RNA molecules

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, the sense and antisense strands of the dsRNA molecule comprise ribonucleotides that are base-paired by non-standard base pairs or that are not base-paired.

Description

[Technical Field]

[0001] This invention relates to novel double-stranded RNA (dsRNA) structures and their use in gene silencing. [Background technology]

[0002] Conventional dsRNA molecules used to induce RNA interference (RNAi) in animal cells are formed by annealing single-stranded sense and antisense RNA, or from self-complementary RNA with a complementary region that enables the formation of a stem-loop structure, also known as hairpin RNA (hpRNA). In plants, transgenes expressing long hairpin RNA have proven highly effective in inducing RNAi. Several studies of long hpRNA have shown that the processed RNA product of the dsRNA, called short interference RNA (siRNA), formed from sequences near the loop end of the hpRNA, is more abundant than the open end of the hpRNA structure. This suggests that Dicer processing of the dsRNA stem of the hpRNA appears to begin from the loop end. The entire loop can be precisely excised from the hpRNA structure, which may result in a dsRNA end to which the RNAase enzyme Dicer can bind and initiate siRNA processing.

[0003] While dsRNA-induced gene silencing has proven to be a beneficial mechanism for altering the phenotypic characteristics of organisms, there is a need for alternative, and preferably improved, dsRNA molecules that can be used in RNAi. [Overview of the project]

[0004] The inventors have devised a novel design for RNAi-inducing molecules, referred to herein as loop-terminated dsRNAs (ledRNAs), which have one or more of the following characteristics: they are easily synthesized, they more readily form dsRNA structures, and they efficiently induce silencing of target genes in eukaryotic cells. LedRNAs are also effective when applied locally to plant leaves.

[0005] In a first aspect, the present invention relates to an RNA molecule comprising a first ribonucleic acid (RNA) component, a second RNA component covalently bonded to the first RNA component, and optionally one or more or all of the following: (i) a binding ribonucleotide sequence covalently bonding the first and second RNA components, (ii) a 5' leader sequence, and (iii) a 3' trailer sequence. The first RNA component consists of a first 5' ribonucleotide, a first RNA sequence, and a first 3' ribonucleotide, in the order of 5' to 3', wherein the first 5' and 3' ribonucleotides form base pairs with each other in the RNA molecule, and the first RNA sequence includes a first sense ribonucleotide sequence of at least 20 consecutive ribonucleotides, a first loop sequence of at least 4 ribonucleotides, and a first antisense ribonucleotide sequence of at least 20 consecutive ribonucleotides, wherein the first antisense ribonucleotide sequence hybridizes with the first sense ribonucleotide sequence in the RNA molecule, and the first antisense ribonucleotide sequence can hybridize to a first region of the target RNA molecule. The second RNA component covalently binds to the first 5' ribonucleotide or the first 3' ribonucleotide, either via a binding ribonucleotide sequence if present, or directly if the binding ribonucleotide sequence is absent. The second RNA component consists of a second 5' ribonucleotide, a second RNA sequence, and a second 3' ribonucleotide, in the order of 5' to 3', wherein the second 5' and 3' ribonucleotides form base pairs with each other in the RNA molecule, and the second RNA sequence includes a second sense ribonucleotide sequence, a second loop sequence of at least four ribonucleotides, and a second antisense ribonucleotide sequence, wherein the second sense ribonucleotide sequence hybridizes with the second antisense ribonucleotide sequence in the RNA molecule. The 5' leader sequence, if present, consists of a ribonucleotide sequence that, if the second RNA component binds to the first 3' ribonucleotide, covalently binds to the first 5' ribonucleotide, or if the second RNA component binds to the first 5' ribonucleotide, covalently binds to the second 5' ribonucleotide. The present invention provides an RNA molecule comprising a ribonucleotide sequence in which, if present, the 3' trailer sequence is covalently bonded to the second 3' ribonucleotide when the second RNA component binds to the first 3' ribonucleotide, or covalently bonded to the first 3' ribonucleotide when the second RNA component binds to the first 5' ribonucleotide.

[0006] In a second aspect, the present invention relates to an RNA molecule comprising a first RNA component, a second RNA component covalently bonded to the first RNA component, and optionally one or more or all of the following: (i) a binding ribonucleotide sequence covalently bonding the first and second RNA components, (ii) a 5' leader sequence, and (iii) a 3' trailer sequence. The first RNA component consists of a first 5' ribonucleotide, a first RNA sequence, and a first 3' ribonucleotide, in the order of 5' to 3', wherein the first 5' and 3' ribonucleotides form base pairs, and the first RNA sequence includes a first sense ribonucleotide sequence, a first loop sequence of at least four 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 consecutive ribonucleotides, thereby ensuring that at least 20 consecutive ribonucleotides of the first sense ribonucleotide sequence are perfectly base-paired with at least 20 consecutive ribonucleotides of the first antisense ribonucleotide sequence, and that at least 20 consecutive ribonucleotides of the first sense ribonucleotide sequence or at least 20 consecutive ribonucleotides of the first antisense ribonucleotide sequence are sequence-identical to each or both of the first region or its complement of the target RNA molecule. The second RNA component, if present, covalently binds to the first 5' ribonucleotide or the first 3' ribonucleotide via a binding ribonucleotide sequence. The second RNA component consists of a second 5' ribonucleotide, a second RNA sequence, and a second 3' ribonucleotide, in the order of 5' to 3', wherein the second 5' and 3' ribonucleotides form base pairs, and the second RNA sequence includes a second sense ribonucleotide sequence, a second loop sequence of at least four ribonucleotides, and a second antisense ribonucleotide sequence, wherein the second sense ribonucleotide sequence forms base pairs with the second antisense ribonucleotide sequence. The 5' leader sequence, if present, consists of a ribonucleotide sequence that, if the second RNA component binds to the first 3' ribonucleotide, covalently binds to the first 5' ribonucleotide, or if the second RNA component binds to the first 5' ribonucleotide, covalently binds to the second 5' ribonucleotide. The present invention provides an RNA molecule comprising a ribonucleotide sequence in which, if present, the 3' trailer sequence is covalently bonded to the second 3' ribonucleotide when the second RNA component binds to the first 3' ribonucleotide, or covalently bonded to the first 3' ribonucleotide when the second RNA component binds to the first 5' ribonucleotide.

[0007] In a preferred embodiment, the RNA molecule of the present invention is a chimeric RNA molecule.

[0008] In a third aspect, the present invention relates to a chimeric ribonucleic acid (RNA) molecule comprising a dsRNA region containing sense ribonucleotide sequences and antisense ribonucleotide sequences that can hybridize with each other to form a double-stranded RNA (dsRNA) region, i) The sense ribonucleotide sequence consists of a first 5' ribonucleotide, a first RNA sequence, and a first 3' ribonucleotide, which are covalently bonded from 5' to 3' in that order. ii) The antisense ribonucleotide sequence consists of a second 5' ribonucleotide, a second RNA sequence, and a second 3' ribonucleotide, which are covalently bonded from 5' to 3' in that order. iii) The first 5' ribonucleotide forms a base pair with the second 3' ribonucleotide to form the terminal base pair of the dsRNA region, iv) The second 5' ribonucleotide forms a base pair with the first 3' ribonucleotide to form the terminal base pair of the dsRNA region, v) Approximately 5% to 40% of the ribonucleotides in the sense ribonucleotide sequence and the antisense ribonucleotide sequence are either base-paired with non-standard base pairs or are not base-paired at all. vi) The dsRNA region does not contain 20 consecutive standard base pairs, vii) The RNA molecule can be processed in eukaryotic cells or in vitro, thereby cleaving the antisense ribonucleotide sequence and generating short antisense RNA (asRNA) molecules with a length of 20-24 ribonucleotides. viii) The RNA molecule, or at least some asRNA molecules, or both, can reduce the expression or activity of target RNA molecules in eukaryotic cells. ix) The present invention provides a chimeric RNA molecule that can be enzymatically produced by transcription in vitro, intracellularly, or both.

[0009] In a fourth aspect, the present invention relates to a chimeric RNA molecule comprising a first RNA component and a second RNA component covalently bonded to the first RNA component, The first RNA component includes a first double-stranded RNA (dsRNA) region comprising a first sense ribonucleotide sequence and a first antisense ribonucleotide sequence that can hybridize with each other to form a first dsRNA region, and a first intervening ribonucleotide sequence of at least four nucleotides that covalently bond the first sense ribonucleotide sequence and the first antisense ribonucleotide sequence. The second RNA component comprises a second sense ribonucleotide sequence, a second antisense ribonucleotide sequence, and a second intervening ribonucleotide sequence of at least four ribonucleotides covalently linking the second sense ribonucleotide sequence and the second antisense ribonucleotide sequence, wherein the second sense ribonucleotide sequence hybridizes with the second antisense ribonucleotide sequence within the RNA molecule. In the first RNA component, i) The first sense ribonucleotide sequence consists of at least 20 consecutive ribonucleotides covalently linked from 5' to 3', the first 5' ribonucleotide, the first RNA sequence, and the first 3' ribonucleotide. ii) The first antisense ribonucleotide sequence consists of at least 20 consecutive ribonucleotides covalently linked from 5' to 3', a second 5' ribonucleotide, a second RNA sequence, and a second 3' ribonucleotide. iii) The first 5' ribonucleotide forms a base pair with the second 3' ribonucleotide, iv) The second 5' ribonucleotide forms a base pair with the first 3' ribonucleotide, v) 5% to 40% of the ribonucleotides in the first sense ribonucleotide sequence and the first antisense ribonucleotide sequence are either base-paired with non-standard base pairs or are not base-paired overall. vi) The first dsRNA region does not contain 20 consecutive standard base pairs. The chimeric RNA molecule can be processed in eukaryotic cells or in vitro, thereby cleaving the first antisense ribonucleotide sequence and generating short antisense RNA (asRNA) molecules with a length of 20-24 ribonucleotides. (a) The chimeric RNA molecule, or at least some asRNA molecules, or both, can reduce the expression or activity of a target RNA molecule in a eukaryotic cell, or (b) The first antisense ribonucleotide sequence comprises a sequence of at least 20 consecutive ribonucleotides whose sequence is at least 50% identical to the complementary region of the target RNA molecule, or (c) Provides a chimeric RNA molecule that is both (a) and (b).

[0010] In the first, second, and fourth embodiments, the first 5' ribonucleotide and the first 3' ribonucleotide of the first RNA component form a base pair. This base pair is defined herein as the terminal base pair of the dsRNA region formed by the autohybridization of the first RNA component. In embodiments in which the first sense ribonucleotide sequence is covalently bonded to the first 5' ribonucleotide without any intervening nucleotides, and the first antisense ribonucleotide sequence is covalently bonded to the first 3' ribonucleotide without any intervening nucleotides, the first 5' ribonucleotide is directly ligated to one of the sense sequence and the antisense sequence, and the first 3' ribonucleotide is directly ligated to the other of the sense sequence and the antisense sequence.

[0011] In a preferred embodiment, at least 20 consecutive ribonucleotides of the first antisense ribonucleotide sequence can all form base pairs with nucleotides in the first region of the target RNA molecule. In one embodiment, the first sense ribonucleotide sequence is covalently bonded to the first 5' ribonucleotide without any intervening nucleotides, or the first antisense ribonucleotide sequence is covalently bonded to the first 3' ribonucleotide without any intervening nucleotides, or both.

[0012] In one embodiment, the RNA molecule comprises a binding ribonucleotide sequence whose sequence is related to a target gene that is at least partially identical to either a region of the target RNA molecule or its complement. In a preferred embodiment, the binding ribonucleotide sequence forms part of one continuous sense sequence together with the sense sequences of the first and second RNA components, or forms part of one continuous antisense sequence together with the antisense sequences of the first and second RNA components. In one embodiment, the RNA molecule comprises a binding ribonucleotide sequence that is less than 20 ribonucleotides. In one embodiment, the binding ribonucleotide sequence hybridizes to the target RNA molecule. In one embodiment, the binding ribonucleotide sequence is identical to a part of the complement of the target RNA molecule. In one embodiment, the binding ribonucleotide sequence is 1 to 10 ribonucleotides.

[0013] In embodiments of the first, second, or fourth aspect, the RNA molecule comprises one or more or all of (i) a binding ribonucleotide sequence that covalently links the first and second RNA components, (ii) a 5' extension sequence, and (iii) a 3' extension sequence, wherein, respectively, the 5' extension sequence, if present, consists of a sequence of ribonucleotides that covalently links to the first RNA component or the second RNA component, and the 3' extension sequence, if present, consists of a sequence of ribonucleotides that covalently links 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 the binding ribonucleotide sequence. In an alternative embodiment, the first RNA component and the second RNA component are directly linked without any binding ribonucleotide sequence present.

[0014] In the embodiments of the first to fourth aspects, the RNA molecule includes two or more sense ribonucleotide sequences that are identical in sequence to regions of the target RNA molecule, and the RNA molecule includes one or more antisense ribonucleotide sequences that base pair with the sense ribonucleotide sequences, and the one or more antisense sequences are complementary, preferably fully complementary, to regions of the target molecule. In one embodiment, the two or more sense ribonucleotide sequences are identical in sequence to different regions of the same target RNA molecule, and may or may not be contiguous within the target RNA molecule. In one embodiment, the two or more sense ribonucleotide sequences are identical in sequence to regions of different target RNA molecules. In one embodiment, the two or more sense ribonucleotide sequences do not have intervening loop sequences, i.e., they are contiguous to the target RNA molecule.

[0015] In the preferred embodiments of the first to fourth aspects, the RNA molecule includes two or more antisense ribonucleotide sequences and sense ribonucleotide sequences that base pair with them, and the antisense sequences are each complementary to regions of the target RNA molecule. The regions of the target RNA molecule that are complementary may or may not be contiguous within the target RNA molecule. In one embodiment, the two or more antisense ribonucleotide sequences are complementary to different regions of the same target RNA molecule. In one embodiment, the second of the two or more antisense ribonucleotide sequences is complementary to a region of a different target RNA molecule than the first of the two or more antisense ribonucleotide sequences. In a preferred embodiment, the two or more antisense ribonucleotide sequences do not have intervening loop sequences, i.e., they are contiguous to the complement of the target RNA molecule. In a preferred embodiment, one or both of the two or more antisense ribonucleotide sequences and sense ribonucleotide sequences base pair along their entire length via standard base pairs, or some standard and some non-standard base pairs, preferably G:U base pairs.

[0016] In one preferred embodiment of the first to fourth aspects, the RNA molecule is a single-stranded ribonucleotide. For example, the RNA molecule may include a 5' end, at least one sense ribonucleotide sequence having a length of at least 21 nucleotides, an antisense ribonucleotide sequence that is fully base-paired with each sense ribonucleotide sequence over at least 21 consecutive nucleotides, at least two loop sequences, and a single-stranded ribonucleotide having a 3' end. The 5' to 3' order may be sense ribonucleotide sequence followed by antisense ribonucleotide sequence, or vice versa. In one embodiment, the 5' and 3' ribonucleotides are adjacent, base-paired with each other, and not directly covalently bonded (see, for example, Figure 1).

[0017] 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, and a second antisense ribonucleotide sequence that hybridizes to a second region of the target RNA, wherein the second region of the target RNA differs from the first region of the target RNA, and the RNA molecule comprises only one sense ribonucleotide sequence that hybridizes to the target RNA, and the two antisense sequences are not contiguous within the RNA molecule. In one embodiment, the first and second regions of the target RNA are contiguous within the target RNA, or they are not contiguous.

[0018] 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 the first region of the target RNA, and a second sense ribonucleotide sequence that is at least 60% identical to the second region of the target RNA, wherein the second region of the target RNA is different from the first region of the target RNA, and the RNA molecule contains only one antisense ribonucleotide sequence that hybridizes to the target RNA, and the two sense sequences are not contiguous within the RNA molecule. In one embodiment, the first and second regions of the target RNA are contiguous within the target RNA molecule, or they are not contiguous. 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 respective regions of the target RNA, i.e., the first sense sequence may be at least 70% identical to its target region, the second sequence at least 80% identical to its target sequence, and so on.

[0019] In one preferred embodiment of the first to fourth aspects, the RNA molecule is a single-stranded ribonucleotide having a 5' end, at least one senseribonucleotide sequence having a length of at least 21 nucleotides, an antisenseribonucleotide sequence that is fully base-paired with each senseribonucleotide sequence over at least 21 consecutive 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 which is a consecutive base-pair having a length of at least 21, containing several non-standard base pairs, most preferably several G:U base pairs, and the double-stranded region contains at least one senseribonucleotide sequence having a length of at least 21 nucleotides.

[0020] In preferred embodiments of the first and second aspects, the second RNA component is i) The second sense ribonucleotide sequence consists of at least 20 consecutive ribonucleotides covalently linked from 5' to 3', a second 5' ribonucleotide, a third RNA sequence, and a third 3' ribonucleotide. ii) The second antisense ribonucleotide sequence consists of at least 20 consecutive ribonucleotides covalently linked from 5' to 3', a third 5' ribonucleotide, a fourth RNA sequence, and a third 3' ribonucleotide. iii) The second 5' ribonucleotide forms a base pair with the second 3' ribonucleotide, iv) Characterized by the third 3' ribonucleotide forming a base pair with the third 5' ribonucleotide, The chimeric RNA molecule can be processed in eukaryotic cells or in vitro, thereby cleaving the second antisense ribonucleotide sequence to generate a short antisense RNA (asRNA) molecule of 20-24 ribonucleotides in length. Most preferably, the asRNA molecule generated from the second antisense sequence can reduce the expression of the target RNA, either without or in combination with the asRNA generated from the first antisense sequence of the first RNA component.

[0021] In a preferred embodiment of the fourth aspect, the second RNA component is i) The second sense ribonucleotide sequence consists of at least 20 consecutive ribonucleotides covalently linked from 5' to 3', a third 5' ribonucleotide, a third RNA sequence, and a third 3' ribonucleotide. ii) The second antisense ribonucleotide sequence consists of at least 20 consecutive ribonucleotides covalently linked from 5' to 3', a fourth 5' ribonucleotide, a fourth RNA sequence, and a fourth 3' ribonucleotide. iii) The third 5' ribonucleotide forms a base pair with the fourth 3' ribonucleotide, iv) Characterized by the third 3' ribonucleotide forming a base pair with the third 5' ribonucleotide, The chimeric RNA molecule can be processed in eukaryotic cells or in vitro, thereby cleaving the second antisense ribonucleotide sequence and generating short antisense RNA (asRNA) molecules with a length of 20-24 ribonucleotides.

[0022] In these preferred embodiments, it is more preferable that 5% to 40% of the ribonucleotides of the second sense ribonucleotide sequence and the second antisense ribonucleotide sequence are, overall, base-paired with non-standard base pairs or not base-paired, and / or the second dsRNA region does not contain 20 consecutive standard 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 are, overall, base-paired with non-standard base pairs or 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-standard base pairs in the second dsRNA region are G:U base pairs. Most preferably, in these embodiments, (a) The chimeric RNA molecule, or at least some asRNA molecules, or both, can reduce the expression or activity of a target RNA molecule in a eukaryotic cell, or (b) The second antisense ribonucleotide sequence comprises at least 20 consecutive ribonucleotide sequences that are at least 50% identical, preferably at least 60%, more preferably at least 70%, even more preferably at least 80%, most preferably at least 90%, or 100% identical to the complementary region of the target RNA molecule, or both of (a) and (b).

[0023] In one embodiment of the first to fourth aspects, the RNA molecule includes a 5' leader sequence or a 5' extension sequence. In one embodiment, the RNA molecule includes a 3' trailer sequence or a 3' extension sequence. In a preferred embodiment, the RNA molecule includes both a 5' leader / extension sequence and a 3' trailer / extension sequence.

[0024] In one embodiment of the first to fourth aspects, each ribonucleotide of the RNA molecule is covalently bonded to two other nucleotides, i.e., it forms a covalently closed circle. Alternatively, the RNA molecule may be shown as dumbbell-shaped (Figure 1), with gaps or nicks in part of the double-stranded structure.

[0025] In one embodiment of the first to fourth aspects, at least one or all loop sequences of the RNA molecule are longer than 20 nucleotides. In a preferred embodiment, at least one loop of the RNA molecule is 4 to 1,000 ribonucleotides long. In a more preferred embodiment, all loops are 4 to 1,000 ribonucleotides long. In a more preferred embodiment, at least one loop of the RNA molecule is 4 to 200 ribonucleotides long. In an even more preferred embodiment, all loops are 4 to 200 ribonucleotides long. In an even more preferred embodiment, at least one loop of the RNA molecule is 4 to 50 ribonucleotides long. In the most preferred embodiment, all loops are 4 to 50 ribonucleotides long. In one embodiment, the eukaryotic cell is a vertebrate cell, and each loop of the RNA molecule is 20 to 50, or 20 to 30 ribonucleotides long.

[0026] In embodiments of the first to fourth aspects, the RNA molecule has no bulge in the double-stranded region, or has one or more bulges. In this context, a bulge is a nucleotide or two or more consecutive nucleotides in a sense or antisense ribonucleotide sequence that is not base-paired in the dsRNA region and does not have a mismatched nucleotide at a corresponding position in the complementary sequence of the dsRNA region. The dsRNA region of the RNA molecule may contain a sense sequence or antisense sequence, or both, that loops out from the dsRNA region when a dsRNA structure is formed, containing a sequence of two or more nucleotides. The loop-out sequence itself may form some internal base pairing, for example, it may form a stem-loop structure.

[0027] In embodiments of the first to fourth aspects, the RNA molecule has no bulge in the double-stranded region, or has one or more bulges. In this context, a bulge is a nucleotide or two or more consecutive nucleotides in a sense or antisense ribonucleotide sequence that is not base-paired in the dsRNA region and does not have a mismatched nucleotide at a corresponding position in the complementary sequence of the dsRNA region. The dsRNA region of the RNA molecule may contain a sense sequence or antisense sequence, or both, that loops out from the dsRNA region when a dsRNA structure is formed, containing a sequence of two or more nucleotides. The loop-out sequence itself may form some internal base pairing, for example, it may form a stem-loop structure.

[0028] 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 of the RNA molecule, or the first and second dsRNA regions, contains one, two, or more nucleotides that are not base-paired in the double-stranded region, or up to 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% of nucleotides that are not base-paired in the double-stranded region.

[0029] In preferred embodiments of the first to fourth aspects of the present invention, the target RNA molecule or RNA molecule, or both, are located within a eukaryotic cell. For example, the eukaryotic cell may 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 different from the cell containing the target RNA, such as a bacterial cell or other microbial cell. Similarly, in one embodiment, the RNA molecule of the present invention is produced in a eukaryotic cell that does not contain the target RNA when the RNA molecule of the present invention is produced, but if the target RNA is, for example, viral RNA or other introduced RNA, the eukaryotic cell containing the RNA molecule of the present invention and / or its processed RNA product may be a host for the target RNA. Such cells may be prophylactically protected from viruses or other introduced RNA.

[0030] In preferred embodiments of the first to fourth aspects, the RNA molecule can be enzymatically produced by transcription in vitro, intracellularly, or both. In one embodiment, the RNA molecule of the present invention is expressed intracellularly, i.e., produced intracellularly 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 that can reside on an intracellular vector or be incorporated into the cellular genome (either the nuclear genome or the cellular plastid DNA). The one or more nucleic acids encoding the RNA molecule may be RNA molecules such as viral vectors.

[0031] Accordingly, in one embodiment, the present invention provides cells containing the RNA molecule described herein. In a preferred embodiment, the present invention provides the RNA molecule described herein, expressed in cells and isolated and / or purified from cells. Accordingly, the present invention provides preparations of RNA molecules isolated according to one or more of the first to fourth embodiments, which are suitable for administration to cells containing or potentially containing target RNA.

[0032] In one embodiment, one or more target RNAs encode a protein. Alternatively, one or more target RNAs do not encode a protein such as rRNA, tRNA, snoRNA, or miRNA.

[0033] In embodiments 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 are either base-paired with non-standard 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-standard base pairs of the dsRNA region or all dsRNA regions of the RNA molecule are G:U base pairs. The G nucleotide of each G:U base pair may independently be in the sense ribonucleotide sequence or, preferably, in the antisense ribonucleotide sequence. With respect to the G nucleotide of the G:U base pair in the dsRNA region, preferably at least 50% are 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% are in the antisense ribonucleotide sequence of the dsRNA region. This feature can be applied to all dsRNA regions within an RNA molecule. In one embodiment, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, more preferably less than 1% of the ribonucleotides in the dsRNA region, or all dsRNA regions of the RNA molecule, are not base-paired, or most preferably all of them are not base-paired. In a preferred embodiment, one-quarter to one-sixth of the ribonucleotides in the dsRNA region, or all dsRNA regions, are non-standard base-paired or not base-paired within the RNA molecule. In a preferred embodiment, the dsRNA region, or all dsRNA regions, do not contain eight consecutive standard base pairs. In alternative embodiments, the dsRNA region includes at least eight consecutive standard base pairs, e.g., 8–12 or 8–14 consecutive standard base pairs. In preferred embodiments, all ribonucleotides in the dsRNA region form base pairs with standard or non-standard base pairs.In one embodiment, one or more ribonucleotides of a sense ribonucleotide sequence, or one or more ribonucleotides of an antisense ribonucleotide sequence, or both, do not form base pairs. In one embodiment, one or more ribonucleotides of each sense ribonucleotide sequence and one or more ribonucleotides of each antisense ribonucleotide sequence do not form base pairs within the RNA molecule of the present invention.

[0034] In one embodiment, the antisense RNA sequences of the first RNA component, or the second RNA component, or both, are sequence-identical to less than 100%, or about 80% to 99.9%, or about 90% to 98%, or about 95% to 98%, preferably 98% to 99.9%, with respect to the complement region of the target RNA molecule, or to two such regions in the target RNA molecule, which may or may not be contiguous. In a preferred embodiment, the antisense RNA sequence is sequence-identical to 100% of the complement region of the target RNA molecule, for example, a region containing the contiguous nucleotides 21, 23, 25, 27, 30, or 32. In one embodiment, the sense or antisense ribonucleotide sequence, or both, has a continuous nucleotide length of 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. When using RNA molecules in plant or fungal cells, or for use in non-vertebrate cells, a length of at least 100 nucleotides is preferred. When using RNA molecules in vertebrate cells, a length of 50 nucleotides or less, for example, 31 to 50 nucleotides in the sense and antisense ribonucleotide sequences in dsRNA 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%, and 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 of an RNA molecule.

[0035] In embodiments of the first to fourth aspects, the first 3' ribonucleotide and the second 5' ribonucleotide of the RNA molecule are covalently linked by a loop sequence consisting of at least 4 ribonucleotides, or 4 to 1,000 ribonucleotides, preferably 4 to 200 ribonucleotides, more preferably 4 to 50 ribonucleotides. In one embodiment, the RNA molecule further comprises a 5' elongation sequence covalently linked to the first 5' ribonucleotide, or a 3' elongation sequence covalently linked to the second 3' ribonucleotide, or both. In one embodiment, the chimeric RNA molecule further comprises a 5' elongation sequence covalently linked to the second 5' ribonucleotide, or a 3' elongation sequence covalently linked to the first 3' ribonucleotide, or both. In this embodiment, the RNA molecule may be produced as a single RNA transcript by transcription from a nucleic acid molecule and then processed to contain two RNA strands, but may contain two separate RNA strands that hybridize to form an RNA molecule.

[0036] The total length of the RNA molecule of the present invention, which is generated as a single-stranded RNA after being cut out by splicing from any intron but before processing of the RNA molecule by Dicer enzyme or other RNAse, is typically 50 to 2000 ribonucleotides, preferably 60 or 70 to 2000 ribonucleotides, more preferably 80 or 90 to 2000 ribonucleotides, and even more preferably 100 or 110 to 2000 ribonucleotides. In preferred embodiments, the minimum length of the RNA molecule is 120, 130, 140, 150, 160, 180, or 200 ribonucleotides, and the maximum length is 400, 500, 600, 700, 800, 900, 1000, 1200, 1400, 1500, or 2000 ribonucleotides. Each combination of the aforementioned minimum and maximum lengths is considered. The generation of RNA molecules of such length can be easily achieved in vitro, or by transcription in cells such as bacteria or other microbial cells, or in eukaryotic cells where the target gene is downregulated.

[0037] In one embodiment of the first to fourth aspects, the chimeric RNA molecule comprises two or more identical or preferably different dsRNA regions.

[0038] In preferred embodiments of the first to fourth aspects, the RNA molecule is expressed in a eukaryotic cell, i.e., produced by intracellular transcription. In these embodiments, processing of RNA molecules of 22 and / or 20 ribonucleotides results in a larger proportion of dsRNA molecules compared to processing similar RNA molecules having a corresponding dsRNA region that fully base-pairs with standard base pairs. That is, the RNA molecules of these embodiments are processed more readily to provide 22 and / or 20 ribonucleotide short antisense RNAs than similar RNA molecules that fully base-pair with standard base pairs, in proportion to the total number of 20-24 nucleotide asRNAs generated from the RNA molecule.

[0039] In one embodiment, the RNA molecule of the present invention comprises a combination of two or more features of the RNA molecules described herein.

[0040] In another embodiment, the present invention provides polynucleotides encoding RNA molecules as described herein, preferably chimeric RNA molecules as described herein. In one embodiment, the polynucleotide is a DNA construct that can be incorporated into a larger DNA molecule such as a chromosome. In one embodiment, the polynucleotide is operably ligated to a promoter that can direct the expression of the RNA molecule in a host cell. The host cell may be a bacterial cell such as E. 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 heterogeneous to the polynucleotide. The polynucleotide encoding the RNA molecule may be a chimeric or recombinant polynucleotide, or an isolated and / or exogenous polynucleotide. In one embodiment, the promoter may function in vitro and may be, for example, a bacteriophage promoter, such as a T7 RNA polymerase promoter or an SP6 RNA polymerase promoter. In one embodiment, the promoter may be an RNA polymerase III promoter such as a U6 promoter or an H1 promoter. In one embodiment, the promoter may be an RNA polymerase II promoter and may 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 that can be excised by splicing during or after the transcription of the polynucleotide in a host cell. In one embodiment, the present invention provides a vector comprising the polynucleotide 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.

[0041] In embodiments where the polynucleotide or vector of the present invention is present in a eukaryotic host cell, preferably a plant, the promoter region of the polynucleotide or vector operably linked to the region encoding the RNA molecule of the present invention has a lower level of methylation compared to the promoter of the corresponding polynucleotide or vector encoding an RNA molecule having a corresponding dsRNA region that fully base-pairs with standard base pairs. In one embodiment, the lower level of methylation is less than 50%, less than 40%, less than 30%, or less than 20% compared to the promoter of the corresponding polynucleotide or vector. In one embodiment, the host cell contains at least two copies of the polynucleotide or vector encoding the RNA molecule of the present invention. In this embodiment, i) The level of reduction in the expression and / or activity of the target RNA molecule in eukaryotic cells is at least the same as that in corresponding eukaryotic cells having a single copy of the polynucleotide or vector, and / or ii) The level of reduction in the expression and / or activity of the target RNA molecule in eukaryotic cells is lower compared to the corresponding cells containing RNA molecules that have a corresponding dsRNA region that fully base pairs with standard base pairs.

[0042] In another embodiment, the present invention provides a host cell comprising an RNA molecule, a polynucleotide, or a vector comprising the same as described herein. 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 non-human or human cell in 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 an alternative embodiment, the host cell is dead.

[0043] In another embodiment, the present invention provides non-human organisms comprising RNA molecules of the present invention, preferably chimeric RNA molecules as described herein, polynucleotides or vectors of the present invention containing them, or host cells containing them. In one embodiment, the non-human organism is transgenic insofar as it contains the polynucleotides of the present invention. In one embodiment, the polynucleotides are stably incorporated into the genome of the non-human organism.

[0044] In another embodiment, the present invention provides a method for generating the RNA molecule of the present invention, the method comprising expressing the polynucleotide of the present invention in a host cell or a cell-free expression system. In this embodiment, the method may or may not further include at least partially purifying the RNA molecule.

[0045] In another embodiment, the present invention provides a method for producing a cell or a non-human organism, preferably a plant or a fungus, the method comprising introducing the polynucleotide or vector of the present invention into a cell, preferably a plant cell or a fungus, preferably such that the polynucleotide or vector or a portion thereof encoding the RNA molecule is stably incorporated into the cell's genome. In one embodiment, the non-human organism is produced from a cell or progeny cell, for example, by regenerating a plant. In one embodiment, the non-human organism is produced by introducing a cell or one or more progeny cells into the non-human organism. Instead of stably incorporating the polynucleotide or vector into the cell's genome, the polynucleotide or vector may be introduced into the cell without incorporating it into the genome, for example, to transiently express an RNA molecule in the cell or organism.

[0046] In another embodiment, the present invention provides an extract of a host cell or organism or a part thereof, wherein the extract comprises the RNA molecule of the present invention, a short-chain RNA molecule (20-24 nt in length) produced by processing the RNA molecule, or both, and / or the polynucleotide or vector of the present invention. In one embodiment, the present invention provides a composition comprising one or more of the RNA molecule of the present invention, a short-chain RNA molecule (20-24 nt in length) produced by processing the RNA molecule, or both, the polynucleotide of the present invention, the vector of the present invention, the 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 administration to humans or other animals. The pharmaceutical composition may be suitable for the prevention or treatment of disease, or for topical applications such as cosmetic applications. In one embodiment, the composition is suitable for application to plants, preferably field plants or plant populations, or insects or insect populations. In one embodiment, the composition is suitable for application to crops, for example, by spraying them in the field.

[0047] In one embodiment, an extract or composition comprising the RNA molecule of the present invention, or a short-chain RNA molecule (20-24 nt in length) 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, thereby helping the RNA molecule, polynucleotide, or vector to be taken up by a cell, such as a cell of an organism. In one embodiment, the compound is a transfection promoter, such as a lipid-containing compound.

[0048] In another embodiment, the present invention provides a method for reducing or downregulating the level and / or activity of a target RNA molecule in a cell or organism, the method comprising delivering one or more RNA molecules of the present invention, or short RNA molecules (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, or a composition of the present invention to a cell or organism. In one embodiment, the target RNA molecule encodes a protein. In one embodiment, the method reduces the level and / or activity of multiple target RNA molecules (the target RNA molecules are different), for example, reducing the level and / or activity of two or more target RNAs associated with sequences from a gene family, etc.

[0049] In another embodiment, the present invention provides a method for controlling a non-human organism, the method comprising delivering one or more RNA molecules of the present invention, or short RNA molecules (20-24 nt in length) produced by processing the RNA molecules, or both, or polynucleotides or vectors of the present invention, host cells of the present invention, extracts produced by the method of the present invention, or compositions of the present invention to a non-human organism, wherein the RNA molecules or short RNA molecules have a harmful effect on the non-human organism. In one embodiment, the non-human organism is, for example, an arthropod such as an insect, or a plant such as grass. In one embodiment, the non-human organism is a plant, and the arthropods feed on the plant or a part of it, thereby controlling the arthropod population.

[0050] In one embodiment, the present invention provides a method for preventing or treating a disease in a subject, the method comprising administering one or more RNA molecules of the present invention, or short 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 method of the present invention, or compositions of the present invention to the subject, wherein the RNA molecule or short RNA molecule has a beneficial effect on at least one symptom of the disease. In one embodiment, the RNA molecule or short RNA molecule, polynucleotide, vector, or composition is 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, a livestock such as a cattle or sheep, or a bird such as a chicken or other poultry.

[0051] In another embodiment, the present invention provides RNA molecules, polynucleotides or vectors, host cells, extracts produced by the method of the present invention, or compositions of the present invention for use in the treatment of a disease in question, wherein the RNA molecule or short RNA molecule has a beneficial effect against at least one symptom of the disease. In one embodiment, the present invention provides a use of the RNA molecule or short RNA molecule produced therefrom, polynucleotides or vectors of the invention, host cells, extracts produced by the method of the present invention, or compositions of the present invention for the production of a pharmacopoeia for the prevention or treatment of a disease in question, wherein the RNA molecule or short RNA molecule produced therefrom has a beneficial effect against at least one symptom of the disease.

[0052] In another embodiment, the present invention provides a kit comprising one or more RNA molecules of the present invention, or short-chain RNA molecules derived therefrom, polynucleotides or vectors of the present invention, host cells of the present invention, extracts produced by the method of the present invention, or compositions of the present invention.

[0053] Any embodiment described herein shall be construed as applicable to any other embodiment with necessary modifications, unless otherwise specified.

[0054] The present invention is not limited in scope by the specific embodiments described herein, which are for illustrative purposes only. Functionally equivalent products, compositions, and methods as described herein are clearly within the scope of the present invention.

[0055] Throughout this specification, unless otherwise specified or unless particularly required by the context, any reference to a single step, composition of a substance, group of steps, or group of compositions of a substance shall be construed as encompassing one or more (i.e., one or more) of these steps, compositions of a substance, groups of steps, or groups of compositions of a substance. The present invention is described below by the following non-limiting examples and with reference to the accompanying drawings. [Brief explanation of the drawing]

[0056] [Figure 1]These are schematic diagrams of two ledRNA molecules. (A) This ledRNA molecule contains a sense sequence that can be considered as two adjacent sense sequences that are covalently linked without an intervening spacer sequence and are identical to the target RNA, an antisense sequence complementary to the sense sequence and divided into two regions, a 5' region and a 3' region, and two loops that separate the sense from the antisense sequence. (B) This ledRNA molecule contains an antisense sequence that can be considered as two adjacent antisense sequences that are covalently linked without an intervening spacer sequence and are identical to the complement of the target RNA, a sense sequence complementary to the antisense sequence and divided into two regions, and two loops that separate the sense from the antisense sequence. RNA molecules generated by transcription, for example, in vitro transcription from a promoter such as the T7 or Sp6 promoter, self-anneal through base pairing between complementary sense and antisense sequences, forming a double-stranded region with loops at both ends and a "nick" in either the antisense or sense sequence. Additional sequences may be attached to the 5' and / or 3' ends as 5'- or 3'-extensions. [Figure 2] ledRNA is more efficient than sense / antisense annealing or hairpin RNA in the formation of dsRNA. Schematic diagrams of three forms of double-stranded RNA molecules are shown: A is conventional dsRNA formed by annealing of two separate strands, B is hairpin RNA with 5'- and 3'-extensions, and C is a ledRNA molecule. The lower panel shows gel electrophoresis images of RNA transcripts of three different RNA molecules targeting either the GUS gene or the GFP gene. [Figure 3] Northern blot hybridization of treated (A and B) and untreated distal (C and D) tissues indicates that ledRNA is more stable than dsRNA and spreads throughout the tobacco leaf tissue. In distal tissues (C and D, upper panel), no dsRNA signal was detected, in contrast to a strong ledRNA signal. [Figure 4]LedRNA treatment induced downregulation of GUS in both the treated region (1) and the untreated region (3) described above. [Figure 5] ledRNA induces silencing of the FAD2.1 gene in the leaves of N. benthamiana. [Figure 6] Northern blot hybridization confirmed strong downregulation of FAD2.1 mRNA by treatment with ledFAD2.1 at 6 and 24 hours. [Figure 7] This is an alignment of the nucleotide sequence of the GUS target gene region (SEQ ID NO: 14) and the sense sequence of the hpGUS[G:U] construct (nucleotides 9-208 of SEQ ID NO: 11). 52 cytidine (C) nucleotides were substituted with thymidine (T) nucleotides. Conserved nucleotides are marked with an asterisk, while substituted C nucleotides are not. [Figure 8] This is an alignment of the nucleotide sequence of the GUS target gene region (SEQ ID NO: 14) and the sense sequence of the hpGUS[1:4] construct (nucleotides 9-208 of SEQ ID NO: 12). Each fourth nucleotide of hpGUS[1:4] was substituted for the corresponding wild-type sense sequence, resulting in the following changes for each fourth nucleotide: C changed to G, G to C, A to T, and T to A. Conserved nucleotides are denoted by an asterisk, substituted G and C are not denoted by an asterisk, and substituted A and T are indicated by a semicolon. [Figure 9]This is an alignment of the nucleotide sequence of the GUS target gene region (SEQ ID NO: 14) and the sense sequence of the hpGUS[2:10] construct (nucleotides 9-208 of SEQ ID NO: 13). The 9th and 10th nucleotides of each block of the 10 nucleotides in hpGUS[2:10] were substituted for the corresponding wild-type sense sequence, resulting in the following changes for each 9th and 10th nucleotide: C changed to G, G to C, A to T, and T to A. Conserved nucleotides are denoted by an asterisk, substituted G and C are not denoted by an asterisk, and substituted A and T are indicated by a semicolon. [Figure 10] This is a schematic diagram showing the structure of a genetic construct encoding a modified hairpin RNA that targets GUS mRNA. [Figure 11] This is a schematic diagram of the vector pWBPPGH used to transform tobacco plants that provide GUS target genes. The T-DNA extends from the right boundary (RB) to the left boundary (LB) of the vector. The selection marker gene for the T-DNA is the 35S-HPT-tm1' gene, which encodes hygromycin resistance. [Figure 12] This is the GUS activity of plants transformed with a construct encoding modified hairpin RNA to reduce the expression of GUS target genes. hp: Control PPGH11 and PPGH24 plants without the 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 plants tested, are shown in parentheses. [Figure 13] (A) Average GUS activity of all transgenic plants: hpGUS[wt] was 59 plants, hpGUS[G:U] was 74 plants, hpGUS[1:4] was 33 plants, and hpGUS[2:10] was 41 plants. (B) Average GUS activity of all silent plants: hpGUS[wt] was 32 plants, hpGUS[G:U] was 71 plants, hpGUS[1:4] was 33 plants, and hpGUS[2:10] was 28 plants. [Figure 14]This refers to the GUS activity of transgenic progeny containing hpGUS[wt], hpGUS[G:U], or hpGUS[1:4]. [Figure 15] This is an autoradiograph of Southern blots of DNA from 16 plants transformed with the hpGUS[G:U] construct. DNA was digested with HindIII and probed with an OCS-T probe before gel electrophoresis. Lane 1: Size marker (lambda DNA digested with HindIII); lanes 2 and 3: DNA from parent plants PPGH11 and PPGH24; lanes 4-19: DNA from 16 different transgenic plants. [Figure 16] This is an autoradiogram of a Northern blot hybridization experiment detecting sense (upper panel) and antisense (lower panel) sRNAs derived from hairpin RNA expressed in transgenic tobacco plants. Lanes 1 and 2 contained RNA obtained from parent plants PPGH11 and PPGH24, which lacked the hpGUS construct. Lanes 3-11 contained RNA from hpGUS[wt] plants, and lanes 12-20 contained RNA from hpGUS[G:U] plants. [Figure 17] This is an autoradiograph of Northern blot hybridization for detecting antisense sRNA from transgenic plants. Lanes 1-10 were derived from hpGUS[wt] plants, and lanes 11-19 were derived from hpGUS[G:U] plants. Antisense sRNAs have mobility corresponding to lengths of 20-24 nt. The blot was reprobeced with antisense against U6 RNA as a lane-loading control. [Figure 18] This is an autoradiograph of repeated Northern blot hybridization for detecting antisense sRNA in transgenic plants. [Figure 19] This study involved DNA methylation analysis of the junction regions of the 35S promoter and sense GUS region in transgenic plant hpGUS constructs. Junction fragments were amplified by PCR with or without pretreatment of plant DNA with McrBC enzyme. [Figure 20] This study analyzes DNA methylation in the 35S promoter region of hpGUS constructs from transgenic plants. The 35S fragment was amplified by PCR with or without pretreatment of plant DNA with McrBC enzyme. [Figure 21] This shows the size distribution and abundance of processed RNA. (A) EIN2 construct. (B) GUS construct. [Figure 22] This is an alignment of the sense sequence of the hpEIN2[G:U] construct (upper row, nucleotides 17-216 of SEQ ID NO: 22) with the nucleotide sequence of the cDNA region corresponding to the A. thaliana EIN2 target gene (lower row, SEQ ID NO: 27). The sense sequence was created by replacing 43 cytidine (C) nucleotides in the wild-type sequence with thymidine (T) nucleotides. Conserved nucleotides are marked with an asterisk, while substituted C nucleotides are not. [Figure 23] This is an alignment of the sense sequence of the hpCHS[G:U] construct (upper sequence, nucleotides 13-212 of SEQ ID NO: 24) with the nucleotide sequence of the cDNA region corresponding to the A. thaliana CHS target gene (SEQ ID NO: 28, lower sequence). The sense sequence was created by replacing 65 cytidine (C) nucleotides in the wild-type sequence with thymidine (T) nucleotides. Conserved nucleotides are marked with an asterisk, while substituted C nucleotides are not. [Figure 24] This is an alignment of the antisense sequence of the hpEIN2[G:U / U:G] construct (upper row, nucleotides 8-207 of SEQ ID NO: 25) with the nucleotide sequence of the complementary region of the A. thaliana EIN2 target gene (lower row, SEQ ID NO: 29). The antisense sequence was created by replacing 49 cytidine (C) nucleotides in the wild-type sequence with thymidine (T) nucleotides. Conserved nucleotides are marked with an asterisk, while substituted C nucleotides are not. [Figure 25]This is an alignment of the antisense sequence of the hpCHS[G:U / U:G] construct (upper row, nucleotides 13-212 of SEQ ID NO: 26) with the nucleotide sequence of the complementary region of the A. thaliana CHS target gene (lower row, SEQ ID NO: 30). The antisense sequence was created by replacing 49 cytidine (C) nucleotides in the wild-type sequence with thymidine (T) nucleotides. Conserved nucleotides are marked with an asterisk, while substituted C nucleotides are not. [Figure 26] This is a schematic diagram of ethylene-insensitive 2 (EIN2) and chalcone synthase (CHS) hpRNA constructs. 35S is the CaMV 35S promoter, and the EIN2 and CHS regions are shown as either wild-type sequences (wt) or G:U modified sequences (G:U). Arrows indicate the direction of the DNA fragment, and right-to-left arrows indicate antisense sequences. Restriction enzyme sites are also shown. [Figure 27] This is the hypocotyl length of transgenic A. thaliana seedlings in an EIN2 assay containing either hpEIN2[wt] or hpEIN2[G:U]. [Figure 28] This is qRT-PCR of CHS mRNA in transgenic A. thaliana transgenic hpCHS[wt] or hpCHS[G:U] constructs, normalized to actin 2 RNA levels. Col-0 is wild-type (non-transgenic) A. thaliana. [Figure 29] This is an autoradiograph of Northern blot hybridization of RNA from plants transformed with hpEIN2[wt] or hpEIN2[G:U]. The upper panel shows the hypocotyl length of the lineage. The autoradiograph shows Northern blots probed with an EIN2 sense probe to detect antisense sRNA. The same blots were reprobed with a U6 RNA probe as a loading control (U6 RNA). [Figure 30] This study analyzes DNA methylation of the 35S promoter and 35S-sense EIN2 sequences in the genomic DNA of transgenic A. thaliana plants. [Figure 31] This refers to the level of DNA methylation in the 5' region of the promoter and hairpin RNA constructs. [Figure 32] The 35S promoter of the least methylated lineage in the hpEIN2[wt] population still shows significant methylation. [Figure 33] The 35S promoter of the G:U hpEIN2 strain shows only weak methylation (less than 10%). [Figure 34] These are ledRNA and hpRNA obtained by silencing the G:U gene in CHO and Vero cells at 72 hours of age. [Figure 35] This is the dumbbell plasmid tested in HeLa cells at 48 hours. [Figure 36] These are examples of possible modifications to dsRNA molecules. [Figure 37] This study describes the decline in aphid performance after feeding with artificial feed supplemented with ledRNA to downregulate the expression of the MpC002 or MpRack-1 gene in the peach aphid. Upper panel (A): Average number of larvae per adult aphid after 10 days of administration of 100 μl of 50 ng / μl ledRNA. Lower panel (B): Percentage of aphids surviving over a 5-day time course after ingestion of 100 μl containing 200 ng / μl of ledRNA for MpC002, MpRack-1, or control ledGFP.

[0057] Sequence listing headings (KEY TO THE SEQUENCE LISTING) Ribonucleotide sequence of SEQ ID NO: 1-GFP ledRNA Ribonucleotide sequence of SEQ ID NO: 2-GUS ledRNA Ribonucleotide sequence of sequence number 3-N.benthamiana FAD2.1 ledRNA Sequence ID 4 - GFP ledRNA encoding nucleotide sequence Sequence ID 5-GUS: Nucleotide sequence encoding ledRNA Sequence ID 6 - Nucleotide sequence encoding N.benthamiana FAD2.1 ledRNA Sequence ID 7 - Nucleotide sequence encoding GFP Nucleotide sequence encoding sequence number 8-GUS Nucleotide sequence encoding SEQ ID NO: 9-N.benthamiana FAD2.1 Nucleotide sequence used to provide the GUS sense region to a construct encoding a hairpin RNA molecule targeting SEQ ID NO: 10-GUS mRNA Sequence ID 11 - Nucleotide sequence used to provide the GUS sense region to a construct encoding the hairpin RNA molecule hpGUS[G:U] Sequence ID 12 - Nucleotide sequence used to provide the GUS sense region to a construct encoding the hairpin RNA molecule hpGUS[1:4] Sequence ID 13 - Nucleotide sequence used to provide the GUS sense region to a construct encoding the hairpin RNA molecule hpGUS[2:10] Nucleotide sequence of nucleotides 781-1020 in the protein-coding region of the SEQ ID NO: 14-GUS gene Ribonucleotide sequence of the hairpin structure (including its loop) of SEQ ID NO: 15-hpGUS[wt]RNA Ribonucleotides of the hairpin structure (including its loop) of SEQ ID NO: 16-hpGUS[G:U]RNA Ribonucleotides of the hairpin structure (including its loop) of SEQ ID NO: 17-hpGUS[1:4]RNA Ribonucleotides of the hairpin structure (including its loop) of SEQ ID NO: 18-hpGUS[2:10]RNA Nucleotide sequence of cDNA corresponding to the sequence number 19-A.thaliana EIN2 gene (accession number NM_120406) Nucleotide sequence of cDNA corresponding to SEQ ID NO: 20-A.thaliana CHS gene (accession number NM_121396, 1703nt) Sequence ID 21 - Nucleotide sequence of a DNA fragment containing a 200nt sense sequence derived from cDNA corresponding to the A. thaliana EIN2 gene adjacent to the restriction enzyme site. The nucleotide sequence of a DNA fragment containing the 200nt sense sequence of EIN2, similar to SEQ ID NO: 21 except that 43C is replaced with T, used to construct SEQ ID NO: 22-hpEIN2[G:U]. Sequence ID 23 - Nucleotide sequence of a DNA fragment containing a 200nt sense sequence derived from cDNA corresponding to the A. thaliana CHS gene adjacent to the restriction enzyme site. The nucleotide sequence of a DNA fragment containing the 200nt sense sequence of CHS, similar to SEQ ID NO: 23 except that 65C is replaced with T, used to construct SEQ ID NO: 24-hpCHS[G:U]. Nucleotide sequence of a DNA fragment containing a 200nt antisense sequence of EIN2 with 50C replaced by T, used in the construction of SEQ ID NO: 25-hpEIN2[G:U / U:G] Nucleotide sequence of a DNA fragment containing a 200nt antisense sequence of CHS with 49C replaced by T, used in the construction of SEQ ID NO: 26-hpCHS[G:U / U:G] Nucleotide sequence of nucleotides 601-900 of the cDNA corresponding to the EIN2 gene (accession number NM_120406) of sequence number 27-A.thaliana Nucleotide sequence of nucleotides 813-1112 of the cDNA corresponding to the CHS gene (accession number NM_121396) of SEQ ID NO: 28-A.thaliana Complementary nucleotide sequences of nucleotides 652-891 of the cDNA corresponding to the EIN2 gene (accession number NM_120406) of sequence number 29-A.thaliana. Complementary nucleotide sequences of nucleotides 804-1103 of the cDNA corresponding to the CHS gene in SEQ ID NO: 30-A.thaliana FANCM I protein-coding region (accession number NM_001333162) of the cDNA of Sequence ID No. 31 - Arabidopsis thaliana. Target region nucleotides 675-1174 (500 nucleotides). FANCM I protein-coding region of the cDNA of sequence number 32-Brassica napus. Target region nucleotides 896-1395 (500 bp) The nucleotide sequence encoding hpFANCM-At[wt], which targets the FANCM I protein-coding region of SEQ ID NO: 33-A.thaliana. FANCM sense sequence, nucleotides 38-537; loop sequence, nucleotides 538-1306; FANCM antisense sequence, nucleotides 1307-1806. The nucleotide sequence encoding hpFANCM-At[G:U], which targets the FANCM I protein-coding region of SEQ ID NO: 34-A.thaliana. FANCM sense sequence, nucleotides 38-537; loop sequence, nucleotides 538-1306; FANCM antisense sequence, nucleotides 1307-1806. The nucleotide sequence encoding hpFANCM-Bn[wt], which targets the FANCM I protein-coding region of SEQ ID NO: 35-B.napus. FANCM sense sequence, nucleotides 34-533; loop sequence, nucleotides 534-1300; FANCM antisense sequence, nucleotides 1301-1800. The nucleotide sequence encoding hpFANCM-Bn[G:U], which targets the FANCM I protein-coding region of SEQ ID NO: 36-B.napus. FANCM sense sequence, nucleotides 34-533; loop sequence, nucleotides 534-1300; FANCM antisense sequence, nucleotides 1301-1800. Sequence ID 37-B.napus: Nucleotide sequence of the protein-coding region of the cDNA corresponding to the DDM1 gene (accession number XR_001278527) The nucleotide sequence of the DNA encoding hpDDM1-Bn[wt], which targets the DDM1 protein-coding region of SEQ ID NO: 38-B.napus. The nucleotide sequence encoding hpDDM1-Bn[G:U], which targets the DDM1 protein-coding region of SEQ ID NO: 39-B.napus. DDM1 sense sequence, nucleotides 35-536; loop sequence, nucleotides 537-1304; DDM1 antisense sequence, nucleotides 1305-1805. Sequence ID 40-EGFP cDNA. Nucleotide sequence of the coding region of sequence number 41-hpEGFP[wt] (in the order of antisense / loop / sense relative to the promoter) The nucleotide sequence of the coding region of hpEGFP[G:U] in which 157C is substituted with T in the sense sequence of SEQ ID NO: 42-EGFP. The nucleotide sequence of the coding region of ledEGFP[wt], where the sense sequence of sequence number 43-EGFP does not have a C-to-T substitution. The nucleotide sequence of the coding region of ledEGFP[G:U], in which 162C is substituted with T in the sense sequence of sequence number 44-EGFP. Sequence ID 45 - A nucleotide sequence used to provide a GUS sense region to a construct encoding the hairpin RNA molecule hpGUS[G:U] without adjacent restriction enzyme sites. Sequence ID 46 - A nucleotide sequence used to provide a GUS sense region to a construct encoding the hairpin RNA molecule hpGUS[1:4] without adjacent restriction enzyme sites. Sequence ID 47 - A nucleotide sequence used to provide a GUS sense region to a construct encoding the hairpin RNA molecule hpGUS[2:10] without adjacent restriction enzyme sites. Sequence ID 48 - A nucleotide sequence of a DNA fragment containing a 200nt sense sequence of EIN2, similar to Sequence ID 21, except that 43C is replaced with T, used to construct hpEIN2[G:U] without adjacent sequences. Sequence ID 49 - A nucleotide sequence of a DNA fragment containing a 200nt sense sequence of CHS, similar to Sequence ID 23, except that 65C is replaced with T, used to construct hpCHS[G:U] without adjacent sequences. Sequence ID 50 - Nucleotide sequence of a DNA fragment containing a 200nt antisense sequence of EIN2 with 50C replaced by T, used to construct hpEIN2[G:U / U:G] without adjacent sequences. Sequence ID 51 - Nucleotide sequence of a DNA fragment containing a 200nt antisense sequence of CHS in which 49C is replaced with T, used for constructing hpCHS[G:U / U:G] without adjacent sequences. Oligonucleotide primer (GUS-WT-F) used for amplification of the GUS sense sequence SEQ ID NO: 52-200bp Oligonucleotide primer (GUS-WT-R) used for amplification of the GUS sense sequence SEQ ID NO: 53-200bp Sequence ID 54 - Oligonucleotide primer (forward) (GUS-GU-F) used to generate hpGUS[G:U] fragments in which all Cs are replaced with Ts. Sequence ID 55 - Oligonucleotide primer (reverse) (GUS-GU-R) used to generate hpGUS[G:U] fragments in which all Cs are replaced with Ts. Sequence ID 56 - Oligonucleotide primer (forward) (GUS-4M-F) used to generate hpGUS[1:4] fragments with substitutions at every nucleotide position 4. Sequence ID 57 - Oligonucleotide primer (reverse) (GUS-4M-R) used to generate hpGUS[1:4] fragments with substitutions at every nucleotide position 4. Oligonucleotide primer (forward) (GUS-10M-F) used to generate hpGUS[2:10] fragments with nucleotide substitutions every 9th and 10th position in sequence number 58. Oligonucleotide primer (reverse) (GUS-10M-R) used to generate hpGUS[2:10] fragments with nucleotide substitutions every 9th and 10th position in sequence number 59. Sequence ID 60 - Nucleotide sequence encoding the forward primer (35S-F3) Sequence ID 61 - Nucleotide sequence encoding the reverse primer (GUSwt-R2) Sequence ID 62 - Nucleotide sequence encoding the forward primer (GUSgu-R2) Sequence ID 63 - Nucleotide sequence encoding the reverse primer (GUS4m-R2) Sequence ID 64 - Nucleotide sequence encoding the forward primer (35S-F2) Sequence ID 65 - Nucleotide sequence (35S-R1) encoding the reverse primer Sequence ID 66 - Oligonucleotide primer (EIN2wt-F) used for amplification of the wild-type 200bp EIN2 sense sequence. Sequence ID 67 - Oligonucleotide primer used for amplification of the wild-type 200bp EIN2 sense sequence (EIN2wt-R) Sequence ID No. 68 - Oligonucleotide primer (CHSwt-F) used for amplification of the wild-type 200 bp CHS sense sequence. Sequence ID 69 - Oligonucleotide primer (CHSwt-R) used for amplification of the wild-type 200 bp CHS sense sequence. Sequence ID 70 - Oligonucleotide primer (forward) (EIN2gu-F) used to generate an hpEIN2[G:U] fragment in which all Cs are replaced with Ts. Sequence ID 71 - Oligonucleotide primer (reverse) (EIN2gu-R) used to generate hpEIN2[G:U] fragments in which all Cs are replaced with Ts. Sequence ID 72 - Oligonucleotide primer (forward) (CHSgu-F) used to generate hpCHS[G:U] fragments in which all Cs are replaced with T. Sequence ID 73 - Oligonucleotide primer (reverse) (CHSgu-R) used to generate hpCHS[G:U] fragments in which all Cs are replaced with Ts. Sequence ID 74 - Oligonucleotide primer (forward) (asEIN2gu-F) used to generate hpEIN2[G:U / U:G] fragments in which all Cs are replaced with Ts. Sequence ID 75 - Oligonucleotide primer (reverse) used to generate hpEIN2[G:U / U:G] fragments in which all Cs are replaced with T (asEIN2gu-R) Sequence ID 76 - Oligonucleotide primer (forward) (asCHSgu-F) used to generate hpCHS[G:U / U:G] fragments in which all Cs are replaced with T. Sequence ID 77 - Oligonucleotide primer (reverse) (asCHSgu-R) used to generate hpCHS[G:U / U:G] fragments in which all Cs are replaced with Ts. Sequence ID 78 - Nucleotide sequence encoding the forward primer (CHS-200-F2) Sequence ID 79 - Nucleotide sequence encoding the reverse primer (CHS-200-R2) Sequence ID 80 - Nucleotide sequence encoding the forward primer (Actin2-For) Sequence ID 81 - Nucleotide sequence encoding a reverse primer (Actin2-Rev) Sequence ID 82 - Nucleotide sequence encoding the forward primer (Top-35S-F2) Sequence ID 83 - Nucleotide sequence encoding the reverse primer (Top-35S-R2) Sequence ID 84 - Nucleotide sequence encoding the forward primer (Link-35S-F2) Sequence ID 85 - Nucleotide sequence encoding the reverse primer (Link-EIN2-R2) Ribonucleotide sequence of SEQ ID NO: 86-sense si22 Ribonucleotide sequence of SEQ ID NO: 87-Antisense SI22 Sequence ID 88 - Forward primer ribonucleotide sequence Sequence ID 89 - Ribonucleotide sequence of reverse primer Sequence ID 90 - Forward primer ribonucleotide sequence Sequence ID 91 - Ribonucleotide sequence of the reverse primer Possible modifications of the SEQ ID NO: 92-dsRNA molecule Nucleotide sequence of cDNA corresponding to the Brassica napus DDM1 gene (accession number XR_001278527). The nucleotide sequence of the chimeric DNA encoding a hairpin RNAi (hpRNA) construct targeting the DDM1 gene in SEQ ID NO: 94-B.napus. The nucleotide sequence of the chimeric DNA encoding a hairpin RNAi (hpRNA) construct containing a G:U base pair, targeting the DDM1 gene of SEQ ID NO: 95-B.napus. The nucleotide sequence of the chimeric DNA encoding the ledRNA construct targeting the DDM1 gene in SEQ ID NO: 96-B.napus. The nucleotide sequence of the cDNA corresponding to the Sequence ID No. 97-A.thaliana FANCM gene (accession number NM_001333162). The nucleotide sequence of the chimeric DNA encoding a hairpin RNAi (hpRNA) construct targeting the FANCM gene in sequence number 98-A.thaliana. The nucleotide sequence of a chimeric DNA encoding a hairpin RNAi (hpRNA) construct containing a G:U base pair, targeting the FANCM gene of sequence number 99-A.thaliana. The nucleotide sequence of chimeric DNA encoding a ledRNA construct targeting the FANCM gene of sequence number 100-A.thaliana. The nucleotide sequence of the cDNA corresponding to the FANCM gene (accession number XM_022719486.1) of sequence number 101-B.napus. The nucleotide sequence of the chimeric DNA encoding a hairpin RNAi (hpRNA) construct targeting the FANCM gene in SEQ ID NO: 102-B.napus. The nucleotide sequence of a chimeric DNA encoding a hairpin RNAi (hpRNA) construct with a G:U base pair, targeting the FANCM gene of SEQ ID NO: 103-B.napus. The nucleotide sequence of the chimeric DNA encoding the ledRNA construct targeting the FANCM gene of SEQ ID NO: 104-B.napus. Sequence ID 105 - The nucleotide sequence of the protein-coding region of the cDNA corresponding to the Nicotiana benthamiana TOR gene. Nucleotide sequence of chimeric DNA encoding a ledRNA construct targeting the TOR gene in SEQ ID NO: 106-N.benthamiana. Sequence ID 107 - Nucleotide sequence of the protein-coding region of the cDNA corresponding to the acetolactate synthase (ALS) gene (accession number LT601589) of barley (Hordeum vulgare). Sequence ID 108 - Nucleotide sequence of chimeric DNA encoding ledRNA that targets the ALS gene in barley (H. vulgare). Sequence ID 109 - Nucleotide sequence of the protein-coding region of the cDNA corresponding to the HvNCED1 gene (accession number AK361999) of barley (Hordeum vulgare). Sequence ID 110 - Nucleotide sequence of the protein-coding region of the cDNA corresponding to the HvNCED2 gene (accession number DQ145931) of barley Hordeum vulgare. Sequence ID 111 - Nucleotide sequence of chimeric DNA encoding ledRNA constructs targeting the NCED1 gene in barley (Hordeum vulgare) and wheat (Triticum aestivum). Sequence ID 112 - Nucleotide sequence of chimeric DNA encoding ledRNA constructs targeting the NCED2 gene in barley (Hordeum vulgare) and wheat (Triticum aestivum). The nucleotide sequence of the protein-coding region of the cDNA corresponding to the barley gene (accession number DQ145933) encoding sequence number 113-ABA-OH-2. Sequence ID 114 - Nucleotide sequence of chimeric DNA encoding ledRNA constructs targeting the ABA-OH-2 gene in barley (Hordeum vulgare) and wheat (Triticum aestivum). The nucleotide sequence of the protein-coding region of the cDNA corresponding to the A.thaliana gene (At5g03280) encoding sequence number 115-EIN2. Nucleotide sequence of chimeric DNA encoding a ledRNA construct targeting the EIN2 gene of sequence number 116-A.thaliana. The nucleotide sequence of the protein-coding region of the cDNA corresponding to the A.thaliana gene (accession number NM_121396) encoding sequence number 117-CHS. Nucleotide sequence of chimeric DNA encoding a ledRNA construct targeting the CHS gene of SEQ ID NO: 118-A.thaliana. The nucleotide sequence of the protein-coding region of the cDNA corresponding to SEQ ID NO: 119-L.angustifolius N-like gene (accession number XM_019604347). Nucleotide sequence of chimeric DNA encoding a ledRNA construct targeting the L. angustifolius N-like gene (SEQ ID NO: 120). The nucleotide sequence of the protein-coding region of the cDNA corresponding to the Vitis pseudoreticulata MLO gene (accession number KR362912), sequence number 121. Sequence ID 122 - Nucleotide sequence of chimeric DNA encoding the first ledRNA construct targeting the Vitis MLO gene. The nucleotide sequence of the protein-coding region of the cDNA corresponding to the MpC002 gene in Sequence ID No. 123 - Myzus persicae. The nucleotide sequence of the protein-coding region of the cDNA corresponding to the MpRack-1 gene in sequence number 124-Myzus persicae. Nucleotide sequence of a chimeric construct encoding ledRNA targeting the M.persicae C002 gene (sequence number 125). Sequence ID 126 - Nucleotide sequence of a chimeric construct encoding ledRNA targeting the Rack-1 gene. Sequence ID 127 - The nucleotide sequence of the cDNA corresponding to the Helicoverpa armigera ABC white gene. Sequence ID 128 - Nucleotide sequence of chimeric DNA encoding a ledRNA construct targeting the ABC transporter white gene of Helicoverpa armigera. The nucleotide sequence of the cDNA corresponding to the sequence number 129-Linepithema humile PBAN-type neuropeptide-like molecule (XM_012368710). Sequence ID 130 - Nucleotide sequence of chimeric DNA encoding a ledRNA construct targeting the PBAN gene (accession number XM_012368710) of the Argentine ant. The nucleotide sequence of chimeric DNA encoding a ledRNA construct that targets the gene encoding the V-type proton ATPase catalytic subunit A (accession number XM_023443547) of SEQ ID NO: 131-L.cuprina. Nucleotide sequence of chimeric DNA encoding a ledRNA construct targeting the gene encoding RNAse 1 / 2 of sequence number 132-L.cuprina. Nucleotide sequence of chimeric DNA encoding a ledRNA construct that targets the gene encoding chitin synthase in SEQ ID NO: 133-L.cuprina. Nucleotide sequence of chimeric DNA encoding a ledRNA construct targeting the gene encoding the ecdysone receptor (EcR) of SEQ ID NO: 134-L. cuprina. Nucleotide sequence of chimeric DNA encoding a ledRNA construct targeting the gene encoding γ-tubulin 1 / 1-like genes in Sequence ID No. 135-L.cuprina. Sequence ID 136 - TaMlo target gene (AF384144). The nucleotide sequence of chimeric DNA encoding a ledRNA construct that targets the gene encoding sequence number 137-TaMlo. The nucleotide sequence of the protein-coding region of the cDNA corresponding to the Vitis pseudoreticulata MLO gene (accession number KR362912), sequence number 138. Sequence ID 139 - Nucleotide sequence of chimeric DNA encoding the first ledRNA construct targeting the Vitis MLO gene. [Modes for carrying out the invention]

[0058] General Techniques and Definitions Unless otherwise specified, all technical and scientific terms used herein should be interpreted as having the same meaning as those commonly understood by those skilled in the art (e.g., in cell culture, molecular genetics, gene silencing, protein chemistry, and biochemistry).

[0059] Unless otherwise specified, the recombinant proteins, cell cultures, and immunological techniques used in this invention are standard procedures well known to those skilled in the art. Such methods are described in J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), TA Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), DMGlover and BDHames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and FMAusubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all revisions to date), Ed Harlow and David Lane (editors), Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988), and JEColigan et al. This is described and explained in various sources, including the literature in *Current Protocols in Immunology*, John Wiley & Sons (including all revisions to date).

[0060] As used herein, the terms “antisense regulatory element,” “antisense ribonucleic acid sequence,” or “antisense RNA sequence” mean an RNA sequence that is at least partially complementary to at least a portion of the target RNA molecule with which it hybridizes. In certain embodiments, an antisense RNA sequence modulates (increases or decreases) the expression or quantity or activity of a target RNA molecule, for example, by reducing the translation of the target RNA molecule. In certain embodiments, an antisense RNA sequence alters the splicing of a target pre-mRNA, generating different splice variants. Exemplary components of antisense sequences include, but are not limited to, oligonucleotides, oligonucleosides, oligonucleotide analogs, oligonucleotide mimetic compounds, and chimeric combinations thereof.

[0061] In the context of this disclosure, the term “antisense activity” is used to refer to any detectable and / or measurable activity resulting from the hybridization of an antisense RNA sequence to its target RNA molecule. Such detection and / or measurement may be direct or indirect. For example, antisense activity is assessed by detecting and / or measuring the amount of target RNA molecule transcript. Antisense activity may also be detected as a phenotypic change associated with the target RNA molecule. As used herein, the term “target RNA molecule” refers to a gene transcript regulated by the antisense RNA sequence according to this disclosure. Thus, 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 transcribed from DNA encoding target proteins, rRNA, tRNA, nuclear small RNA, and miRNA (including, but not limited to, pre-mRNA and mRNA or portions thereof), including their precursor forms. The target RNA may be the genomic RNA of a pathogen or pest, such as a virus, or RNA molecules derived therefrom, such as a replicated form of a viral pathogen, or transcripts thereof. For example, the target RNA molecule may be an endogenous gene (or mRNA transcribed from a gene), or a gene that is introduced or can be introduced into a eukaryotic cell whose expression is associated with a particular phenotype, trait, disorder, or disease state, or RNA from a nucleic acid molecule from an infectious agent. In one example, the target RNA molecule is located in a eukaryotic cell. In another example, the target RNA molecule encodes a protein. In this context, antisense activity can be assessed by detecting and / or measuring the amount of the target protein through activity such as enzymatic activity, or through non-enzymatic function, or through a phenotype associated with that function. As used herein, the term “target protein” refers to the protein regulated by the antisense RNA sequence according to this disclosure.

[0062] In certain embodiments, antisense activity is evaluated by detecting and / or measuring the amount of target RNA molecules and / or cleaved target RNA molecules and / or selectively spliced ​​target RNA molecules.

[0063] Antisense activity can be detected or measured using various methods. For example, antisense activity can be detected or evaluated by comparing the activity of a particular sample and comparing that activity to the activity of a control sample.

[0064] In the context of this disclosure, the term “target” is used to refer to the association of an antisense RNA sequence with a specific target RNA molecule or a specific region of nucleotides within a target RNA molecule. For example, the antisense RNA sequence according to this disclosure shares complementarity with at least one region of a target RNA molecule. In this context, the term “complementarity” refers to a ribonucleotide sequence that can form base pairs with a sequence of ribonucleotides on a target RNA molecule via hydrogen bonds between bases on the ribonucleotide. For example, in RNA, adenine (A) is complementary to uracil (U), and guanine (G) is complementary to cytosine (C).

[0065] In certain embodiments, “complementary base” refers to a ribonucleotide of an antisense RNA sequence that can base-pair with a ribonucleotide of a sense RNA sequence in the RNA molecule of the present invention or its target RNA molecule. For example, if a ribonucleotide at a particular position in the antisense RNA sequence can hydrogen bond with a ribonucleotide at a particular position in the target RNA molecule, the hydrogen bond sites between the antisense RNA sequence and the target RNA molecule are considered complementary in that ribonucleotide. In contrast, the term “non-complementary” refers to a pair of ribonucleotides that do not hydrogen bond with each other or otherwise do not support hybridization. The term “complementary” can also be used to refer to the ability of an antisense RNA sequence to hybridize to another nucleic acid via complementarity. In certain embodiments, the RNA sequence and its target are complementary if a sufficient number of corresponding positions in each molecule are occupied by ribonucleotides that bind to each other, enabling a stable association between the antisense RNA sequence and the sense RNA sequence of the RNA molecule and / or target RNA molecule of the present invention. Those skilled in the art will recognize that the inclusion of mismatches is possible without excluding the ability of the antisense RNA sequence and target to remain associated. Therefore, antisense RNA sequences that may contain up to about 20% mismatched (i.e., not complementary to the corresponding nucleotide of the target) nucleotides are described herein. Preferably, the antisense compound contains or has no mismatches, or about 15% or less, more preferably about 10% or less, and most preferably 5% or less. The remaining ribonucleotides are complementary or otherwise do not inhibit hybridization (e.g., G:U or A:G pairs) between the antisense RNA sequence and the sense RNA sequence or target RNA molecule. Those skilled 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.

[0066] As used herein, “chimeric RNA molecule” refers to any RNA molecule not found naturally in nature. For example, the chimeric RNA molecules disclosed herein are modified to create a mismatch in the dsRNA region(s). For example, a chimeric RNA molecule may be modified to convert cytosine to uracil. For example, a chimeric RNA molecule is modified by treatment with a bisulfite for a sufficient amount of time and under conditions to convert unmethylated cytosine to uracil.

[0067] Those skilled in the art will understand that various ribonucleotide combinations can form base pairs. Both standard and non-standard base pairings are contemplated in this disclosure. In one example, base pairing may include A:T or G:C in a DNA molecule, or U:A or G:C in an RNA molecule. In another example, base pairing may include A:G or G:T or U:G.

[0068] As used in this disclosure, the term “standard base pairing” means base pairing between two nucleotides that is A:T or G:C for deoxyribonucleotides, or A:U or G:C for ribonucleotides.

[0069] As used in this disclosure, the term “non-standard base pairing” means, in the context of two DNA or two RNA sequences, an interaction between two nucleotide bases other than standard base pairing. For example, non-standard base pairing includes pairing between G and U (G:U) or between A and G (A:G). Examples of non-standard base pairing include purine-purine or pyrimidine-pyrimidine. In the context of this disclosure, the most common non-standard base pairing is G:U. Other less desirable examples of non-standard base pairing are A:C, G:T, G:G, and A:A.

[0070] This disclosure refers to RNA components that “hybridize” across a series of ribonucleotides. Those skilled in the art will understand that terms such as “hybridize” and “hybridize” are used to describe molecules that anneal based on complementary nucleic acid sequences. Such molecules do not need to be 100% complementary for hybridization (i.e., they do not need to “perfectly base-pair”). For example, there may be one or more mismatches in sequence complementarity. In one example, the RNA components defined herein hybridize under stringent hybridization conditions. The term “stringent hybridization conditions” refers to parameters well known in the art, including variations in hybridization temperature with respect to the length of the RNA molecule. Ribonucleotide hybridization parameters can be found in references summarizing such methods, Sambrook, et al. (above) and Ausubel, et al. (above). For example, the stringent hybridization conditions used herein may refer to hybridization at 65°C in a hybridization buffer (3.5xSSC, 0.02% Ficoll, 0.02% polyvinylpyrrolidone, 0.02% bovine serum albumin (BSA), 2.5 mM NaH2PO4 (pH 7), 0.5% SDS, 2 mM EDTA), followed by one or more washes at 50°C in 0.2xSSC, 0.01% BSA. Shorter RNA components, such as RNA sequences of 20-24 nucleotides in length, hybridize under lower stringency conditions. The term "low stringency hybridization conditions" refers to parameters well known in the art, including variations in hybridization temperature depending on the length of the RNA molecule.For example, the low stringency hybridization conditions used herein may refer to hybridization at 42°C in a hybridization buffer (3.5xSSC, 0.02% Ficoll, 0.02% polyvinylpyrrolidone, 0.02% bovine serum albumin (BSA), 2.5mM NaH2PO4 (pH 7), 0.5% SDS, 2mM EDTA), followed by one or more washes at 30°C in 0.2xSSC, 0.01% BSA.

[0071] The present invention also encompasses RNA components that “fully base-pair” across a sequence of ribonucleotides. The term “fully base-pair” is used in the context of this disclosure to refer to a sequence of consecutive ribonucleotides that form base pairs. A sequence of fully base-paired ribonucleotides does not contain any nucleotides with gaps or no base-pairing within that sequence. The term “sequential” is used to refer to a sequence of ribonucleotides. The ribonucleotides constituting a sequence are linked by a sequence of phosphodiester bonds, with each ribonucleotide directly bonded to the next ribonucleotide.

[0072] The RNA molecule of the present invention comprises a sense sequence and a corresponding antisense sequence. The relationship between these sequences is defined herein. The sequence relationships and activity of the antisense sequence with respect to the target RNA molecule are also defined herein.

[0073] In the context of this disclosure, the term “covalently bonded” is used to refer to the bond between the first and second RNA components or any RNA sequence or ribonucleotide. As those skilled in the art will understand, a covalent bond or linkage is a chemical bond involving the sharing of electron pairs between atoms. In one example, the first and second RNA components or sense RNA sequence and antisense RNA sequence are covalently bonded as part of a single RNA chain that can be folded by self-complementarity. In this example, the components are covalently bonded across one or more ribonucleotides by phosphodiester bonds.

[0074] In the context of this disclosure, the term “hybridization” means the pairing of complementary polynucleotides by base pairing of complementary bases. While not limited to specific mechanisms, the most common mechanisms of pairing include hydrogen bonding (which may be Watson-Crick hydrogen bonding) between complementary ribonucleotides.

[0075] As used herein, the phrase "RNA molecules have harmful effects on non-human organisms" or similar phrases means that the target RNA molecule of the molecule is present in a non-human organism, and exposure of cells 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 compared to similar cells lacking the RNA molecule. In one embodiment, the target RNA molecule encodes a protein important for growth, reproduction, or survival. For example, if the non-human organism is a crop pest or pathogen, or an animal pest or pathogen, RNA molecules may have adverse effects on feeding by the pest or pathogen, cell apoptosis, cell differentiation and development, sexual reproduction ability or desire, myogenesis, muscle spasms, muscle contraction, juvenile hormone formation, juvenile hormone regulation, ion regulation and transport, maintenance of cell membrane potential, amino acid biosynthesis, amino acid degradation, spermatogenesis, pheromone synthesis, pheromone sensing, antenna formation, wing formation, leg formation, oogenesis, larval maturation, digestive enzyme formation, hemolymph synthesis, hemolymph maintenance, neurotransmission, larval transition, pupation, emergence from pupation, cell division, energy metabolism, respiration, chitin metabolism, and cytoskeletal structure formation. In another example, if the non-human organism is grass, RNA molecules may have adverse effects on amino acid biosynthesis, photosynthesis, fatty acid synthesis, cell membrane integrity, pigment synthesis, or growth.

[0076] Where used herein, the phrase "the RNA molecule has a beneficial effect on at least one symptom of a disease" or similar phrase means that the target RNA of the molecule is present in the 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 compared to similar cells lacking the RNA molecule. In one embodiment, the target RNA encodes a protein that plays a role in the presence of a disease. In one embodiment, the disease is cancer or cancerous disease, infectious disease, cardiovascular disease, neurological disease, prion disease, inflammatory disease, autoimmune disease, lung disease, kidney disease, liver disease, mitochondrial disease, endocrine disease, reproductive disorders and conditions, and any other manifestation that may respond to levels of gene products expressed in cells or organisms.

[0077] The RNA molecules and compositions comprising the same according to this disclosure can be administered to subjects. Terms such as “subject,” “patient,” or “individual” are interchangeable terms in this disclosure depending on the context. In one example, the subject is a mammal. Mammals may be companion animals such as dogs or cats, or domestic animals such as horses or cattle. In one example, the subject is a human. For example, the subject may be an adult. In another example, the subject may be a child. In another example, the subject may be a young adult. In another example, the RNA molecules and compositions comprising the same according to this disclosure can be administered to insects. In another example, the RNA molecules and compositions comprising the same according to this disclosure can be administered to plants. In another example, the RNA molecules and compositions comprising the same according to this disclosure can be administered to fungal cells or populations.

[0078] The term "and / or," for example, "X and / or Y," should be understood to mean either "X and Y" or "X or Y," and should be interpreted as explicitly supporting both meanings or either one of them.

[0079] As used herein, the term “about” means ±20%, more preferably ±10%, of the specified value, unless otherwise specified.

[0080] Throughout this specification, the word “comprise,” or variations such as “comprises” or “comprising,” shall be understood to mean that they include the elements, integers, or steps, or groups of elements, integers, or steps described herein, but not any other elements, integers, or steps, or groups of elements, integers, or steps.

[0081] RNA molecule In certain embodiments, the RNA molecule of the present invention comprises a first RNA component covalently bonded to a second RNA component. In preferred embodiments, the RNA molecule self-hybridizes or folds to form a “dumbbell” or ledRNA structure (see, for example, Figure 1). In one embodiment, the molecule is as follows: - Covalently binding ribonucleotide sequences for the first and second RNA components, -5' leader sequence, and -3' Trailer Arrangement, further including one or more of the following.

[0082] In one embodiment, the first RNA component comprises, in the order of 5' to 3', a first 5' ribonucleotide, a first RNA sequence, and a first 3' ribonucleotide, wherein the first 5' and 3' ribonucleotides form base pairs with each other in the RNA molecule, and the first RNA sequence includes a first sense ribonucleotide sequence of at least 20 consecutive ribonucleotides, a first loop sequence of at least 4 ribonucleotides, and a first antisense ribonucleotide sequence of at least 20 consecutive ribonucleotides, wherein the first antisense ribonucleotide sequence hybridizes with the first sense ribonucleotide sequence in the RNA molecule, and the first antisense ribonucleotide sequence can hybridize to a first region of the target RNA molecule.

[0083] In another embodiment, the first RNA component comprises, in 5' to 3' order, a first 5' ribonucleotide, a first RNA sequence, and a first 3' ribonucleotide, wherein the first 5' and 3' ribonucleotides base-pair with each other in the RNA molecule, and the first RNA sequence includes a first sense ribonucleotide sequence of at least 20 consecutive ribonucleotides, a first loop sequence of at least 4 ribonucleotides, and a first antisense ribonucleotide sequence of at least 20 consecutive ribonucleotides, wherein the first antisense ribonucleotide sequence is perfectly base-paired with the first sense ribonucleotide sequence in the RNA molecule, and the first antisense ribonucleotide sequence is sequence-identical to the complement of the first region of the target RNA molecule. Examples of the first RNA component in these two embodiments are schematically shown in the left half of Figure 1A or the right half of Figure 1B.

[0084] In another embodiment, the first RNA component comprises a first 5' ribonucleotide, a first RNA sequence, and a first 3' ribonucleotide, wherein the first 5' and 3' ribonucleotides are base-paired with each other in the first RNA component, and the first RNA sequence comprises a first sense ribonucleotide sequence, a first loop sequence of at least four 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 consecutive ribonucleotides, so that at least 20 consecutive ribonucleotides of the first sense ribonucleotide sequence are perfectly base-paired with at least 20 consecutive ribonucleotides of the first antisense ribonucleotide sequence, and the at least 20 consecutive ribonucleotides of the first sense ribonucleotide sequence are substantially identical in sequence to a first region of the target RNA molecule.

[0085] In these embodiments, the base pair formed between the first 5' ribonucleotide and the first 3' ribonucleotide is considered the terminal base pair of the dsRNA region formed by the autohybridization of the first RNA component, i.e., it defines the end of the dsRNA region.

[0086] In one embodiment, the first sense sequence has substantial sequence identity with respect to a region of the target RNA, and this identity may be with respect to a sequence 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 consecutive ribonucleotides, preferably at least 20 consecutive ribonucleotides, are sequencely identical by 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%. In another embodiment, at least 15, at least 16, at least 17, at least 18, or at least 19 consecutive ribonucleotides of the first sense ribonucleotide sequence and the first region of the target RNA molecule are 100% identical. In one embodiment, the first 3, 4, 5, 6, or 7 ribonucleotides from the 5' end of the first sense ribonucleotide sequence are 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%, or at least 99% identical to the target RNA molecule.

[0087] In one embodiment, at least 20 consecutive ribonucleotides of the first sense ribonucleotide sequence and the first region of the target RNA molecule 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. 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 may be 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 target RNA molecule. In another embodiment, at least 20 consecutive ribonucleotides of the first sense ribonucleotide sequence and the first region of the target RNA molecule are 100% identical.

[0088] In one embodiment, the first antisense sequence has substantially sequence identity with respect to the complement of the region of the target RNA, and this identity may be with respect to a complement sequence less than 20 nucleotides long. In one embodiment, at least 15, at least 16, at least 17, at least 18, or at least 19 consecutive ribonucleotides, preferably at least 20 consecutive ribonucleotides, of the first antisense ribonucleotide sequence and the complement of the first region of the target RNA molecule are sequencely identical by 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%. In another embodiment, at least 15, at least 16, at least 17, at least 18, or at least 19 consecutive ribonucleotides of the first antisense ribonucleotide sequence and the complement of the first region of the target RNA molecule are 100% identical. In one embodiment, the first 3, 4, 5, 6, or 7 ribonucleotides from the 5' end of the first antisense ribonucleotide sequence are 100% identical to the complement of 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%, or at least 99% identical to the complement of the target RNA molecule.

[0089] In one embodiment, at least 20 consecutive ribonucleotides of the first antisense ribonucleotide sequence and the complement of the first region of the target RNA molecule 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. In this embodiment, the first 3, first 4, first 5, first 6, or first 7 ribonucleotides are 100% identical to the complement of 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%, or at least 99% identical to the complement of the target RNA molecule. In another embodiment, at least 20 consecutive ribonucleotides of the first antisense ribonucleotide sequence and the first region of the target RNA molecule are 100% identical.

[0090] In another embodiment, the second RNA component consists of a second 5' ribonucleotide, a second RNA sequence, and a second 3' ribonucleotide, in the order of 5' to 3', wherein the second 5' and 3' ribonucleotides form a base pair, and the second RNA sequence includes a second sense ribonucleotide sequence, a second loop sequence of at least four ribonucleotides, and a second antisense ribonucleotide sequence, wherein the second sense ribonucleotide sequence forms a base pair 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 the autohybridization of the second RNA component.

[0091] In one embodiment, the RNA molecule includes a 5' leader sequence or 5' extension sequence, which may arise as a result of transcription from a promoter in a genetic construct, from the transcription start site to the beginning of the polynucleotide encoding the rest of the RNA molecule. In embodiments using RNAse processing, this 5' leader sequence or 5' extension sequence is preferably relatively short compared to the rest of the molecule and can be removed from the RNA molecule after transcription. The 5' leader sequence or 5' extension sequence may be mostly unpaired or may contain one or more stem-loop structures. In this embodiment, the 5' leader sequence may consist of a sequence of ribonucleotides that covalently bind to the first 5' ribonucleotide if the second RNA component binds to the first 3' ribonucleotide, or covalently bind to the second 5' ribonucleotide if the second RNA component binds to the first 5' ribonucleotide. In one embodiment, the 5' leader sequence is at least 10, at least 20, at least 30, at least 100, and at least 200 ribonucleotides long, preferably with 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 function as an elongation sequence for amplifying the RNA molecule via a suitable amplification reaction. In an embodiment, the elongation sequence may facilitate polymerase-mediated amplification.

[0092] In another embodiment, the RNA molecule includes a 3' trailer sequence or a 3' extension sequence, which may result from transcription continuing to a transcription termination or polyadenylation signal in the construct encoding the RNA molecule. The 3' trailer sequence or 3' extension sequence may include a polyA tail. In embodiments using RNAse processing, this 3' trailer sequence or 3' extension sequence is preferably relatively short compared to the rest of the molecule and can be removed from the RNA molecule after transcription. The 3' trailer sequence or 3' extension sequence may be mostly unpaired or may contain one or more stem-loop structures. In this embodiment, the 3' trailer sequence may consist of a sequence of ribonucleotides that covalently bind to the second 3' ribonucleotide if the second RNA component binds to the first 3' ribonucleotide, or covalently bind to the first 3' ribonucleotide if the second RNA component binds to the first 5' ribonucleotide. In one embodiment, the 3' leader sequence is at least 10, at least 20, at least 30, at least 100, and at least 200 ribonucleotides long, preferably with a maximum length of 250 ribonucleotides. In another embodiment, the 3' leader sequence is at least 50 ribonucleotides long. In one embodiment, the 3' trailer sequence can function as an elongation sequence for amplifying the RNA molecule via a suitable amplification reaction. In an embodiment, the elongation sequence may facilitate polymerase-mediated amplification.

[0093] In one embodiment, all but two ribonucleotides are covalently bonded to two other nucleotides; that is, the RNA molecule consists of only one RNA strand having a self-complementary region, and therefore has only one 5' terminal nucleotide and only one 3' terminal nucleotide. In another embodiment, all but four ribonucleotides are covalently bonded to two other nucleotides; that is, the RNA molecule consists of two RNA strands having a self-complementary region that hybridizes, and therefore has only two 5' terminal nucleotides and only two 3' terminal nucleotides. In yet another embodiment, each ribonucleotide is covalently bonded to two other nucleotides; that is, the RNA molecule is circular and has a self-complementary region, and therefore has neither a 5' terminal nucleotide nor a 3' terminal nucleotide.

[0094] In one embodiment, the double-stranded region of an RNA molecule may contain one or more bulges arising from unpaired nucleotides in the sense RNA sequence or antisense RNA sequence, or both. In one embodiment, the RNA molecule contains a series of bulges. In embodiments, the double-stranded region of an RNA molecule may have 2, 3, 4, 5, 6, 7, 8, 9, 10 or more bulges. Each bulge may independently contain 1, 2 or more unpaired nucleotides, or as many as 10 nucleotides. Longer sequences may loop out from the sense or antisense sequence of the dsRNA region, which may either form base pairs internally or remain unpaired. In another embodiment, the double-stranded region of an RNA molecule does not contain bulges, i.e., is fully base-paired along the entire length of the dsRNA region.

[0095] In another embodiment, the first sense ribonucleotide sequence is covalently bonded to the first 5' ribonucleotide without any intervening nucleotides, or the first antisense ribonucleotide sequence is covalently bonded 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, and at least 10 intervening nucleotides. It is understood that such intervening nucleotides are sequence-independent of the target RNA molecule but may help stabilize base pairing of adjacent sense and antisense sequences.

[0096] In another embodiment, 20 consecutive nucleotides of the first sense ribonucleotide sequence are covalently bonded to the first 5' ribonucleotide without any intervening nucleotides, and 20 consecutive nucleotides of the first antisense ribonucleotide sequence are covalently bonded 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, and at least 10 intervening nucleotides. The intervening nucleotides may base-pair as part of the double-stranded region of the RNA molecule but are sequence-independent of the target RNA. The intervening nucleotides may help to increase the stability of the double-stranded region or to retain two ends of the RNA molecule, leaving either a 5' or 3' end, or both, unbase-paired.

[0097] In one embodiment, the first and second RNA components mentioned above include binding ribonucleotide sequences. In one embodiment, the binding ribonucleotide sequence functions as a spacer between a first sense ribonucleotide sequence whose sequence is substantially identical to that of a first region of the target RNA molecule and other components of the molecule. For example, the binding ribonucleotide sequence may function as a spacer between this region and a loop. In another embodiment, the RNA molecule includes a plurality of sense ribonucleotide sequences whose sequences are substantially identical to those of a first region of the target RNA molecule, and binding ribonucleotide sequences that function as spacers 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, and at least 10 ribonucleotide sequences, each substantially identical to that of a first region of the target RNA molecule, are provided in the RNA molecule, each separated from others by binding ribonucleotide sequences.

[0098] In one embodiment, the RNA molecule mentioned above includes a 5' leader sequence. In one embodiment, the 5' leader sequence consists of a sequence of ribonucleotides that covalently bind to the first 5' ribonucleotide if the second RNA component binds to the first 3' ribonucleotide, or covalently bind to the second 5' ribonucleotide if the second RNA component binds to the first 5' ribonucleotide. In one embodiment, the RNA molecule is modified at its 5' or 3' end by the attachment of, for example, a lipid group such as cholesterol, or a vitamin such as biotin, or a polypeptide. Such modifications may help the uptake of the RNA molecule into a eukaryotic cell in which the RNA functions.

[0099] In one embodiment, the binding ribonucleotide sequence is less than 100 ribonucleotides long. In one embodiment, the binding ribonucleotide sequence is less than 50 ribonucleotides long. In one embodiment, the binding ribonucleotide sequence is less than 20 ribonucleotides long. In one embodiment, the binding ribonucleotide sequence is less than 10 ribonucleotides long. In one embodiment, the binding ribonucleotide sequence is less than 5 ribonucleotides long. In one embodiment, the binding ribonucleotide sequence is 1 to 100 ribonucleotides long. In one embodiment, the binding ribonucleotide sequence is 1 to 50 ribonucleotides long. In one embodiment, the binding ribonucleotide sequence is 1 to 20 ribonucleotides long. In one embodiment, the binding ribonucleotide sequence is 1 to 10 ribonucleotides long. In one embodiment, the binding ribonucleotide sequence is 1 to 5 ribonucleotides long. In one embodiment, the ribonucleotides in the binding ribonucleotide sequence do not form base pairs. In a preferred embodiment, all ribonucleotides in the binding ribonucleotide sequence are base-paired, or all ribonucleotides except 1, 2, or 3 are base-paired.

[0100] In one embodiment, the first or second RNA component includes a hairpin structure. In a preferred embodiment, the first and second RNA components each include a hairpin structure. In these embodiments, the hairpin structure may be a stem-loop. Thus, in one embodiment, the RNA molecule may include first and second RNA components each containing a hairpin structure, which is covalently linked by a linker sequence. See, for example, Figure 1. In one embodiment, the linker sequence is one or more unpaired ribonucleic acids. In one embodiment, the linker sequence is 1 to 10 unpaired ribonucleotides.

[0101] In one embodiment, the RNA molecule has a double hairpin structure, i.e., a "ledRNA structure" or "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, either the first 3' ribonucleotide and the second 5' ribonucleotide, or the second 3' ribonucleotide and the first 5' ribonucleotide, are covalently linked. In this embodiment, the other 5' / 3' ribonucleotides are separable by a nick (i.e., discontinuous in a dsRNA molecule where there is no phosphodiester bond between the 5' / 3' ribonucleotides). An embodiment of this type of arrangement is shown in Figure 1B. In another embodiment, each 5' / 3' ribonucleotide is separable by a loop. The lengths of the 5' leader and 3' trailer sequences may be the same or different. In embodiments, the 5' leader may be approximately 5, 10, 15, 20, 25, 50, 100, 200, or 500 ribonucleotides longer than the 3' trailer sequence, or vice versa.

[0102] In embodiments where the RNA molecule has a double hairpin structure, the second hairpin (in addition to the first hairpin structure) includes a sense RNA sequence and an antisense RNA sequence, each substantially identical in sequence to a region or complement of the target RNA molecule. In one embodiment, each hairpin has a set of ribonucleotides substantially identical in sequence to a region of the same target RNA molecule. In another embodiment, each hairpin has a set of ribonucleotides substantially identical in sequence to different regions of the same target RNA molecule. In yet another embodiment, each hairpin has a set of ribonucleotides substantially identical in sequence to regions of different target RNA molecules, i.e., the RNA molecule can be used to reduce the expression and / or activity of two target RNA molecules that may be unrelated in sequence.

[0103] In the double hairpin structure of the RNA molecule, the order of the sense and antisense RNA sequences in each hairpin can be either sense-to-antisense or antisense-to-sense, independently in the order of 5' to 3'. In a preferred embodiment, the order of the sense and antisense sequences in the double hairpin structure of the RNA molecule is either antisense-sense-sense-antisense (Figure 1A) with two consecutive sense sequences, or sense-antisense-antisense-sense (Figure 1B) with two consecutive antisense sequences.

[0104] In one embodiment, the RNA molecule may include, in 5' to 3' order, a 5' leader sequence, a first loop, a sense RNA sequence, a second loop, and a 3' trailer sequence, the 5' and 3' leader sequences covalently bound to the sense strand to form a dsRNA sequence. In one embodiment, the 5' leader and 3' trailer sequences are not covalently bound to each other. In one embodiment, the 5' leader and 3' trailer sequences are separated by a nick. In one embodiment, the 5' leader and 3' trailer sequences are ligated together to provide a closed RNA molecule. In another embodiment, the 5' leader and 3' trailer sequences are separated by a loop.

[0105] In the context of this disclosure, the term “loop” is used to refer to a loop structure in the RNA molecules disclosed herein, formed by a series of non-complementary ribonucleotides. Loops generally follow a series of base pairs between first and second RNA components, or connect sense RNA sequences and antisense RNA sequences of one or both of the first and second RNA components. In one embodiment, all loop ribonucleotides are non-complementary, and the loop is generally a short loop of 4 to 10 ribonucleotides. In other embodiments, some ribonucleotides within one or more loops are complementary and can form base pairs within the loop sequence, but only if these base pair formations enable the formation of the loop structure. For example, at least 5%, at least 10%, or at least 15% of the loop ribonucleotides are complementary. Embodiments of loops include stem loops or hairpins, pseudoknots, and tetraloops.

[0106] In one embodiment, the RNA molecule contains only two loops, and in another embodiment, the RNA molecule contains at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten loops, preferably up to ten loops. For example, the RNA molecule may contain four loops.

[0107] In this disclosure, loops of various sizes are envisioned. For example, a loop may contain 4, 5, 6, 7, 8, 9, 10, 11, or 12 ribonucleotides. In other embodiments, a loop may contain 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 longer, for example, containing 50, 100, 150, 200, or 300 ribonucleotides. In one embodiment, a loop contains 160 ribonucleotides. In another embodiment, less preferably, but provided that the loop does not interfere with the hybridization of sense and antisense RNA sequences, the loop may contain 200, 500, 700, or 1,000 ribonucleotides. In one embodiment, each loop has the same number of ribonucleotides. For example, a loop may be 100 to 1,000 ribonucleotides long. For example, a loop may be 600 to 1,000 ribonucleotides long. For example, the loop can be 4 to 1,000 ribonucleotides. For example, the loop preferably has 4 to 50 ribonucleotides. In another embodiment, the loop contains a different number of ribonucleotides.

[0108] In another embodiment, one or more loops include introns that can be excised from an RNA molecule by splicing. In one embodiment, the introns are derived from plant genes. Exemplary introns include intron 3 of maize alcohol dehydrogenase 1 (Adh1) (GenBank: AF044293), intron 4 of soybean β-conglycinin alpha subunit (GenBank: AB051865), and 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).

[0109] 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, or at least 10 consecutive base pairs, which may be standard base pairs or may include one or more non-standard base pairs. In other embodiments, which are less preferred, particularly in vertebrate cells, the loop may be at the end of at least 20, 30, 50, 100, 200, 500, or more consecutive base pairs.

[0110] In another embodiment, the RNA molecule comprises two or more sense ribonucleotide sequences and antisense ribonucleotide sequences that form base pairs with them, each of which is identical in sequence to a region of the target RNA molecule. For example, the RNA molecule may contain 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more sense ribonucleotide sequences and antisense ribonucleotide sequences that form perfect base pairs with them, each of which is independently identical in sequence to a region of the target RNA molecule. In this embodiment, one or more or all of the sequences can be separated by binding ribonucleotide sequences. In this embodiment, one or more or all of the sequences can be separated by loops.

[0111] In one embodiment, two or more senseribonucleotide sequences are identical to different regions of the same target RNA molecule. For example, the sequences may be identical to at least 2, at least 3, at least 4, at least 5, and at least 6 regions of the same target molecule. In another embodiment, two or more senseribonucleotide sequences are identical. In one embodiment, two or more senseribonucleotide sequences are identical to the same region of the same target RNA molecule. In another embodiment, two or more senseribonucleotide sequences are identical to different target RNA molecules. In an embodiment, the sequences may be identical to at least 2, at least 3, at least 4, at least 5, and at least 6 regions of different target molecules.

[0112] In another embodiment, two or more senseribonucleotide sequences do not have an intervening loop (spacer) sequence.

[0113] In one embodiment, the RNA molecule has a 5' end, at least one sense ribonucleotide sequence at least 21 nucleotides long, an antisense ribonucleotide sequence that is perfectly base-paired with each sense ribonucleotide sequence over at least 21 consecutive nucleotides, at least two loop sequences, and a single-stranded ribonucleotide having a 3' end. In this embodiment, the 5' and 3' ribonucleotides are not directly covalently bonded, but rather are base-paired and positioned adjacent to each other.

[0114] In another embodiment, consecutive base pairs of RNA components are separated by at least one gap. In one embodiment, the “gap” is provided by a non-paired ribonucleotide. In another embodiment, the “gap” is provided by an unligated 5' leader sequence and / or 3' trailer sequence. In this embodiment, this gap may also be referred to as the “unligated gap.” Mismatches and unligated gaps may be located at various positions on the RNA molecule. In one embodiment, the unligated gap may follow immediately after an antisense sequence. In another embodiment, the unligated gap may be near a loop on the RNA molecule. In yet another embodiment, the unligated gap is located approximately equidistant between at least two loops.

[0115] In one embodiment, the RNA molecule is produced from a single-stranded RNA. In one embodiment, the single-stranded RNA is not circularly closed and includes, for example, an unligated gap. In another embodiment, the RNA molecule is a circularly closed molecule. The closed molecule can be produced by ligating the aforementioned RNA molecule, which includes an unligated gap, for example, using an RNA ligase.

[0116] In another embodiment, the RNA molecule includes a 5'- or 3' or both elongation sequence. For example, the RNA molecule may include a 5' elongation sequence covalently bonded to a first 5' ribonucleotide. In another embodiment, the RNA molecule includes a 3' elongation sequence covalently bonded to a second 3' ribonucleotide. In yet another embodiment, the RNA molecule includes a 5' elongation sequence covalently bonded to a first 5' ribonucleotide and a 3' elongation sequence covalently bonded to a second 3' ribonucleotide.

[0117] In another embodiment, the RNA molecule includes a 5' elongation sequence covalently bonded to a second 5' ribonucleotide. In another embodiment, the RNA molecule includes a 3' elongation sequence covalently bonded to a first 3' ribonucleotide. In yet another embodiment, the RNA molecule includes a 5' elongation sequence covalently bonded to a second 5' ribonucleotide and a 3' elongation sequence covalently bonded to a first 3' ribonucleotide.

[0118] In another embodiment, the RNA molecule is as follows: - A 5' elongated sequence covalently bonded to the first 5' ribonucleotide, - A 3' elongated sequence covalently bonded to the second 3' ribonucleotide, - A 5' elongated sequence covalently bonded to the first 5' ribonucleotide, and a 3' elongated sequence covalently bonded to the second 3' ribonucleotide, - A 5' elongated sequence covalently bonded to the second 5' ribonucleotide, - A 3' elongated sequence covalently bonded to the first 3' ribonucleotide, -It may contain one or more of the following: a 5' extension sequence covalently bonded to the second 5' ribonucleotide and a 3' extension sequence covalently bonded to the first 3' ribonucleotide.

[0119] Non-standard base pairing In one embodiment, the RNA molecule of the present invention comprises senseribonucleotide sequences and antisenseribonucleotide sequences that can hybridize with each other to form a double-stranded (ds)RNA region having several non-standard base pairs, i.e., a combination of standard and non-standard base pairs. In one embodiment, the RNA molecule of the present invention comprises two or more senseribonucleotide sequences that can each hybridize with a region of one (consecutive) antisenseribonucleotide sequence to form a dsRNA region having several non-standard base pairs. See, for example, Figure 1B. In one embodiment, the RNA molecule of the present invention comprises two or more antisenseribonucleotide sequences that can each hybridize with a region of one (consecutive) senseribonucleotide sequence to form a dsRNA region having several non-standard base pairs. See, for example, Figure 1A. In one embodiment, the RNA molecule of the present invention comprises two or more antisense sense ribonucleotide sequences and two or more sense ribonucleotide sequences, wherein each antisense ribonucleotide sequence can hybridize to an antisense ribonucleotide sequence to form two or more dsRNA regions, one or both of which contain some non-standard base pairs.

[0120] In the following embodiments, the full-length dsRNA region of the RNA molecule of the present invention (i.e., the entire dsRNA region) is considered a contextual feature if there is only one (consecutive) dsRNA region, or if the RNA molecule has two or more dsRNA regions, for each of the dsRNA regions of the RNA molecule. In one embodiment, at least 5% of the base pairs of the dsRNA region are non-standard base pairings. In one embodiment, at least 6% of the base pairs of the dsRNA region are non-standard base pairings. In one embodiment, at least 7% of the base pairs of the dsRNA region are non-standard base pairings. In one embodiment, at least 8% of the base pairs of the dsRNA region are non-standard base pairings. In one embodiment, at least 9% or 10% of the base pairs of the dsRNA region are non-standard base pairings. In one embodiment, at least 11% or 12% of the base pairs of the dsRNA region are non-standard base pairings. In one embodiment, at least 15% or about 15% of the base pairs of the dsRNA region are non-standard base pairings. In one embodiment, at least 20% or about 20% of the base pairs in the dsRNA region are non-standard base pairs. In one embodiment, at least 25% or about 25% of the base pairs in the dsRNA region are non-standard base pairs. In one embodiment, at least 30% or about 30% of the base pairs in the dsRNA region are non-standard base pairs. In each of these embodiments, it is preferable that up to 40% of the base pairs in the dsRNA region are non-standard base pairs, more preferably up to 35% of the base pairs in the dsRNA region are non-standard base pairs, and even more preferably up to 30% of the base pairs in the dsRNA region are non-standard base pairs. In one embodiment, less preferable, about 35% of the base pairs in the dsRNA region are non-standard base pairs. In one embodiment, even less preferable, about 40% of the base pairs in the dsRNA region are non-standard base pairs. In each of the above embodiments, the dsRNA region may or may not contain one or more non-base-pairing ribonucleotides in either the sense sequence or the antisense sequence, or both.

[0121] In one embodiment, 10% to 40% of the base pairs in the dsRNA region of the RNA molecule of the present invention are non-standard base pairs. In one embodiment, 10% to 35% of the base pairs in the dsRNA region are non-standard base pairs. In one embodiment, 10% to 30% of the base pairs in the dsRNA region are non-standard base pairs. In one embodiment, 10% to 25% of the base pairs in the dsRNA region are non-standard base pairs. In one embodiment, 10% to 20% of the base pairs in the dsRNA region are non-standard base pairs. In one embodiment, 10% to 15% of the base pairs in the dsRNA region are non-standard base pairs. In one embodiment, 15% to 30% of the base pairs in the dsRNA region are non-standard base pairs. In one embodiment, 15% to 25% of the base pairs in the dsRNA region are non-standard base pairs. In one embodiment, 15% to 20% of the base pairs in the dsRNA region are non-standard base pairs. In one embodiment, 5% to 30% of the base pairs in the dsRNA region are non-standard base pairs. In one embodiment, 5% to 25% of the base pairs in the dsRNA region are non-standard base pairs. In one embodiment, 5% to 20% of the base pairs in the dsRNA region are non-standard base pairs. In one embodiment, 5% to 15% of the base pairs in the dsRNA region are non-standard base pairs. In one embodiment, 5% to 10% of the base pairs in the dsRNA region are non-standard base pairs. In each of the above embodiments, the dsRNA region may or may not contain one or more non-base-pairing ribonucleotides in either the sense sequence or the antisense sequence, or both.

[0122] In one embodiment, the dsRNA region of the RNA molecule of the present invention includes 20 consecutive base pairs, and at least one of the 20 consecutive base pairs is a non-standard base pair. In one embodiment, the dsRNA region includes 20 consecutive base pairs, and at least two of the 20 consecutive base pairs are non-standard base pairs. In one embodiment, the dsRNA region includes 20 consecutive base pairs, and at least three of the 20 consecutive base pairs are non-standard base pairs. In one embodiment, the dsRNA region includes 20 consecutive base pairs, and at least four of the 20 consecutive base pairs are non-standard base pairs. In one embodiment, the dsRNA region includes 20 consecutive base pairs, and at least five of the 20 consecutive base pairs are non-standard base pairs. In one embodiment, the dsRNA region includes 20 consecutive base pairs, and at least six of the 20 consecutive base pairs are non-standard base pairs. In one embodiment, the dsRNA region includes 20 consecutive base pairs, of which at least 7 are non-standard base pairs. In another embodiment, the dsRNA region includes 20 consecutive base pairs, of which at least 8 are non-standard base pairs. In yet another embodiment, the dsRNA region includes 20 consecutive base pairs, of which at least 9 are non-standard base pairs. In each of these embodiments, it is preferable that up to 10 of the 20 consecutive base pairs in the dsRNA region are non-standard base pairs, more preferably up to 9 of the dsRNA region are non-standard base pairs, even more preferably up to 8 of the dsRNA region are non-standard base pairs, even more preferably up to 7 of the dsRNA region are non-standard base pairs, and most preferably up to 6 of the dsRNA region are non-standard base pairs. Preferably, in the above embodiments, the non-standard base pairs include at least one G:U base pair, and more preferably, all of the non-standard base pairs are G:U base pairs. Preferably, the features of the above embodiments are applied to each of the 20 consecutive base pairs present in the RNA molecule of the present invention.

[0123] In one embodiment, the dsRNA region of the RNA molecule of the present invention includes 21 consecutive base pairs, and at least one of the 21 consecutive base pairs is a non-standard base pair. In one embodiment, the dsRNA region includes 21 consecutive base pairs, and at least two of the 21 consecutive base pairs are non-standard base pairs. In one embodiment, the dsRNA region includes 21 consecutive base pairs, and at least three of the 21 consecutive base pairs are non-standard base pairs. In one embodiment, the dsRNA region includes 21 consecutive base pairs, and at least four of the 21 consecutive base pairs are non-standard base pairs. In one embodiment, the dsRNA region includes 21 consecutive base pairs, and at least five of the 21 consecutive base pairs are non-standard base pairs. In one embodiment, the dsRNA region includes 21 consecutive base pairs, and at least six of the 21 consecutive base pairs are non-standard base pairs. In one embodiment, the dsRNA region includes 21 consecutive base pairs, of which at least 7 are non-standard base pairs. In another embodiment, the dsRNA region includes 21 consecutive base pairs, of which at least 8 are non-standard base pairs. In yet another embodiment, the dsRNA region includes 21 consecutive base pairs, of which at least 9 are non-standard base pairs. In each of these embodiments, it is preferable that up to 10 of the 21 consecutive base pairs in the dsRNA region are non-standard base pairs, more preferably up to 9 of the dsRNA region are non-standard base pairs, even more preferably up to 8 of the dsRNA region are non-standard base pairs, even more preferably up to 7 of the dsRNA region are non-standard base pairs, and most preferably up to 6 of the dsRNA region are non-standard base pairs. Preferably, in the above embodiments, the non-standard base pairs include at least one G:U base pair, and more preferably, all of the non-standard base pairs are G:U base pairs. Preferably, the features of the above embodiments are applied to each of the 21 consecutive base pairs present in the RNA molecule of the present invention.

[0124] In one embodiment, the dsRNA region of the RNA molecule of the present invention includes 22 consecutive base pairs, and at least one of the 22 consecutive base pairs is a non-standard base pair. In one embodiment, the dsRNA region includes 22 consecutive base pairs, and at least two of the 22 consecutive base pairs are non-standard base pairs. In one embodiment, the dsRNA region includes 22 consecutive base pairs, and at least three of the 22 consecutive base pairs are non-standard base pairs. In one embodiment, the dsRNA region includes 22 consecutive base pairs, and at least four of the 22 consecutive base pairs are non-standard base pairs. In one embodiment, the dsRNA region includes 22 consecutive base pairs, and at least five of the 22 consecutive base pairs are non-standard base pairs. In one embodiment, the dsRNA region includes 22 consecutive base pairs, and at least six of the 22 consecutive base pairs are non-standard base pairs. In one embodiment, the dsRNA region includes 22 consecutive base pairs, of which at least 7 are non-standard base pairs. In another embodiment, the dsRNA region includes 22 consecutive base pairs, of which at least 8 are non-standard base pairs. In yet another embodiment, the dsRNA region includes 22 consecutive base pairs, of which at least 9 are non-standard base pairs. In each of these embodiments, it is preferable that up to 10 of the 22 consecutive base pairs in the dsRNA region are non-standard base pairs, more preferably up to 9 of the dsRNA region are non-standard base pairs, even more preferably up to 8 of the dsRNA region are non-standard base pairs, even more preferably up to 7 of the dsRNA region are non-standard base pairs, and most preferably up to 6 of the dsRNA region are non-standard base pairs. Preferably, in the above embodiments, the non-standard base pairs include at least one G:U base pair, and more preferably, all of the non-standard base pairs are G:U base pairs. Preferably, the features of the above embodiments are applied to each of the 22 consecutive base pairs present in the RNA molecule of the present invention.

[0125] In one embodiment, the dsRNA region of the RNA molecule of the present invention includes 23 consecutive base pairs, and at least one of the 23 consecutive base pairs is a non-standard base pair. In one embodiment, the dsRNA region includes 23 consecutive base pairs, and at least two of the 23 consecutive base pairs are non-standard base pairs. In one embodiment, the dsRNA region includes 23 consecutive base pairs, and at least three of the 23 consecutive base pairs are non-standard base pairs. In one embodiment, the dsRNA region includes 23 consecutive base pairs, and at least four of the 23 consecutive base pairs are non-standard base pairs. In one embodiment, the dsRNA region includes 23 consecutive base pairs, and at least five of the 23 consecutive base pairs are non-standard base pairs. In one embodiment, the dsRNA region includes 23 consecutive base pairs, and at least six of the 23 consecutive base pairs are non-standard base pairs. In one embodiment, the dsRNA region includes 23 consecutive base pairs, of which at least 7 are non-standard base pairs. In another embodiment, the dsRNA region includes 23 consecutive base pairs, of which at least 8 are non-standard base pairs. In yet another embodiment, the dsRNA region includes 23 consecutive base pairs, of which at least 9 are non-standard base pairs. In each of these embodiments, it is preferable that up to 10 of the 23 consecutive base pairs in the dsRNA region are non-standard base pairs, more preferably up to 9 of the dsRNA region are non-standard base pairs, even more preferably up to 8 of the dsRNA region are non-standard base pairs, even more preferably up to 7 of the dsRNA region are non-standard base pairs, and most preferably up to 6 of the dsRNA region are non-standard base pairs. Preferably, in the above embodiments, the non-standard base pairs include at least one G:U base pair, and more preferably, all of the non-standard base pairs are G:U base pairs. Preferably, the features of the above embodiments are applied to each of the 23 consecutive base pairs present in the RNA molecule of the present invention.

[0126] In one embodiment, the dsRNA region of the RNA molecule of the present invention includes 24 consecutive base pairs, and at least one of the 24 consecutive base pairs is a non-standard base pair. In one embodiment, the dsRNA region includes 24 consecutive base pairs, and at least two of the 24 consecutive base pairs are non-standard base pairs. In one embodiment, the dsRNA region includes 24 consecutive base pairs, and at least three of the 24 consecutive base pairs are non-standard base pairs. In one embodiment, the dsRNA region includes 24 consecutive base pairs, and at least four of the 24 consecutive base pairs are non-standard base pairs. In one embodiment, the dsRNA region includes 24 consecutive base pairs, and at least five of the 24 consecutive base pairs are non-standard base pairs. In one embodiment, the dsRNA region includes 24 consecutive base pairs, and at least six of the 24 consecutive base pairs are non-standard base pairs. In one embodiment, the dsRNA region includes 24 consecutive base pairs, of which at least 7 are non-standard base pairs. In another embodiment, the dsRNA region includes 24 consecutive base pairs, of which at least 8 are non-standard base pairs. In yet another embodiment, the dsRNA region includes 24 consecutive base pairs, of which at least 9 are non-standard base pairs. In each of these embodiments, it is preferable that up to 10 of the 24 consecutive base pairs in the dsRNA region are non-standard base pairs, more preferably up to 9 of the dsRNA region are non-standard base pairs, even more preferably up to 8 of the dsRNA region are non-standard base pairs, even more preferably up to 7 of the dsRNA region are non-standard base pairs, and most preferably up to 6 of the dsRNA region are non-standard base pairs. Preferably, in the above embodiments, the non-standard base pairs include at least one G:U base pair, and more preferably, all of the non-standard base pairs are G:U base pairs. Preferably, the features of the above embodiments are applied to each of the 24 consecutive base pairs present in the RNA molecule of the present invention.

[0127] In the following embodiments, the full-length dsRNA region of the RNA molecule of the present invention (i.e., the entire dsRNA region) is considered a contextual feature if there is only one (consecutive) dsRNA region, or if the RNA molecule has two or more dsRNA regions, for each of the dsRNA regions of the RNA molecule. In one embodiment, the dsRNA region does not contain 20 consecutive standard base pairs, i.e., all subregions of the 20 consecutive base pairs contain at least one non-standard base pair, preferably at least one G:U base pair. In one embodiment, the dsRNA region does not contain 19 consecutive standard base pairs. In one embodiment, the dsRNA region does not contain 18 consecutive standard base pairs. In one embodiment, the dsRNA region does not contain 17 consecutive standard base pairs. In one embodiment, the dsRNA region does not contain 16 consecutive standard base pairs. In one embodiment, the dsRNA region does not contain 15 consecutive standard base pairs. In one embodiment, the dsRNA region does not contain 14 consecutive standard base pairs. In one embodiment, the dsRNA region does not contain 13 consecutive standard base pairs. In one embodiment, the dsRNA region does not contain 12 consecutive standard base pairs. In one embodiment, the dsRNA region does not contain 11 consecutive standard base pairs. In one embodiment, the dsRNA region does not contain 10 consecutive standard base pairs. In one embodiment, the dsRNA region does not contain 9 consecutive standard base pairs. In one embodiment, the dsRNA region does not contain 8 consecutive standard base pairs. In one embodiment, the dsRNA region does not contain 7 consecutive standard base pairs. In the above embodiments, the longest subregion of consecutive standard base pairs in the dsRNA region of an RNA molecule, or any dsRNA region of an RNA molecule, is preferably 5, 6, or 7 consecutive standard base pairs, i.e., shorter than the lengths mentioned. Each of the above features of embodiments is preferably combined with the following features in an RNA molecule. In one embodiment, the dsRNA region contains 10 to 19 or 20 consecutive base pairs. In a preferred embodiment, the dsRNA region includes 12 to 19 or 20 consecutive base pairs.In one embodiment, the dsRNA region contains 14 to 19 or 20 consecutive base pairs. In these embodiments, the dsRNA region contains 15 consecutive base pairs. In one embodiment, the dsRNA region contains 16, 17, 18, or 19 consecutive base pairs. In one embodiment, the dsRNA region contains 20 consecutive base pairs. Preferably, in the above embodiments, the consecutive base pairs contain at least one non-standard base pair including at least one G:U base pair, and more preferably, all non-standard base pairs within the region of consecutive base pairs are G:U base pairs.

[0128] In one embodiment, the dsRNA region includes subregions of four standard base pairs adjacent to a non-standard base pair, i.e., at least one, preferably one or two (two or fewer) non-standard base pairs adjacent to each end of the four standard base pairs. In one embodiment, the dsRNA region includes two subregions for each of the four standard base pairs adjacent to a non-standard base pair. In one embodiment, the dsRNA region includes three subregions for each of the four standard base pairs adjacent to a non-standard base pair. In one embodiment, the dsRNA region includes four or five subregions for each of the four standard base pairs adjacent to a non-standard base pair. In one embodiment, the dsRNA region includes six or seven subregions for each of the four standard base pairs adjacent to a non-standard base pair. In one embodiment, the dsRNA region includes eight to ten subregions for each of the four standard base pairs adjacent to a non-standard base pair. In one embodiment, the dsRNA region includes eleven to fifteen subregions for each of the four standard base pairs adjacent to a non-standard base pair. In one embodiment, the dsRNA region includes subregions of 2 to 50 each of four standard base pairs adjacent to a non-standard base pair. In another embodiment, the dsRNA region includes subregions of 2 to 40 each of four standard base pairs adjacent to a non-standard base pair. In yet another embodiment, the dsRNA region includes subregions of 2 to 30 each of four standard base pairs adjacent to a non-standard base pair. In yet another embodiment, the dsRNA region includes subregions of 2 to 20 each of four standard base pairs adjacent to a non-standard base pair. Preferably, in the above embodiments, the non-standard base pair includes at least one G:U base pair, and more preferably, all non-standard base pairs adjacent to consecutive standard base pairs within a subregion are G:U base pairs. In variations of the above embodiments, one or both of the adjacent non-standard base pairs are replaced with non-basepaired ribonucleotides in the sense sequence, antisense sequence, or both sequences for part or all of the subregion. In the above embodiments, it is readily apparent that the maximum number of subregions is determined by the length of the dsRNA region of the RNA molecule.

[0129] In one embodiment, the dsRNA region includes subregions of five standard base pairs adjacent to a non-standard base pair. In one embodiment, the dsRNA region includes two subregions for each of the five standard base pairs adjacent to a non-standard base pair. In one embodiment, the dsRNA region includes three subregions for each of the five standard base pairs adjacent to a non-standard base pair. In one embodiment, the dsRNA region includes four or five subregions for each of the five standard base pairs adjacent to a non-standard base pair. In one embodiment, the dsRNA region includes six or seven subregions for each of the five standard base pairs adjacent to a non-standard base pair. In one embodiment, the dsRNA region includes eight to ten subregions for each of the five standard base pairs adjacent to a non-standard base pair. In one embodiment, the dsRNA region includes eleven to fifteen subregions for each of the five standard base pairs adjacent to a non-standard base pair. In one embodiment, the dsRNA region includes two to fifty subregions for each of the five standard base pairs adjacent to a non-standard base pair. In one embodiment, the dsRNA region includes subregions of 2 to 50 each of five standard base pairs adjacent to a non-standard base pair. In another embodiment, the dsRNA region includes subregions of 2 to 30 each of five standard base pairs adjacent to a non-standard base pair. In yet another embodiment, the dsRNA region includes subregions of 2 to 20 each of five standard base pairs adjacent to a non-standard base pair. Preferably, in the above embodiments, the non-standard base pair includes at least one G:U base pair, and more preferably, all non-standard base pairs adjacent to consecutive standard base pairs within a subregion are G:U base pairs. In variations of the above embodiments, one or both of the adjacent non-standard base pairs are replaced with non-basepaired ribonucleotides in the sense sequence, antisense sequence, or both sequences for part or all of the subregions. In the above embodiments, it is readily apparent that the maximum number of subregions is determined by the length of the dsRNA region of the RNA molecule.

[0130] In one embodiment, the dsRNA region includes subregions of six standard base pairs adjacent to a non-standard base pair. In one embodiment, the dsRNA region includes two subregions for each of the six standard base pairs adjacent to a non-standard base pair. In one embodiment, the dsRNA region includes three subregions for each of the six standard base pairs adjacent to a non-standard base pair. In one embodiment, the dsRNA region includes four or five subregions for each of the six standard base pairs adjacent to a non-standard base pair. In one embodiment, the dsRNA region includes six or seven subregions for each of the six standard base pairs adjacent to a non-standard base pair. In one embodiment, the dsRNA region includes eight to ten subregions for each of the six standard base pairs adjacent to a non-standard base pair. In one embodiment, the dsRNA region includes eleven to sixteen subregions for each of the six standard base pairs adjacent to a non-standard base pair. In one embodiment, the dsRNA region includes two to sixteen subregions for each of the six standard base pairs adjacent to a non-standard base pair. In one embodiment, the dsRNA region includes subregions of 2 to 60 each of six standard base pairs adjacent to a non-standard base pair. In another embodiment, the dsRNA region includes subregions of 2 to 30 each of six standard base pairs adjacent to a non-standard base pair. In yet another embodiment, the dsRNA region includes subregions of 2 to 20 each of six standard base pairs adjacent to a non-standard base pair. Preferably, in the above embodiments, the non-standard base pair includes at least one G:U base pair, and more preferably, all non-standard base pairs adjacent to consecutive standard base pairs within a subregion are G:U base pairs. In variations of the above embodiments, one or both of the adjacent non-standard base pairs are replaced with non-basepaired ribonucleotides in the sense sequence, antisense sequence, or both sequences for part or all of the subregions. In the above embodiments, it is readily apparent that the maximum number of subregions is determined by the length of the dsRNA region of the RNA molecule.

[0131] In one embodiment, the dsRNA region includes a subregion of 10 consecutive base pairs, with base pairs 2-4 being non-standard base pairs. In another embodiment, the dsRNA region includes 10 consecutive base pairs, each containing 2 subregions, with base pairs 2-4 being non-standard base pairs. In yet another embodiment, the dsRNA region includes 10 consecutive base pairs, each containing 3 subregions, with base pairs 2-4 being non-standard base pairs. In yet another embodiment, the dsRNA region includes 10 consecutive base pairs, each containing 4 subregions, with base pairs 2-4 being non-standard base pairs. In yet another embodiment, the dsRNA region includes 10 consecutive base pairs, each containing 5 subregions, with base pairs 2-4 being non-standard base pairs. In yet another embodiment, the dsRNA region includes 10 consecutive base pairs, each containing 10 subregions, with base pairs 2-4 being non-standard base pairs. In one embodiment, the dsRNA region consists of 15 consecutive base pairs, each containing 4 sub-regions, with 2-6 of the 15 consecutive base pairs being non-standard base pairs. In another embodiment, the dsRNA region consists of 10 consecutive base pairs, each containing 2-50 sub-regions, with 2-4 of the 10 consecutive base pairs being non-standard base pairs. In yet another embodiment, the dsRNA region consists of 10 consecutive base pairs, each containing 2-40 sub-regions, with 2-4 of the 10 consecutive base pairs being non-standard base pairs. In yet another embodiment, the dsRNA region consists of 10 consecutive base pairs, each containing 2-30 sub-regions, with 2-4 of the 10 consecutive base pairs being non-standard base pairs. In yet another embodiment, the dsRNA region consists of 10 consecutive base pairs, each containing 2-20 sub-regions, with 2-4 of the 10 consecutive base pairs being non-standard base pairs. Preferably, in the above embodiments, the non-standard base pairs include at least one G:U base pair, and more preferably, all non-standard base pairs in the subregion are G:U base pairs. In variations of the above embodiments, one or more of 2-4 or 2-6 non-standard base pairs are replaced with non-basepaired ribonucleotides in the sense sequence, antisense sequence, or both sequences for some or all of the subregions. In the above embodiments, it is readily apparent that the maximum number of subregions is determined by the length of the dsRNA region of the RNA molecule.

[0132] In one embodiment, the ratio of standard base pairs to non-standard base pairs within a dsRNA region is 2.5:1 to 3.5:1, for example, about 3:1. In another embodiment, the ratio of standard base pairs to non-standard base pairs within a dsRNA region is 3.5:1 to 4.5:1, for example, about 4:1. In yet another embodiment, the ratio of standard base pairs to non-standard base pairs within a dsRNA region is 4.5:1 to 5.5:1, for example, about 5:1. In yet another embodiment, the ratio of standard base pairs to non-standard base pairs within a dsRNA region is 5.5:1 to 6.5:1, for example, about 6:1. Different dsRNA regions of different RNA molecules may have different ratios.

[0133] In the embodiments described above, the non-standard base pairs in the dsRNA region(s) of the RNA molecule are preferably all G:U base pairs. In one embodiment, at least 99% of the non-standard base pairs are G:U base pairs. In one embodiment, at least 98% of the non-standard base pairs are G:U base pairs. In one embodiment, at least 97% of the non-standard base pairs are G:U base pairs. In one embodiment, at least 95% of the non-standard base pairs are G:U base pairs. In one embodiment, at least 90% of the non-standard base pairs are G:U base pairs. In one embodiment, 90-95% of the non-standard base pairs are G:U base pairs. For example, if there are 10 non-standard base pairs, at least 9 (90%) are G:U base pairs.

[0134] The dsRNA region containing non-standard base pairs (or more) comprises an antisense sequence of 20 consecutive nucleotides that act 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 a target RNA molecule in a eukaryotic cell. In one embodiment, the dsRNA region comprises 2, 3, 4, or 5 antisense regulatory elements that are complementary to the same target RNA molecule (i.e., to different regions of the same target RNA molecule) or to different target RNA molecules.

[0135] In one embodiment, when the sense and antisense sequences hybridize, one or more ribonucleotides of the sense ribonucleotide sequence, or one or more ribonucleotides of the antisense ribonucleotide sequence, or both, do not form base pairs in the dsRNA region. In this embodiment, the dsRNA region does not contain any loop sequences to which the sense and antisense sequences are covalently bonded. One or more ribonucleotides of the dsRNA region or subregion may not form base pairs. Therefore, in this embodiment, the sense strand of the dsRNA region does not fully base pair with its corresponding antisense strand.

[0136] Furthermore, in one embodiment, the total number of ribonucleotides in the sense sequence(s) and the total number of ribonucleotides in the antisense sequence(s) may not be the same, but preferably they are the same, in combination with any of the features of the above embodiments. In one embodiment, the total number of ribonucleotides in the sense ribonucleotide sequence(s) of the dsRNA region is 90% to 110% of the total number of ribonucleotides in the antisense ribonucleotide sequence(s). In one embodiment, the total number of ribonucleotides in the sense ribonucleotide sequence(s) is 95% to 105% of the total number of ribonucleotides in the antisense ribonucleotide sequence(s). In one embodiment, the chimeric RNA molecule of this disclosure may include one or more structural elements such as internal or terminal bulges or loops. Various embodiments of bulges and loops have been described above. In one embodiment, the dsRNA region is separated by structural elements such as bulges or loops. In one embodiment, the dsRNA region is separated by intervening (spacer) sequences. Some of the ribonucleotides in the spacer sequence may base-pair with other ribonucleotides in the RNA molecule, for example, other ribonucleotides in the spacer sequence, or they may not base-pair in the RNA molecule, or some of each may base-pair. In one embodiment, the dsRNA region is attached to a terminal loop. In one embodiment, the dsRNA region is adjacent to a terminal loop.

[0137] In one embodiment, if the dsRNA region of the RNA molecule of the present invention has at least three non-standard base pairs in any subregion of five consecutive base pairs, the non-standard base pairs are not consecutive and are separated by one or more standard base pairs; that is, the dsRNA region does not have three or more consecutive non-standard base pairs. In one embodiment, the dsRNA region does not have four or more consecutive non-standard base pairs. For example, in one embodiment, the dsRNA region includes at least three non-standard base pairs in a 10-base-pair subregion, and each non-standard base pair is separated by four standard base pairs.

[0138] In one embodiment, the RNA molecule of the present invention comprises two or more dsRNA regions. For example, the RNA molecule comprises two, three, four, five, six, seven, eight, nine, ten or more dsRNA regions. In this example, one or more or all of the dsRNA regions may include the characteristics exemplified above, such as the number of non-standard base pairing and / or antisense regulatory elements.

[0139] Silencing activity The RNA molecules of this disclosure possess antisense activity because they contain a sense ribonucleotide sequence that is substantially complementary to the region of the target RNA molecule. For example, the ribonucleotide sequence is substantially complementary to the region of the target RNA molecule in eukaryotic cells. In one example, the target RNA molecule may be in a bacterial cell, a fungal cell, a plant cell, an insect cell, or an animal cell. Such components of the RNA molecule as defined herein may also be referred to as “antisense regulatory elements.” “Substantially complementary” means that the sense ribonucleotide sequence may have insertions, deletions, and individual point mutations compared to the complement of the target RNA molecule in eukaryotic cells. Preferably, the homology between the sense ribonucleotide sequence having 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 contain about 15, about 16, about 17, about 18, about 19 or more consecutive nucleotides whose sequence is identical to the first region of the target RNA molecule in eukaryotic cells. In another example, a senseribonucleotide sequence may contain approximately 20 consecutive nucleotides whose sequence is identical to that of the first region of a target RNA molecule in eukaryotic cells.

[0140] "Antisense activity" is used in the context of this disclosure to refer to an antisense regulatory element from an RNA molecule, as defined herein, that modulates (increases or decreases) the expression of a target RNA molecule.

[0141] In various examples, the antisense regulatory elements according to this disclosure may include multiple monomer subunits bound together by a binding group. Examples include primers, probes, antisense compounds, antisense oligonucleotides, external guide sequence (EGS) oligonucleotides, alternative splicers, gapmers, siRNAs, and microRNAs. Thus, the RNA molecules according to this disclosure may include antisense regulatory elements having single-stranded, double-stranded, cyclic, branched, or hairpin structures. In one example, the antisense sequence may include structural elements such as internal or terminal bulges or loops.

[0142] In one example, the RNA molecule of this disclosure includes a chimeric oligomeric component, such as a chimeric oligonucleotide. For example, the RNA molecule may include differently modified nucleotides, mixed-skeleton antisense oligonucleotides, or combinations thereof. In one example, the chimeric oligomeric compound may include at least one region modified to result in increased resistance to nuclease degradation, increased cellular uptake, and / or increased binding affinity to a target RNA molecule.

[0143] Antisense regulatory elements can have various lengths. Throughout various examples, the present disclosure provides antisense regulatory elements consisting of X-Y linked bases, 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 (where X < Y). For example, in certain embodiments, the present disclosure provides the following: 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, 14-21,14-28, 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, 17-22, 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, 23-2 4. Provides an antisense modulating element containing 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 binding bases.

[0144] The RNA molecules according to this disclosure may contain multiple antisense regulatory elements. For example, an RNA molecule may contain at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, and at least 10 antisense regulatory elements. In one example, the antisense regulatory elements are identical. In this example, the RNA molecule may contain copies of at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, and at least 10 antisense regulatory elements. In another example, the RNA molecules according to this disclosure may contain different antisense regulatory elements. For example, antisense regulatory elements may be provided to target multiple genes in pathways such as lipid biosynthesis. In this example, the RNA molecule may contain at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, and at least 10 different antisense regulatory elements.

[0145] The antisense regulatory elements described herein can regulate (increase or decrease) the expression or quantity of various target RNA molecules. For example, the target RNA molecules are fatty acid biosynthesis genes. Examples of such genes include genes encoding acetyltransacylase, acyl transport proteins ("acyl carrier proteins"), desaturases such as stearyl desaturase or microsomal D12-desaturase, particularly the Fad2-1 gene, malonyltransacylase, 3-ketoacyl-ACP synthetase, 3-keto-ACP reductase, enoyl-ACP hydrolase, acyl-ACP thioesterase, and enoyl-ACP reductase. In one example, the target RNA molecule is the FAD2 gene (e.g., as described by Genbank accession numbers: AF124360 (Brassica carinata), AF042841 (Brassica rapa), L26296 (Arabidopsis thaliana), A65102 (Corylus avellana)). For example, the target RNA molecule could be the FAD2.1 gene. In another example, the target RNA molecule could be the FAD2.2 gene. In yet another example, the target RNA molecules could be both the FAD2.1 and FAD2.2 genes.Examples of other genes involved in the modification of lipid composition that may be target RNA molecules are known in the field (Shure M 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 GM and Somerville CR (1993) Curr Opin Biotech 4(2):152-8; US5,530,192 and WO94 / 18337).

[0146] In another example, 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 (for example, nematode resistance genes are summarized in WO93 / 10251 and WO94 / 17194).

[0147] In another example, the target RNA molecule is disease-related. For instance, the target RNA molecule may 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, cyclin kinases, and their inhibitors.

[0148] In another example, the target RNA molecule is associated with delayed fruit maturation. Delayed fruit maturation can be achieved by reducing the gene expression of genes selected from the group consisting of, for example, polygalacturonase, pectin esterase, β-(1-4) glucanase (cellulase), β-galactanase (β-galactosidase), or genes for carotenoid biosynthesis, such as prephytoene or phytoene biosynthesis, such as phytoene desaturase, including genes for ethylene biosynthesis, such as 1-aminocyclopropane-1-carboxylic acid synthase.

[0149] In another example, target RNA molecules are associated with delaying the symptoms of aging. Preferred target RNA molecules include cinnamoyl-CoA:NADPH reductase or cinnamoyl alcohol dehydrogenase. Further target RNA molecules are described (in WO1995 / 07993).

[0150] In another example, target RNA molecules are associated with modifying the fiber content of foods, preferably seeds. For instance, RNA molecules may reduce the expression of caffeate O-methyltransferase or cinnamoyl alcohol dehydrogenase.

[0151] RNA molecules are encoded by nucleic acids. Those skilled in the art will understand from the foregoing description that this disclosure also provides isolated nucleic acids and component portions thereof that encode the RNA molecules disclosed herein. For example, nucleic acids comprising the sequences described in any one or more of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, and 9. The nucleic acids may be partially purified after expression in a host cell. The term “partially purified” is used to refer to RNA molecules that have been typically separated from lipids, nucleic acids, other peptides, and other relevant contaminants in the host cell. Preferably, the partially purified polynucleotides are free of at least 60%, more preferably at least 75%, and more preferably at least 90% of the other components associated with them.

[0152] In another example, the polynucleotides relating to this disclosure are heterologous polynucleotides. The term “heterologous polynucleotide” is well understood in the art and refers to a polynucleotide that is not endogenous in cells, or a native polynucleotide whose native sequence has been altered, or a native polypeptide whose expression has been quantitatively altered as a result of cell manipulation by recombinant DNA technology.

[0153] In another example, the polynucleotides according to this disclosure are synthetic polynucleotides. For example, polynucleotides may be produced using existing nucleic acid sequence-independent techniques such as DNA printing and oligonucleotide synthesis. In yet another example, polynucleotides are produced from xenonucleic acids.

[0154] In one example, the polynucleotide disclosed herein encodes an RNA precursor molecule containing an intron in at least one loop sequence that can be cleaved by splicing during transcription of the polynucleotide in a host cell. In another example, the loop sequence contains two, three, four, five, or more introns. The disclosure also provides expression constructs, such as DNA constructs, containing isolated nucleic acids of the disclosure operably ligated to a promoter. In one example, such isolated nucleic acids and / or expression constructs are provided in cells or non-human organisms. In one example, the isolated nucleic acids are stably incorporated into the genome of a cell or non-human organism. Various examples of suitable expression constructs, promoters, and cells containing them are described below.

[0155] The synthesis of RNA molecules according to this disclosure can be achieved using various methods known in the art. The Examples section provides examples of in vitro synthesis. In these examples, constructs containing the RNA molecules disclosed herein are restricted at the 3' end, precipitated, purified, and quantified. RNA synthesis can be carried out in bacterial culture medium after transformation of HT115 electrocompetent cells and induction of RNA synthesis using the T7, IPTG system.

[0156] Recombinant vectors One embodiment of the present invention includes a recombinant vector that comprises at least one RNA molecule as defined herein and is capable of delivering the RNA molecule to a host cell. The recombinant vector includes an expression vector. The recombinant vector comprises a heterologous polynucleotide sequence, i.e., a polynucleotide sequence that is not naturally found adjacent to the RNA molecule as defined herein, and is preferably derived from a different species. The vector may be either RNA or DNA, and is typically a viral vector derived from a virus, or a plasmid.

[0157] Various viral vectors can be used to deliver and mediate the expression of RNA molecules according to this disclosure. The selection of a viral vector generally depends on various parameters such as the target cells or tissues for delivery, the transduction efficiency of the vector, and pathogenicity. In one example, the viral vector is integrated into the chromatin of a host cell (e.g., lentivirus). In another example, the viral vector persists in the cell nucleus, mainly as an extrachromosomal episome (e.g., adenovirus). Examples of these types of viral vectors include oncoretroviruses, lentiviruses, adeno-associated viruses, adenoviruses, herpesviruses, and retroviruses.

[0158] Plasmid vectors typically contain additional nucleic acid sequences that facilitate the selection, amplification, and transformation of expression cassettes in prokaryotic cells (e.g., pUC-derived vectors, pGEM-derived vectors, or binary vectors containing one or more T-DNA regions). These additional nucleic acid sequences may include origins of replication that provide vector self-replication, selection marker genes (preferably encoding antibiotic or herbicide resistance), multiple unique cloning sites providing multiple insertion points for genes encoded in nucleic acid sequences or constructs, and sequences that facilitate transformation of prokaryotic and eukaryotic cells (particularly plants).

[0159] As used herein, “operably linked” refers to a functional relationship between two or more nucleic acid (e.g., DNA) fragments. Typically, it refers to a functional relationship between a transcriptional regulatory element (promoter) and a transcribed sequence. For example, a promoter is operably linked to the coding sequence of an RNA molecule as defined herein if it stimulates or modulates the transcription of the coding sequence in a suitable cell. Generally, promoter transcriptional regulatory elements operably linked to a transcribed sequence are physically contiguous to the transcribed sequence; i.e., they are cis-acting. However, some transcriptional regulatory elements, such as enhancers, do not need to be physically contiguous or located in close proximity to the coding sequence whose transcription is enhanced.

[0160] If multiple promoters exist, each promoter may be independent of and identical or different from others.

[0161] To facilitate the identification of transformants, the recombinant vector preferably includes a selective marker gene or a screenable marker gene. A “marker gene” means a gene that confers a distinct phenotype to cells expressing the marker gene, thus allowing such transformed cells to be distinguished from cells lacking the marker. A selective marker gene confers a trait that can be “selected” based on resistance to a selective agent (e.g., herbicides, antibiotics). A screenable marker gene (or reporter gene) confers a trait that can be identified through observation or testing, i.e., “screening” (e.g., β-glucuronidase, luciferase, GFP, or other enzymatic activity not present in non-transformed cells).Exemplary selection markers for selecting plant transformants include, but are not limited to, the hyg gene encoding hygromycin B resistance, the neomycin phosphotransferase (nptII) gene conferring resistance to kanamycin and paromomycin, for example, the glutathione-S-transferase gene from rat liver conferring resistance to glutathione-derived herbicides as described in EP256223, the glutamine synthetase gene that, when overexpressed, confers resistance to glutamine synthetase inhibitors such as phosphinothricin as described in WO87 / 05327, for example, the acetyltransferase gene from Streptomyces viridochromogenes conferring resistance to the selective agent phosphinothricin as described in EP275957, e.g., Hinchee et al. Examples include genes encoding 5-enolshikimic acid-3-phosphate synthase (EPSPS) that confer resistance to N-phosphonomethylglycine as described in al. (1988), such as the bar gene that confers resistance to bialaphos as described in WO91 / 02071, nitrilase genes such as bxn from Klebsiella ozaenae that confer resistance to bromoxynil (Stalker et al., 1988), dihydrofolate reductase (DHFR) genes that confer resistance to methotrexate (Thillet et al., 1988), mutant acetolactate synthase genes (ALS) (EP154, 204) that confer resistance to imidazolinone, sulfonylurea, or other ALS inhibitory chemicals, mutant anthranilate synthase genes that confer resistance to 5-methyltryptophan, or darapone dehalogenase genes that confer resistance to herbicides.

[0162] Preferably, the recombinant vector is stably integrated into the genome of a cell, such as a plant cell. Therefore, the recombinant vector may contain appropriate elements that enable the vector to be integrated into the genome or into the chromosome of a cell.

[0163] Expression vector Where used herein, “expression vector” is a DNA vector capable of transforming host cells and resulting in the expression of RNA molecules as defined herein. The expression vectors of the present invention include regulatory sequences such as transcriptional regulatory sequences, translational regulatory sequences, origins of replication, and other regulatory sequences that are compatible with host cells and control the expression of RNA molecules according to this disclosure. In particular, the expression vectors of the present invention include transcriptional regulatory sequences. Transcriptional regulatory sequences are sequences that control the initiation, elongation, and termination of transcription. Particularly important transcriptional regulatory sequences are those that control transcription initiation, such as promoters, enhancers, operators, and repressor sequences. The selection of regulatory sequences used depends on the target organism, such as the plant and / or target organ or tissue. Such regulatory sequences may be obtained from any eukaryote, such as a plant or plant virus, or may be chemically synthesized.

[0164] 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 contain one or more cloned plant genes and dominant selection markers, for example, under transcriptional control of 5' and 3' regulatory sequences. Such plant expression vectors may also contain promoter regulatory regions (e.g., regulatory regions that are inductively or constitutively, environmentally or developmentally regulated, or that control cell or tissue-specific expression), transcription initiation sites, ribosome binding sites, transcription termination sites, and / or polyadenylation signals.

[0165] The vectors of the present invention may also be used to produce RNA molecules as defined herein in cell-free expression systems, such systems are well known in the art.

[0166] In one example, a polynucleotide encoding an RNA molecule according to this disclosure is operably ligated to a promoter that can directly express the RNA molecule in a host cell. In one example, the promoter functions in vitro. In one example, the promoter is an RNA polymerase promoter. For example, the promoter may be an RNA polymerase III promoter. In another example, the promoter may be an RNA polymerase II promoter. However, the choice of promoter may depend on the target organism, such as the plant, insect, and / or target organ or tissue. Exemplary mammalian promoters include CMV, EF1a, 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 polyhedra. Many constitutive promoters that are active in plant cells are also described. Promoter suitable for constitutive expression in plants includes, but is not limited to, the cauliflower mosaic virus (CaMV) 35S promoter, the Figwort mosaic virus (FMV) 35S, the photoinducible promoter from the small subunit (SSU) of ribulose-1,5-bis-phosphate carboxylase, the rice cytoplasmic triose phosphate isomerase promoter, the Arabidopsis adenine phosphoribosyltransferase promoter, the rice actin 1 gene promoter, the mannopin synthase and octopine synthase promoters, the Adh promoter, the sucrose synthase promoter, the R gene complex promoter, and the chlorophyll α / β-binding protein gene promoter. These promoters have been used to create DNA vectors expressed in plants; see, for example, WO84 / 02913. All of these promoters have been used to create various types of recombinant DNA vectors expressible in plants.

[0167] For expression in plant source tissues such as leaves, seeds, roots, or stems, the promoters used in the present invention are preferably those that exhibit relatively high expression in these specific tissues. For this purpose, a selection may be made from many promoters of tissue- or cell-specific or enhanced gene expression. Examples of such promoters reported in the literature include the pea chloroplast glutamine synthetase GS2 promoter, the wheat chloroplast fructose-1,6-biphosphatase promoter, the potato nuclear photosynthesis ST-LS1 promoter, and the Arabidopsis thaliana serine / threonine kinase promoter and glucoamylase (CHS) promoter. Additionally, the promoters include the ribulose-1,5-bisphosphate carboxylase promoter from Eastern larch (Larix laricina), the Cab gene and Cab6 promoter from pine, the Cab-1 gene promoter from wheat, the Cab-1 gene promoter from spinach, the Cab 1R gene promoter from rice, the pyruvate orthophosphate dikinase (PPDK) promoter from Zea mays, the tobacco Lhcb1*2 gene promoter, and the Arabidopsis thaliana Suc2 sucrose-H 30 Symporter promoters and promoters of thylakoid membrane protein genes derived from spinach (PsaD, PsaF, PsaE, PC, FNR, AtpC, AtpD, Cab, RbcS) have been reported to be active in photosynthetically active tissues. Other promoters of chlorophyll α / β binding proteins, such as the promoters of the LhcB and PsbP genes derived from white mustard (Sinapis alba), may also be used in this invention.

[0168] Various plant gene promoters regulated in response to environmental, hormonal, chemical, and / or developmental signals, including promoters regulated by (1) heat, (2) light (e.g., pea RbcS-3A promoter, maize RbcS promoter), (3) hormones such as abscisic acid, (4) wounds (e.g., WunI), or (5) chemicals, e.g., methyl jasmonate, salicylic acid, steroid hormones, alcohols, Safeners (WO97 / 06269), can also be used for the expression of RNA-binding protein genes in plant cells, or (6) organ-specific promoters may also be advantageous.

[0169] As used herein, the term “plant storage organ-specific promoter” refers to a promoter that preferentially induces gene transcription in plant storage organs compared to other plant tissues. For expression in plant sink tissues such as potato tubers, tomato fruits, or seeds of soybeans, canola, cotton, Zea may, wheat, rice, and barley, the promoters used in the present invention preferably have relatively high expression in these specific tissues. The β-conglycinin promoter, or other seed-specific promoters such as napin, zein, linine, and phaseolin promoters can be used. Root-specific promoters may also be used. An example of such a promoter is the acid chitinase gene promoter. Expression in root tissue was also achievable by utilizing the root-specific subdomain of the identified CaMV 35S promoter.

[0170] In particularly preferred embodiments, the promoter induces expression in tissues and organs where lipid biosynthesis occurs. Such promoters can act in seed development at appropriate times to modify the lipid composition in the seed. Promoters preferred for seed-specific expression include: 1) promoters derived from genes encoding enzymes involved in lipid biosynthesis and seed accumulation, such as desaturases and elongases; 2) promoters derived from genes encoding seed storage proteins; and 3) promoters derived from genes encoding enzymes involved in carbohydrate biosynthesis and seed accumulation. Appropriate seed-specific promoters include the rapeseed oil gene promoter (US5,608,152), the Vicia faba USP promoter (Baumlein et al., 1991), the Arabidopsis oleosin promoter (WO98 / 45461), the Phaseolus vulgaris phaseolin promoter (US5,504,200), the Brassica Bce4 promoter (WO91 / 13980), or the Legmin B4 promoter (Baumlein et al., 1992), as well as promoters that result in seed-specific expression in monocotyledonous plants such as maize, barley, wheat, rye, and rice. Appropriate promoters of note are the barley lpt2 or lpt1 gene promoters (WO 95 / 15389 and WO 95 / 23230), or the promoters described in WO 99 / 16890 (promoters derived from the barley hordein gene, rice glutelin gene, rice lysine gene, rice prolamin gene, wheat gliadin gene, wheat glutelin gene, maize zein gene, oat glutelin gene, sorghum casilin gene, and rye gisekarin gene). Other promoters include those described in Broun et al. (1998), Potenza et al. (2004), US 20070192902, and US 20030159173. In one embodiment, a seed-specific promoter is preferentially expressed in a defined part of the seed, such as the cotyledon(s) or endosperm.Examples of cotyledon-specific promoters include, but are not limited to, the FP1 promoter (Ellerstrom et al., 1996), the pea legumin promoter (Perrin et al., 2000), and the pea phytohemagglutinin 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 further embodiments, seed-specific promoters are not expressed in the embryo and / or after seed germination, or are expressed only at low levels.

[0171] In another embodiment, the plant storage organ-specific promoter is the fruit-specific promoter. Examples include, but are not limited to, the polygalacturonase, E8 and Pds promoters in tomato, and the ACC oxidase promoter in apple (see Potenza et al., 2004 for a review). In a preferred embodiment, the promoter preferentially induces expression in the edible part of the fruit, such as the pith, compared to the peel or seeds within the fruit.

[0172] In one embodiment, the inducible promoter is the Aspergillus nidulans alc system. Examples of inducible expression systems that can be used instead of the Aspergillus nidulans alc system are described in the reviews Padidam (2003) and Corrado and Karali (2009). In another embodiment, the inducible promoter is a safer inducible promoter, such as the maize ln2-1 or ln2-2 promoter (Hershey and Stoner, 1991), and safer inducible promoters are the maize GST-27 promoter (Jepson et al., 1994) or the soybean GH2 / 4 promoter (Ulmasov et al., 1995).

[0173] In another embodiment, the inducible promoter is, for example, the senescence-inducible promoter SAG12 and SAG13 from Arabidopsis (Gan, 1995; Gan and Amasino, 1995), or the senescence-inducible promoter LSC54 from Brassica napus (Buchanan-Wollaston, 1994). Such promoters show increased expression around the time of senescence in plant tissues, particularly leaves.

[0174] For expression in vegetative tissues, leaf-specific promoters such as the ribulose bisphosphate carboxylase (RBCS) promoter can be used. For example, the tomato RBCS1, RBCS2, and RBCS3A genes are expressed in leaves and photon crops (Meier et al., 1997). The ribulose bisphosphate carboxylase promoter described by Matsuoka et al. (1994), which is expressed at nearly exclusively high levels in mesophyll cells of the leaf blade and leaf sheath, can be used. Another leaf-specific promoter is the light-harvesting chlorophyll a / b binding protein gene promoter (see Shiina et al., 1997). The Arabidopsis thaliana myb-related gene promoter (Atmyb5), described by Li et al. (1996), is leaf-specific. The Atmyb5 promoter is expressed in the trichomes, stipules, and epidermal cells of developing leaves at the margins of young rosettes and stem leaves, as well as in immature seeds. Alternatively, the leaf promoter identified in maize by Busk et al. (1997) can be used.

[0175] In some cases, for example, when LEC2 or BBM are recombinantly expressed, it may be desirable that the transgene is not expressed at high levels. An example of a promoter that can be used in such situations is a truncated napin A promoter that preserves the seed-specific expression pattern but has reduced expression levels (Tan et al., 2011).

[0176] The 5' untranslated leader sequence may be derived from a promoter selected to express a heterologous gene sequence of the RNA molecule of this disclosure, or may be heterologous to the coding region of the enzyme produced, and may be specifically modified as needed to increase mRNA translation. For a review of the optimization of transgene expression, see Koziel et al. (1996). The 5' untranslated region can be obtained from plant virus RNA (in particular tobacco mosaic virus, tobacco etch virus, maize mosaic virus, alfalfa mosaic virus), from appropriate eukaryotic genes, plant genes (wheat and maize chlorophyll a / b binding protein gene leaders), or from synthetic gene sequences. The present invention is not limited to constructs derived from a 5' untranslated sequence in which the untranslated region has a promoter sequence. Leader sequences can also be derived from unrelated promoter or coding sequences. Useful leader sequences in the context of the present invention include maize Hsp70 leaders (US5,362,865 and US5,859,347) and the TMV omega element.

[0177] Transcription termination is achieved by a 3' untranslated DNA sequence operably linked to the target RNA molecule in the expression vector. The 3' untranslated region of the recombinant DNA molecule contains a polyadenylation signal that functions in plants, adding an adenylate nucleotide to the 3' end of the RNA. The 3' untranslated region can be obtained from a variety of genes expressed in plant cells. The 3' untranslated region of nopalin synthase, the 3' untranslated region of the pea subunit Rubisco gene, and the 3' untranslated region of the soybean 7S seed storage protein gene are commonly used for this purpose. The 3' transcribed untranslated region containing a polyadenylation signal from the Agrobacterium tumor induction (Ti) plasmid gene is also suitable.

[0178] transfer nucleic acid The transfer nucleic acid may be used to deliver an exogenous polynucleotide to a cell and comprises one, preferably two, boundary sequences and one or more RNA molecules of interest. The transfer nucleic acid may or may not encode a selection marker. Preferably, the transfer nucleic acid forms part of a binary vector within a bacterium, the binary vector further comprising elements that enable replication of the vector within the bacterium, selection of bacterial cells containing the binary vector, or maintenance. Upon transfer into eukaryotic cells, the transfer nucleic acid component of the binary vector can be incorporated into the eukaryotic cell genome, and transient expression experiments only allow for intracellular expression.

[0179] As used herein, the term “extrachromosomal translocation nucleic acid” refers to nucleic acid molecules that can be translocated from bacteria, such as Agrobacterium species, into eukaryotic cells, such as plant leaf cells. Extrachromosomal translocation nucleic acids are well-known genetic elements in which the translocated nucleic acid, and subsequently the nucleotide sequences contained within its boundaries, can be incorporated into the genome of the recipient cell. In this regard, the translocated nucleic acid is typically flanked by two “boundary” sequences, but in some cases, a single boundary at the first end may be used, and the second end of the translocated nucleic acid is randomly generated during the translocation process. The target RNA molecule is typically located between the left boundary-like sequence and the right boundary-like sequence of the translocated nucleic acid. The RNA molecule contained within the translocated nucleic acid can be operably linked to a variety of different promoter and terminator regulatory elements that promote its expression, i.e., the transcription and / or translation of the RNA molecule. Imported DNA (T-DNA) from Agrobacterium species, such as Agrobacterium tumefaciens or Agrobacterium rhizogenes, and their artificial variants / mutants, are perhaps the most characteristic examples of imported nucleic acids. Another example is P-DNA ("plant-DNA") containing plant-derived T-DNA boundary-like sequences.

[0180] As used herein, “T-DNA” refers to the T-DNA from the Agrobacterium tumefaciens Ti plasmid, or the T-DNA from the Agrobacterium rhizogenes Ri plasmid, or variants thereof that function for the translocation of DNA into plant cells. The T-DNA may include the entire T-DNA with both right and left boundary sequences, but only the minimum sequence required for cis-translocation, i.e., the right T-DNA boundary sequence, is necessary. The T-DNA of the present invention is inserted into the target RNA molecule somewhere between the right boundary sequence and the left boundary sequence (if present). Sequences encoding factors required in trans-translocation of the T-DNA into plant cells, such as vir genes, may be inserted into the T-DNA, present in the same replicon as the T-DNA, or preferably in trans-trans-a compatible replicon of the Agrobacterium host. Such “binary vector systems” are well known in the art. As used herein, "P-DNA" refers to an immobilized nucleic acid isolated from a plant genome or its artificial variant / mutant, which contains a T-DNA boundary-like sequence at each end or at only one end.

[0181] As used herein, the “boundary” sequence of the translocated nucleic acid may be isolated from a selected organism such as a plant or bacterium, or may be an artificial variant / mutant thereof. The boundary sequence may facilitate and facilitate the translocation of the RNA molecule to which it is ligated, and may facilitate its integration into the recipient cell genome. In one embodiment, the boundary sequence is 10 to 80 bp long. Boundary sequences of T-DNA from Agrobacterium species are well known in the art, including those described by Lacroix et al. (2008).

[0182] Originally, only Agrobacterium species were used for gene transfer into plant cells. However, currently, numerous identified and developed systems exist that function in a similar manner to Agrobacterium species. Recently, several non-Agrobacterium species have been genetically modified to be competent for gene transfer (Chung et al., 2006; Broothaerts et al., 2005). These include Rhizobium species NGR234, Sinorhizobium meliloti, and Mezorhizobium loti.

[0183] The direct transfer of eukaryotic expression plasmids from bacteria to eukaryotic hosts was first achieved decades ago by fusing mammalian cells with protoplasts of Escherichia coli that carried the plasmids (Schaffner, 1980). Since then, the number of bacteria capable of delivering genes to mammalian cells has steadily increased (Weiss, 2003), and has been discovered independently by four groups (Sizemore et al. 1995; Courvalin et al., 1995; Powell et al., 1996; Darji et al., 1997).

[0184] As used herein, the terms “transfection,” “transformation,” and their variations are generally interchangeable. A “transfected” or “transformed” cell may be manipulated to introduce a desired RNA molecule(s), or may be a progeny cell derived therefrom.

[0185] Recombinant cells The present invention also provides recombinant cells, such as recombinant bacterial cells, fungal cells, plant cells, insect cells, or animal cells, or combinations thereof, which are host cells transformed with one or more RNA molecules or vectors as defined herein. Preferred cells of the present invention include any cells that can be transformed with RNA molecules or recombinant vectors according to this disclosure. In one example, the transformed host cell is dead.

[0186] Recombinant cells may be cells in culture, in vitro cells, or cells in organisms such as plants, or in organs such as seeds or leaves. Preferably, the cells are in plants, and more preferably in plant seeds. In one embodiment, the recombinant cells are non-human cells. Thus, in one example, this disclosure relates to a non-human organism comprising one or more or all of the RNA molecules disclosed herein.

[0187] In one example, the cells are insect cells. In another example, the insect cells are derived from Trichoplusia.

[0188] Another example of a suitable host cell is the electrocompetent HT115 cell.

[0189] The host cells into which the RNA molecule(s) are introduced may be either untransformed cells(s) or cells already transformed with at least one nucleic acid. Such nucleic acids may or may not be involved in lipid synthesis. The host cells of the present invention can endogenously (i.e., naturally) express the RNA molecule(s) as defined herein, in which case recombinant cells derived therefrom have an enhanced ability to produce the RNA molecule(s) or can produce the RNA molecule(s) only after being transformed with at least one RNA molecule(s) as defined herein. In one example, the cells are cells that can be used to produce lipids. In one embodiment, the recombinant cells of the present invention have an enhanced ability to produce nonpolar lipids such as TAGs.

[0190] The host cells of this disclosure may be any cell capable of expressing at least one RNA molecule described herein, and include bacteria, fungi (including yeast), parasites, arthropods, animal and plant cells. Examples 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 include E. coli containing the E. coli K-12 derivative, Salmonella typhi, Salmonella typhimurium containing attenuated strains, Spodoptera frugiperda, Trichoplusia ni, and non-tumor-forming mouse myoblast G8 cells (e.g., ATCC CRL 1246). Additional suitable mammalian cell hosts include other kidney cell lines, other fibroblast cell lines (e.g., human, mouse, or chicken embryo fibroblast cell lines), myeloma cell lines, Chinese hamster ovary cells, mouse NIH / 3T3 cells, LMTK cells, and / or HeLa cells.

[0191] In a preferred embodiment, the plant cell is a seed cell, particularly a cell in the cotyledon or endosperm of a seed. In one embodiment, the cell is an animal cell. The animal cell may be, for example, a non-human animal cell, a non-human vertebrate cell, a non-human mammal cell, or any type of animal such as an aquatic animal like a fish or crustacean, an invertebrate, or an insect. Examples of algal cells useful 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.

[0192] Transgenic plants The present invention also provides a plant comprising one or more exogenous RNA molecules as defined herein, cells as disclosed herein, vectors as disclosed herein, or a combination thereof. When used as a noun, “plant” refers to the entire plant, and when used as a noun, “part thereof” refers to plant organs (e.g., leaves, stems, roots, flowers, fruits), single cells (e.g., pollen), seed parts such as seeds, embryos, endosperm, blastodisc or seed coat, plant tissues such as vascular tissue, plant cells and their offspring. As used herein, part thereof includes plant cells.

[0193] As used herein, the terms “in a plant” and “in the plant” in the context of modifications to plants mean that the modification occurred in at least a portion of the plant, including cases where the modification occurred in the entire plant, and do not exclude cases where the modification occurred in only one or more parts of the plant, rather than all parts of the plant. For example, a tissue-specific promoter may be expressed only in specific parts of a plant, but is said to be expressed “in a plant.” Similarly, “a transcription factor polypeptide that increases the expression of one or more glycolytic and / or fatty acid biosynthesis genes in a plant” means that the increased expression occurs in at least a portion of the plant.

[0194] As used herein, the term “plant” is used in its broadest sense and includes all organisms in the plant kingdom, including red algae, brown algae, and green algae. It also includes, but is not limited to, flowering plants, grasses, crops, or grains (e.g., oilseeds, maize, soybeans), fodder or forage, fruit or vegetable plants, herbaceous plants, woody plants, or any species of tree. It is not intended to limit “plant” to any particular structure. It also refers to single-celled plants (e.g., microalgae). With respect to a plant, the term “part of it” refers to plant cells and their offspring, multiple plant cells, structures present at any stage of plant development, or plant tissue. Such structures include, but are not limited to, leaves, stems, flowers, fruits, nuts, roots, seeds, seed coats, and embryos. The term "plant tissue" includes differentiated and undifferentiated tissues of plants, including those present in leaves, stems, flowers, fruits, nuts, roots, and seeds, such as embryonic tissue, endosperm, epidermal tissue (e.g., epidermis, periderm), vascular tissue (e.g., xylem, phloem), or basal tissue (including parenchymal cells, plaque cells, and / or plastinum cells), and cells in culture (e.g., single cells, protoplasts, callus, embryos, etc.). Plant tissue may be in planta, in organ culture, tissue culture, or cell culture.

[0195] Different amounts of 18:3 and 16:3 fatty acids have been found in glycolipids of different plant species. This is because fatty acids with three double bonds are usually always C 18 The atomic length is 18:3 in plants, and C 16 Fatty acids and C 18This is used to distinguish 18:3 plants from 16:3 plants that contain both fatty acids. In 18:3 chloroplasts, the enzymatic activity catalyzing the conversion of phosphatidate to diacylglycerol and diacylglycerol to monogalactosyldiacylglycerol (MGD) is significantly lower than in 16:3 chloroplasts. In the leaves of 18:3 plants, chloroplasts synthesize stearoyl-ACP2 in the stroma, introducing the first double bond into the saturated hydrocarbon chain and hydrolyzing the thioester. The released oleic acid is transported across the chloroplast envelope to the membrane of the eukaryotic portion of the cell, possibly the endoplasmic reticulum, where it is incorporated into PC. The PC-bound oleoyl group is desaturated in these membranes and then returned to the chloroplast. The MGD-bound acyl group is a substrate for introducing a third double bond to produce MGD with two linolenoyl residues. This galactolipid is characteristic of 18:3 plants, such as Asteraceae and Fabaceae. For example, in photosynthetically active cells of 16:3 plants, represented by members of the Apiaceae and Brassicaceae families, two pathways operate in parallel to provide thylakoids containing MGD. Cooperative “eukaryotic” sequences are added to varying degrees by a “prokaryotic” pathway. The reaction is confined to chloroplasts, and esterification to MGD results in a typical arrangement of acyl groups and its complete desaturation. The prokaryotic DAG skeleton holds C16:0 and its desaturated product at position C-2, where C18: fatty acids are excluded. Position C-1 is occupied by C18 fatty acids and slightly by the C16 group. The similarity between the DAG skeleton of cyanobacteria-derived lipids and that synthesized by the chloroplast-confmed pathway in 16:3 plants suggests a phylogenetic relationship and justifies the term prokaryotic.

[0196] As used herein, the terms “vegetative tissue” or “vegetative plant part” refer to any plant tissue, organ, or part other than the organs for sexual reproduction of a plant. The organs for sexual reproduction of a plant are specifically the seed-carrying organs, flowers, pollen, fruits, and seeds. Vegetative tissue and parts include at least the leaves, stems (including the bolts and tillers, but excluding the heads), tubers, and roots of a plant, but excluding the flowers, pollen, seeds including the seed coat, embryo, and endosperm, fruits including the mesocarp tissue, seed-bearing pods, and seed-bearing heads. In one embodiment, the vegetative plant part of a plant is the epiphytic plant part. In another or further embodiment, the vegetative plant part is a green part such as a leaf or stem.

[0197] A “transgenic plant” or a variant thereof refers to a plant containing an introduced gene not found in the wild-type plant of the same species, subspecies, or variety. Transgenic plants as defined in the context of this invention include plants and their offspring that have been genetically modified using recombination techniques to induce the production of at least one polypeptide as defined herein in a desired plant or a portion thereof. The term “transgenic plant portion” has a corresponding meaning.

[0198] The terms “seed” and “grain” are used interchangeably herein. “Grain” refers to mature grain, such as harvested grain or grain still on a plant but ready for harvest, but depending on the context, it may also refer to grain after absorption or germination. Mature grain typically has a moisture content of less than about 18%. In preferred embodiments, 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 an immature seed typically found in the reproductive structure of a plant after fertilization or flowering, but may also refer to such an immature seed isolated from the plant. Mature seed typically has a moisture content of less than about 12%.

[0199] As used herein, the term "plant storage organ" refers to a part of a plant specialized for storing energy, for example, in the form of proteins, carbohydrates, or lipids. Examples of plant storage organs are seeds, fruits, tubers, and rhizomes. A preferred plant storage organ of the present invention is a seed.

[0200] As used herein, the term "phenotypically normal" refers to a genetically modified plant or a part thereof, such as a transgenic plant, or a storage organ such as a seed, tuber, or fruit of the present invention, whose ability to grow and reproduce is not significantly reduced compared to an unmodified plant or a part thereof. Preferably, the biomass, growth rate, germination rate, size of the storage organ, size of the seed, and / or number of viable seeds produced is at least 90% of that of a plant lacking the recombinant polynucleotide when grown under the same conditions. This term does not include plant characteristics that may differ from wild-type plants but do not affect the utility of plants for commercial purposes, such as the ballerina phenotype of seedling leaves. In one embodiment, a genetically modified plant or a part thereof that is phenotypically normal comprises a recombinant polynucleotide encoding a silencing suppressor operably linked to a plant storage organ-specific promoter and has essentially the same ability to grow or reproduce as the corresponding plant or a part thereof that does not contain the polynucleotide.

[0201] The plants provided or intended for use in the implementation of the present invention include both monocots and dicots. In preferred embodiments, the plants of the present invention are crops (e.g., cereals and legumes, maize, wheat, potatoes, rice, sorghum, millet, cassava, barley), or legumes such as soybeans, beans, or peas. The plants may be cultivated for the production of edible roots, tubers, leaves, stems, flowers, or fruits. The plants may be vegetable plants, in which the nutrient-rich parts are used as food. The plants of the present invention include Acrocomia aculeata, Arabidopsis thaliana, Aracinis hypogaea, Astrocaryum murumuru, Astrocaryum vulgare, Attalea geraensis, Indaia-rateiro, Attalea humilis, Attalea Brassica species such as oleifera (andaia), Attalea phalerata (uricuri), Attalea speciosa (babassu), Avena sativa (oat), Beta vulgaris (sugar beet), Brassica carinata, Brassica juncea, Brassica napobrassica, Brassica napus (canola), Camelina sativa (cannabis), Cannabis sativa (hemp), Carthamus tinctorius (safflower), Caryocar brasiliense (peky), Cocos nucifera (coconut), Crambe abyssinica (Abyssinian kale), Cucumis melo (melon), Elaeis guineensis (African palm), Glycine max (soybean), Gossypium hirsutum (cotton), Helianthus species such as Helianthus annuus (sunflower), Hordeum vulgare (barley), Jatropha curcas (acorn tung tree), Joannesia princeps (arara nut-tree), LemnaLemna species such as aequinoctialis, Lemna disperma, Lemna ecuadoriensis, Lemna gibba (swollen duckweed), Lemna japonica, Lemna minor, Lemna minuta, Lemna obscura, Lemna paucicostata, Lemna perpusilla, Lemna tenera, Lemna trisulca, Lemna turionifera, Lemna valdiviana, Lemna yungensis, etc. (duckweed), Licania rigida, Linum usitatissimum, Lupinus angustifolius, Mauritia flexuosa, Maximiliana maripa, Miscanthus species such as Miscanthus x giganteus and Miscanthus sinensis, Nicotiana Nicotiana species (tobacco) such as tabacum or Nicotiana benthamiana, Oenocarpus bacaba (bacaba-do-azeite), Oenocarpus bataua (pataua), Oenocarpus distichus (bacaba-de-leque), Oryza species (rice) such as Oryza sativa and Oryza glaberrima, Panicum virgatum (switchgrass), Paraqueiba paraensis (mari), Persea amencana (avocado), Pongamia pinnata (black avocado), Populus trichocarpa, Ricinus communis (caster), Saccharum species (sugarcane), Sesamum indicum (sesame), Solanum tuberosum (potato), Sorghum bicolor, Sorghum vulgare, Theobroma Sorghum species such as grandiforum (cupuaçu), Trifolium species, Trithrinax brasiliensis (Brazilian hedge palm), TriticumIt can be Triticum species such as aestivum (wheat), Zea mays (maize), alfalfa (lucerne), Secale cereale (rye), Ipomoea batatas (sweet potato), Manihot esculenta (cassava), Coffea species (coffee), Ananas comosus (pineapple), Citrus species (citrus tree), Theobroma cacao (cacao), Camellia sinensis (tea), Musa species (banana), Persea americana (avocado), Ficus casica (fig), Psidium guajava (guava), Mangifer indica (mango), Olea europaea (olive), Carica papaya (papaya), Anacardium occidentale (cashew), Macadamia intergrifolia (macadamia), and Prunus amygdalus (almond). For example, the plant of the present disclosure can be Nicotiana benthamiana.

[0202] Other preferred plants include, in addition to the above, C4 grasses such as Andropogon gerardi, Bouteloua curtipendula, B. gracilis, Buchloe dactyloides, Schizachyrium scoparium, Sorghastrum nutans, Sporobolus cryptandrus, C3 grasses such as Elymus canadensis, the legumes Lespedeza capitata, and Petalostemum villosum, forb Aster azureus, and woody plants such as Quercus ellipsoidalis and Q. macrocarpa. Other preferred plants include C3 grasses.

[0203] In a preferred embodiment, the plant is an angiosperm.

[0204] In one embodiment, the plant is an oilseed plant, preferably an oilseed crop. As used herein, “oilseed plant” refers to a plant species used for the commercial production of lipids derived from the plant's seeds. Oilseed plants may be, for example, rapeseed (such as canola), maize, sunflower, safflower, soybean, sorghum, flax, or sugar beet. Furthermore, oilseed plants may be other Brassica, cotton, peanut, poppy, rutabaga, mustard, castor bean, sesame, safflower, Jatropha curcas, or nut-producing plants. Plants may produce high levels of lipids in their fruits, such as olives, oil palms, or coconuts. Horticultural plants to which the present invention can be applied include lettuce, endive, or vegetable Brassica, including cabbage, broccoli, or cauliflower. The present invention may be applied to tobacco, melon, carrot, strawberry, tomato, or pepper.

[0205] In a preferred embodiment, the transgenic plant is homozygous for all introduced genes (transgenes), so its offspring are not segregated for the desired phenotype. The transgenic plant may also be heterozygous for the introduced transgene(s), preferably uniformly heterozygous for the transgenes in F1 offspring grown from hybrid seeds, for example. Such plants may offer advantages such as hybrid vigor, which is well known in the art.

[0206] Transformation The RNA molecules disclosed herein can be stably introduced into the host cells and / or non-human organisms such as plants. For the sake of avoidance of misunderstanding, the examples in this disclosure include the plants stably transformed with the RNA molecules disclosed herein. As used herein, the terms “stably transformed” and their variant forms mean that an RNA molecule or the nucleic acid encoding it is incorporated into the genome of a cell and transferred into progeny cells during cell division without the need for active selection of their presence. Stable transformants, or their progeny, can be identified and selected by any means known in the art, such as Southern blotting of chromosomal DNA or in situ hybridization of genomic DNA.

[0207] 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).

[0208] In one embodiment, a plant can be transformed by topically applying an RNA molecule according to the present disclosure to the plant or a part thereof. For example, the RNA molecule may be provided as a formulation containing a suitable carrier and can be applied by spraying, spraying, or other means to the surface of the plant or a part thereof. Thus, in an example, the method of the present disclosure includes introducing the RNA molecule disclosed herein into a plant, and the method includes topically applying a composition containing the RNA molecule to the plant or a part thereof.

[0209] Because DNA can be introduced into cells of whole plant tissues, plant organs, or tissue culture explants for transient expression or stable integration of DNA into the plant cell genome, Agrobacterium-mediated translocation is a widely applicable system for introducing genes into plant cells. For example, the floral dip (in planta) method may be used. The use of Agrobacterium-mediated plant integration vectors for introducing DNA into plant cells is well known in the art. The region of DNA to be translocated is defined by a boundary sequence, and intervening DNA (T-DNA) is usually inserted into the plant genome. Due to the ease and clear nature of gene translocation, this is an optimal method.

[0210] Available acceleration methods include, for example, microprojectile bombardment. One example of a method for delivering transformed nucleic acid molecules to plant cells is microprojectile bombardment. This method was reviewed by Yang et al., Particle Bombardment Technology for Gene Transfer, Oxford Press, Oxford, England (1994). Non-biological particles (microprojectiles) that can be coated with nucleic acids can be delivered by propulsion to, for example, cells of an immature embryo. Exemplary particles include those made of tungsten, gold, platinum, etc.

[0211] Alternatively, plastids can be stably transformed. Disclosed methods for plastid transformation in higher plants include particle gun delivery of DNA containing a selection marker and targeting of DNA to the plastid genome by homologous recombination (US5,451,513, US5,545,818, US5,877,402, US5,932479, and WO99 / 05265). Other cell transformation methods may also be used, including, but are not limited to, the introduction of DNA into plants by direct DNA transfer into pollen, direct injection of DNA into the reproductive organs of plants, or rehydration of a dried embryo after direct injection of DNA into cells of an immature embryo.

[0212] The regeneration, development, and cultivation of plants from single plant protoplast transformants or from various transformed explants are well known in the art (Weissbach et al., In: Methods for Plant Molecular Biology, Academic Press, San Diego, Calif., (1988)). This regeneration and growth process typically involves a step of selecting transformed cells, which are then cultured from the normal embryonic stage to the rooted seedling 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.

[0213] The development or regeneration of plants containing foreign exogenous genes is well known in the art. Preferably, the regenerated plants self-pollinate to provide homozygous transgenic plants. Otherwise, pollen obtained from the regenerated plants is crossed with seed-cultivated plants of agriculturally important strains. Conversely, pollen from these important strains is used to pollinate the regenerated plants. The transgenic plants of the present invention containing the desired polynucleotides are cultivated using methods well known to those skilled in the art.

[0214] To confirm the presence of the transgene in transgenic cells and plants, polymerase chain reaction (PCR) amplification or Southern blotting analysis can be performed using methods known to those skilled in the art. The expression product of the transgene can be detected by any of a variety of methods, depending on the nature of the product, including Northern blotting hybridization, Western blotting, and enzyme assays. Once transgenic plants are obtained, they can be grown to produce plant tissue or parts having the desired phenotype. The plant tissue or parts can be harvested, and / or seeds can be recovered. The seeds can serve as a source for growing further plants with tissue or parts having the desired characteristics. Preferably, vegetative plant parts are harvested when the yield of nonpolar lipids is highest. In one embodiment, vegetative plant parts are harvested around the time of flowering or after the start of flowering. Preferably, plant parts are harvested when senescence begins (usually indicated by yellowing and drying of the leaves).

[0215] Transgenic plants formed using Agrobacterium or other transformation methods typically contain a single locus on one chromosome. Such transgenic plants are sometimes referred to as hemizygous for the added gene(s). More preferable are transgenic plants that are homozygous for the added gene(s), i.e., transgenic plants containing two added genes (one gene at the same locus on each chromosome in a chromosome pair). Homozygous transgenic plants can be obtained by self-fertilizing a hemizygous transgenic plant, germinating some of the resulting seeds, and analyzing the resulting plants for the gene of interest.

[0216] It is also understood that two different transgenic plants containing two independently separated exogenous genes or loci can be crossed (mating) to produce offspring containing both sets of genes or loci. Appropriate self-pollination of F1 offspring can produce plants that are homozygous for both exogenous genes or loci. Backcrossing to parent plants and outcrossing with non-transgenic plants are also intended, as are vegetative propagation. Similarly, transgenic plants can be crossed with second plants containing genetic modifications, such as offspring containing both mutant genes and identified transgenes and genetic modifications. Descriptions of various traits and other crossing methods commonly used for crops can be found in Fehr, In: Breeding Methods for Cultivar Development, Wilcox J. ed., American Society of Agronomy, Madison Wis. (1987).

[0217] formulation The RNA molecules described herein can be provided in various formulations. For example, the RNA molecules may be in the form of a solid, ointment, gel, cream, powder, paste, suspension, colloid, foam, or aerosol. The solid form may include powders, dispersible ("wettable"), granules, pellets, pills, pastels, tablets, filling films (including seed coatings), etc. In one example, the composition is in the form of a concentrate.

[0218] In one example, the RNA molecule may be provided as a topical formulation. In another example, the formulation stabilizes the RNA molecule in and / or in vivo. For example, the RNA molecule may be provided as a lipid formulation. For example, the RNA molecule may be provided as a liposome. In another example, the formulation includes a transfection enhancer.

[0219] In one example, the RNA molecule can be incorporated into a formulation suitable for application to a field. In one example, the field includes plants. Suitable plants include crops (e.g., grains and legumes, corn, wheat, potato, tapioca, rice, sorghum, millet, cassava, barley, or fava bean), or leguminous plants. The plants can be cultivated for the production of edible roots, tubers, leaves, stems, flowers or fruits. In one example, the crop is a cereal plant. Examples of cereal plants include, but are not limited to, wheat, barley, sorghum oats, and rye. In these examples, the RNA molecule can be formulated for administration to a plant or any part of a plant by any suitable method. For example, the composition can be formulated for administration to the leaves, stems, roots, fruit vegetables, grains and / or legumes of a plant. In one instance, the RNA molecule is formulated for administration to the leaves of a plant and is sprayable on the leaves of the plant.

[0220] Depending on the desired formulation, the RNA molecules described herein can be formulated using a variety of other agents. Exemplary agents include one or more of a suspending agent, flocculant, base, buffer, bittering agent, flavorant, preservative, propellant, thixotropic agent, cryoprotectant, and colorant.

[0221] In other examples, the RNA molecule formulation can include an insecticide, pesticide, fungicide, antibiotic, repellent, antiparasitic agent, antiviral agent, or nematicide.

[0222] In another example, the RNA molecule can be incorporated into a pharmaceutical composition. Such a composition typically includes the RNA molecule described herein and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" includes solvents, dispersion media, coating agents, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into the composition.

[0223] Pharmaceutical compositions are formulated to suit their intended route of administration. Examples of routes of administration include parenteral administration, such as intravenous, intradermal, subcutaneous, inhalation, transdermal (topical), transmucosal, oral, and rectal administration.

[0224] In one embodiment, the active compound is prepared with a carrier that protects the compound from rapid elimination from the body, such as a controlled-release formulation including implants and microencapsulation delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. For example, liposome suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared, for example, according to methods known to those skilled in the art as described in US4,522,811.

[0225] The RNA molecules disclosed herein may be provided in kits or packs. For example, the RNA molecules disclosed herein may be packaged in a suitable container with written instructions for producing the cells or organisms or treating the conditions described above.

[0226] Methods for controlling non-human organisms In one example, the RNA molecules of this disclosure can be used to control non-human organisms such as insects. Such use includes administering the RNA molecules of this disclosure by various means. In one example, the RNA molecules of this disclosure may be provided as insect feed for ingestion by insects. In another example, the RNA molecules may be sprayed onto insects as needed. In yet another example, the RNA molecules may be sprayed onto plants or crops to protect them from insects. Exemplary crops include cotton, corn, tomato, chickpea, pigeon pea, alfalfa, rice, sorghum, and cowpea.

[0227] In one example, RNA molecules can be provided to alter the behavior of insects. In another example, RNA molecules can be provided to kill insects. In yet another example, RNA molecules can be provided to reduce the reproductive capacity of insects. 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 fabae). Further exemplary insect targets include spiders, mosquitoes, ectoparasites, flies, spider mites, thrips, ticks, mites, ants, cockroaches, termites, crickets including house crickets, silverfish, booklice, beetles, earwigs, mosquitoes, and fleas. Other exemplary insect targets include agricultural pests. Examples include sap-feeding organisms such as stink bugs and aphids, chewing insects such as caterpillars, rhinoceros beetles, and grubs, rasping insects such as thrips and slugs, flies such as moths and fruit flies, and grain pests such as longhorn beetles, weevils, and gypsy moths.

[0228] In one embodiment, the insect is a sap-sucking insect. In this example, the RNA molecule may have antisense activity against MpC002 and / or MpRack-1. In one embodiment, the sap-sucking insect is an aphid. In another embodiment, the aphid is Myzus persicae.

[0229] In one embodiment, the insect target is an ant (e.g., Linepithema humile), a cotton bollworm or corn ear worm (Helicoverpa armigera), or a blowfly (e.g., Lucilia cuprina). In one embodiment, the target insect is Helicoverpa armigera, and the RNA molecule has antisense activity against the ABC transporter white gene (ABC white). In another embodiment, the target insect is Linepithema humile, and the RNA molecule has antisense activity against the pheromone biosynthesis-activating neuropeptide (PBAN). In yet another embodiment, the target insect is Lucilia cuprina, and the RNA molecule has antisense activity against 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 γ-tubulin 1 / 1-like proteins.

[0230] In the embodiments described above, the compositions and RNA molecules disclosed herein may be provided in a dispenser. In one example, the dispenser is a trap or lure. In one embodiment, the trap and / or lure contains bait comprising the RNA molecule(s) disclosed herein.

[0231] In one embodiment, the disclosure encompasses a method for controlling insect behavior, which includes applying RNA molecules disclosed herein to insects by spraying, scattering, or other means. In this embodiment, the RNA molecules can be applied directly to insects by spraying, scattering, or other means. In another embodiment, the RNA molecules can be applied to plants or crops by spraying, scattering, or other means before insects emerge.

[0232] In one embodiment of the present invention, the insect or spider may belong to the following orders: Acari, Arachnida, Anoplura, Blattodea, Coleoptera, Collembola, Dermaptera, Dictyoptera, Diplura, Diptera, Embioptera, Ephemeroptera, Grylloblatodea, Hem iptera, Heteroptera, Homoptera, Hymenoptera, Isoptera, Lepidoptera, Mallophaga, Mecoptera, Neurooptera, Odonata, Orthoptera, Phasmida, Phithiraptera, Plecoptera, Protura, Psocoptera, Siphonaptera, Siphunculata, Thysanura, Sternorrhyncha, Strepsiptera, Thysanoptera, Trichoptera, Zoraptera, and Zygentoma.

[0233] In preferred but non-limiting embodiments of the present invention, insects or spiders are selected from the group consisting of: (1) Acari: Mites including Ixodida (ticks); (2) Arachnida: Spiders including Araneae and Opiliones (blind spiders), such as Latrodectus mactans (black widow spider) and Loxosceles recluse (web spider); (3) Anoplura: Louses such as Pediculus humanus (human louse); (4) Blattodea: Cockroaches such as Blatella germanica, Periplaneta americana and Periplaneta australiasiae of the genus Periplaneta, Blatta orientalis of the genus Blatta, and Supella longipalpa of the genus Supella. The most preferred target is the German cockroach (Blatella germanica).(5) Coleoptera: Beetles, for example, Bostrichoidea family; Dendroctonus species (black turpentine beetle, southern pine beetle, IPS enclave beetle); carpet beetles (Anthrenus species, Attagenus species); Old House Borer (Cerambycidae family: Hylotrupes bajulus); Anobium punctatum; Tribolium species (false flour beetle); Trogoderma granarium (red carpet beetle); Oryzaephilus (6) Dermaptera: Dermaptera (Elymidae) (7) Diptera: Mosquitoes (Culicidae) and flies (Brachycera), e.g., Anophelinae, e.g., Anopheles species and Culicinae, e.g., Aedes fulvus; Tabandae, e.g., Tabanus punctifer (horse horsefly), Glossina morsitans morsitans (tsetse fly), drain flies (Psychodidae) and Calyptratae, e.g., Musca domestica (house fly), flesh flies (Sarcophagidae), etc. (8) Heteroptera: Insects, such as Cimex lectularius (bed bug) (9) Hymenoptera: Bees (Apocrita), including ants (Formicoidea), honeybees (Apoidea): Solenopsis invicta (fire ant), Monomorium pharaonis (house ant), Camponotus species (carpenter ant), Iasius niger (small black ant), tetramorium caespitum (brown ant), Myrmica rubra (red ant), Formica species (formia ant), Crematogaster lineolata (acrobatic ant), Iridomyrmex humilis (Argentine ant), Pheidole species (soldier ant), Dasymutilla occidentalis (velvet ant), etc.(10) Isoptera: Termites, e.g., Amitermes floridensis (Florida darkwing termite), Reticulitermes flavipes (eastern termite), R. hesperus (Western termite), Coptotermes formosanus (Formosan termite), Incisitermes minor (American drywood termite), Neotermes connexus (Forest tree termite), and Termitidae (11) Lepidoptera: Moths, e.g., Tineidae & Oecophoridae, e.g., Tineola bisselliella (wood moth), and Pyralidae, e.g., Pyralis farinalis (oak leaf moth), etc. (12) Psocoptera: Flat booklice (Psocids) (13) Siphonaptera: fleas, e.g., Pulex irritans(14)Sternorrhyncha: Aphids (Aphididae)(15)Zygentoma: Silverfish, e.g., Thermobia domestica and Lepisma saccharina.

[0234] Other target insects or spiders include household insects, exoparasites, and insects and / or spiders related to public health and sanitation, and include, but are not limited to, flies, spider mites, thrips, ticks, mites, ants (e.g., by targeting PBAN), cockroaches, termites, crickets including house crickets, silverfish, booklice, beetles, earwigs, mosquitoes, and fleas. More preferred targets include cockroaches (Blattodea), for example, but not limited to, Blatella species (e.g., Blatella germanica (German cockroach)), Periplaneta species (e.g., Periplaneta americana (American cockroach) and Periplaneta australiasiae (Small-banded cockroach)), Blatta species (e.g., Blatta orientalis (Eastern cockroach)), and Supella species (e.g., Supella longipalpa (Brown-banded cockroach)); ants (Formicoidea), for example, but not limited to, Solenopsis species (e.g., Solenopsis invicta (Fire ant)), Monomorium species (e.g., Monomorium pharaonis (House ant)), Camponotus species (e.g., Camponotus (Giant ant)), and lasius species (e.g., lasius niger (small black ant), Tetramorium species (e.g., Tetramorium caespitum), Myrmica species (e.g., Myrmica rubra), Formica species (formia ant), Crematogaster species (e.g., Crematogaster lineolata), Iridomyrmex species (e.g., Iridomyrmex humilis), Pheidole species (soldier ant), and Dasymutilla species (e.g., Dasymutilla occidentalis);Termites (Isoptera and / or Termitidae), for example, but not limited to, the Amitermes species (e.g., Amitermes floridensis (Florida darkwing termite)), Reticulitermes species (e.g., Reticulitermes flavipes (Eastern termite), Reticulitermes hesperus (Western termite)), Coptotermes species (e.g., Coptotermes formosanus (Formosan termite)), Incisitermes species (e.g., Incisitermes minor (American drywood termite)), and Neotermes species (e.g., Neotermes connexus (Forest tree termite)).

[0235] In one embodiment, the target RNA encodes insect acetolactate synthase.

[0236] When delivered and / or expressed in plants, the RNA molecules of the present invention can have a wide range of desired properties that affect, for example, agricultural traits, insect resistance (by targeting genes such as MpC002, MpRack-1, and ABC transporter genes), disease resistance (by targeting genes such as LanR), herbicide resistance, sterility, and grain characteristics. Target RNA molecules may be involved in the metabolism of oils, starches, carbohydrates, nutrients, etc., or in the synthesis of proteins, peptides, fatty acids, lipids, recombination frequency (by targeting genes such as DDM1 and FANCM), waxes, oils (by targeting genes such as TOR), starches, sugars, carbohydrates, aromas, 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.

[0237] In certain cases, plants produce enzymes for oil production in plants such as Brassica, e.g., rapeseed or sunflower, safflower, flax, cotton, soybean, or maize; enzymes involved in starch synthesis in plants such as potatoes, maize, and cereals, e.g., wheat, barley, or rice; enzymes that synthesize natural drugs, e.g., pharmaceutical or veterinary products; or increased levels of proteins that are themselves.

[0238] In another embodiment, the RNA molecule of the present invention is found in the following species: Altemaria; Armillaria mellae; Arthrobotrys oligosporus; Blumeria graminis (by targeting the Mlo gene using the RNA molecule described in Example 17), Boletus granulatus; Botritis cinerea; Botrytis fabae; Candida albicans; Claviceps purpurea; Cronarium ribicola; Epicoccum purpurescens; Epidermophyton floccosum; Fomes annosus; Fusarium oxysporum; Gaeumannomyces graminis var. tritici; Glomerella cingulata; Gymnosporangium juniperi-virginianae; Microsporum canis; Monilinia fructicola; Physoderma alfalfae; Phytopthera infestans; Pityrosporum orbiculare (Malassezia furfur); Polyporus This relates to preventive or therapeutic measures for infections caused by fungal pathogens selected from the group consisting of *Sulphureus*, *Puccinia* species, *Saccharomyces cerevisiae*, *Septoria apiicola*, *Trichophyton rubrum*, *T. mentagrophytes*, *Ustilago* species, *Venturia inaequalis*, and *Verticillium dahliae*.

[0239] Examples of conditions requiring treatment The RNA molecules disclosed herein can be used in a variety of ways. In some examples, this disclosure relates to methods for treating cancer, which involve administering the RNA molecules disclosed herein. The term “cancer” refers to or describes a mammalian physiological condition typically characterized by uncontrolled cell growth / proliferation. Examples of cancer include, but are not limited to, carcinomas, lymphomas, blastomas, sarcomas, and leukemias or lymphoid tumors. 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 including gastrointestinal cancer and gastrointestinal stromal carcinoma, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urinary tract cancer, hepatocellular carcinoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, melanoma, superficial spreading melanoma, lentiginous malignant melanoma, acral lentiginous melanoma, and nodules. Examples include melanoma, multiple myeloma, and B-cell lymphoma (including low-grade / follicular non-Hodgkin lymphoma (NHL), mantle cell lymphoma, AIDS-related lymphoma; and Waldenström macroglobulinemia), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic myeloblastic leukemia, and post-transplant lymphoproliferative disorders (PTLD), as well as abnormal angiogenesis, edema (such as that associated with brain tumors), Meggs syndrome, brain and head and neck cancers, and associated metastases. Therefore, in examples, this disclosure relates to methods for treating breast cancer, ovarian cancer, colon cancer, prostate cancer, lung cancer, brain cancer, skin cancer, liver cancer, gastric cancer, pancreatic cancer, or hematological cancers.

[0240] In other examples, 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 examples, the methods described herein are used to treat cancers associated with mutations in genes related to DNA mismatch repair, such as MSH2, MLH1, PMS2, and related genes. In other examples, the methods described herein are used to treat cancers with silencing DNA repair genes such as BRCA1, MLH1, or RAD51B, RAD51C, or RAD51D.

[0241] In other examples of this disclosure, the methods described herein are used to kill cells with damaged 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 Examples include CPIK1, CHK2, FANCA, FANCB, FANCC, FANCD1, FANCD2, FANCE, FANCF, FANCG, FANCC, FANCD1, FANCD2, FANCE, FANCF, FANCG, RAD1, and RAD9. For example, the methods described herein are used to kill cells having mutant tumor suppressor genes. For instance, cells may have one or more mutations in BRCA1 or BRCA2.

[0242] In other examples of this disclosure, the methods described herein are used to treat cells transformed with a virus. In other examples of this disclosure, the methods described herein are used to kill cells transformed with a latent virus. Examples of latent viruses include CMV, EBV, herpes simplex virus (types 1 and 2), and varicella-zoster virus. In other examples of this disclosure, the methods described herein are used to treat active viral infections caused by viruses that cause cancer, immunodeficiency, hepatitis, encephalitis, pneumonia, or respiratory disease. Exemplary viruses include parvovirus, poxvirus, and herpesvirus.

[0243] In other examples of the disclosure, the methods described herein are used to treat Zika virus, Colorado tick fever (caused by cortivirus, an RNA virus), West Nile fever (encephalitis, caused by a flavivirus that occurs mainly in the Middle East and Africa), yellow fever, rabies (caused by a number of different strains of neurotropic viruses of the Rhabdoviridae family), viral hepatitis, gastroenteritis (virus) - acute viral gastroenteritis caused by Norwalk and Norwalk-like viruses, rotavirus, calicivirus, and astrovirus, polio, influenza (influenza) caused by orthomyxoviruses capable of frequent antigenic mutation, measles (measles), paramyxoviridae, mumps, respiratory syndromes including viral pneumonia, acute respiratory syndromes including acute obstructive laryngitis caused by various viruses collectively referred to as acute respiratory viruses, and respiratory diseases caused by respiratory syncytial viruses. [Examples]

[0244] Example 1. Materials and Method Synthesis of genetic constructs To design a typical ledRNA construct, a region of target RNA approximately 100–1000 nucleotides long, typically 400–600 nucleotides, was identified. In one example, the 5' half of the sequence and a 130 nt adjacent region, as well as the 3' half and a 130 nt adjacent region, were oriented in the antisense direction relative to the promoter. These sequences were interrupted by a 400–600 nucleotide sense target sequence (Figure 1A). The resulting construct is modified to have a promoter, such as a T7 or SP6 RNA polymerase promoter, before the 5' end, and a restriction enzyme cleavage site at the 3' end, allowing for transcription termination in vitro.

[0245] For transcription in cells such as bacterial cells, promoter and terminator sequences were incorporated to promote expression as a transgene, for example, using an inducible promoter. The lengths of the double-stranded regions and loop sequences can be varied. Constructs were created using standard cloning methods or commissioned from a commercial service provider.

[0246] RNA synthesis After digestion with restriction enzymes to linearize the DNA at the 3' end, the 5' and 3' arms of the ledRNAi transcript were annealed to a central target sequence, which is a molecule containing a central stem or double-stranded region with a single nick and terminal loop, by transcription using RNA polymerase. The central sequence can be oriented in the sense or antisense direction relative to the promoter (Figures 1A and 1B, respectively).

[0247] In in vitro synthesis, construct DNA was digested at the 3' restriction site using appropriate restriction enzymes, followed by precipitation, purification, and quantification. RNA synthesis was achieved using RNA polymerase according to the manufacturer's instructions. The derived RNA was resuspended in annealing buffer (25 mM Tris-HCl, pH 8.0, 10 mM MgCl2) using DEPC-treated water to inactivate trace amounts of RNAse. The yield and integrity of the RNA produced by this method were determined by nanodrop analysis and gel electrophoresis, respectively (Figure 2).

[0248] LedRNA synthesis was achieved in bacterial cells by introducing the construct into E. coli strain HT115. The transformed cell culture was induced with IPTG (0.4 mM) to express T7 RNA polymerase, resulting in transcription of the ledRNA construct. RNA extraction and purification from bacterial cells were performed essentially as described in Timmons et al. (2001).

[0249] For RNA transcription using Cy3 labeling, the ribonucleotide (rNTP) mix contained 10 mM ATP, GTP, CTP, 1.625 mM UTP, and 8.74 mM Cy3-UTP, respectively. The transcription reaction was incubated at 37°C for 2.5 hours. 160 μl of the transcription reaction was transferred to an Eppendorf tube, 17.7 μl of turbo DNase buffer and 1 μl of turbo DNA were added, and the DNA was digested by incubation at 37°C for 10 minutes. Next, 17.7 μl of Turbo DNase inactivation solution was added, mixed, and incubated at room temperature for 5 minutes. The mixture was centrifuged for 2 minutes, and the supernatant was transferred to a new RNAse-free Eppendorf tube. 1.5 μl samples of each transcription reaction were subjected to electrophoresis on a gel to test the quality of the RNA product. Generally, depending on the construct, a single RNA band with a size of 500 bp to 1000 bp was observed. RNA was precipitated by adding 88.5 μl of 7.5 M ammonium acetate and 665 μl of cold 100% ethanol to each tube. The tubes were cooled to -20°C for several hours or overnight, then centrifuged at 4°C for 30 minutes. The supernatant was carefully removed, and the RNA pellet was washed with 1 ml of 70% ethanol (prepared with nuclease-free water) at -20°C and centrifuged. The pellet was dried, and the purified RNA was resuspended in 50 μl of 1x RNAi annealing buffer. RNA concentration was measured using the nanodrop method and stored at -80°C until use.

[0250] Example 2. Design of ledRNA As schematically shown in Figure 1A, a typical ledRNA molecule includes a sense sequence that can be considered as two adjacent sense sequences covalently linked and identical to the target RNA, an antisense sequence complementary to the sense sequence and divided into two regions, and two loops that separate the sense from the antisense sequence. Thus, the DNA construct encoding this form of ledRNA includes, in 5' to 3' order, 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, then a second antisense region complementary to the region toward the 3' end of the target RNA, and finally, means for terminating transcription. In this arrangement, the two antisense sequences are adjacent to the sense sequence and the loop sequence. Upon transcription, the two regions of the antisense sequence anneal with the sense sequence to form a dsRNA stem with two adjacent loops.

[0251] In a morphology distinct from, but related to, ledRNA, the sense sequence is divided into two regions, while the two antisense regions remain as a single sequence (Figure 1B). Therefore, the DNA construct encoding this second morphology of ledRNA includes, in 5' to 3' order, a promoter for transcription of the ledRNA coding region, a first sense region identical to the region oriented toward 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 region oriented toward the 5' end of the target RNA, and finally, means for terminating transcription. In this arrangement, the two sense sequences are adjacent to the antisense sequence and the loop sequence.

[0252] While we do not wish to be limited by theory, because these ledRNA structures have closed loops at both ends, they are formed between single-stranded sense RNA and antisense RNA, and are more resistant to exonucleases than open-ended dsRNAs without loops, and similarly compared to hairpin RNAs with only a single loop. In addition, we hypothesize that the loops at both ends of the dsRNA stem allow Dicer to efficiently access both ends, thereby increasing the efficiency of dsRNA processing and silencing of sRNA.

[0253] As a first example, genetic constructs for in vitro transcription were created using T7 or SP6 RNA polymerase to form a ledRNA target gene encoding GFP or GUS. The ledGFP construct contained the following regions in the following 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 GFP CDS, the sense sequence corresponding to nucleotides 131-591 of the GFP CDS, the second antisense loop corresponding to nucleotides 731-592 of the GFP CDS, and the second half of the antisense sequence corresponding to nucleotides 591-359 of the GFP CDS.

[0254] The ledGUS construct contained the following regions in the following order: the first half of the antisense sequence corresponding to nucleotides 609-357 of GUS CDS (SEQ ID NO: 8), the first antisense loop corresponding to nucleotides 356-197 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, and the second half of the antisense sequence corresponding to nucleotides 861-610 of GUS CDS.

[0255] To create separate-strand sense / antisense GUS dsRNAs (conventional dsRNAs), the same target sequences corresponding to nucleotides 357–860 of GUS CDS were ligated between T7 and the SP6 promoter of the pGEM-T Easy vector. The sense and antisense strands were transcribed separately using T7 or SP6 polymerase, respectively. The transcripts were mixed, the mixture was heated to denature the RNA strands, and then annealed with annealing buffer.

[0256] Example 3. Stability of ledRNA The ability of ledRNA to form dsRNA structures was compared with open-ended dsRNA (i.e., without loops and formed by annealing of separate single-stranded sense and antisense RNAs) and long hpRNA. LedRNA, long hpRNA, and mixtures of sense and antisense RNA were denatured by boiling, annealed in annealing buffer (250 mM Tris-HCl, pH 8.0, and 100 mM MgCl2), and then subjected to electrophoresis on 1.0% agarose gels under non-denaturing conditions.

[0257] As shown in Figure 2, both GUS-ledRNA and GFP-ledRNA yielded dominant RNA bands with the 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 showed only weak bands for dsRNA, indicating that most of the sense and antisense RNAs did not immediately anneal to each other to form dsRNA. The hairpin RNA sample showed two prominent bands, indicating that only a portion of the transcript formed the predicted-led

[0258] The ability of ledRNA to remain on the leaf surface and spread was also compared with that of dsRNA. When GUS ledRNA (ledGUS) was applied to the lower part of the surface of tobacco leaves, it was easily detected in the upper part of untreated leaves after 24 hours (Figure 3). However, another strand of GUS dsRNA (dsGUS) was not detected in the upper part of untreated leaves (Figure 3). This result indicates that ledRNA is more resistant to degradation than dsRNA and therefore can spread within the plant leaf tissue.

[0259] Example 4. Testing of ledRNA by local delivery The ability of ledRNAs 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 constructs encoding the ledRNAs are shown in SEQ ID NOs: 7, 8, 4, and 5, respectively. The ribonucleotide sequences of the encoded RNA molecules are provided as SEQ ID NOs: 1 (GFP ledRNA) and 2 (GUS ledRNA).

[0260] To facilitate reproducible and uniform application of ledRNA to the leaf surface, ledRNA at concentrations 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 adaxial surface of leaves using a soft paintbrush. Leaf samples were collected 6 hours and 3 days after ledRNA application for analysis of target gene silencing.

[0261] Application of ledRNA to GFP in N. benthamiana leaves and to GUS in N. tabacum leaves resulted in a clear decrease of 20–40% and 40–50% in mRNA (GFP) or protein activity (GUS) levels of each target gene 6 hours post-treatment. However, in this experiment, the decrease did not persist to day 3 post-treatment. The inventors considered that the observation at day 3 was likely due to some nonspecific response of the transgene to the dissipation of dsRNA treatment or ledRNA. However, in another experiment, GUS silencing was detected 24 hours after ledRNA treatment in both the treated leaf region and the distal untreated leaf region (Figure 4).

[0262] Example 5. LedRNA-induced silencing of endogenous target genes In a further example, the ledRNA was designed to target the mRNA encoded by the endogenous gene, namely the FAD2.1 gene of N. benthamiana. The sequence of the target FAD2.1 mRNA and the sequence of the construct encoding ledFAD2.1 are shown in SEQ ID NOs. 9 and 6, respectively. The ribonucleotide sequence of the encoded RNA molecule is provided as SEQ ID NO: 3 (N. benthamiana FAD2.1 ledRNA).

[0263] The FAD2.1 ledRNA construct consisted of the first half of the antisense sequence corresponding to nucleotides 678-379 of FAD2.1 CDS (Niben101Scf09417g01008.1), a first antisense loop corresponding to nucleotides 378-242nt of FAD2.1 CDS, a corresponding sense sequence from 379-979nt, a second antisense loop corresponding to nucleotides 1115-980nt, and the latter half of the antisense sequence corresponding to nucleotides 979-679nt of FAD2.1 CDS.

[0264] The ledGUS RNA from the aforementioned example was used in parallel as a negative control. In the initial experiment, target gene silencing was assayed for both FAD2.1 mRNA levels and C18:1 fatty acid accumulation levels (Figure 5). The activity level of the related gene, FAD2.2, was also assayed. For each sample, approximately 3 μg of total RNA was treated with DNase and reverse transcribed at 50°C for 50 minutes using oligo dT primers. The reaction was stopped at 85°C for 5 minutes and diluted to 120 μl with water. Using a rotor gene PCR instrument, the relative expression of FAD2.1 and FAD2.2 mRNA was analyzed three times for each 5 μl sample, referencing the housekeeping gene actin, using gene-specific primers. In subsequent experiments, Northern blot hybridization was used to confirm silencing of the FAD2.1 gene by locally applied ledFAD2.1 RNA (Figure 6).

[0265] FAD2.1 mRNA levels were significantly reduced, becoming barely detectable in leaf tissue treated with ledRNA at 2, 4, and 10 hours (Figure 5). The reason for the less pronounced decrease in FAD2.1 mRNA levels at 6 hours remains unclear. Repeated experiments, shown in Figure 6, demonstrated strong FAD2.1 downregulation at both 6 and 24 hours, particularly at 24 hours. The related FAD2.2 gene, which has sequence homology to FAD2.1, also showed downregulation at 2 and 4 hours with ledRNA (Figure 5).

[0266] 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 assayed in leaf tissue treated with ledRNA. At 2, 4, and 6 hours, the accumulation of oleic acid (18:1) was clearly increased in the ledRNA-treated leaf tissue, which indicates a decrease in the amount of FAD2 enzyme (Figure 5). Thus, both qRT-PCR and fatty acid composition assays demonstrated that ledRNA induces silencing of the FAD2.1 gene.

[0267] Example 6.G: Design and testing of hairpin RNA containing U base pairs or mismatched nucleotides Modified hairpin RNA targeting GUS RNA Reporter genes, such as the gene encoding the enzyme β-glucuronidase (GUS), provide a simple and convenient assay system that can be used to measure the efficiency of gene silencing in eukaryotic cells, including plant cells (Jefferson et al., 1987). Therefore, the inventors designed, manufactured, and tested several modified hairpin RNAs for their ability to reduce the expression of the GUS gene as a target gene, using a gene delivery approach to deliver the hairpin RNA to cells, and compared the modified hairpins with conventional hairpin RNA. The conventional hairpin RNA used as a control in the experiments had a double-stranded region with a continuous base pair length of 200, where all base pairs were standard base pairs, i.e., G:C and A:U base pairs without G:U base pairs, and the double-stranded region targeting the 200nt region of the same GUS mRNA molecule as the modified hairpin RNA had no non-base-pairing nucleotides (mismatches). The sense and antisense sequences forming the double-stranded regions were covalently linked by a spacer sequence containing a PDK intron (Helliwell et al., 2005; Smith et al., 2000), and after splicing the intron from the primary transcript, an RNA loop of 39 or 45 nucleotides in length (depending on the cloning strategy used) was obtained. The DNA fragments used for the antisense sequences were flanked by an XhoI-BamHI restriction site at the 5' end and a HindIII-KpnI restriction site at the 3' end for easy cloning into the expression cassette, and each sense sequence was flanked by both the XhoI and KpnI restriction sites. The 200 bp dsRNA region of each hairpin RNA contained a 200 nucleotide antisense sequence that was fully complementary to the wild-type GUS sequence within the protein-coding region in both the control and modified hairpins. This antisense sequence, corresponding to nucleotides 13-212 of SEQ ID NO: 10, was complementary to nucleotides 804-1003 of the GUS open reading frame (ORF) (the cDNA sequence provided as SEQ ID NO: 8). Therefore, the GUS target mRNA was over 1900 nt in length.The 200-nucleotide length of the sense and antisense sequences was chosen as short enough to be reasonably convenient for the synthesis of DNA fragments using synthetic oligonucleotides, yet long enough to provide multiple sRNA molecules upon processing with Dicer. Because it is part of an ORF, this sequence was unlikely to contain a potential splice site or transcription termination site.

[0268] Preparation of genetic constructs A 200 bp 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), each containing XhoI and BamHI sites or HindIII and KpnI sites, respectively. These restriction enzyme sites were then introduced into the 5' and 3' positions of the GUS sequence. The amplified fragments were inserted into the vector pGEM-T Easy, and the correct nucleotide sequence was confirmed by sequencing. The GUS fragment was excised by digestion with BamHI and HindIII and inserted into the BamHI / HindIII site of pKannibal (Helliwell and Waterhouse, 2005), which is an antisense insertion of the GUS sequence into the operably linked CaMV e35S promoter (Grave, 1992) and the ocs gene polyadenylation / transcription terminator (Ocs-T). The resulting vector, named pMBW606, contained a 35S::PDK intron::antisense GUS::Ocs-T expression cassette in the order of 5' to 3'. This vector was an intermediate vector used as the base vector for assembling four hpRNA constructs.

[0269] hpGUS[wt] constructs containing only standard base pairs To prepare a vector called hpGUS[wt] encoding a standard base-paired hairpin RNA molecule to be used as a control in experiments, a 200 bp GUS PCR fragment was excised from a pGEM-T Easy plasmid using XhoI and KpnI and inserted into the XhoI / KpnI site between the 35S promoter and PDK intron of pMBW606. This generated a vector called pMBW607 containing a 35S::sense GUS[wt]::PDK intron::antisense GUS::OCS-T expression cassette. This cassette was excised by digestion with NotI and inserted into the NotI site of pART27 (Gleave, 1992) to create the vector called hpGUS[wt], encoding a standard base-pairing hairpin RNA targeting GUS mRNA.

[0270] Upon self-annealing by hybridization of a 200 nt sense and antisense sequence, this hairpin yielded a double-stranded region of 200 consecutive base pairs corresponding to the GUS sequence. The sense and antisense sequences of the expression cassette are adjacent to the BamHI and HindIII restriction sites located at the 5' and 3' ends, respectively, compared to the GUS sense sequence. Upon transcription, the nucleotides corresponding to these sites could also hybridize, extending the double-stranded region by 6 bp at both ends. After transcription of the expression cassette and splicing of the PDK intron from the primary transcript, the hairpin RNA structure prior to processing by Dicer or other RNAse was predicted to have a 39-nucleotide loop structure. The nucleotide sequence of the hairpin RNA structure including that loop is provided as SEQ ID NO: 15, and the free energy of folding was predicted to be -471.73 kcal / mol. Therefore, this was an energetically stable hairpin structure. The free energy was calculated using "RNAfold" (http: / / rna.tbi.univie.ac.at / cgi-bin / RNAWebSuite / RNAfold.cgi) based on the nucleotide sequence after splicing from the PDK intron sequence.

[0271] When transcribed from an expression cassette containing a 35S promoter and OCS-T terminator, the resulting hairpin RNA was embedded into a larger RNA molecule with 8 nucleotides added to the 5' end and approximately 178 nucleotides added to the 3' end without a poly(A) tail. Since the same promoter-terminator design was used for the modified hairpin RNA, these molecules also exhibited these elongations at the 5' and 3' ends. Therefore, the length of the hairpin RNA molecule after splicing of the PDH intron was approximately 630 nucleotides.

[0272] hpGUS[G:U] constructs containing G:U base pairs DNA fragments containing the same 200-nucleotide sense sequence, but in which all 52 cytidine nucleotides (C) in the corresponding wild-type GUS region were replaced with thymidine nucleotides (T), were assembled by annealing the duplicate oligonucleotides GUS-GU-F (SEQ ID NO: 54) and GUS-GU-R (SEQ ID NO: 55) and performing 3' end PCR extension using high-fidelity LongAmp Taq polymerase (New England Biolabs, catalog number M0323). The amplified DNA fragments were inserted into a pGEM-T Easy vector, and the correct nucleotide sequence (SEQ ID NO: 11) was confirmed by sequencing. Next, the DNA fragments containing the modified sequence were excised by digestion with XhoI and KpnI and inserted into the XhoI / KpnI site of the base vector pMBW606. This generated a construct called pMBW608, containing the expression cassette 35S::sense GUS[G:U]::PDK intron::antisense GUS::OCS-T. This expression cassette was excised by NotI digestion and inserted into the NotI region of pART27 to create a vector called hpGUS[G:U] that encodes a hairpin RNA molecule with G:U base pairing.

[0273] This cassette encoded a hairpin RNA targeting GUS mRNA. Upon self-annealing by hybridization of a 200nt sense and antisense sequence, it contained 52 G:U base pairs (instead of the G:C base pairs in hpGUS[wt]) and 148 standard base pairs, meaning 26% of the nucleotides in the double-stranded region were G:U base pairs. The 148 standard base pairs of hpGUS[G:U] were identical to those of the control hairpin RNA, containing 49 U:A base pairs, 45 A:U base pairs, and 54 G:C base pairs at their corresponding positions. The longest stretch of consecutive standard base pairs in the double-stranded region was 9 base pairs. Thus, the antisense nucleotide sequence of hpGUS[G:U] was identical in length (200nt) and sequence to the antisense sequence of the control hairpin RNA hpGUS[wt]. Following transcription of the expression cassette and splicing of PDK introns from the primary transcript, the hairpin RNA structure prior to processing by Dicer or other RNAse was predicted to have a 45-nucleotide loop structure. The nucleotide sequence of the hairpin structure containing that loop is provided as SEQ ID NO: 16, and its folding free energy was predicted to be -331.73 kcal / mol. For hpGUS[wt], this was therefore an energetically stable hairpin structure despite the 52 G:U base pairs being individually even weaker than the G:C base pairs of hpGUS[wt].

[0274] Figure 7 shows the alignment between the modified GUS sense sequence (nucleotides 9-208 of SEQ ID NO: 11) and the corresponding region of the GUS target gene (SEQ ID NO: 14).

[0275] hpGUS[1:4] construct containing mismatched nucleotides for each fourth nucleotide DNA fragments containing the same 200bp sense sequence were designed and assembled, but with substitutions at every fourth nucleotide of the corresponding wild-type GUS sequence. Each fourth nucleotide in each 4-nucleotide block (at positions such as 4, 8, 12, 16, and 20) was substituted by changing C to G, G to C, A to T, and T to A, while other nucleotides were left unchanged. All of these substitutions were transversion substitutions, which were expected to have a greater destabilizing effect on the resulting hairpin RNA structure than transition substitutions. The DNA fragments were assembled by annealing the duplicate oligonucleotides GUS-4M-F (SEQ ID NO: 56) and GUS-4M-R (SEQ ID NO: 57) and performing 3' end PCR extension using LongAmp Taq polymerase. The amplified DNA fragments were inserted into a pGEM-T Easy vector, and the correct nucleotide sequence (SEQ ID NO: 12) was confirmed by sequencing. Next, the DNA fragment containing the modified sequence was excised by digestion with XhoI and KpnI and inserted into the XhoI / KpnI region of the base vector pMBW606. This generated a construct called pMBW609, which contained the expression cassette 35S::sense GUS[1:4]::PDK intron::antisense GUS::OCS-T. This expression cassette was excised by NotI digestion and inserted into the NotI region of pART27 to create a vector called hpGUS[1:4] encoding a 1:4 mismatch hairpin RNA molecule.

[0276] This cassette encodes a hairpin RNA targeting GUS mRNA. Upon self-annealing by hybridization of the sense and antisense sequences, there was a 50-nucleotide mismatch in the 200-nt antisense sequence (including a nucleotide mismatch at position 200). The double-stranded region of the hairpin RNA, excluding position 200, had 150 standard base pairs and 49 mismatched nucleotide pairs over the 199-nt sense and antisense sequence length; i.e., 24.6% of the nucleotides in the double-stranded region were predicted to be mismatched (not involving base pairing). After transcription of the expression cassette and splicing of the PDK intron from the primary transcript, the hairpin RNA structure prior to processing by Dicer or other RNAse was predicted to have a 45-nucleotide loop structure. The nucleotide sequence of the hairpin structure including that loop is provided as SEQ ID NO: 17, and its folding free energy was predicted to be -214.05 kcal / mol. For hpGUS[wt], this was therefore an energetically stable hairpin structure despite the mismatched nucleotides.

[0277] Figure 8 shows the alignment between the modified GUS sense sequence (nucleotides 9-208 of SEQ ID NO: 12) and the corresponding region of the GUS target gene (SEQ ID NO: 14).

[0278] hpGUS[2:10] construct in which nucleotides 9 and 10 of the 10 nucleotides are mismatched. DNA fragments containing the same 200bp sense sequence were designed and assembled, but with substitutions at the 9th and 10th nucleotides of the corresponding wild-type GUS sequence. Each 10-nucleotide block's 9th and 10th nucleotides (positions 9, 10, 19, 20, 29, 30, etc.) were substituted by changing C to G, G to C, A to T, and T to A, while other nucleotides remained unchanged. The DNA fragments were assembled by annealing the duplicate oligonucleotides GUS-10M-F (SEQ ID NO: 58) and GUS-10M-R (SEQ ID NO: 59), followed by 3' end PCR extension using LongAmp Taq polymerase. The amplified DNA fragments were inserted into pGEM-T Easy, and the correct nucleotide sequence (SEQ ID NO: 13) was confirmed by sequencing. Next, the DNA fragment containing the modified sequence was excised by digestion with XhoI and KpnI and inserted into the XhoI / KpnI site of the base vector pMBW606. This generated a construct called pMBW610, which contained the expression cassette 35S::senseGUS[2:10]::PDK intron::antisenseGUS::OCS-T. This expression cassette was excised by NotI digestion and inserted into the NotI site of pART27 to create a vector called hpGUS[2:10] encoding a 2:10 mismatched hairpin RNA molecule.

[0279] This cassette encoded a hairpin RNA targeting GUS mRNA. Upon self-annealing by hybridization of sense and antisense sequences, there were 50 nucleotide mismatches in the 200 nt antisense sequence (including nucleotide mismatches at positions 199 and 200). The double-stranded region of the hairpin RNA had 160 standard base pairs and 19 dinucleotide mismatches over the 198 nt sense and antisense sequence length, excluding positions 199 and 200. This predicted that 19.2% of the nucleotides in the double-stranded region were mismatched (not involving base pairing). The 160 base pairs of hpGUS[2:10] were identical to those of the control hairpin RNA, containing 41 U:A base pairs, 34 A:U base pairs, 42 G:C base pairs, and 43 C:G base pairs at their corresponding positions. Following transcription of the expression cassette and splicing of PDK introns from the primary transcript, the hairpin RNA structure prior to processing by Dicer or other RNAse was predicted to have a 45-nucleotide loop structure. The nucleotide sequence of the hairpin structure containing that loop is provided as SEQ ID NO: 18, and its folding free energy was predicted to be -302.78 kcal / mol. For hpGUS[wt], this was therefore an energetically stable hairpin structure despite the bulge from the stem of the hairpin structure and the predicted mismatched nucleotides.

[0280] Figure 9 shows the alignment between the modified GUS sense sequence (nucleotides 9-208 of SEQ ID NO: 13) and the corresponding region of the GUS target gene (SEQ ID NO: 14).

[0281] Four genetic constructs for the expression of control and modified hairpin RNA are schematically shown in Figure 10.

[0282] Example 7. Testing of modified hairpin RNA in transgenic plants The efficacy of the four hairpin RNA constructs described above was tested using Nicotiana tabacum (tobacco) plants transformed with the GUS target gene. Specifically, the target plants were derived from two homozygous, independent transgenic lines, PPGH11 and PPGH24, each containing a single copy insertion of the GUS transgene from the vector pWBPPGH, schematically shown in Figure 11. The GUS gene in the T-DNA of pWBPPGH contained a GUS coding region (nucleotides 7-1812 of SEQ ID NO: 8) operably ligated to the 1.3 kb promoter of the phloem protein 2 (PP2) gene of Cucurbita pepo L. cv. Autumn Gold (Wang et al., 1994; Wang, 1994). The construct pWBPPGH was created by excising the PP2 promoter and 5'UTR and 54 nucleotides of the PP2 protein-coding region from lambda genome clone CPP1.3 encoding the first 18 amino acids of PP2 (Wang, 1994), fusing this fragment with a GUS-coding sequence beginning with a nucleotide encoding the third amino acid of GUS, and generating an N-terminal fusion polypeptide with GUS activity. The pPP2::GUS:Nos-T cassette was inserted into pWBVec2a (Wang et al., 1998) to produce pWBPPGH, which was used to transform Nicotiana tabacum cv.Wisconsin 38 plants using leaf-disk transformation via Agrobacterium tumefaciens, sorted for hygromycin resistance (Ellis et al., 1987). GUS activity was similar in homozygous offspring plants of the two transgenic lines PPGH11 and PPGH24. GUS expression in both transgenic plants was not limited to the phloem but was present in most plant tissues. Therefore, GUS expression from the PP2 promoter in these plants appeared to be constitutive.There are two reasons for selecting PP2-GUS plants as test plants: i) PP2-GUS plants exhibit constitutively high levels of GUS expression, almost identical to 35S-GUS plants; and ii) the PP2 promoter is an endogenous PP2 gene promoter derived from Cucurbita pepo, having a different sequence from the 35S promoter used to drive hpRNA transgene expression, and was not subject to transcriptional corepression by the incoming 35S promoter.

[0283] Using the Agrobacterium-mediated leaf disk method (Ellis et al., 1987), PPGH11 and PPGH24 plants were transformed using all four hairpin RNA constructs (Example 6) with 50 mg / L kanamycin as the selective agent. This selective system, using kanamycin, a different agent from hygromycin previously used to introduce pWBPPGH T-DNA, was observed to produce only transformed plants, with no non-transformed plants being regenerated. The regenerated transgenic plants containing hpGUS construct-derived T-DNA were transferred to soil for greenhouse growth and maintained for approximately 4 weeks before assaying GUS activity. Upon assay, the transgenic plants were healthy and vigorously growing, and their appearance was identical to untransformed control plants and parental PPGH11 and PPGH24 plants. In total, 59 transgenic plants were obtained transformed with T-DNA encoding hpGUS[wt], 74 plants were obtained transformed with T-DNA encoding hpGUS[G:U], 33 plants were obtained transformed with T-DNA encoding hpGUS[1:4], and 41 plants were obtained transformed with T-DNA encoding hpGUS[2:10].

[0284] GUS expression levels were measured using a fluorescent 4-methylumbelliferyl β-D-glucuronide (MUG) assay according to a modified kinetic method described in Chen et al. (2005) (Jefferson et al., 1987). Plants were evaluated by taking leaf samples approximately 1 cm in diameter from three different leaves of each plant and selecting well-grown, healthy, green leaves. Care was taken to ensure that test plants were at the same growth and developmental stage as control plants. In each assay, 5 μg of protein extracted from each leaf sample was used, and the cleavage rate of MUG was measured as described in Chen et al. (2005).

[0285] Representative data are shown in Figure 12, illustrating the GUS activity (MUG units in the assay) of each independent transgenic plant. Because the hpGUS[wt] construct data showed that some plants exhibited strong silencing, at least a 90% decrease in activity, and other weaker silencing, in this situation, the plants were classified into two categories, and a 10% GUS activity level compared to the control plant was selected as the activity level for comparing different constructs.

[0286] The genetic construct encoding standard base-pairing hpGUS[wt] induced potent GUS silencing in 32 (54.2%) of the 59 transgenic plants tested, using a 10% activity level as a benchmark for potent silencing. All other 27 plants showed reduced GUS activity, but retained over 10% of the enzyme activity compared to control plants, suggesting weak silencing under these conditions. Transgenic plants with this construct exhibited a wide range of GUS gene silencing (Figure 12), with activity remaining at less than 1% to approximately 80%, typical of conventional hairpin designs (Smith et al., 2000).

[0287] In contrast, the hpGUS[G:U] construct induced consistent and uniform silencing across independent transgenic lines, with 71 out of 74 plants (95.9%) exhibiting potent GUS silencing. Again, in contrast, all 33 hpGUS[1:4] plants tested showed reduced GUS activity levels, with only 8 (24%) exhibiting less than 10% GUS activity compared to control plants, and the other 25 classified as having weak silencing. These results indicate that this construct induced a weaker but more uniform level of GUS downregulation across transgenic lines. The hpGUS[2:10] construct functioned similarly to the hpGUS[wt] construct, inducing good levels of silencing in some lines (28 out of 41, i.e., 68.3%), while the remaining 13 plants exhibited little to no GUS silencing.

[0288] When only silenced lines (with less than 10% remaining activity) were used for comparison and the average GUS activity was calculated, hpGUS[wt] plants showed the highest degree of average silencing, followed by hpGUS[G:U] plants and hpGUS[2:10] plants (Figure 13). hpGUS[1:4] plants showed the smallest average decrease in GUS activity. The degree of GUS silencing showed a good correlation with the thermodynamic stability of the predicted hpRNA structure derived from four different hpRNA constructs (Example 6).

[0289] To test whether the difference persists in offspring plants, representative transgenic plants containing both the homozygous target GUS gene and the hpGUS transgene (hemizytic) were self-fertilized. Since kanamycin-resistant offspring plants were selected from the hpGUS line, null isolates lacking the hpGUS transgene were removed. This ensured that the hpGUS transgene was present in all offspring in either a homozygous or heterozygous state. The offspring plants were assayed for GUS activity, and representative data are shown in Figure 14. Offspring containing the hpGUS[wt] transgene clearly fall into two categories: those exhibiting strong GUS silencing and those showing weak or no silencing. These classifications correlate well with the phenotype of the previous generation, indicating that the degree of target gene silencing is heritable. All plants from the tested hpGUS[G:U] line consistently showed strong silencing, while plants from the hpGUS[1:4] line consistently showed weak silencing. The inventors concluded that the phenotype observed in the parent generation is generally maintained in the offspring plants.

[0290] Southern blot hybridization experiment of transgenic plants The remarkable and unexpected uniformity of potent gene silencing observed in numerous independent transgenic plants generated by the hpGUS[G:U] construct was not only significant but also astonishing. We sought to establish whether any explanation other than the effects induced by hpGUS[G:U]RNA contributed to this uniformity of silencing. To test whether multiple transgenic plants arose from intended 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). In Southern blot hybridization, approximately 10 μg of DNA from each plant sample was digested with HindIII enzyme, separated by gel electrophoresis on a 1% agarose gel in TBE buffer, and blotted onto a Hybond-N+ membrane using the capillary method (Sambrook et al., 1989). The membrane in the OCS-T terminator region 32 The probe was hybridized with a P-labeled DNA fragment overnight at 42°C. This probe was selected because it hybridized to the hpGUS[G:U] transgene but not to GUS target genes that lacked the OCS-T terminator sequence. The membrane was washed with high stringency to retain the probe visualized by PhosphoImager.

[0291] The autoradiographs of the hybridized blots are shown in Figure 15. Each lane showed one to five or six hybridized bands. No two lanes showed the same pattern; that is, the autoradiographs showed that each of the 16 representative hpGUS[G:U] plants had a different pattern of hybridized HindIII fragments and therefore originated from different transgene insertions. The inventors concluded that the uniform GUS silencing observed in hpGUS[G:U] lines was not due to similar transgene insertion patterns in the plants, but rather 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 are not necessary to obtain potent gene silencing, and a single copy of the transgene is sufficient.

[0292] Northern blot hybridization experiment of transformed plants To determine whether hpGUS[G:U]RNA was processed in the same manner as control hairpin RNA in transgenic plants, Northern blot hybridization experiments were performed on RNA isolated from the leaves of transgenic plants. Northern blot experiments were conducted to detect shorter RNAs (sRNA, approximately 21–24 nucleotides in length) resulting from the Dicer processing of hairpin RNA. The experiments were performed on short-chain RNAs isolated from transgenic hpGUS[wt] and hpGUS[G:U] plants, including GUS target genes expressed as (sense) mRNA. Nine plants were selected for sRNA analysis for each construct. In the hpGUS[wt] transgenic population, plants exhibiting both weak and strong GUS silencing were included. Short RNA samples were isolated using the hot phenol method (Wang et al., 2008), and Northern blot hybridization was performed along with gel electrophoresis of the RNA samples under denaturing conditions, according to Wang et al. (2008). The probes used were 32P-labeled RNAs corresponding to either the sense sequence or antisense sequence corresponding to nucleotides 804-1003 of SEQ ID NO: 8.

[0293] Figure 16 shows autoradiographs of Northern blots detecting sense sRNA molecules derived from hairpin RNA by hybridizing with an antisense probe (upper panel), or detecting antisense sRNA by hybridizing with a sense probe (lower panel). At the bottom, a figure shows qualitative scores of GUS expression levels compared to control plants lacking the hpGUS construct. Hybridization to short RNAs of approximately 20-25 nucleotides was observed based on the mobility of sRNA compared to RNA of known length in other experiments. The hpGUS[wt] lines showed varying levels of sRNA accumulation. This was observed for both sense and antisense sRNA, although the antisense sRNA band was not as distinct as the sense band. Since the hpGUS[wt] plants contained both an hpGUS transgene expressing both sense and antisense sequences corresponding to a 200 nt target region, and a GUS target gene expressing a full-length sense gene, the sense sRNA could have been generated from either hairpin RNA or target mRNA. In hpGUS[wt] plants, there appeared to be a negative correlation between sRNA levels and the degree of GUS silencing. For example, the two plants shown in lanes 4 and 5 accumulated relatively large amounts of sRNA but showed only moderate GUS downregulation. In contrast, the two plants shown in lanes 7 and 8 exhibited strong GUS silencing but had relatively low levels of accumulated sRNA.

[0294] In contrast to hpGUS[wt] plants, and consistent with the relatively uniform range of silencing by the hpGUS[G:U] construct, hpGUS[G:U] plants accumulated a uniform amount of antisense sRNA throughout the entire lineage. Furthermore, the degree of GUS silencing appeared to correlate well with the amount of antisense sRNA. Sense sRNA was barely detectable in these plants. This suggested that the RNA probe used in Northern blot hybridization, being transcribed from a wild-type GUS sequence, had a lower level of complementarity to the hpGUS[G:U]-derived sense sRNA, in which all C nucleotides were replaced with U nucleotides, thus allowing only lower stringency hybridization. However, this experiment did not rule out the possibility that hpGUS[G:U] RNA was processed to produce less sense sRNA or was degraded more rapidly.

[0295] Northern blot hybridization experiments were repeated, this time using only sense probes to detect antisense sRNA. The autoradiographs are shown in Figure 17. Again, the generation of antisense sRNA from the hpGUS[wt] construct showed a negative correlation with GUS activity (top panel of Figure 17). Strongly silenced plants produced high levels of antisense sRNA (lanes 1, 3, 5, 8, and 10), while weakly or unsilenced plants did not produce a hybridization signal in this experiment (lanes 2, 4, 6, 7, and 9). In very clear contrast, plants expressing hpGUS[G:U] produced much less but consistent amounts of antisense sRNA. The observation that strongly silenced plants expressing hpGUS[G:U] accumulated significantly lower levels of sRNA than strongly silenced plants expressing hpGUS[wt] was intriguing and suggested to the inventors that hpGUS[wt] is processed by a different mechanism in plants but was as effective as the hpGUS[wt] construct. Further observations in this experiment provided a clue that the two relatively weak antisense bands of the hpGUS[G:U] plants appeared to have the same mobility as the second and fourth bands observed in the antisense sRNA bands of hpGUS[wt]. This was confirmed in further experiments described below. The inventors hypothesized that the four bands of sRNA from hpGUS[wt] represented 24, 22, 21, and 20mer, and that the hpGUS[G:U] RNA was processed to primarily produce 22 and 20mer antisense sRNA.

[0296] A key and clear conclusion from the above data is that the hpGUS[G:U]RNA molecule was processed by one or more Dicer enzymes to produce sRNA, particularly antisense sRNA, which is thought to be a mediator of RNA interference in the presence of various proteins such as Argonaut. The observed production of antisense sRNA also meant that sense sRNA was produced, but the experiments did not distinguish between degradation / instability of sense sRNA or poor detection of sense sRNA due to insufficient hybridization with the probe used. From these experiments, the inventors also concluded that there are clear differences in the processing of hpGUS[wt] and hpGUS[G:U]RNA molecules. This indicates that the molecules were recognized differently by one or more Dicer enzymes.

[0297] Example 8. Analysis of sRNA from transgenic plants expressing modified hairpin RNA. Another Northern blot hybridization experiment was performed to detect antisense sRNA from hpGUS[G:U] plants and compare its size to that of hpGUS[wt]. The autoradiograph is shown in Figure 18. This time, the size difference between the two antisense sRNA bands of hpGUS[G:U] and the two major bands of hpGUS[wt] was more pronounced. This is best seen by comparing the mobility of the bands in adjacent lanes 9 and 10 in Figure 18. This result confirmed that the two hairpin RNAs were processed separately by one or more Dicers in the plant.

[0298] To further investigate this, short-chain RNA populations from hpGUS[wt] and hpGUS[G:U] were analyzed by deep sequencing of the total linker-amplified sRNAs isolated from plants. The frequency of sRNAs mapped to the double-stranded region of hairpin RNA was determined. The length distribution of such sRNAs was also determined. The results showed an increased frequency of 22-mer antisense RNAs from the hpGUS[G:U] construct compared to the hpGUS[wt] construct. The increased proportion of 22nt-length sRNAs indicated a shift in the treatment of the hpGUS[G:U] hairpin with Dicer-2 compared to hpGUS[wt].

[0299] Example 9. DNA methylation analysis of plant transgenes Observations of the variability in the range 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, raised the question for the inventors whether the two plant populations differed in the level of DNA methylation of target GUS genes. Sequence-specific 24-mer sRNA is thought to be involved in promoting DNA methylation of reverse repeat structures in plants (Dong et al., 2011). Therefore, the inventors tested the level of DNA methylation in the 35S promoter region of GUS transgenes, particularly hairpin-encoding genes (silencing genes), in hpGUS plants.

[0300] To do this, we used the DNA methylation-dependent endonuclease McrBC. McrBC is methylcytosine ( m C) A commercially available endonuclease that cleaves DNA containing bases along one or both strands of double-stranded DNA (Stewart et al., 2000). McrBC is 5'(G or A) m C 3', preferably G mIt recognizes a site on DNA composed of two half-sites in the form of C. These half-sites may be hundreds of base pairs apart, but the optimal spacing is 55 to about 100 bp. Double-stranded DNA with such linked G m C dinucleotides on both strands functions as the best substrate. McrBC activity depends on one or both of the methylated GC dinucleotides. Since plant DNA can be methylated at C in the CG, CHG, or CHH sequences (H represents A, C, or T) (Zhang et al., 2018), digestion of DNA using McrBC followed by PCR amplification of gene-specific sequences can be used to detect the presence or absence of m C in specific DNA sequences of the plant genome. In this assay, PCR amplification of methylated McrBC-digested genomic DNA results in a decrease in the amount of amplification product compared to unmethylated DNA, but the same amount of PCR product as untreated DNA is obtained if the DNA is not methylated.

[0301] Genomic DNA was isolated from plants containing the hpGUS[wt], hpGUS[G:U], or hpGUS[1:4] construct, in addition to the target GUS gene, by standard methods (Draper and Scott, 1988). The purified DNA samples were treated with McrBC (catalog number M0272; New England Biolabs, Massachusetts) according to the manufacturer's instructions, including the presence of Mg 2+ ions and GTP. Briefly, approximately 1 μg of genomic DNA was digested with McrBC overnight in a 30 μl reaction volume. The digested DNA samples were diluted to 100 μl and the target region was PCR amplified as follows.

[0302] <000123'3>The processed DNA samples were used in PCR reactions with the following primers: For hpGUS[wt] 35S-GUS junction sequences: Forward primer (35S-F3), 5'-TGGCTCCTACAAATGCCATC-3' (SEQ ID NO: 60); Reverse primer (GUSwt-R2), 5'-CARRAACTRTTCRCCCTTCAC-3' (SEQ ID NO: 61). For hpGUS[G:U] 35S-GUS junction sequences: Forward primer (GUSgu-R2), 5'-CAAAAACTATTCACCCTTCAC-3' (SEQ ID NO: 62), Reverse primer (GUS4m-R2), CACRAARTRTACRCRCTTRAC (SEQ ID NO: 63). For the 35S promoter sequences of both constructs: forward primer (35S-F2), 5'-GAGGATCTAACAGAACTCGC-3' (SEQ ID NO: 64); reverse primer (35S-R1), 5'-CTCTCCAAATGAAATGAACTTCC-3' (SEQ ID NO: 65). In both cases, R=A or G, Y=C or T. The PCR reaction was performed for 35 cycles under the following cycling conditions: annealing at 94°C for 1 minute, 94°C for 30 seconds, 55°C for 45 seconds, extension at 68°C for 1 minute, and final extension at 68°C for 5 minutes. The PCR amplification products were electrophoresed and the intensity of the bands was quantified.

[0303] Representative results are shown in Figures 19 and 20. In the 35S-GUS junction region, which includes 200 bp of the 35S promoter sequence containing the transcription start site, most hpGUS[wt] plants showed significant levels of DNA methylation. Within the hpGUS[wt] plant population, individual plants with high levels of GUS activity, i.e., less silencing, appeared to have greater methylation in the promoter-GUS sense junction region. The results were similar in the 35S promoter region. In contrast, most hpGUS[G:U] and hpGUS[1:4] plants showed weak DNA methylation at the 35S-GUS junction. We hypothesize that this proximal promoter sequence is important for transgene expression, and that methylation in this region is likely to reduce the expression of silencing constructs via transgene transcription silencing (TGS). This is referred to as "self-silencing."

[0304] General Considerations Regarding Examples 6-9 Disrupting the reverse repeat DNA structure in the transgene enhances its stability. Both populations of hpGUS[wt] and hpGUS[2:10] transgenic plants exhibited a broad range of target gene silencing. In contrast, populations including hpGUS[G:U] and hpGUS[1:4] plants both showed relatively uniform GUS silencing in many independent lines, with strong silencing observed in the former construct and relatively weak but significant reduction in gene activity observed in the latter construct. In hairpin RNA from the [G:U] and [1:4] constructs, approximately 25% of the nucleotides in the sense and antisense sequences were involved in a uniformly distributed sequence mismatch across the G:U base pair or the entire 200-nucleotide sense / antisense sequence. Due to the sequence differences between the sense and antisense sequences, the mismatch or reverse repeat structure of the DNA construct between the sense and antisense "arms" was thought to significantly disrupt the reverse repeat DNA structure. Repetitive DNA structures can induce DNA methylation and silencing in various organisms (Hsieh and Fire, 2000). The hpGUS[2:10] construct also contained mismatches between the sense and antisense regions, but each 2bp mismatch between the sense and antisense sequences was flanked by an 8bp consecutive match, suggesting that the mismatches may not disrupt the reverse repeat DNA structure to the same extent as 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 at least partially due to the disruption of the reverse repeat DNA structure, resulting in less methylation and thus reduced self-silencing of the two transgenes. Another advantage of mismatches between the sense and antisense DNA regions is that the cloning of reverse repeats in E. coli has been facilitated because bacteria tend to delete or rearrange complete reverse repeats.

[0305] The thermodynamic stability of hpRNA is important for the degree of target gene silencing. When comparing only strongly silenced transgenic lines, hpGUS[wt] plants exhibited the greatest degree of downregulation of target genes, 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 highest stability, followed by hpGUS[G:U], hpGUS[2:10], and hpGUS[1:4] hairpins. The inventors hypothesized that the more stable the hairpin RNA structure, the greater the degree of target gene silencing it could induce. This was also favorable to longer double-stranded RNA structures than shorter ones. The formation of stable double-stranded RNA was considered necessary for efficient Dicer processing. The experimental results described herein demonstrate another significant advantage of G:U base-pairing constructs over constructs containing nearly simple mismatched nucleotides, such as hpGUS[1:4]: both types of constructs disrupted the reverse repeat DNA structure, reducing self-silencing; however, at the RNA level, hpGUS[G:U]RNA was more stable due to the ability of G and U to base-pair. A combination of two types of modifications is also considered beneficial, containing G:U base pairs and some mismatched nucleotides in the double-stranded RNA structure, but with at least 2, 3, 4, or 5 times more nucleotides associated with G:U base pairs than mismatches.

[0306] hpGUS[G:U]RNA was efficiently processed by Dicer. One of the key questions revealed in these experiments was whether mismatched or G:U base-paired hpRNAs could be processed into short-chain RNA (sRNA) by Dicer. Strong silencing in hpGUS[G:U] plants, as well as in 1:4 and 2:10 mismatched hpRNA plants, indicated that these hairpin RNA structures were processed by Dicer. This was confirmed in the [G:U] molecule by sRNA Northern blot hybridization, which readily detected antisense sRNA. Furthermore, the degree of GUS silencing in hpGUS[G:U] plants showed a good correlation with the amount of accumulated antisense sRNA. Short-chain RNA deep sequencing analysis of two selected lines (only one in hpGUS[wt]) confirmed that hpGUS[G:U] plants, such as hpGUS[wt] plants, produced abundant sRNA, while hpGUS[1:4] plants also produced sRNA, but in much smaller quantities (Figure 21). Low levels of sRNA in hpGUS[1:4] plants were consistent with relatively low GUS silencing efficiency, suggesting that low thermodynamic stability of the dsRNA stem of hpGUS[1:4] RNA reduces Dicer processing efficiency. The degree of GUS silencing correlated relatively poorly with sRNA levels in hpGUS[wt] constructs, and some strongly silenced lines were found to contain relatively small amounts of sRNA. This suggests that GUS silencing in some hpGUS[wt] lines is at least partially due to transcriptional silencing rather than PTGS by sRNA. We have recognized that self-silencing of hairpin-encoding genes, including methylation of gene sequences such as promoter regions, can be mitigated by using modified hairpin RNA constructs, particularly G:U constructs.

[0307] G:U and 1:4 hpRNA transgenes showed reduced DNA methylation in the proximal 35S promoter region. McrBC digestive PCR analysis revealed that the hpGUS[G:U] and hpGUS[1:4] transgenic populations showed reduced DNA methylation levels at the 240 bp 35S sequence near the transcription start site (TSS) compared to the hpGUS[wt] population. This result demonstrated to the inventors that complete disruption of the reverse repeat structure, either by C-to-T modification (in hpGUS[G:U]) or 25% nucleotide mismatch (in hpGUS[1:4]) in the sense sequence, minimizes transcriptional self-silencing of the hpRNA transgene. This was consistent with the uniformity of GUS gene silencing observed in the hpGUS[G:U] and hpGUS[1:4] populations compared to the hpGUS[wt] population. The inventors recognized that the hpGUS[G:U] construct was more ideal than the hpGUS[1:4] construct for reduced promoter methylation and transcriptional self-silencing because it had fewer or missing cytosine nucleotides in the sense sequence, and therefore did not induce DNA methylation that could have spread to the promoter.

[0308] Example 10. Design and testing of hairpin RNA containing G:U base pairs targeting endogenous genes. Modified hairpin RNA targeting EIN2 and CHS RNA Because the G:U modified hairpin RNAs appeared to induce more consistent and uniform silencing of target genes compared to the conventional hairpin RNAs described above, the inventors sought to test whether the improved design also reduced the expression of endogenous genes. Therefore, the inventors designed, fabricated, and tested several [G:U] modified hairpin RNA constructs targeting either the EIN2 or CHS gene, or both, which were endogenous genes of Arabidopsis thaliana selected as examples of target genes to be attempted to silencing. The EIN2 gene (SEQ ID NO: 19) encodes ethylene-insensitive protein 2 (EIN2), a regulatory protein that is a central factor in the ethylene-regulated signaling pathway, a plant signaling molecule, and the CHS gene (SEQ ID NO: 20) encodes chalcone synthase (CHS), an enzyme involved in anthocyanin production in the seed coat of A. thaliana. Another G:U modified construct was generated that simultaneously targeted both the EIN2 and CHS genes, and the EIN2 and CHS...

Claims

1. An RNA molecule comprising a first RNA component, a second RNA component covalently bonded to the first RNA component, and optionally one or all of the following: (i) a binding ribonucleotide sequence covalently bonding the first and second RNA components, (ii) a 5' leader sequence, and (iii) a 3' trailer sequence, The first RNA component comprises, in the order of 5' to 3', a first 5' ribonucleotide, a first RNA sequence, and a first 3' ribonucleotide, wherein the first 5' and 3' ribonucleotides form base pairs with each other in the first RNA component, and the first RNA sequence includes a first sense ribonucleotide sequence of at least 40 consecutive ribonucleotides, a first loop sequence of at least 4 ribonucleotides, and a first antisense ribonucleotide sequence of at least 40 consecutive ribonucleotides, wherein the first antisense ribonucleotide sequence hybridizes with the first sense ribonucleotide sequence in the RNA molecule, and the 40 consecutive ribonucleotides of the first antisense ribonucleotide sequence are perfectly complementary to the first region of the target RNA molecule and can form base pairs with it. The second RNA component, if present, covalently binds to the first 5' ribonucleotide or the first 3' ribonucleotide via the binding ribonucleotide sequence, or directly if the binding ribonucleotide sequence is absent. The second RNA component comprises, in the order of 5' to 3', a second 5' ribonucleotide, a second RNA sequence, and a second 3' ribonucleotide, wherein the second 5' and 3' ribonucleotides form base pairs with each other in the RNA molecule, and the second RNA sequence includes a second sense ribonucleotide sequence of at least 21 nucleotides in length, a second loop sequence of at least 4 ribonucleotides, and a second antisense ribonucleotide sequence of at least 21 nucleotides in length, wherein the second sense ribonucleotide sequence hybridizes with the second antisense ribonucleotide sequence over the length of at least 21 consecutive nucleotides in the RNA molecule. The 5' leader sequence, if present, consists of a ribonucleotide sequence that, when the second RNA component binds to the first 3' ribonucleotide, covalently binds to the first 5' ribonucleotide, or when the second RNA component binds to the first 5' ribonucleotide, covalently binds to the second 5' ribonucleotide. The 3' trailer sequence, if present, consists of a ribonucleotide sequence that, when the second RNA component binds to the first 3' ribonucleotide, covalently binds to the second 3' ribonucleotide, or when the second RNA component binds to the first 5' ribonucleotide, covalently binds to the first 3' ribonucleotide. The RNA molecule is a single-stranded ribonucleotide including a 5' end and a 3' end. The aforementioned target RNA molecule is from an arthropod cell or a plant cell. 5% to 40% of the ribonucleotides in the first sense ribonucleotide sequence and the first antisense ribonucleotide sequence are formed by non-standard base pairs. RNA molecule.

2. The RNA molecule according to claim 1, wherein 10% to 40% of the total ribonucleotides of the first sense ribonucleotide sequence and the first antisense ribonucleotide sequence are base-paired by non-standard base pairs.

3. The RNA molecule according to claim 1, wherein 10% to 35% of the total ribonucleotides of the first sense ribonucleotide sequence and the first antisense ribonucleotide sequence are base-paired by non-standard base pairs.

4. The RNA molecule according to claim 1, wherein 15% to 30% of the ribonucleotides in the first sense ribonucleotide sequence and the first antisense ribonucleotide sequence are base-paired by non-standard base pairs.

5. The RNA molecule according to any one of claims 1 to 4, wherein the non-standard base pair is a G:U base pair.

6. The RNA molecule according to any one of claims 1 to 5, wherein both the second sense ribonucleotide sequence and the second antisense ribonucleotide sequence have a length of at least 40 consecutive nucleotides.

7. (a) The first and second antisense ribonucleotide sequences are adjacent to the first and second sense ribonucleotide sequences such that the first and second sense ribonucleotide sequences are located between the first and second antisense ribonucleotide sequences, or (b) The first and second sense ribonucleotide sequences are adjacent to the first and second antisense ribonucleotide sequences such that the first and second antisense ribonucleotide sequences are located between the first and second sense ribonucleotide sequences.

8. An RNA molecule according to any one of claims 1 to 7, characterized by one or more of the following: (i) The first sense ribonucleotide sequence is covalently bonded to the first 5' ribonucleotide without any intervening nucleotides, or the first antisense ribonucleotide sequence is covalently bonded to the first 3' ribonucleotide without any intervening nucleotides, or both; (ii) The RNA molecule comprises the binding ribonucleotide sequence having fewer than 20 ribonucleotides; (iii) The binding ribonucleotide sequence hybridizes to the target RNA molecule; (iv) The binding ribonucleotide sequence is identical to a part of the complement of the target RNA molecule; (v) The binding ribonucleotide sequence is 1 to 10 ribonucleotides long; (vi) The RNA molecule comprises two or more sense ribonucleotide sequences that are identical in sequence to the region of the target RNA molecule, and an antisense ribonucleotide sequence that forms a perfect base pair therewith; (vii) The first and second sense ribonucleotide sequences are identical in sequence to different regions of the same target RNA molecule; (viiii) The first and second senseribonucleotide sequences are identical in sequence to regions of different target RNA molecules; (ix) The first and second sense ribonucleotide sequences do not have an intervening loop sequence; (x) The first and second antisense ribonucleotide sequences are perfectly base-paired with the first and second sense ribonucleotide sequences, respectively, and are complementary to the regions of the target RNA molecule; (xi) The first and second antisense ribonucleotide sequences are complementary to different regions of the same target RNA molecule; (xi) The second antisense ribonucleotide sequence is complementary to a different region of the target RNA molecule than the first antisense ribonucleotide sequence; (xiii) (a) The first 5' ribonucleotide and the second 3' ribonucleotide are adjacent, each forming a base pair, but not directly covalently bonded to each other, or (b) The second 5' ribonucleotide and the first 3' ribonucleotide are adjacent, each forming a base pair, but not directly covalently bonded to each other; (xiv) The first antisense ribonucleotide sequence hybridizes to a first region of the target RNA molecule, the second antisense ribonucleotide sequence hybridizes 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, the first and second sense ribonucleotide sequences are contiguous, and the two antisense sequences are not contiguous within the RNA molecule; (xv) The first sense ribonucleotide sequence is at least 60% identical to the first region of the target RNA molecule, the second sense ribonucleotide sequence is at least 60% identical to the 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, the first and second antisense ribonucleotide sequences are contiguous and hybridize to the target RNA molecule, and the two sense sequences are not contiguous within the RNA molecule; (xvi) The RNA molecule comprises the 5' leader sequence, the 3' trailer sequence, or both; (xvii) At least one or all of the loop sequences in the RNA molecule are longer than 20 nucleotides; and (xviiii) The RNA molecule has no bulge, or has one or more bulges, or the double-stranded region of the RNA molecule contains one, two or more nucleotides that are not base-paired in the double-stranded region.

9. The RNA molecule according to any one of claims 1 to 8, wherein the target RNA molecule is located in a plant cell.

10. An RNA molecule according to any one of claims 1 to 9, which is produced in a cell by transcription from a genetic construct.

11. An isolated and / or exogenous polynucleotide or vector encoding an RNA molecule according to any one of claims 1 to 10.

12. A host cell or plant or a portion thereof comprising an RNA molecule according to any one of claims 1 to 10 and one or both of the polynucleotides or vectors according to claim 11.

13. A method for generating an RNA molecule according to any one of claims 1 to 10, wherein the method comprises expressing the polynucleotide according to claim 11 in a host cell or a cell-free expression system.

14. A method for producing the plant described in claim 12, wherein the method comprises introducing the polynucleotide described in claim 11 into a cell to stably incorporate it into the genome of the cell, and producing the plant from the cell.

15. An extract of a host cell or plant according to claim 12, wherein the extract comprises an RNA molecule according to any one of claims 1 to 10 and / or a polynucleotide or vector according to claim 11.

16. A composition comprising one or more of the RNA molecule described in any one of claims 1 to 10, the polynucleotide or vector described in claim 11, the host cell or plant described in claim 12, or the extract described in claim 15, and one or more suitable carriers.

17. A composition for downregulating the level and / or activity of a target RNA molecule in a plant or arthropod, wherein the composition comprises one or more of the RNA molecule described in any one of claims 1 to 10, the polynucleotide or vector described in claim 11, the host cell described in claim 12, the extract described in claim 15, or the composition described in claim 16.

18. A method for controlling a non-human organism, the method comprising delivering one or more of the RNA molecule described in any one of claims 1 to 10, the polynucleotide or vector described in claim 11, the host cell described in claim 12, the extract described in claim 15, or the composition described in claim 16 to the non-human organism, wherein the RNA molecule has a harmful effect on the non-human organism, and the non-human organism is a plant or an arthropod.

19. An RNA molecule according to any one of claims 1 to 10, a polynucleotide or vector according to claim 11, a host cell or plant according to claim 12, an extract according to claim 15, or a composition according to claim 16, for use in the prevention or treatment of a target disease, wherein the RNA molecule has a beneficial effect on at least one symptom of the disease or on the prevention of the disease.

20. A method for introducing an RNA molecule into a plant, wherein the method comprises applying a composition containing the RNA molecule described in any one of claims 1 to 10 to the plant or a part thereof.

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