO: 54). Vertical dashes show identity of nucleotides, gaps show the 77 C to T substitutions.

[0372] Figure 21: Alignment of the sense DNA sequences for hpHa7[Con] (top row; SEQ ID NO: 59) and hpHa7[G:U] (SEQ ID NO: 60). Vertical dashes show identity of nucleotides, gaps show the 77 C to T substitutions. Figure 22: Alignment of the sense DNA sequences for hpHa8[Con] (top row; SEQ ID NO: 65) and hpHa8[G:U] (SEQ ID NO: 66). Vertical dashes show identity of nucleotides, gaps show the 77 C to T substitutions.

[0373] Figure 23: Relative expression levels of candidate target genes in H. argimera larvae in neonates (Od) and at 1, 3, 5 and 8 days post hatching. Expression levels were normalised to expression of the EFl gene.

[0374] Figure 24: Upper panel: Photograph of an agarose gel after electrophoresis and staining for RNA of samples of extracted RNA from artificial diet material supplemented with hairpin RNAs transcribed from the hpAChE[Con] (hpCon) or hpAChE[G:U] (hpGU) genetic constructs, or without the RNA (Control), in the absence (-) or in the presence (+) of H. armigera larvae. Lower panel: Autoradiograph of the Northern blot of the same extracted RNA, probed for the hpAChE RNA.

[0375] Figure 25: Left hand panel. Autoradiograph of a Northern blot of extracted RNA from leaves painted with hairpin RNAs transcribed from the hpPBAN[Con] (hpCon) or hpPBAN[G:U] (hpGU) genetic constructs, or without the RNA (Control). The bands for in vitro transcribed RNA show the position of the full-length transcripts. Right hand panel. Autoradiograph of the Northern blot of RNA extracted from larvae fed for 24 hr on the painted leaves, probed for the hpPBAN RNA.

[0376] Figure 26: Quantitation by RT-PCR of target gene transcript levels in H. armigera larvae after feeding them on pakchoi leaves painted with in vitro transcribed hpRNA[G:U] RNA at the stated amount per 0.5 ml per leaf. Larvae were fed for three days (upper and middle panels) or six days (lower panels). Expression levels were normalised to expression of the EFl gene, as before.

[0377] Figure 27: Mortality rates for H. armigera larvae fed on N. benthamiana leaves expressing hairpin RNAs for 7 days.

[0378] Figure 28: Representative northern blot hybridisations of RNA from TO plants transformed with a genetic construct for expression of hpHal[G:U] or hpHal[Con] (upper panel), hpHa6[G:U] or hpHa6[Con] (middle and lower panels). The positions of the presumed full-length hairpin RNA and the loop fragment are arrowed. The lane labelled W38 had RNA from an untransformed plant.

[0379] Figure 29: Average leaf damage scores at day 7 for leaf pieces from transgenic plants transformed with constructs expressing hpHal[G:U] or hpHa6[G:U] RNA molecules compared to the corresponding, canonically basepaired hpHal[Con] RNA molecules or the control hpGFP[G:U] molecules. Each bar represents the average score for an independently transformed TO plant.

[0380] Figure 30: Northern blot hybridization shows clear downregulation of Ha6 mRNA in Helicoverpa armigera that have fed for 6 days on transgenic hpHa6[G:U] tobacco leaves. This is in contrast to insects fed on the hpHa6[Con] lines (labelled hpHal[WT]), showing significantly less Ha6 downregulation. Each RNA sample was extracted from approximately 5 insects that fed on leaves of the same TO plant line.

[0381] Figure 31: Schematic for construction by GoldenGate methods of a binary vector having sequences for a Gemini Virus (GV) construct for expression of RNAi molecules in plant cells. The DNA fragments are mixed, treated with restriction enzyme Bsal and T4 DNA ligase to insert the four components in the order shown into the backbone vector pICH47742rc, replacing the lacZ sequence.

[0382] Figure 32: Northern blot analysis of production in plant cells of a modified hairpin RNA molecule using a Gemini Virus (GV) expression construct compared to a non-replicative vector (NV) expressing the same RNA molecule, after transient expression in N. benthamiana leaves. The right-hand lane shows the RNA produced in a stably transformed tobacco plant. The CMV 2b silencing suppressor protein was either present (+) or absent (-) as indicated. The lower panel shows the stained RNA in the agarose gel prior to blotting, as loading control.

[0383] Figure 33: Schematic of asymmetric hairpin RNA (hpRNA) molecules transcribed from genetic constructs comprising sense sequences containing regularly spaced single, double or triple nucleotide deletions, targeting a transgene encoding GUS for silencing. The construct encoding hpGUS[Con] had the wild-type sense sequence from the GUS target region, whereas the construct encoding hpGUS[G:U] had the same sense sequence except that each C nucleotide was substituted with a T nucleotide, producing G:U non- canonical basepairs in the hpGUS[G:U]; these two constructs were used as controls. The siRNAs produced from each hpGUS molecule are shown schematically toward the left, indicating hybridised sense (upper) and antisense (lower) strands. The free energy of folding of each of the transcripts (kcal / mol) are shown at the right. Downward arrowheads indicate the positions of the unpaired nucleotides that bulge out from the dsRNA regions, and the X symbols in the siRNAs indicate positions of deletions in the modified sense sequences of the siRNAs.

[0384] Figure 34: Alignment of the nucleotide sequence (sense sequence) of a region of the GUS gene (upper sequence GUS[Con]; SEQ ID NO: 2) targeted by a hpRNA and the modified sense sequences (lower sequence) of the hpGUS[A22] construct (GUS[A22]; SEQ ID NO: 95), the hpGUS[A23] construct (GUS[A23]; SEQ ID NO: 96), the hpGUS [A24-1] construct (GUS[A24-1]; SEQ ID NO: 97), the hpGUS [A24-2] construct (GUS[A24-2]; SEQ ID NO: 98) or the hpGUS[A24-3] construct (GUS[A24-3]; SEQ ID NO: 99). Retained nucleotides are asterisked, nucleotides deleted in the modified sense sequences of the hpGUS construct are indicated with a dash.

[0385] Figure 35: Expression analysis. (A) Northern blot of small antisense RNAs expressed from the hpGUS transgenes agroinfiltrated into N. benthamiana leaves, probed with the sense sequence from the hpGUS RNA molecules. The lower panel of A shows the same Northern blot hybridised with a probe that was complementary to mirl68 as a loading control. (B) Northern blot of small antisense RNAs produced in hpGUS transgenic tobacco lines. The lower panel of B shows the same Northern blot hybridised with a probe that was complementary to U6 rRNA as a loading control. (C) Northern blot of small sense RNAs produced in hpGUS transgenic tobacco lines. The lower panel of C shows the same Northern blot hybridised with a probe that was complementary to mirl68 as a loading control. (D) GUS activity of 14 different TO transgenic plants for each genetic construct, measured by MUG assay of leaf samples.

[0386] Figure 36: Deep sequencing analysis of doubly transgenic tobacco plants transformed with a GUS target gene and a hpGUS construct, as indicated above each frame. (A) Reads (reads per million) for sRNAs of 19-25 nucleotides length mapped to the 200-nucleotide target region of the GUS gene. (B) Reads (reads per million) for sRNAs of 19-25 nucleotides length downstream of the target region, The rightward three frames in the upper panel are expanded in the middle panel. The bars show the frequencies of sense or antisense sRNAs which are 19, 20, 21, 22, 23, 24 or 25 nucleotides in length, per million reads in the total sRNA populations. Error bars = s.e.m. Figure 37: Deep sequencing reads for the total number of sRNAs of 19-25 nucleotides length mapping to the GUS target transcript, where the sRNAs were produced in tobacco plants transformed with a GUS target gene and a hpGUS construct as indicated. The plots show the positions in the GUS transcript where the sRNAs mapped to and the frequency at each position. The dotted lines show the position of the 200-nucleotide target region.

[0387] Figure 38: Alignment of the nucleotide sequence (sense sequence) of a region of the EIN2 gene (upper sequence EIN2[Con]; SEQ ID NO: 110) targeted by a hpRNA and the modified sense sequences (lower sequence) of the hpEIN2[A22] construct (EIN2[A22]; SEQ ID NO: 111), the hpEIN2[A23] construct (EIN2[A23]; SEQ ID NO: 112), the hpEIN2[A24-l] construct (EIN2[A24-1]; SEQ ID NO: 113), the hpEIN2[A24-2] construct (EIN2[A24-2]; SEQ ID NO: 114) or the hpEIN2[A24-3] construct (EIN2[A24- 3]; SEQ ID NO: 115). Retained nucleotides are asterisked, nucleotides deleted in the modified sense sequences of the hpEIN2 construct are indicated with a dash.

[0388] Figure 39: Analysis of transformed plants (A) Average hypocotyl lengths for A. thaliana seedlings transformed with asymmetric hpEIN2 constructs, compared to the untransformed seedlings (wildtype) or the conventional hpEIN2[Con] construct as a control. (B) Normalised EIN2 transcript levels in the same batches of transformed plants. Error bars = s.e.m.

[0389] Figure 40: Deep sequencing reads for transgenic A. thaliana plants transformed with a hpEIN2 construct, as indicated above each frame. (A) Reads (reads per million) for sRNAs of 19-25 nucleotides length mapped to the 200-nucleotide target region of the EIN2 gene. (B) Reads (reads per million) for sRNAs of 19-25 nucleotides length downstream of the target region. The frames labelled WT were for the wild-type (untransformed) control plants, and hpEIN2[Con] were for plants transformed with the conventional hairpin construct. The bars show the frequencies of sense or antisense sRNAs which are 19, 20, 21, 22, 23, 24 or 25 nucleotides in length, per million reads in the total sRNA populations. Error bars = s.e.m.

[0390] Figure 41: Deep sequencing reads of sRNAs were produced in A. thaliana plants transformed with a hpEIN2 construct as indicated. Deep sequencing reads for the total number of sRNAs of 19-25 nucleotides length mapping to the EIN2 target transcript. The plots show the positions in the EIN2 transcript where the sRNAs mapped to and the frequency at each position. The dotted lines show the position of the 200-nucleotide target region.

[0391] Figure 42: Alignment of the nucleotide sequence of a chimeric sense sequence formed by joining 300 nucleotides of CMV 2b gene with 300 nucleotides of the cDNA from PVY (upper sequence; SEQ ID NO: 117) and the modified sense sequence (lower sequence; SEQ ID NO: 119) of the hpCMV / PVY[A22] construct. Retained nucleotides are asterisked, nucleotides deleted in hpCMV / PVY[A22] are indicated with a dash.

[0392] Figure 43: Northern blot analyses. Panel A: Northern blot of RNA from plants infected with CMV, probed for viral RNAs, showing the positions of CMV RNA3 and RNA4(CP). Panel B. Photo of the stained gel for Panel A prior to blotting, showing the loading of the lanes and the rRNA bands. Panel C. Northern blot of sRNAs hybridizing to the target region of the CMV, showing the positions of 21nt, 22nt and 24nt bands. Panel D. The same blot as in Panel C but hybridized with a probe to detect rRNA U6, for loading control. The lanes are labelled with the construct in the transgenic plants from which the RNAs were prepared; WT = wild-type, non-transgenic plants as control. The left-hand most WT lanes were from wild-type control plants that were not infected with CMV.

[0393] Figure 44. Alignment of the chimeric sense sequence formed by joining to regions of the B. napus DDM1 gene (upper sequence DDMl[Con]; SEQ ID NO: 122) included in the control construct hpDDMl[Con] and in ledDDMl[Con], and the modified sense sequence (lower sequence) of the ledDDMl [A22] construct (SEQ ID NO: 125). Identical nucleotides are shown by vertical strokes; nucleotides deleted in the modified sense sequence of the ledDDMl [A22] construct are indicated with a dash.

[0394] Figure 45. qRT-PCR measurement of DDM1 mRNA transcript abundance in B. napus floral tissue after treatment with ledDDMl [Con] (ledDDMl) or ledDDMl [A22] (ledDDMl [22nt]) RNAs.

[0395] Figure 46: Demonstration of root uptake and systemic movement of ledDDMl [Con] molecules after topical application. The canola roots were in contact with the RNA solution for six hours before being analysed for uptake of ledDDMl [Con] and reduction of DDM1 mRNA in root and cotyledons. Figure 47: Systemic silencing and movement of ledRNA molecules taken up through the roots. (A) 1% agarose gel demonstrating integrity and amount of the ledGUS (control), ledDDMlfCon] and ledDDMl[A22] molecules. (B) Initial experiment demonstrating enhanced silencing of ledDDMl[A22] compared to ledDDMlfCon], (C) Northern blot demonstrating uptake of the RNAi molecules into the roots and movement into the cotyledons. D) qRT-PCR demonstrating reduction of DDM1 mRNA in roots and cotyledons.

[0396] Figure 48: Demonstration of enhanced silencing and systemic movement of RNA molecules from cotyledon into adjacent tissues. (C) Northern blot of RNA from untreated cotyledons and roots showing presence of RNA molecules in those organs. (D) Reduction of the target mRNA at 6hr, 24hr and 6 days as measured by qRT-PCR.

[0397] Figure 49: Alignment of the modified sense sequence (SEQ ID NO: 139), used in NbSu Construct 2 having the A22 modification (NbSu-A22), with the wild-type sense sequence (NbSu-WT; SEQ ID NO: 129). The positions of the single nucleotide deletions shown as dashes.

[0398] Figure 50: Alignment of the modified sense sequence (SEQ ID NO: 140), used in NbSu Constructs 3 and 4 having the A22 and A to G substitution modifications (NbSu-A22AG), with the wild-type sense sequence (NbSu-WT; SEQ ID NO: 129). The positions of the single nucleotide deletions shown as dashes and the substitutions in grey.

[0399] Figure 51: Alignment of the modified antisense sequence (SEQ ID NO: 141), used in NbSu Construct 4 having the C to T substitutions (NbSu-A22CT) with the unmodified antisense sequence (NbSu-AS; SEQ ID NO: 130) used in Constructs 1, 2 and 3. The positions of the 24 nucleotide substitutions shown in grey.

[0400] Figure 52: Schematic design of genetic constructs to express a hairpin RNA molecule (Panel A) or ledRNA molecule (Panel B) either in vitro using a T7 RNA Polymerase promoter or in vivo using a CaMV 35S promoter. The linear arrangements show the DNA elements of the genetic constructs, the lower schematic structures show the hpRNA or ledRNA molecules after transcription of the genetic constructs, splicing of the Cat-1 introns when transcribed in a eukaryotic cell, and folding of the RNA strand by hybridisation between complementary sense and antisense strands to produce the hpRNA or ledRNA molecules. The 5' and 3' ends of the RNA molecules are indicated. There is a single-strand nick between the 3' and 5' ends of the ledRNA (not shown). The GGG trinucleotide at the 5' end was added to provide for efficient in vitro transcription using T7 RNA Polymerase, with the complementary CCC trinucleotide inserted at the 3' end of the hairpin molecule or into the antisense strand of the ledRNA construct.

[0401] Figure 53: Schematic representations of structures of asymmetric RNA molecules. Showing a precursor RNA molecule (A) in the upper part of each panel A-Z and the resultant double-stranded product RNA molecules (P) after cleavage by a Dicer in the lower part. The extent of the double- stranded region (B) comprising the first RNA strand

[0402] (D) and second RNA strand (F) is shown, joined by a linking RNA sequence (Loop(L)). In each panel A-Z, the double-stranded region (B) can be extended leftward and / or rightward by addition of ribonucleotides, as desired. The position of the loop at the righthand end of each precursor molecule implies a 5' to 3' order of the sequences as (D)- loop-(F), whereas if the loop (L) is present at the left-hand end of the double- stranded region to join the strands, the 5' to 3' order would be (F)-loop-(D); this is a possible variation for all panels A-Z. The arbitrary position and length of the first RNA sequence

[0403] (E) within the first RNA strand (D) and the second RNA sequence (G) within the second RNA strand (F) are shown — the position and length of (E) and (G) within (D) and (F), respectively, can be changed arbitrarily, for example can be considered to occupy the whole of (D) and (F), respectively. Ribonucleotides are shown as N, N' or B. N and N' with downward strokes represent basepairs when the first RNA strand (D) and second RNA strand (F) hybridise, independently forming either canonical basepairs or non- canonical basepairs such as G:U basepairs. Ribonucleotides shown as B bulge out from the double-stranded structure. Each horizontal dash represents the absence of a ribonucleotide opposite a B ribonucleotide, engineered through either the deletion of a ribonucleotide at the dash position or the insertion of the ribonucleotide shown as B. Each B opposite another B represents a mismatched (non-basepaired) ribonucleotide pair. Vertical arrows represent exemplary positions for cleavage by a Dicer, at either closed arrows or open arrows. The positions of cleavage can be moved leftward or rightward within the double-stranded region (B), with a first cleavage anywhere between the loop (L) and the first bulged nucleotide, provided at least some of product molecules (P) are produced having the same lengths of sense RNA sequence (H) and antisense RNA sequence (J) i.e. 21 / 22-mer, 21 / 23-mer or 21 / 24-mers. When Dicer cleaves at the exemplary positions shown with closed arrows, the product RNA molecules (P) are shown below the long, closed arrow; analogously the cleavage at the positions shown by open arrows produces product RNA molecules (P) shown below the long, open arrow. Panels A-E and F-H represent exemplary structures that produce at least some 21 / 22-mer siRNAs (P), panels I-N and O-Q represent exemplary structures that produce at least some 21 / 23-mer siRNAs (P), and panels R-V and U-Z represent exemplary structures that produce at least some 21 / 24-mer siRNAs (P). Panels F-H, O-Q and U-Z represent shorter structures where a ribonuclease such as Drosha is capable of cleaving off the 5' leader and 3' trailer sequences. In panels F-H, O-Q and U-Z, the double- stranded regions can be extended by basepairing between ribonucleotides N2 and N3, and further between ribonucleotides N 1 and N4, or extended even further. In each of panels A-Z, the positions of the non-basepaired nucleotide(s) B can be varied as desired. Product RNA molecules (P) have two-ribonucleotide 3' overhangs that are not basepaired. The 5' and 3' ends are shown. Panel A: the first RNA sequence (E) has one non-basepaired ribonucleotide and the second RNA sequence (G) has two non-basepaired ribonucleotides, including a mismatched pair, forming two bulges in the double- stranded region. Panel B: the first RNA sequence (E) has one non-basepaired ribonucleotide and the second RNA sequence (G) has two non-basepaired ribonucleotides, including a mismatched pair, forming one bulge in the double-stranded region. Panel C: the first RNA sequence (E) has one non-basepaired ribonucleotide and the second RNA sequence (G) has two non-basepaired ribonucleotides, forming three single -ribonucleotide bulges in the double-stranded region. Panel D: the first RNA sequence (E) has no non- basepaired ribonucleotides and the second RNA sequence (G) has one non-basepaired ribonucleotide, forming one bulge in the double- stranded region. Panel E: shows an extended double- stranded structure where the modifications are concatemerized. Each first RNA sequence (E) has no non-basepaired ribonucleotides and each second RNA sequence (G) has one non-basepaired ribonucleotide, forming one bulge in the doublestranded region about every 22 ribonucleotides. The bulges are separated by 21 contiguous basepairs, but that number can be varied to provide an average separation of the bulges of about 21 basepairs. Panel F: the first RNA sequence (E) has one non- basepaired ribonucleotide and the second RNA sequence (G) has two non-basepaired ribonucleotides, including a mismatched pair, not counting ribonucleotides N1-N4 which may or may not be basepaired, forming two bulges in the double-stranded region. Panel G: the first RNA sequence (E) has one non-basepaired ribonucleotide and the second RNA sequence (G) has two non-basepaired ribonucleotides, not counting ribonucleotides N1-N4 which may or may not be basepaired, forming three bulges in the double-stranded region. Panel H: the first RNA sequence (E) has no non-basepaired ribonucleotides and the second RNA sequence (G) has one non-basepaired ribonucleotide, not counting ribonucleotides N1-N4 which may or may not be basepaired, forming one bulge in the double- stranded region. Panel I: the first RNA sequence (E) has one non-basepaired ribonucleotide and the second RNA sequence (G) has two non-basepaired ribonucleotides, including a mismatched pair, forming two bulges in the double- stranded region. Produces at least some 21 / 23-mers. Panel J: the first RNA sequence (E) has one non-basepaired ribonucleotide and the second RNA sequence (G) has three non-basepaired ribonucleotides, forming four bulges in the double-stranded region. Produces at least some 21 / 23-mers. Panel K: the first RNA sequence (E) has no non-basepaired ribonucleotides and the second RNA sequence (G) has two non-basepaired ribonucleotides, forming a single di -ribonucleotide bulge in the double-stranded region. Produces at least some 21 / 23-mers. Panel L: the first RNA sequence (E) has no non-basepaired ribonucleotides and the second RNA sequence (G) has two non-basepaired ribonucleotides, forming two single-ribonucleotide bulges in the double-stranded region. Produces at least some 21 / 23-mers. Panel M: shows an extended double-stranded structure where the modifications are concatemerized. Each first RNA sequence (E) has no non-basepaired ribonucleotides and each second RNA sequence (G) has two non-basepaired ribonucleotides, forming two bulges in the double- stranded region, then concatemerized. The bulges are each separated by 10 or 11 contiguous basepairs, but that number can be varied to provide a separation of two bulges about, on average, every 23 ribonucleotides. Produces at least some 21 / 23-mers. Panel N: shows an extended double- stranded structure where the modifications are concatemerized. Each first RNA sequence (E) has no non-basepaired ribonucleotides and each second RNA sequence (G) has two non-basepaired ribonucleotides, forming a di-ribonucleotide bulge in double-stranded region, then concatemerized. The bulges are each separated by 21 contiguous basepairs, but that number can be varied to provide a separation between bulges of, on average, about 21 basepairs. Produces at least some 21 / 23-mers. A hybrid structure between those shown in panels M and N would have single and diribonucleotide bulges. Panel O: the first RNA sequence (E) has one non-basepaired ribonucleotide and the second RNA sequence (G) has three non-basepaired ribonucleotides, including a mismatched pair, not counting ribonucleotides N1-N4 which may or may not be basepaired, forming two bulges in the double-stranded region. Produces at least some 21 / 23-mers. Panel P: the first RNA sequence (E) has no non- basepaired ribonucleotides and the second RNA sequence (G) has two non-basepaired ribonucleotides, not counting ribonucleotides N1-N4 which may or may not be basepaired, forming a di-ribonucleotide bulge in the double- stranded region. Produces at least some 21 / 23-mers. Panel Q: the first RNA sequence (E) has no non-basepaired ribonucleotides and the second RNA sequence (G) has two non-basepaired ribonucleotides, not counting ribonucleotides N1-N4 which may or may not be basepaired, forming two single-ribonucleotide bulges in the double-stranded region. Produces at least some 21 / 23-mers. Panel R: the first RNA sequence (E) has one nonbasepaired ribonucleotide and the second RNA sequence (G) has four non-basepaired ribonucleotides, including a mismatched pair, forming two bulges in the double- stranded region including a tri-ribonucleotide bulge. Produces at least some 21 / 24-mers. Panel S: the first RNA sequence (E) has one non-basepaired ribonucleotide and the second RNA sequence (G) has four non-basepaired ribonucleotides, forming five bulges in the doublestranded region, all single-ribonucleotide bulges. Produces at least some 21 / 24-mers. Panel T: extended double- stranded region, showing how multiple structures can be formed for product RNA molecules (P). The first RNA sequence (E) has no non- basepaired ribonucleotides and the second RNA sequence (G) in each case has three non- basepaired ribonucleotides, forming one tri-ribonucleotide bulge and three singleribonucleotide bulges in the double- stranded region. Produces at least some 21 / 24-mers. Panel U: extended double-stranded region, showing how multiple structures can be formed for product RNA molecules (P). The first RNA sequence (E) has no non- basepaired ribonucleotides and the second RNA sequence (G) in each case has three non- basepaired ribonucleotides, forming one tri-ribonucleotide bulge, a single -ribonucleotide bulge and a di-ribonucleotide bulge in the double- stranded region. The order of these bulges can be re-arranged, provided the second RNA sequence (G) in each case has, on average, three non-basepaired ribonucleotides about every 24 ribonucleotides. Produces at least some 21 / 24-mers. Panel V: extended form, concatemerized. The first RNA sequence (E) has no non-basepaired ribonucleotides and the second RNA sequence (G) has three non-basepaired ribonucleotides, forming a tri-ribonucleotide bulge, on average, about every 24 ribonucleotides in the double- stranded region. Produces at least some 21 / 24-mers. Panel W: the first RNA sequence (E) has one non-basepaired ribonucleotide and the second RNA sequence (G) has four non-basepaired ribonucleotides, including a mismatched pair, not counting ribonucleotides N1-N4 which may or may not be basepaired, forming two bulges in the double-stranded region. Produces at least some 21 / 24-mers. Panel X: the first RNA sequence (E) has no non-basepaired ribonucleotides and the second RNA sequence (G) has three non-basepaired ribonucleotides, not counting ribonucleotides N1-N4 which may or may not be basepaired, forming a single tri-ribonucleotide bulge in the double-stranded region. Produces at least some 21 / 24- mers. Panel Y: the first RNA sequence (E) has no non-basepaired ribonucleotides and the second RNA sequence (G) has three non-basepaired ribonucleotide, not counting ribonucleotides N 1-N4 which may or may not be basepaired, forming two bulges in the double-stranded region, including one single ribonucleotide bulge and the other a diribonucleotide bulge. Produces at least some 21 / 24-mers. Panel Z: the first RNA sequence (E) has no non-basepaired ribonucleotides and the second RNA sequence (G) has three non-basepaired ribonucleotide, not counting ribonucleotides N1-N4 which may or may not be basepaired, forming three single-ribonucleotide bulges in the doublestranded region. Produces at least some 21 / 24-mers.

[0404] Figure 54. Variant chimeric RNA molecules having additional target transcript sequences. The loop sequence of hpNbSu[Con] (Example 24) was substituted with a 400nt unmodified sense sequence to make Construct 27.1 encoding hpNbSu[Con]- Loopl, or a chimeric 500nt sense sequence to make Construct 27.2 encoding hpNbSu[Con]-Loop2. In analogous fashion, the loop sequence of hpNbSu[G:U] was substituted with the 400nt unmodified sense sequence to make Construct 27.3 encoding hpNbSu[G:U]-Loopl, and the loop sequence of hpNbSu[A22] was substituted with the 400nt unmodified sense sequence to make Construct 27.4 encoding hpNbSu[A22]-Loop 1 or with the chimeric 500nt sense sequence to make Construct 27.5 encoding hpNbSu[A22]-Loop2. The hpNbSu[A22] molecule comprising the Cat-1 intron as a loop sequence was further modified by adding the chimeric 500nt sequence to the 3' end of the antisense sequence as a 3' extension, generating Construct 27.6 encoding hpNbSu[A22]-ext.

[0405] Figure 55. Resistance of plants to fungal infection. The graph shows the mean percentage of transgenic plants with yellowing or necrotic leaves 16 days post inoculation with F. oxysporum. Mean taken of two replicates of 10 plants per pot for each line.

[0406] Figure 56. Representative northern blot hybridisation of RNA from T1 transgenic tobacco (W38) or N. benthamiana (Nb) plants transformed with a genetic construct for expression of hpHa5[Con] or hpHa5[G:U]. The position of the presumed full-length hairpin RNA is arrowed. The lane labelled W38 had RNA from an untransformed tobacco plant.

[0407] KEY TO THE SEQUENCE LISTING

[0408] SEQ ID NO: 1. Nucleotide sequence of the protein coding region of the cDNA corresponding to the mRNA encoding GUS; 1812nt. SEQ ID NO: 2. Nucleotide sequence of the GUS target sequence, included as the sense sequence for a conventional construct encoding hpGUS[Con]; 200nt.

[0409] SEQ ID NO: 3. Nucleotide sequence of the sense sequence for the construct encoding hpGUS[G:U], containing 52 C to T substitutions relative to SEQ ID NO: 2; 200nt.

[0410] SEQ ID NO: 4. Nucleotide sequence (RNA) of the hairpin structure of the hpGUS[Con] RNA, including its loop sequence after splicing of the intron and the restriction enzyme sites used for cloning purposes. The sense sequence is nucleotides 7-206 and the antisense sequence is nucleotides 258-457; 463nt.

[0411] SEQ ID NO: 5. Nucleotide sequence (RNA) of the hairpin structure of the hpGUS[G:U] RNA, including its loop sequence after splicing of the intron and the restriction enzyme sites used for cloning purposes. The sense sequence is nucleotides 7-206 and the antisense sequence is nucleotides 246-451; 457nt.

[0412] SEQ ID NO: 6. Nucleotide sequence encoding stem-loop structure of hairpin RNAs hpGUS93-l and hpGUS93-2. Nucleotides 1-11 and 285-295, restriction enzyme sites; nucleotides 8-100 are the 93-nucleotide sense sequence corresponding to nucleotides 512-604 of SEQ ID NO: 1, nucleotides 101-195 are a spacer sequence corresponding to nucleotides 610-700 of SEQ ID NO: 1, and nucleotides 196-288 are the 93-nucleotide antisense sequence complementary to nucleotides 512-604 of SEQ ID NO: 1; 295nt.

[0413] SEQ ID NO: 7. Amino acid sequence of Helicoverpa armigera Pheromone Biosynthesis Activating Neuropeptide (PBAN) polypeptide; NCBI Reference Sequence: XP_021199198; 174aa.

[0414] SEQ ID NO: 8. Nucleotide sequence of a cDNA for Helicoverpa armigera PBAN, through to translation stop codon; Genbank Accession No. XM_021343523.1 (LOCI 10382821). The protein coding region corresponds to nucleotides 36-560; 560nt.

[0415] SEQ ID NO: 9. Nucleotide sequence of a 280-nucleotide target region of the RNA for H. armigera PBAN; 280nt. SEQ ID NO: 10. Nucleotide sequence of a modified 280-nucleotide sense region corresponding to the RNA for H. armigera PBAN, having 64 cytosines substituted with thymidines; 280nt.

[0416] SEQ ID NO: 11. Nucleotide sequence of a DNA molecule encoding hpPBAN[Con] for targeting the transcript of the gene encoding H. armigera PBAN. The order of elements are: Restriction sites for BamHI (nucleotides 1-6), Xhol (6-11), T7 promoter (12-31), PBAN antisense sequence (32-311), loop sequence (312-461), PBAN sense sequence (462-741), restriction sites for Smal (742-747) and Hindlll (748-754). The region of the mRNA used as the sense sequence was nucleotides 36-315, the loop sequence was nucleotides 316-465; 754nt.

[0417] SEQ ID NO: 12. Nucleotide sequence encoding hpPBAN[G:U] targeting the transcript of the gene encoding H. armigera PBAN. The order of elements are: restriction sites for BamHI (nucleotides 1-6), Xhol (6-11), T7 promoter (12-31), PBAN antisense sequence (32-311), loop sequence (312-461), PBAN sense sequence (462-741), restriction sites for Smal (742-747) and t dIII (748-754); 754nt.

[0418] SEQ ID NO: 13. Nucleotide sequence of the protein coding region of a construct p35S- CMV-2b encoding a CMV 2b silencing suppressor polypeptide; 333nt.

[0419] SEQ ID NO: 14. Amino acid sequence of the CMV 2b silencing suppressor polypeptide (Goto et al., 2007); 11 laa.

[0420] SEQ ID NO: 15. Amino acid sequence of H. armigera acetylcholinesterase AChEl; NCBI Accession No. AAM90333.1 (Ren et al., 2002); 646aa.

[0421] SEQ ID NO: 16. Nucleotide sequence of a cDNA for H. armigera acetylcholinesterase mRNA; GenBank Accession No. AF369793.1; Ren et al., (2002). The protein coding region is nucleotides 316-2259; 255 Int.

[0422] SEQ ID NO: 17. Nucleotide sequence of a 494 -nucleotide target region of the cDNA for the mRNA encoding H. armigera acetylcholinesterase, corresponding to nucleotides 895-1388 of SEQ ID NO: 16; 494nt. SEQ ID NO: 18. Nucleotide sequence of a modified 494 nucleotide sense region corresponding to the RNA for H. armigera acetylcholinesterase, having 86 cytosines substituted with thymidines relative to the wild-type sequence; 494nt.

[0423] SEQ ID NO: 19. Nucleotide sequence of a DNA molecule encoding hpAChE[Con] for targeting the transcript of the AChEl gene encoding H. armigera acetylcholinesterase. The order of elements are: Restriction sites for BamHI (nucleotides 1-6), Xhol (6-11), T7 promoter (12-31), AChE antisense sequence (32-525), loop sequence (526-787), AChE sense sequence (788-1281), restriction sites for Smal (1282-1287) and Hindlll (1288-1293); 1294nt.

[0424] SEQ ID NO: 20. Nucleotide sequence of a DNA molecule encoding hpAChE[G:U] for targeting the transcript of the AChEl gene encoding H. armigera acetylcholinesterase. The order of elements are: Restriction sites for BamHI (nucleotides 1-6), Xhol (6-11), T7 promoter (12-31), AChE antisense sequence (32-525), loop sequence (526-787), modified AChE sense sequence (788-1281), restriction sites for Smal (1282-1287) and Hzndm (1288-1293); 1294nt.

[0425] SEQ ID NO: 21. Amino acid sequence of El. armigera ecdysone receptor EcR (Hal); NCBI Genbank Accession No. ASK12085.1; 584aa.

[0426] SEQ ID NO: 22. Nucleotide sequence of a cDNA for El. armigera ecdysone receptor EcR mRNA; GenBank Accession No. KY328717.1. The protein coding region is nucleotides 255-2009; 2407 nt.

[0427] SEQ ID NO: 23. Nucleotide sequence of a 300-nucleotide target region of the cDNA for a mRNA encoding El. armigera ecdysone receptor EcR, corresponding to nucleotides 1240-1539 of SEQ ID NO: 22; 300nt.

[0428] SEQ ID NO: 24. Nucleotide sequence of a modified 300-nucleotide sense region corresponding to the RNA for El. armigera ecdysone receptor, having 73 cytosines substituted with thymidines (24.3%) relative to the wild-type sequence; 300nt.

[0429] SEQ ID NO: 25. Nucleotide sequence of a DNA molecule encoding hpHal[Con] for targeting the transcript of the EcR gene encoding El. armigera ecdysone receptor. The order of elements: Restriction site for Xhol (nucleotides 3-8), T7 promoter (9-28), EcR (Hal) antisense sequence (29-328), loop sequence (329-478), EcR sense sequence (479- 778), restriction sites for Smal (779-784) and Xbal (787-792); 794nt.

[0430] SEQ ID NO: 26. Nucleotide sequence of a DNA molecule encoding hpHal[G:U] for targeting the transcript of the EcR gene encoding H. armigera ecdysone receptor. The order of elements are: Restriction site for Xhol (nucleotides 3-8), T7 promoter (9-28), EcR (Hal) antisense sequence (29-328), loop sequence (329-478), modified EcR sense sequence (479-778), restriction sites for Smal (779-784) and Xbal (787-792); 794nt.

[0431] SEQ ID NO: 27. Amino acid sequence of H. armigera coatomer beta subunit (Ha2); NCBI Genbank Accession No. XP_021194683.1 (LOCI 10379367); encoded by transcript variant X2; 950aa.

[0432] SEQ ID NO: 28. Nucleotide sequence of a cDNA for H. armigera coatomer beta subunit mRNA; GenBank Accession No. XM_021339008.1. The protein coding region is nucleotides 88-2940; 3057nt.

[0433] SEQ ID NO: 29. Nucleotide sequence of a 300-nucleotide target region of the cDNA for the mRNA encoding H. armigera coatomer-P, corresponding to nucleotides 1632-1931 of SEQ ID NO: 28; 300nt.

[0434] SEQ ID NO: 30. Nucleotide sequence of a modified 300-nucleotide sense region corresponding to the RNA for H. armigera coatomer-P, having 73 cytosines substituted with thymidines (24.3%) relative to the wild-type sequence; 300nt.

[0435] SEQ ID NO: 31. Nucleotide sequence of a DNA molecule encoding hpHa2[Con] for targeting the transcript of the coatomer beta gene encoding H. armigera coatomer beta subunit. The order of elements are: Restriction site for Xhol (nucleotides 3-8), T7 promoter (9-28), Ha2 antisense sequence (29-328), loop sequence (329-478), Ha2 sense sequence (479-778), restriction sites for Smal (779-784) and Xbal (787-792); 794nt.

[0436] SEQ ID NO: 32. Nucleotide sequence of a DNA molecule encoding hpHa2[G:U] for targeting the transcript of the coatomer beta gene encoding H. armigera coatomer beta subunit. The order of elements are: Restriction site for Xhol (nucleotides 3-8), T7 promoter (9-28), Ha2 antisense sequence (29-328), loop sequence (329-478), modified Ha2 sense sequence (479-778), restriction sites for Smal (779-784) and Xbal (787-792); 794nt.

[0437] SEQ ID NO: 33. Amino acid sequence of H. armigera molt-regulating transcription factor (HR3), referred to herein as Ha3; NCBI Genbank Accession No. ACH86113.1; 556aa.

[0438] SEQ ID NO: 34. Nucleotide sequence of the protein coding region of a cDNA for H. armigera molt-regulating transcription factor (Ha3) mRNA; GenBank Accession No. FJ009448.1. The protein coding region is nucleotides 1-1671; 1671nt.

[0439] SEQ ID NO: 35. Nucleotide sequence of a 300-nucleotide target region of the cDNA for the mRNA encoding H. armigera Molt-regulating transcription factor (Ha3), corresponding to nucleotides 48-347 of SEQ ID NO: 34; 300nt.

[0440] SEQ ID NO: 36. Nucleotide sequence of a modified 300-nucleotide sense region corresponding to the RNA for H. armigera Molt-regulating transcription factor (Ha3), having 75 cytidines substituted with thymidines (25.0%) relative to the wild-type sequence; 300nt.

[0441] SEQ ID NO: 37. Nucleotide sequence of a DNA molecule encoding hpHa3[Con] for targeting the transcript of the Molt-regulating transcription factor gene encoding H. armigera Molt-regulating transcription factor. The order of elements are: Restriction site for TzoI (nucleotides 3-8), T7 promoter (9-28), Ha3 antisense sequence (29-328), loop sequence (329-478), Ha3 sense sequence (479-778), restriction sites for Smal (779-784) and Xbal (787-792); 794nt.

[0442] SEQ ID NO: 38. Nucleotide sequence of a DNA molecule encoding hpHa3[G:U] for targeting the transcript of the Molt-regulating transcription factor gene encoding H. armigera Molt-regulating transcription factor. The order of elements are: Restriction site for TzoI (nucleotides 3-8), T7 promoter (9-28), Ha3 antisense sequence (29-328), loop sequence (329-478), modified Ha3 sense sequence (479-778), restriction sites for Smal (779-784) and Xbal (787-792); 794nt. SEQ ID NO: 39. Amino acid sequence of H. armigera V-type proton ATPase catalytic subunit A (Ha4); NCBI Genbank Accession No. XP_021181049.1 (LOCI 10369820), encoded by transcript variant XI; 620aa.

[0443] SEQ ID NO: 40. Nucleotide sequence of a cDNA for H. armigera V-type proton ATPase catalytic subunit A mRNA; GenBank Accession No. XM_021325374.1 (LOCI 10369820), transcript variant XL The protein coding region is nucleotides 144- 2006; 2645nt.

[0444] SEQ ID NO: 41. Nucleotide sequence of a 300-nucleotide target region of the cDNA for the mRNA encoding H. armigera V-type proton ATPase catalytic subunit A, corresponding to nucleotides 164-463 of SEQ ID NO: 40; 300nt.

[0445] SEQ ID NO: 42. Nucleotide sequence of a modified 300-nucleotide sense region corresponding to the RNA for H. armigera V-type proton ATPase catalytic subunit A, having 77 cytosines substituted with thymidines (25.7%) relative to the wild-type sequence; 300nt.

[0446] SEQ ID NO: 43. Nucleotide sequence of a DNA molecule encoding hpHa4[Con] for targeting the transcript of the V-type proton ATPase catalytic subunit A gene encoding H. armigera V-type proton ATPase catalytic subunit A. The order of elements are: Restriction site for Xhol (nucleotides 3-8), T7 promoter (9-28), Ha4 antisense sequence (29-328), loop sequence (329-478), Ha4 sense sequence (479-778), restriction sites for Smal (779-784) and Xbal (787-792); 794nt.

[0447] SEQ ID NO: 44. Nucleotide sequence of a DNA molecule encoding hpHa4[G:U] for targeting the transcript of the V-type proton ATPase catalytic subunit A gene encoding H. armigera V-type proton ATPase catalytic subunit A. The order of elements are: Restriction site for Xhol (nucleotides 3-8), T7 promoter (9-28), Ha3 antisense sequence (29-328), loop sequence (329-478), modified Ha3 sense sequence (479-778), restriction sites for Smal (779-784) and Xbal (787-792); 794nt.

[0448] SEQ ID NO: 45. Amino acid sequence of H. armigera trypsin-like serine protease (Ha5); NCBI Genbank Accession No. ACJ66841.1; 299aa. The signal peptide consists of the first 20 amino acids. The trypsin-like serine protease domain is amino acids 28- 274; 299aa. SEQ ID NO: 46. Nucleotide sequence of a cDNA for H. armigera trypsin-like serine protease mRNA; GenBank Accession No. EU874846.1. The protein coding region is nucleotides 32-931; 1025nt.

[0449] SEQ ID NO: 47. Nucleotide sequence of a 300-nucleotide target region of the cDNA for the mRNA encoding H. armigera trypsin-like serine protease, corresponding to nucleotides 621-920 of SEQ ID NO: 46; 300nt.

[0450] SEQ ID NO: 48. Nucleotide sequence of a modified 300-nucleotide sense region corresponding to the RNA for H. armigera Trypsin-like serine protease, having 75 cytosines substituted with thymidines (25.0%) relative to the wild-type sequence; 300nt.

[0451] SEQ ID NO: 49. Nucleotide sequence of a DNA molecule encoding hpHa5[Con] for targeting the transcript of the Trypsin-like serine protease gene encoding H. armigera Trypsin-like serine protease. The order of elements are: Restriction site for Xhol (nucleotides 3-8), T7 promoter (9-28), Ha5 antisense sequence (29-328), loop sequence (329-478), Ha5 sense sequence (479-778), restriction sites for Smal (779-784) and ?al (787-792); 794nt.

[0452] SEQ ID NO: 50. Nucleotide sequence of a DNA molecule encoding hpHa5[G:U] for targeting the transcript of the Trypsin-like serine protease gene encoding H. armigera Trypsin-like serine protease. The order of elements are: Restriction site for Xhol (nucleotides 3-8), T7 promoter (9-28), Ha5 antisense sequence (29-328), loop sequence (329-478), modified Ha5 sense sequence (479-778), restriction sites for Smal (779-784) and Xbal (787-792); 794nt.

[0453] SEQ ID NO: 51. Amino acid sequence of H. armigera synaptic vesicle glycoprotein 2C- like, encoded by transcript variant X2 of gene LOCI 10370333 (Ha6); NCBI Genbank Accession No. XP_021181756.1; 557aa.

[0454] SEQ ID NO: 52. Nucleotide sequence of a cDNA for H. armigera synaptic vesicle glycoprotein 2C-like mRNA; GenBank Accession No. XM_021326081. The protein coding region is nucleotides 386-2059; 2661nt. SEQ ID NO: 53. Nucleotide sequence of a 300-nucleotide target region of the cDNA for the mRNA encoding H. armigera synaptic vesicle glycoprotein 2C-like, corresponding to nucleotides 1010-1309 of SEQ ID NO: 52; 300nt.

[0455] SEQ ID NO: 54. Nucleotide sequence of a modified 300-nucleotide sense region corresponding to the RNA for H. armigera synaptic vesicle glycoprotein 2C-like, having 77 cytosines substituted with thymidines (25.7%) relative to the wild-type sequence; 300nt.

[0456] SEQ ID NO: 55. Nucleotide sequence of a DNA molecule encoding hpHa6[Con] for targeting the transcript of the synaptic vesicle glycoprotein 2C-like gene encoding H. armigera synaptic vesicle glycoprotein 2C-like. The order of elements are: Restriction site for Xhol (nucleotides 3-8), T7 promoter (9-28), Ha6 antisense sequence (29-328), loop sequence (329-478), Ha6 sense sequence (479-778), restriction sites for Smal (779-784) and Xbal (787-792); 794nt.

[0457] SEQ ID NO: 56. Nucleotide sequence of a DNA molecule encoding hpHa6[G:U] for targeting the transcript of the synaptic vesicle glycoprotein 2C-like gene encoding H. armigera synaptic vesicle glycoprotein 2C-like. The order of elements are: Restriction site for Xhol (nucleotides 3-8), T7 promoter (9-28), Ha6 antisense sequence (29-328), loop sequence (329-478), modified Ha6 sense sequence (479-778), restriction sites for Smal (779-784) and Xbal (787-792); 794nt.

[0458] SEQ ID NO: 57. Amino acid sequence of H. armigera Troponin C, encoded by transcript variant X2 of gene (LOCI 10380220) (Ha7); NCBI Genbank Accession No. XP_021195809.1; 151aa.

[0459] SEQ ID NO: 58. Nucleotide sequence of a cDNA for H. armigera Troponin C mRNA; GenBank Accession No. XM_021340134.1 (LOCI 10380220). The protein coding region is nucleotides 132-587; 955nt.

[0460] SEQ ID NO: 59. Nucleotide sequence of a 300-nucleotide target region of the cDNA for the mRNA encoding H. armigera Troponin C, corresponding to nucleotides 140-439 of SEQ ID NO: 58; 300nt. SEQ ID NO: 60. Nucleotide sequence of a modified 300-nucleotide sense region corresponding to the RNA for H. armigera Troponin C, having 77 cytosines substituted with thymidines (25.7%) relative to the wild-type sequence; 300nt.

[0461] SEQ ID NO: 61. Nucleotide sequence of a DNA molecule encoding hpHa7[Con] for targeting the transcript of the Troponin C gene encoding H. armigera Troponin C. The order of elements are: Restriction site for Xhol (nucleotides 3-8), T7 promoter (9-28), Ha7 antisense sequence (29-328), loop sequence (329-476), Ha7 sense sequence (477- 776), restriction sites for Smal (777-782) and Xbal (785-790); 792nt.

[0462] SEQ ID NO: 62. Nucleotide sequence of a DNA molecule encoding hpHa7[G:U] for targeting the transcript of the Troponin C gene encoding H. armigera Troponin C. The order of elements are: Restriction site for Xhol (nucleotides 3-8), T7 promoter (9-28), Ha7 antisense sequence (29-328), loop sequence (329-476), modified Ha7 sense sequence (477-776), restriction sites for Smal (777-782) and Xbal (785-790); 792nt.

[0463] SEQ ID NO: 63. Amino acid sequence of H. armigera Titin, encoded by transcript variant X6 of gene (LOCI 10380881) (Ha8); NCBI Genbank Accession No. XP_021196691. l; 4213aa.

[0464] SEQ ID NO: 64. Nucleotide sequence of a cDNA for H. armigera Titin mRNA; GenBank Accession No. XM_021341016.1 (LOCI 10380881). The protein coding region is nucleotides 365-13006; 13053nt.

[0465] SEQ ID NO: 65. Nucleotide sequence of a 300-nucleotide target region of the cDNA for the mRNA encoding H. armigera Titin, corresponding to nucleotides 520-819 of SEQ ID NO: 64; 300nt.

[0466] SEQ ID NO: 66. Nucleotide sequence of a modified 300-nucleotide sense region corresponding to the RNA for H. armigera Titin, having 77 cytosines substituted with thymidines (25.7%) relative to the wild-type sequence; 300nt.

[0467] SEQ ID NO: 67. Nucleotide sequence of a DNA molecule encoding hpHa8[Con] for targeting the transcript of the Titin gene encoding H. armigera Titin. The order of elements are: Restriction site for Xhol (nucleotides 3-8), T7 promoter (9-28), Ha8 antisense sequence (29-328), loop sequence (329-476), Ha8 sense sequence (477-776), restriction sites for Smal (777-782) and Xbal (785-790); 793nt.

[0468] SEQ ID NO: 68. Nucleotide sequence of a DNA molecule encoding hpHa8[G:U] for targeting the transcript of the Titin gene encoding H. armigera Titin. The order of elements are: Restriction site for Xhol (nucleotides 3-8), T7 promoter (9-28), Ha8 antisense sequence (29-328), loop sequence (329-476), modified Ha8 sense sequence (477-776), restriction sites for Smal (777-782) and Xbal (785-790); 793nt.

[0469] SEQ ID NOs: 69-90. Oligonucleotide primers.

[0470] SEQ ID NO: 91. Nucleotide sequence of the DNA fragment for the LIR module comprising the LIR from BeYDV; Bsal restriction sites are present at nucleotides 9-14 and 319-324, spanning the LIR region, including the invariant 9 nucleotides of the LIR at positions 179-187; 332nt.

[0471] SEQ ID NO: 92. Nucleotide sequence of the DNA fragment for the Pr / UTR module comprising the CaMV e35S promoter and TMV 5’UTR regions, Fragment number EN38509. Bsal restriction sites are present at nucleotides 9-14 and 864-869, the e35S promoter at nucleotides 13-767 and the TMV 5’UTR at positions 796-852; the translation start ATG for the S module polypeptide is at nucleotides 860-862; 877nt.

[0472] SEQ ID NO: 93. Nucleotide sequence of the DNA fragment for the T module comprising the CaMV 35S Tm transcription terminator and SIR / Rep / RepA / LIR regions from Gemini Virus BeYDV, used in GV vectors herein, number EN38511. Bsal restriction sites are present at nucleotides 9-14 and 1766-1771, the 35S Tm at nucleotides 20-223, the SIR at positions 224-375, the Rep / RepA coding region in reverse orientation from nucleotides 1466 to 376, and an LIR at positions 1467-1760. The thymidine at nucleotide position 1469 was replaced with a guanosine. Nucleotide position 1469 is a G in GVc and an A for GVt; 1779nt.

[0473] SEQ ID NO: 94. Nucleotide sequence of the DNA fragment encoding a hpHal[G:U] molecule, used in construction of a GV vector for expressing the RNA molecule in plant cells. Bsal restriction sites are present at nucleotides 9-14 and 812-817, the Hal antisense sequence at nucleotides 35-334, the Hal sense sequence having 73 cytosines substituted with thymidines (24.3%) relative to the wild-type sequence at nucleotides 485-784, and the sequence encoding the loop at nucleotides 335-484; 825nt.

[0474] SEQ ID NO: 95. DNA sequence of a modified sense sequence from the GUS gene as used in the hpGUS[A22] construct; 191nt.

[0475] SEQ ID NO: 96. DNA sequence of a modified sense sequence from the GUS gene as used in the hpGUS[A23] construct; 183nt.

[0476] SEQ ID NO: 97. DNA sequence of a modified sense sequence from the GUS gene as used in the hpGUS[A24-l] construct; 177nt.

[0477] SEQ ID NO: 98. DNA sequence of a modified sense sequence from the GUS gene as used in the hpGUS[A24-2] construct; 176nt.

[0478] SEQ ID NO: 99. DNA sequence of a modified sense sequence from the GUS gene as used in the hpGUS[A24-3] construct; 176nt.

[0479] SEQ ID NO: 100. DNA sequence encoding the hpGUS[Con] hairpin RNA molecule. Nucleotides 1-11, 212-223, 1018-1029 and 1230-1235 correspond to restriction enzyme sites from the cloning vector, nucleotides 12-211 correspond to the GUS sense sequence, nucleotides 232-998 correspond to a PDK intron, and nucleotides 1030-1229 correspond to the GUS antisense sequence. The other nucleotides flanking the intron were from the cloning vector; 1235nt.

[0480] SEQ ID NO: 101. DNA sequence corresponding to the hpGUS[Con] hairpin RNA molecule after removal of the PDK intron by splicing, excluding additional 5' and 3' extensions coming from the expression vector. Nucleotides 1-11, 212-223, 251-262 and 463-468 correspond to restriction enzyme sites from the cloning vector, nucleotides 12- 211 correspond to the GUS sense sequence, and nucleotides 263-462 correspond to the GUS antisense sequence. Nucleotides 224-250 were from the cloning vector and form part of the loop of hpGUS[Con]; 468nt.

[0481] SEQ ID NO: 102. DNA sequence corresponding to the hpGUS[G:U] hairpin RNA molecule after removal of the PDK intron by splicing, excluding additional 5' and 3' extensions coming from the expression vector. Nucleotides 1-6, 207-212, 240-251 and I l l

[0482] 452-457 correspond to restriction enzyme sites from the cloning vector, nucleotides 7- 206 correspond to the modified GUS sense sequence, and nucleotides 252-451 correspond to the GUS antisense sequence. Nucleotides 213-239 were from the cloning vector and form part of the loop of hpGUS[G:U]; 457nt.

[0483] SEQ ID NO: 103 DNA sequence corresponding to the hpGUS[A22] hairpin RNA molecule after removal of the PDK intron by splicing, excluding additional 5' and 3' extensions coming from the expression vector. Nucleotides 1-6, 198-203, 231-242 and 443-448 correspond to restriction enzyme sites from the cloning vector, nucleotides 7- 197 correspond to the modified GUS sense sequence, and nucleotides 243-442 correspond to the GUS antisense sequence. Nucleotides 204-230 were from the cloning vector and form part of the loop of hpGUS[A22]; 448nt.

[0484] SEQ ID NO: 104. DNA sequence corresponding to the hpGUS[A23] hairpin RNA molecule after removal of the PDK intron by splicing, excluding additional 5' and 3' extensions coming from the expression vector. Nucleotides 1-6, 190-195, 223-234 and 435-440 correspond to restriction enzyme sites from the cloning vector, nucleotides 7- 189 correspond to the modified GUS sense sequence, and nucleotides 235-434 correspond to the GUS antisense sequence. Nucleotides 196-222 were from the cloning vector and form part of the loop of hpGUS[A23]; 440nt.

[0485] SEQ ID NO: 105 DNA sequence corresponding to the hpGUS[A24-l] hairpin RNA molecule after removal of the PDK intron by splicing, excluding additional 5' and 3' extensions coming from the expression vector. Nucleotides 1-6, 184-189, 217-228 and 429-434 correspond to restriction enzyme sites from the cloning vector, nucleotides 7- 183 correspond to the modified GUS sense sequence, and nucleotides 229-428 correspond to the GUS antisense sequence. Nucleotides 190-216 were from the cloning vector and form part of the loop of hpGUS[A24-l]; 434nt.

[0486] SEQ ID NO: 106. DNA sequence corresponding to the hpGUS[A24-2] hairpin RNA molecule after removal of the PDK intron by splicing, excluding additional 5' and 3' extensions coming from the expression vector. Nucleotides 1-6, 183-188, 216-227 and 428-433 correspond to restriction enzyme sites from the cloning vector, nucleotides 7- 182 correspond to the modified GUS sense sequence, and nucleotides 228-427 correspond to the GUS antisense sequence. Nucleotides 189-215 were from the cloning vector and form part of the loop of hpGUS[A24-2]; 433nt. SEQ ID NO: 107. DNA sequence corresponding to the hpGUS[A24-3] hairpin RNA molecule after removal of the PDK intron by splicing, excluding additional 5' and 3' extensions coming from the expression vector. Nucleotides 1-6, 183-188, 216-227 and 428-433 correspond to restriction enzyme sites from the cloning vector, nucleotides 7- 182 correspond to the modified GUS sense sequence, and nucleotides 228-427 correspond to the GUS antisense sequence. Nucleotides 189-215 were from the cloning vector and form part of the loop of hpGUS[A24-3]; 433nt.

[0487] SEQ ID NO: 108. Nucleotide sequence of the cDNA corresponding to the A. thaliana EIN2 gene, Accession No. NM_120406. Nucleotides 629-4513 correspond to the protein coding region; 4851 nt.

[0488] SEQ ID NO: 109. Nucleotide sequence of the cDNA corresponding to A. thaliana CHS gene, Accession No. NM_121396, Nucleotides 287-1471 correspond to the protein coding region; 1703 nt.

[0489] SEQ ID NO: 110. DNA sequence of a 200-nucleotide target region of the A. thaliana EIN2 gene, in sense orientation, used as a target sequence; corresponding to nucleotides 654-853 of SEQ ID NO: 108; 200nt.

[0490] SEQ ID NO: 111. Nucleotide sequence of the sense fragment in the construct encoding hpEIN2[A22], generated by deleting every 22nd nucleotide from the EIN2 wild-type sense sequence used in the conventional hairpin construct; 191nt.

[0491] SEQ ID NO: 112. Nucleotide sequence encoding the sense sequence in the construct encoding hpEIN2[A23], generated by deleting every 11th nucleotide from the EIN2 wild-type sense sequence used in the conventional hairpin construct; 183nt.

[0492] SEQ ID NO: 113. Nucleotide sequence encoding the sense sequence in the construct encoding hpEIN2[A24-l], generated by deleting every 7th or 8th nucleotide from the EIN2 wild-type sense sequence used in the conventional hairpin construct; 177nt.

[0493] SEQ ID NO: 114. Nucleotide sequence encoding the sense sequence in the construct encoding hpEIN2[A24-2], generated by deleting three nucleotides per 24 nucleotides from the EIN2 wild-type sense sequence used in the conventional hairpin construct, by alternating 1 nt and 2nt deletions; 176nt.

[0494] SEQ ID NO: 115. Nucleotide sequence encoding the sense sequence in the construct encoding hpEIN2[A24-3], generated by deleting every 22nd, 23rd and 24th nucleotides from each 24-nucleotide window in the EIN2 wild-type sense sequence used in the conventional hairpin construct; 176nt.

[0495] SEQ ID NO: 116. Nucleotide sequence of a DNA fragment comprising a 200-nt sense sequence from the cDNA corresponding to A. thaliana CHS gene, used as a target sequence. The sense sequence corresponds to nucleotides 863-1062 of the cDNA sequence (SEQ ID NO: 109); 200nt.

[0496] SEQ ID NO: 117. Nucleotide sequence of a DNA fragment consisting of a 300-nt sense sequence from CMV 2b gene joined to a 300-nt sense sequence from the cDNA for PVY, used as a target sequence. The PVY sense sequence corresponds to nucleotides 6217- 6516 of Accession No. NC_001616; 600nt.

[0497] SEQ ID NO: 118. Nucleotide sequence of a DNA fragment encoding hpCMV / PVY[Con]. Nucleotides 1-18 and 1482-1499 corresponding to restriction sites from the cloning vector, nucleotides 26-42 correspond to a T7 RNA Polymerase promoter for in vitro transcription, nucleotides 46-345 correspond to a CMV 2b sense sequence, nucleotides 346-645 correspond to a PVY sense sequence, nucleotides 652- 841 correspond to a CAT-1 intron, nucleotides 842-1141 correspond to a PVY antisense sequence, and nucleotides 1142-1441 correspond to a CMV 2b antisense sequence; 1493nt.

[0498] SEQ ID NO: 119. Nucleotide sequence of a DNA fragment comprising a modified sense sequence from a CMV 2b gene with single nucleotide deletions spaced on average about every 22 nucleotides, joined to a modified sense sequence from the cDNA for PVY with single nucleotide deletions spaced on average about every 22 nucleotides, used as the sense sequence in the genetic construct encoding hpCMV / PVY[A22]; 574nt.

[0499] SEQ ID NO: 120. Nucleotide sequence of a DNA fragment encoding hpCMV / PVY[A22], Nucleotides 1-12 and 1406-1417 corresponding to restriction sites from the cloning vector, nucleotides 13-29 correspond to a T7 RNA Polymerase promoter for in vitro transcription, nucleotides 30-319 correspond to a modified CMV 2b sense sequence, nucleotides 320-606 correspond to a modified PVY sense sequence, nucleotides 613-802 correspond to a CAT-1 intron, nucleotides 803-1102 correspond to a PVY antisense sequence, and nucleotides 1103-1399 correspond to a CMV 2b antisense sequence; 1416nt.

[0500] SEQ ID NO: 121. Nucleotide sequence of the protein coding region of the cDNA corresponding to the BnaA07g37430D-l DDM1 gene (LOC106391353) on the A07 chromosome of B. napus. The protein coding region is nucleotides 145-2469; 2653nt.

[0501] SEQ ID NO: 122. Nucleotide sequence of a chimeric sense sequence corresponding to two regions of the B. napus DDM1 gene transcript, joined together. This sequence corresponds to nucleotides 648-959 and nucleotides 2029-2218 of SEQ ID NO: 121; 502nt.

[0502] SEQ ID NO: 123. Nucleotide sequence of DNA encoding hpDDMl[Con] targeting RNA transcripts of the four DDM1 genes of B. napus. Restriction enzyme sites, nucleotides 1-14 and 1807-1824; T7 RNA polymerase promoter with GGG trinucleotide, nucleotides 15-34; DDM1 chimeric sense sequence, nucleotides 35-536; loop sequence from pHellsgate 8 cloning vector comprising the PDK1 intron, nucleotides 537-1304; DDM1 chimeric antisense sequence, nucleotides 1305-1806; 1824nt.

[0503] SEQ ID NO: 124. Nucleotide sequence of DNA encoding ledDDMl[Con] targeting RNA transcripts of the four DDM1 genes of B. napus. Restriction enzyme sites, nucleotides 1-14 and 1296-1312; T7 RNA polymerase promoter with GGG trinucleotide, nucleotides 16-35; DDM1 antisense sequence, nucleotides 36-281; loop sequence, nucleotides 282-413; DDM1 chimeric sense sequence, nucleotides 414-912; loop sequence, nucleotides 913-1042; DDM1 antisense sequence, nucleotides 1043- 1292; 1314nt.

[0504] SEQ ID NO: 125. Nucleotide sequence of a chimeric sense sequence corresponding to two regions of the B. napus DDM1 gene transcript, joined together, having the A22 modifications; 480nt.

[0505] SEQ ID NO: 126. Nucleotide sequence of DNA encoding ledDDMl[A22] targeting RNA transcripts of the four DDM1 genes of B. napus. Restriction enzyme sites, nucleotides 1-14 and 1296-1312; T7 RNA polymerase promoter with GGG trinucleotide, nucleotides 16-35; DDM1 antisense sequence, nucleotides 36-281; loop sequence, nucleotides 282-411; DDM1 chimeric sense sequence with A22 modification, nucleotides 412-891; loop sequence, nucleotides 892-1020; DDM1 antisense sequence, nucleotides 1021-1270; 1292nt.

[0506] SEQ ID NO: 127. Nucleotide sequence of a cDNA encoding magnesium-chelatase subunit CHL1 in N. benthamiana (Nbv5.1tr6204879). The protein coding sequence corresponds to nucleotides 141-1424; 1691nt.

[0507] SEQ ID NO: 128. Amino acid sequence of a magnesium-chelatase subunit CHL1 in N. benthamiana (Nbv5.1tr6204879); 427aa.

[0508] SEQ ID NO: 129. Nucleotide sequence of a selected sense sequence used as a target sequence for the NbSu mRNA. This sequence corresponds to nucleotides 463-854 of SEQ ID NO: 127; 392nt.

[0509] SEQ ID NO: 130. Nucleotide sequence of an antisense sequence complementary to SEQ ID NO: 129; 392nt.

[0510] SEQ ID NO: 131. Nucleotide sequence of Construct 24.1 encoding hpNbSufCon] RNA, a symmetric hairpin RNA targeting the Su transcript in N. benthamiana. The order and position of the elements are: T7 promoter (nucleotides 1-17) — sense sequence (nucleotides 21-412) — linker comprising a Cat-1 intron (nucleotides 413-608) — antisense sequence (nucleotides 609-1000) — Smal restriction enzyme site (nucleotides 1001-1006); 1006nt.

[0511] SEQ ID NO: 132. Nucleotide sequence of Construct 24.2 encoding hpNbSu[A22] RNA, an asymmetric hairpin RNA with the A22 modification, targeting the Su transcript in N. benthamiana. The order and position of the elements are: T7 promoter (nucleotides 1- 17) — sense sequence (nucleotides 21-394) — linker comprising a Cat-1 intron (nucleotides 395-590) — antisense sequence (nucleotides 591-982) — Smal restriction enzyme site (nucleotides 983-988); 988nt.

[0512] SEQ ID NO: 133. Nucleotide sequence of Construct 24.3 encoding hpNbSu[A22AG]

[0513] RNA, an asymmetric hairpin RNA with the A22 modification and 38 A to G substitutions in the sense sequence, targeting the Su transcript in N. benthamiana. The order and position of the elements are: T7 promoter (nucleotides 1-17) — sense sequence (nucleotides 21-394) — linker comprising a Cat-1 intron (nucleotides 395-590) — antisense sequence (nucleotides 591-982) — Smal restriction enzyme site (nucleotides 983-988); 988nt.

[0514] SEQ ID NO: 134. Nucleotide sequence of Construct 24.4 encoding hpNbSu[A22CT] RNA, an asymmetric hairpin RNA with the A22 modification, 38 A to G substitutions in the sense sequence and 24 C to T substitutions in the antisense sequence, targeting the Su transcript in A. benthamiana. The order and position of the elements are: T7 promoter (nucleotides 1-17) — sense sequence (nucleotides 21-394) - linker comprising a Cat-1 intron (nucleotides 395-590) — antisense sequence (nucleotides 591-982) — Smal restriction enzyme site (nucleotides 983-988); 988nt.

[0515] SEQ ID NO: 135. Nucleot...

Claims

CLAIMS1. A precursor RNA molecule comprising at least one double- stranded RNA region, wherein the double-stranded RNA region comprises(a) a first RNA strand which comprises a first RNA sequence of at least 24 contiguous ribonucleotides, and(b) a second RNA strand which comprises a second RNA sequence of at least 24 contiguous ribonucleotides, wherein the first RNA sequence hybridises to the second RNA sequence by basepairing between the at least 24 contiguous ribonucleotides of the first RNA sequence and the at least 24 contiguous ribonucleotides of the second RNA sequence, forming the double-stranded RNA region, wherein the first RNA sequence along the full length of the double-stranded RNA region comprises a guanine (G) ribonucleotide content such that 36-55%, preferably 40.1-55%, of the ribonucleotides of the first RNA sequence are G ribonucleotides, and wherein between 10% and 40%, preferably between 15% and 40%, or more preferably between 20% and 40%, of the ribonucleotides of the first RNA sequence and the second RNA sequence, in total, are basepaired in G:U basepairs, and optionally wherein the first RNA strand and second RNA strand are covalently linked by a linking RNA sequence.

2. A precursor RNA molecule comprising at least one double- stranded RNA region, wherein the double-stranded RNA region comprises(a) a first RNA strand which comprises a first RNA sequence of at least 21 contiguous ribonucleotides, preferably at least 24 contiguous ribonucleotides, and(b) a second RNA strand which comprises a second RNA sequence of at least 21 contiguous ribonucleotides, preferably at least 24 contiguous ribonucleotides, wherein the first RNA strand and second RNA strand are covalently linked by a linking RNA sequence, wherein the first RNA sequence hybridises to the second RNA sequence by basepairing between the at least 21 or at least 24 contiguous ribonucleotides of the first RNA sequence and the at least 21 or at least 24 contiguous ribonucleotides of the second RNA sequence, forming the double- stranded RNA region,wherein the first RNA sequence along the full length of the double-stranded RNA region comprises an adenine (A) ribonucleotide content such that less than 10%, preferably less than 8%, more preferably less than 6%, even more preferably less than 4% or less than 2%, most preferably zero of the ribonucleotides of the first RNA sequence are A ribonucleotides, optionally wherein the double-stranded RNA region comprises a guanine (G) ribonucleotide content such that 36-55%, preferably 40.1-55%, of the ribonucleotides of the first RNA sequence are G ribonucleotides, and optionally wherein between 10% and 40%, preferably between 15% and 40%, or more preferably between 20% and 40%, of the ribonucleotides of the first RNA sequence and the second RNA sequence, in total, are basepaired in G:U basepairs.

3. The precursor RNA molecule of claim 1 or claim 2, wherein the double-stranded RNA region comprises(a) a first RNA strand which comprises a first RNA sequence of at least 42, at least 46 or at least 48 contiguous ribonucleotides, and(b) a second RNA strand comprises a second RNA sequence of at least 42, at least 46 or at least 48 contiguous ribonucleotides, wherein the first RNA sequence hybridises to the second RNA sequence by basepairing between the at least 42, at least 46 or at least 48 contiguous ribonucleotides of the first RNA sequence and the at least 42, at least 46 or at least 48 contiguous ribonucleotides of the second RNA sequence, forming the double-stranded RNA region.

4. The precursor RNA molecule according to any one of claims 1 to 3 which is cleaved in a eukaryotic cell, preferably a plant cell, to produce double-stranded product RNA molecules each independently consisting of a sense RNA sequence of 24 contiguous ribonucleotides from the first RNA sequence and an antisense RNA sequence of 24 contiguous ribonucleotides from the second RNA sequence, or an increased amount of such product RNA molecules relative to a corresponding control RNA molecule having only canonical basepairing, wherein ribonucleotides 1 to 22 of the sense RNA sequence basepair with ribonucleotides 1 to 22 of the antisense RNA sequence in each of the double-stranded product RNA molecules, wherein the basepairs in each of the double- stranded product RNA molecules comprise, independently, 1, 2, 3, 4, 5, 6, 7, 8 or 9 G:U basepairs, preferably 2-9 G:U basepairs,wherein ribonucleotides 23 and 24 of the sense RNA sequence in each of the double-stranded product RNA molecules form a 2 -ribonucleotide unpaired 3' overhang, and 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.

5. The precursor RNA molecule according to any one of claims 1 to 4, which is cleaved in a eukaryotic cell, preferably a plant cell, to produce double-stranded product RNA molecules each independently consisting of a sense RNA sequence of 23 contiguous ribonucleotides from the first RNA sequence and an antisense RNA sequence of 23 contiguous ribonucleotides from the second RNA sequence, or an increased amount of such product RNA molecules relative to a corresponding control RNA molecule having only canonical basepairing, wherein ribonucleotides 1 to 21 of the sense RNA sequence basepair with ribonucleotides 1 to 21 of the antisense RNA sequence in each of the double-stranded product RNA molecules, wherein the basepairs in each of the double- stranded product RNA molecules comprise, independently, 1, 2, 3, 4, 5, 6, 7, 8 or 9 G:U basepairs, preferably 2-9 G:U basepairs, wherein ribonucleotides 22 and 23 of the sense RNA sequence in each of the double-stranded product RNA molecules form a 2-ribonucleotide unpaired 3' overhang, and 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.

6. The precursor RNA molecule according to any one of claims 1 or 3 to 5, comprising an A ribonucleotide content of the first RNA sequence along the full length of the dsRNA region, or the first and second RNA sequences, in total, wherein the A ribonucleotide content is less than 10%, preferably less than 8%, more preferably less than 6%, even more preferably less than 4% or less than 2% or most preferably zero, and / or the A ribonucleotide content of the first RNA sequence or the first and second RNA sequences, in total, is not more than 1, not more than 2, not more than 3, not more than 4, not more than 5, not more than 6, not more than 7, not more than 8, not more than 9 or not more than 10 A ribonucleotides.

7. A precursor RNA molecule comprising at least one double- stranded RNA region, wherein:(i) the double- stranded RNA region comprises:(a) a first RNA strand which comprises a first RNA sequence of at least 23 contiguous ribonucleotides, and(b) a second RNA strand which comprises a second RNA sequence of at least 24 contiguous ribonucleotides, wherein the first RNA sequence hybridises to the second RNA sequence by basepairing between at least 22 of the at least 23 contiguous ribonucleotides of the first RNA sequence and at least 22 of the at least 24 ribonucleotides of the second RNA sequence, forming the double- stranded RNA region, wherein 1 or 2 ribonucleotides of the at least 24 ribonucleotides of the second RNA sequence are not basepaired to ribonucleotides of the first RNA sequence, and 0 or 1 ribonucleotides, respectively, of the first RNA sequence are not basepaired to ribonucleotides of the second RNA sequence, wherein the non -basepaired ribonucleotides form 1, 2 or 3 bulges in the doublestranded RNA region, wherein the first RNA sequence along the full length of the double-stranded RNA region comprises a guanine (G) ribonucleotide content such that 36-55%, preferably 40.1-55%, of the ribonucleotides of the first RNA sequence are G ribonucleotides, and wherein between 10% and 40%, preferably between 10% and 35%, or more preferably between 20% and 40%, of the ribonucleotides of the first RNA sequence and the second RNA sequence, in total, are basepaired in G:U basepairs.

8. The precursor RNA molecule of claim 7, wherein the first RNA sequence comprises at least 46 contiguous ribonucleotides, and the second RNA sequence comprises at least 48 contiguous ribonucleotides, wherein the first RNA sequence hybridises to the second RNA sequence by basepairing between at least 44 of the at least 46 contiguous ribonucleotides of the first RNA sequence and at least 44 of the at least 48 ribonucleotides of the second RNA sequence, forming the double- stranded RNA region, wherein 2, 3, or 4 ribonucleotides of the 48 ribonucleotides of the second RNA sequence are not basepaired to ribonucleotides of the first RNA sequence, and 0, 1 or 2 ribonucleotides, respectively, of the first RNA sequence are not basepaired to ribonucleotides of the second RNA sequence, and wherein the non-basepaired ribonucleotides form bulges in the double-stranded RNA region.

9. The precursor RNA molecule of claim 7 or claim 8 which is cleaved in a eukaryotic cell, preferably a plant cell, to produce double- stranded product RNA molecules each independently consisting of a sense RNA sequence of 23 contiguous ribonucleotides from the first RNA sequence and an antisense RNA sequence of 24 contiguous ribonucleotides from the second RNA sequence, or an increased amount of such product RNA molecules relative to a corresponding control RNA molecule having only canonical basepairing, wherein at least 20 of ribonucleotides 1 to 21 of the sense RNA sequence basepair with at least 20 ribonucleotides from ribonucleotides 1 to 22 of the antisense RNA sequence, independently, in each of the double- stranded product RNA molecules, wherein the basepairs in each of the double- stranded product RNA molecules comprise, independently, 1, 2, 3, 4, 5, 6, 7, or 8 G:U basepairs, preferably 2-8 G:U basepairs, wherein 1 or 2 ribonucleotides of ribonucleotides 3-20 of the second RNA sequence are not basepaired to ribonucleotides of the first RNA sequence, and 0 or 1 ribonucleotides, respectively, of ribonucleotides 3-19 of the first RNA sequence are not basepaired to ribonucleotides of the second RNA sequence, wherein the non -basepaired ribonucleotides form 1, 2 or 3 bulges in the doublestranded product RNA molecules, wherein ribonucleotides 22 and 23 of the sense RNA sequence in each of the double-stranded product RNA molecules form a 2 -ribonucleotide unpaired 3' overhang, and 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.

10. The precursor RNA molecule according to any one of claims 7 to 9, which is cleaved in a eukaryotic cell, preferably a plant cell, to produce double-stranded product RNA molecules each independently consisting of a sense RNA sequence of 22 contiguous ribonucleotides from the first RNA sequence and an antisense RNA sequence of 23 contiguous ribonucleotides from the second RNA sequence, or an increased amount of such product RNA molecules relative to a corresponding control RNA molecule having only canonical basepairing, wherein at least 19 ribonucleotides from ribonucleotides 1 to 20 of the sense RNA sequence basepair with at least 19 ribonucleotides from ribonucleotides 1 to 21 of the antisense RNA sequence in each of the double- stranded product RNA molecules,wherein the basepairs in each of the double- stranded product RNA molecules comprise, independently, 1, 2, 3, 4, 5, 6, 7, or 8 G:U basepairs, preferably 2-8 G:U basepairs, wherein 1 or 2 ribonucleotides of ribonucleotides 3-19 of the second RNA sequence are not basepaired to ribonucleotides of the first RNA sequence, and 0 or 1 ribonucleotides, respectively, of ribonucleotides 3-18 of the first RNA sequence are not basepaired to ribonucleotides of the second RNA sequence, wherein the non -basepaired ribonucleotides form 1, 2 or 3 bulges in the doublestranded product RNA molecules, wherein ribonucleotides 21 and 22 of the sense RNA sequence in each of the double-stranded product RNA molecules form a 2 -ribonucleotide unpaired 3' overhang, and 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.

11. A precursor RNA molecule comprising at least one double- stranded RNA region, wherein:(i) the double- stranded RNA region comprises:(a) a first RNA strand which comprises a first RNA sequence of at least 22 contiguous ribonucleotides, and(b) a second RNA strand which comprises a second RNA sequence of at least 24 contiguous ribonucleotides, wherein the first RNA sequence hybridises to the second RNA sequence by basepairing between at least 21 of the at least 22 contiguous ribonucleotides of the first RNA sequence and at least 21 of the at least 24 ribonucleotides of the second RNA sequence, forming the double- stranded RNA region, wherein 2 or 3 ribonucleotides of the at least 24 ribonucleotides of the second RNA sequence are not basepaired to ribonucleotides of the first RNA sequence, and 0 or 1 ribonucleotides, respectively, of the first RNA sequence are not basepaired to ribonucleotides of the second RNA sequence, wherein the non-basepaired ribonucleotides form bulges in the double-stranded RNA region, wherein the first RNA sequence along the full length of the double-stranded RNA region comprises a guanine (G) ribonucleotide content such that 36-55%, preferably 40.1-55%, of the ribonucleotides of the first RNA sequence are G ribonucleotides, andwherein between 10% and 40%, preferably between 10% and 35%, or more preferably between 20% and 40%, of the ribonucleotides of the first RNA sequence and the second RNA sequence, in total, are basepaired in G:U basepairs.

12. The precursor RNA molecule of claim 11, wherein the first RNA sequence comprises at least 44 contiguous ribonucleotides, and the second RNA sequence comprises at least 48 contiguous ribonucleotides, wherein the first RNA sequence hybridises to the second RNA sequence by basepairing between at least 42 of the at least 44 contiguous ribonucleotides of the first RNA sequence and at least 42 of the at least 48 ribonucleotides of the second RNA sequence, forming the double- stranded RNA region, wherein 4, 5 or 6 ribonucleotides of the 48 ribonucleotides of the second RNA sequence are not basepaired to ribonucleotides of the first RNA sequence, and 0, 1 or 2 ribonucleotides, respectively, of the first RNA sequence are not basepaired to ribonucleotides of the second RNA sequence, and wherein the non-basepaired ribonucleotides form bulges in the double-stranded RNA region.

13. The precursor RNA molecule of claim 11 or claim 12 which is cleaved in a eukaryotic cell, preferably a plant cell, to produce double- stranded product RNA molecules each independently consisting of a sense RNA sequence of 22 contiguous ribonucleotides from the first RNA sequence and an antisense RNA sequence of 24 contiguous ribonucleotides from the second RNA sequence, or an increased amount of such product RNA molecules relative to a corresponding control RNA molecule having only canonical basepairing, wherein at least 19 of ribonucleotides 1 to 20 of the sense RNA sequence basepair with at least 19 ribonucleotides from ribonucleotides 1 to 22 of the antisense RNA sequence, independently, in each of the double- stranded product RNA molecules, wherein the basepairs in each of the double- stranded product RNA molecules comprise, independently, 1, 2, 3, 4, 5, 6, 7, or 8 G:U basepairs, preferably 2-8 G:U basepairs, wherein 2 or 3 ribonucleotides of ribonucleotides 3-20 of the second RNA sequence are not basepaired to ribonucleotides of the first RNA sequence, and 0 or 1 ribonucleotides, respectively, of ribonucleotides 3-18 of the first RNA sequence are not basepaired to ribonucleotides of the second RNA sequence,wherein the non -basepaired ribonucleotides form 1, 2 or 3 bulges in the doublestranded product RNA molecules, wherein ribonucleotides 21 and 22 of the sense RNA sequence in each of the double-stranded product RNA molecules form a 2 -ribonucleotide unpaired 3' overhang, and 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.

14. The precursor RNA molecule according to any one of claims 11 to 13, which is cleaved in a eukaryotic cell, preferably a plant cell, 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, or an increased amount of such product RNA molecules relative to a corresponding control RNA molecule having only canonical basepairing, wherein at least 18 ribonucleotides from ribonucleotides 1 to 19 of the sense RNA sequence basepair with at least 18 ribonucleotides from ribonucleotides 1 to 21 of the antisense RNA sequence in each of the double- stranded product RNA molecules, wherein the basepairs in each of the double- stranded product RNA molecules comprise, independently, 1, 2, 3, 4, 5, 6, 7, or 8 G:U basepairs, preferably 2-8 G:U basepairs, wherein 2 or 3 ribonucleotides of ribonucleotides 3-19 of the second RNA sequence are not basepaired to ribonucleotides of the first RNA sequence, and 0 or 1 ribonucleotides, respectively, of ribonucleotides 3-17 of the first RNA sequence are not basepaired to ribonucleotides of the second RNA sequence, wherein the non-basepaired ribonucleotides form bulges in the double-stranded product RNA molecules, 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 22 and 23 of the antisense RNA sequence in each of the double-stranded product RNA molecules form a 2-ribonucleotide unpaired 3' overhang.

15. A precursor RNA molecule comprising at least one double- stranded RNA region, wherein:(i) the double- stranded RNA region comprises:(a) a first RNA strand which comprises a first RNA sequence of at least 21 contiguous ribonucleotides, and(b) a second RNA strand which comprises a second RNA sequence of at least 24 contiguous ribonucleotides, wherein the first RNA sequence hybridises to the second RNA sequence by basepairing between at least 20 of the at least 21 contiguous ribonucleotides of the first RNA sequence and at least 20 of the at least 24 ribonucleotides of the second RNA sequence, forming the double- stranded RNA region, wherein 3 or 4 ribonucleotides of the at least 24 ribonucleotides of the second RNA sequence are not basepaired to ribonucleotides of the first RNA sequence, and 0 or 1 ribonucleotides, respectively, of the first RNA sequence are not basepaired to ribonucleotides of the second RNA sequence, wherein the non-basepaired ribonucleotides form bulges in the double-stranded RNA region, wherein the first RNA sequence along the full length of the double-stranded RNA region comprises a guanine (G) ribonucleotide content such that 36-55%, preferably 40.1-55%, of the ribonucleotides of the first RNA sequence are G ribonucleotides, and wherein between 10% and 40%, preferably between 10% and 35%, or more preferably between 20% and 40%, of the ribonucleotides of the first RNA sequence and the second RNA sequence, in total, are basepaired in G:U basepairs.

16. The precursor RNA molecule of claim 15, wherein the first RNA sequence comprises at least 42 contiguous ribonucleotides, and the second RNA sequence comprises at least 48 contiguous ribonucleotides, wherein the first RNA sequence hybridises to the second RNA sequence by basepairing between at least 40 of the at least 42 contiguous ribonucleotides of the first RNA sequence and at least 40 of the at least 48 ribonucleotides of the second RNA sequence, forming the double- stranded RNA region, wherein 6, 7 or 8 ribonucleotides of the 48 ribonucleotides of the second RNA sequence are not basepaired to ribonucleotides of the first RNA sequence, and 0, 1 or 2 ribonucleotides, respectively, of the first RNA sequence are not basepaired to ribonucleotides of the second RNA sequence, and wherein the non-basepaired ribonucleotides form bulges in the double-stranded RNA region.

17. The precursor RNA molecule of claim 15 or claim 16, which is cleaved in a eukaryotic cell, preferably a plant cell, to produce double- stranded product RNA molecules each independently consisting of a sense RNA sequence of 21 contiguousribonucleotides from the first RNA sequence and an antisense RNA sequence of 24 contiguous ribonucleotides from the second RNA sequence, or an increased amount of such product RNA molecules relative to a corresponding control RNA molecule having only canonical basepairing, wherein at least 18 of ribonucleotides 1 to 19 of the sense RNA sequence basepair with at least 18 ribonucleotides from ribonucleotides 1 to 22 of the antisense RNA sequence, independently, in each of the double- stranded product RNA molecules, wherein the basepairs in each of the double- stranded product RNA molecules comprise, independently, 1, 2, 3, 4, 5, 6, 7, or 8 G:U basepairs, preferably 2-8 G:U basepairs, wherein 3 or 4 ribonucleotides of ribonucleotides 3-20 of the second RNA sequence are not basepaired to ribonucleotides of the first RNA sequence, and 0 or 1 ribonucleotides, respectively, of ribonucleotides 3-17 of the first RNA sequence are not basepaired to ribonucleotides of the second RNA sequence, wherein the non-basepaired ribonucleotides form bulges in the double-stranded product RNA molecules, 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 23 and 24 of the antisense RNA sequence in each of the double-stranded product RNA molecules form a 2-ribonucleotide unpaired 3' overhang.

18. The precursor RNA molecule according to any one of claims 15 to 17 which is cleaved in a eukaryotic cell, preferably a plant cell, to also produce double-stranded product RNA molecules each independently consisting of a sense RNA sequence of 20 contiguous ribonucleotides from the first RNA sequence and an antisense RNA sequence of 23 contiguous ribonucleotides from the second RNA sequence, or an increased amount of such product RNA molecules relative to a corresponding control RNA molecule having only canonical basepairing, wherein at least 17 ribonucleotides from ribonucleotides 1 to 18 of the sense RNA sequence basepair with at least 17 ribonucleotides from ribonucleotides 1 to 21 of the antisense RNA sequence in each of the double- stranded product RNA molecules, wherein the basepairs in each of the double- stranded product RNA molecules comprise, independently, 1, 2, 3, 4, 5, 6, 7, or 8 G:U basepairs, preferably 2-8 G:U basepairs, wherein 3 or 4 ribonucleotides of ribonucleotides 3-19 of the second RNA sequence are not basepaired to ribonucleotides of the first RNA sequence, and 0 or 1ribonucleotides, respectively, of ribonucleotides 3-16 of the first RNA sequence are not basepaired to ribonucleotides of the second RNA sequence, wherein the non-basepaired ribonucleotides form bulges in the double-stranded product RNA molecules, wherein ribonucleotides 19 and 20 of the sense RNA sequence in each of the double-stranded product RNA molecules form a 2 -ribonucleotide unpaired 3' overhang, and 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.

19. The precursor RNA molecule according to any one of claims 7 to 18, comprising an A ribonucleotide content of the first RNA sequence along the full length of the dsRNA region, or the first and second RNA sequences, in total, wherein the A ribonucleotide content is less than 10%, preferably less than 8%, more preferably less than 6%, even more preferably less than 4% or less than 2% or most preferably zero, and / or the A ribonucleotide content of the first RNA sequence is not more than 1, not more than 2, not more than 3, not more than 4, not more than 5, not more than 6, not more than 7, not more than 8, not more than 9 or not more than 10 A ribonucleotides.

20. The precursor RNA molecule according to any one of claims 7 to 19, wherein less than 20%, less than 18%, less than 16%, less than 14%, less than 12%, less than 10%, less than 8%, less than 6%, less than 4%, or less than 2%, but in each case at least one ribonucleotide, of the ribonucleotides of the dsRNA region are non-basepaired.

21. The precursor RNA molecule according to any one of claims 7 to 20, comprising (i) one or more double- stranded region(s) which comprise bulges which are evenly spaced apart along most or all of each double- stranded region, and / or (ii) the precursor RNA molecule has a single linking RNA sequence, thereby forming a hairpin RNA (hpRNA) structure, or the precursor RNA molecule comprises two double- stranded regions and two linking RNA sequences, forming a ledRNA structure.

22. A double-stranded RNA molecule, consisting of a sense RNA sequence of 24 contiguous ribonucleotides and an antisense RNA sequence of 24 contiguous ribonucleotides, wherein ribonucleotides 1 to 22 of the sense RNA sequence each basepair with a ribonucleotide of ribonucleotides 1 to 22 of the antisense RNA sequence,wherein ribonucleotides 23 and 24 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 the sense RNA sequence and / or the double-stranded product RNA molecule comprises a guanine (G) ribonucleotide content such that 36-55%, preferably 40.1-55%, of the ribonucleotides of the sense RNA sequence are G ribonucleotides, and wherein between 10% and 40%, preferably between 15% and 40%, or more preferably between 20% and 40%, of the ribonucleotides of the sense RNA sequence and the antisense RNA sequence, in total, are basepaired in G:U basepairs.

23. A double-stranded RNA molecule, consisting of a sense RNA sequence of 23 contiguous ribonucleotides and an antisense RNA sequence of 23 contiguous ribonucleotides, wherein ribonucleotides 1 to 21 of the sense RNA sequence each basepair with a ribonucleotide of ribonucleotides 1 to 21 of the antisense RNA sequence, wherein ribonucleotides 22 and 23 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 the sense RNA sequence and / or the double-stranded product RNA molecule comprises a guanine (G) ribonucleotide content such that 36-55%, preferably 40.1-55%, of the ribonucleotides of the sense RNA sequence are G ribonucleotides, and wherein between 10% and 40%, preferably between 15% and 40%, or more preferably between 20% and 40%, of the ribonucleotides of the sense RNA sequence and the antisense RNA sequence, in total, are basepaired in G:U basepairs.

24. A double-stranded RNA molecule, consisting of a sense RNA sequence of 23 contiguous ribonucleotides and an antisense RNA sequence of 24 contiguous ribonucleotides, wherein ribonucleotides 1 to 21 of the sense RNA sequence each basepair with a ribonucleotide of ribonucleotides 1 to 22 of the antisense RNA sequence, wherein one of ribonucleotides 3 to 20 of the antisense RNA sequence is nonbasepaired, forming a bulge in the double- stranded RNA molecule, wherein ribonucleotides 22 and 23 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 the sense RNA sequence and / or the double-stranded product RNA molecule comprises a guanine (G) ribonucleotide content such that 36-55%, preferably 40.1-55%, of the ribonucleotides of the sense RNA sequence are G ribonucleotides, and wherein between 10% and 40%, preferably between 15% and 40%, or more preferably between 20% and 40%, of the ribonucleotides of the sense RNA sequence and the antisense RNA sequence, in total, are basepaired in G:U basepairs.

25. A double-stranded RNA molecule, consisting of a sense RNA sequence of 23 contiguous ribonucleotides and an antisense RNA sequence of 24 contiguous ribonucleotides, wherein 20 ribonucleotides of ribonucleotides 1 to 21 of the sense RNA sequence each basepair with a ribonucleotide of ribonucleotides 1 to 22 of the antisense RNA sequence, wherein one ribonucleotide of ribonucleotides 3 to 19 of the sense RNA sequence and two ribonucleotides of ribonucleotides 3 to 20 of the antisense RNA sequence are non-basepaired, forming one or more bulges in the double- stranded RNA molecule, wherein ribonucleotides 22 and 23 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 the sense RNA sequence and / or the double-stranded product RNA molecule comprises a guanine (G) ribonucleotide content such that 36-55%, preferably 40.1-55%, of the ribonucleotides of the sense RNA sequence are G ribonucleotides, and wherein between 10% and 40%, preferably between 15% and 40%, or more preferably between 20% and 40%, of the ribonucleotides of the sense RNA sequence and the antisense RNA sequence, in total, are basepaired in G:U basepairs.

26. A double-stranded RNA molecule, consisting of a sense RNA sequence of 22 contiguous ribonucleotides and an antisense RNA sequence of 24 contiguous ribonucleotides, wherein ribonucleotides 1 to 20 of the sense RNA sequence each basepair with a ribonucleotide of ribonucleotides 1 to 22 of the antisense RNA sequence, wherein two of ribonucleotides 3 to 20 of the antisense RNA sequence are non- basepaired, forming one or two bulges in the double-stranded RNA molecule, wherein ribonucleotides 21 and 22 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 the sense RNA sequence and / or the double-stranded product RNA molecule comprises a guanine (G) ribonucleotide content such that 36-55%, preferably 40.1-55%, of the ribonucleotides of the sense RNA sequence are G ribonucleotides, and wherein between 10% and 40%, preferably between 15% and 40%, or more preferably between 20% and 40%, of the ribonucleotides of the sense RNA sequence and the antisense RNA sequence, in total, are basepaired in G:U basepairs.

27. A double-stranded RNA molecule, consisting of a sense RNA sequence of 22 contiguous ribonucleotides and an antisense RNA sequence of 24 contiguous ribonucleotides, wherein 19 ribonucleotides of ribonucleotides 1 to 20 of the sense RNA sequence each basepair with a ribonucleotide of ribonucleotides 1 to 22 of the antisense RNA sequence, wherein one ribonucleotide of ribonucleotides 3 to 17 of the sense RNA sequence and three ribonucleotides of ribonucleotides 3 to 20 of the antisense RNA sequence are non-basepaired, forming one or more bulges in the double- stranded RNA molecule, wherein ribonucleotides 21 and 22 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 the sense RNA sequence and / or the double-stranded product RNA molecule comprises a guanine (G) ribonucleotide content such that 36-55%, preferably 40.1-55%, of the ribonucleotides of the sense RNA sequence are G ribonucleotides, and wherein between 10% and 40%, preferably between 15% and 40%, or more preferably between 20% and 40%, of the ribonucleotides of the sense RNA sequence and the antisense RNA sequence, in total, are basepaired in G:U basepairs.

28. A double-stranded RNA molecule, consisting of a sense RNA sequence of 21 contiguous ribonucleotides and an antisense RNA sequence of 24 contiguous ribonucleotides, wherein 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 three ribonucleotides of ribonucleotides 3 to 18 of the antisense RNA sequence are non-basepaired, forming one, two or three bulges 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 the sense RNA sequence and / or the double-stranded product RNA molecule comprises a guanine (G) ribonucleotide content such that 36-55%, preferably 40.1-55%, of the ribonucleotides of the sense RNA sequence are G ribonucleotides, and wherein between 10% and 40%, preferably between 15% and 40%, or more preferably between 20% and 40%, of the ribonucleotides of the sense RNA sequence and the antisense RNA sequence, in total, are basepaired in G:U basepairs.

29. A double-stranded RNA molecule, consisting of a sense RNA sequence of 21 contiguous ribonucleotides and an antisense RNA sequence of 24 contiguous ribonucleotides, wherein 18 ribonucleotides 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 one ribonucleotide of ribonucleotides 3 to 17 of the sense RNA sequence and four ribonucleotides of ribonucleotides 3 to 20 of the antisense RNA sequence are non-basepaired, forming one or more bulges 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 the sense RNA sequence and / or the double-stranded product RNA molecule comprises a guanine (G) ribonucleotide content such that 36-55%, preferably 40.1-55%, of the ribonucleotides of the sense RNA sequence are G ribonucleotides, and wherein between 10% and 40%, preferably between 15% and 40%, or more preferably between 20% and 40%, of the ribonucleotides of the sense RNA sequence and the antisense RNA sequence, in total, are basepaired in G:U basepairs.

30. A population of multiple, different double- stranded RNA molecules comprising double-stranded RNA molecules according to any one of claims 22 to 29, or any combination thereof, wherein the different, double- stranded RNA molecules comprise different lengths of the sense RNA sequence, antisense RNA sequence, or both, or produced from both a symmetrical precursor RNA molecule and an asymmetric precursor RNA molecule.

31. The population of multiple, different double-stranded RNA molecules of claim 30, wherein(i) more antisense RNA sequences in the population of multiple, different doublestranded RNA molecules consist of 24 ribonucleotides than consist of 21 ribonucleotides, or(ii) more antisense RNA sequences in the population of multiple, different doublestranded RNA molecules consist of 23 ribonucleotides than consist of 21 ribonucleotides, or both (i) and (ii).

32. 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 21, or that produces the double- stranded product RNA molecules according to any one of claims 22 to 29 or the population of double-stranded product RNA molecules of claim 30 or claim 31, optionally wherein 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, and optionally a polyadenylation region / transcription terminator or a transcription termination sequence.

33. 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 a eukaryotic cell, the method comprising i) producing a precursor RNA molecule according to any one of claims 1 to 21, and / or a double- stranded product RNA molecule according to any one of claims 22 to 29, or a population of multiple, different precursor RNA molecules according to claim 30 or claim 31, ii) determining the ability of the precursor RNA molecule or double-stranded RNA molecule, or members of a population of multiple, different precursor RNA molecules, or the population of multiple, different double- stranded product RNA molecules, to reduce the amount and / or activity of the target RNA molecule of interest, optionally wherein step i) comprises producing the precursor RNA molecule in the eukaryotic cell, wherein the precursor RNA molecule is cleaved in the eukaryotic cell by one or more ribonucleases (RNases) to produce the double- stranded product RNA molecule or the population of multiple, different double- stranded product RNA molecules.

34. A host cell, preferably a eukaryotic cell, comprising one or more or all of a precursor RNA molecule according to any one of claims 1 to 21, a polynucleotide encoding the precursor RNA molecule, the double- stranded RNA molecule according to any one of claims 22 to 29, and / or the population of multiple, different double- stranded RNA molecules of claim 30 or claim 31, preferably wherein the cell is a non-human cell or a eukaryotic cell in vitro.

35. A non-human organism, or a part thereof, comprising one or more or all of a precursor RNA molecule according to any one of claims 1 to 21, a polynucleotide encoding the precursor RNA molecule, the double- stranded RNA molecule according to any one of claims 22 to 29, and / or the population of multiple, different double- stranded product RNA molecules of claim 30 or claim 31, a cell of claim 34, preferably a transgenic non-human organism or part thereof, being transgenic for a polynucleotide of claim 32, and / or wherein the polynucleotide is stably integrated into the genome of the organism or part thereof.

36. A method of producing the cell of claim 34, the method comprising introducing into a cell one or more or all of the precursor RNA molecule of any one of claims 1 to 21, the double- stranded product RNA molecule according to any one of claims 22 to 29, the population of multiple, different double-stranded product RNA molecules of claim 30 or claim 31, and the polynucleotide or the vector of claim 32.

37. A method of producing a non-human organism or a part thereof of claim 35, the method comprising introducing one or more or all of the precursor RNA molecule according to of any one of claims 1 to 21, the double- stranded product RNA molecule according to any one of claims 22 to 29, the population of multiple, different doublestranded product RNA molecules of claim 30 or claim 31, and the polynucleotide or the vector of claim 32, into a cell and generating the non-human organism from the cell.

38. A method of producing the precursor RNA molecule of any one of claims 1 to 21, the double-stranded product RNA molecule according to any one of claims 22 to 29, and / or the population of multiple, different double-stranded product RNA molecules of claim 30 or claim 31, the method comprising expressing the polynucleotide or vector of claim 32 in a host cell or cell-free expression system.

39. An extract of a cell of claim 34, wherein the extract comprises one or more or all of the precursor RNA molecule, the double-stranded product RNA molecule, the population of multiple, different double- stranded RNA molecules, and the polynucleotide or the vector.

40. A method for increasing the number of double-stranded product RNA molecules according to any one of claims 22 to 29 in a eukaryotic cell or organism, or increasing the amount of a population of double- stranded product RNA molecules of claim 30 or claim 31 in the cell or organism, comprising expressing in the cell or organism a polynucleotide or a vector of claim 32, or contacting the cell or organism with the precursor RNA molecule according to any one of claims 1 to 21, the double- stranded product RNA molecule according to any one of claims 22 to 29 or the population of double-stranded product RNA molecules of claim 30 or claim 31.

41. A composition comprising one or more or all of the precursor RNA molecule of any one of claims 1 to 21, the double-stranded product RNA molecule according to any one of claims 22 to 29, the population of multiple, different double-stranded product RNA molecules of claim 30 or claim 31, the polynucleotide or the vector of claim 32, the cell of claim 34, the non-human organism or part thereof of claim 35, and the extract of claim 39.

42. 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 21, the double-stranded product RNA molecule according to any one of claims 22 to 29, the population of multiple, different double- stranded product RNA molecules of claim 30 or claim 31, the polynucleotide or vector of claim 32, the extract of claim 39, or the composition of claim 41, 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.

43. 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 RNAmolecules according to any one of claims 1 to 21, multiple double-stranded product RNA molecules according to any one of claims 22 to 29, populations of multiple, different double-stranded product RNA molecules of claim 30 or claim 31, polynucleotides or vectors of claim 32, extracts of claim 39, and compositions of claim 41, wherein the multiple precursor RNA molecules, double- stranded product RNA molecules or populations of multiple double- stranded product 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.

44. 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 21, the double- stranded product RNA molecule according to any one of claims 22 to 29, the population of multiple, different double-stranded product RNA molecules of claim 30 or claim 31, the polynucleotide or the vector of claim 32, the extract of claim 39, and the composition of claim 41.

45. The method of claim 44, wherein one or more or all of the precursor RNA molecule, the double- stranded product RNA molecule, the population of double- stranded product 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.

46. 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 21, the double-stranded product RNA molecule according to any one of claims 22 to 29, the population of different double- stranded product RNA molecules of claim 30 or claim 31, the polynucleotide or vector of claim 32, the extract of claim 39, and the composition of claim 41, (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 productRNA molecule or population of different double- stranded product RNA molecules has an effect on the pest or pathogen, and optionally (iv) if the precursor RNA molecule, double-stranded product RNA molecule or population of different double- stranded product 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.

47. 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 21, the double-stranded product RNA molecule according to any one of claims 22 to 29, the population of multiple, different double-stranded product RNA molecules of claim 30 or claim 31, the extract of claim 39, and the composition of claim 41, (ii) testing the pest or pathogen for an effect of the precursor RNA molecule, double-stranded product RNA molecule or population of double- stranded product RNA molecules, and optionally (iii) selecting an RNA molecule based on results from step (ii), thereby identifying the RNA molecule.

48. 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 21, the double-stranded product RNA molecule according to any one of claims 22 to 29, the population of double- stranded product RNA molecules of claim 30 or claim 31, the cell of claim 34, the non-human organism or part thereof of claim 35, the extract of claim 39, and the composition of claim 41.

49. 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 21, the double-stranded product RNA molecule according to any one of claims 22 to 29, the population of double- stranded product RNA molecules of claim 30 or claim 31, the polynucleotide or vector of claim 32, the cell of claim 34, the non-human organism or part thereof of claim 35, the extract of claim 39, and the composition of claim 41, wherein the precursor RNA molecule, double-stranded product RNA molecule or population of double- stranded product RNA molecules 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.

50. 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 21, the double- stranded product RNA molecule according to any one of claims 22 to 29, the population of double-stranded product RNA molecules of claim 30 or claim 31, the polynucleotide or vector of claim 32, the cell of claim 34, the non-human organism or part thereof of claim 35, the extract of claim 39, and the composition of claim 41, preferably wherein one or more or all of the precursor RNA molecule, the double- stranded product RNA molecule, the population of double- stranded product 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 animal or a plant.

51. One or more or all of the precursor RNA molecule according to any one of claims 1 to 21, the double-stranded product RNA molecule according to any one of claims 22 to 29, the population of double- stranded product RNA molecules of claim 30 or claim 31, the polynucleotide or vector of claim 32, the cell of claim 34, the non-human organism or part thereof of claim 35, the extract of claim 39, and the composition of claim 41, for use in treating a disease in a subject, wherein the double-stranded product RNA molecule or population has a beneficial effect on at least one symptom of the disease.

52. Use of one or more or all of the precursor RNA molecule according to any one of claims 1 to 21, the double- stranded product RNA molecule according to any one of claims 22 to 29, the population of double-stranded product RNA molecules of claim 30 or claim 31, the polynucleotide or vector of claim 32, the cell of claim 34, the non-human organism or part thereof of claim 35, the extract of claim 39, and the composition of claim 41 in the manufacture of a medicament for treating a disease.

53. A kit comprising one or more or all of the precursor RNA molecule according to any one of claims 1 to 21, the double-stranded product RNA molecule according to any one of claims 22 to 29, the population of double- stranded product RNA molecules of claim 30 or claim 31, the polynucleotide or vector of claim 32, the cell of claim 34, the non-human organism or part thereof of claim 35, the extract of claim 39, and the composition of claim 41.

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