RNA molecule

Abstract

The present invention relates to novel double-stranded RNA (dsRNA) structures and their use in gene silencing. In one embodiment, the dsRNA comprises a double hairpin (dumbbell) structure. In another embodiment, a portion of the sense strand and the antisense strand of the dsRNA molecule contains ribonucleotides that are base-paired or not base-paired in a non-canonical base pair.

Description

[0001] This application is a divisional application with an application date of September 17, 2018, application number 201880072998.2 (PCT application number PCT / AU2018 / 051015), and invention name “RNA molecule”. Technical Field

[0002] The present invention relates to novel double-stranded RNA (dsRNA) structures and their use in gene silencing. Background Art

[0003] Conventional dsRNA molecules for inducing RNA interference (RNAi) in animal cells are formed by annealing single-stranded sense and antisense RNA or self-complementary RNA, which has a complementary region that allows the formation of a stem-loop structure, also known as hairpin RNA (hpRNA). In plants, transgenics expressing long hairpin RNAs have been shown to be highly effective in inducing RNAi. Some studies on long hpRNAs have shown that the processed RNA products of short interfering RNAs (siRNAs) formed by sequences near the ends of the hpRNA loop are more abundant relative to the open ends of the hpRNA structure, suggesting that Dicer processing of the dsRNA stems of hpRNAs appears to start from the ends of the loops. The entire loop can be precisely excised from the hpRNA structure, which can provide dsRNA ends for the RNase Dicer to bind and initiate siRNA processing.

[0004] While dsRNA-induced gene silencing has proven to be a valuable tool for altering the phenotype of an organism, there is a need for alternative, preferably improved, dsRNA molecules that can be used for RNAi. Summary of the invention

[0005] The inventors have conceived a new design of RNAi-inducing molecules, referred to herein as loop-terminal dsRNA (ledRNA), which have one or more of the following features: easy to synthesize, more easily form dsRNA structures and induce effective silencing of target genes in eukaryotic cells. When applied topically to plant leaves, ledRNA is also effective.

[0006] In a first aspect, the present invention provides a ribonucleic acid (RNA) molecule comprising a first RNA component, a second RNA component covalently linked to the first RNA component, and optionally one or more or all of the following: (i) a linking ribonucleotide sequence covalently linked to the first and second RNA components, (ii) a 5' leader sequence and (iii) a 3' trailer sequence,

[0007] wherein the first RNA component consists of a first 5' ribonucleotide, a first RNA sequence, and a first 3' ribonucleotide in a 5' to 3' order, wherein the first 5' and 3' ribonucleotides in the RNA molecule are base paired with each other, wherein the first RNA sequence comprises a first sense ribonucleotide sequence of at least 20 contiguous ribonucleotides, a first loop sequence of at least 4 ribonucleotides, and a first antisense ribonucleotide sequence of at least 20 contiguous ribonucleotides, wherein the first antisense ribonucleotide sequence hybridizes with the first sense ribonucleotide sequence in the RNA molecule, wherein the first antisense ribonucleotide sequence is capable of hybridizing with a first region of a target RNA molecule,

[0008] wherein the second RNA component is covalently linked to the first 5' ribonucleotide or the first 3' ribonucleotide via the linking ribonucleotide sequence if present, or directly if the linking ribonucleotide sequence is absent,

[0009] wherein the second RNA component consists of a second 5' ribonucleotide, a second RNA sequence, and a second 3' ribonucleotide in a 5' to 3' order, wherein the second 5' and 3' ribonucleotides are base paired with each other in the RNA molecule, wherein the second RNA sequence comprises a second sense ribonucleotide sequence, a second loop sequence of at least 4 ribonucleotides, and a second antisense ribonucleotide sequence, wherein the second sense ribonucleotide sequence hybridizes with the second antisense ribonucleotide sequence in the RNA molecule;

[0010] wherein the 5' leader sequence, if present, consists of a ribonucleotide sequence covalently linked to the first 5' ribonucleotide if the second RNA component is linked to the first 3' ribonucleotide, or covalently linked to the second 5' ribonucleotide if the second RNA component is linked to the first 5' ribonucleotide, and

[0011] wherein the 3' trailer sequence, if present, consists of a ribonucleotide sequence that is covalently linked to the second 3' ribonucleotide if the second RNA component is linked to the first 3' ribonucleotide, or that is covalently linked to the first 3' ribonucleotide if the second RNA component is linked to the first 5' ribonucleotide.

[0012] In a second aspect, the present invention provides an RNA molecule comprising a first RNA component, a second RNA component covalently linked to the first RNA component, and optionally one or more or all of the following: (i) a linking ribonucleotide sequence covalently linked to the first and second RNA components, (ii) a 5' leader sequence and (iii) a 3' trailer sequence,

[0013] wherein the first RNA component consists of a first 5' ribonucleotide, a first RNA sequence, and a first 3' ribonucleotide in 5' to 3' order, wherein the first 5' and 3' ribonucleotides are base paired, wherein the first RNA sequence comprises a first sense ribonucleotide sequence, a first loop sequence of at least 4 ribonucleotides, and a first antisense ribonucleotide sequence, wherein the first sense ribonucleotide sequence and the first antisense ribonucleotide sequence each consist of at least 20 contiguous ribonucleotides, wherein the at least 20 contiguous ribonucleotides of the first sense ribonucleotide sequence are completely base paired with at least 20 contiguous ribonucleotides of the first antisense ribonucleotide sequence, wherein the at least 20 contiguous ribonucleotides of the first sense ribonucleotide sequence or the at least 20 contiguous ribonucleotides of the first antisense ribonucleotide sequence are identical in sequence to a first region of a target RNA molecule or its complement, or both, respectively,

[0014] wherein, if the linking ribonucleotide sequence is present, the second RNA component is covalently linked to the first 5' ribonucleotide or the first 3' ribonucleotide via the linking ribonucleotide sequence,

[0015] wherein the second RNA component consists of a second 5' ribonucleotide, a second RNA sequence, and a second 3' ribonucleotide in a 5' to 3' order, wherein the second 5' and 3' ribonucleotides are base paired, wherein the second RNA sequence comprises a second sense ribonucleotide sequence, a second loop sequence of at least 4 ribonucleotides, and a second antisense ribonucleotide sequence, wherein the second sense ribonucleotide sequence is base paired with the second antisense ribonucleotide sequence,

[0016] wherein the 5' leader sequence, if present, consists of a ribonucleotide sequence covalently linked to the first 5' ribonucleotide if the second RNA component is linked to the first 3' ribonucleotide, or covalently linked to the second 5' ribonucleotide if the second RNA component is linked to the first 5' ribonucleotide, and

[0017] wherein the 3' trailer sequence, if present, consists of a ribonucleotide sequence that is covalently linked to the second 3' ribonucleotide if the second RNA component is linked to the first 3' ribonucleotide, or that is covalently linked to the first 3' ribonucleotide if the second RNA component is linked to the first 5' ribonucleotide.

[0018] In a preferred embodiment, the RNA molecule of the invention is a chimeric RNA molecule.

[0019] In a third aspect, the present invention provides a chimeric ribonucleic acid (RNA) molecule comprising a double-stranded RNA (dsRNA) region comprising a sense ribonucleotide sequence and an antisense ribonucleotide sequence that are capable of hybridizing to each other to form a dsRNA region, wherein

[0020] i) the sense ribonucleotide sequence consists of a first 5' ribonucleotide, a first RNA sequence and a first 3' ribonucleotide covalently linked in a 5' to 3' order,

[0021] ii) the antisense ribonucleotide sequence consists of a second 5' ribonucleotide, a second RNA sequence and a second 3' ribonucleotide covalently linked in a 5' to 3' order,

[0022] iii) said first 5' ribonucleotide base pairs with said second 3' ribonucleotide base to form a terminal base pair of said dsRNA region,

[0023] iv) said second 5' ribonucleotide base pairs with said first 3' ribonucleotide base to form a terminal base pair of said dsRNA region,

[0024] v) about 5% to about 40% of the ribonucleotides of the sense ribonucleotide sequence and the antisense ribonucleotide sequence as a whole are base paired with non-canonical base pairs or are not base paired,

[0025] vi) the dsRNA region does not contain 20 contiguous canonical base pairs,

[0026] vii) the RNA molecule is capable of being processed in a eukaryotic cell or in vitro, whereby the antisense ribonucleotide sequence is cleaved to produce a short antisense RNA (asRNA) molecule of 20-24 ribonucleotides in length,

[0027] viii) the RNA molecule or at least some of the asRNA molecules, or both, are capable of reducing the expression or activity of a target RNA molecule in the eukaryotic cell, and

[0028] ix) The RNA molecule can be produced enzymatically by transcription in vitro or in a cell or both.

[0029] In a fourth aspect, the invention provides a chimeric RNA molecule comprising a first RNA component and a second RNA component covalently linked to the first RNA component,

[0030] wherein the first RNA component comprises a first double-stranded RNA (dsRNA) region and a first intervening ribonucleotide sequence of at least 4 nucleotides, wherein the first double-stranded RNA (dsRNA) region comprises a first sense ribonucleotide sequence and a first antisense ribonucleotide sequence that can hybridize with each other to form the first dsRNA region, and the first intervening ribonucleotide sequence covalently connects the first sense ribonucleotide sequence and the first antisense ribonucleotide sequence,

[0031] wherein the second RNA component comprises a second sense ribonucleotide sequence, a second antisense ribonucleotide sequence, and a second intervening ribonucleotide sequence of at least 4 ribonucleotides, wherein the second intervening ribonucleotide sequence is covalently linked to the second sense ribonucleotide sequence and the second antisense ribonucleotide sequence, wherein the second sense ribonucleotide sequence in the RNA molecule hybridizes with the second antisense ribonucleotide sequence,

[0032] Wherein in the first RNA component,

[0033] i) the first sense ribonucleotide sequence consists of at least 20 contiguous ribonucleotides covalently linked in 5' to 3' order, a first 5' ribonucleotide, a first RNA sequence and a first 3' ribonucleotide,

[0034] ii) the first antisense ribonucleotide sequence consists of at least 20 contiguous ribonucleotides covalently linked in 5' to 3' order, a second 5' ribonucleotide, a second RNA sequence, and a second 3' ribonucleotide,

[0035] iii) a first 5' ribonucleotide base pair with a second 3' ribonucleotide,

[0036] iv) a second 5' ribonucleotide base pair with a first 3' ribonucleotide,

[0037] v) 5% to 40% of the ribonucleotides of the first sense ribonucleotide sequence and the first antisense ribonucleotide sequence as a whole are base paired with non-canonical base pairs or are not base paired, and

[0038] vi) said first dsRNA region does not comprise 20 contiguous canonical base pairs,

[0039] wherein the chimeric RNA molecule is capable of being processed in a eukaryotic cell or in vitro, whereby the first antisense ribonucleotide sequence is cleaved to produce a short antisense RNA (asRNA) molecule of 20-24 ribonucleotides in length, and wherein

[0040] (a) the chimeric RNA molecule or at least some of the asRNA molecules, or both, are capable of reducing the expression or activity of a target RNA molecule in the eukaryotic cell, or

[0041] (b) the first antisense ribonucleotide sequence comprises a sequence of at least 20 contiguous ribonucleotides that are at least 50% identical in sequence to a complementary region of the target RNA molecule, or

[0042] (c) Both (a) and (b).

[0043] In the first, second and fourth aspects, the first 5' ribonucleotide and the first 3' ribonucleotide of the first RNA component are base paired to form a base pair. The base pair is defined herein as the terminal base pair of the dsRNA region formed by self-hybridization of the first RNA component. In embodiments where the first sense ribonucleotide sequence is covalently linked to the first 5' ribonucleotide without any intervening nucleotides and the first antisense ribonucleotide sequence is covalently linked to the first 3' ribonucleotide without any intervening nucleotides, the first 5' ribonucleotide is directly linked to one of the sense sequence and the antisense sequence and the first 3' ribonucleotide is directly linked to the other of the sense sequence and the antisense sequence.

[0044] In a preferred embodiment, at least 20 contiguous ribonucleotides of the first antisense ribonucleotide sequence are capable of base pairing with nucleotides of the first region of the target RNA molecule. In one embodiment, the first sense ribonucleotide sequence is covalently linked to the first 5' ribonucleotide without any intervening nucleotides, or the first antisense ribonucleotide sequence is covalently linked to the first 3' ribonucleotide without any intervening nucleotides, or both.

[0045] In one embodiment, the RNA molecule comprises a connecting ribonucleotide sequence, wherein the connecting ribonucleotide sequence is related to the target gene in sequence, or is at least partially identical to or complementary to a region of the target RNA molecule. In a preferred embodiment, the connecting ribonucleotide sequence forms a portion of an adjacent sense sequence together with the sense sequence in the first and second RNA components, or forms a portion of an adjacent antisense sequence together with the antisense sequence in the first and second RNA components. In one embodiment, the RNA molecule comprises a connecting ribonucleotide sequence, wherein the connecting ribonucleotide sequence is less than 20 ribonucleotides. In one embodiment, the connecting ribonucleotide sequence hybridizes with the target RNA molecule. In one embodiment, the connecting ribonucleotide sequence is identical to a portion of the complement of the target RNA molecule. In one embodiment, the connecting ribonucleotide sequence is between 1 and 10 ribonucleotides.

[0046] In the first, second or fourth aspect embodiments, the RNA molecule comprises one or more or all of the following: (i) a linking ribonucleotide sequence covalently linking the first and second RNA components, (ii) a 5' extension sequence and (iii) a 3' extension sequence, wherein respectively, if the 5' extension sequence is present, it consists of a ribonucleotide sequence covalently linked to the first RNA component or the second RNA component, and wherein if the 3' extension sequence is present, it consists of a ribonucleotide sequence covalently linked to the second RNA component or the first RNA component. In one embodiment, the first RNA component and the second RNA component are covalently linked via a linking ribonucleotide sequence. In another embodiment, the first RNA component and the second RNA component are directly linked without any linking ribonucleotide sequence.

[0047] In the embodiments of the first to fourth aspects, the RNA molecule comprises two or more sense ribonucleotide sequences, each sense ribonucleotide sequence is identical in sequence to a region of the target RNA molecule, and the RNA molecule comprises one or more antisense ribonucleotide sequences based on pairing with the sense ribonucleotide sequence, wherein one or more antisense sequences are complementary to the region of the target molecule, preferably completely complementary. In one embodiment, two or more sense ribonucleotide sequences are identical in sequence to different regions of the same target RNA molecule, which may or may not be adjacent in the target RNA molecule. In one embodiment, two or more sense ribonucleotide sequences are identical in sequence to regions of different target RNA molecules. In one embodiment, two or more sense ribonucleotide sequences do not have intervening loop sequences, i.e., they are adjacent relative to the target RNA molecule.

[0048] In the preferred embodiment of the first to fourth aspects, the RNA molecule comprises two or more antisense ribonucleotide sequences and a sense ribonucleotide sequence with base pairing, and the antisense sequence is complementary to the region of the target RNA molecule. The target RNA molecule and its complementary region may be or may not be adjacent in the target RNA molecule. In one embodiment, the two or more antisense ribonucleotide sequences are complementary to the different regions of the same target RNA molecule. In one embodiment, the second one of the two or more antisense ribonucleotide sequences is complementary to the region of the target RNA molecule that is different from the first one of the two or more antisense ribonucleotide sequences. In a preferred embodiment, two or more antisense ribonucleotide sequences do not have an intervening loop sequence, i.e., they are adjacent relative to the complementary sequence of the target RNA molecule. In a preferred embodiment, one or both of the two or more antisense ribonucleotide sequences and the sense ribonucleotide sequence are base paired along their full length by a canonical base pair, or by some canonical and some non-canonical base pairs, preferably G:U base pairs are base paired.

[0049] In a preferred embodiment of the first to fourth aspects, the RNA molecule is a single-stranded ribonucleotide. For example, the RNA molecule can comprise a single-stranded ribonucleotide with a 5' end, at least one sense ribonucleotide sequence of at least 21 nucleotides in length, an antisense ribonucleotide sequence that is fully base-paired with each sense ribonucleotide sequence on at least 21 adjacent nucleotides, at least two loop sequences, and a 3' end. The 5' to 3' order can be a sense ribonucleotide sequence, followed by an antisense ribonucleotide sequence, or vice versa. In one embodiment, the ribonucleotide at the 5' end and the ribonucleotide at the 3' end are adjacent, and each base is paired and not directly covalently bound, see, e.g. Figure 1 .

[0050] In another embodiment of the first to fourth aspects, the RNA molecule comprises a first antisense ribonucleotide sequence that hybridizes to a first region of the target RNA; a second antisense ribonucleotide sequence that hybridizes to a second region of the target RNA, the second region of the target RNA being different from the first region of the target RNA; and the RNA molecule comprises only one sense ribonucleotide sequence that hybridizes to the target RNA, wherein the two antisense sequences are not adjacent in the RNA molecule. In one embodiment, the first and second regions of the target RNA are adjacent in the target RNA. Alternatively, they are not adjacent.

[0051] In another embodiment of the first to fourth aspects, the RNA molecule comprises a first sense ribonucleotide sequence that is at least 60% identical to a first region of the target RNA; and a second sense ribonucleotide sequence that is at least 60% identical to a second region of the target RNA, the second region of the target RNA being different from the first region of the target RNA; and the RNA molecule comprises only one antisense ribonucleotide sequence that hybridizes with the target RNA, wherein the two sense sequences are not adjacent in the RNA molecule. In one embodiment, the first and second regions of the target RNA are adjacent in the target RNA molecule. Alternatively, they are not adjacent. In a preferred embodiment, the first and second sense ribonucleotide sequences are each independently at least 70%, at least 80%, at least 90%, at least 95% or at least 99% identical to the corresponding regions of the target RNA, i.e., the first sense sequence may be at least 70% identical to its target region, the second sequence may be at least 80% identical to its target sequence, and so on.

[0052] In preferred embodiments of the first to fourth aspects, the RNA molecule is a single-stranded ribonucleotide having a 5' end, at least one sense ribonucleotide sequence of at least 21 nucleotides in length, an antisense ribonucleotide sequence that is fully base paired with each sense ribonucleotide sequence over at least 21 adjacent nucleotides, at least two loop sequences, and a 3' end. In a more preferred embodiment, the base pairing in the RNA molecule is contained in a double-stranded region of at least 21 adjacent base pairs in length, the double-stranded region includes some non-canonical base pairs, most preferably some G:U base pairs, and the double-stranded region includes at least one sense ribonucleotide sequence of at least 21 nucleotides in length.

[0053] In preferred embodiments of the first and second aspects, the second RNA component is characterized by:

[0054] i) the second sense ribonucleotide sequence consists of at least 20 contiguous second 5' ribonucleotides, a third RNA sequence and a third 3' ribonucleotide covalently linked in 5' to 3' order,

[0055] ii) the second antisense ribonucleotide sequence consists of at least 20 contiguous ribonucleotides covalently linked in 5' to 3' order, a third 5' ribonucleotide, a fourth RNA sequence and a second 3' ribonucleotide,

[0056] iii) a second 5' ribonucleotide base pair with a second 3' ribonucleotide,

[0057] iv) a third 3' ribonucleotide base pair with a third 5' ribonucleotide,

[0058] Wherein the chimeric RNA molecule is capable of being processed in a eukaryotic cell or in vitro, whereby the second antisense ribonucleotide sequence is cleaved to produce a short antisense RNA (asRNA) molecule of 20-24 ribonucleotides in length, and wherein most preferably, in the absence of or in combination with an asRNA produced from the first antisense sequence of the first RNA component, the asRNA molecule produced from the second antisense sequence is capable of reducing the expression of the target RNA.

[0059] In a preferred embodiment of the fourth aspect, the second RNA component is characterized in that:

[0060] i) the second sense ribonucleotide sequence consists of at least 20 contiguous third 5' ribonucleotides, a third RNA sequence and a third 3' ribonucleotide covalently linked in 5' to 3' order,

[0061] ii) the second antisense ribonucleotide sequence consists of at least 20 contiguous ribonucleotides covalently linked in 5' to 3' order, a fourth 5' ribonucleotide, a fourth RNA sequence and a fourth 3' ribonucleotide,

[0062] iii) a third 5' ribonucleotide base pair with a fourth 3' ribonucleotide,

[0063] iv) a third 3' ribonucleotide base pair with a third 5' ribonucleotide,

[0064] wherein the chimeric RNA molecule is capable of being processed in a eukaryotic cell or in vitro, whereby the second antisense ribonucleotide sequence is cleaved to produce a short antisense RNA (asRNA) molecule of 20-24 ribonucleotides in length, and wherein

[0065] In these preferred embodiments, more preferably, 5% to 40% of the ribonucleotides of the second sense ribonucleotide sequence and the second antisense ribonucleotide sequence as a whole are base paired with non-canonical base pairs or are not base paired, and / or the second dsRNA region does not contain 20 adjacent canonical base pairs. More preferably, about 12%, about 15%, about 18%, about 21%, about 24%, or 15% to 30%, or even more preferably 16% to 25% of the ribonucleotides of the second sense ribonucleotide sequence and the second antisense ribonucleotide sequence as a whole are base paired with non-canonical base pairs, or are not base paired. In preferred embodiments, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or 100% of the non-canonical base pairs of the second dsRNA region are G:U base pairs. Most preferably, in these embodiments,

[0066] (a) the chimeric RNA molecule or at least some of the asRNA molecules, or both, are capable of reducing the expression or activity of a target RNA molecule in the eukaryotic cell, or

[0067] (b) the second antisense ribonucleotide sequence comprises a sequence of at least 20 contiguous ribonucleotides that are at least 50% identical, preferably at least 60% identical, more preferably at least 70% identical, even more preferably at least 80% identical in sequence to the complementary region of the target RNA molecule, and most preferably at least 90% identical or 100% identical to the complementary region of the target RNA molecule, or

[0068] (c) Both (a) and (b).

[0069] In the embodiments of the first to fourth aspects, the RNA molecule comprises a 5' leader sequence or a 5' extension sequence. In one embodiment, the RNA molecule comprises a 3' trailer sequence or a 3' extension sequence. In a preferred embodiment, the RNA molecule comprises a 5' leader / extension sequence and a 3' trailer / extension sequence.

[0070] In the embodiments of the first to fourth aspects, each ribonucleotide of the RNA molecule is covalently linked to two other nucleotides, i.e., it is a covalent closed ring. Alternatively, the RNA molecule can be represented as a dumbbell ( Figure 1 ), but with a gap or break in a portion of the double-stranded structure.

[0071] In the embodiment of the first to fourth aspects, at least one or all of the loop sequences of the RNA molecule are longer than 20 nucleotides. In a preferred embodiment, the length of at least one loop of the RNA molecule is 4 to 1,000 ribonucleotides. In a more preferred embodiment, the length of all loops is 4 to 1,000 ribonucleotides. In a more preferred embodiment, the length of at least one loop of the RNA molecule is 4 to 200 ribonucleotides. In an even more preferred embodiment, the length of all loops is 4 to 200 ribonucleotides. In an even more preferred embodiment, the length of at least one loop of the RNA molecule is 4 to 50 ribonucleotides. In a most preferred embodiment, the length of all loops is 4-50 ribonucleotides. In one embodiment, the eukaryotic cell is a vertebrate cell, and the length of each loop of the RNA molecule is 20 to 50, or 20 to 30 ribonucleotides.

[0072] In the embodiment of the first to fourth aspects, the RNA molecule does not have, or has one or two or more protuberances in the double-stranded region. In this article, a protuberance is a nucleotide in a sense or antisense ribonucleotide sequence, or two or more adjacent nucleotides, which is not base-paired in the dsRNA district and does not have mismatched nucleotides in the corresponding position in the complementary sequence in the dsRNA district. The dsRNA district of the RNA molecule can be included in a sense or antisense sequence or a sequence that exceeds 2 or 3 nucleotides in both, and when the dsRNA structure is formed, it is looped from the dsRNA district. The looped sequence itself can form some internal base pairs, and for example it itself can form a stem-loop structure.

[0073] In the embodiment of the first to fourth aspects, the RNA molecule does not have, or has one or two or more protuberances in the double-stranded region. In this article, a protuberance is a nucleotide in a sense or antisense ribonucleotide sequence, or two or more adjacent nucleotides, which is not base-paired in the dsRNA district and does not have mismatched nucleotides in the corresponding position in the complementary sequence in the dsRNA district. The dsRNA district of the RNA molecule can be included in a sense or antisense sequence or a sequence that exceeds 2 or 3 nucleotides in both, and when the dsRNA structure is formed, it is looped from the dsRNA district. The looped sequence itself can form some internal base pairs, and for example it itself can form a stem-loop structure.

[0074] In one embodiment, the RNA molecule has three, four or more loops. In a preferred embodiment, the RNA molecule has only two loops. In one embodiment, the first double-stranded region or the first dsRNA region and the second dsRNA region of the RNA molecule include one or two or more nucleotides that are not base-paired in the double-stranded region, or at most 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% of the nucleotides that are not base-paired in the double-stranded region.

[0075] In a preferred embodiment of the first to fourth aspects, the target RNA molecule of the present invention or the RNA molecule or both are in a eukaryotic cell. For example, the eukaryotic cell can be a plant cell, an animal cell or a fungal cell. In one embodiment, the eukaryotic cell is a fungal cell. In one embodiment, the RNA molecule of the present invention is produced in a cell such as a bacterial cell or other microbial cell, and the cell is different from the cell comprising the target RNA. Similarly, in one embodiment, when producing the RNA molecule of the present invention, the RNA molecule of the present invention is produced in a eukaryotic cell that does not comprise the target RNA, but if the target RNA is, for example, a viral RNA or other introduced RNA, the eukaryotic cell comprising the RNA molecule of the present invention and / or the RNA product of its processing can become a host of the target RNA. Such cells can be preventively protected against viral or other introduced RNA.

[0076] In preferred embodiments of the first to fourth aspects, the RNA molecule can be prepared enzymatically by transcription in vitro or in a cell or both. In one embodiment, the RNA molecule of the present invention is expressed in a cell, i.e., produced in a cell by transcription from one or more nucleic acids encoding the RNA molecule. The one or more nucleic acids encoding the RNA molecule are preferably DNA molecules, which may be present on a vector in the cell or integrated into the genome of the cell, the nuclear genome of the cell, or the plastid DNA of the cell. The one or more nucleic acids encoding the RNA molecule may also be RNA molecules, such as viral vectors.

[0077] Therefore, in one embodiment, the invention provides cells comprising RNA molecules as described herein. In preferred embodiments, the invention provides RNA molecules as described herein that are expressed in cells and have been isolated and / or purified from cells. Therefore, the invention provides preparations of RNA molecules isolated according to one or more of the first to fourth aspects, which are suitable for administration to cells comprising a target RNA or potentially comprising a target RNA.

[0078] In one embodiment, the one or more target RNAs encode proteins. Alternatively, the one or more target RNAs do not encode proteins, such as rRNA, tRNA, snoRNA or miRNA.

[0079] In the embodiment of the first to fourth aspects, about 12%, about 15%, about 18%, about 21%, about 24%, or about 15% to about 30%, or preferably about 16% to about 25% of the ribonucleotides of the sense ribonucleotide sequence and antisense ribonucleotide sequence forming the dsRNA region as a whole are base paired or not base paired with non-canonical base pairs. In preferred embodiments, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97% or 100% of the non-canonical base pairs in all dsRNA regions in the dsRNA region or RNA molecule are G:U base pairs. The G nucleotides in each G:U base pair can be independently in the sense ribonucleotide sequence or preferably in the antisense ribonucleotide sequence. About the G nucleotide in the G:U base pair of the dsRNA region, preferably at least 50% is in the antisense ribonucleotide sequence, more preferably at least 60% or 70%, even more preferably at least 80% or 90%, and most preferably at least 95% is in the antisense ribonucleotide sequence of the dsRNA region. This feature can be applied to all dsRNA regions in RNA molecules. In one embodiment, less than 25%, less than 20%, less than 15%, less than 10%, preferably less than 5%, more preferably less than 1% or most preferably no ribonucleotide is base paired in the RNA molecule. In a preferred embodiment, in the dsRNA region or in the dsRNA region in general, every 4 to every 1 of the 6 ribonucleotides form non-standard base pairs or are not base paired in the RNA molecule. In a preferred embodiment, the dsRNA region or the dsRNA region generally does not include 8 adjacent standard base pairs. In another embodiment, the dsRNA region includes at least 8 adjacent standard base pairs, such as 8-12 or 8-14 adjacent standard base pairs. In preferred embodiments, all ribonucleotides in the dsRNA region are base paired with canonical base pairs or non-canonical base pairs. In one embodiment, one or more ribonucleotides of the sense ribonucleotide sequence or one or more ribonucleotides of the antisense ribonucleotide sequence, or both, are not base paired. In one embodiment, in the RNA molecule of the present invention, one or more ribonucleotides of each sense ribonucleotide sequence and one or more ribonucleotides of each antisense ribonucleotide sequence are not base paired.

[0080] In one embodiment, the antisense RNA sequence of the first RNA component or the second RNA component or both is less than 100% identical in sequence to a region of the target RNA molecule or the complement of two such regions, or about 80% to 99.9% identical, or about 90% to 98% identical, or about 95% to 98% identical, preferably 98% to 99.9% identical, and the two regions may or may not be adjacent in the target RNA molecule. In a preferred embodiment, the antisense RNA sequence is 100% identical in sequence to the complementary region of the target RNA molecule, the region being, for example, a region comprising 21, 23, 25, 27, 30 or 32 adjacent nucleotides. In one embodiment, the sense or antisense ribonucleotide sequence or both are at least 40, at least 50, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, at least about 1,000, or about 100 to about 1,000 contiguous nucleotides in length. When RNA molecules are used in plant cells or fungal cells or for non-vertebrate cells, a length of at least 100 nucleotides is preferred. When RNA molecules are used in vertebrate cells, the length of the sense and antisense ribonucleotide sequences in the dsRNA is 50 nucleotides or less, for example, 31 to 50 nucleotides is preferred. In one embodiment, the number of ribonucleotides in the sense ribonucleotide sequence is about 90% to about 110%, preferably 95% to 105%, more preferably 98% to 102%, even more preferably 99% to 101% of the number of ribonucleotides in the antisense ribonucleotide sequence. In the most preferred embodiment, the number of ribonucleotides in the sense ribonucleotide sequence is the same as the number of ribonucleotides in the antisense ribonucleotide sequence.These features can be applied to each dsRNA region in an RNA molecule.

[0081] In the embodiments of the first to fourth aspects, the first 3' ribonucleotide and the second 5' ribonucleotide in the RNA molecule are covalently linked by a loop sequence consisting of at least 4 ribonucleotides, or 4 to 1,000 ribonucleotides, or preferably 4 to 200 ribonucleotides, more preferably 4 to 50 ribonucleotides. In one embodiment, the RNA molecule further comprises a 5' extension sequence covalently linked to the first 5' ribonucleotide or a 3' extension sequence covalently linked to the second 3' ribonucleotide, or both. In one embodiment, the chimeric RNA molecule further comprises a 5' extension sequence covalently linked to the second 5' ribonucleotide or a 3' extension sequence covalently linked to the first 3' ribonucleotide, or both. In this embodiment, the RNA molecule comprises two separate RNA chains that hybridize to form the RNA molecule, although it may have been produced by transcribing from a nucleic acid molecule into a single RNA transcript and then being processed to comprise two RNA chains.

[0082] After splicing out from any introns but before any processing of the RNA molecule by Dicer enzyme or other RNase, the total length of the RNA molecule of the present invention produced as single-stranded RNA is typically 50 to 2000 ribonucleotides, preferably 60 or 70 to 2000 ribonucleotides, more preferably 80 or 90 to 2000 ribonucleotides, even more preferably 100 or 110 to 2000 ribonucleotides. In a preferred embodiment, the minimum length of the RNA molecule is 120, 130, 140, 150, 160, 180 or 200 nucleotides, and the maximum length is 400, 500, 600, 700, 800, 900, 1000, 1200, 1400, 1500 or 2000 ribonucleotides. Each combination of the above minimum and maximum lengths can be considered. The production of RNA molecules of such lengths can be readily accomplished by in vitro transcription or transcription in cells such as bacteria or other microbial cells or in eukaryotic cells in which the target gene is down-regulated.

[0083] In an embodiment of the first to fourth aspects, the chimeric RNA molecule comprises two or more identical or preferably different dsRNA regions.

[0084] In preferred embodiments of the first to fourth aspects, the RNA molecule is expressed in a eukaryotic cell, i.e., produced by transcription in the cell. In these embodiments, a greater proportion of dsRNA molecules are formed by processing RNA molecules of 22 and / or 20 ribonucleotides in length, compared to the processing of similar RNA molecules having a corresponding dsRNA region that is completely base-paired with canonical base pairs. That is, the RNA molecules of these embodiments are more easily processed than similar RNA molecules whose dsRNA region is completely base-paired with canonical base pairs, to provide 22- and / or 20-ribonucleotide short antisense RNAs, as part of the total 20-24 nucleotide asRNA produced by the RNA molecule.

[0085] In one embodiment, an RNA molecule of the invention comprises a combination of two or more features of the RNA molecules described herein.

[0086] On the other hand, the present invention provides polynucleotides encoding RNA molecules described herein, preferably chimeric RNA molecules described herein. In one embodiment, the polynucleotide is a DNA construct that can be integrated into a larger DNA molecule such as a chromosome. In one embodiment, the polynucleotide is operably linked to a promoter capable of directing the expression of the RNA molecule in a host cell. The host cell can be: a bacterial cell, such as Escherichia coli; a fungal cell, such as a yeast cell; or a eukaryotic cell, such as a plant cell or an animal cell. In one embodiment, the promoter is heterologous relative to the polynucleotide. The polynucleotide encoding the RNA molecule can be a chimeric or recombinant polynucleotide, or an isolated and / or exogenous polynucleotide. In one embodiment, the promoter can function in vitro, such as a bacteriophage promoter, such as a T7 RNA polymerase promoter or an SP6 RNA polymerase promoter. In one embodiment, the promoter is an RNA polymerase III promoter, such as a U6 promoter or an H1 promoter. In one embodiment, the promoter is an RNA polymerase II promoter, which can be a constitutive promoter, a tissue-specific promoter, a developmentally regulated promoter, or an inducible promoter. In one embodiment, the polynucleotide encodes an RNA precursor molecule containing an intron in at least one loop sequence, which can be spliced ​​out during or after transcription of the polynucleotide in a host cell. In one embodiment, the invention provides a vector comprising the polynucleotide as described herein. In one embodiment, the vector is a viral vector. In one embodiment, the vector is a plasmid vector, such as a binary vector suitable for use with Agrobacterium tumefaciens.

[0087] In one embodiment, wherein the polynucleotide or vector of the invention is in a eukaryotic host cell, preferably in a plant, the promoter region of the polynucleotide or vector is operably linked to the region encoding the RNA molecule of the invention, and has a lower level of methylation compared to the promoter of the corresponding polynucleotide or vector encoding the RNA molecule having the corresponding dsRNA region that is fully base paired with the canonical base pairs. In one embodiment, the lower level of methylation is less than 50%, less than 40%, less than 30% or less than 20% when compared to the promoter of the corresponding polynucleotide or vector. In one embodiment, the host cell comprises at least two copies of the polynucleotide or vector encoding the RNA molecule of the invention. In this embodiment:

[0088] i) the expression and / or activity of the target RNA molecule in the eukaryotic cell is reduced to at least the same level relative to a corresponding eukaryotic cell having a single copy of the polynucleotide or vector, and / or

[0089] ii) expression and / or activity of the target RNA molecule in the eukaryotic cell is reduced to a lower level when compared to a corresponding cell comprising an RNA molecule having a corresponding dsRNA region that is fully base paired with canonical base pairs.

[0090] On the other hand, the invention provides host cells comprising RNA molecules described herein, polynucleotides described herein, or vectors comprising them. In one embodiment, the host cell is a non-human cell, such as a bacterial cell, a fungal cell, a plant cell, or a non-human animal cell. In one embodiment, the cell is a human cell in a non-human cell or a cell culture. In one embodiment, the cell is a eukaryotic cell, such as a cell other than an animal cell. In one embodiment, the cell is a microbial cell such as a prokaryotic cell. In one embodiment, the host cell is alive. In another embodiment, the host cell is dead.

[0091] In another aspect, the invention provides a non-human organism comprising an RNA molecule of the invention, preferably a chimeric RNA molecule as described herein, a polynucleotide or vector of the invention comprising it, or a host cell comprising it. In one embodiment, the non-human organism is transgenic, as long as it comprises a polynucleotide of the invention. In one embodiment, the polynucleotide is stably integrated into the genome of the non-human organism.

[0092] On the other hand, the invention provides a method for producing the RNA molecules of the invention, the method comprising expressing the polynucleotides of the invention in a host cell or a cell-free expression system. In this embodiment, the method may further comprise at least partially purifying or not purifying the RNA molecules.

[0093] On the other hand, the invention provides a method for producing a cell or a non-human organism, preferably a plant or a fungus, the method comprising importing a polynucleotide or a vector of the present invention into a cell, preferably a plant cell or a fungus, preferably such that the polynucleotide or vector encoding the RNA molecule or a portion thereof is stably integrated into the genome of the cell. In one embodiment, the non-human organism is produced, for example, from a cell or a daughter cell by regenerating a plant. In one embodiment, a non-human organism is produced by importing a cell or one or more daughter cells into a non-human organism. As an alternative to stably integrating a polynucleotide or a vector into the cell genome, a polynucleotide or a vector can be introduced into a cell without integrating the polynucleotide or the vector into the genome, for example, to transiently express an RNA molecule in a cell or an organism.

[0094] On the other hand, the present invention provides an extract of a host cell or organism of the present invention or a part thereof, wherein the extract comprises an RNA molecule of the present invention, a small RNA molecule (20-24 nt in length) produced by processing the RNA molecule, or both, and / or a polynucleotide or vector of the present invention. In one embodiment, the present invention provides a composition comprising one or more RNA molecules of the present invention, a small RNA molecule (20-24 nt in length) produced by processing the RNA molecule, or both; a polynucleotide of the present invention; a vector of the present invention; a host cell of the present invention; or an extract produced by the method of the present invention; and one or more suitable carriers. In one embodiment, the composition is a pharmaceutical composition, such as a composition suitable for application to humans or other animals. The pharmaceutical composition may be suitable for the prevention or treatment of a disease, or for topical application, such as a cosmetic application. In one embodiment, the composition is suitable for application to plants, preferably plants or plant groups in a field, or to insects or insect groups. In one embodiment, the composition is suitable for application to crops, such as by spraying to crop plants in a field.

[0095] In one embodiment, the extract or composition comprising the RNA molecule of the invention or the small RNA molecule (length 20-24 nt) produced by processing the RNA molecule or both further comprises at least one compound that enhances the stability of the RNA molecule or polynucleotide and / or vector, wherein the at least one compound facilitates the uptake of the RNA molecule, polynucleotide or vector by cells such as cells of an organism. In one embodiment, the compound is a transfection facilitator, such as a lipid-containing compound.

[0096] In another aspect, the invention provides a method for reducing or down-regulating the level and / or activity of a target RNA molecule in a cell or organism, the method comprising delivering to the cell or organism one or more RNA molecules of the invention or small RNA molecules (20-24 nt in length) produced by processing the RNA molecules, or both; polynucleotides of the invention; vectors of the invention or compositions of the invention. In one embodiment, the target RNA molecule encodes a protein. In one embodiment, the method reduces the level and / or activity of more than one target RNA molecule, for example, reduces the level and / or activity of two or more target RNAs, the target RNA molecules are different, and the level and / or activity of the target RNAs are related, for example, in sequences from a gene family.

[0097] On the other hand, the present invention provides a method for controlling a non-human organism, the method comprising delivering to the non-human organism one or more RNA molecules of the present invention or small RNA molecules (20-24 nt in length) produced by processing the RNA molecules, or both; polynucleotides or vectors of the present invention; host cells of the present invention; extracts produced by the methods of the present invention; or compositions of the present invention, wherein the RNA molecules or small RNA molecules have a harmful effect on the non-human organism. In one embodiment, the non-human organism is an arthropod, such as an insect; or a plant, such as a weed. In one embodiment, the non-human organism is a plant, and the arthropod ingests the plant or a part thereof, thereby controlling the arthropod.

[0098] In one embodiment, the present invention provides a method for preventing or treating a disease in a subject, the method comprising administering to the subject one or more RNA molecules of the present invention or small RNA molecules (20-24 nt in length) produced by processing of RNA molecules, or both; polynucleotides or vectors of the present invention; host cells of the present invention; extracts produced by the methods of the present invention; or compositions of the present invention, wherein the RNA molecules or small RNA molecules have a beneficial effect on at least one symptom of the disease. In one embodiment, the RNA molecules or small RNA molecules, polynucleotides, vectors or compositions are administered topically, orally or by injection. In one embodiment, the subject is a vertebrate. In one embodiment, the vertebrate is a mammal, such as a human, livestock such as a cow or sheep; or a bird, such as a chicken and other poultry.

[0099] In another aspect, the present invention provides the use of the RNA molecules of the present invention, the polynucleotides or vectors of the present invention, the host cells of the present invention, the extracts produced by the methods of the present invention, or the compositions of the present invention for treating a disease in a subject, wherein the RNA molecules or small RNA molecules have a beneficial effect on at least one symptom of the disease. In one embodiment, the present invention provides the use of the RNA molecules of the present invention or small RNA molecules produced therefrom, the polynucleotides or vectors of the present invention, the host cells of the present invention, the extracts produced by the methods of the present invention, or the compositions of the present invention for the manufacture of a medicament for preventing or treating a disease in a subject, wherein the RNA molecules or small RNA molecules produced therefrom have a beneficial effect on at least one symptom of the disease.

[0100] In another aspect, the invention provides a kit comprising one or more of the following: an RNA molecule of the invention or a small RNA molecule produced thereby, a polynucleotide or a vector of the invention, a host cell of the invention, an extract produced by a method of the invention, or a composition of the invention.

[0101] Unless specifically stated otherwise, any embodiment herein should be understood to be applicable mutatis mutandis to any other embodiment.

[0102] The present invention is not to be limited in scope by the specific embodiments described herein, which are intended for purposes of illustration only. Functionally equivalent products, compositions and methods are clearly within the scope of the present invention as described herein.

[0103] Throughout the specification, reference to an individual step, composition of matter, group of steps or group of compositions of matter shall be considered to include both one and multiple (ie one or more) those steps, compositions of matter, groups of steps or groups of compositions of matter unless specifically stated otherwise or the context requires otherwise.

[0104] The invention is now described by way of the following non-limiting examples and with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0105] Figure 1 Schematic diagram of two ledRNA molecules. (A) The ledRNA molecule comprises a sense sequence, an antisense sequence, and two loops, wherein the sense sequence can be considered as two adjacent sense sequences, covalently linked without an intervening spacer sequence, and having identity with a target RNA, the antisense sequence is complementary to the sense sequence and is divided into two regions, a 5' region and a 3' region, and the two loops separate the sense sequence from the antisense sequence. (B) The ledRNA molecule comprises an antisense sequence, a sense sequence, and two loops: the antisense sequence can be considered as two adjacent antisense sequences, covalently linked without an intervening spacer sequence, and having identity with the complement of a target RNA, the sense sequence is complementary to the antisense sequence and is divided into two regions, and the two loops separate the sense sequence from the antisense sequence. The RNA molecule is produced by transcription, for example by in vitro transcription from a promoter such as the T7 or Sp6 promoter, by self-annealing through base pairing between complementary sense and antisense sequences to form a double-stranded region with a loop at each end and a "gap" in the antisense or sense sequence. Additional sequences may be attached to the 5' and / or 3' ends as 5' or 3' extension sequences.

[0106] Figure 2 Compared with sense / antisense annealing or hairpin RNA, ledRNA is more efficient in forming dsRNA. Schematic diagrams of three forms of double-stranded RNA molecules are shown: A, conventional dsRNA formed by annealing of two separate strands; B, hairpin RNA with 5' or 3' extension sequence; and C, ledRNA molecule. The lower panel shows photos after gel electrophoresis of RNA transcripts of three types of RNA molecules targeting the GUS gene or GFP gene.

[0107] Figure 3Northern blot hybridization of treated (A and B) and untreated distal (C and D) tissues shows that ledRNA is more stable than dsRNA and spreads through tobacco leaf tissues. In contrast to the strong ledRNA signal, no dsRNA signal can be detected in distal tissues (C and D, upper panels).

[0108] Figure 4 .ledRNA treatment induced GUS downregulation in both the treated area (1) and the untreated area (3) mentioned above.

[0109] Figure 5 .ledRNA induces FAD2.1 gene silencing in N. benthamiana leaves.

[0110] Figure 6 Northern blot hybridization confirmed that FAD2.1 mRNA was strongly down-regulated by treatment with ledFAD2.1 at 6 and 24 hours.

[0111] Figure 7 The nucleotide sequence of the GUS target gene region (SEQ ID NO: 14) was aligned with the sense sequence of the hpGUS [G:U] construct (nucleotides 9 to 208 of SEQ ID NO: 11). 52 cytosine (C) nucleotides were substituted with thymine (T) nucleotides. The retained nucleotides are marked with an asterisk, and the substituted C is not marked with an asterisk.

[0112] Figure 8 The nucleotide sequence of the GUS target gene region (SEQ ID NO: 14) was aligned with the sense sequence of the hpGUS [1:4] construct (nucleotides 9 to 208 of SEQ ID NO: 12). Every fourth nucleotide in hpGUS [1:4] was substituted relative to the corresponding wild-type sense sequence, whereby for every fourth nucleotide, C was changed to G, G was changed to C, A was changed to T, and T was changed to A. The retained nucleotides are marked with an asterisk, the substituted G and C are not marked with an asterisk, and the substituted A and T are marked with a semicolon.

[0113] Fig. 9The nucleotide sequence of the GUS target gene region (SEQ ID NO: 14) was aligned with the sense sequence of the hpGUS [2: 10] construct (nucleotides 9 to 208 of SEQ ID NO: 13). Relative to the corresponding wild-type sense sequence, every "9th" and "10th" nucleotide in each segment of 10 nucleotides in hpGUS [2: 10] was substituted, whereby for every "9th" and "10th" nucleotide, C was changed to G, G was changed to C, A was changed to T, and T was changed to A. The retained nucleotides are marked with an asterisk, the substituted G and C are not marked with an asterisk, and the substituted A and T are marked with a semicolon.

[0114] Fig.10 . Schematic diagram showing the structure of the genetic construct encoding the modified hairpin RNA targeting GUS mRNA.

[0115] Fig.11 . Schematic diagram of the vector pWBPPGH, which is used to transform tobacco plants and provide the GUS target gene. The T-DNA extends from the right border (RB) to the left border (LB) of the vector. The selectable marker gene on the T-DNA is the 35S-HPT-tm1' gene that encodes hygromycin resistance.

[0116] Fig.12 GUS activity in plants transformed with constructs encoding modified hairpin RNA for reducing the expression of the GUS target gene. No hp: Control PPGH11 and PPGH24 plants without hpGUS construct. The number of plants showing less than 10% GUS activity compared to the corresponding control PPGH11 or PPGH24 plants and the percentage of such plants relative to the number of tested plants are given in parentheses.

[0117] Fig.13 (A) Average GUS activity of all transgenic plants: 59 for hpGUS[wt], 74 for hpGUS[G:U], 33 for hpGUS[1:4], 41 for hpGUS[2:10]. (B) Average GUS activity of all silenced plants (32 for hpGUS[wt], 71 for hpGUS[G:U], 33 for hpGUS[1:4], 28 for hpGUS[2:10]).

[0118] Fig.14 . GUS activity of transgenic progeny plants containing hpGUS[wt], hpGUS[G:U] or hpGUS[1:4].

[0119] Fig.15.Autoradiograph of a Southern blot of DNA from 16 plants transformed with the hpGUS[G:U] construct. DNA was digested with HindIII and probed with OCS-T probe before gel electrophoresis. Lane 1: size marker (λ DNA digested with HindIII); Lanes 2 and 3, DNA from parental plants PPGHII and PPGH24; Lanes 4-19: DNA from 16 different transgenic plants.

[0120] Fig.16 Autoradiographs of Northern blot hybridization experiments detecting sense (upper panels) and antisense (lower panels) sRNA from hairpin RNA expressed in transgenic tobacco plants. Lanes 1 and 2 contain RNA obtained from parental plants PPGHII and PPGH24 lacking the hpGUS construct. Lanes 3-11 contain RNA from hpGUS[wt] plants, and lanes 12-20 contain RNA from hpGUS[G:U] plants.

[0121] Fig.17 Autoradiograph of a Northern blot hybridization detecting antisense sRNA from transgenic plants. Lanes 1-10 are from hpGUS[wt] plants and lanes 11-19 are from hpGUS[G:U] plants. Antisense sRNAs have mobilities corresponding to lengths of 20-24 nt. The blot was reprobed with antisense RNA to U6 RNA as a lane loading control.

[0122] Fig.18 . Autoradiograph of repeated Northern blot hybridizations detecting antisense sRNA from transgenic plants.

[0123] Fig.19 DNA methylation analysis of the junction region between the 35S promoter and the sense GUS region in the hpGUS construct in transgenic plants. The junction fragment was PCR amplified with (+) or without (-) McrBC enzyme before treating the plant DNA.

[0124] Fig. 20 DNA methylation analysis of the 35S promoter region in the hpGUS construct in transgenic plants. Before treating plant DNA with McrBC enzyme, PCR amplification of the 35S fragment was performed with (+) or without (-).

[0125] Fig.21 . Size distribution and abundance of processed RNA. (A) EIN2 construct. (B) GUS construct.

[0126] Fig. 22Alignment of the sense sequence of the .hpEIN2[G:U] construct (upper sequence, nucleotides 17 to 216 of SEQ ID NO: 22) and the nucleotide sequence of the cDNA region corresponding to the EIN2 target gene of A. thaliana (lower sequence, SEQ ID NO: 27). The sense sequence was obtained by replacing 43 cytosine (C) nucleotides in the wild-type sequence with thymine (T) nucleotides. The retained nucleotides are marked with an asterisk, and the substituted C is not marked with an asterisk.

[0127] Fig.23 Alignment of the sense sequence of the .hpCHS[G:U] construct (upper sequence, nucleotides 13 to 212 of SEQ ID NO:24) and the nucleotide sequence corresponding to the cDNA region of the CHS target gene of Arabidopsis thaliana (SEQ ID NO:28, in reverse order). The sense sequence was obtained by replacing 65 cytosine (C) nucleotides in the wild-type sequence with thymine (T) nucleotides. The retained nucleotides are marked with an asterisk, and the replaced C is not marked with an asterisk.

[0128] Fig.24 Alignment of the antisense sequence of the .hpEIN2[G:U / U:G] construct (upper sequence, nucleotides 8 to 207 of SEQ ID NO: 25) and the nucleotide sequence corresponding to the complement region of the Arabidopsis thaliana EIN2 target gene and (lower sequence, SEQ ID NO: 29). The antisense sequence was obtained by replacing 49 cytosine (C) nucleotides in the wild-type sequence with thymine (T) nucleotides. The retained nucleotides are marked with an asterisk, and the replaced C is not marked with an asterisk.

[0129] Fig.25 Alignment of the antisense sequence of the .hpCHS[G:U / U:G] construct (upper sequence, nucleotides 13 to 212 of SEQ ID NO:26) and the nucleotide sequence corresponding to the complement region of the A.thaliana CHS target gene (lower sequence, SEQ ID NO:30). The antisense sequence was obtained by replacing 49 cytosine (C) nucleotides in the wild-type sequence with thymine (T) nucleotides. The retained nucleotides are marked with an asterisk, and the replaced C is not marked with an asterisk.

[0130] Fig.26 Schematic diagram of ethylene insensitive 2 (EIN2) and chalcone synthase (CHS) hpRNA constructs. 35S: CaMV35S promoter; EIN2 and CHS regions are shown as wild-type sequence (wt) or G:U modified sequence (G:U). Arrows indicate the orientation of DNA fragments, and arrows from right to left indicate antisense sequences. Restriction enzyme sites are also shown.

[0131] Fig. 27 Hypocotyl length of transgenic Arabidopsis thaliana seedlings containing hpEIN2[wt] or hpEIN2[G:U] in the EIN2 assay.

[0132] Fig.28 For CHS mRNA in transgenic A. thaliana, qRT-PCR normalized the transgenesis of hpCHS[wt] or hpCHS[G:U] constructs to the level of Actin 2 RNA. Col-0 is wild-type (non-transgenic) A. thaliana.

[0133] Fig.29 Autoradiographs of Northern blot hybridizations of RNA from plants transformed with hpEIN2[wt] or hpEIN2[G:U]. The upper panel shows the hypocotyl length of the lines. The autoradiographs show Northern blots probed with the EIN2 sense probe to detect antisense sRNA. The same blots were reprobed with the U6 RNA probe as a loading control (U6 RNA).

[0134] Fig.30 DNA methylation analysis of 35S promoter and 35S-sense EIN2 sequences in genomic DNA in transgenic Arabidopsis (A. thaliana) plants.

[0135] Fig.31 .DNA methylation levels in the promoter and 5' regions of the hairpin RNA constructs.

[0136] Fig.32 The 35S promoter in the least methylated line of the .hpEIN2[wt] population still showed significant methylation.

[0137] Fig.33 .G: The 35S promoter in the U hpEIN2 line showed only weak methylation (P<10%).

[0138] Fig.34 .ledRNA and hpRNA with G:U gene silencing in CHO and Vero cells at 72 hours.

[0139] Fig.35 .The dumbbell-shaped plasmid was tested in HeLa cells at 48 hours.

[0140] Fig.36 Examples of possible modifications of dsRNA molecules.

[0141] Fig.37Aphids' performance decreased after feeding artificial diet supplemented with ledRNA to downregulate the expression of MpC002 or MpRack-1 genes in green peach aphids. Upper panel (A): Average number of nymphs per adult aphid after a ten-day period with 100 μl of 50 ng / μl ledRNA. Lower panel (B): Percentage of aphids surviving over a five-day time course after feeding 100 μl of 200 ng / μl ledRNA containing MpC002, MpRack-1 or control ledGFP.

[0142] Sequence Listing Keywords

[0143] SEQ ID NO: 1—Ribonucleotide sequence of GFP ledRNA.

[0144] SEQ ID NO: 2—Ribonucleotide sequence of GUS ledRNA.

[0145] SEQ ID NO:3—Ribonucleotide sequence of N. benthamiana FAD2.1 ledRNA.

[0146] SEQ ID NO:4—Nucleotide sequence encoding GFP ledRNA.

[0147] SEQ ID NO:5—Nucleotide sequence encoding GUS ledRNA.

[0148] SEQ ID NO:6—Nucleotide sequence encoding N. benthamiana FAD2.1 ledRNA.

[0149] SEQ ID NO:7—Nucleotide sequence encoding GFP.

[0150] SEQ ID NO:8—Nucleotide sequence encoding GUS.

[0151] SEQ ID NO:9—Nucleotide sequence encoding N. benthamiana FAD2.1.

[0152] SEQ ID NO: 10—Nucleotide sequence used to provide the GUS sense region for a construct encoding a hairpin RNA molecule that targets GUS mRNA.

[0153] SEQ ID NO: 11—Nucleotide sequence used to provide the GUS sense region for a construct encoding the hairpin RNA molecule hpGUS[G:U].

[0154] SEQ ID NO: 12—Nucleotide sequence used to provide the GUS sense region for a construct encoding the hairpin RNA molecule hpGUS[1:4].

[0155] SEQ ID NO: 13—Nucleotide sequence used to provide the GUS sense region for a construct encoding the hairpin RNA molecule hpGUS[2:10].

[0156] SEQ ID NO: 14 - Nucleotide sequence of nucleotides 781-1020 of the protein coding region of the GUS gene.

[0157] SEQ ID NO: 15 - Ribonucleotide sequence of the hairpin structure (including its loop) of hpGUS [wt] RNA.

[0158] SEQ ID NO: 16 - Ribonucleotides of the hairpin structure (including the loop) of hpGUS[G:U] RNA.

[0159] SEQ ID NO: 17—Ribonucleotides of the hairpin structure (including the loop) of hpGUS[1:4] RNA.

[0160] SEQ ID NO: 18—Ribonucleotides of the hairpin structure (including the loop) of hpGUS[2:10] RNA.

[0161] SEQ ID NO: 19—Nucleotide sequence corresponding to the cDNA of the A. thaliana EIN2 gene, Accession No. NM_120406.

[0162] SEQ ID NO:20—Nucleotide sequence corresponding to the cDNA of A. thaliana CHS gene, accession number NM_121396, 1703 nt.

[0163] SEQ ID NO:21—Nucleotide sequence of a DNA fragment comprising a 200 nt sense sequence from the cDNA corresponding to the A. thaliana EIN2 gene flanked by restriction enzyme sites.

[0164] SEQ ID NO:22—Nucleotide sequence of a DNA fragment containing 200 nt sense sequence of EIN2, identical to SEQ ID NO:21, except that 43 C's were replaced with T's, used to construct hpEIN2[G:U].

[0165] SEQ ID NO:23—Nucleotide sequence of a DNA fragment comprising a 200 nt sense sequence from the cDNA corresponding to A. thaliana CHS flanked by restriction enzyme sites.

[0166] SEQ ID NO:24—Nucleotide sequence of a DNA fragment containing the 200 nt sense sequence of CHS, identical to SEQ ID NO:23, except that 65 C's are replaced by T's, used to construct hpCHS[G:U].

[0167] SEQ ID NO:25—Nucleotide sequence of a DNA fragment containing the 200 nt antisense sequence of EIN2, except that 50 C's were substituted for T's, used to construct hpEIN2[G:U / U:G].

[0168] SEQ ID NO:26—Nucleotide sequence of a DNA fragment containing the 200 nt antisense sequence of CHS, except that 49 C's were substituted for T's, used to construct hpCHS[G:U / U:G].

[0169] SEQ ID NO:27—Nucleotide sequence corresponding to nucleotides 601-900 of the cDNA of the A. thaliana EIN2 gene (Accession No. NM_120406).

[0170] SEQ ID NO:28—Nucleotide sequence corresponding to nucleotides 813-1112 of the cDNA of the A. thaliana CHS gene (Accession No. NM_121396).

[0171] SEQ ID NO:29—Nucleotide sequence corresponding to the complement of nucleotides 652-891 of the cDNA of the A. thaliana EIN2 gene (Accession No. NM_120406).

[0172] SEQ ID NO:30—Nucleotide sequence corresponding to the complement of nucleotides 804-1103 of the cDNA of the A. thaliana CHS gene.

[0173] SEQ ID NO: 31 - FANCM I protein coding region of cDNA of Arabidopsis thaliana, accession number NM_001333162. Target region nucleotides 675-1174 (500 nucleotides)

[0174] SEQ ID NO: 32 - FANCM I protein coding region of Brassica napus cDNA. Target region nucleotides 896-1395 (500 bp)

[0175] SEQ ID NO:33—Nucleotide sequence encoding hpFANCM-At[wt], which targets the FANCM I protein coding region of Arabidopsis thaliana. FANCM sense sequence, nucleotides 38-537; loop sequence, nucleotides 538-1306; FANCM antisense sequence, nucleotides 1307-1806.

[0176] SEQ ID NO:34—Nucleotide sequence encoding hpFANCM-At[G:U], which targets the FANCM I protein coding region of Arabidopsis thaliana. FANCM sense sequence, nucleotides 38-537; loop sequence, nucleotides 538-1306; FANCM antisense sequence, nucleotides 1307-1806.

[0177] SEQ ID NO:35—Nucleotide sequence encoding hpFANCM-Bn[wt], which targets the FANCM I protein coding region of B. napus. FANCM sense sequence, nucleotides 34-533; loop sequence, nucleotides 534-1300; FANCM antisense sequence, nucleotides 1301-1800.

[0178] SEQ ID NO:36—Nucleotide sequence encoding hpFANCM-Bn[G:U], which targets the FANCM I protein coding region of B. napus. FANCM sense sequence, nucleotides 34-533; loop sequence, nucleotides 534-1300; FANCM antisense sequence, nucleotides 1301-1800.

[0179] SEQ ID NO:37—Nucleotide sequence corresponding to the protein coding region of the cDNA of B. napus DDM1 gene; Accession No. XR_001278527.

[0180] SEQ ID NO:38—Nucleotide sequence of DNA encoding hpDDM1-Bn[wt], which targets the DDM1 protein coding region of B. napus.

[0181] SEQ ID NO:39—Nucleotide sequence encoding hpDDM1-Bn[G:U], which targets the DDM1 protein coding region of B. napus. DDM1 sense sequence, nucleotides 35-536; loop sequence, nucleotides 537-1304; DDM1 antisense sequence, nucleotides 1305-1805.

[0182] SEQ ID NO:40—EGFP cDNA.

[0183] SEQ ID NO:41 - Nucleotide sequence of the coding region of hpEGFP[wt], antisense / loop / sense relative to the promoter order.

[0184] SEQ ID NO:42 - Nucleotide sequence of the coding region of hpEGFP[G:U] having 157 C to T substitutions in the EGFP sense sequence.

[0185] SEQ ID NO:43 - Nucleotide sequence of the coding region of ledEGFP[wt] without the C to T substitution in the EGFP sense sequence.

[0186] SEQ ID NO:44 - Nucleotide sequence of the coding region of ledEGFP[G:U] having 162 C to T substitutions in the EGFP sense sequence.

[0187] SEQ ID NO:45—Nucleotide sequence used to provide the GUS sense region for a construct encoding the hairpin RNA molecule hpGUS[G:U] not flanked by restriction enzyme sites.

[0188] SEQ ID NO:46—Nucleotide sequence used to provide the GUS sense region for a construct encoding the hairpin RNA molecule hpGUS[1:4] not flanked by restriction enzyme sites.

[0189] SEQ ID NO:47—Nucleotide sequence used to provide the GUS sense region for a construct encoding the hairpin RNA molecule hpGUS[2:10] not flanked by restriction enzyme sites.

[0190] SEQ ID NO:48—Nucleotide sequence of a DNA fragment comprising the 200 nt sense sequence of EIN2, identical to SEQ ID NO:21, except that 43 C's are replaced by T's, used to construct hpEIN2[G:U] without flanking sequences.

[0191] SEQ ID NO:49—Nucleotide sequence of a DNA fragment containing the 200 nt sense sequence of CHS, identical to SEQ ID NO:23, except that 65 C's are replaced by T's, used to construct hpCHS without flanking sequences [G:U].

[0192] SEQ ID NO:50—Nucleotide sequence of a DNA fragment containing the 200 nt antisense sequence of EIN2, except that 50 C's were substituted for T's, used to construct hpEIN2 without flanking sequences [G:U / U:G].

[0193] SEQ ID NO:51—Nucleotide sequence of a DNA fragment containing the 200 nt antisense sequence of CHS, except that 49 C's were substituted for T's, used to construct the hpCHS [G:U / U:G] sequence without flanking sequences.

[0194] SEQ ID No: 52—Oligonucleotide primer for amplifying 200 bp GUS sense sequence (GUS-WT-F)

[0195] SEQ ID No: 53 - Oligonucleotide primer for amplifying 200 bp GUS sense sequence (GUS-WT-R)

[0196] SEQ ID NO:54—Oligonucleotide primer (forward) for production of hpGUS[G:U] fragments in which each C is replaced by a T (GUS-GU-F)

[0197] SEQ ID NO:55—Oligonucleotide primer (reverse) for production of hpGUS[G:U] fragments in which each C is replaced by a T (GUS-GU-R)

[0198] SEQ ID NO:56—Oligonucleotide primer (forward) for production of hpGUS[1:4] fragments in which every 4th nucleotide is substituted (GUS-4M-F)

[0199] SEQ ID NO:57—Oligonucleotide primer (reverse) for production of hpGUS[1:4] fragments in which every 4th nucleotide is substituted (GUS-4M-R)

[0200] SEQ ID NO:58—Oligonucleotide primer (forward) for production of hpGUS[2:10] fragment in which every 9th and 10th nucleotides are substituted (GUS-10M-F)

[0201] SEQ ID NO:59 - Oligonucleotide primer (reverse) for production of hpGUS[2:10] fragment in which every 9th and 10th nucleotides are substituted (GUS-10M-R)

[0202] SEQ ID No: 60 - Nucleotide sequence encoding forward primer (35S-F3)

[0203] SEQ ID NO:61 - Nucleotide sequence encoding reverse primer (GUSwt-R2)

[0204] SEQ ID NO:62—Nucleotide sequence encoding forward primer (GUSgu-R2)

[0205] SEQ ID NO:63—Nucleotide sequence encoding reverse primer (GUS4m-R2)

[0206] SEQ ID No: 64 - Nucleotide sequence encoding forward primer (35S-F2)

[0207] SEQ ID NO:65 - Nucleotide sequence encoding reverse primer (35S-R1)

[0208] SEQ ID NO:66—Oligonucleotide primer for amplification of wild-type 200 bp EIN2 sense sequence (EIN2wt-F)

[0209] SEQ ID NO:67—Oligonucleotide primer for amplification of the wild-type 200 bp EIN2 sense sequence (EIN2wt-R)

[0210] SEQ ID No: 68 - Oligonucleotide primer for amplification of wild-type 200 bp CHS sense sequence (CHSwt-F)

[0211] SEQ ID No: 69 - Oligonucleotide primer for amplification of wild-type 200 bp CHS sense sequence (CHSwt-R)

[0212] SEQ ID NO:70—Oligonucleotide primer (forward) for producing a hpEIN2[G:U] fragment in which each C is replaced by a T (EIN2gu-F)

[0213] SEQ ID NO:71 - Oligonucleotide primer (reverse) for producing a hpEIN2[G:U] fragment in which each C is replaced by a T (EIN2gu-R)

[0214] SEQ ID NO:72—Oligonucleotide primer (forward) for producing hpCHS[G:U] fragments in which each C is replaced by a T (CHSgu-F)

[0215] SEQ ID NO:73 - Oligonucleotide primer (reverse) for producing hpCHS[G:U] fragments in which each C is replaced by a T (CHSgu-R)

[0216] SEQ ID NO:74—Oligonucleotide primer (forward) for generating a hpEIN2[G:U / U:G] fragment in which each C is replaced by a T (asEIN2gu-F)

[0217] SEQ ID NO:75—Oligonucleotide primer (reverse) for producing a hpEIN2[G:U / U:G] fragment in which each C is replaced by a T (asEIN2gu-R)

[0218] SEQ ID NO:76 - Oligonucleotide primer (forward) for producing hpCHS[G:U / U:G] fragments in which each C is replaced by a T (asCHSgu-F)

[0219] SEQ ID NO:77 - Oligonucleotide primer (reverse) for production of hpCHS[G:U / U:G] fragments in which each C is replaced by a T (asCHSgu-R)

[0220] SEQ ID No: 78 - Nucleotide sequence encoding forward primer (CHS-200-F2)

[0221] SEQ ID NO:79 - Nucleotide sequence encoding reverse primer (CHS-200-R2)

[0222] SEQ ID No: 80 - Nucleotide sequence encoding forward primer (Actin2-For)

[0223] SEQ ID NO:81 - Nucleotide sequence encoding reverse primer (Actin2-Rev)

[0224] SEQ ID NO:82—Nucleotide sequence encoding forward primer (Top-35S-F2)

[0225] SEQ ID NO:83—Nucleotide sequence encoding reverse primer (Top-35S-R2)

[0226] SEQ ID NO:84—Nucleotide sequence encoding forward primer (Link-35S-F2)

[0227] SEQ ID NO:85—Nucleotide sequence encoding reverse primer (Link-EIN2-R2)

[0228] SEQ ID NO: 86 - sense si22 - ribonucleotide sequence

[0229] SEQ ID NO: 87 - Antisense si22-ribonucleotide sequence

[0230] SEQ ID NO: 88—Ribonucleotide sequence of forward primer

[0231] SEQ ID NO: 89—Ribonucleotide sequence of reverse primer

[0232] SEQ ID NO:90—Ribonucleotide sequence of forward primer

[0233] SEQ ID NO:91—Ribonucleotide sequence of reverse primer

[0234] SEQ ID NO:92 - Possible modifications of dsRNA molecules

[0235] SEQ ID NO:93—Nucleotide sequence corresponding to the cDNA of the B. napus DDM1 gene (Accession No. XR_001278527).

[0236] SEQ ID NO:94—Nucleotide sequence of chimeric DNA encoding a hairpin RNAi (hpRNA) construct targeting the B. napus DDM1 gene.

[0237] SEQ ID NO:95—Nucleotide sequence encoding a chimeric DNA hairpin RNAi (hpRNA) construct targeting B. napus DDM1 gene with G:U base pairs.

[0238] SEQ ID NO:96—Nucleotide sequence of chimeric DNA encoding a ledRNA construct targeting the B. napus DDM1 gene.

[0239] SEQ ID NO:97—Nucleotide sequence corresponding to the cDNA of the A. thaliana FANCM gene (Accession No. NM_001333162).

[0240] SEQ ID NO:98—Nucleotide sequence of chimeric DNA encoding a hairpin RNAi (hpRNA) construct targeting the Arabidopsis thaliana FANCM gene.

[0241] SEQ ID NO:99—Nucleotide sequence encoding a chimeric DNA hairpin RNAi (hpRNA) construct targeting the Arabidopsis thaliana FANCM gene with G:U base pairs.

[0242] SEQ ID NO: 100—Nucleotide sequence of a chimeric DNA encoding a ledRNA construct targeting the Arabidopsis thaliana (A. thaliana) FANCM gene.

[0243] SEQ ID NO: 101—Nucleotide sequence corresponding to the cDNA of the B. napus FANCM gene (Accession No. XM_022719486.1).

[0244] SEQ ID NO: 102—Nucleotide sequence of chimeric DNA encoding a hairpin RNAi (hpRNA) construct targeting the B. napus FANCM gene.

[0245] SEQ ID NO: 103—Nucleotide sequence encoding a chimeric DNA hairpin RNAi (hpRNA) construct targeting the B. napus FANCM gene with G:U base pairs.

[0246] SEQ ID NO: 104—Nucleotide sequence of chimeric DNA encoding a ledRNA construct targeting the B. napus FANCM gene.

[0247] SEQ ID NO: 105—Nucleotide sequence corresponding to the protein coding region of the cDNA of the Nicotiana benthamiana TOR gene.

[0248] SEQ ID NO: 106—Nucleotide sequence of a chimeric DNA encoding a ledRNA construct targeting the Nicotiana benthamiana TOR gene.

[0249] SEQ ID NO: 107: Nucleotide sequence corresponding to the protein coding region of the cDNA of the acetolactate synthase (ALS) gene of barley (Hordeum vulgare) (Accession No. LT601589).

[0250] SEQ ID NO: 108—Nucleotide sequence of a chimeric DNA encoding a ledRNA construct targeting the barley (H. vulgare) ALS gene.

[0251] SEQ ID NO: 109: Nucleotide sequence corresponding to the protein coding region of the cDNA of the barley (Hordeum vulgare) HvNCED1 gene (Accession No. AK361999).

[0252] SEQ ID NO: 110—Nucleotide sequence corresponding to the protein coding region of the cDNA corresponding to the Hordeum vulgare HvNCEDD2 gene (Accession No. DQ145931).

[0253] SEQ ID NO: 111—Nucleotide sequence of chimeric DNA encoding a ledRNA construct targeting the NCED1 gene of barley (Hordeum vulgare) and wheat (Triticum aestivum).

[0254] SEQ ID NO: 112—Nucleotide sequence of chimeric DNA encoding a ledRNA construct targeting the NCED2 gene of barley (Hordeum vulgare) and wheat (Triticum aestivum).

[0255] SEQ ID NO: 113: Nucleotide sequence corresponding to the protein coding region of the cDNA of the barley gene encoding ABA-OH-2 (Accession No. DQ145933).

[0256] SEQ ID NO: 114—Nucleotide sequence of chimeric DNA encoding a ledRNA construct targeting the ABA-OH-2 gene of barley (Hordeum vulgare) and wheat (Triticum aestivum).

[0257] SEQ ID NO: 115—Nucleotide sequence corresponding to the cDNA protein coding region of the A. thaliana gene (At5g03280) encoding EIN2.

[0258] SEQ ID NO: 116—Nucleotide sequence of a chimeric DNA encoding a ledRNA construct targeting the Arabidopsis thaliana EIN2 gene.

[0259] SEQ ID NO: 117: Nucleotide sequence corresponding to the protein coding region of the cDNA of the A. thaliana gene encoding CHS (Accession No. NM_121396).

[0260] SEQ ID NO: 118—Nucleotide sequence of chimeric DNA encoding a ledRNA construct targeting the A. thaliana CHS gene.

[0261] SEQ ID NO: 119: Nucleotide sequence corresponding to the protein coding region of the cDNA encoding the L. angustifolius N-like gene (Accession No. XM_019604347).

[0262] SEQ ID NO: 120—Nucleotide sequence of chimeric DNA encoding a ledRNA construct targeting a L. angustifolius N-like gene.

[0263] SEQ ID NO: 121—Nucleotide sequence corresponding to the protein coding region of the cDNA of the Vitis pseudoreticulata MLO gene (Accession No. KR362912).

[0264] SEQ ID NO: 122—Nucleotide sequence of a chimeric DNA encoding a first ledRNA construct targeting the Vitis sphenanthenica MLO gene.

[0265] SEQ ID NO: 123—Nucleotide sequence corresponding to the protein coding region of the cDNA of the Myzus persicae MpC002 gene.

[0266] SEQ ID NO: 124—Nucleotide sequence corresponding to the protein coding region of the cDNA of the MpRack-1 gene of Myzus persicae.

[0267] SEQ ID NO: 125—Nucleotide sequence encoding a chimeric construct of ledRNA targeting the M. persicae C002 gene.

[0268] SEQ ID NO: 126—Nucleotide sequence of a chimeric construct encoding a ledRNA targeting the M. persicae Rack-1 gene.

[0269] SEQ ID NO: 127—Nucleotide sequence corresponding to the cDNA of the Helicoverpa armigera ABCwhite gene.

[0270] SEQ ID NO: 128—Nucleotide sequence of chimeric DNA encoding a ledRNA construct targeting the ABC transporter gene of Helicoverpa armigera.

[0271] SEQ ID NO: 129—Nucleotide sequence corresponding to the cDNA of the Argentine ant (Linepithema humile) PBAN-type neuropeptide-like (Accession No. XM_012368710).

[0272] SEQ ID NO: 130: Nucleotide sequence of chimeric DNA encoding a ledRNA construct targeting the PBAN gene in Argentine ants (Accession No. XM_012368710).

[0273] SEQ ID NO: 131: Nucleotide sequence of a chimeric DNA encoding a ledRNA construct targeting the gene encoding the catalytic subunit A of the V-type proton ATPase of L. cuprina (Accession No. XM_023443547).

[0274] SEQ ID NO: 132—Nucleotide sequence of a chimeric DNA encoding a ledRNA construct targeting the gene encoding RNAse 1 / 2 of L. cuprina.

[0275] SEQ ID NO: 133—Nucleotide sequence of a chimeric DNA encoding a ledRNA construct targeting the gene encoding the chitin synthase of L. cuprina.

[0276] SEQ ID NO: 134—Nucleotide sequence of a chimeric DNA encoding a ledRNA construct targeting the gene encoding the ecdysone receptor (EcR) of L. cuprina.

[0277] SEQ ID NO: 135—Nucleotide sequence of a chimeric DNA encoding a ledRNA construct targeting the gene encoding gamma-tubulin 1 / 1 of L. cuprina.

[0278] SEQ ID NO: 136—TaMlo target gene (AF384144).

[0279] SEQ ID NO: 137—Nucleotide sequence of a chimeric DNA encoding a ledRNA construct targeting a gene encoding TaMlo.

[0280] SEQ ID NO: 138—Nucleotide sequence corresponding to the protein coding region of the cDNA of the Vitis pseudoreticulata MLO gene (Accession No. KR362912).

[0281] SEQ ID NO: 139—Nucleotide sequence of a chimeric DNA encoding a first ledRNA construct targeting the Vitis sphenanthera MLO gene. DETAILED DESCRIPTION

[0282] General techniques and definitions

[0283] Unless specifically defined otherwise, all technical and scientific terms used herein should be considered to have the same meanings as commonly understood by one of ordinary skill in the art (eg, cell culture, molecular genetics, gene silencing, protein chemistry, and biochemistry).

[0284] Unless otherwise indicated, the recombinant protein, cell culture, and immunological techniques utilized in the present invention are standard methods well known to those skilled in the art. This technique is described throughout the literature in sources such as: J. Perbal, APractical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989), TA Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press(1991),DMGlover and BDHames(editors),DNACloning:A Practical Approach,Volumes 1-4,IRL Press(1995and 1996),andF.M.Ausubel et al.(editors),Current Protocols in Molecular Biology,GreenePub.Associates and Wiley-Interscience(1988,including all updates untilpresent),Ed Harlow and David Lane(editors)Antibodies:A Laboratory Manual,ColdSpring Harbor Laboratory, (1988), and JEColigan et al. (editors) CurrentProtocols in Immunology, John Wiley&Sons (including all updates until present).

[0285] The term "antisense regulatory element" or "antisense ribonucleic acid sequence" or "antisense RNA sequence" as used herein refers to an RNA sequence that is at least partially complementary to at least a portion of a target RNA molecule to which it hybridizes. In certain embodiments, the antisense RNA sequence regulates (increases or decreases) the expression or amount of the target RNA molecule or its activity, for example by reducing the translation of the target RNA molecule. In certain embodiments, the antisense RNA sequence alters the splicing of the target precursor mRNA, resulting in different splicing variants. Exemplary components of antisense sequences include, but are not limited to, oligonucleotides, oligonucleosides, oligonucleotide analogs, oligonucleotide mimetics, and chimeric combinations of these.

[0286] The term "antisense activity" is used in the context of the present invention to refer to any detectable and / or measurable activity attributed to the hybridization of an antisense RNA sequence with its target RNA molecule. This detection and / or measurement can be direct or indirect. In one embodiment, antisense activity is assessed by detecting and / or measuring the amount of a target RNA molecule transcript. Antisense activity can also be detected as a phenotypic change associated with a target RNA molecule. As used herein, the term "target RNA molecule" refers to a gene transcript regulated by an antisense RNA sequence according to the present invention. Therefore, a "target RNA molecule" can be any RNA molecule whose expression or activity can be regulated by an antisense RNA sequence. Exemplary target RNA molecules include, but are not limited to, RNA (including but not limited to pre-mRNA and mRNA or parts thereof), rRNA, tRNA, small nuclear RNA and miRNA transcribed from a DNA encoding a target protein, including their precursor forms. The target RNA can be a genomic RNA of a pathogen or pest such as a virus, or an RNA molecule derived therefrom, such as a replication form of a viral pathogen, or its transcript. For example, the target RNA molecule can be an RNA from an endogenous gene (or from an mRNA transcribed from the gene), or is introduced or can be introduced into a gene of a eukaryotic cell whose expression is related to a specific phenotype, a trait, a disease or a disease state, or a nucleic acid molecule from an infectious agent. In one embodiment, the target RNA molecule is in a eukaryotic cell. In another embodiment, the target RNA molecule encodes a protein. In this article, antisense activity can be assessed by detecting and / or measuring the amount of the target protein, for example, by its activity such as enzymatic activity, or by a function different from that of an enzyme, or by a phenotype related to its function. As used herein, the term "target protein" refers to a protein regulated by an antisense RNA sequence according to the present invention.

[0287] In certain embodiments, antisense activity is assessed by detecting and / or measuring the amount of target RNA molecules and / or cleaved target RNA molecules and / or alternatively spliced ​​target RNA molecules.

[0288] Antisense activity can be detected or measured using a variety of methods. For example, antisense activity can be detected or assessed by comparing the activity in a particular sample and comparing that activity to the activity of a control sample.

[0289] The term "targeting" is used in the context of the present invention to refer to the association of an antisense RNA sequence with a specific target RNA molecule or a specific nucleotide region within a target RNA molecule. In one embodiment, the antisense RNA sequence according to the present invention shares complementarity with at least one region of a target RNA molecule. In this article, the term "complementarity" refers to a ribonucleotide sequence that can be base-paired with a ribonucleotide sequence on a target RNA molecule through hydrogen bonds between bases on a ribonucleotide. For example, in RNA, adenine (A) is complementary to uracil (U), and guanine (G) is complementary to cytosine (C).

[0290] In certain embodiments, "complementary bases" refer to ribonucleotides of antisense RNA sequences that can be paired with ribonucleotide bases of sense RNA sequences in RNA molecules of the present invention or their target RNA molecules. For example, if a ribonucleotide at a certain position of the antisense RNA sequence can hydrogen bond with a ribonucleotide at a certain position of the target RNA molecule, the position of hydrogen bonding between the antisense RNA sequence and the target RNA molecule is considered to be complementary on the ribonucleotide. In contrast, the term "non-complementary" refers to a pair of ribonucleotides that do not form hydrogen bonds with each other or otherwise support hybridization. The term "complementary" can also be used to refer to the ability of an antisense RNA sequence to hybridize with another nucleic acid through complementarity. In certain embodiments, when a sufficient number of corresponding positions in each molecule are occupied by ribonucleotides, the RNA sequence and its target are complementary to each other, and the ribonucleotides can bind to each other to allow the stable association between the antisense RNA sequence and the sense RNA sequence in the RNA molecule of the present invention and / or the target RNA molecule. Those skilled in the art recognize that mismatches can be included without eliminating the ability of the antisense RNA sequence and the target to remain associated. Thus, antisense RNA sequences are described herein that may contain up to about 20% mismatched nucleotides (i.e., not complementary to the corresponding nucleotides of the target sequence). Preferably, the antisense compound contains no more than about 15%, more preferably no more than about 10%, most preferably no more than 5% or no mismatches. The remaining ribonucleotides are complementary or do not disrupt the hybridization between the antisense RNA sequence and the sense RNA sequence or the target RNA molecule (e.g., G:U or A:G pairs). One of ordinary skill in the art will recognize that the antisense RNA sequences described herein are at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% (completely) complementary to at least one region of the target RNA molecule.

[0291] As used herein, "chimeric RNA molecule" refers to any RNA molecule that does not exist in nature. In one embodiment, the chimeric RNA molecule disclosed herein has been modified to produce a mismatch in a region of the dsRNA. For example, the chimeric RNA molecule can be modified to convert cytosine to uracil. In one embodiment, the chimeric RNA molecule has been modified by treatment with bisulfite for a time and under conditions sufficient to convert unmethylated cytosine to uracil.

[0292] Those skilled in the art will appreciate that various ribonucleotide combinations can base pair. The present invention contemplates both canonical and non-canonical base pairs. In one embodiment, base pairing can include A:T or G:C in a DNA molecule or U:A or G:C in an RNA molecule. In another embodiment, base pairing can include A:G or G:T or U:G.

[0293] The term "canonical base pairing" as used in the present invention means base pairing between two nucleotides, which is A:T or G:C for deoxyribonucleotides, or A:U or G:C for ribonucleotides.

[0294] The term "non-canonical base pairing" as used in the present invention means the interaction between the bases of two nucleotides except the canonical base pairing in the context of two DNA or two RNA sequences. For example, non-canonical base pairing includes pairing between G and U (G:U) or between A and G (A:G). Embodiments of non-canonical base pairing include purine-purine or pyrimidine-pyrimidine. In the context of the present invention, the most common non-canonical base pairing is G:U. Other embodiments of non-canonical base pairs are less preferably A:C, G:T, G:G and A:A.

[0295] The present invention relates to RNA components that "hybridize" across a series of ribonucleotides. Those skilled in the art will understand that terms such as "hybridization" and "hybridization" are used to describe molecules based on the annealing of complementary nucleic acid sequences. Such molecules do not need to be 100% complementary in order to hybridize (i.e., they do not need "perfect base pairs". For example, there may be one or more mismatches in sequence complementarity. In one embodiment, the RNA components defined herein hybridize under stringent hybridization conditions. The term "stringent hybridization conditions" refers to parameters familiar to the art, including the variation of hybridization temperature with the length of the RNA molecule. Ribonucleotide hybridization parameters can be found in the references compiled for these methods, Sambrook et al. (see above), and Ausubel et al. (see above). For example, stringent hybridization conditions used herein can refer to hybridization buffer (3.5×SSC, 0.02% polysucrose, 0.02% polyvinyl pyrrolidone, 0.02% bovine serum albumin (BSA), 2.5mM NaH2PO4 (pH7), 0.5% SDS, 2mM The hybridization conditions are described in detail below. The hybridization conditions are described in detail below. The hybridization conditions are described in detail below. The hybridization conditions are described in detail below. The hybridization conditions are described in detail below. The hybridization conditions are described in detail below. The hybridization conditions are described in detail below. The hybridization conditions are described in detail below.

[0296] The present invention also encompasses RNA components that span "perfect base pairs" of adjacent ribonucleotides. The term "perfect base pairs" is used in the context of the present invention to refer to a series of adjacent ribonucleotide base pairs. A complete base pairing series of adjacent ribonucleotides does not contain gaps or non-base pairing nucleotides within the series. The term "adjacent" is used to refer to a series of ribonucleotides. A ribonucleotide comprising an adjacent series will be connected by a continuous series of phosphodiester bonds, with each ribonucleotide directly bonded to the next.

[0297] The RNA molecules of the present invention comprise sense sequences and corresponding antisense sequences. The relationship between these sequences is defined herein. The sequence relationship and activity of the antisense sequences and target RNA molecules are also defined herein.

[0298] The term "covalently linked" is used in the context of the present invention to refer to the connection between the first and second RNA components or any RNA sequence or ribonucleotides. As will be appreciated by those skilled in the art, a covalent link or bond is a chemical bond involving shared electron pairs between atoms. In one embodiment, the first and second RNA components or the sense RNA sequence and the antisense RNA sequence are covalently linked as part of a single RNA strand that can be folded back on itself by self-complementarity. In this embodiment, the components are covalently linked across one or more ribonucleotides via phosphodiester bonds.

[0299] In the context of the present invention, the term "hybridization" means pairing of complementary polynucleotides by base pairing of complementary bases. Although not limited to a specific mechanism, the most common pairing mechanism involves hydrogen bonding between complementary ribonucleotides, which may be Watson-Crick hydrogen bonding.

[0300] As used herein, the phrase "an RNA molecule has a deleterious effect on a non-human organism" or similar phrases means that a target RNA molecule of the molecule is present in a non-human organism, and exposing a cell expressing the target RNA molecule to the target RNA molecule results in a decrease in the level and / or activity of the target RNA molecule when compared to the same cell lacking the RNA molecule. In one embodiment, the target RNA molecule encodes a protein that is important for growth, reproduction, or survival. As an example, if the non-human organism is a crop pest or pathogen, or an animal pest or pathogen, the RNA molecule may have a deleterious effect on the pest's or pathogen's feeding, apoptosis, cell differentiation and development, sexual reproduction ability or desire, muscle formation, muscle twitching, muscle contraction, juvenile hormone formation, juvenile hormone regulation, ion regulation and transport, maintenance of cell membrane potential, amino acid biosynthesis, amino acid degradation, sperm formation, pheromone synthesis, pheromone sensing, antenna formation, wing formation, leg formation, egg formation, larval maturation, digestive enzyme formation, hemolymph synthesis, hemolymph maintenance, neurotransmission, larval stage transition, pupation, emergence from pupation, cell division, energy metabolism, respiration, chitin metabolism, formation of cytoskeletal structure. In another example, the non-human organism is a weed, and the RNA molecule has a deleterious effect on amino acid biosynthesis, photosynthesis, fatty acid synthesis, cell membrane integrity, pigment synthesis, or growth.

[0301] As used herein, the phrase "the RNA molecule has a beneficial effect on at least one symptom of a disease" or similar phrases means that the target RNA of the molecule is present in a subject, and exposure of cells expressing the target RNA to the RNA molecule results in a decrease in the level and / or activity of the target RNA when compared to the same cells lacking the RNA molecule. In one embodiment, the target RNA encodes a protein that exerts its effect in the presence of a disease. In one embodiment, the disease is cancer or a cancerous disease, an infectious disease, a cardiovascular disease, a neurological disease, a prion disease, an inflammatory disease, an autoimmune disease, a lung disease, a kidney disease, a liver disease, a mitochondrial disease, an endocrine disease, a reproductive-related disease and disorder, and any other indication that may be responsive to the level of a gene product expressed in a cell or organism.

[0302] The RNA molecules according to the present invention and compositions comprising the RNA molecules can be applied to a subject. Terms such as "subject", "patient" or "individual" are terms that can be used interchangeably in the present invention in context. In one embodiment, the subject is a mammal. The mammal can be a pet, such as a dog or a cat, or a domestic animal, such as a horse or a cow. In one embodiment, the subject is a human. For example, the subject can be an adult. In another embodiment, the subject can be a child. In another embodiment, the subject can be a teenager. In another embodiment, the RNA molecules according to the present invention and compositions comprising them can be applied to insects. In another embodiment, the RNA molecules according to the present invention and compositions comprising the RNA molecules can be applied to plants. In another embodiment, the RNA molecules according to the present invention and compositions comprising them can be applied to fungal cells or colonies.

[0303] The term "and / or", such as "X and / or Y", should be understood to mean "X and Y" or "X or Y", and should be understood to provide clear support for both meanings or both meanings.

[0304] As used herein, unless otherwise indicated to the contrary, the term "about" means + / - 20%, more preferably + / - 10% of the specified value.

[0305] Throughout the specification, the word "comprise" or variations such as "comprises" or "comprising" will be understood to imply the inclusion of stated elements, integers or steps, or groups of elements, integers or steps, but not the exclusion of any other elements, integers or steps, or groups of elements, integers or steps.

[0306] RNA molecules

[0307] In certain embodiments, the RNA molecules of the invention comprise a first RNA component covalently linked to a second RNA component. In preferred embodiments, the RNA molecules self-hybridize or fold to form a "dumbbell" or ledRNA structure, e.g., see Figure 1 In one embodiment, the molecule further comprises one or more of the following:

[0308] - a linking ribonucleotide sequence which covalently links the first and second RNA components;

[0309] -5' leader sequence; and,

[0310] -3' trailer sequence.

[0311] In one embodiment, the first RNA component consists of a first 5' ribonucleotide, a first RNA sequence, and a first 3' ribonucleotide in 5' to 3' order, wherein the first 5' and 3' ribonucleotides in the RNA molecule are base paired with each other, wherein the first RNA sequence comprises a first sense ribonucleotide sequence of at least 20 contiguous ribonucleotides, a first loop sequence of at least 4 ribonucleotides, and a first antisense ribonucleotide sequence of at least 20 contiguous ribonucleotides, wherein the first antisense ribonucleotide sequence hybridizes with the first sense ribonucleotide sequence in the RNA molecule, and wherein the first antisense ribonucleotide sequence is capable of hybridizing to a first region of a target RNA molecule.

[0312] In another embodiment, the first RNA component consists of a first 5' ribonucleotide, a first RNA sequence, and a first 3' ribonucleotide in a 5' to 3' order, wherein the first 5' and 3' ribonucleotides in the RNA molecule are base paired with each other, wherein the first RNA sequence comprises a first sense ribonucleotide sequence of at least 20 adjacent ribonucleotides, a first loop sequence of at least 4 ribonucleotides, and a first antisense ribonucleotide sequence of at least 20 adjacent ribonucleotides, wherein the first antisense ribonucleotide sequence is completely base paired with the first sense ribonucleotide sequence in the RNA molecule, wherein the first antisense ribonucleotide sequence is identical to the sequence of the complement of the first region of the target RNA molecule. Examples of the first RNA component of these two embodiments are schematically shown in Figure 1 The left half of A or Figure 1 In the right half of B.

[0313] In another embodiment, the first RNA component consists of a first 5' ribonucleotide, a first RNA sequence, and a first 3' ribonucleotide, wherein the first 5' and 3' ribonucleotides in the first RNA component are base paired with each other, wherein the first RNA sequence comprises a first sense ribonucleotide sequence, a first loop sequence of at least 4 ribonucleotides, and a first antisense ribonucleotide sequence, wherein the first sense ribonucleotide sequence and the first antisense ribonucleotide sequence each have at least 20 consecutive ribonucleotides, wherein the at least 20 consecutive ribonucleotides of the first sense ribonucleotide sequence are completely base paired with the at least 20 consecutive ribonucleotides of the first antisense ribonucleotide sequence, and wherein the at least 20 consecutive ribonucleotides of the first sense ribonucleotide sequence are substantially identical in sequence to the first region of the target RNA molecule.

[0314] In these embodiments, the base pair formed between the first 5' ribonucleotide and the first 3' ribonucleotide is considered to be the terminal base pair of the dsRNA region formed by self-hybridization of the first RNA component, ie, it defines the terminus of the dsRNA region.

[0315] In one embodiment, the first sense sequence has substantial sequence identity with a region of the target RNA, which identity may be a sequence of less than 20 nucleotides in length. In one embodiment, at least 15, at least 16, at least 17, at least 18, or at least 19 contiguous ribonucleotides, preferably at least 20 contiguous ribonucleotides of the first sense ribonucleotide sequence are at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or 99% identical in sequence to the first region of the target RNA molecule. In another embodiment, at least 15, at least 16, at least 17, at least 18, at least 19 contiguous ribonucleotides of the first sense ribonucleotide sequence are 100% identical to the first region of the target RNA molecule. In one embodiment, the first 3, first 4, first 5, first 6 or first 7 ribonucleotides from the 5' end of the first sense ribonucleotide sequence are 100% identical to a region of the target RNA molecule, and the remaining ribonucleotides are at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the target RNA molecule.

[0316] In one embodiment, at least 20 contiguous ribonucleotides of the first sense ribonucleotide sequence are at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the first region of the target RNA molecule. Similarly, in this embodiment, the first 3, first 4, first 5, first 6, or first 7 ribonucleotides may be 100% identical to the region of the target RNA molecule, and the remaining ribonucleotides are at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the target RNA molecule. In another embodiment, at least 20 contiguous ribonucleotides of the first sense ribonucleotide sequence are 100% identical to the first region of the target RNA molecule.

[0317] In one embodiment, the first antisense sequence has substantial sequence identity with the complement of the region of the target RNA, which identity may be an identity with a sequence of less than 20 nucleotides in length of the complement. In one embodiment, at least 15, at least 16, at least 17, at least 18 or at least 19 adjacent ribonucleotides, preferably at least 20 adjacent ribonucleotides of the first antisense ribonucleotide sequence are at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or 99% identical in sequence to the complement of the first region of the target RNA molecule. In another embodiment, at least 15, at least 16, at least 17, at least 18, at least 19 adjacent ribonucleotides of the first antisense ribonucleotide sequence are 100% identical to the complement of the first region of the target RNA molecule. In one embodiment, the first 3, first 4, first 5, first 6 or first 7 ribonucleotides from the 5' end of the first antisense ribonucleotide sequence are 100% identical to the complementary sequence of the target RNA molecule region, and the remaining ribonucleotides are at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the complement of the target RNA molecule.

[0318] In one embodiment, at least 20 adjacent ribonucleotides of the first antisense ribonucleotide sequence are at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the complement of the first region of the target RNA molecule. Similarly, in this embodiment, the first 3, first 4, first 5, first 6 or first 7 ribonucleotides are 100% identical to the complement of the target RNA molecule region, and the remaining ribonucleotides are at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the complement of the target RNA molecule. In another embodiment, at least 20 adjacent ribonucleotides of the first antisense ribonucleotide sequence are 100% identical to the first region of the target RNA molecule.

[0319] In another embodiment, the second RNA component consists of a second 5' ribonucleotide, a second RNA sequence, and a second 3' ribonucleotide in a 5' to 3' order, wherein the second 5' and 3' ribonucleotides are base paired, wherein the second RNA sequence comprises a second sense ribonucleotide sequence, a second loop sequence of at least 4 ribonucleotides, and a second antisense ribonucleotide sequence, wherein the second sense ribonucleotide sequence is base paired with the second antisense ribonucleotide sequence. In this embodiment, the base pair formed between the second 5' ribonucleotide and the second 3' ribonucleotide is considered to be the terminal base pair of the dsRNA region formed by self-hybridization of the second RNA component.

[0320] In one embodiment, the RNA molecule comprises a 5' leader sequence or a 5' extension sequence, which can be produced as a result of transcription from the promoter in the genetic construct, from the start site of transcription to the polynucleotide of the remainder of the coding RNA molecule. Preferably, the 5' leader sequence or the 5' extension sequence is relatively short compared to the remainder of the molecule, and can be removed from the RNA molecule after transcription, for example, by RNA enzyme treatment. The 5' leader sequence or the 5' extension sequence can be mostly non-base paired, or can contain one or more stem-loop structures. In this embodiment, the 5' leader sequence can be composed of a ribonucleotide sequence, and if the second RNA component is connected to the first 3' ribonucleotide, the ribonucleotide sequence is covalently linked to the first 5' ribonucleotide, or if the second RNA component is connected to the first 5' ribonucleotide, the ribonucleotide sequence is covalently linked to the second 5' ribonucleotide. In one embodiment, the 5' leader sequence is at least 10, at least 20, at least 30, at least 100, at least 200 ribonucleotides long, preferably to a maximum length of 250 ribonucleotides. In another embodiment, the 5' leader sequence is at least 50 ribonucleotides long. In one embodiment, the 5' leader sequence can be used as an extension sequence for amplifying RNA molecules by a suitable amplification reaction. For embodiments, the extension sequence can promote amplification by a polymerase.

[0321] In another embodiment, the RNA molecule comprises a 3' tail sequence or a 3' extension sequence, which may be produced due to continuous transcription until the transcription termination or polyadenylation signal in the construct encoding the RNA molecule. The 3' tail sequence or the 3' extension sequence may comprise a poly A (polyA) tail. Preferably, the 3' tail sequence or the 3' extension sequence is relatively short compared to the remainder of the molecule, and can be removed from the RNA molecule after transcription, for example, by RNA enzyme treatment. The 3' tail sequence or the 3' extension sequence can be mostly non-base paired, or can contain one or more stem-loop structures. In this embodiment, the 3' tail sequence can be composed of a ribonucleotide sequence, and if the second RNA component is connected to the first 3' ribonucleotide, the ribonucleotide sequence is covalently linked to the second 3' ribonucleotide, or if the second RNA component is connected to the first 5' ribonucleotide, the ribonucleotide sequence is covalently linked to the first 3' ribonucleotide. In one embodiment, the 3' leader sequence is at least 10, at least 20, at least 30, at least 100, at least 200 ribonucleotides long, preferably to a maximum length of 250 ribonucleotides. In another embodiment, the 3' leader sequence is at least 50 ribonucleotides long. In one embodiment, the 3' trailing sequence can be used as an extension sequence for amplifying RNA molecules by a suitable amplification reaction. For embodiments, the extension sequence can promote amplification by a polymerase.

[0322] In one embodiment, all nucleotides except two ribonucleotides are covalently linked to two other nucleotides, i.e., the RNA molecule consists of only one RNA strand with a self-complementary region, and therefore has only one 5' terminal nucleotide and one 3' terminal nucleotide. In another embodiment, all nucleotides except four ribonucleotides are covalently linked to two other nucleotides, i.e., the RNA molecule consists of two RNA strands with hybridized complementary regions, and therefore has only two 5' terminal nucleotides and two 3' terminal nucleotides. In another embodiment, each ribonucleotide is covalently linked to two other nucleotides, i.e., the RNA molecule is circular and has a self-complementary region, and therefore has no 5' terminal nucleotide and 3' terminal nucleotide.

[0323] In one embodiment, the double-stranded region of RNA molecule can include one or more protuberances produced by unpaired nucleotides in sense RNA sequence or antisense RNA sequence or both. In one embodiment, RNA molecule includes a series of protuberances. For embodiment, the double-stranded region of RNA molecule can have 2,3,4,5,6,7,8,9,10 or more protuberances. Each protuberance can be one, two or more unpaired nucleotides independently, up to 10 nucleotides. Longer sequence can loop out sense or antisense sequence in dsRNA district, and it can be in internal base pairing or keep unpaired. In another embodiment, the double-stranded region of RNA molecule does not include protuberances, i.e., complete base pairing along the total length in dsRNA district.

[0324] In another embodiment, the first sense ribonucleotide sequence is covalently linked to the first 5' ribonucleotide without any intervening nucleotides, or the first antisense ribonucleotide sequence is covalently linked to the first 3' ribonucleotide without any intervening nucleotides, or both. In another embodiment, there are at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 intervening nucleotides. It should be understood that such intervening nucleotides are not related to the target RNA molecule in sequence, but can help stabilize the base pairing of adjacent sense and antisense sequences.

[0325] In another embodiment, 20 adjacent nucleotides of the first sense ribonucleotide sequence are covalently linked to the first 5' ribonucleotide without any intervening nucleotides, and 20 adjacent nucleotides of the first antisense ribonucleotide sequence are covalently linked to the first 3' ribonucleotide without any intervening nucleotides. In another embodiment, there are at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 intervening nucleotides. Intervening nucleotides can be used as part of the double-stranded region of the RNA molecule for base pairing, but are not related to the target RNA in sequence. They can help provide increased stability to the double-stranded region or bind the two ends of the RNA molecule together without leaving unpaired 5' or 3' ends, or both.

[0326] In one embodiment, the above-mentioned first and second RNA components include connecting ribonucleotide sequences. In one embodiment, the connecting ribonucleotide sequence is used as a spacer between the first sense ribonucleotide sequence and other components of the molecule, and the first sense ribonucleotide sequence is substantially identical to the first district of the target RNA molecule in sequence. For example, the connecting ribonucleotide sequence can be used as a spacer between the district and the ring. In another embodiment, the RNA molecule includes a plurality of sense ribonucleotide sequences and connecting ribonucleotide sequences, and the sense ribonucleotide sequence is substantially identical to the first district of the target RNA molecule in sequence, and the connecting ribonucleotide sequence serves as a spacer between these sequences. In one embodiment, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 ribonucleotide sequences that are substantially identical to the first district of the target RNA molecule in sequence are provided in the RNA molecule, and each ribonucleotide sequence is separated from each other by connecting ribonucleotide sequences.

[0327] In one embodiment, the RNA molecule comprises a 5' leader sequence. In one embodiment, the 5' leader sequence consists of a ribonucleotide sequence that is covalently linked to the first 5' ribonucleotide if the second RNA component is linked to the first 3' ribonucleotide, or covalently linked to the second 5' ribonucleotide if the second RNA component is linked to the first 5' ribonucleotide. In one embodiment, the RNA molecule has a modified 5' or 3' end, for example, by a linked lipid group such as cholesterol, or a vitamin such as biotin, or a polypeptide. Such modifications can help the RNA molecule to be taken into a eukaryotic cell where the RNA will work.

[0328] In one embodiment, the length of the connecting ribonucleotide sequence is less than 100 ribonucleotides. In one embodiment, the length of the connecting ribonucleotide sequence is less than 50 ribonucleotides. In one embodiment, the length of the connecting ribonucleotide sequence is less than 20 ribonucleotides. In one embodiment, the length of the connecting ribonucleotide sequence is less than 10 ribonucleotides. In one embodiment, the length of the connecting ribonucleotide sequence is less than 5 ribonucleotides. In one embodiment, the length of the connecting ribonucleotide sequence is 1 to 100 ribonucleotides. In one embodiment, the length of the connecting ribonucleotide sequence is 1 to 50 ribonucleotides. In one embodiment, the length of the connecting ribonucleotide sequence is 1 to 20 ribonucleotides. In one embodiment, the length of the connecting ribonucleotide sequence is 1 to 10 ribonucleotides. In one embodiment, the length of the connecting ribonucleotide sequence is 1 to 5 ribonucleotides. In one embodiment, the ribonucleotides connecting the ribonucleotide sequence are not base paired. In a preferred embodiment, the ribonucleotides connecting the ribonucleotide sequence are all base paired, or are all base paired except 1, 2 or 3 ribonucleotides.

[0329] In one embodiment, the first or second RNA component comprises a hairpin structure. In a preferred embodiment, the first and second RNA components each comprise a hairpin structure. In these embodiments, the hairpin structure can be a stem-loop. Thus, in one embodiment, the RNA molecule can comprise a first and a second RNA component each comprising a hairpin structure, wherein the hairpin is covalently attached via a linker sequence. For example, see Figure 1 In one embodiment, the linker sequence is one or more unpaired ribonucleic acids (RNA). In one embodiment, the linker sequence is 1 to 10 unpaired ribonucleotides.

[0330] In one embodiment, the RNA molecule has a double hairpin structure, i.e., a "ledRNA structure" or a "dumbbell structure". In this embodiment, the first hairpin is the first RNA component and the second hairpin is the second RNA component. In these embodiments, the first 3' ribonucleotide and the second 5' ribonucleotide, or the second 3' ribonucleotide and the first 5' ribonucleotide, but not both, are covalently linked. In this embodiment, the other 5' / 3' ribonucleotides can be separated by a cleft (i.e., a discontinuity in the dsRNA molecule in which there is no phosphodiester bond between the 5' / 3' ribonucleotides). Figure 1B shows an example of this type of arrangement. In another embodiment, the corresponding 5' / 3' ribonucleotides can be separated by a loop. The length of the 5' leader sequence and the 3' trailing sequence can be the same or different. For embodiments, the 5' leader can be about 5, 10, 15, 20, 25, 50, 100, 200, 500 ribonucleotides longer than the 3' trailing sequence, and vice versa.

[0331] In the embodiment of the RNA molecule having a double hairpin structure, the second hairpin (in addition to the first hairpin structure) comprises a sense RNA sequence and an antisense RNA sequence, and its sequence is substantially the same as the region of the target RNA molecule or its complement, respectively. In one embodiment, each hairpin has a series of ribonucleotides that are substantially the same in sequence as the region of the same target RNA molecule. In one embodiment, each hairpin has a series of ribonucleotides that are substantially the same in sequence as different regions of the same target RNA molecule. In one embodiment, each hairpin has a series of ribonucleotides that are substantially the same in sequence as the regions of different target RNA molecules, that is, the RNA molecule can be used to reduce the expression and / or activity of two target RNA molecules that are not related in sequence.

[0332] In each hairpin structure of the double hairpin structure of the RNA molecule, the order of the sense and antisense RNA sequences in each hairpin structure can be independently sense then antisense, or antisense then sense in the 5' to 3' order. In a preferred embodiment, the order of the sense and antisense sequences in the double hairpin structure of the RNA molecule is antisense-sense-sense-antisense ( Figure 1 A), or sense-antisense-antisense-sense ( Figure 1 B).

[0333] In one embodiment, in 5' to 3' order, the RNA molecule can include a 5' leader sequence, a first loop, a sense RNA sequence, a second loop and a 3' tail sequence, wherein the 5' and 3' leader sequences are covalently bound to the sense strand to form a dsRNA sequence. In one embodiment, the 5' leader sequence and the 3' tail sequence are not covalently bound to each other. In one embodiment, the 5' leader sequence and the 3' tail sequence are separated by a rift. In one embodiment, the 5' leader sequence and the 3' tail sequence are connected together to provide an RNA molecule with a closed structure. In another embodiment, the 5' leader sequence and the 3' tail sequence are separated by a loop.

[0334] The term "loop" used in the context of the present invention refers to a ring structure formed by a series of non-complementary ribonucleotides in the RNA molecule disclosed herein. The loop usually follows a series of base pairs between the first and second RNA components or connects the sense RNA sequence and the antisense RNA sequence in one or both of the first and second RNA components. In one embodiment, usually for the shorter loop of 4-10 ribonucleotides, all ring ribonucleotides are non-complementary. In other embodiments, some ribonucleotides in one or more loops are complementary and can base pair in the loop sequence, as long as these base pairs can form a ring structure. For example, at least 5%, at least 10% or at least 15% of the ring ribonucleotides are complementary. The embodiment of the ring includes stem loop or hairpin, pseudoknot and four rings.

[0335] In one embodiment, the RNA molecule comprises only two loops. In another embodiment, the RNA molecule comprises at least two, at least three, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10 loops, preferably at most 10 loops. For example, the RNA molecule may comprise 4 loops.

[0336] The present invention contemplates loops of various sizes. For example, the loop may contain 4, 5, 6, 7, 8, 9, 10, 11 or 12 ribonucleotides. In other embodiments, the loop contains 15, 20, 25 or 30 nucleotides. In one embodiment, one or all of the loop sequences are longer than 20 nucleotides. In other embodiments, the loop is larger, for example, containing 50, 100, 150, 200 or 300 ribonucleotides. In one embodiment, the loop contains 160 ribonucleotides. In another less preferred embodiment, the loop contains 200, 500, 700 or 1,000 ribonucleotides, provided that the loop does not interfere with the hybridization of sense and antisense RNA sequences. In one embodiment, each loop has the same number of ribonucleotides. For example, the loop may have a length of 100 to 1,000 ribonucleotides. For example, the loop may have a length of 600 to 1,000 ribonucleotides. For example, the loop may have 4 to 1,000 ribonucleotides. For example, the loop preferably has 4-50 ribonucleotides. In another embodiment, the loop comprises a different number of ribonucleotides.

[0337] In another embodiment, one or more loops comprise introns that can be spliced ​​out of the RNA molecule. In one embodiment, the introns are from plant genes. Exemplary introns include intron 3 of corn alcohol dehydrogenase 1 (Adhl) (GenBank: AF044293), intron 4 of soybean β-conglycinin alpha subunit (GenBank: AB051865); one of the introns of the pea rbcS-3A gene (GenBank: X04333) of the ribulose-1,5-bisphosphate carboxylase (RBC) small subunit. Other embodiments of suitable introns are discussed in (McCullough and Schuler, 1997; Smith et al., 2000).

[0338] In various embodiments, the loop may be at the end of at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 contiguous base pairs, which may be canonical base pairs or may include one or more non-canonical base pairs. In other embodiments, particularly for vertebrate cells, less preferably, the loop may be at the end of at least 20, 30, 50, 100, 200, 500 or more contiguous base pairs.

[0339] In another embodiment, the RNA molecule comprises two or more sense ribonucleotide sequences and antisense ribonucleotide sequences based completely on their base pairing, each of which is identical in sequence to the region of the target RNA molecule. For example, the RNA molecule may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more sense ribonucleotide sequences and antisense ribonucleotide sequences that are completely paired therewith, and the sense ribonucleotide sequences are each independently identical in sequence to the region of the target RNA molecule. In this embodiment, any one or more or all of the sequences may be separated by connecting ribonucleotide sequences. In this embodiment, any one or more or all of the sequences may be separated by loops.

[0340] In one embodiment, two or more sense ribonucleotide sequences are identical in sequence to different regions of the same target RNA molecule. For example, the sequence may be identical to at least 2, at least 3, at least 4, at least 5, or at least 6 regions of the same target molecule. In another embodiment, two or more sense ribonucleotide sequences are identical in sequence. In one embodiment, two or more sense ribonucleotide sequences are identical in sequence to the same region of the same target RNA molecule. In another embodiment, two or more sense ribonucleotide sequences are identical in sequence to different target RNA molecules. For embodiments, the sequence may be identical to at least 2, at least 3, at least 4, at least 5, or at least 6 regions of different target molecules.

[0341] In another embodiment, the two or more sense ribonucleotide sequences have no intervening loop (spacer) sequence.

[0342] In one embodiment, the RNA molecule may comprise a single-stranded ribonucleotide having a 5' end, at least one sense ribonucleotide sequence of at least 21 nucleotides in length, an antisense ribonucleotide sequence that is fully base paired with each sense ribonucleotide sequence over at least 21 adjacent nucleotides, at least two loop sequences, and a 3' end. In this embodiment, the ribonucleotide at the 5' end and the ribonucleotide at the 3' end are not directly covalently bound, but are adjacent to each base-paired position.

[0343] In another embodiment, the adjacent base pairs of the RNA component are spaced at least one gap. In one embodiment, the "gap" is provided by unpaired ribonucleotides. In another embodiment, the "gap" is provided by unconnected 5' leader sequences and / or 3' trailing sequences. In this embodiment, the gap can be referred to as an "unconnected gap". Mismatches and unconnected gaps can be located at different positions of the RNA molecule. For embodiments, unconnected gaps can be immediately after the antisense sequence. In another embodiment, unconnected gaps can be close to the loops of the RNA molecule. In another embodiment, unconnected gaps are positioned to be approximately equidistant between at least two loops.

[0344] In one embodiment, the RNA molecule is produced by single-stranded RNA. In one embodiment, the single strand is not circularly closed, for example, comprises an unconnected gap. In another embodiment, the RNA molecule is a circular closed molecule. Closed molecules can be produced by, for example, connecting the above-mentioned RNA molecules comprising an unconnected gap with RNA ligase.

[0345] In another embodiment, the RNA molecule comprises a 5'- or 3'-extension sequence or both. For example, the RNA molecule may comprise a 5' extension sequence covalently linked to a first 5' ribonucleotide. In another embodiment, the RNA molecule comprises a 3' extension sequence covalently linked to a second 3' ribonucleotide. In another embodiment, the RNA molecule comprises a 5' extension sequence covalently linked to a first 5' ribonucleotide and a 3' extension sequence covalently linked to a second 3' ribonucleotide.

[0346] In another embodiment, the RNA molecule comprises a 5' extension sequence covalently linked to a second 5' ribonucleotide. In another embodiment, the RNA molecule comprises a 3' extension sequence covalently linked to a first 3' ribonucleotide. In another embodiment, the RNA molecule comprises a 5' extension sequence covalently linked to a second 5' ribonucleotide and a 3' extension sequence covalently linked to a first 3' ribonucleotide.

[0347] In another embodiment, the RNA molecule may comprise one or more of the following:

[0348] - a 5' extension sequence covalently linked to the first 5' ribonucleotide;

[0349] - a 3' extension sequence covalently linked to a second 3' ribonucleotide;

[0350] - a 5' extension sequence covalently linked to the first 5' ribonucleotide and a 3' extension sequence covalently linked to the second 3' ribonucleotide;

[0351] - a 5' extension sequence covalently linked to a second 5' ribonucleotide;

[0352] - a 3' extension sequence covalently linked to the first 3' ribonucleotide;

[0353] - a 5' extension sequence covalently linked to the second 5' ribonucleotide and a 3' extension sequence covalently linked to the first 3' ribonucleotide.

[0354] Non-canonical base pairing

[0355] In one embodiment, the RNA molecule of the invention comprises a sense ribonucleotide sequence and an antisense ribonucleotide sequence that are capable of hybridizing to each other to form a double-stranded (ds) RNA region with some non-canonical base pairing, i.e., a combination of canonical and non-canonical base pairing. In one embodiment, the RNA molecule of the invention comprises two or more sense ribonucleotide sequences, each of which is capable of hybridizing to a region of a (adjacent) antisense ribonucleotide sequence to form a dsRNA region with some non-canonical base pairing. See, e.g. Figure 1 B. In one embodiment, the RNA molecule of the invention comprises two or more antisense sense ribonucleotide sequences, each of which is capable of hybridizing to a region of a (contiguous) sense ribonucleotide sequence to form a dsRNA region with some non-canonical base pairing. See, e.g. Figure 1 A. In one embodiment, the RNA molecule of the invention comprises two or more antisense sense ribonucleotide sequences and two or more sense ribonucleotide sequences, wherein each antisense ribonucleotide sequence is capable of hybridizing with an antisense ribonucleotide sequence to form two or more dsRNA regions, one or both dsRNA regions comprising some non-canonical base pairing.

[0356] In the following embodiments, if there is only one (adjacent) dsRNA district, the full length (i.e., the entire dsRNA district) of the dsRNA district of the RNA molecule of the present invention is considered to be the context of the feature, or if there are two or more dsRNA districts in the RNA molecule, it is considered to be the context of each dsRNA district of the RNA molecule. In one embodiment, at least 5% of the base pairs in the dsRNA district are non-canonical base pairs. In one embodiment, at least 6% of the base pairs in the dsRNA district are non-canonical base pairs. In one embodiment, at least 7% of the base pairs in the dsRNA district are non-canonical base pairs. In one embodiment, at least 8% of the base pairs in the dsRNA district are non-canonical base pairs. In one embodiment, at least 9% or 10% of the base pairs in the dsRNA district are non-canonical base pairs. In one embodiment, at least 11% or 12% of the base pairs in the dsRNA district are non-canonical base pairs. In one embodiment, at least 15% or about 15% of the base pairs in the dsRNA district are non-canonical base pairs. In one embodiment, at least 20% or about 20% of the base pairs in the dsRNA district are non-standard base pairs. In one embodiment, at least 25% or about 25% of the base pairs in the dsRNA district are non-standard base pairs. In one embodiment, at least 30% or about 30% of the base pairs in the dsRNA district are non-standard base pairs. In each of these embodiments, preferably at most 40% of the base pairs in the dsRNA district are non-standard base pairs, more preferably at most 35% of the base pairs in the dsRNA district are non-standard base pairs, and more preferably at most 30% of the base pairs in the dsRNA district are non-standard base pairs. In a less preferred embodiment, about 35% of the base pairs in the dsRNA district are non-standard base pairs. In one embodiment, even more less preferably, about 40% of the base pairs in the dsRNA district are non-standard base pairs. In each of the above embodiments, the dsRNA district may include or not include one or more non-base paired ribonucleotides in the sense sequence or antisense sequence or both.

[0357] In one embodiment, 10%-40% of the base pairs in the dsRNA region of the RNA molecule of the present invention are non-canonical base pairs. In one embodiment, 10%-35% of the base pairs in the dsRNA region are non-canonical base pairs. In one embodiment, 10%-30% of the base pairs in the dsRNA region are non-canonical base pairs. In one embodiment, 10%-25% of the base pairs in the dsRNA region are non-canonical base pairs. In one embodiment, 10%-20% of the base pairs in the dsRNA region are non-canonical base pairs. In one embodiment, 10%-15% of the base pairs in the dsRNA region are non-canonical base pairs. In one embodiment, 15%-30% of the base pairs in the dsRNA region are non-canonical base pairs. In one embodiment, 15%-25% of the base pairs in the dsRNA region are non-canonical base pairs. In one embodiment, 15%-20% of the base pairs in the dsRNA region are non-canonical base pairs. In one embodiment, 5%-30% of the base pairs in the dsRNA district are non-canonical base pairs. In one embodiment, 5%-25% of the base pairs in the dsRNA district are non-canonical base pairs. In one embodiment, 5%-20% of the base pairs in the dsRNA district are non-canonical base pairs. In one embodiment, 5%-15% of the base pairs in the dsRNA district are non-canonical base pairs. In one embodiment, 5%-10% of the base pairs in the dsRNA district are non-canonical base pairs. In each of the above-mentioned embodiments, the dsRNA district may include or may not include one or more non-base paired ribonucleotides in the sense sequence or antisense sequence or both.

[0358] In one embodiment, the dsRNA district of the RNA molecule of the present invention comprises 20 adjacent base pairs, wherein at least one base pair in the 20 adjacent base pairs is a non-canonical base pair. In one embodiment, the dsRNA district comprises 20 adjacent base pairs, wherein at least 2 base pairs in the 20 adjacent base pairs are non-canonical base pairs. In one embodiment, the dsRNA district comprises 20 adjacent base pairs, wherein at least 3 base pairs in the 20 adjacent base pairs are non-canonical base pairs. In one embodiment, the dsRNA district comprises 20 adjacent base pairs, wherein at least 4 base pairs in the 20 adjacent base pairs are non-canonical base pairs. In one embodiment, the dsRNA district comprises 20 adjacent base pairs, wherein at least 5 base pairs in the 20 adjacent base pairs are non-canonical base pairs. In one embodiment, the dsRNA district comprises 20 adjacent base pairs, wherein at least 6 base pairs in the 20 adjacent base pairs are non-canonical base pairs. In one embodiment, the dsRNA district comprises 20 adjacent base pairs, and wherein at least 7 base pairs in 20 adjacent base pairs are non-conventional base pairs. In one embodiment, the dsRNA district comprises 20 adjacent base pairs, and wherein at least 8 base pairs in 20 adjacent base pairs are non-conventional base pairs. In one embodiment, the dsRNA district comprises 20 adjacent base pairs, and wherein at least 9 base pairs in 20 adjacent base pairs are non-conventional base pairs. In each of these embodiments, preferably in the dsRNA district, 10 of the 20 adjacent base pairs are non-conventional base pairs, more preferably in the dsRNA district, 9 of the non-conventional base pairs, more preferably in the dsRNA district, 8 of the non-conventional base pairs, even more preferably in the dsRNA district, 7 of the non-conventional base pairs, and most preferably in the dsRNA district, 6 of the non-conventional base pairs. Preferably, in the above embodiments, the non-canonical base pairs comprise at least one G:U base pair, and more preferably all non-canonical base pairs are G:U base pairs. Preferably, the features of the above embodiments apply to each of the 20 contiguous base pairs present in the RNA molecule of the present invention.

[0359] In one embodiment, the dsRNA district of the RNA molecule of the present invention comprises 21 adjacent base pairs, wherein at least one base pair in the 21 adjacent base pairs is a non-canonical base pair. In one embodiment, the dsRNA district comprises 21 adjacent base pairs, wherein at least 2 base pairs in the 21 adjacent base pairs are non-canonical base pairs. In one embodiment, the dsRNA district comprises 21 adjacent base pairs, wherein at least 3 base pairs in the 21 adjacent base pairs are non-canonical base pairs. In one embodiment, the dsRNA district comprises 21 adjacent base pairs, wherein at least 4 base pairs in the 21 adjacent base pairs are non-canonical base pairs. In one embodiment, the dsRNA district comprises 21 adjacent base pairs, wherein at least 5 base pairs in the 21 adjacent base pairs are non-canonical base pairs. In one embodiment, the dsRNA district comprises 21 adjacent base pairs, wherein at least 6 base pairs in the 21 adjacent base pairs are non-canonical base pairs. In one embodiment, the dsRNA district comprises 21 adjacent base pairs, and wherein at least 7 base pairs in the 21 adjacent base pairs are non-conventional base pairs. In one embodiment, the dsRNA district comprises 21 adjacent base pairs, and wherein at least 8 base pairs in the 21 adjacent base pairs are non-conventional base pairs. In one embodiment, the dsRNA district comprises 21 adjacent base pairs, and wherein at least 9 base pairs in the 21 adjacent base pairs are non-conventional base pairs. In each of these embodiments, preferably in the dsRNA district, 10 of the 21 adjacent base pairs are non-conventional base pairs, more preferably in the dsRNA district, 9 of the non-conventional base pairs, more preferably in the dsRNA district, 8 of the non-conventional base pairs, even more preferably in the dsRNA district, 7 of the non-conventional base pairs, and most preferably in the dsRNA district, 6 of the non-conventional base pairs. Preferably, in the above embodiments, the non-canonical base pairs comprise at least one G:U base pair, and more preferably all non-canonical base pairs are G:U base pairs. Preferably, the features of the above embodiments apply to each of the 21 contiguous base pairs present in the RNA molecule of the present invention.

[0360] In one embodiment, the dsRNA district of the RNA molecule of the present invention comprises 22 adjacent base pairs, wherein at least one base pair in the 22 adjacent base pairs is a non-canonical base pair. In one embodiment, the dsRNA district comprises 22 adjacent base pairs, wherein at least 2 base pairs in the 22 adjacent base pairs are non-canonical base pairs. In one embodiment, the dsRNA district comprises 22 adjacent base pairs, wherein at least 3 base pairs in the 22 adjacent base pairs are non-canonical base pairs. In one embodiment, the dsRNA district comprises 22 adjacent base pairs, wherein at least 4 base pairs in the 22 adjacent base pairs are non-canonical base pairs. In one embodiment, the dsRNA district comprises 22 adjacent base pairs, wherein at least 5 base pairs in the 22 adjacent base pairs are non-canonical base pairs. In one embodiment, the dsRNA district comprises 22 adjacent base pairs, wherein at least 6 base pairs in the 22 adjacent base pairs are non-canonical base pairs. In one embodiment, the dsRNA district comprises 22 adjacent base pairs, and wherein at least 7 base pairs in the 22 adjacent base pairs are non-conventional base pairs. In one embodiment, the dsRNA district comprises 22 adjacent base pairs, and wherein at least 8 base pairs in the 22 adjacent base pairs are non-conventional base pairs. In one embodiment, the dsRNA district comprises 22 adjacent base pairs, and wherein at least 9 base pairs in the 22 adjacent base pairs are non-conventional base pairs. In each of these embodiments, preferably in the dsRNA district, 10 of the 22 adjacent base pairs are non-conventional base pairs, more preferably in the dsRNA district, 9 of the non-conventional base pairs, more preferably in the dsRNA district, 8 of the non-conventional base pairs, even more preferably in the dsRNA district, 7 of the non-conventional base pairs, and most preferably in the dsRNA district, 6 of the non-conventional base pairs. Preferably, in the above embodiments, the non-canonical base pairs comprise at least one G:U base pair, and more preferably all non-canonical base pairs are G:U base pairs. Preferably, the features of the above embodiments apply to each of the 22 contiguous base pairs present in the RNA molecule of the present invention.

[0361] In one embodiment, the dsRNA district of the RNA molecule of the present invention comprises 23 adjacent base pairs, wherein at least one base pair in the 23 adjacent base pairs is a non-canonical base pair. In one embodiment, the dsRNA district comprises 23 adjacent base pairs, wherein at least 2 base pairs in the 23 adjacent base pairs are non-canonical base pairs. In one embodiment, the dsRNA district comprises 23 adjacent base pairs, wherein at least 3 base pairs in the 23 adjacent base pairs are non-canonical base pairs. In one embodiment, the dsRNA district comprises 23 adjacent base pairs, wherein at least 4 base pairs in the 23 adjacent base pairs are non-canonical base pairs. In one embodiment, the dsRNA district comprises 23 adjacent base pairs, wherein at least 5 base pairs in the 23 adjacent base pairs are non-canonical base pairs. In one embodiment, the dsRNA district comprises 23 adjacent base pairs, wherein at least 6 base pairs in the 23 adjacent base pairs are non-canonical base pairs. In one embodiment, the dsRNA district comprises 23 adjacent base pairs, and wherein at least 7 base pairs in the 23 adjacent base pairs are non-conventional base pairs. In one embodiment, the dsRNA district comprises 23 adjacent base pairs, and wherein at least 8 base pairs in the 23 adjacent base pairs are non-conventional base pairs. In one embodiment, the dsRNA district comprises 23 adjacent base pairs, and wherein at least 9 base pairs in the 23 adjacent base pairs are non-conventional base pairs. In each of these embodiments, preferably in the dsRNA district, 10 of the 23 adjacent base pairs are non-conventional base pairs, more preferably in the dsRNA district, 9 of the non-conventional base pairs, more preferably in the dsRNA district, 8 of the non-conventional base pairs are also more preferably in the dsRNA district, 7 of the non-conventional base pairs are even more preferably in the dsRNA district, and 6 of the non-conventional base pairs are most preferably in the dsRNA district. Preferably, in the above embodiments, the non-canonical base pairs comprise at least one G:U base pair, and more preferably all non-canonical base pairs are G:U base pairs. Preferably, the features of the above embodiments apply to each of the 23 contiguous base pairs present in the RNA molecule of the present invention.

[0362] In one embodiment, the dsRNA district of the RNA molecule of the present invention comprises 24 adjacent base pairs, wherein at least one base pair in the 24 adjacent base pairs is a non-canonical base pair. In one embodiment, the dsRNA district comprises 24 adjacent base pairs, wherein at least 2 base pairs in the 24 adjacent base pairs are non-canonical base pairs. In one embodiment, the dsRNA district comprises 24 adjacent base pairs, wherein at least 3 base pairs in the 24 adjacent base pairs are non-canonical base pairs. In one embodiment, the dsRNA district comprises 24 adjacent base pairs, wherein at least 4 base pairs in the 24 adjacent base pairs are non-canonical base pairs. In one embodiment, the dsRNA district comprises 24 adjacent base pairs, wherein at least 5 base pairs in the 24 adjacent base pairs are non-canonical base pairs. In one embodiment, the dsRNA district comprises 24 adjacent base pairs, wherein at least 6 base pairs in the 24 adjacent base pairs are non-canonical base pairs. In one embodiment, the dsRNA district comprises 24 adjacent base pairs, and wherein at least 7 base pairs in the 24 adjacent base pairs are non-conventional base pairs. In one embodiment, the dsRNA district comprises 24 adjacent base pairs, and wherein at least 8 base pairs in the 24 adjacent base pairs are non-conventional base pairs. In one embodiment, the dsRNA district comprises 24 adjacent base pairs, and wherein at least 9 base pairs in the 24 adjacent base pairs are non-conventional base pairs. In each of these embodiments, preferably in the dsRNA district, 10 of the 24 adjacent base pairs are non-conventional base pairs, more preferably in the dsRNA district, 9 of the non-conventional base pairs, more preferably in the dsRNA district, 8 of the non-conventional base pairs are also more preferably in the dsRNA district, 7 of the non-conventional base pairs are even more preferably in the dsRNA district, and 6 of the non-conventional base pairs are most preferably in the dsRNA district. Preferably, in the above embodiments, the non-canonical base pairs comprise at least one G:U base pair, and more preferably all non-canonical base pairs are G:U base pairs. Preferably, the features of the above embodiments apply to each of the 24 contiguous base pairs present in the RNA molecule of the present invention.

[0363] In the following embodiments, if there is only one (adjacent) dsRNA district, the full length (i.e., the entire dsRNA district) of the dsRNA district of the RNA molecule of the present invention is considered to be the context of the feature, or if there are two or more dsRNA districts in the RNA molecule, it is considered to be the context of each dsRNA district of the RNA molecule. In one embodiment, the dsRNA district does not include 20 adjacent canonical base pairs, i.e., each sub-region of 20 adjacent base pairs includes at least one non-canonical base pair, preferably at least one G:U base pair. In one embodiment, the dsRNA district does not include 19 adjacent canonical base pairs. In one embodiment, the dsRNA district does not include 18 adjacent canonical base pairs. In one embodiment, the dsRNA district does not include 17 adjacent canonical base pairs. In one embodiment, the dsRNA district does not include 16 adjacent canonical base pairs. In one embodiment, the dsRNA district does not include 15 adjacent canonical base pairs. In one embodiment, the dsRNA district does not include 14 adjacent canonical base pairs. In one embodiment, the dsRNA district does not include 13 adjacent canonical base pairs. In one embodiment, the dsRNA district does not include 12 adjacent canonical base pairs. In one embodiment, the dsRNA district does not include 11 adjacent canonical base pairs. In one embodiment, the dsRNA district does not include 10 adjacent canonical base pairs. In one embodiment, the dsRNA district does not include 9 adjacent canonical base pairs. In one embodiment, the dsRNA district does not include 8 adjacent canonical base pairs. In one embodiment, the dsRNA district does not include 7 adjacent canonical base pairs. In the above-mentioned embodiments, the longest sub-region of the adjacent canonical base pairing of each dsRNA district in the dsRNA district of the preferred RNA molecule or the RNA molecule is 5,6 or 7 adjacent canonical base pairings, i.e., towards the shorter length. Each feature of the above-mentioned embodiment is preferably combined in the RNA molecule with the following features. In one embodiment, the dsRNA district includes 10-19 or 20 adjacent base pairs. In a preferred embodiment, the dsRNA region comprises 12-19 or 20 adjacent base pairs. In one embodiment, the dsRNA region comprises 14-19 or 20 adjacent base pairs. In these embodiments, the dsRNA region comprises 15 adjacent base pairs. In one embodiment, the dsRNA region comprises 16, 17, 18 or 19 adjacent base pairs. In one embodiment, the dsRNA region comprises 20 adjacent base pairs. Preferably, in the above-mentioned embodiments, the adjacent base pairs comprise at least one non-canonical base pair including at least one G:U base pair, and more preferably, all non-canonical base pairs in the adjacent base pair region are G:U base pairs.

[0364] In one embodiment, the dsRNA region comprises a subregion of 4 standard base pairs of flank non-canonical base pairs, i.e., at least 1, preferably 1 or 2 (no more than 2) non-canonical base pairs adjacent to each end of 4 canonical base pairs. In one embodiment, the dsRNA region comprises 2 subregions, each of which is 4 standard base pairs of flank non-canonical base pairs. In one embodiment, the dsRNA region comprises 3 subregions, each of which is 4 standard base pairs of flank non-canonical base pairs. In one embodiment, the dsRNA region comprises 4 or 5 subregions, each of which is 4 standard base pairs of flank non-canonical base pairs. In one embodiment, the dsRNA region comprises 6 or 7 subregions, each of which is 4 standard base pairs of flank non-canonical base pairs. In one embodiment, the dsRNA region comprises 8 to 10 subregions, each of which is 4 standard base pairs of flank non-canonical base pairs. In one embodiment, the dsRNA district comprises 11 to 15 sub-districts, each of which is 4 standard base pairs of flank non-canonical base pairs. In one embodiment, the dsRNA district comprises 2 to 50 sub-districts, each of which is 4 standard base pairs of flank non-canonical base pairs. In one embodiment, the dsRNA district comprises 2 to 40 sub-districts, each of which is 4 standard base pairs of flank non-canonical base pairs. In one embodiment, the dsRNA district comprises 2 to 30 sub-districts, each of which is 4 standard base pairs of flank non-canonical base pairs. In one embodiment, the dsRNA district comprises 2 to 20 sub-districts, each of which is 4 standard base pairs of flank non-canonical base pairs. Preferably, in the above-mentioned embodiment, the non-canonical base pair comprises at least one G:U base pair, and more preferably, all non-canonical base pairs of the canonical base pairs flanked adjacent in the sub-district are G:U base pairs. In a variation of the above embodiment, for some or all of the subregions, one or both of the flanking non-canonical base pairs are replaced by non-base-paired ribonucleotides in the sense sequence, the antisense sequence, or in both sequences. It will be readily appreciated that in the above embodiment, the maximum number of subregions is determined by the length of the dsRNA region in the RNA molecule.

[0365] In one embodiment, the dsRNA district comprises a subregion of 5 standard base pairs of flank non-canonical base pairs. In one embodiment, the dsRNA district comprises 2 subregions, each of which is 5 standard base pairs of flank non-canonical base pairs. In one embodiment, the dsRNA district comprises 3 subregions, each of which is 5 standard base pairs of flank non-canonical base pairs. In one embodiment, the dsRNA district comprises 4 or 5 subregions, each of which is 5 standard base pairs of flank non-canonical base pairs. In one embodiment, the dsRNA district comprises 6 or 7 subregions, each of which is 5 standard base pairs of flank non-canonical base pairs. In one embodiment, the dsRNA district comprises 8 to 10 subregions, each of which is 5 standard base pairs of flank non-canonical base pairs. In one embodiment, the dsRNA district comprises 11 to 15 subregions, each of which is 5 standard base pairs of flank non-canonical base pairs. In one embodiment, the dsRNA district comprises 2 to 50 sub-districts, and each sub-district is 5 standard base pairs of the non-standard base pairs of the side joints. In one embodiment, the dsRNA district comprises 2 to 50 sub-districts, and each sub-district is 5 standard base pairs of the non-standard base pairs of the side joints. In one embodiment, the dsRNA district comprises 2 to 30 sub-districts, and each sub-district is 5 standard base pairs of the non-standard base pairs of the side joints. In one embodiment, the dsRNA district comprises 2 to 20 sub-districts, and each sub-district is 5 standard base pairs of the non-standard base pairs of the side joints. Preferably, in the above-mentioned embodiment, the non-standard base pair comprises at least one G:U base pair, and more preferably, all non-standard base pairs of the standard base pairs of the side joints adjacent in the sub-district are G:U base pairs. In the variant of the above-mentioned embodiment, for some or all of the sub-districts, one or two of the non-standard base pairs of the side joints are replaced by non-base paired ribonucleotides in sense sequence, antisense sequence or in two sequences. It will be readily appreciated that in the above embodiments, the maximum number of subregions is determined by the length of the dsRNA region in the RNA molecule.

[0366] In one embodiment, the dsRNA region comprises a subregion of 6 standard base pairs of flank non-canonical base pairs. In one embodiment, the dsRNA region comprises 2 subregions, each of which is 6 standard base pairs of flank non-canonical base pairs. In one embodiment, the dsRNA region comprises 3 subregions, each of which is 6 standard base pairs of flank non-canonical base pairs. In one embodiment, the dsRNA region comprises 4 or 5 subregions, each of which is 6 standard base pairs of flank non-canonical base pairs. In one embodiment, the dsRNA region comprises 6 or 7 subregions, each of which is 6 standard base pairs of flank non-canonical base pairs. In one embodiment, the dsRNA region comprises 8 to 10 subregions, each of which is 6 standard base pairs of flank non-canonical base pairs. In one embodiment, the dsRNA region comprises 11 to 16 subregions, each of which is 6 standard base pairs of flank non-canonical base pairs. In one embodiment, the dsRNA district comprises 2 to 60 sub-districts, and each sub-district is 6 standard base pairs of the non-standard base pairs of the side joints. In one embodiment, the dsRNA district comprises 2 to 60 sub-districts, and each sub-district is 6 standard base pairs of the non-standard base pairs of the side joints. In one embodiment, the dsRNA district comprises 2 to 30 sub-districts, and each sub-district is 6 standard base pairs of the non-standard base pairs of the side joints. In one embodiment, the dsRNA district comprises 2 to 20 sub-districts, and each sub-district is 6 standard base pairs of the non-standard base pairs of the side joints. Preferably, in the above-mentioned embodiment, the non-standard base pair comprises at least one G:U base pair, and more preferably, all non-standard base pairs of the standard base pairs of the side joints adjacent in the sub-district are G:U base pairs. In the variant of the above-mentioned embodiment, for some or all of the sub-districts, one or two of the non-standard base pairs of the side joints are replaced by non-base-paired ribonucleotides in sense sequence, antisense sequence or in two sequences. It will be readily appreciated that in the above embodiments, the maximum number of subregions is determined by the length of the dsRNA region in the RNA molecule.

[0367] In one embodiment, the dsRNA district comprises the subdistrict of 10 adjacent base pairs, wherein 2-4 base pairs are non-canonical base pairs. In one embodiment, the dsRNA district comprises 2 subdistricts, each subdistrict is 10 adjacent base pairs, wherein 2-4 of 10 adjacent base pairs are non-canonical base pairs. In one embodiment, the dsRNA district comprises 3 subdistricts, each of which is 10 adjacent base pairs, wherein 2-4 of 10 adjacent base pairs are non-canonical base pairs. In one embodiment, the dsRNA district comprises 4 subdistricts, each of which is 10 adjacent base pairs, wherein 2-4 of 10 adjacent base pairs are non-canonical base pairs. In one embodiment, the dsRNA district comprises 5 subdistricts, each of which is 10 adjacent base pairs, wherein 2-4 of 10 adjacent base pairs are non-canonical base pairs. In one embodiment, the dsRNA district comprises 10 sub-districts, each of which is 10 adjacent base pairs, wherein 2-4 of the 10 adjacent base pairs are non-canonical base pairs. In one embodiment, the dsRNA district comprises 4 sub-districts, each of which is 15 adjacent base pairs, wherein 2-6 of the 15 adjacent base pairs are non-canonical base pairs. In one embodiment, the dsRNA district comprises 2-50 sub-districts, each of which comprises 10 adjacent base pairs, wherein 2-4 of the 10 adjacent base pairs are non-canonical base pairs. In one embodiment, the dsRNA district comprises 2-40 sub-districts, each of which comprises 10 adjacent base pairs, wherein 2-4 of the 10 adjacent base pairs are non-canonical base pairs. In one embodiment, the dsRNA district comprises 2-30 sub-districts, each of which comprises 10 adjacent base pairs, wherein 2-4 of the 10 adjacent base pairs are non-canonical base pairs. In one embodiment, the dsRNA region comprises 2-20 subregions, each subregion comprises 10 adjacent base pairs, wherein 2-4 of the 10 adjacent base pairs are non-canonical base pairs. Preferably, in the above-mentioned embodiment, the non-canonical base pair comprises at least one G:U base pair, and more preferably all non-canonical base pairs in the subregion are G:U base pairs. In a variant of the above-mentioned embodiment, for some or all of the subregions, one or more of 2-4 or 2-6 non-canonical base pairs are replaced by non-base-paired ribonucleotides in a sense sequence, an antisense sequence, or in two sequences. It is easy to understand that in the above-mentioned embodiment, the maximum number of subregions is determined by the length of the dsRNA region in the RNA molecule.

[0368] In one embodiment, the ratio of canonical to non-canonical base pairs in a dsRNA region is between 2.5:1 and 3.5:1, such as about 3:1. In one embodiment, the ratio of canonical to non-canonical base pairs in a dsRNA region is between 3.5:1 and 4.5:1, such as about 4:1. In one embodiment, the ratio of canonical to non-canonical base pairs in a dsRNA region is between 4.5:1 and 5.5:1, such as about 5:1. In one embodiment, the ratio of canonical to non-canonical base pairs in a dsRNA region is between 5.5:1 and 6.5:1, such as about 6:1. Different dsRNA regions in an RNA molecule can have different ratios.

[0369] In the above-mentioned embodiment, the non-canonical base pairs in the dsRNA region of the RNA molecule are preferably all G:U base pairs. In one embodiment, at least 99% of the non-canonical base pairs are G:U base pairs. In one embodiment, at least 98% of the non-canonical base pairs are G:U base pairs. In one embodiment, at least 97% of the non-canonical base pairs are G:U base pairs. In one embodiment, at least 95% of the non-canonical base pairs are G:U base pairs. In one embodiment, at least 90% of the non-canonical base pairs are G:U base pairs. In one embodiment, 90-95% of the non-canonical base pairs are G:U base pairs. For example, if there are 10 non-canonical base pairs, at least 9 (90%) are G:U base pairs.

[0370] The dsRNA region comprising non-canonical base pairing comprises an antisense sequence of 20 adjacent nucleotides, which serves as an antisense regulatory element. In one embodiment, the antisense regulatory element is at least 80%, preferably at least 90%, more preferably at least 95% or most preferably 100% complementary to the target RNA molecule in the eukaryotic cell. In one embodiment, the dsRNA region comprises 2, 3, 4 or 5 antisense regulatory elements complementary to the same target RNA molecule (i.e., complementary to different regions of the same target RNA molecule) or complementary to different target RNA molecules.

[0371] In one embodiment, when sense and antisense sequences are hybridized, one or more ribonucleotides of the sense ribonucleotide sequence or one or more ribonucleotides of the antisense ribonucleotide sequence or both are not base paired in the dsRNA region. In this embodiment, the dsRNA region does not include any loop sequence that covalently connects the sense and antisense sequences. One or more ribonucleotides in a dsRNA region or subregion may not be base paired. Therefore, in this embodiment, the sense strand in a dsRNA region is not completely base paired with its corresponding antisense strand.

[0372] In addition, in one embodiment and optionally in combination with any of the features of the above embodiments, the total number of ribonucleotides in the sense sequence and the total number of ribonucleotides in the antisense sequence may not be the same, although preferably they are the same. In one embodiment, the total number of ribonucleotides in the sense ribonucleotide sequence of the dsRNA region is 90%-110% of the total number of ribonucleotides in the antisense ribonucleotide sequence. In one embodiment, the total number of ribonucleotides in the sense ribonucleotide sequence is 95%-105% of the total number of ribonucleotides in the antisense ribonucleotide sequence. In one embodiment, the chimeric RNA molecule of the present invention may comprise one or more structural elements, such as internal or terminal protuberances or loops. Various embodiments of protuberances and loops are discussed above. In one embodiment, the dsRNA region is separated by a structural element such as a protuberance or loop. In one embodiment, the dsRNA region is separated by an intervening (spacer) sequence. Some ribonucleotides of the spacer sequence may base pair with other ribonucleotides in the RNA molecule, such as with other ribonucleotides in the spacer sequence, or they may not base pair in the RNA molecule, or some of them may not base pair. In one embodiment, the dsRNA region is linked to the terminal loop.In one embodiment, the dsRNA region is flanked by the terminal loop.

[0373] In one embodiment, when the dsRNA region of the RNA molecule of the present invention has at least 3 non-canonical base pairs in any subregion of 5 adjacent base pairs, the non-canonical base pairs are not adjacent, but are separated by one or more canonical base pairs, i.e., the dsRNA region does not have 3 or more adjacent non-canonical base pairs. In one embodiment, the dsRNA region does not have 4 or more adjacent non-canonical base pairs. For example, in one embodiment, the dsRNA region comprises at least 3 non-canonical base pairs in a subregion of 10 base pairs, wherein each non-canonical base pair is separated by 4 canonical base pairs.

[0374] In one embodiment, the RNA molecule of the present invention comprises more than one dsRNA region. For example, the RNA molecule comprises 2, 3, 4, 5, 6, 7, 8, 9, 10 or more dsRNA regions. In this embodiment, one or more or all of the dsRNA regions may comprise the properties exemplified above, such as non-canonical base pairing and / or several antisense regulatory elements.

[0375] Silencing activity

[0376] The RNA molecules of the present invention have antisense activity because they contain sense ribonucleotide sequences that are substantially complementary to the regions of the target RNA molecules. For example, the ribonucleotide sequence is substantially complementary to the regions of the target RNA molecules in eukaryotic cells. In one embodiment, the target RNA molecules can be in bacterial cells, fungal cells, plant cells, insect cells or animal cells. Such components of RNA molecules defined herein can be referred to as "antisense regulatory elements". "Substantially complementary" means that the sense ribonucleotide sequence can have insertions, deletions and single point mutations compared to the complementary sequences of the target RNA molecules in eukaryotic cells. Preferably, the homology between the sense ribonucleotide sequence with antisense activity and the target RNA molecule is at least 80%, preferably at least 90%, preferably at least 95%, and most preferably 100%. For example, the sense ribonucleotide sequence may include about 15, about 16, about 17, about 18, about 19 or more adjacent nucleotides having the same sequence as the first region of the target RNA molecule in the eukaryotic cell. In another embodiment, the sense ribonucleotide sequence may include about 20 adjacent nucleotides having the same sequence as the first region of the target RNA molecule in the eukaryotic cell.

[0377] "Antisense activity" is used in the context of the present invention to refer to an antisense regulatory element from an RNA molecule as defined herein, which modulates (increases or decreases) the expression of a target RNA molecule.

[0378] In various embodiments, the antisense regulatory element according to the present invention may include multiple monomer subunits connected together by a linking group. Embodiments include primers, probes, antisense compounds, antisense oligonucleotides, external guide sequence (EGS) oligonucleotides, alternate splicers, gapmers, siRNA and microRNA. Therefore, the RNA molecule according to the present invention may include antisense regulatory elements with single-stranded, double-stranded, circular, branched or hairpin structures. In one embodiment, the antisense sequence can contain structural elements, such as internal or terminal protuberances or loops.

[0379] In one embodiment, the RNA molecules of the present invention comprise chimeric oligomeric components, such as chimeric oligonucleotides. For example, the RNA molecules may comprise differently modified nucleotides, mixed backbone antisense oligonucleotides, or combinations thereof. In one embodiment, the chimeric oligomeric compound may comprise at least one modified region to confer increased resistance to nuclease degradation, increased cellular uptake, and / or increased binding affinity to the target RNA molecule.

[0380] Antisense regulatory elements can have a variety of lengths. In various embodiments, the present invention provides antisense regulatory elements composed of bases connected by X-Y, where X and Y are each independently selected from 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50 (provided that X < Y). For example, in certain embodiments, the present invention provides antisense regulatory elements comprising 8-9, 8-10, 8-11, 8-12, 8-13, 8-14, 8-15, 8-16, 8-17, 8-18, 8-19, 8-20, 8-21, 8-22, 8-23, 8-24, 8-25, 8-26, 8-27, 8-28, 8-29, 8-30, 9-10, 9-11, 9-12, 9-13, 9-14, 9-15, 9-16, 9-17, 9-18, 9-19, 9-20, 9-21, 9-22, 9-23, 9-24, 9-25, 9-26, 9-27, 9-28, 9-29, 9-30, 10-11, 10-12, 10-13, 10-14, 10-15, 10-16, 10-17, 10-18, 10-19, 10-20, 10-21, 10-22, 10-23, 10-24, 10-25, 10-26, 10-27, 10-28, 10-29, 10-30, 11-12, 11-13, 11-14, 11-15, 11-16, 11-17, 11-18, 11-19, 11-20, 11-21, 11-22, 11-23, 11-24, 11-25, 11-26, 11-27, 11-28, 11-29, 11-30, 12-13, 12-14, 12-15, 12-16, 12-17, 12-18, 12-19, 12-20, 12-21, 12-22, 12-23, 12-24, 12-25, 12-26, 12-27, 12-28, 12-29, 12-30, 13-14, 13-15, 13-16, 13-17, 13-18, 13-19, 13-20, 13-21, 13-22, 13-23, 13-24, 13-25, 13-26, 13-27, 13-28, 13-29, 13-30, 14-15, 14-16, 14-17, 14-18, 14-19, 14-20, 14-21, 14-22, 14-23, 14-24, 14-25, 14-26, 14-27, 14-28, 14-29, 14-30, 15-16, 15-17, 15-18, 15-19,15-20, 15-21, 15-22, 15-23, 15-24, 15-25, 15-26, 15-27, 15-28, 15-29, 15-30, 16-17, 16-18, 16-19, 16-20, 16-21, 16-22, 16-23, 16-24, 16-25, 16-26, 16-27, 16-28, 16-29, 16-30, 17-18, 17-19, 17-20, 17-21, 1 7-22, 17-23, 17-24, 17-25, 17-26, 17-27, 17-28, 17-29, 17-30, 18-19, 18-20, 18-21, 18-22, 18-23, 18-24, 18-25, 18-26, 18-27, 18-28, 18-29, 18-30, 19-20, 19-21, 19-22, 19-23, 19-24, 19-25, 19-26, 19-29, 19- 28, 19-29, 19-30, 20-21, 20-22, 20-23, 20-24, 20-25, 20-26, 20-27, 20-28, 20-29, 20-30, 21-22, 21-23, 21-24, 21-25, 21-26, 21-27, 21-28, 21-29, 21-30, 22-23, 22-24, 22-25, 22-26, 22-27, 22-28, 22-29, 22-30 , 23-24, 23-25, 23-26, 23-27, 23-28, 23-29, 23-30, 24-25, 24-26, 24-27, 24-28, 24-29, 24-30, 25-26, 25-27, 25-28, 25-29, 25-30, 26-27, 26-28, 26-29, 26-30, 27-28, 27-29, 27-30, 28-29, 28-30, or 29-30 linked bases. ,

[0381] RNA molecules according to the present invention can comprise multiple antisense regulatory elements. For example, RNA molecules can comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 antisense regulatory elements. In one embodiment, the antisense regulatory elements are identical. In this embodiment, RNA molecules can comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 copies of antisense regulatory elements. In another embodiment, RNA molecules according to the present invention can comprise different antisense regulatory elements. For example, antisense regulatory elements can be provided to target multiple genes in pathways such as lipid biosynthesis. In this embodiment, RNA molecules can comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 different antisense regulatory elements.

[0382] Antisense regulatory elements according to the present invention can regulate (increase or decrease) the expression or amount of various target RNA molecules. In one embodiment, the target RNA molecule is a fatty acid biosynthesis gene. Examples of such genes include genes encoding: acetyltransferase; acyl transporter (e.g., "acyl carrier protein"); desaturase, such as stearoyl desaturase or microsomal D12-desaturase, particularly Fad2-1 gene; malonyltransferase; -ketoacyl-ACP synthase; 3-keto-ACP reductase; enoyl-ACP hydratase; thioesterase, such as acyl-ACP thioesterase; enoyl-ACP reductase. In one embodiment, the target RNA molecule is a FAD2 gene (e.g., those described by Genbank accession number:). Unpublished: AF124360 (Brassica carinata), AF042841 (Brassica rapa), L26296 (Arabidopsis thaliana), A65102 (Brassica napus). For example, the target RNA molecule can be the FAD2.1 gene. In another embodiment, the target RNA molecule can be the FAD2.2 gene. In another embodiment, the target RNA molecule can be the FAD2.1 and FAD2.2 genes. Examples of other genes involved in modifying lipid complexes that can be target RNA molecules are known in the art (Shure et al. (1983) Cell 35:225-233; Preiss et al. (1987) Tailoring Genes for Crop Improvement (Bruening et al., eds.), Plenum Press, S. 133-152; Gupta et al. (1988) Plant Mol. Biol. 10:215-224; Olive et al. (1989) Plant Mol Biol 12:525-538; Bhattacharyya et al. (1990) Cell 60:155-122; Dunwell JM (2000) J Exp Botany 51 Spec No:487-96; Brar DS et al. (1996) Biotech Genet. Eng Rev 13:167-79; Kishore G M and Somerville CR (1993) Curr Opin Biotech 4(2):152-8; US 5,530,192 and WO 94 / 18337).

[0383] In another embodiment, the target RNA molecule is an arthropod gene, such as an insect gene transcript. Examples of such genes include chitin synthase genes, such as CHS1 and / or CHS2 or other genes that control insect activity, behavior, reproduction, growth and / or development. Various essential genes for various pathogens are known to those skilled in the art (e.g., nematode resistance genes are summarized in WO 93 / 10251, WO 94 / 17194).

[0384] In another embodiment, the target RNA molecule is associated with a disease. For example, the target RNA molecule can be an oncogene or tumor suppressor gene transcript. Exemplary oncogenes include ABL1, BCL1, BCL2, BCL6, CBFA2, CBL, CSF1R, ERBA, ERBB, EBRB2, FGR, FOS, FYN, HRAS, JUN, LCK, LYN, MYB, MYC, NRAS, RET or SRC. Exemplary tumor suppressor genes include BRCA1 or BRCA2; adhesion molecules; cell cycle protein kinases and their inhibitors.

[0385] In another embodiment, the target RNA molecule is associated with a delay in fruit ripening. Delaying fruit ripening can be achieved, for example, by reducing gene expression selected from the group consisting of polygalacturonase, pectinesterase, β-(1-4)-glucanase (cellulase), β-galactosidase (β-galactosidase); ethylene biosynthesis genes, such as 1-aminocyclopropane-1-carboxylic acid synthase; genes for carotenoid biosynthesis, such as, for example, genes for the biosynthesis of pre-phytoene or phytoene.

[0386] In another embodiment, the target RNA molecule is associated with the delay of aging symptoms. Suitable target RNA molecules include cinnamoyl-CoA:NADPH reductase or cinnamoyl alcohol dehydrogenase. Target RNA molecules are further described in WO1995 / 07993.

[0387] In another embodiment, the target RNA molecule is associated with the modification of the fiber content in a food, preferably a seed. For example, the RNA molecule can reduce the expression of caffeic acid O-methyltransferase or cinnamoyl alcohol dehydrogenase.

[0388] Nucleic acid encoding RNA molecules

[0389] Those skilled in the art will appreciate from the foregoing description that the present invention also provides isolated nucleic acids encoding RNA molecules disclosed herein and their components. For example, nucleic acids comprising any one or more of the sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9. The nucleic acid may be partially purified after expression in a host cell. The term "partially purified" is used to refer to RNA molecules that have been generally separated from lipids, nucleic acids, other peptides and other contaminating molecules with which the RNA molecules are associated in the host cell. Preferably, the partially purified polynucleotide is at least 60% free, more preferably at least 75% free, and more preferably at least 90% free of other components associated with it.

[0390] In another embodiment, the polynucleotide according to the present invention is a heterologous polynucleotide. The term "heterologous polynucleotide" is well known in the art and refers to a polynucleotide that is not endogenous to a cell, or a natural polynucleotide whose native sequence has been altered, or a natural polypeptide whose expression is quantitatively altered due to manipulation of cells by recombinant DNA technology.

[0391] In another embodiment, polynucleotides according to the present invention are synthetic polynucleotides. For example, techniques that do not require pre-existing nucleotide sequences, such as DNA printing and oligonucleotide synthesis, can be used to produce polynucleotides. In another embodiment, polynucleotides are produced by heterologous nucleic acids.

[0392] In one embodiment, polynucleotide encoding RNA precursor molecules disclosed herein include introns in at least one loop sequence, and the introns can be spliced ​​out during transcription of polynucleotides in host cells. In another embodiment, the loop sequence includes two, three, four, five or more introns. The present invention also provides expression constructs, such as DNA constructs comprising isolated nucleic acids of the present invention operably connected to a promoter. In one embodiment, such isolated nucleic acids and / or expression constructs are provided in cells or non-human organisms. In one embodiment, isolated nucleic acids are stably integrated into the genome of cells or non-human organisms. Various embodiments of suitable expression constructs, promoters and cells comprising them are discussed below.

[0393] The synthesis of RNA molecules according to the present invention can be achieved using various methods known in the art. The Examples section provides an example of in vitro synthesis. In this example, a construct comprising an RNA molecule disclosed herein is subjected to 3' end restriction, precipitation, purification and quantification. RNA synthesis can be achieved in bacterial culture after transforming HT115 electrocompetent cells and inducing RNA synthesis using the T7, IPTG system.

[0394] Recombinant vector

[0395] One embodiment of the present invention includes a recombinant vector comprising at least one RNA molecule defined herein and capable of delivering the RNA molecule to a host cell. Recombinant vectors include expression vectors. Recombinant vectors contain heterologous polynucleotide sequences, i.e. polynucleotide sequences that are not naturally adjacent to the RNA molecule defined herein, which are preferably derived from different species. The vector may be RNA or DNA, and is typically a viral vector or plasmid derived from a virus.

[0396] Various viral vectors can be used for delivering and mediating the expression of RNA molecules according to the present invention. The selection of viral vectors generally depends on various parameters, for example, the cells or tissues for delivery, the transduction efficiency and pathogenicity of the vector. In one embodiment, the viral vector is integrated into the host cell chromatin (e.g., slow virus). In another embodiment, the viral vector is mainly retained in the nucleus as an extrachromosomal episome (e.g., adenovirus). The embodiments of these types of viral vectors include tumor retroviruses, slow viruses, adeno-associated viruses, adenoviruses, herpes viruses, and retroviruses.

[0397] Plasmid vectors typically include additional nucleic acid sequences that provide easy selection, amplification and transformation of expression cassettes in prokaryotic cells, such as pUC derived vectors, pGEM derived vectors or binary vectors containing one or more T-DNA regions. Additional nucleic acid sequences include an origin of replication that provides for autonomous replication of the vector, preferably a selectable marker gene encoding antibiotic or herbicide resistance, a unique multiple cloning site that provides multiple sites for inserting nucleic acid sequences or genes encoded in the nucleic acid construct, and sequences that enhance transformation of prokaryotic and eukaryotic (particularly plant) cells.

[0398] As used herein, "operably linked" refers to the functional relationship between two or more nucleic acid (e.g., DNA) fragments. Typically, it refers to the functional relationship between a transcriptional regulatory element (promoter) and a transcribed sequence. For example, if a promoter stimulates or regulates the transcription of a coding sequence in a suitable cell, the promoter is operably linked to the coding sequence of an RNA molecule defined herein. Typically, promoter transcriptional regulatory elements operably linked to a transcribed sequence are physically adjacent to the transcribed sequence, i.e., they are cis-acting. However, some transcriptional regulatory elements such as enhancers do not need to be physically adjacent or located near the coding sequence whose transcription is enhanced.

[0399] When multiple promoters are present, each promoter may independently be the same or different.

[0400] For the ease of identifying transformants, recombinant vectors desirably comprise selectable or screenable marker genes." marker gene " refers to the gene that gives the cell different phenotypes of expressing this marker gene, therefore allows the cell of this conversion to be distinguished from the cell without this mark. Selectable marker gene gives a kind of proterties, and people can " select " this proterties based on the resistance to selection agent (for example, herbicide, antibiotic etc.). Screenable marker gene (or reporter gene) gives the proterties that can be identified by observation or test, that is, by " screening " (for example, other enzyme activities that do not exist in beta-glucuronidase, luciferase, GFP or unconverted cells). Exemplary selectable markers for selecting plant transformants include, but are not limited to, the hyg gene encoding hygromycin B resistance; the neomycin phosphotransferase (nptII) gene that confers kanamycin and paromomycin resistance; the glutathione-S-transferase gene from rat liver that confers resistance to glutathione-derived herbicides, as described, for example, in EP 256 223; the glutamine synthase gene that, when overexpressed, confers resistance to glutamine synthase inhibitors such as phosphinothricin, as described in WO 256 223. 87 / 05327; an acetyltransferase gene from Streptomyces viridopromogenes, which confers resistance to the selective agent phosphinothricin, as described, for example, in EP 275 957; a gene encoding 5-enolisothiocyanate-3-phosphate synthase (EPSPS), which confers tolerance to N-phosphonomethylglycine, as described, for example, in Hinchee et al. (1988); a bar gene, which confers resistance to bialaphos, as described, for example, in WO 91 / 02071; a nitrilase gene, for example from Klebsiella ozaenae), which confers resistance to bromoxynil (Stalker et al., 1988); a dihydrofolate reductase (DHFR) gene, which confers resistance to methotrexate (Thillet et al., 1988); a mutant acetolactate synthase gene (ALS), which confers resistance to imidazolinones, sulfonylureas or other ALS-inhibiting chemicals (see EP 154,204); a mutant anthranilate synthase gene, which confers resistance to 5-methyltryptophan; or a dalapon dehalogenase gene, which confers herbicide resistance.

[0401] Preferably, the recombinant vector is stably integrated into the genome of a cell such as a plant cell. Thus, the recombinant vector may comprise appropriate elements that allow the vector to be incorporated into the genome or cell chromosome.

[0402] Expression vector

[0403] As used herein, "expression vector" is a DNA vector capable of transforming a host cell and affecting the expression of an RNA molecule defined herein. The expression vector of the present invention comprises regulatory sequences, such as transcription control sequences, translation control sequences, replication origins, and other regulatory sequences that are compatible with host cells and control the expression of the RNA molecule according to the present invention. In particular, the expression vector of the present invention includes a transcription control sequence. A transcription control sequence is a sequence that controls the initiation, extension and termination of transcription. Particularly important transcription control sequences are those that control the initiation of transcription, such as promoters, enhancers, operators and repressor sequences. The selection of the regulatory sequence used depends on the target organism, such as the plant and / or target organ or tissue of interest. Such a regulatory sequence can be obtained from any eukaryotic organism, such as a plant or plant virus, or can be chemically synthesized.

[0404] Exemplary vectors suitable for stable transfection of plant cells or establishment of transgenic plants are described, for example, in Pouwels et al., Cloning Vectors: A Laboratory Manual, 1985, supp. 1987, Weissbach and Weissbach, Methods for Plant Molecular Biology, Academic Press, 1989, and Gelvin et al., Plant Molecular Biology Manual, Kluwer Academic Publishers, 1990. Typically, plant expression vectors include, for example, one or more cloned plant genes under the transcriptional control of 5' and 3' regulatory sequences and dominant selectable markers. Such plant expression vectors may also include promoter regulatory regions (e.g., regulatory regions that regulate inducible or constitutive, environmental or developmental, or cell or tissue specific expression), transcription initiation start sites, ribosome binding sites, transcription termination sites, and / or polyadenylation signals.

[0405] The vectors of the invention may also be used to produce RNA molecules as defined herein in cell-free expression systems, such systems being well known in the art.

[0406] In one embodiment, the polynucleotide encoding the RNA molecule according to the present invention is operably connected to a promoter capable of directly expressing the RNA molecule in a host cell. In one embodiment, the promoter works in vitro. In one embodiment, the promoter is an RNA polymerase promoter. For example, the promoter can be an RNA polymerase III promoter. In another embodiment, the promoter can be an RNA polymerase II promoter. However, the selection of the promoter can depend on the target organism, such as the plant, insect and / or tissue of interest. Exemplary mammalian promoters include CMV, EF1α, SV40, PGK1, Ubc, human beta actin, CAG, TRE, UAS, CaMKIIa, CAL1, 10, TEF1, GDS, ADH1, CaMV35S, Ubi, H1 and U6. Exemplary insect promoters include Ac5 and polyhedrin. Many constitutive promoters active in plant cells have been described. Suitable promoters for constitutive expression in plants include, but are not limited to, cauliflower mosaic virus (CaMV) 35S promoter, figwort mosaic virus (FMV) 35S, light-induced promoter from the small subunit (SSU) of ribulose-1,5-bisphosphate carboxylase, rice cytoplasmic trisaccharide phosphate isomerase promoter, Arabidopsis adenine phosphoribosyltransferase promoter, rice actin 1 gene promoter, mannopine synthase and octopine synthase promoters, Adh promoter, sucrose synthase promoter, R gene complex promoter, chlorophyll α / α binding protein gene promoter. These promoters have been used to produce DNA vectors that have been expressed in plants, see, for example, WO 84 / 02913. All of these promoters have been used to produce various types of plant-expressible recombinant DNA vectors.

[0407] In order to express in the source tissue of plant such as leaf, seed, root or stem, it is preferred that the promoter of the present invention has relatively high expression in these specific tissues. For this reason, the gene with tissue or cell specificity or enhanced expression can be selected from many promoters. The embodiment of this type of promoter reported in the document comprises the chloroplast glutamine synthase GS2 promoter from pea, the chloroplast fructose-1 from wheat, the 6-bisphosphatase promoter from the nuclear photosynthesis ST-LSI promoter from potato, the serine / threonine kinase promoter from Arabidopsis thaliana and the glucoamylase (CHS) promoter. The promoters of ribulose-1,5-bisphosphate carboxylase from oriental larch (Larix laricina), Cab gene and Cab6 from pine, Cab-1 gene from wheat, Cab-1 gene from spinach, Cab 1R gene from rice, pyruvate phosphate dikinase (PPDK) from maize (Zea mays), Lhcb1*2 gene from tobacco, Suc2 sucrose-H2 from Arabidopsis thaliana, and sucrose-H2 from rice (Arabidopsis thaliana) have also been reported. 30 Symporter promoter, promoters of thylakoid membrane protein genes (PsaD, PsaF, PsaE, PC, FNR, AtpC, AtpD, Cab, RbcS) of spinach. Other promoters of chlorophyll α / β-binding protein can also be used in the present invention, such as the promoters of the LhcB gene and the PsbP gene from white mustard (Sinapis alba).

[0408] A variety of plant gene promoters that are regulated in response to environmental, hormonal, chemical, and / or developmental signals can also be used to express RNA binding protein genes in plant cells, including by (1) heat; (2) light (e.g., pea RbcS-3A promoter, corn RbcS promoter); (3) hormones, such as abscisic acid; (4) damage (e.g., WunI); or (5) chemicals, such as methyl jasmonate, salicylic acid, steroid hormones, alcohol,

[0409] (Safeners) (WO 97 / 06269); or (6) organ-specific promoters may also be advantageously used.

[0410] As used herein, the term "plant storage organ specific promoter" refers to a promoter that preferentially directs transcription of genes in plant storage organs when compared to other plant tissues. For expression in the sink tissues of plants, such as tubers of potato plants, fruits of tomatoes, or seeds of soybeans, rapeseed, cotton, maize, wheat, rice, and barley, it is preferred that the promoter used in the present invention has relatively high expression in these specific tissues. The β-conglycinin promoter can be used, or other seed-specific promoters such as rapeseed protein, zein, linin, and phaseolin promoters. Root-specific promoters can also be used. An example of such a promoter is the promoter of the acid chitinase gene. Expression in root tissue can also be achieved by utilizing the root-specific subdomain of the CaMV 35S promoter that has been identified.

[0411] In a particularly preferred embodiment, the promoter directs expression in tissues and organs where lipid biosynthesis occurs. Such promoters can act at the appropriate time in seed development to modify the lipid composition in the seed. Preferred promoters for seed-specific expression include: 1) promoters from genes encoding enzymes involved in lipid biosynthesis and accumulation in seeds (such as desaturases and elongases), 2) promoters from genes encoding seed storage proteins, and 3) promoters from genes encoding enzymes involved in carbohydrate biosynthesis and accumulation in seeds. Suitable seed-specific promoters are the promoter of the napus protein gene of rapeseed ( US 5,608,152 ), the Vicia faba USP promoter ( Baumlein et al., 1991 ), the Arabidopsis oleosin promoter ( WO 98 / 45461 ), the phaseolin promoter ( US 5,504,200 ) of bean ( WO 98 / 45461 ), the Brassica Bce4 promoter ( WO 91 / 13980 ) or the legumin B4 promoter ( Baumlein et al., 1992 ) and promoters which lead to seed-specific expression in monocotyledonous plants such as maize, barley, wheat, rye, rice, etc. Suitable promoters of note are the promoters of the barley lpt2 or lpt1 genes (WO 95 / 15389 and WO 95 / 23230), or the promoters described in WO 99 / 16890 (promoters from hordein genes, rice glutelin genes, rice oryzin genes, rice prolamin genes, wheat prolamin genes, wheat glutelin genes, zein genes, oat glutelin genes, sorghum kasirin genes, rye secalin genes). Other promoters include those described in Broun et al., (1998), Potenza et al., (2004), US20070192902 and US20030159173. In one embodiment, the seed-specific promoter is preferably expressed in a defined part of the seed, such as the cotyledon or endosperm. Examples of cotyledon-specific promoters include, but are not limited to, the FPI promoter (Ellerstrom et al., 1996), the pea legumin promoter (Perrin et al., 2000); and the bean lectin promoter (Perrin et al., 2000). Examples of endosperm-specific promoters include, but are not limited to, the maize zein-1 promoter (Chikwamba et al., 2003), the rice glutelin-1 promoter (Yang et al., 2003); the barley D-hordein promoter (Horvath et al., 2000); and the wheat HMW glutenin promoter (Alvarez et al., 2000). In another embodiment, the seed-specific promoter is not expressed or is expressed only at a low level after embryo and / or seed germination.

[0412] In another embodiment, the plant storage organ specific promoter is a fruit specific promoter. Examples include, but are not limited to, the tomato polygalacturonase E8 and Pds promoters, and the apple ACC oxidase promoter (for review, see Potenza et al., 2004). In a preferred embodiment, the promoter preferentially directs expression in the edible portion of the fruit, such as the pith of the fruit, relative to the peel of the fruit or the seeds within the fruit.

[0413] In one embodiment, the inducible promoter is the Aspergillus nidulans alc system. The examples of inducible expression systems that can be used to replace the Aspergillus nidulans alc system are described in the reviews of Padidam (2003) and Corrado and Karali (2009). In another embodiment, the inducible promoter is a safener inducible promoter, such as corn ln2-1 or ln2-2 promoter (Hershey and Stoner, 1991), and the safener inducible promoter is corn GST-27 promoter (Jepson et al., 1994) or soybean GH2 / 4 promoter (Ulmasov et al., 1995).

[0414] In another embodiment, the inducible promoter is a senescence-inducible promoter, such as the senescence-inducible promoters SAG (senescence-associated gene) 12 and SAG13 from Arabidopsis (Gan, 1995; Gan and Amasino, 1995) and LSC54 from Brassica napus (Buchanan-Wollaston, 1994). Such promoters show increased expression at about the onset of senescence in plant tissues, particularly leaves.

[0415] For expression in vegetative tissues, leaf-specific promoters can be used, such as the ribulose bisphosphate carboxylase (RBCS) promoter. For example, tomato RBCS1, RBCS2 and RBCS3A genes are expressed in leaves and lightly grown seedlings (Meier et al., 1997). The ribulose bisphosphate carboxylase promoter described by Matsuoka et al. (1994) can be used, which is almost exclusively expressed at high levels in the mesophyll cells of leaves and sheaths. Another leaf-specific promoter is the promoter of the light-collecting chlorophyll a / b binding protein gene (see Shiina et al., 1997). The Arabidopsis thaliana myb-related gene promoter (Atmby5) described by Li et al. (1996) is leaf-specific. The Atmyb5 promoter is expressed in the leaf hairs, stipules and epidermal cells of the young rosette and cauliflower leaf margins, as well as in immature seeds. The leaf promoter identified in corn by Busk et al. (1997) can also be used.

[0416] In some cases, such as when LEC2 or BBM is expressed recombinantly, it may be desirable that the transgene is not expressed at high levels. An example of a promoter that can be used in this case is a truncated napin A promoter that retains a seed-specific expression pattern but has reduced expression levels (Tan et al., 2011).

[0417] The 5' non-translated leader sequence can be derived from a promoter of a heterologous gene sequence selected to express the RNA molecule of the present invention, or can be heterologous relative to the coding region of the enzyme to be produced, and can be specifically modified to increase the translation of the mRNA if desired. For a review of optimizing transgenic expression, see Koziel et al., (1996). The 5' non-translated region can also be obtained from plant viral RNA (tobacco mosaic virus, tobacco etch virus, corn dwarf mosaic virus, alfalfa mosaic virus, etc.) of a suitable eukaryotic gene, plant gene (wheat and corn chlorophyll a / b binding protein gene leader sequence) or synthetic gene sequence. The present invention is not limited to constructs in which the non-translated region is derived from the 5' non-translated sequence of the accompanying promoter sequence. The leader sequence can also be derived from an unrelated promoter or coding sequence. Useful leader sequences in the context of the present invention include corn Hsp70 leader sequences (US 5,362,865 and US 5,859,347) and TMV ω elements.

[0418] Termination of transcription is achieved by being operably connected to the 3' non-translated DNA sequence of the RNA molecule of interest in the expression vector. The 3' non-translated region of the recombinant DNA molecule contains a polyadenylation signal, which works in plants to cause adenylate nucleotides to be added to the 3' end of the RNA. The 3' non-translated region can be obtained from various genes expressed in plant cells. The 3' non-translated region of nopaline synthase, the 3' non-translated region of the pea small subunit Rubisco gene, and the 3' non-translated region of the soybean 7S seed storage protein gene are usually used in this capacity. The 3' transcription and non-translated region containing the polyadenylic acid signal of Agrobacterium (Agrobacterium) tumor induction (Ti) plasmid gene are also suitable.

[0419] Transfer RNA

[0420] Transfer nucleic acid can be used for exogenous polynucleotide delivery to cell and comprises one, preferably two edge sequences and one or more RNA molecules of interest. Transfer nucleic acid can encode or not encode selectable marker. Preferably, transfer nucleic acid forms a part of binary vector in bacterium, wherein binary vector further comprises elements that allow vector to replicate in bacterium, select or maintain bacterial cells containing binary vector. After transfer to eukaryotic cells, the transfer nucleic acid component of binary vector can be integrated into the genome of eukaryotic cells, or for transient expression experiments, can only be expressed in cells.

[0421] As used herein, the term "extrachromosomal transfer nucleic acid" refers to a nucleic acid molecule that can be transferred from a bacterium (such as Agrobacterium species) to a eukaryotic cell such as a plant leaf cell. Extrachromosomal transfer nucleic acid is a genetic element that is known as an element that can be transferred, and the nucleotide sequence contained in its edge is subsequently integrated into the genome of the recipient cell. In this respect, transfer nucleic acid is usually flanked by two "edge" sequences, although a single edge at one end can be used in some cases and the second end of the transfer nucleic acid is randomly generated during the transfer process. The RNA molecule of interest is usually located between the left edge sample sequence and the right edge sample sequence of the transfer nucleic acid. The RNA molecule contained in the transfer nucleic acid can be operably connected with a variety of different promoters and terminator regulatory elements, and the regulatory elements promote its expression, i.e., the transcription of the RNA molecule and / or the translation of the RNA molecule. Transferring DNA from Agrobacterium species (T-DNAs), such as Agrobacterium tumefaciens or Agrobacterium rhizogenes and their artificial variants / mutants, may be the best characterized embodiment of the transfer nucleic acid. Another embodiment is P-DNA ("plant-DNA"), which comprises T-DNA border-like sequences from plants.

[0422] As used herein, "T-DNA" refers to the T-DNA of Agrobacterium tumefaciens Ti plasmid or Agrobacterium rhizogenes Ri plasmid, or a variant thereof for transferring DNA into a plant cell. The T-DNA may comprise a complete T-DNA, including right and left border sequences, but only needs to comprise the minimum sequence required for cis transfer, i.e., the right T-DNA border sequence. The T-DNA of the present invention has been inserted into them, and the RNA molecule of interest is inserted anywhere between the right and left border sequences (if present). The sequence encoding the trans factors (such as vir genes) required for the T-DNA transfer into a plant cell may be inserted into the T-DNA, or may be present on the same replicon as the T-DNA, or preferably be trans on a compatible replicon in an Agrobacterium host. Such a "binary vector system" is well known in the art. As used herein, "P-DNA" refers to a transfer nucleic acid isolated from a plant genome or a man-made variant / mutant thereof, and comprises a T-DNA border-like sequence at each end or at only one end.

[0423] As used herein, the "edge" sequence of a transfer nucleic acid can be isolated from a selected organism, such as a plant or bacterium, or is an artificial variant / mutant thereof. The edge sequence promotes and facilitates the transfer of the RNA molecule connected thereto, and can promote its integration in the recipient cell genome. In one embodiment, the length of the border sequence is 10-80bp. Agrobacterium species T-DNA edge sequences are well known in the art and include those described in Lacroix et al. (2008).

[0424] While traditionally only Agrobacterium species have been used to transfer genes into plant cells, a large number of systems have now been identified / developed that function in a similar manner to Agrobacterium species. Several non-Agrobacterium species have recently been genetically modified to have gene transfer capabilities (Chung et al., 2006; Broothaerts et al., 2005). These include Rhizobium species NGR234, Sinorhizobium meliloti, and Mezorhizobium loti.

[0425] Direct transfer of eukaryotic expression plasmids from bacteria to eukaryotic hosts was first achieved decades ago by fusion of protoplasts of mammalian cells and plasmid-carrying Escherichia coli (Schaffner, 1980). Since then, the number of bacteria capable of delivering genes into mammalian cells has steadily increased (Weiss, 2003), with independent discoveries by four groups (Sizemore et al., 1995; Courvalin et al., 1995; Powell et al., 1996; Darji et al., 1997).

[0426] As used herein, the terms "transfection," "transformation," and variations thereof are generally used interchangeably. A "transfected" or "transformed" cell may have been manipulated to introduce an RNA molecule of interest, or may be a progeny cell derived therefrom.

[0427] Recombinant cells

[0428] The present invention also provides recombinant cells, such as recombinant bacterial cells, fungal cells, plant cells, insect cells or animal cells, which are host cells transformed with one or more RNA molecules or vectors defined herein or a combination thereof. Suitable cells of the present invention include any cells that can be transformed with RNA molecules or recombinant vectors according to the present invention. In one embodiment, the transformed host cell is dead.

[0429] The recombinant cell can be a cell in culture, a cell in vitro, or a cell in an organism such as a plant, or a cell in an organ such as a seed or leaf. Preferably, the cell is in a plant, more preferably in a seed of a plant. In one embodiment, the recombinant cell is a non-human cell. Therefore, in one embodiment, the present invention relates to a non-human organism comprising one or more or all of the RNA molecules disclosed herein.

[0430] In one embodiment, the cell is an insect cell. In one embodiment, the insect cell is derived from Trichoplusia.

[0431] Another example of a suitable host cell is electrocompetent HT115 cells.

[0432] The host cell of the RNA molecule can be an untransformed cell or a cell transformed with at least one nucleic acid. Such nucleic acid can be related to lipid synthesis, or unrelated. The host cell of the present invention can be endogenously (that is, naturally) capable of expressing the RNA molecule defined herein, in which case the recombinant cell derived therefrom has the enhanced ability to produce RNA molecules, or can only produce the ability of the RNA molecules after being transformed with at least one RNA molecule defined herein. In one embodiment, the cell is a cell that can be used to produce lipids. In one embodiment, the recombinant cell of the present invention has the enhanced ability to produce non-polar lipids such as TAG.

[0433] Host cells of the present invention can be any cell capable of expressing at least one RNA molecule described herein, and include bacteria, fungi (including yeast), parasites, arthropods, animals and plant cells. Embodiments of host cells include Salmonella, Escherichia, Bacillus, Listeria, Saccharomyces, Spodoptera, Mycobacteria, Trichoplusia, Agrobacterium, BHK (baby hamster kidney) cells, MDCK cells, CRFK cells, CV-1 cells, COS (e.g., COS-7) cells and Vero cells. Further examples of host cells are E. coli, including E. coli K-12 derivatives; Salmonella typhi; Salmonella typhimurium, including attenuated strains; Spodoptera frugiperda; Spodoptera frugiperda; and non-tumorigenic mouse myoblast G8 cells (e.g., ATCC CRL 1246). Other suitable mammalian cell hosts include other kidney cell lines, other fibroblast cell lines (e.g., human, mouse or chicken embryonic fibroblast cell lines), myeloma cell lines, Chinese hamster ovary cells, mouse NIH / 3T3 cells, LMTK cells, and / or HeLa cells.

[0434] In a preferred embodiment, the plant cell is a seed cell, in particular a cell in the cotyledon or endosperm of a seed. In one embodiment, the cell is an animal cell. The animal cell can be any type of animal, such as, for example, a non-human animal cell, a non-human vertebrate cell, a non-human mammal cell, or a cell of an aquatic animal such as a fish or crustacean, an invertebrate, an insect, etc. Examples of algae cells used as host cells of the present invention include, for example, Chlamydomonas species (e.g., Chlamydomonas reinhardtii), Dunaliella species, Haematococcus species, Chlorella species, Thraustochytrium species, Schizochytrium species, and Volvox species.

[0435] Transgenic plants

[0436] The present invention also provides plants comprising one or more exogenous RNA molecules defined herein, cells according to the present invention, vectors according to the present invention, or combinations thereof. When used as a noun, the term "plant" refers to a complete plant, while the term "part thereof" refers to a plant organ (e.g., leaves, stems, roots, flowers, fruits, seeds); a single cell (e.g., pollen); a seed; a seed part, such as an embryo, endosperm, embryonic disc, or seed coat; a plant tissue, such as a vascular tissue; a plant cell and its progeny. As used herein, a plant part comprises a plant cell.

[0437] As used herein, in the context of modifying a plant, the terms "in a plant" and "in the plant" mean that the modification has occurred in at least a portion of the plant, including situations where the modification has occurred in the entire plant, and do not exclude situations where the modification occurs in only one or more but not all parts of the plant. For example, a tissue-specific promoter is said to be expressed "in a plant" even though it may only be expressed in certain parts of the plant. Similarly, "a transcription factor polypeptide that increases the expression of one or more glycolytic and / or fatty acid biosynthetic genes in a plant" means that the increased expression occurs in at least a portion of the plant.

[0438] As used herein, the term "plant" is used in its broadest sense, including any organism in the plant kingdom. Also included are red algae, brown algae, and green algae. It includes, but is not limited to, any kind of flowering plants, grasses, crops, or cereals (e.g., oilseeds, corn, soybeans), feed or forage, fruit or vegetable plants, herbs, woody plants, or trees. This does not mean that plants are limited to any particular structure. It also refers to unicellular plants (e.g., microalgae). The term "parts thereof" with respect to plants refers to plant cells and their progeny, multiple plant cells, structures present in any stage of plant development, or plant tissues. Such structures include, but are not limited to, leaves, stems, flowers, fruits, nuts, roots, seeds, seed coats, embryos. The term "plant tissue" includes differentiated and undifferentiated tissues of plants, including those present in leaves, stems, flowers, fruits, nuts, roots, seeds, e.g., embryonic tissue, endosperm, dermal tissue (e.g., epidermis, periderm), vascular tissue (e.g., xylem, phloem), or ground tissue (comprising parenchyma, collenchyma, and / or mesenchymal cells), and cells in culture (e.g., single cells, protoplasts, callus, embryos, etc.). Plant tissue can be a plant, organ culture, tissue culture, or cell culture.

[0439] 18:3 and 16:3 fatty acids are found in varying amounts in glycolipids of different plants. This is used to distinguish fatty acids with 3 double bonds, which are usually always C 18 The 18:3 plant atom contains C 16 and C 1816:3 plants of fatty acids. In 18:3 chloroplasts, the enzyme activity that catalyzes the conversion of phosphatidic acid to diacylglycerol and diacylglycerol to monogalactosyldiacylglycerol (MGD) is significantly lower than that of 16:3 chloroplasts. In 18:3 leaves, chloroplasts synthesize stearoyl ACP2 in the matrix, introduce the first double bond in the saturated hydrocarbon chain, and then hydrolyze the thioester. The released oleate is exported across the chloroplast envelope into the membrane of the eukaryotic part of the cell, probably the endoplasmic reticulum, where it is incorporated into PC. The oleoyl group connected to PC is desaturated in these membranes and then moved back into the chloroplast. The acyl group connected to MGD is a substrate for introducing the third double bond to produce MGD with two linolenic acid residues. This galactolipid is a characteristic of 18:3 plants such as Asteraceae and Fabaceae. In photosynthetically active cells of 16:3 plants, such as those represented by members of the Apiaceae and Brassicaceae, two pathways run in parallel to provide thylakoids with MGD. The synergistic "eukaryotic" sequence is supplemented to varying degrees by the "prokaryotic" pathway. Its reaction is limited to chloroplasts and results in a typical arrangement of acyl groups and their complete desaturation once esterified to MGD. The prokaryotic DAG skeleton carries C16:0 and its desaturated products in which C-2 position is excluded from C18: fatty acids. The C-1 position is occupied by C18 fatty acids and to a lesser extent by C16 groups. The similarity of the DAG skeleton of cyanobacterial lipids in 16:3 plants to the DAG skeleton synthesized by the chloroplast transport pathway illustrates this phylogenetic relationship and proves the existence of prokaryotic cells.

[0440] As used herein, the term "vegetative tissue" or "vegetative plant part" is any plant tissue, organ or part other than an organ used for sexual reproduction of a plant. Organs for sexual reproduction of a plant are specifically seed-bearing organs, flowers, pollen, fruits and seeds. Vegetative tissue and parts include at least plant leaves, stems (including bulbs and tillers, but not including the head), tubers and roots, but not including flowers, pollen, seeds including seed coats, embryos and endosperms, fruits including mesocarp tissue, pods with seeds and heads with seeds. In one embodiment, the vegetative part of a plant is an above-ground plant part. In another or further embodiment, the vegetative plant part is a green part, such as a leaf or stem.

[0441] A "transgenic plant" or variants thereof refers to a plant containing a transgene not found in wild-type plants of the same species, variety or cultivar. A transgenic plant as defined in the context of the present invention includes plants and their progeny which have been genetically modified using recombinant techniques to produce at least one polypeptide as defined herein in the desired plant or part thereof. Transgenic plant parts have a corresponding meaning.

[0442] The terms "seed" and "grain" are used interchangeably herein. "Grain" refers to mature grain, such as harvested grain or grain still on the plant but ready for harvest, but may also refer to grain after imbibition or germination, depending on the context. Mature grain typically has a moisture content of less than about 18%. In a preferred embodiment, the moisture content of the grain is at a level generally considered safe for storage, preferably 5%-15%, 6%-8%, 8%-10%, or 10%-15%. As used herein, "developing seed" refers to a seed before maturity, typically found in the reproductive structure of a plant after fertilization or flowering, but may also refer to such a seed before maturity that has been separated from a plant. Mature seeds typically have a moisture content of less than about 12%.

[0443] As used herein, the term "plant storage organ" refers to a part of a plant that is specialized for storing energy in the form of, for example, proteins, carbohydrates, lipids. Examples of plant storage organs are seeds, fruits, tuberous roots and tubers. Preferred plant storage organs of the present invention are seeds.

[0444] As used herein, the term "phenotypically normal" refers to a genetically modified plant or part thereof, such as a transgenic plant, or a storage organ of the present invention, such as a seed, tuber or fruit, that does not have significantly reduced growth and reproductive capacity compared to an unmodified plant or part thereof. Preferably, when grown under the same conditions, the biomass, growth rate, germination rate, storage organ size, seed size and / or number of surviving seeds produced is not less than 90% of the biomass, growth rate, germination rate, storage organ size, seed size and / or number of surviving seeds of a plant lacking the recombinant polynucleotide. The term does not include plant characteristics that may differ from wild-type plants but do not affect the usefulness of the plant for commercial purposes, such as the ballerina phenotype of seedling leaves. In one embodiment, the phenotypically normal genetically modified plant or part thereof comprises a recombinant polynucleotide encoding a silencing suppressor that is operably linked to a plant storage organ-specific promoter and has substantially the same growth or reproductive capacity as a corresponding plant or part thereof that does not comprise the polynucleotide.

[0445] Plant provided by the invention or intended for implementation of the present invention comprises monocotyledon and dicotyledon.In preferred embodiments, plant of the present invention is crops (for example, cereals and dry beans, corn, wheat, potato, rice, sorghum, millet, cassava, barley) or beans such as soybean, kidney bean or pea.Described plant can be grown for producing edible root, tuber, leaf, stem, flower or fruit.Plant can be the vegetable plant that its nutrition part is used as food. The plant of the present invention can be: Acrocomia aculeata, Arabidopsis thaliana, Aracinishypogaea, Astrocaryum murumuru, Astrocaryum vulgare, Attalea geraensis, Attalea humilis, Attalea oleifera (andaiá), Attalea phalerata (uricuri), Attalea speciosa, Avena sativa, Beta vulgaris, Brassica sp, such as Brassica carinata, Brassica juncea, Brassica napobrassica, Brassica napus, Camelina sativa, Carthamus tinctorius, Brazilian nectarine (Caryocar brasiliense), coconut (Cocos nucifera), Ethiopian two-sectioned shepherd's purse (Crambe abyssinica), melon (Cucumis melo), African palm (Elaeis guineensis), soybean (Glycine max), cotton (Gossypium hirsutum), Helianthus species such as sunflower (Helianthus annuus), barley (Hordeum vulgare), Jatropha curcas, andada tree (Joannesia princeps), Lemna species such as Lemna aequinoctialis, Lemna disperma, Lemna ecuadoriensis, Lemna gibba, Lemna japonica, Lemna minor,Lemnaminuta, Lemna obscura, Lemna paucicostata, Lemna perpusilla, Lemna Lemnaminuta tenera, Lemna trisulca, Lemna turionifera, Lemnavaldiviana, Lemna yungensis, Licania rigida, Linumusitatissimum, Lupinus angustifolius, Mauritia flexuosa, Maximiliana maripa, Miscanthus species, such as Miscanthusx giganteus and Miscanthus sinensis, Nicotiana species, such as Nicotiana tabacum or Nicotiana benthamiana benthamiana, Oenocarpus bacaba, Oenocarpus bataua, Oenocarpus distichus, Oryza species such as Oryza sativa and Oryza glaberrima, Panicum virgatum, Paraqueiba paraensis, Perseaamencana, Pongamia spp., Populus trichocarpa, Ricinus communis, Saccharum species, Sesamumindicum, Solanum tuberosum, Sorghum species such as Sorghum bicolor, Sorghum vulgare, Theobroma grandiforum, Trifolium species, Brazilian needle palm (Trithrinax brasiliensis), Triticum species such as wheat (Triticum aestivum), maize (Zeamays), alfalfa (Medicago sativa), rye (Secale cerale), sweet potato (Lopmoea batatus), cassava (Manihot esculenta),Coffee (Cofea) species, pineapple (Anana comosus), citrus (Citrus) species, cocoa (Theobroma cacao), tea (Camellia senensis), banana (Musa) species, avocado (Perseaamericana), fig (Ficus casica), guava (Psidium guajava), mango (Mangifer indica), olive (Olea europaea), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamia intergrifolia) and almond (Prunus amygdalus). For example, the plant of the present invention can be Nicotiana benthamiana. ,

[0446] Other preferred plants include C4 grasses, for example, Andropogongerardi, Bouteloua curtipendula, B. gracilis, Buchloedactyloides, Schizachyrium scoparium, Sorghastrum nutans, Sporobolus creptandrus, C3 grasses, such as Elymus canadensis, Lespedeza capitata and Petalostemum villosum, Aster azureus, and woody plants, such as Quercus ellipsoidalis and Q. macrocarpa, in addition to those mentioned above. Other preferred plants include C3 grasses.

[0447] In a preferred embodiment, the plant is an angiosperm.

[0448] In one embodiment, plant is an oilseed plant, preferably an oilseed crop plant. As used herein, "oilseed plant" is a plant species for commercial production of lipids from the seeds of a plant. Oilseed plants can be, for example, oilseed rape (e.g., rapeseed), corn, sunflower, safflower, soybean, sorghum, flax (linseed) or beet. In addition, oilseed plants can be other Brassica, cotton, peanut, Brassica, mustard, castor, sesame, safflower, Jatropha curcas or plants that produce nuts. The plant can produce high levels of lipids in its fruit, such as olive, oil palm or coconut. Horticultural plants that can apply the present invention are lettuce, cabbage or vegetable Brassica, including cabbage, broccoli or cauliflower. The present invention can be applied to tobacco, melons, carrots, strawberries, tomatoes or peppers.

[0449] In a preferred embodiment, the transgenic plant is homozygous for each gene (transgene) that has been introduced, so that its offspring do not segregate for the desired phenotype. The transgenic plant can also be heterozygous for the introduced transgene, preferably uniformly heterozygous for the transgene, for example in the F1 progeny that has been grown from hybrid seeds. Such plants can provide advantages well known in the art, such as hybrid vigor.

[0450] Conversion

[0451] The RNA molecules disclosed herein can be stably introduced into the above-mentioned host cells and / or non-human organisms, such as plants. For the avoidance of doubt, embodiments of the present invention encompass the above-mentioned plants stably transformed with the RNA molecules disclosed herein. As used herein, the terms "stable transformation", "stably transformed" and variants thereof refer to the integration of RNA molecules or nucleic acids encoding RNA molecules into the genome of the cell so that they are transferred to daughter cells during cell division without the need for positive selection of their presence. Stable transformants or their progeny can be identified by any method known in the art, such as Southern blotting on chromosomal DNA, or in situ hybridization of genomic DNA, so that they can be selected.

[0452] Transgenic plants can be produced using techniques known in the art, such as those generally described in Slater et al., Plant Biotechnology - The Genetic Manipulation of Plants, Oxford University Press (2003), and Christou and Klee, Handbook of Plant Biotechnology, John Wiley and Sons (2004).

[0453] In one embodiment, plants can be transformed by topically applying the RNA molecules according to the present invention to plants or parts thereof. For example, the RNA molecules can be provided as a formulation with a suitable carrier and sprayed, dusted or otherwise applied to the surface of a plant or part thereof. Thus, in one embodiment, the methods of the present invention encompass the introduction of RNA molecules disclosed herein into plants, the methods comprising topically applying a composition comprising the RNA molecules to the plant or part thereof.

[0454] Agrobacterium-mediated transfer is a widely applicable system for introducing genes into plant cells, because DNA can be introduced into cells in whole plant tissues, plant organs or explants in tissue culture for transient expression or for stable integration of DNA in plant cell genomes. For example, the flower dip method (in planta) can be used. It is well known in the art to use plant integration vectors mediated by Agrobacterium to introduce DNA into plant cells. The DNA region to be transferred is limited by the edge sequence, and the intervening DNA (T-DNA) is usually inserted into the plant genome. Due to the simplicity and clear nature of gene transfer, this method is selected.

[0455] Acceleration methods that can be used include, for example, microparticle bombardment, etc. One embodiment of a method for delivering a transforming nucleic acid molecule to a plant cell is microparticle bombardment. This method has been reviewed in Yang et al., Particle Bombardment Technology for Gene Transfer, Oxford Press, Oxford, England (1994). Non-biological particles (microparticles) that can be coated with nucleic acids and delivered to, for example, cells of immature embryos by a propulsive force. Exemplary particles include those composed of tungsten, gold, platinum, etc.

[0456] In another method, plasmids can be stably transformed. The disclosed method for plasmid transformation in higher plants includes the delivery of a particle gun containing a selectable marker DNA and targeting DNA to a plasmid genome by homologous recombination (US5,451,513, US 5,545,818, US 5,877,402, US 5,932479, and WO 99 / 05265). Other methods of cell transformation can also be used, including but not limited to by directly transferring DNA to pollen, by directly injecting DNA into the reproductive organs of plants, or by directly injecting DNA into the cells of immature embryos, and then rehydrating the dried embryos, thereby importing DNA into plants.

[0457] The regeneration, development and culture of plants from single plant protoplast transformants or from various transformed explants are well known in the art (Weissbach et al., in: Methods in Plant Molecular Biology, Academic Press, San Diego, CA, (1988)). Such regeneration and growth processes generally include the following steps: selecting transformed cells, culturing those individualized cells through the usual stages of embryonic development from the rooted plantlet stage. Transgenic embryos and seeds are similarly regenerated. The resulting transgenic rooted shoots are then planted in a suitable plant growth medium, such as soil.

[0458] The development or regeneration of plants containing foreign genes is well known in the art. Preferably, the regenerated plants are self-pollinated to provide homozygous transgenic plants. Otherwise, the pollen obtained from the regenerated plants is crossed with seed-grown plants of agronomically important lines. Instead, pollen from plants of these important lines is used to pollinate the regenerated plants. Transgenic plants of the present invention containing the desired polynucleotides are cultivated using methods well known to those skilled in the art.

[0459] In order to confirm the presence of transgenic in transgenic cells and plants, polymerase chain reaction (PCR) amplification or Southern blot analysis can be performed using methods known to those skilled in the art. The expression product of transgenic can be detected in any of a variety of ways, depending on the nature of the product, and includes Northern blot hybridization, protein blot and enzyme assay. Once transgenic plants are obtained, they can be grown to produce plant tissues or parts with desired phenotypes. Plant tissues or plant parts can be harvested, and / or seeds can be collected. Seeds can be used as the source of additional plants with tissues or parts with desired characteristics. Preferably, the vegetative plant parts are harvested when the output of non-polar lipids is the highest. In one embodiment, the vegetative plant parts are harvested approximately when flowering or after flowering begins. Preferably, the plant parts are harvested approximately when aging begins, usually showing yellowing and drying of leaves.

[0460] Transgenic plants formed using Agrobacterium or other transformation methods typically contain a single locus on one chromosome. Such transgenic plants can be considered hemizygous for the added gene. More preferred are transgenic plants that are homozygous for the added gene, i.e., transgenic plants that contain two added genes, one gene at the same locus on each chromosome of a chromosome pair. Homozygous transgenic plants can be obtained by fertilizing the hemizygous transgenic plants themselves, germinating some of the resulting seeds, and analyzing the resulting plants for the gene of interest.

[0461] It should also be understood that two different transgenic plants containing two independently separated foreign genes or loci can also be hybridized (mating) to produce offspring containing two groups of genes or loci. The selfing of the appropriate F1 offspring can produce a plant that is isozygous for the foreign gene or locus. Backcrossing with the parental plant and outcrossing with non-transgenic plants, as well as asexual reproduction are also contemplated. Similarly, transgenic plants can be hybridized with a second plant comprising a genetic modification such as a mutant gene and offspring containing both the identified transgenic and genetic modifications. The description of other breeding methods commonly used for different traits and crops can be found in Fehr, In: Breeding Methods for Cultivar Development, Wilcox J. American Society of Agronomy (Breeding Methods for Cultivar Development), Madison, Wisconsin, USA (1987).

[0462] preparation

[0463] RNA molecules according to the present invention can be provided as various formulations. For example, the RNA molecules can be in the form of solids, ointments, gels, creams, powders, pastes, suspensions, colloids, foams or aerosols. Solid forms can include powders, pulvis, granules, microspheres, pills, lozenges, tablets, filled films (including seed coatings), etc., which can be water dispersible. In one embodiment, the composition is in the form of a concentrate.

[0464] In one embodiment, the RNA molecule can be provided as a topical formulation. In one embodiment, the formulation stabilizes the RNA molecule in the formulation and / or in vivo. For example, the RNA molecule can be provided in a lipid formulation. For example, the RNA molecule can be provided in a liposome. In one embodiment, the formulation comprises a transfection facilitator.

[0465] In one embodiment, RNA molecules can be incorporated into a formulation suitable for field application. In one embodiment, the field includes plants. Suitable plants include crops (e.g., cereals and beans, corn, wheat, potato, cassava, rice, sorghum, soybean, cassava, barley or pea), or leguminous plants). The plant can be grown for producing edible roots, tubers, leaves, stems, flowers or fruits. In one embodiment, the crop plant is a cereal plant. The embodiment of cereal plants includes but is not limited to wheat, barley, sorghum oats and rye. In these embodiments, the RNA molecule can be formulated for application to any part of a plant or plant in any suitable manner. For example, the composition can be formulated for application to leaves, stems, roots, fruits, vegetables, grains and / or buds of a plant. In one embodiment, the RNA molecule is formulated for application to the leaves of a plant, and can be sprayed on the leaves of a plant.

[0466] Depending on the desired formulation, the RNA molecules described herein can be formulated with a variety of other agents. Exemplary agents include one or more of suspending agents, coalescing agents, bases, buffers, bittering agents, flavoring agents, preservatives, propellants, thixotropic agents, antifreeze agents, and coloring agents.

[0467] In other embodiments, the RNA molecule preparation may comprise an insecticide, a pesticide, a fungicide, an antibiotic, an anthelmintic, an antiparasitic agent, an antiviral agent, or a nematicide.

[0468] In another embodiment, the RNA molecules can be incorporated into pharmaceutical compositions. Such compositions generally include the RNA molecules described herein and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" includes solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. that are compatible with drug administration. Supplementary active compounds can also be incorporated into the composition.

[0469] A pharmaceutical composition is formulated to be compatible with its intended route of administration.Examples of routes of administration include parenteral, such as intravenous, intradermal, subcutaneous, inhalation, transdermal (topical), transmucosal, oral, and rectal administration.

[0470] In one embodiment, the active compound is prepared together with a carrier that protects the compound from rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. For example, liposomal suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in US 4,522,811.

[0471] The RNA molecules according to the present invention can be provided in a kit or package. For example, the RNA molecules disclosed herein can be packaged in a suitable container with written instructions for use in producing the above-mentioned cells or organisms or treating a condition.

[0472] Methods for controlling non-human organisms

[0473] In one embodiment, the RNA molecules according to the present invention can be used to control non-human organisms, such as insects. These uses involve applying the RNA molecules according to the present invention using various methods. In one embodiment, the RNA molecules according to the present invention can be provided as insect baits for insect ingestion. In another embodiment, the RNA molecules can be sprayed on insects as needed. In another embodiment, the RNA molecules can be sprayed on plants or crops to protect the plants or crops from insects. Exemplary crops include cotton, corn, tomatoes, chickpeas, pigeon peas, alfalfa, rice, sorghum and cowpeas.

[0474] In one embodiment, RNA molecules can be provided to modify insect behavior. In another embodiment, RNA molecules can be provided to kill insects. In another embodiment, RNA molecules can be provided to reduce insect fertility. Exemplary insect targets include household insects. Other exemplary insect targets include sap-sucking insects such as aphids (e.g., Myzus persicae, Metopolophium dirhodum, Rhopalosiphum padi, Aphis glycines, Aphis glycines. Additional exemplary insect targets include arachnids, mosquitoes, ectoparasites, flies, spider mites, thrips, ticks, red fowl mites, ants, cockroaches, termites, crickets including house crickets, silverfish, booklice, beetles, midges, mosquitoes, and fleas. Other exemplary insect targets include agricultural pests. Examples include sap feeders, such as stink bugs and aphids, chewing insects such as caterpillars, beetles, worms, filing insects such as thrips and slugs, moths, flies such as fruit flies, grain pests such as grain borers, weevils, and grain moths.

[0475] In one embodiment, the insect is a sap-sucking insect. In this example, the RNA molecule may have antisense activity to MpC002 and / or MpRack-1. In one embodiment, the sap-sucking insect is an aphid. In another embodiment, the aphid is Myzus persicae.

[0476] In one embodiment, the insect target is an ant (e.g., Argentine ant (Linepithema humile)), cotton bollworm or corn earworm (Helicoverpa armigera) or fly (e.g., Lucilia cuprina). In one embodiment, the target insect is cotton bollworm (Helicoverpa armigera) and the RNA molecule has antisense activity to the ABC transporter white gene (ABCwhite). In another embodiment, the target insect is Argentine ant (Linepithema humile) and the RNA molecule has antisense activity to pheromone biosynthesis activating neuropeptide (PBAN). In another embodiment, the target insect is Lucilia cuprina and the RNA molecule has antisense activity to one or more genes encoding proteins selected from the group consisting of V-type proton ATPase catalytic subunit A, RNase 1 / 2, chitin synthase, ecdysone receptor and gamma-tubulin 1 / 1-like.

[0477] In the above embodiments, the compositions and RNA molecules disclosed herein can be provided in a dispenser. In one embodiment, the dispenser is a trap or bait. In one embodiment, the trap and / or bait comprises a bait containing one or more RNA molecules disclosed herein.

[0478] In one embodiment, the invention includes a method of controlling insect behavior comprising spraying, dusting, or otherwise applying the RNA molecules disclosed herein to the insect. In this embodiment, the RNA molecules can be sprayed, dusted, or otherwise applied directly to the insect. In another embodiment, the RNA molecules can be sprayed, dusted, or otherwise applied to plants or crops prior to insect infestation.

[0479] In one embodiment of the present invention, the insect or arachnid may belong to the following orders: Acari, Arachnida, Anoplura, Blattodea, Coleoptera, Collembola, Dermaptera, Dictyoptera, Dipura, Emboptera, Ephemeroptera, Gryllobatodea, Hemiptera, Heteroptera, Homoptera, Hymenoptera, Isoptera, Lepidoptera. ), Mallophaga, Mecoptera, Neuroptera, Odonata, Orthoptera, Phasmida, Phithiraptera, Plecoptera, Protura, Pso coptera), Siphunculata, Thysanura, Sternorrhynchoa, Strepsiptera, Thysanoptera, Trichoptera, Zoraptera and Zygentoma.

[0480] In a preferred but non-limiting embodiment of the present invention, the insect or arachnid is selected from the group consisting of: (1) Acari: mites, including the order Ixodida (2) Arachnida: spiders and whiteflies, examples include: black widow (Latrodectus mactans) and brown loxosceles recluse (3) Anoplura: lice, such as human lice (Pediculus humanus) (4) Blattodea: cockroaches, including German cockroach (Blatella germanica), Periplaneta, including American cockroach (Periplaneta americana) and Australian cockroach (Periplaneta australiasiae), Blatta, including Oriental cockroach (Blatta orientalis) and Brown-banded cockroach (Supella longipalpa). The most preferred target is the German cockroach (Blatella germanica). (5) Coleoptera: Beetles, examples include: family of powderpost beetles (family of Bostricholidae); species of the genus Dendroctonus (black turpentine beetle, southern rosin beetle, IPS carving beetle); carpet beetles (Anthrenus, Attagenus); house beetles (family of the family Cerambicyidae: Hylotrupes bajulus; furniture beetle (Anobium punctatum); Tribolium; Trogoderma granarium; toothed beetle (Oryzaephilus)). sarinamensis, etc. (bookworms) (6) Dermaptera: the family of the family Earwigs (7) Diptera: mosquitoes (Culicidae) and flies (Brachycera), examples are: Anophelinae, for example Anopheles and Culicinae, such as Aedes fulvus; Tabanidae, for example Tabanus punctifer, Glossina morsitans morsitans, Psychodidae and Calyptratae, such as Musca domestica,(8) Heteroptera: Bed bugs, such as Cimex lectularius (9) Hymenoptera: Apocrita, including Formicoidea, Apoidea: Fire ant (Solenopsis invicta), Pharaoh ant (Monomorium pharaonis), Camponotus species, Iasius niger, Tetramorium caespitum, Myrmica rubra, Formica species, Crematogaster lineolata species, Iridomyrmex humilis, Pheidole species, Dasymutilla occidentalis) etc. (10) Isoptera: Termites, examples include: Florida black-winged subterranean termite (Amitermes floridensis), Oriental subterranean termite (Reticulitermes flavipes), Western subterranean termite (R. hesperus), Formosan white-skinned termite (Coptotermes formosanus), Western drywood termite (Incisitermes minor), forest termite (Neotermes connexus) and Termitidae (Termitidae) (11) Lepidoptera: Moths, examples include: Tineidae and Oecophoridae, for example, the common clothes moth (Tineola bisselliella), and Pyrolidae, for example Pyrolis farinalis, etc. (12) Psoralea: Psocids (13) Siphonaptera: fleas, for example Pulex irritans (14) Sternorrhyncha: Aphididae (15) Zygentoma: silverfish, examples are: Thermobia domestica and Lepisma saccharina;

[0481] Other target insects or arachnids include household insects, external parasites and insects and / or arachnids relevant to public health and healthcare, such as but not limited to flies, spider mites, thrips, ticks, red poultry mites, ants (e.g., by targeting PBAN), cockroaches, termites, crickets including house crickets, silverfish, book lice, beetles, earwigs, mosquitoes and fleas. More preferred targets are cockroaches (Blattodea), such as but not limited to Blatella species (e.g., Blatella germanica), Periplaneta species (e.g., Periplaneta americana, Periplaneta australiasiae); Blatta species (e.g., Blatta orientalis) and Supella species (e.g., Supella longipalpa);The superfamily Formicoidea, such as, but not limited to, Solenopsis species (e.g., Solenopsis invicta), Monomorium species (e.g., Monomorium pharaonis), Camponotus species (e.g., Camponotus), Lasius (e.g., Iasius niger), Tetramorium spp. (e.g., Tetramorium caespitum), Myrmica (e.g., Myrmica rubra), Formica species, Crematogaster (e.g., Crematogaster lineolata), Crematogaster (e.g., Iridomyrmex humilis), Pheidole species, and Dasymutilla (e.g., Dasymutilla occidentalis) termites (Isoptera and / or Termitidae), such as, but not limited to, Termitidae (e.g., Amitermes floridensis), Reticulitermes (e.g., Reticulitermes flavipes, Reticulitermes hesperus, Coptotermes (e.g., Coptotermes formosanus), Incisitermes (e.g., Incisitermes minor), Neotermes (e.g., Neotermes connexus).

[0482] In one embodiment, the target RNA encodes an insect lactate synthase.

[0483] When delivered and / or expressed in plants, the RNA molecules of the invention can have a wide range of desired properties that affect, for example, agronomic traits, insect resistance (e.g., by targeting genes such as MpC002, MpRack-1, and ABC transporter genes), disease resistance (e.g., by targeting genes such as LanR), herbicide resistance, sterility, grain characteristics, etc. The target RNA molecules may be involved in the metabolism of oils, starches, carbohydrates, nutrients, etc., or may be involved in the synthesis of proteins, peptides, fatty acids, lipids, recombination frequencies (by targeting genes such as DDM1 and FANCM), waxes, oils (by targeting genes such as TOR), starches, sugars, carbohydrates, flavors, odors, toxins, carotenoids, hormones (by targeting genes such as EIN2, NCED1, and NCED2), polymers, flavonoids (by targeting genes such as chalcone synthase), storage proteins, phenolic acids, alkaloids, lignin, tannins, cellulose, glycoproteins, glycolipids, etc.

[0484] In particular embodiments, the plant produces: increased levels of enzymes for oil production in plants, such as Brassicas plants, for example rapeseed or sunflower, safflower, flax, cotton, soybean or corn; enzymes involved in starch synthesis in plants, such as potatoes, corn and cereals, such as wheat, barley or rice; synthetic enzymes, or proteins themselves, natural drugs, such as pharmaceuticals or veterinary products.

[0485] In another embodiment, the RNA molecules of the invention are intended to prophylactically or therapeutically treat infection with a fungal pathogen selected from the group consisting of: Altemaria species; Armillaria mellae; Armillaria mellae; Blumeria graminis (by targeting the Mlo gene using RNA molecules as described in Example 17), Boletus granulatus; Botritis cinerea; Botrytis fabae; Candida albicans; Claviceps purpurea; Cronartium ribicola; Epicoccum purpurescens; Epidermophyton floccosum; Fomes annosus; Fusarium oxysporum; oxysporum; Gaeumannomyces graminis var. tritici; Glomerella cingulata; Gymnosporangium juniperi-virginianae; Microsporum canis; Monilinia fructicola; Physoderma alfalfae; Phytoptherainfestans; Pityrosporum orbiculare; Polyporus sulphureus; Puccinia spp.; Saccharomyces cerevisiae; Septoria apiicola; Trichophyton rubrum rubrum; T. mentagrophytes; Ustilago spp.; Venturia inaequalis; and Venturia inaequalis.

[0486] Exemplary Conditions to be Treated

[0487] RNA molecules according to the present invention can be used in methods for various conditions. In some embodiments, the present invention relates to a method for treating cancer, which method comprises administering the RNA molecules disclosed herein. The term "cancer" refers to or describes a mammalian physiological condition that is generally characterized by unregulated cell growth / proliferation. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More specific examples of such cancers include, but are not limited to, squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer (including small cell lung cancer), non-small cell lung cancer, lung adenocarcinoma and lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastric cancer or gastric cancer (including gastrointestinal cancer and gastrointestinal stromal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, visceral urinary tract cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, melanoma, superficial spreading melanoma, Lentigo maligna melanoma, acral lentiginous melanoma, nodular melanoma, multiple myeloma and B-cell lymphoma (including low-grade / follicular non-Hodgkin lymphoma (NHL)); mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's macroglobulinemia); chronic lymphocytic leukemia (CLL); acute lymphocytic leukemia (ALL); hairy cell leukemia; chronic myeloid leukemia; and post-transplant lymphoproliferative disorder (PTLD) and abnormal vascular proliferation associated with keloids, edema (e.g., edema associated with brain tumors), Meig's syndrome, brain cancer and head and neck cancer, and related metastases. Thus, in one embodiment, the present invention relates to methods of treating breast, ovarian, colon, prostate, lung, brain, skin, liver, stomach, pancreas or blood-based cancers.

[0488] In other embodiments, the methods described herein are used to treat cancers associated with mutations in BRCA1, BRCA2, PALB2, OR RAD51B, RAD51C, RAD51D or related genes. In other embodiments, the methods described herein are used to treat cancers associated with mutations in DNA mismatch repair-related genes such as MSH2, MLH1, PMS2 and related genes. In other embodiments, the methods described herein are used to treat cancers with silenced DNA repair genes such as BRCA1, MLH1, OR RAD51B, RAD51C, OR RAD51D.

[0489] In other embodiments of the present invention, the methods described herein are used to kill cells with impaired DNA repair processes. For example, cells with impaired DNA repair may abnormally express genes involved in DNA repair, DNA synthesis, or homologous recombination. Exemplary genes include XRCC1, ADPRT (PARP-1), ADPRTL2, (PARP-2), POLYMERASE BETA, CTPS, MLH1, MSH2, FANCD2, PMS2, p53, p21, PTEN, RPA, RPAl, RPA2, RPA3, XPD, ERCC1, XPF, MMS19, RAD51, RAD51B, RAD51C, RAD51D, DMC1, XRCCR, XRCC3, BRCA1, BRCA2, PALB2, RAD52, RAD54, RAD50, MREU, NB51, WRN, BLM, KU70, KU80, ATM, ATR CPIK1, HK2, FANCA, FANCB, FANCC, FANCD1, FANCD2, FANCE, FANCF, FANCG, FANCC, FANCD1, FANCD2, FANCE, FANCF, FANCG, RAD1, and RAD9. In one embodiment, the methods described herein are used to kill cells with mutant tumor suppressor genes. For example, the cells can have one or more mutations in BRCA1 or BRCA2.

[0490] In other embodiments of the present invention, the methods described herein are used to treat cells transformed by viruses. In other embodiments of the present invention, the methods described herein are used to kill cells transformed by latent viruses. Exemplary latent viruses include CMV, EBV, herpes simplex virus (type I and type II) and varicella zoster virus. In other embodiments of the present invention, the methods described herein are used to treat active viral infections caused by viruses that cause cancer, immunodeficiency, hepatitis, encephalitis, pneumonia or respiratory diseases. Exemplary viruses include parvovirus, poxvirus, herpes virus.

[0491] In other embodiments of the invention, the methods described herein are used to treat Zika virus, Colorado tick fever (caused by the RNA virus Coltivirus), West Nile fever (encephalitis, caused by a flavivirus that occurs primarily in the Middle East and Africa), yellow fever, rabies (caused by different strains of the Rhabdoviridae family), viral hepatitis, gastroenteritis (viral) - acute viral gastroenteritis caused by Norwalk and Norwalk-like viruses, rotaviruses, Caliciviruses and Astroviruses, poliomyelitis, influenza (Flu) caused by orthomyxoviruses that are capable of undergoing frequent antigenic variation, measles (Rubella), Paramyxoviridae, mumps, respiratory syndromes, including pneumonia and acute respiratory syndrome, including laryngitis caused by various viruses collectively known as acute respiratory viruses, and respiratory diseases caused by respiratory syncytial virus.

[0492] Example

[0493] Example 1: Materials and Methods

[0494] Synthesis of gene constructs

[0495] To design a typical ledRNA construct, a target RNA region of about 100-1000 nucleotides, typically 400-600 nucleotides in length, is identified. In one embodiment, the 5' half of the sequence and about 130 nt of the flanking region and similarly the 3' half and 130 nt of the flanking region are oriented in the antisense direction relative to the promoter. These sequences are interrupted by a sense target sequence of 400-600 nucleotides ( Figure 1 A) The 5' end of the resulting construct is preceded by a promoter, such as the T7 or SP6 RNA polymerase promoter, and the 3' end is engineered to include a restriction enzyme cleavage site to allow for termination of in vitro transcription.

[0496] For transcription in cells such as bacterial cells, an inducible promoter is used, for example, to introduce promoter and terminator sequences to promote expression as a transgene. The double-stranded region and loop sequence lengths may vary. Constructs are prepared using standard cloning methods or ordered from commercial service providers.

[0497] RNA synthesis

[0498] After digestion with a restriction enzyme to linearize the DNA at the 3' end, transcription using RNA polymerase generates 5' and 3' arms of the ledRNAi transcript that anneal to the central target sequence. The molecule contains a central stem or double-stranded region with a single nick and terminal loops. The central sequence can be oriented in the sense or antisense direction relative to the promoter (respectively). Figure 1 A, Figure 1 B).

[0499] For in vitro synthesis, DNA of the construct was digested at the 3' restriction site using an appropriate restriction enzyme, precipitated, purified and quantified. RNA synthesis was achieved using RNA polymerase according to the manufacturer's instructions. The ledRNA was resuspended in annealing buffer (25 mM Tris-HCL, pH 8.0, 10 mM MgCl2) using DEPC-treated water to inactivate any traces of RNases. The yield and integrity of RNA produced by this method were determined by nanodroplet analysis and gel electrophoresis (see respectively). Figure 2 ).

[0500] Synthesis of ledRNA in bacterial cells was achieved by introducing the construct into E. coli strain HT115. Transformed cell cultures were induced with IPTG (0.4 mM) to express T7 RNA polymerase, providing transcription of the ledRNA construct. RNA was extracted from bacterial cells and purified essentially as described by Timmons et al. (2001).

[0501] For Cy3-labeled RNA transcription, the ribonucleotide (rNTP) mixture contains 10mM each of ATP, GTP, CTP, 1.62mM UTP and 8.74mM Cy3-UTP. The transcription reaction was incubated at 37°C for 2.5 hours. The transcription reaction (160μl) was transferred to an Eppendorf tube, 17.7μl turbo DNase buffer and 1μl turbo DNA were added, and incubated at 37°C for 10 minutes to digest the DNA. Then 17.7μl Turbo DNAse inactivation solution was added, mixed, and incubated at room temperature for 5min. The mixture was centrifuged for 2 minutes and the supernatant was transferred to a new Eppendorf tube without RNAse. 1.5μl samples of each transcription reaction were electrophoresed on a gel to test the quality of the RNA product. Typically, depending on the construct, an RNA band of 500bp to 1000bp in size was observed. Precipitate the RNA by adding 88.5 μl 7.5 M ammonium acetate and 665 μl cold 100% ethanol to each tube. Chill the tubes to -20°C for several hours or overnight and then centrifuge at 4°C for 30 minutes. Carefully remove the supernatant and wash the RNA pellet with 1 ml 70% ethanol (prepared with nuclease-free water) at -20°C and centrifuge. Dry the pellet and resuspend the purified RNA in 50 μl 1× RNAi annealing buffer. Measure RNA concentration using the nanodrop method and store at -80°C until use.

[0502] Example 2: Design of ledRNA

[0503] like Figure 1As shown schematically in A, a typical ledRNA molecule comprises a sense sequence that can be considered as two adjacent sense sequences covalently linked and identical to the target RNA, an antisense sequence that is complementary to the sense sequence and divided into two regions, and two loops that separate the sense sequence from the antisense sequence. Therefore, the DNA construct encoding this form of ledRNA comprises a promoter for transcription of the ledRNA coding region, a first antisense region complementary to the region toward the 5' end of the target RNA, a first loop sequence, a sense sequence, a second loop sequence, and then a second antisense region complementary to the 3' end region of the target RNA, and finally a means of terminating transcription. In this arrangement, two antisense sequences are located on the flanks of the sense sequence and the loop sequence. When transcribed, the two regions of the antisense sequence anneal with the sense sequence to form a dsRNA stem with two flanking loops.

[0504] In another but related form of ledRNA, the sense sequence is divided into two regions, while the two antisense regions remain as a single sequence ( Figure 1 B). Thus, the DNA construct encoding this second form of ledRNA comprises, in 5' to 3' order, a promoter for transcription of the ledRNA coding region, a first sense region identical to the region towards the 3' end of the target RNA, a first loop sequence, an antisense sequence, a second loop sequence, then a second sense region identical to the 5' end of the target RNA, and finally a means for terminating transcription. In this arrangement, the two sense sequences flank the antisense sequence and the loop sequence.

[0505] Without wishing to be bound by theory, these ledRNA structures will be more resistant to exonucleases than open-ended dsRNAs formed between single-stranded sense and antisense RNAs without loops, and also more resistant to exonucleases than hairpin RNAs with only a single loop, because of the closed loops at each end. In addition, the inventors contemplate that the loops at both ends of the dsRNA stem will allow Dicer to effectively access both ends, thereby enhancing the processing of dsRNA into sRNA and silencing efficiency.

[0506] As a first example, a genetic construct for in vitro transcription was prepared using T7 or SP6 RNA polymerase to form LedRNA targeting a gene encoding GFP or GUS. The ledGFP construct comprises the following regions in order: the first half of the antisense sequence corresponding to nucleotides 358-131 of the GFP coding sequence (CDS) (SEQ ID NO: 7), the first antisense loop corresponding to nucleotides 130-1 of the GFPCDS, the sense sequence corresponding to nucleotides 131-591 of the GFP CDS, the second antisense loop corresponding to nucleotides 731-592 of the GFPCDS, and the second half of the antisense sequence corresponding to nucleotides 591-359 of the GFPCDS.

[0507] The ledGUS construct comprises the following regions in sequence: the first half of the antisense sequence corresponding to nucleotides 609-357 of GUS CDS (SEQ ID NO: 8); and the first antisense loop corresponding to nucleotides 356 to 197 of GUS CDS, the sense sequence corresponding to nucleotides 357-860 of GUS CDS, the second antisense loop corresponding to nucleotides 1029-861 of GUS CDS; the second half of the antisense sequence corresponding to nucleotides 861-610 of GUS CDS.

[0508] To prepare separate strands of sense / antisense GUS dsRNA (conventional dsRNA), the same target sequence corresponding to nucleotides 357-860 of the GUS CDS was ligated between T7 and SP6 promoters in the pGEM-TEasy vector. The sense and antisense strands were transcribed using T7 or SP6 polymerase, respectively, and the transcripts were mixed and the mixture was heated to denature the RNA strands before annealing in annealing buffer.

[0509] Example 3: Stability of ledRNA

[0510] The ability of ledRNA to form dsRNA structures was compared with open-ended dsRNA (i.e., without loops, formed by annealing of separate single-stranded sense and antisense RNAs) and long hpRNA. The mixture of ledRNA, long hpRNA, and sense and antisense RNAs was denatured by boiling and annealed in annealing buffer (250 mM Tris-HCL, pH 8.0 and 100 mM MgCl2 / buffer) and then electrophoresed in a 1.0% agarose gel under non-denaturing conditions.

[0511] like Figure 2 As shown, both GUS ledRNA and GFP ledRNA give dominant RNA bands with expected mobility for double-stranded molecules, indicating the formation of the predicted ledRNA structure. This is in contrast to the mixture of sense and antisense RNAs, which only show a weak dsRNA band, indicating that most sense and antisense RNAs do not easily anneal to each other to form dsRNA. The hairpin RNA sample gives two prominent bands, indicating that only part of the transcript forms the predicted hairpin RNA structure. Therefore, ledRNA is the most effective in forming the predicted dsRNA structure.

[0512] The ability of ledRNA to stay and diffuse on the leaf surface was also compared with dsRNA. When applied to the lower part of the tobacco leaf surface, GUS ledRNA (ledGUS) could be readily detected in the upper part of untreated tobacco leaves after 24 hours ( Figure 3 However, single strand GUS dsRNA (dsGUS) could not be detected in the upper part of untreated leaves ( Figure 3 ). This result suggests that ledRNA is more resistant to degradation than dsRNA and is therefore able to spread within plant leaf tissue.

[0513] Example 4: Testing ledRNA by local delivery

[0514] The ability of ledRNA to induce RNAi after local delivery was tested in Nicotiana benthamiana and Nicotiana tabacum plants expressing GFP or GUS reporter genes, respectively. The sequences of the GFP and GUS target sequences and the ledRNA encoding constructs are shown in SEQ ID NOs: 7, 8, 4, and 5, respectively. The ribonucleotide sequences of the encoded RNA molecules are shown in SEQ ID NOs: 1 (GFPLedRNAGFP ledRNA) and 2 (GUS ledRNA).

[0515] To facilitate reproducible and uniform application of ledRNA to the leaf surface, ledRNA at a concentration of 75-100 μg / ml in 25 mM Tris-HCL, pH 8.0, 10 mM MgCl2 and Silwet 77 (0.05%) was applied to the subaxial surface of the leaf using a soft paint brush. Leaf samples were taken 6 hours and 3 days after application of ledRNA for analysis of targeted gene silencing.

[0516] Application of ledRNA for GFP in N. benthamiana leaves and for GUS in Nicotiana tabacum resulted in a significant reduction of 20-40% and 40-50% of the activity of the corresponding target genes at the mRNA (GFP) or protein activity (GUS) levels 6 hours after treatment. However, in this experiment, the reduction was not sustained 3 days after treatment. The inventors believe that the observation at 3 days may be due to some non-specific response of the transgene to the dsRNA treatment or the amount of ledRNA dissipated. However, in a separate experiment, GUS silencing was detected in both treated and distal untreated leaf areas 24 hours after ledRNA treatment ( Figure 4 ).

[0517] Example 5. ledRNA-induced silencing of endogenous target genes

[0518] In another embodiment, ledRNA is designed to target mRNA encoded by the endogenous gene, FAD2.1 gene of N. benthamiana. The sequences of the target FAD2.1 mRNA and ledFAD2.1 encoding construct are shown in SEQ ID NO: 9 and 6, respectively. The ribonucleotide sequence of the encoded RNA molecule is provided as SEQ ID NO: 3 (N. benthamiana FAD2.1 ledRNA).

[0519] The FAD2.1 ledRNA construct consists of the following: the first half of the antisense sequence corresponding to nucleotides 678-379 of the FAD2.1 CDS (Niben101Scf09417g01008.1); the first antisense loop corresponding to nt378-242 of the FAD2.1 CDS; the sense sequence corresponding to nt379-979; the second antisense loop corresponding to nt1115-980; and the second half of the antisense sequence corresponding to nt979-nt679 of the FAD2.1 CDS.

[0520] In parallel, ledGUS RNA from the previous example was used as a negative control. In the first experiment, the FAD2.1 mRNA level and the accumulated C18:1 fatty acid ( Figure 5 ). The activity level of the related gene FAD2.2 was also determined. For each sample, approximately 3 μg of total RNA was treated with DNase and reverse transcribed using oligo dT primers at 50°C for 50 minutes. The reaction was terminated at 85°C for 5 minutes and diluted to 120 μl with water. Using a rotor gene PCR instrument, 5 μl of each sample was analyzed in triplicate for relative expression of FAD2.1 and FAD2.2 mRNA using gene-specific primers for the housekeeping gene actin. In subsequent experiments, Northern blot hybridization was also used to confirm the silencing of the FAD2.1 gene by topically applied ledFAD2.1 RNA ( Figure 6 ).

[0521] At the 2, 4, and 10 h time points, FAD2.1 mRNA was significantly reduced to almost undetectable levels in leaf tissue treated with ledRNA ( Figure 5 ). In this experiment, it is unclear why the level of FAD2.1 mRNA did not decrease as much at the 6 hour time point. Figure 6 In the replicate experiments shown, strong FAD2.1 downregulation occurred at 6 and 24 hours, particularly at the 24 hour time point. The related FAD2.2 gene, which has sequence homology to FAD2.1, also showed downregulation by ledRNA at the 2 and 4 hour time points ( Figure 5 ).

[0522] Since FAD2.1 and FAD2.2 encode fatty acid Δ12 desaturases that desaturate oleic acid to linoleic acid, the levels of these fatty acids were determined in leaf tissue treated with ledRNA. At the 2, 4, and 6 hour time points, there was a significant increase in oleic acid (18:1) accumulation in ledRNA treated leaf tissue, suggesting that reduced amounts of the FAD2 enzyme ( Figure 5 ). Therefore, qRT-PCR and fatty acid composition analysis showed ledRNA-induced FAD2.1 gene silencing.

[0523] Example 6: Design and testing of hairpin RNAs containing G:U base pairs or mismatched nucleotides

[0524] Modified hairpin RNA targeting GUS RNA

[0525] The reporter gene of the gene such as encoding β-glucuronidase (GUS) provides a simple and convenient determination system, which can be used to measure the gene silencing efficiency in eukaryotic cells including plant cells (Jefferson et al., 1987). Therefore, the inventors designed, produced and tested some modified hairpin RNAs to reduce the ability of GUS gene expression as the target gene, provided the hairpin RNA to the cells using a gene delivery method, and compared the modified hairpin RNA with a conventional hairpin RNA. The conventional hairpin RNA used as a control in the experiment has a double-stranded region of 200 adjacent base pairs in length, wherein all base pairs are standard base pairs, i.e., G:C and A:U base pairs, without any G:U base pairs in the double-stranded region, and without any non-base paired nucleotides (mismatches), targeting the 200nt region identical to the modified hairpin RNA of the GUS mRNA molecule. The sense and antisense sequences forming the double-stranded region were covalently linked by a spacer sequence that included the PDK intron (Helliwell et al., 2005; Smith et al., 2000), providing an RNA loop of 39 or 45 nucleotides in length (depending on the cloning strategy used) after splicing of the introns from the primary transcript. The DNA fragments for the antisense sequence were flanked by XhoI-BamHI restriction sites at the 5' end and HindIII-KpnI restriction sites at the 3' end to facilitate cloning into the expression cassette, with each sense sequence flanked by XhoI and KpnI restriction sites, respectively. For the control hairpin and the modified hairpin, the 200 bp dsRNA region of each hairpin RNA included a 200 nucleotide antisense sequence that was fully complementary to the wild-type GUS sequence from within the protein coding region. The antisense sequence, corresponding to nucleotides 13-212 of SEQ ID NO: 10, is the complement of nucleotides 804-1003 of the GUS open reading frame (ORF) (the cDNA sequence is provided as SEQ ID NO: 8). The length of the GUS target mRNA is therefore greater than 1900 nt. The length of 200 nucleotides for the sense and antisense sequences was chosen to be small enough to reasonably facilitate the synthesis of DNA fragments using synthetic oligonucleotides, but also long enough to provide multiple sRNA molecules after Dicer processing. As part of the ORF, the sequence is unlikely to contain cryptic splice sites or transcription termination sites.

[0526] Preparation of genetic constructs

[0527] 200 bp of the GUS ORF sequence was PCR amplified using oligonucleotide primer pairs GUS-WT-F (SEQ ID NO: 52) and GUS-WT-R (SEQ ID NO: 53) containing XhoI and BamHI sites or HindIII and KpnI, respectively, to introduce these restriction enzyme sites 5' and 3' to the GUS sequence. The amplified fragment was inserted into the vector pGEM-T Easy and the correct nucleotide sequence was verified by sequencing. The GUS sequence was inserted in the antisense orientation relative to the operably linked CaMV e35S promoter (Grave, 1992), and the ocs gene polyadenylation / transcription terminator (Ocs-T) by excising the GUS fragment with BamHI and HindIII digestion and inserting it into the BamHI / HindIII site of pKannibal (Helliwell and Waterhouse, 2005). The resulting vector was named pMBW606 and contained the 35S::PDK intron::antisense GUS::Ocs-T expression cassette in 5' to 3' order. This vector was an intermediate vector used as a base vector for assembling the four hpRNA constructs.

[0528] Construct hpGUS[wt] with canonical base pairs only

[0529] To prepare a vector with only canonical base pairs, encoding a hairpin RNA molecule used as a control, named hpGUS[wt], a 200 bp GUS PCR fragment with XhoI and KpnI was excised from the pGEM-T Easy plasmid and inserted into the XhoI / KpnI site between the 35S promoter and the PDK intron in pMBW606. This produced a vector named pMBW607, which contains a 35S::sense GUS[wt]::PDK intron::antisense GUS::OCS-T expression cassette. The cassette was excised by digestion with NotI and inserted into the NotI site of pART27 (Gleave, 1992), resulting in a vector named hpGUS[wt], which encodes a canonical base-paired hairpin RNA targeting GUS mRNA.

[0530] When self-annealed by hybridization of the 200nt sense and antisense sequences, the hairpin has a double-stranded region of 200 adjacent base pairs corresponding to the GUS sequence. Relative to the GUS sense sequence, the sense and antisense sequences in the expression cassette are flanked by BamHI and HindIII restriction enzyme sites located at the 5' and 3' ends, respectively. When transcribed, the nucleotides corresponding to these sites are also able to hybridize, extending the double-stranded region by 6bp at each end. After transcription of the expression cassette and splicing of the PDK intron from the primary transcript, the hairpin RNA structure is predicted to have a 39-nucleotide loop structure before any processing by Dicer or other RNases. The nucleotide sequence of the hairpin RNA structure including its loop is provided in SEQ ID NO: 15, and its folding free energy is predicted to be -471.73 kcal / mol. Therefore, this is an energetically stable hairpin structure. The free energy was calculated based on the nucleotide sequence after splicing of the PDK intron sequence using “RNAfold” (http: / / rna.tbi.univie.ac.at / cgi-bin / RNAWebSuite / RNAfold.cgi).

[0531] When transcribed from an expression cassette with a 35S promoter and an OCS-T terminator, the resulting hairpin RNA is embedded in a larger RNA molecule, adding 8 nucleotides at the 5' end and approximately 178 nucleotides at the 3' end, without taking into account any poly(A) tail added to the 3' end. Since the modified hairpin RNAs use the same promoter-terminator design, these molecules also have these extensions at the 5' and 3' ends. Therefore, the length of the hairpin RNA molecule after splicing the PDH intron is approximately 630 nucleotides.

[0532] Construct containing G:U base pair hpGUS[G:U]

[0533] A DNA fragment containing the same 200 nucleotide sense sequence, but with all 52 cytosine nucleotides (C) of the corresponding wild-type GUS region replaced by thymine nucleotides (T) was assembled by annealing overlapping oligonucleotides GUS-GU-F (SEQ ID NO: 54) and GUS-GU-R (SEQ ID NO: 55) and PCR extension of the 3' end using high-fidelity LongAmp Taq polymerase (New England Biolabs, catalog number M0323). The amplified DNA fragment was inserted into the pGEM-T Easy vector and the correct nucleotide sequence was verified by sequencing. (SEQ ID NO: 11). The DNA fragment containing the modified sequence was then excised by digestion with XhoI and KpnI and inserted into the XhoI / KpnI site of the basic vector pMBW606. This generated a construct named pMBW608, which contains the expression cassette 35S::sense GUS[G:U]::PDK intron::antisense GUS::OCS-T. The expression cassette was excised by NotI digestion and inserted into the NotI site of pART27, resulting in a vector named hpGUS[G:U], which encodes a G:U base-paired hairpin RNA molecule.

[0534] This cassette encodes a hairpin RNA targeting GUS mRNA, which, when self-annealed by hybridization of the 200nt sense and antisense sequences, has 52 G:U base pairs (instead of G:C base pairs in hpGUS[wt]) and 148 canonical base pairs, i.e., 26% of the nucleotides in the double-stranded region involve G:U base pairs. The 148 canonical base pairs in hpGUS[G:U] are the same as those in the control hairpin RNA, including 49 U:A base pairs, 45 A:U base pairs and 54 G:C base pairs at the corresponding positions. The longest stretch of the double-stranded region adjacent to the canonical base pairing is 9 base pairs. Therefore, the antisense nucleotide sequence of hpGUS[G:U] is identical in length (200nt) and sequence to the antisense sequence of the control hairpin RNA hpGUS[wt]. After transcription of the expression cassette and splicing of the PDK intron from the primary transcript, the hairpin RNA structure is predicted to have a 45-nucleotide loop structure before any processing by Dicer or other RNases. The nucleotide sequence of the hairpin structure including its loop is provided as SEQ ID NO: 16, and its folding free energy is predicted to be -331.73 kcal / mol. For hpGUS [wt], although the 52 G:U base pairs are much weaker than the G:C base pairs in hpGUS [wt], this is therefore an energetically stable hairpin structure.

[0535] Figure 7An alignment of the modified GUS sense sequence (nucleotides 9-208 of SEQ ID NO: 11) with the corresponding region of the GUS target gene (SEQ ID NO: 14) is shown.

[0536] Construct hpGUS[1:4] containing a mismatched nucleotide every fourth nucleotide

[0537] DNA fragments containing the same 200 bp sense sequence but in which every 4th nucleotide of the corresponding wild-type GUS sequence was replaced were designed and assembled. Every 4th nucleotide of each grouping of 4 nucleotides (nucleotides at positions 4, 8, 12, 16, 20, etc.) was replaced by changing C' to G', G' to C', A' to T' and T' to A', leaving the other nucleotides unchanged. These substitutions are all translocation substitutions, which are expected to have a greater destabilizing effect on the resulting hairpin RNA structure than transition substitutions. The DNA fragments were assembled by annealing overlapping oligonucleotides GUS-4M-F (SEQ ID NO: 56) and GUS-4M-R (SEQ ID NO: 57) and performing PCR extension of the 3' end using LongAmpTaq polymerase. The amplified DNA fragments were inserted into the pGEM-T Easy vector and the correct nucleotide sequence was verified by sequencing. (SEQ ID NO: 12). The DNA fragment containing the modified sequence was then excised by digestion with XhoI and KpnI and inserted into the XhoI / KpnI site of the basic vector pMBW606. This resulted in a construct named pMBW609, which contained the expression cassette 35S::sense GUS[1:4]::PDK intron::antisense GUS::OCS-T. The expression cassette was excised by digestion with NotI and inserted into the NotI site of pART27, resulting in a vector named hpGUS[1:4], which encodes a 1:4 mismatched hairpin RNA molecule.

[0538] The cassette encodes a hairpin RNA that targets GUS mRNA and, when self-annealed by hybridization of the sense and antisense sequences, has mismatches for 50 nucleotides of the 200 nt antisense sequence, including a mismatch for the nucleotide at position 200. In addition to position 200, the double-stranded region of the hairpin RNA has 150 canonical base pairs and 49 mismatched nucleotide pairs over the sense and antisense sequences of 199 nt in length, i.e., 24.6% of the nucleotides in the double-stranded region are predicted to be mismatched (not involving base pairs). After transcription of the expression cassette and splicing of the PDK intron from the primary transcript, the hairpin RNA structure is predicted to have a 45-nucleotide loop structure before any processing by Dicer or other RNases. The nucleotide sequence of the hairpin structure including its loop is provided as SEQ ID NO: 17, and its folding free energy is predicted to be -214.05 kcal / mol. For hpGUS [wt], this is therefore an energetically stable hairpin structure, except for the mismatched nucleotides.

[0539] Figure 8 An alignment of the modified GUS sense sequence (nucleotides 9-208 of SEQ ID NO: 12) with the corresponding region of the GUS target gene (SEQ ID NO: 14) is shown.

[0540] The construct hpGUS[2:10], in which 9 and 10 of the 10 nucleotides were mismatched

[0541] Design and assemble DNA fragments containing the same 200 bp sense sequence but in which every ninth and tenth nucleotide of the corresponding wild-type GUS sequence is replaced. Every ninth and tenth nucleotide of each grouping of 10 nucleotides (nucleotides at positions 9, 10, 19, 20, 29, 30, etc.) is replaced by changing C' to G', G' to C', A' to T' and T' to A', leaving the other nucleotides unchanged. The DNA fragments are assembled by annealing overlapping oligonucleotides GUS-10M-F (SEQ ID NO: 58) and GUS-10M-R (SEQ ID NO: 59) and performing PCR extension of the 3' end using LongAmp Taq polymerase. The amplified DNA fragments are inserted into pGEM-T Easy and the correct nucleotide sequence is verified by sequencing. (SEQ ID NO: 13). The DNA fragment containing the modified sequence is then excised by digestion with XhoI and KpnI and inserted into the XhoI / KpnI site of the basic vector pMBW606. This generated a construct named pMBW610, which contains the expression cassette 35S::sense GUS[2:10]::PDK intron::antisense GUS::OCS-T. The expression cassette was excised by NotI digestion and inserted into the NotI site of pART27, resulting in a vector named hpGUS[2:10], which encodes a 2:10 mismatched hairpin RNA molecule.

[0542] The cassette encodes a hairpin RNA targeting GUS mRNA, which has mismatches for 50 nucleotides of the 200nt antisense sequence, including mismatches for nucleotides at positions 199 and 200, when self-annealed by hybridization of the sense and antisense sequences. In addition to positions 199 and 200, the double-stranded region of the hairpin RNA has 160 canonical base pairs and 19 dinucleotide mismatches over the sense and antisense sequences of 198nt in length, i.e., 19.2% of the nucleotides in the double-stranded region are predicted to be mismatched (not involving base pairs). The 160 base pairs in hpGUS[2:10] are the same as those in the control hairpin RNA, including 41 U:A base pairs, 34 A:U base pairs, and 42 G:C and 43 G:C base pairs at the corresponding positions. After transcription of the expression cassette and splicing of the PDK intron from the primary transcript, the hairpin RNA structure is predicted to have a 45 nucleotide loop structure before any processing by Dicer or other RNases. The nucleotide sequence of the hairpin structure including its loop is provided as SEQ ID NO: 18, and its folding free energy is predicted to be -302.78 kcal / mol. For hpGUS [wt], this is therefore an energetically stable hairpin structure, except for the mismatched nucleotides that are expected to bulge out from the stem of the hairpin structure.

[0543] Fig. 9Shown is an alignment of the modified GUS sense sequence (nucleotides 9-208 of SEQ ID NO: 13) with the corresponding region of the GUS target gene (SEQ ID NO: 14).

[0544] Fig.10 Four genetic constructs used to express control and modified hairpin RNAs are shown schematically.

[0545] Example 7: Detection of modified hairpin RNA in transgenic plants

[0546] Plants of the common tobacco (Nicotiana tabacum) species transformed with the GUS target gene were used to test the efficacy of the above four hairpin RNA constructs. Specifically, the target plants were from two homozygous independent transgenic lines PPGH11 and PPGH24, each containing a single copy insertion of the GUS transgene from the vector pWBPPGH, such as Fig.11As shown schematically. The GUS gene in the T-DNA of pWBPPGH has a GUS coding region (nucleotides 7-1812 of SEQ ID NO: 8) operably linked to a 1.3 kb long promoter of the phloem protein 2 (PP2) gene from Cucurbita pepo L. cv. Autumn Gold (Wang et al., 1994; Wang, 1994). The construct pWBPPGH was constructed by excising the PP2 promoter plus 5'UTR and 54 nucleotides of the PP2 protein coding region (encoding the first 18 amino acids of PP2) from the lambda genomic clone CPP1.3 (Wang, 1994), and fused the fragment with the GUS coding sequence starting from the nucleotide encoding the third amino acid of GUS to produce an N-terminal fusion polypeptide with GUS activity. pPP2:: The GUS: Nos-T box was inserted into pWBVec2a (Wang et al., 1998) to generate pWBPPGH, which was used to transform plants of Nicotiana tabacum cv. Wisconsin 38 using Agrobacterium tumefaciens-mediated leaf disc transformation (Ellis et al., 1987), selecting for resistance to hygromycin. The GUS activity in the homozygous progeny plants of the two transgenic lines PPGH11 and PPGH24 was similar. The GUS expression in the two transgenic plants was not limited to the phloem, but was present in most tissues of the plant. Therefore, in these plants, the GUS expression from the PP2 promoter appears to be constitutive. PP2-GUS plants were chosen as test plants for two reasons: i) they gave about the same constitutively high level of GUS expression as 35S-GUS plants; ii) the PP2 promoter is derived from the endogenous PP2 gene promoter of Cucurbita pepo, which has a different sequence from the 35S promoter used to drive hpRNA transgene expression and is therefore not transcriptionally co-repressed by the imported 35S promoter.

[0547] All 4 kinds of hairpin RNA constructs (Example 6) were used to transform PPGH11 and PPGH24 plants using the leaf disk method (Ellis et al., 1987) mediated by Agrobacterium, using 50mg / L kanamycin as a selection agent. This selection system using kanamycin as a different reagent from the hygromycin used previously for importing the T-DNA of pWBPPGH was observed to produce only transformed plants, without untransformed plants being regenerated. The regenerated transgenic plants containing the T-DNA from the hpGUS construct were transferred to soil for growth in a greenhouse and kept for about 4 weeks before measuring GUS activity. When measured, the transgenic plants were healthy and actively grown, and were identical in appearance to untransformed control plants and parental PPGHII and PPGH24 plants. In total, 59 transgenic plants transformed with T-DNA encoding hpGUS[wt] were obtained, 74 plants transformed with T-DNA encoding hpGUS[G:U] were obtained, 33 plants transformed with T-DNA encoding hpGUS[1:4] were obtained, and 41 plants transformed with T-DNA encoding hpGUS[2:10] were obtained.

[0548] GUS expression levels were determined using the fluorescent 4-methylumbelliferyl β-D-glucuronide (MUG) assay (Jefferson et al., 1987) following a modified kinetic method described by Chen et al. (2005). Plants were assayed by collecting leaf samples of approximately 1 cm in diameter from 3 different leaves per plant, selecting well-swollen, healthy, and green leaves. Note that the test plants were at the same growth and development stage as the control plants. Each assay used 5 μg of protein extracted from each leaf sample, and the cleavage rate of MUG was measured as described by Chen et al. (2005).

[0549] Representative data are shown in Fig.12 In the Figures, the GUS activity (MUG units in the assay) of each independent transgenic plant is shown. Because the data for the hpGUS[wt] construct showed that some plants showed strong silencing, with activity reduced by at least 90%, and others were weaker, in this article, 10% GUS activity relative to control plants was chosen as the activity level used to classify plants into two categories and compare different constructs.

[0550] Using a 10% activity level as a benchmark for strong silencing, the genetic construct encoding canonically base-paired hpGUS [wt] induced strong GUS silencing in 32 of the 59 transgenic plants tested (54.2%). The other 27 plants all showed reduced GUS activity, but retained more than 10% enzyme activity relative to the control plants and are therefore considered herein to show weak silencing. Transgenic plants with this construct showed a wide range of degrees of GUS gene silencing ( Fig.12 ), ranging from less than 1% to about 80% activity retention, which is typical for conventional hairpin structures (Smith et al., 2000).

[0551] In stark contrast, the hpGUS[G:U] construct induced consistent and uniform silencing across independent transgenic lines, with 71 of 74 plants tested (95.9%) showing strong GUS silencing. Again, unlike all 33 hpGUS[1:4] plants tested, which showed reduced levels of GUS activity, only 8 (24%) produced <10% GUS activity relative to control plants, and the other 25 were classified as having weaker silencing. These results suggest that this construct induces weaker but more uniform levels of GUS downregulation across transgenic lines. The hpGUS[2:10] construct behaved more like the hpGUS[wt] construct, inducing good levels of silencing in some lines (28 of 41, or 68.3%) and little or no GUS silencing in the remaining 13 plants.

[0552] When only silenced lines (residual activity <10%) were used for comparison and average GUS activity was calculated, hpGUS[wt] plants showed the highest average degree of silencing, followed by hpGUS[G:U] plants and hpGUS[2:10] plants ( Fig.13 ). HPGUS[1:4] plants had the smallest average reduction in GUS activity. The extent of GUS silencing showed a good correlation with the thermodynamic stability of the predicted hpRNA structures derived from four different hpRNA constructs (Example 6).

[0553] To test whether these differences persist in progeny plants, representative transgenic plants containing the target GUS gene (which is homozygous) and the hpGUS transgene (hemizygous) were self-pollinated. Progeny plants from the hpGUS line were selected for kanamycin resistance, thus discarding any ineffective segregants lacking the hpGUS transgene. This ensured that the hpGUS transgene was present in all progeny in either a homozygous or heterozygous state. Progeny plants were assayed for GUS activity, and representative data are shown in Fig.14Progeny containing the hpGUS[wt] transgene clearly fell into two categories, those with strong GUS silencing and those showing weak or no silencing. These categories correlated well with the phenotypes of the previous generation, indicating that the extent of target gene silencing is heritable. All plants in the hpGUS[G:U] lines tested consistently showed strong silencing, while plants in the hpGUS[1:4] lines consistently showed weaker silencing. The inventors concluded that the phenotypes observed in the parental generation are generally maintained in the progeny plants.

[0554] Southern blot hybridization experiments of transformed plants

[0555] The uniformity of strong gene silencing observed in a large number of independent transgenic plants generated with the hpGUS[G:U] construct was surprising and unexpected. The inventors sought to determine whether any explanation other than the effect caused by the hpGUS[G:U] RNA was responsible for the uniformity of silencing. To test whether multiple transgenic plants were generated from the expected independent transformation events, Southern blot hybridization experiments were performed on DNA isolated from 18 representative transgenic plants containing the hpGUS[G:U] construct. DNA was isolated from leaf tissue using the hot phenol method described by Wang et al. (2008). For Southern blot hybridization, approximately 10 μg of DNA from each plant sample was digested with HindIII enzyme, separated by gel electrophoresis in 1% agarose gel in TBE buffer, and blotted onto Hybond-N+ membrane (Sambrook et al., 1989) using a capillary method. The membrane was incubated at 42°C with 1% agarose gel from the OCS-T terminator region. 32 P-labeled DNA fragments were hybridized overnight. This probe was selected because it hybridizes to the hpGUS[G:U] transgene but not to the GUS target gene, which does not have an OCS-T terminator sequence. The membrane was washed at high stringency and the retained probe was visualized using a phosphoimager.

[0556] The autoradiograph of the hybridization blot is shown in Fig.15 Each lane showed 1-5 or 1-6 hybridizing bands. No two lanes showed the same pattern, i.e., the autoradiograph showed that each of the 16 representative hpGUS[G:U] plants had a different pattern of post-hybridization HindIII fragments, and therefore came from different transgene insertions. The inventors concluded that the uniform GUS silencing observed for the hpGUS[G:U] lines was not due to a similar transgene insertion pattern in the plants, and that the uniformity of silencing was caused by the structure of the hpGUS[G:U] RNA. The inventors also concluded that multiple copies of the hpGUS[G:U] transgene were not required to obtain strong gene silencing; a single copy of the transgene was sufficient.

[0557] Northern blot hybridization experiments of transformed plants

[0558] To determine whether hpGUS[G:U] RNA is processed in the same manner as control hairpin RNA in transgenic plants, Northern blot hybridization experiments were performed on RNA isolated from leaves of transgenic plants. Northern blot experiments were performed to detect shorter RNAs (sRNAs, about 21-24 nucleotides in length) produced after Dicer-processing of hairpin RNA. Experiments were performed on small RNAs isolated from transgenic HPGUS[wt] and HPGUS[G:U] plants, which also contained the GUS target gene expressed as (sense) mRNA. Nine plants were selected for sRNA analysis for each construct. For the hpGUS[wt] transgenic population, plants showing weak GUS silencing and some plants showing strong GUS silencing were included. Small RNA samples were isolated using the hot phenol method (Wang et al., 2008), and Northern blot hybridization was performed according to the description of Wang et al. (2008), and gel electrophoresis of RNA samples was performed under denaturing conditions. The probe used was 32P-labeled RNA corresponding to the sense sequence or antisense sequence corresponding to nucleotides 804-1003 of SEQ ID NO:8.

[0559] Fig.16 Shown are autoradiograms of Northern blots hybridized with antisense probes (upper panels) to detect sense sRNA molecules derived from hairpin RNAs, or hybridized with sense probes to detect antisense sRNAs (lower panels). At the bottom, the figure shows qualitative scoring of GUS expression levels relative to control plants lacking hpGUS constructs. In other experiments, hybridization with small RNAs of approximately 20-25 nucleotides was observed based on the mobility of sRNAs compared to RNAs of known length. The hpGUS[wt] lines showed a range of variation in sRNA accumulation. This was observed for both sense and antisense sRNAs, although the antisense sRNA bands were not as clear as the sense bands. Since the hpGUS[wt] plants contain both hpGUS transgenes expressing sense and antisense sequences corresponding to the 200nt target region and a GUS target gene expressing the full-length sense gene, sense sRNAs can be generated from either hairpin RNAs or target mRNAs. The sRNA levels in hpGUS[wt] are negatively correlated with the degree of GUS silencing. For example, the two plants represented in lanes 4 and 5 accumulated relatively more sRNA but showed only a moderate degree of GUS downregulation. In contrast, the two plants presented in lanes 7 and 8 had strong GUS silencing but accumulated relatively low levels of sRNA.

[0560] In contrast to hpGUS[wt] plants and consistent with the relatively consistent degree of silencing of the hpGUS[G:U] construct, hpGUS[G:U] plants accumulated consistent amounts of antisense sRNA across lines. In addition, the degree of GUS silencing appeared to show good correlation with the amount of antisense sRNA. Almost no sense sRNA was detected in these plants. This is expected because the RNA probe used in the Northern blot hybridization is transcribed from the wild-type GUS sequence and therefore has a lower level of complementarity to sense sRNA from hpGUS[G:U], in which all C nucleotides are replaced by U nucleotides, allowing only lower stringency hybridization. However, this experiment does not rule out the possibility of processing hpGUS[G:U] RNA to produce less sense sRNA or to degrade them more quickly.

[0561] The Northern blot hybridization experiment was repeated, this time using only the sense probe to detect the antisense sRNA; the autoradiogram showed Fig.17 Again, the production of antisense sRNA from the hpGUS[wt] construct was negatively correlated with GUS activity ( Fig.17 The strong silenced plants produced high levels of antisense sRNA (lanes 1, 3, 5, 8 and 10), while plants that showed only weak or no silencing did not produce hybridization signals in this experiment (lanes 2, 4, 6, 7 and 9). In contrast, plants expressing hpGUS[G:U] produced much lower but consistent amounts of antisense sRNA. It is interesting to observe that plants expressing strong silence of hpGUS[G:U] accumulate much lower sRNA levels than plants expressing strong silence of hpGUS[wt] and suggest to the inventors that hpGUS[wt] is processed by different mechanisms in plants, but is still as effective as the hpGUS[wt] construct. Further observations in this experiment provide clues, namely that the two relatively weak antisense bands for hpGUS[G:U] plants appear to have the same mobility as the second and fourth bands observed for the antisense sRNA bands from hpGUS[wt]. This is confirmed in further experiments described below. The inventors hypothesized that the four bands of sRNA from hpGUS[wt] represent 24-, 22-, 21-, and 20-mers, and that hpGUS[G:U] RNA is primarily processed to generate 22- and 20-mer antisense sRNAs.

[0562] An important clear conclusion from the above data is that the hpGUS[G:U] RNA molecules are processed by one or more Dicer enzymes to produce sRNAs, particularly antisense sRNAs, which are thought to be mediators of RNA interference in the presence of various proteins such as Argonaute. The observed production of antisense sRNAs suggests that sense sRNAs are also produced, but the experiment does not distinguish between degradation / instability of sense sRNAs or lack of detection of sense sRNAs due to insufficient hybridization with the probes used. From these experiments, the inventors also concluded that there are clear differences between hpGUS[wt] and hpGUS[G:U] RNA molecules in their processing. This suggests that the molecules are recognized differently by one or more Dicers.

[0563] Example 8: sRNA analysis of transgenic plants expressing modified hairpin RNA

[0564] Another Northern blot hybridization experiment was performed to detect antisense sRNAs from hpGUS[G:U] plants and compare their sizes with those produced by hpGUS[wt]. Fig.18 At this time, the size difference of the two antisense sRNA bands from hpGUS[G:U] was more obvious compared with the two major bands from hpGUS[wt]. Fig.18 This can be best seen by the mobility of the bands in adjacent lanes 9 and 10. This result confirms that the two hairpin RNAs are differentially processed by one or more Dicers in plants.

[0565] To investigate this further, the small RNA populations from hpGUS[wt] and hpGUS[G:U] were analyzed by deep sequencing of total, adaptor-amplifiable sRNAs isolated from the plants. The frequency of sRNAs mapping to the double-stranded region of the hairpin RNA was determined. And their length distribution was determined. The results showed that the frequency of 22-mer antisense RNAs was increased in the hpGUS[G:U] construct compared to the hpGUS[wt] construct. The increase in the proportion of sRNAs with a length of 22 nt indicates that Dicer-2 has altered processing of the hpGUS[G:U] hairpin relative to hpGUS[wt].

[0566] Example 9: DNA methylation analysis of plant transgenics

[0567] The observation of variability in the degree of GUS silencing conferred by hpGUS[wt] and the detection of antisense 24-mer sRNA in hpGUS[wt] plants, but not in hpGUS[G:U] plants, led the inventors to question whether the two plant populations differed in the level of DNA methylation of the target GUS gene. Sequence-specific 24-mer sRNAs are thought to be involved in promoting DNA methylation of inverted repeat structures in plants (Dong et al., 2011). Therefore, the inventors tested the DNA methylation level of the GUS transgene in hpGUS plants, in particular the DNA methylation level of the 35S promoter region of the hairpin encoding gene (silencer gene).

[0568] For this purpose, the DNA methylation-dependent endonuclease McrBC was used. McrBC is a commercially available endonuclease that cleaves methylcytosine-containing ( m McrBC recognizes two sites on DNA, which are 5' (G or A) m C 3' consists of two half sites, preferably G m C. These half sites can be separated by hundreds of base pairs, but the optimal spacing is 55 to about 100 bp. Double-stranded DNA with such linked GmC dinucleotides on both strands is the best substrate. McrBC activity depends on one or both GC dinucleotides being methylated. Since plant DNA can be methylated at C in CG, CHG, or CHH sequences, where H represents A, C, or T (Zhang et al., 2018), digestion of DNA with McrBC and subsequent PCR amplification of gene-specific sequences can be used to detect specific DNA sequences in plant genomes. m The presence or absence of C. In this assay, PCR amplification of methylated McrBC-digested genomic DNA produces reduced amounts of amplification products compared to unmethylated DNA, but if the DNA is unmethylated, the same amount of PCR product will be produced as untreated DNA.

[0569] Genomic DNA was isolated from plants containing hpGUS[wt], hpGUS[G:U] or hpGUS[1:4] constructs in addition to the target GUS gene by standard methods (Draper and Scott, 1988). The Mg required for endonuclease activity was included according to the manufacturer's instructions. 2+ions and GTP, purified DNA samples were treated with McrBC (Catalog No. M0272; New England Biolabs, Massachusetts). In summary, approximately 1 μg of genomic DNA was digested overnight with McrBC in a 30 μl reaction volume. The digested DNA samples were diluted to 100 μl and PCR amplified the region of interest as follows.

[0570] The treated DNA samples were used for PCR reactions using the following primers. For the 35S-GUS junction sequence of hpGUS[wt]: forward primer (35S-F3), 5'-TGGCTCCTACAAATGCCATC-3' (SEQ ID NO: 60); reverse primer (GUSwt-R2, 5'-CARRAACTRTTCRCCCTTCAC-3' (SEQ ID NO: 61). For the 35S-GUS junction sequence of hpGUS[G:U]: forward primer (GUSgu-R2), 5'-CAAAAACTATTCACCCTTCAC-3' (SEQ ID NO: 62); reverse primer (GUS4m-R2), CACRAARTRTACRCRCTTRAC (SEQ ID NO: 63). For the 35S promoter sequence of both constructs: forward primer (35S-F2, reverse, 5'-GAGGATCTAACAGAACTCGC-3' (SEQ ID NO: 64); reverse primer (35S-R1), 5'-CTCTCCAAATGAAATGAACTTCC-3' (SEQ ID NO: 65). In each case, R = A or G, Y = C or T. The PCR reaction was performed using the following cycling conditions: 94°C for 1 minute, followed by 35 cycles of 94°C for 30 seconds, 55°C annealing for 45 seconds, 68°C extension for 1 minute, and a final extension at 68°C for 5 minutes. The PCR amplification products were subjected to electrophoresis and the intensity of the bands was quantified.

[0571] Representative results are shown in Fig.19 and 20For the 35S-GUS junction region, which includes 200 bp of the 35S promoter sequence that contains the transcription start site, most hpGUS[wt] plants showed significant levels of DNA methylation. Within the hpGUS[wt] plant population, individual plants that maintained higher levels of GUS activity, i.e., less silencing, appeared to have more methylation of the promoter-GUS sense junction region. The results for the 35S promoter region were similar. In contrast, most hpGUS[G:U] and hpGUS[1:4] plants showed weaker DNA methylation at the 35S-GUS junction. The inventors believe that this proximal promoter sequence is important for the expression of the transgene, and that methylation in this region will likely reduce the expression of the silencing construct through transcriptional gene silencing (TGS) of the transgene. This is called "self-silencing."

[0572] General Discussion of Examples 6-9

[0573] Disruption of inverted repeat DNA structure in transgenics enhances their stability

[0574] Both the hpGUS[wt] and hpGUS[2:10] transgenic plant populations showed a broad range of target gene silencing. In contrast, two populations containing hpGUS[G:U] and hpGUS[1:4] plants showed relatively uniform GUS silencing in many independent lines, with strong silencing observed for the former construct and relatively weak but still significant reductions in gene activity observed for the latter construct. In the hairpin RNAs from the [G:U] and [1:4] constructs, approximately 25% of the nucleotides in the sense and antisense sequences participated in G:U base pairs or in sequence mismatches that were evenly distributed over the 200 nucleotide sense / antisense sequence. Due to the sequence differences between the sense and antisense sequences, it is believed that DNA construct mismatches between the sense and antisense "arms" or reverse request structures significantly disrupt the inverted repeat DNA structure. Repeated DNA structures can attract DNA methylation and silencing in various organisms (Hsieh and Fire, 2000). The hpGUS[2:10] construct also contains mismatches in the sense and antisense regions, but each of the 2-bp mismatches between the sense and antisense sequences is flanked by 8-bp adjacent mismatches, so the mismatches may not disrupt the inverted repeat DNA structure as they do in the [G:U] and [1:4] transgenes. Therefore, the uniformity of GUS silencing induced by hpGUS[G:U] and hpRNA[1:4] may be due, at least in part, to the disruption of the inverted repeat DNA structure, which results in lower methylation and thus reduced auto-silencing of both transgenes. Another benefit of mismatches between the sense and antisense DNA regions is that it aids in the cloning of inverted repeats in E. coli, as bacteria tend to delete or rearrange perfect inverted repeats.

[0575] Thermodynamic stability of hpRNA is an important factor affecting the degree of target gene silencing

[0576] When only the strongly silenced transgenic lines were compared, the hpGUS[wt] plants had the greatest degree of target gene downregulation, followed by hpGUS[G:U], hpGUS[2:10] and hpGUS[1:4]. RNAFold analysis predicted that the hpGUS[wt] hairpin RNA structure had the lowest free energy, i.e., the greatest stability, followed by the hpGUS[G:U], hpGUS[2:10] and hpGUS[1:4] hairpins. The inventors believe that the more stable the hairpin RNA structure, the greater the degree of target gene silencing it can induce. This also favors longer double-stranded RNA structures rather than shorter double-stranded RNA structures. It is believed that effective Dicer processing requires stable double-stranded RNA formation. The experimental results described herein indicate another important advantage of G:U base-paired constructs over constructs containing most simple mismatched nucleotides such as hpGUS[1:4]. Although both types of constructs disrupt the inverted repeat DNA structure, which reduces self-silencing, at the RNA level, hpGUS[G:U] RNA is more stable due to the ability of G and U to form base pairs. Combinations of both types of modifications are also considered beneficial, including G:U base pairs and some mismatched nucleotides in the double-stranded RNA structure, but with at least 2, 3, 4 or even 5 times more nucleotides involved in the G:U base pairs than in the mismatches.

[0577] Dicer efficiently processes hpGUS[G:U]RNA

[0578] An important question answered in these experiments is whether mismatched or G:U base-paired hpRNA can be processed into small RNAs (sRNAs) by Dicer. Strong silencing in hpGUS[G:U] plants and 1:4 and 2:10 mismatched hpRNA plants suggests that these hairpin RNA structures are processed by Dicer. This was confirmed by sRNA Northern blot hybridization for easy detection of antisense sRNAs for [G:U] molecules. In addition, the degree of GUS ...

Claims

1. An RNA molecule comprising a first RNA component, a second RNA component covalently linked to the first RNA component, and optionally one or more or all of the following: (i) a linking ribonucleotide sequence covalently linked to the first and second RNA components, (ii) a 5' leader sequence and (iii) a 3' trailer sequence, wherein the first RNA component consists of a first 5' ribonucleotide, a first RNA sequence, and a first 3' ribonucleotide in a 5' to 3' order, wherein the first 5' and 3' ribonucleotides in the first RNA component are base paired with each other, wherein the first RNA sequence comprises a first sense ribonucleotide sequence of at least 21 contiguous ribonucleotides, a first loop sequence of at least 4 ribonucleotides, and a first antisense ribonucleotide sequence of at least 21 contiguous ribonucleotides, wherein the at least 21 contiguous ribonucleotides of the first antisense ribonucleotide sequence in the RNA molecule are base paired with the at least 21 contiguous ribonucleotides of the first sense ribonucleotide sequence, wherein the first antisense ribonucleotide sequence is capable of hybridizing to a first region of a target RNA molecule, in, the second RNA component is covalently linked to the first 5' ribonucleotide or the first 3' ribonucleotide via the linking ribonucleotide sequence if present, or directly if the linking ribonucleotide sequence is absent, wherein the second RNA component consists of, in 5′ to 3′ order, a second 5′ ribonucleotide, a second RNA sequence, and a second 3′ ribonucleotide, wherein the second 5′ and 3′ ribonucleotides are base paired with each other in the RNA molecule, wherein the second RNA sequence comprises a second antisense ribonucleotide sequence of at least 21 contiguous ribonucleotides, a second loop sequence of at least 4 ribonucleotides, and a second sense ribonucleotide sequence of at least 21 contiguous ribonucleotides, wherein in the RNA molecule the second sense ribonucleotide sequence hybridizes with the second antisense ribonucleotide sequence; wherein the 5' leader sequence, if present, consists of a ribonucleotide sequence covalently linked to the first 5' ribonucleotide if the second RNA component is linked to the first 3' ribonucleotide, or covalently linked to the second 5' ribonucleotide if the second RNA component is linked to the first 5' ribonucleotide, wherein the 3' trailer sequence, if present, consists of a ribonucleotide sequence covalently linked to the second 3' ribonucleotide if the second RNA component is linked to the first 3' ribonucleotide, or covalently linked to the first 3' ribonucleotide if the second RNA component is linked to the first 5' ribonucleotide, wherein the RNA molecule consists of a single ribonucleotide chain comprising a 5' end and a 3' end, and wherein (a) when a first sense ribonucleotide sequence hybridizes with a first antisense ribonucleotide sequence, 5% to 40% of the ribonucleotides of the first sense ribonucleotide sequence and the first antisense ribonucleotide sequence are base paired with non-canonical base pairs, or (b) when a second sense ribonucleotide sequence hybridizes with a second antisense ribonucleotide sequence, 5% to 40% of the ribonucleotides of the second sense ribonucleotide sequence and the second antisense ribonucleotide sequence are base paired with non-canonical base pairs, or both (a) and (b).

2. The RNA molecule of claim 1, wherein (a) the first sense ribonucleotide sequence and the first antisense ribonucleotide sequence are at least 40 contiguous nucleotides in length, or (b) the second sense ribonucleotide sequence and the second antisense ribonucleotide sequence are at least 40 contiguous nucleotides in length, or both (a) and (b).

3. The RNA molecule of claim 1, wherein at least 21 contiguous ribonucleotides of the first antisense ribonucleotide sequence are capable of base pairing with nucleotides in the first region of the target RNA molecule.

4. The RNA molecule of claim 3, wherein at least 21 contiguous ribonucleotides of the first antisense ribonucleotide sequence are identical to the complement of the first region of the target RNA molecule.

5. The RNA molecule of claim 1, wherein (i) the first and second antisense ribonucleotide sequences flank the first and second sense ribonucleotide sequences, or (ii) the first and second sense ribonucleotide sequences flank the first and second antisense ribonucleotide sequences.

6. The RNA molecule of claim 1, characterized by one or more of the following: (i) at least 21 contiguous ribonucleotides of the first sense ribonucleotide sequence are identical in sequence to the first region of the target RNA molecule, (ii) a first sense ribonucleotide sequence is covalently linked to the first 5' ribonucleotide without any intervening nucleotides, or a first antisense ribonucleotide sequence is covalently linked to the first 3' ribonucleotide without any intervening nucleotides, or both, (iii) the RNA molecule comprises a linker ribonucleotide sequence, wherein the linker ribonucleotide sequence is less than 20 ribonucleotides in length, (iv) the linking ribonucleotide sequence hybridizes with the target RNA molecule, (v) the linking ribonucleotide sequence is identical to a portion of the complement of the target RNA molecule, (vi) the length of the connecting ribonucleotide sequence is between 1 and 10 ribonucleotides, (vii) at least 21 ribonucleotides of the first sense ribonucleotide sequence and at least 21 ribonucleotides of the second sense ribonucleotide sequence are identical in sequence to different regions of the same target RNA molecule, (viii) at least 21 ribonucleotides of the first sense ribonucleotide sequence and at least 21 ribonucleotides of the second sense ribonucleotide sequence are identical in sequence to regions of different target RNA molecules, (ix) at least 21 ribonucleotides of the second antisense ribonucleotide sequence are completely base paired with at least 21 ribonucleotides of the second sense ribonucleotide sequence, (x) at least 21 ribonucleotides of the first antisense ribonucleotide sequence and at least 21 ribonucleotides of the second antisense ribonucleotide sequence are complementary to different regions of the same target RNA molecule, (xi) the second antisense ribonucleotide sequence is complementary to a region of the target RNA molecule that is different from the first antisense ribonucleotide sequence, (xii) the first 5' ribonucleotide and the second 3' ribonucleotide are adjacent in the RNA molecule, each base-paired and not directly covalently bound to each other, or the second 5' ribonucleotide and the first 3' ribonucleotide are adjacent in the RNA molecule, each base-paired and not directly covalently bound to each other, (xiii) the first and second antisense ribonucleotide sequences are separated by a linker ribonucleotide sequence, (xiv) the first sense ribonucleotide sequence is at least 60% identical to a first region of the target RNA molecule, the second sense ribonucleotide sequence is at least 60% identical to a second region of the target RNA molecule, the second region of the target RNA molecule is different from the first region of the target RNA molecule, wherein the first and second antisense ribonucleotide sequences are linked by a linker ribonucleotide sequence, (xv) the RNA molecule has a 5' leader sequence or a 3' trailer sequence, or both, (xvi) at least one loop sequence, or the first and second loop sequences, in the RNA molecule is longer than 20 nucleotides, (xvii) the RNA molecule has only two loops, and (xviii) at least one loop, or all loops, are between 4 and 50 ribonucleotides in length.

7. The RNA molecule of claim 1, wherein (a) the first antisense ribonucleotide sequence has 80% to 99.9% sequence identity with the complement of the first target region of the target RNA molecule, or (b) the second antisense ribonucleotide sequence independently has 80% to 99.9% sequence identity with the complement of the second target region of the target RNA molecule, or both (a) and (b).

8. The RNA molecule of claim 1, wherein at least 21 ribonucleotides of the first antisense ribonucleotide sequence and at least 21 ribonucleotides of the second antisense ribonucleotide sequence are complementary to different regions of the same target RNA molecule.

9. The RNA molecule of claim 1, wherein at least 21 ribonucleotides of the first antisense ribonucleotide sequence and at least 21 ribonucleotides of the second antisense ribonucleotide sequence are complementary to regions of different target RNA molecules.

10. The RNA molecule of claim 1, wherein when the sense and antisense ribonucleotide sequences hybridize, between 15% and 30% of the ribonucleotides of (a) the first sense ribonucleotide sequence and the first antisense ribonucleotide sequence as a whole, and (b) the second sense ribonucleotide sequence and the second antisense ribonucleotide sequence as a whole, are base paired in non-canonical base pairs.

11. The RNA molecule of claim 10, wherein when the sense and antisense ribonucleotide sequences hybridize, between 16% and 25% of the ribonucleotides of (a) the first sense ribonucleotide sequence and the first antisense ribonucleotide sequence as a whole, or (b) the second sense ribonucleotide sequence and the second antisense ribonucleotide sequence as a whole, or both (a) and (b) are base paired in non-canonical base pairs.

12. The RNA molecule of claim 1, wherein (a) the first sense ribonucleotide sequence and the first antisense ribonucleotide sequence are each 40 to 1,000 nucleotides in length, and (b) the second sense ribonucleotide sequence and the second antisense ribonucleotide sequence are each 40 to 1,000 nucleotides in length.

13. The RNA molecule of claim 1, wherein all non-canonical base pairs in the RNA molecule are G:U base pairs.

14. The RNA molecule of claim 1, wherein the RNA molecule is produced in a cell.

15. The RNA molecule of claim 14, wherein the RNA molecule is produced in a plant cell or a fungal cell.

16. The RNA molecule of claim 14, wherein the RNA molecule is produced in a yeast cell.

17. The RNA molecule of claim 1, wherein the RNA molecule is produced in a cell-free expression system.

18. The RNA molecule of claim 1, wherein the target RNA molecule is in a plant cell, a fungal cell, or an animal cell.

19. The RNA molecule of claim 1, wherein the target RNA molecule is located in an arthropod cell.

20. The RNA molecule of claim 1, wherein the target RNA molecule is located in a plant cell.

21. The RNA molecule of claim 1, wherein the target RNA molecule is located in a eukaryotic cell, wherein the eukaryotic cell is different from the cell that produced the RNA molecule.

22. The RNA molecule of claim 1, wherein the RNA molecule comprises a bulge, or two or more bulges, in the first sense ribonucleotide sequence or the first antisense ribonucleotide sequence, or both, each bulge comprising one or more unpaired nucleotides, wherein the dsRNA region lacks a non-base paired nucleotide at a corresponding position in the complementary sequence opposite to the one or more unpaired nucleotides.

23. The RNA molecule of claim 1, characterized in that One or more of the following: (i) either (a) one out of every four to six ribonucleotides in the first sense ribonucleotide sequence forms a non-canonical base pair with a ribonucleotide in the first antisense ribonucleotide sequence, or (b) one out of every four to six ribonucleotides in the second sense ribonucleotide sequence forms a non-canonical base pair with a ribonucleotide in the second antisense ribonucleotide sequence, or both (a) and (b), (ii) either (a) the region of the dsRNA formed by the first sense ribonucleotide sequence and the first antisense ribonucleotide sequence lacks eight contiguous canonical base pairs, or (b) the region of the dsRNA formed by the second sense ribonucleotide sequence and the second antisense ribonucleotide sequence lacks eight contiguous canonical base pairs, or both (a) and (b), (iii) the first or second antisense ribonucleotide sequence, or both, independently have less than 100% sequence identity to the complement of the first or second target region of the target RNA molecule, respectively, (iv) the first or second antisense ribonucleotide sequence, or both, have 100% sequence identity with the first or second target region of the target RNA molecule, respectively, (v) either (a) the number of ribonucleotides in the first sense ribonucleotide sequence is 90% to 110% of the number of ribonucleotides in the first antisense ribonucleotide sequence, or (b) the number of ribonucleotides in the second sense ribonucleotide sequence is 90% to 110% of the number of ribonucleotides in the second antisense ribonucleotide sequence, or both (a) and (b), and (vi) either (a) the number of ribonucleotides in the first sense ribonucleotide sequence is the same as the number of ribonucleotides in the first antisense ribonucleotide sequence, or (b) the number of ribonucleotides in the second sense ribonucleotide sequence is the same as the number of ribonucleotides in the second antisense ribonucleotide sequence, or both (a) and (b).

24. An RNA molecule comprising a first RNA component, a second RNA component covalently linked to the first RNA component, and optionally one or more or all of the following: (i) a linking ribonucleotide sequence covalently linked to the first and second RNA components, (ii) a 5' leader sequence and (iii) a 3' trailer sequence, wherein the first RNA component consists of a first 5' ribonucleotide, a first RNA sequence, and a first 3' ribonucleotide in a 5' to 3' order, wherein the first 5' and 3' ribonucleotides in the first RNA component are base paired with each other, wherein the first RNA sequence comprises a first sense ribonucleotide sequence of at least 21 contiguous ribonucleotides, a first loop sequence of at least 4 ribonucleotides, and a first antisense ribonucleotide sequence of at least 21 contiguous ribonucleotides, wherein the first antisense ribonucleotide sequence hybridizes with the first sense ribonucleotide sequence in the RNA molecule, wherein the first antisense ribonucleotide sequence is capable of hybridizing with a first region of a target RNA molecule, in, if said linking ribonucleotide sequence is present, said second RNA component is covalently linked to said first 5' ribonucleotide or said first 3' ribonucleotide via said linking ribonucleotide sequence, or if said linking ribonucleotide sequence is absent, said second RNA component is covalently linked to said first 5' ribonucleotide or said first 3' ribonucleotide directly, wherein the second RNA component consists of a second 5' ribonucleotide, a second RNA sequence, and a second 3' ribonucleotide in 5' to 3' order, wherein the second 5' and 3' ribonucleotides in the RNA molecule are base paired with each other, wherein the second RNA sequence comprises a second antisense ribonucleotide sequence of at least 21 contiguous ribonucleotides, a second loop sequence of at least 4 ribonucleotides, and a second sense ribonucleotide sequence of at least 21 contiguous ribonucleotides, wherein the second sense ribonucleotide sequence hybridizes with the second antisense ribonucleotide sequence in the RNA molecule, wherein the 5' leader sequence, if present, consists of a ribonucleotide sequence covalently linked to the first 5' ribonucleotide if the second RNA component is linked to the first 3' ribonucleotide, or covalently linked to the second 5' ribonucleotide if the second RNA component is linked to the first 5' ribonucleotide, wherein the 3' trailer sequence, if present, consists of a ribonucleotide sequence covalently linked to the second 3' ribonucleotide if the second RNA component is linked to the first 3' ribonucleotide, or covalently linked to the first 3' ribonucleotide if the second RNA component is linked to the first 5' ribonucleotide, wherein the RNA molecule consists of a single ribonucleotide chain comprising a 5' end and a 3' end, and wherein the RNA molecule comprises a bulge or two or more bulges in the first sense ribonucleotide sequence or the first antisense ribonucleotide sequence or both, each bulge comprising one or more unpaired nucleotides, wherein the dsRNA region lacks a non-base paired nucleotide at a corresponding position in the complementary sequence relative to the one or more unpaired nucleotides.

25. The RNA molecule of claim 24, wherein the first antisense ribonucleotide sequence comprises one bulge, or two or more bulges.

26. The RNA molecule of claim 24, wherein at least 21 contiguous ribonucleotides of the first antisense ribonucleotide sequence are capable of base pairing with nucleotides in the first region of the target RNA molecule.

27. The RNA molecule of claim 26, wherein at least 21 contiguous ribonucleotides of the first antisense ribonucleotide sequence are identical to the complement of the first region of the target RNA molecule.

28. The RNA molecule of claim 24, wherein (a) the first sense ribonucleotide sequence and the first antisense ribonucleotide sequence are at least 40 nucleotides in length, or (b) the second sense ribonucleotide sequence and the second antisense ribonucleotide sequence are each at least 40 nucleotides in length, or both (a) and (b).

29. The RNA molecule of claim 24, wherein at least 21 ribonucleotides of the first antisense ribonucleotide sequence and at least 21 ribonucleotides of the second antisense ribonucleotide sequence are complementary to different regions of the same target RNA molecule.

30. The RNA molecule of claim 24, wherein at least 21 ribonucleotides of the first antisense ribonucleotide sequence and at least 21 ribonucleotides of the second antisense ribonucleotide sequence are complementary to regions of different target RNA molecules.

31. The RNA molecule of claim 24, wherein (a) when the first sense ribonucleotide sequence hybridizes with the first antisense ribonucleotide sequence, 5% to 40% of the ribonucleotides in the first sense ribonucleotide sequence and the first antisense ribonucleotide sequence are base paired with non-canonical base pairs, and (b) when the second sense ribonucleotide sequence hybridizes with the second antisense ribonucleotide sequence, 5% to 40% of the ribonucleotides in the second sense ribonucleotide sequence and the second antisense ribonucleotide sequence are base paired with non-canonical base pairs.

32. The RNA molecule of claim 31, wherein when the sense and antisense ribonucleotide sequences are hybridized, (a) 15% to 30% of the ribonucleotides in the first sense ribonucleotide sequence and the first antisense ribonucleotide sequence, and (b) 15% to 30% of the ribonucleotides in the second sense ribonucleotide sequence and the second antisense ribonucleotide sequence are base paired with non-canonical base pairs.

33. The RNA molecule of claim 32, wherein when the sense and antisense ribonucleotide sequences are hybridized, 16% to 25% of the ribonucleotides in (a) the first sense ribonucleotide sequence and the first antisense ribonucleotide sequence as a whole, or (b) the second sense ribonucleotide sequence and the second antisense ribonucleotide sequence as a whole, or in both (a) and (b) are base paired with non-canonical base pairs.

34. The RNA molecule of claim 24, wherein (a) the first sense ribonucleotide sequence and the first antisense ribonucleotide sequence are each 40 to 1,000 nucleotides in length, and (b) the second sense ribonucleotide sequence and the second antisense ribonucleotide sequence are each 40 to 1,000 nucleotides in length.

35. The RNA molecule of claim 24, wherein all non-canonical base pairs in the RNA molecule are G:U base pairs.

36. The RNA molecule of claim 24, wherein the RNA molecule is produced in a plant cell or a fungal cell.

37. The RNA molecule of claim 24, wherein the RNA molecule is produced in a yeast cell.

38. The RNA molecule of claim 24, wherein the target RNA molecule is in a plant cell, a fungal cell, or an animal cell.

39. The RNA molecule of claim 24, wherein the target RNA molecule is located in an arthropod cell.

40. The RNA molecule of claim 24, wherein all ribonucleotides in the dsRNA region formed by the second sense ribonucleotide sequence and the second antisense ribonucleotide sequence are base paired.

41. The RNA molecule of claim 24, wherein (a) the first antisense ribonucleotide sequence is 80% to 99.9% identical in sequence to the complement of the first target region of the target RNA molecule, or (b) the second antisense ribonucleotide sequence is independently 80% to 99.9% identical in sequence to the complement of the second target region of the target RNA molecule, or both (a) and (b).

42. The RNA molecule of claim 24, characterized in that One or more of the following: (i) either (a) one out of every four to six ribonucleotides in the first sense ribonucleotide sequence forms a non-canonical base pair with a ribonucleotide in the first antisense ribonucleotide sequence, or (b) one out of every four to six ribonucleotides in the second sense ribonucleotide sequence forms a non-canonical base pair with a ribonucleotide in the second antisense ribonucleotide sequence, or both (a) and (b), (ii) either (a) the region of the dsRNA formed by the first sense ribonucleotide sequence and the first antisense ribonucleotide sequence lacks eight contiguous canonical base pairs, or (b) the region of the dsRNA formed by the second sense ribonucleotide sequence and the second antisense ribonucleotide sequence lacks eight contiguous canonical base pairs, or both (a) and (b), (iii) the first or second antisense ribonucleotide sequence, or both, independently have less than 100% sequence identity to the complement of the first or second target region of the target RNA molecule, respectively, (iv) the first or second antisense ribonucleotide sequence, or both, have 100% sequence identity with the first or second target region of the target RNA molecule, respectively, (v) either (a) the number of ribonucleotides in the first sense ribonucleotide sequence is 90% to 110% of the number of ribonucleotides in the first antisense ribonucleotide sequence, or (b) the number of ribonucleotides in the second sense ribonucleotide sequence is 90% to 110% of the number of ribonucleotides in the second antisense ribonucleotide sequence, or both (a) and (b), and (vi) either (a) the number of ribonucleotides in the first sense ribonucleotide sequence is the same as the number of ribonucleotides in the first antisense ribonucleotide sequence, or (b) the number of ribonucleotides in the second sense ribonucleotide sequence is the same as the number of ribonucleotides in the second antisense ribonucleotide sequence, or both (a) and (b).

43. An isolated and / or exogenous polynucleotide, or a vector comprising the polynucleotide, encoding the RNA molecule of any one of claims 1 to 42, wherein the polynucleotide is operably linked to a promoter capable of directing expression of the RNA molecule in a host cell.

44. A host microorganism comprising (a) an RNA molecule according to any one of claims 1 to 42, or (b) an exogenous polynucleotide or vector encoding the RNA molecule, or both (a) and (b).

45. An in vitro cell comprising (a) the RNA molecule of any one of claims 1 to 42, or (b) an exogenous polynucleotide or vector encoding the RNA molecule, or both (a) and (b).

46. ​​A host cell comprising (a) an RNA molecule according to any one of claims 1 to 42, or (b) an exogenous polynucleotide or vector encoding the RNA molecule, or both (a) and (b), wherein the cell is dead or incapable of reproduction.

47. The host cell of claim 46, wherein the exogenous polynucleotide is stably integrated into the genome of the cell.

48. A method of producing the RNA molecule of any one of claims 1 to 42, the method comprising expressing a polynucleotide encoding the RNA molecule in a host cell or a cell-free expression system.

49. A method for producing a transgenic plant cell, the method comprising introducing an exogenous polynucleotide encoding the RNA molecule of any one of claims 1 to 42 into the plant cell, such that the exogenous polynucleotide is stably integrated into the genome of the transgenic plant cell.

50. A method for producing a host cell, an in vitro cell, or a plant or a part thereof, wherein the host cell, the in vitro cell, or the plant or a part thereof comprises (a) an RNA molecule of any one of claims 1 to 42, or (b) an exogenous polynucleotide or a vector encoding the RNA molecule, or both (a) and (b), the method comprising contacting the cell, the plant or a part thereof with the RNA molecule, the exogenous polynucleotide or the vector.

51. A composition comprising an RNA molecule according to any one of claims 1 to 42 and one or more suitable carriers.

52. An extract of a host cell, wherein the extract comprises an RNA molecule according to any one of claims 1 to 42, and / or a polynucleotide or vector encoding the RNA molecule.

53. A method for down-regulating the level and / or activity of a target RNA molecule in an arthropod or plant, the method comprising delivering to the arthropod or plant an RNA molecule according to any one of claims 1 to 42.

54. A method for down-regulating the level and / or activity of a target RNA molecule in an arthropod, the method comprising delivering to the arthropod a host microorganism according to claim 44 or a plant cell comprising an RNA molecule according to any one of claims 1 to 42.

55. A method for down-regulating the level and / or activity of a target RNA molecule in an arthropod, the method comprising delivering the host cell of claim 46 to the arthropod.

56. A method of controlling an arthropod or a plant, the method comprising delivering to the arthropod or plant an RNA molecule according to any one of claims 1 to 42, wherein the RNA molecule has a deleterious effect on the arthropod or plant.

57. A method of controlling an arthropod, the method comprising delivering the host microorganism of claim 44 to the arthropod, wherein the RNA molecule has a deleterious effect on the arthropod.

58. A method of controlling an arthropod, the method comprising delivering to the arthropod the host cell of claim 46, wherein the RNA molecule has a deleterious effect on the arthropod.

59. The method of claim 57, used to control insects.

60. The method of claim 59, wherein the RNA molecule is produced in a plant or part thereof and the insect consumes the plant or part thereof, thereby controlling the insect.

61. The method of claim 56, wherein the RNA molecule is delivered as a formulation with a suitable carrier and sprayed, dusted or otherwise applied to the surface of a plant or part thereof.

62. An RNA molecule according to any one of claims 1 to 42 for use in preventing or treating a disease in a subject, wherein the RNA molecule has a beneficial effect on at least one symptom of the disease or prevents the disease.

63. An RNA molecule according to any one of claims 1 to 42, for use in the manufacture of a medicament for preventing or treating a disease in a subject, wherein the RNA molecule has a beneficial effect on at least one symptom of the disease.

64. A polynucleotide or vector encoding an RNA molecule according to any one of claims 1 to 42 for use in the manufacture of a medicament for preventing or treating a disease in a subject, wherein the RNA molecule has a beneficial effect on at least one symptom of the disease.

65. The host microorganism of claim 44, for use in the manufacture of a medicament for preventing or treating a disease in a subject, wherein the RNA molecule has a beneficial effect on at least one symptom of the disease.

66. The host cell of claim 46, for use in the manufacture of a medicament for preventing or treating a disease in a subject, wherein the RNA molecule has a beneficial effect on at least one symptom of the disease.

67. The RNA molecule of claim 63, wherein the RNA molecule or the drug is administered topically, orally or by injection.

68. A method of introducing an RNA molecule into a plant, the method comprising topically applying to the plant or part thereof a composition comprising an RNA molecule according to any one of claims 1 to 42.

Images