RNA interference agents for GST-π gene modulation

Abstract

To provide compounds, compositions and methods for modulating the expression of human GST-π using RNA interference.SOLUTION: The RNA interference molecules can be used in methods for preventing or treating diseases such as malignant tumors. Provided are a range of siRNA structures, comprising one or more nucleotides being modified or chemically-modified. Advantageous structures include siRNAs with 2'-deoxy nucleotides located in the seed region, as well as other nucleotide modifications.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to the field of biopharmaceuticals and therapeutic agents composed of nucleic acid-based molecules. More particularly, the present invention relates to compounds and compositions that utilize RNA interference (RNAi) to modulate the expression of human GST-π and their use.

[0002] This application includes a sequence listing that was electronically submitted as an ASCII file created on December 20, 2015, named ND5123385WO_SL.txt, which is 100,000 bytes in size, and the entire sequence listing is incorporated herein by reference.

Background Art

[0003] It has been found that various human cancer tissues correlate with the emergence of the mutant KRAS gene. In some cases, the tissue also shows an increase in the expression level of glutathione S-transferase Pi (GST-π) (Miyanishi et al., Gastroenterology, 2001, Vol. 121:865-874, abstract). For example, an increase in serum GST-π levels was observed in patients with various gastrointestinal malignancies (Niitsu et al., Cancer, 1989, Vol.63, No.2, pp.317-323, abstract).

[0004] GST-π is a member of the GST family of enzymes that play a role in detoxification by catalyzing the conjugation of hydrophobic compounds with electrophilic compounds having reduced glutathione. GST-π expression can be decreased by siRNA in vitro (Niitsu et al., US 2014 / 0315975 A1). However, existing siRNA agents have many drawbacks, such as insufficient activity, off-target effects, lack of serum stability, and lack of in vivo efficacy or effect.

Summary of the Invention

Problems to be Solved by the Invention

[0005] There is an urgent need for compositions and methods for regulating the expression of genes associated with cancer. In particular, therapeutic agents based on the inhibition of GST-π expression require very potent and stable siRNA sequences and structures that can reduce off-target effects.

[0006] There is a need for siRNA sequences, compounds, and structures for regulating GST-π expression for use in the treatment of diseases such as malignant tumors.

Means for Solving the Problems

[0007] The present invention relates to compounds, compositions, and methods for regulating the expression of human GST-π using RNA interference.

[0008] In some embodiments, the present invention provides molecules for RNA interference gene silencing of GST-π.

[0009] In further embodiments, the structures, molecules, and compositions of the present invention can be used in methods for preventing or treating diseases associated with GST-π, including malignant tumors, or for improving the symptoms of conditions or disorders associated with GST-π.

[0010] Embodiments of the present invention include the following: A nucleic acid molecule for inhibiting the expression of GST-π comprising a sense strand and an antisense strand, wherein the strands form a double-stranded region. The nucleic acid molecule may be an siRNA molecule for inhibiting the expression of GST-π and may contain one or more nucleotides that are modified or chemically modified.

[0011] In some embodiments, the nucleic acid siRNA molecules for inhibiting the expression of GST-π may comprise 2'-deoxynucleotides, 2'-O-alkyl-substituted nucleotides, 2'-deoxy-2'-fluoro-substituted nucleotides, or any combination thereof. In certain embodiments, the 2'-deoxynucleotides may be present in the seed region of the siRNA molecule. In certain aspects, the siRNA molecule for inhibiting the expression of GST-π can have deoxynucleotides at multiple positions of the antisense strand.

[0012] The nucleic acid molecules of the present invention have an IC50 of less than 300 pM and can advantageously inhibit GST-π mRNA expression. In certain embodiments, the nucleic acid molecule can inhibit the expression level of GST-π mRNA in vivo to at least 25% upon a single administration of the molecule. In some embodiments, the nucleic acid molecule can reduce the passenger strand off-target activity or reduce it by at least 50-fold, or at least 100-fold.

[0013] Embodiments of the present invention further provide a pharmaceutical composition containing an siRNA molecule and a pharmaceutically acceptable carrier. In some embodiments, the carrier can be a lipid molecule or a liposome. The present invention includes a vector containing a nucleic acid molecule or a cell.

[0014] Also, the present invention contemplates a method for treating a disease associated with GST-π expression by administering a composition containing siRNA to a subject in need thereof. Here, the disease includes malignant tumors, cancers, cancers caused by cells expressing mutant KRAS, sarcomas, or carcinomas. BRIEF DESCRIPTION OF THE DRAWINGS

[0015]

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[0016] The present invention relates to compounds, compositions, and methods for nucleic acid-based therapeutic agents for modulating the expression of GST-π.

[0017] In some embodiments, the present invention provides molecules active in RNA interference, as well as structures and compositions capable of silencing the expression of GST-π.

[0018] The structures and compositions of the present disclosure can be used for the prevention or treatment of various diseases such as malignant tumors.

[0019] In further embodiments, the present invention provides compositions for the delivery and uptake of one or more therapeutic RNAi molecules of the present invention, and methods of using the same. The RNA-based compositions of the present invention can be used in methods for preventing or treating cancerous malignancies.

[0020] The therapeutic compositions of the present invention comprise nucleic acid molecules that are active in RNA interference. The therapeutic nucleic acid molecules can target GSTP1 (GST-π) for gene silencing.

[0021] In various embodiments, the present invention provides a series of molecules that are active as small interfering RNAs (siRNAs) and can regulate or silence GST-π gene expression.

[0022] The siRNAs of the present invention can be used to prevent or treat malignancies.

[0023] Embodiments of the present invention further provide a vehicle, formulation, or lipid nanoparticle formulation for delivering the siRNAs of the present invention to a subject in need of prevention or treatment of a malignancy. The present invention further contemplates a method of administering siRNAs as a therapeutic agent to a mammal.

[0024] The therapeutic molecules and compositions of the present invention can be used for RNA interference aimed at preventing or treating GST-π-related diseases by administering the compound or composition to a subject in need thereof.

[0025] The methods of the present invention can utilize the compounds of the present invention for preventing or treating malignancies.

[0026] In some aspects, malignancies can be present in various diseases such as cancers that highly express GST-π, cancers resulting from cells expressing mutant KRAS, sarcomas, fibrosarcomas, malignant fibrous histiocytomas, liposarcomas, rhabdomyosarcomas, leiomyosarcomas, angiosarcomas, Kaposi's sarcomas, lymphangiosarcomas, synovial sarcomas, chondrosarcomas, osteosarcomas, and carcinomas, among others.

[0027] In certain embodiments, the methods of the invention utilize the compounds of the invention for the prevention or treatment of malignant tumors and cancer in any organ. This includes, for example, brain tumors, head and neck cancers, breast cancers, lung cancers, esophageal cancers, gastric cancers, duodenal cancers, colorectal cancers, liver cancers, pancreatic cancers, gallbladder cancers, bile duct cancers, kidney cancers, urethral cancers, bladder cancers, prostate cancers, testicular cancers, uterine cancers, ovarian cancers, skin cancers, leukemias, malignant lymphomas, epithelial malignant tumors, and non-epithelial malignant tumors.

[0028] In certain embodiments, the combination of therapeutic molecules of the invention can be used to silence or inhibit GST-π gene expression.

[0029] The invention provides a series of RNAi molecules, each molecule having a polynucleotide sense strand and a polynucleotide antisense strand, each strand of the molecule being 15 - 30 nucleotides in length, a 15 - 30 nucleotide contiguous region of the antisense strand being complementary to the sequence of the mRNA encoding GST-π, at least a portion of the sense strand being complementary to at least a portion of the antisense strand, and the molecule having a double-stranded region 15 - 30 nucleotides in length.

[0030] The RNAi molecules of the invention can have a 15 - 30 nucleotide contiguous region of the antisense strand, which is complementary to the sequence of the mRNA encoding GST-π and is located in the double-stranded region of the molecule.

[0031] In some embodiments, the RNAi molecule can have a 15 - 30 nucleotide contiguous region of the antisense strand that is complementary to the sequence of the mRNA encoding GST-π.

[0032] Embodiments of the invention can further provide a method for preventing, treating, or ameliorating one or more symptoms of a malignant tumor, or a method for reducing the risk of progression of a malignant tumor, or a method for delaying the onset of a malignant tumor in a mammal in need thereof.

[0033] GST-π and RNAi molecules The nucleic acid sequence of exemplary target human glutathione S-transferase (human GST-π) mRNA is disclosed in GenBank accession number NM_000852.3 (hGSTP1) and is 986 nucleotides in length.

[0034] Those skilled in the art will understand that the reported sequences may change over time and that they can incorporate the necessary changes into the nucleic acid molecules herein accordingly.

[0035] Embodiments of the present invention can provide compositions and methods for gene silencing of GST-π expression using small nucleic acid molecules. Nucleic acid molecules include molecules active in RNA interference (RNAi molecules), short interfering RNAs (siRNAs), double-stranded RNAs (dsRNAs), microRNAs (miRNAs) and short hairpin RNA (shRNA) molecules, as well as DNA-directed RNA (ddRNA), Piwi-interacting RNAs (piRNAs) and repeat-associated siRNAs (rasiRNAs). Such molecules can mediate RNA interference against GST-π gene expression.

[0036] The compositions and methods disclosed herein can also be used in the treatment of various types of malignancies in a subject.

[0037] The nucleic acid molecules and methods of the present invention are used to down-regulate the expression of the gene encoding GST-π.

[0038] The compositions and methods of the present invention can comprise one or more nucleic acid molecules, which alone or in combination can regulate or control the expression of GST-π protein and / or the gene encoding GST-π protein, for example, diseases such as malignancies, and proteins and / or genes encoding GST-π involved in the maintenance and / or development of GST-π-related conditions or diseases.

[0039] The compositions and methods of the present invention are described with reference to an exemplary sequence of GST-π. Those skilled in the art will understand that various aspects and embodiments of the present invention are directed to variants such as the related GST-π gene, sequence, or homolog genes and transcript variants, as well as polymorphisms including single nucleotide polymorphisms (SNPs) associated with the GST-π gene.

[0040] In some embodiments, the compositions and methods of the present invention can provide double-stranded short interfering nucleic acid (siRNA) molecules that down-regulate the expression of the GST-pi gene, such as the human GST-pi.

[0041] The RNAi molecules of the present invention can target GST-π and can target any homologous sequence that can provide additional target sequences, for example, using complementary sequences or by incorporating non-standard base pairs such as mismatches and / or wobble base pairs.

[0042] When mismatches are identified, nucleic acid molecules that target two or more gene sequences can be generated using non-standard base pairs such as mismatches and / or wobble bases.

[0043] For example, nucleic acid molecules can be generated that can target sequences for different GST-π targets having sequence homology using non-standard base pairs such as UU and CC base pairs. Thus, the RNAi molecules can target nucleotide sequences conserved among homologous genes and can inhibit the expression of two or more genes using a single RNAi molecule.

[0044] In some aspects, the compositions and methods of the present invention include RNAi molecules that are active against GST-π mRNA, and the RNAi molecules include sequences complementary to any mRNA encoding the GST-π sequence.

[0045] In some embodiments, the RNAi molecules of the present disclosure can be active against GST-π RNA, and the RNAi molecules include sequences complementary to RNAs encoding mutant GST-π, for example, RNAs of mutant GST-pi genes known in the art to be associated with malignancies.

[0046] In further embodiments, the RNAi molecules of the present invention can include nucleotide sequences capable of mediating the silencing of GST-π gene expression.

[0047] The nucleic acid molecule for inhibiting the expression of GST-π can have a sense strand and an antisense strand, where the strands form a double-stranded region. The nucleic acid molecule can have one or more nucleotides modified or chemically modified within the double-stranded region, including modifications as known in the art. Any nucleotide within the overhang of the siRNA can also be modified or chemically modified.

[0048] In some embodiments, 2'-deoxynucleotides are preferred as the modified or chemically modified nucleotides. In further embodiments, the modified or chemically modified nucleotides include 2'-O-alkyl-substituted nucleotides, 2'-deoxy-2'-fluoro-substituted nucleotides, phosphorothioate nucleotides, locked nucleotides, or any combination thereof.

[0049] In certain embodiments, a structure having an antisense strand containing deoxynucleotides at multiple positions is preferred. The multiple positions are one of the following: each of positions 4, 6, and 8 from the 5' end of the antisense strand; each of positions 3, 5, and 7 from the 5' end of the antisense strand; each of positions 1, 3, 5, and 7 from the 5' end of the antisense strand; each of positions 3 to 8 from the 5' end of the antisense strand; and each of positions 5 to 8 from the 5' end of the antisense strand. Any of these structures can be combined with one or more 2'-deoxy-2'-fluoro-substituted nucleotides within the double-stranded region.

[0050] The nucleic acid molecule of the present invention can inhibit the expression of GST-π mRNA with an advantageous IC50 of less than about 300 pM, or less than about 200 pM, or less than about 100 pM, or less than about 50 pM.

[0051] Furthermore, the nucleic acid molecule can inhibit the expression level of GST-π mRNA in vivo to at least 25% with a single administration.

[0052] In the present invention, a pharmaceutical composition is contemplated that can comprise one or more of the siRNAs described herein in combination with a pharmaceutically acceptable carrier. Any suitable carrier, lipid molecule, nanoparticle or liposome known in the art (any of which can encapsulate the siRNA molecule) can be used.

[0053] The present invention discloses a method for treating a disease associated with GST-π expression, the method comprising administering to a subject in need a composition comprising one or more siRNAs. The diseases to be treated can include, inter alia, malignant tumors, cancers, cancers caused by cells expressing mutant KRAS, sarcomas and carcinomas.

[0054] Examples of the RNAi molecules of the present invention targeting GST-π mRNA are shown in Table 1.

[0055] [Table 1] TIFF2025102833000003.tif227164

[0056] Highlights of Table 1: Capital letters A, G, C and U refer to riboA, riboG, riboC and riboU, respectively. Lowercase letters a, u, g, c, t mean 2'-deoxy-A, 2'-deoxy-U, 2'-deoxy-G, 2'-deoxy-C and deoxythymidine, respectively.

[0057] Examples of the RNAi molecules of the present invention targeting GST-π mRNA are shown in Table 2.

[0058]

Table 2

[0059] Highlights of Table 2: Capital letters A, G, C, and U mean ribo A, ribo G, ribo C, and ribo U, respectively. Lowercase letters a, u, g, c, t mean 2'-deoxy-A, 2'-deoxy-U, 2'-deoxy-G, 2'-deoxy-C, and deoxythymidine (dT = T = t), respectively. Underlines mean 2'-OMe substituents, e.g., U ... Lowercase f means 2'-deoxy-2'-fluoro substitution, e.g., fU is 2'-deoxy-2'-fluoro-U. N is A, C, G, U, U , a, c, g, u, t, or a modified nucleotide, an inverted nucleotide, or a chemically modified nucleotide. The letter “s” means a phosphorothioate bond.

[0060] Examples of the RNAi molecules of the present invention targeting GST-π mRNA are shown in Table 3.

[0061]

Table 3

[0062] Highlights of Table 3: Capital letters A, G, C, and U mean ribo A, ribo G, ribo C, and ribo U, respectively. Lowercase letters a, u, g, c, t mean 2'-deoxy-A, 2'-deoxy-U, 2'-deoxy-G, 2'-deoxy-C, and deoxythymidine (dT = T = t), respectively. Underlines mean 2'-OMe substituents, e.g., U ... Lowercase f means 2'-deoxy-2'-fluoro substitution, e.g., fU is 2'-deoxy-2'-fluoro-U. N is A, C, G, U, U , a, c, g, u, t, or a modified nucleotide, an inverted nucleotide, or a chemically modified nucleotide.

[0063] Examples of the RNAi molecules of the present invention targeting GST-π mRNA are shown in Table 4.

[0064] [Table 4]

[0065] Highlights of Table 4: Capital letters A, G, C, and U mean ribo A, ribo G, ribo C, and ribo U, respectively. Lowercase letters a, u, g, c, t mean 2'-deoxy-A, 2'-deoxy-U, 2'-deoxy-G, 2'-deoxy-C, and deoxythymidine (dT = T = t), respectively. Underlines mean 2'-OMe substituents, for example U . Lowercase f means 2'-deoxy-2'-fluoro substitution, for example fU is 2'-deoxy-2'-fluoro-U. N is A, C, G, U, U , a, c, g, u, t, or a modified nucleotide, an inverted nucleotide, or a chemically modified nucleotide.

[0066] Examples of the RNAi molecules of the present invention targeting GST-π mRNA are shown in Table 5.

[0067] [Table 5]

[0068] Highlights of Table 5: Capital letters A, G, C, and U mean ribo A, ribo G, ribo C, and ribo U, respectively. Lowercase letters a, u, g, c, t mean 2'-deoxy-A, 2'-deoxy-U, 2'-deoxy-G, 2'-deoxy-C, and deoxythymidine (dT = T = t), respectively. Underlines mean 2'-OMe substituents, for example U . Lowercase f means 2'-deoxy-2'-fluoro substitution, for example fU is 2'-deoxy-2'-fluoro-U. N is A, C, G, U, U , a, c, g, u, t, or a modified nucleotide, an inverted nucleotide, or a chemically modified nucleotide.

[0069] Table 6 shows examples of the RNAi molecules of the present invention targeting GST-π mRNA.

[0070] [Table 6]

[0071] Highlights of Table 6: Capital letters A, G, C, and U mean ribo-A, ribo-G, ribo-C, and ribo-U, respectively. Lowercase letters a, u, g, c, t mean 2'-deoxy-A, 2'-deoxy-U, 2'-deoxy-G, 2'-deoxy-C, and deoxythymidine (dT = T = t), respectively. Underlines mean 2'-OMe substituents, for example U ... Lowercase f means 2'-deoxy-2'-fluoro substitution, for example fU is 2'-deoxy-2'-fluoro-U. N is A, C, G, U, U ..., a, c, g, u, t, or a modified nucleotide, an inverted nucleotide, or a chemically modified nucleotide.

[0072] In some embodiments, the present invention provides a range of nucleic acid molecules. Here, a) the molecule has a polynucleotide sense strand and a polynucleotide antisense strand, b) each strand of the molecule is 15 to 30 nucleotides in length, c) a continuous region of 15 to 30 nucleotides of the antisense strand is complementary to the sequence of the mRNA encoding GST-π, d) at least a part of the sense strand is complementary to at least a part of the antisense strand, and the molecule has a double-stranded region 15 to 30 nucleotides in length.

[0073] In some embodiments, the nucleic acid molecule can have a continuous region of 15 to 30 nucleotides of the antisense strand that is complementary to the sequence of the mRNA encoding GST-π located in the double-stranded region of the molecule.

[0074] In further embodiments, the nucleic acid molecule can have a continuous region of 15 to 30 nucleotides of the antisense strand that is complementary to the sequence of the mRNA encoding GST-π.

[0075] In certain embodiments, each strand of the nucleic acid molecule can be 18 to 22 nucleotides in length. The double-stranded region of the nucleic acid molecule can be 19 nucleotides in length.

[0076] In another form, the nucleic acid molecule has a polynucleotide sense strand and a polynucleotide antisense strand linked as a single strand and can form a double-stranded region linked by a loop at one end.

[0077] Some embodiments of the nucleic acid molecules of the present disclosure can have blunt ends. In certain embodiments, the nucleic acid molecule can have one or more 3' overhangs.

[0078] The present invention provides a series of nucleic acid molecules that are RNAi molecules active against gene silencing. The nucleic acid molecules of the present invention can be dsRNA, siRNA, microRNA or shRNA active against DNA silencing, and DNA-directed RNA (ddRNA), Piwi-interacting RNA (piRNA) or related repeat siRNA (rasiRNA). The nucleic acid molecule can be active against inhibiting the expression of GST-π.

[0079] Embodiments of the present invention further provide nucleic acid molecules having an IC50 for knockdown of GST-π of less than 100 pM.

[0080] Further embodiments of the present invention provide nucleic acid molecules having an IC50 for knockdown of GST-π of less than 50 pM.

[0081] The present invention further contemplates a composition comprising one or more of the nucleic acid molecules of the present invention together with a pharmaceutically acceptable carrier. In certain embodiments, the carrier can be a lipid molecule or a liposome.

[0082] The compounds and compositions of the present invention are useful in methods for preventing or treating GST-π-related diseases by administering the compound or composition to a subject in need thereof.

[0083] The method of the present invention can utilize the compounds of the present invention for preventing or treating malignant tumors. Malignant tumors can exist in various diseases. Such diseases include, for example, cancers associated with GST-π expression, cancers caused by cells expressing mutant KRAS, sarcomas, fibrosarcomas, malignant fibrous histiocytomas, liposarcomas, rhabdomyosarcomas, leiomyosarcomas, angiosarcomas, Kaposi's sarcomas, lymphangiosarcomas, synovial sarcomas, chondrosarcomas, osteosarcomas, carcinomas, brain tumors, head and neck cancers, breast cancers, lung cancers, esophageal cancers, gastric cancers, duodenal cancers, appendiceal cancers, colorectal cancers, rectal cancers, liver cancers, pancreatic cancers, gallbladder cancers, bile duct cancers, anal cancers, kidney cancers, urethral cancers, bladder cancers, prostate cancers, testicular cancers, uterine cancers, ovarian cancers, skin cancers, leukemias, malignant lymphomas, epithelial malignant tumors, and non-epithelial malignant tumors.

[0084] Modified and chemically modified siRNA Embodiments of the present invention include modified or chemically modified siRNA molecules, which can provide enhanced properties for therapeutic uses such as increased gene silencing activity and efficacy. The present invention provides modified or chemically modified siRNA molecules that have excellent serum stability and further reduced off-target effects without losing the activity and efficacy of siRNA molecules for gene regulation and gene silencing. In some aspects, the present invention provides siRNAs having various combinations of modifications or chemical modifications that enhance the stability and effectiveness of siRNA.

[0085] In some embodiments, the siRNA molecules of the present invention can have passenger strand off-target activity that is reduced by at least 10-fold, or at least 20-fold, or at least 30-fold, or at least 50-fold, or at least 100-fold.

[0086] As used herein, the terms modification and chemical modification mean changes made to the structure of the naturally occurring nucleotides of siRNA or the nucleic acid structure, including siRNAs having one or more nucleotide analogs, modified nucleotides, non-standard nucleotides, non-naturally occurring nucleotides, and combinations thereof.

[0087] In some embodiments, the number of modified structures or chemically modified structures in the siRNA can include all of the structural components and / or all of the nucleotides of the siRNA molecule.

[0088] Examples of modified and chemically modified siRNAs include modifications of the sugar moiety of the nucleotide, modifications of the nucleobase of the nucleotide, modifications of the nucleic acid backbone or linkage, modifications of the structure of the (one or more) nucleotides at the ends of the siRNA strand, and combinations thereof.

[0089] Examples of modified and chemically modified siRNAs include siRNAs having substituent modifications at the 2'-carbon of the sugar.

[0090] Examples of modified and chemically modified siRNAs include siRNAs having modifications at the 5'-end, 3'-end, or both ends of the strand.

[0091] Examples of modified and chemically modified siRNAs include siRNAs having modifications that result in intermolecular complementary mismatches.

[0092] Examples of modified and chemically modified siRNAs include siRNAs having a 5'-propylamine terminus, 5'-phosphorylated terminus, 3'-puromycin terminus, or 3'-biotin terminus group.

[0093] Examples of modified and chemically modified siRNAs include siRNAs having 2'-fluoro-substituted ribonucleotides, 2'-OMe-substituted ribonucleotides, 2'-deoxyribonucleotides, 2'-amino-substituted ribonucleotides, 2'-thio-substituted ribonucleotides.

[0094] Examples of modified and chemically modified siRNAs include siRNAs having one or more 5-halouridine, 5-halocytidine, 5-methylcytidine, ribothymidine, 2-aminopurine, 2,6-diaminopurine, 4-thiouridine, or 5-aminoallyluridine.

[0095] Examples of modified and chemically modified siRNAs include siRNAs having one or more phosphorothioate groups.

[0096] Examples of modified and chemically modified siRNAs include siRNAs having one or more 2'-fluoro-substituted ribonucleotides, 2'-fluorouridine, 2'-fluorocytidine, 2'-deoxyribonucleotides, 2'-deoxyadenosine or 2'-deoxyguanosine.

[0097] Examples of modified and chemically modified siRNAs include siRNAs having one or more phosphorothioate linkages.

[0098] Examples of modified and chemically modified siRNAs include siRNAs having one or more alkylenediol linkages, oxy-alkylthio linkages or oxycarbonyloxy linkages.

[0099] Examples of modified and chemically modified siRNAs include siRNAs having one or more deoxy-basic groups, inosine, N3-methyl-uridine, N6,N6-dimethyl-adenosine, pseudouridine, purine ribonucleosides and ribavirin.

[0100] Examples of modified and chemically modified siRNAs include siRNAs having one or more 3' or 5' inverted terminal groups.

[0101] Examples of modified and chemically modified siRNAs include siRNAs having one or more 5-(2-amino)propyluridine, 5-bromouridine, adenosine, 8-bromoguanosine, 7-deaza-adenosine or N6-methyladenosine.

[0102] Methods for modulating GST-π and treating malignancies Embodiments of the present invention can provide RNAi molecules that can be used to down-regulate or inhibit the expression of GST-π and / or GST-π protein.

[0103] In some embodiments, the RNAi molecules of the invention can be used to down - regulate or inhibit the expression of GST - π and / or GST - π protein resulting from GST - π haplotype polymorphisms that may be associated with diseases or conditions such as malignancies.

[0104] Monitoring of GST - π protein or mRNA levels can be used to characterize gene silencing and to determine the effectiveness of the compounds and compositions of the invention.

[0105] The RNAi molecules of the present disclosure can be used alone or in combination with other siRNAs to regulate the expression of one or more genes.

[0106] The RNAi molecules of the present disclosure can be used alone or in combination, or in combination with other known drugs, for the prevention or treatment of diseases associated with GST - π (including malignancies), or for the improvement of symptoms or conditions.

[0107] The RNAi molecules of the invention can be used to regulate or inhibit the expression of GST - π in a sequence - specific manner.

[0108] The RNAi molecules of the present disclosure can include a guide strand in which a series of consecutive nucleotides is at least partially complementary to GST - π mRNA.

[0109] In certain embodiments, malignancies can be treated by RNA interference using the RNAi molecules of the invention.

[0110] Treatment of malignancies can be characterized in suitable cell - based models, as well as in ex vivo or in vivo animal models.

[0111] Treatment of malignancies is characterized by determining the level of GST - π mRNA or the level of GST - π protein in cells of the affected tissue.

[0112] The treatment of malignant tumors is characterized by non-invasive medical scans of the affected organ or tissue.

[0113] Embodiments of the present invention can include methods for preventing, treating, or ameliorating the symptoms of GST-π related diseases or conditions in a subject in need thereof.

[0114] In some embodiments, a method for preventing, treating, or ameliorating the symptoms of a malignant tumor in a subject includes modulating the expression of the GST-π gene in the subject or organism by administering an RNAi molecule of the present invention to the subject.

[0115] In some embodiments, the present invention contemplates a method of down-regulating the expression of the GST-π gene in a cell or organism by contacting the cell or organism with an RNAi molecule of the present invention.

[0116] Embodiments of the present invention include siRNA molecules of Tables 1-6 modified or chemically modified according to the above-described exemplification.

[0117] RNA interference RNA interference (RNAi) refers to sequence-specific post-transcriptional gene silencing in animals mediated by short interfering RNAs (siRNAs). See, for example, Zamore et al., Cell, 2000, Vol. 101, pp. 25-33; Fire et al., Nature, 1998, Vol. 391, pp. 806-811; Sharp, Genes & Development, 1999, Vol. 13, pp. 139-141.

[0118] The RNAi response within cells can be induced by double-stranded RNA (dsRNA), but the mechanism is not yet fully understood. Certain dsRNAs within cells can be acted upon by the Dicer enzyme, a ribonuclease III enzyme. See, e.g., Zamore et al., Cell, 2000, Vol. 101, pp. 25-33; Hammond et al., Nature, 2000, Vol. 404, pp. 293-296. Dicer can process dsRNA into shorter fragments of dsRNA known as siRNA.

[0119] Generally, siRNA can be about 21 to about 23 nucleotides in length and can include a base pair duplex region about 19 nucleotides in length.

[0120] RNAi is involved in an endonuclease complex known as the RNA-induced silencing complex (RISC). siRNA has an antisense or guide strand that enters the RISC complex and mediates cleavage of a single-stranded RNA target having a sequence complementary to the antisense strand of the siRNA duplex. The other strand of the siRNA is the passenger strand. Cleavage of the target RNA occurs in the middle of the region complementary to the antisense strand of the siRNA duplex (see, e.g., Elbashir et al., Genes & Development, 2001, Vol. 15, pp. 188-200).

[0121] As used herein, the term "sense strand" refers to the nucleotide sequence of an siRNA molecule that is partially or completely complementary to at least a portion of the corresponding antisense strand of the siRNA molecule. The sense strand of an siRNA molecule can include a nucleic acid sequence having homology to the target nucleic acid sequence.

[0122] As used herein, the term "antisense strand" refers to the nucleotide sequence of an siRNA molecule that is partially or completely complementary to at least a portion of the target nucleic acid sequence. The antisense strand of an siRNA molecule can include a nucleic acid sequence complementary to at least a portion of the corresponding sense strand of the siRNA molecule.

[0123] RNAi molecules can down-regulate or knockdown gene expression by mediating RNA interference in a sequence-specific manner. See, for example, Zamore et al., Cell, 2000, Vol. 101, pp. 25-33; Elbashir et al., Nature, 2001, Vol. 411, pp. 494-498; Kreutzer et al., WO2000 / 044895; Zernicka-Goetz et al., WO2001 / 36646; Fire et al., WO1999 / 032619; Plaetinck et al., WO2000 / 01846; Mello et al., WO2001 / 029058.

[0124] As used herein, the terms “inhibit,” “down-regulate,” or “reduce” with respect to gene expression mean that the expression of a gene or the level of an mRNA molecule encoding one or more proteins, or the activity of one or more of the encoded proteins, is lower than the activity observed in the absence of the RNAi molecule or siRNA of the present invention. For example, the level of expression, the level of mRNA, or the level of encoded protein activity may be reduced by at least 1%, or at least 10%, or at least 20%, or at least 50%, or at least 90%, or more, compared to the activity observed in the absence of the RNAi molecule or siRNA of the present invention.

[0125] RNAi molecules can also be used to knockdown viral gene expression, thus affecting viral replication.

[0126] RNAi molecules can be made from separate polynucleotide strands: a sense strand or passenger strand, and an antisense strand or guide strand. The guide strand and the passenger strand are at least partially complementary. The guide strand and the passenger strand can form a double-stranded region having from about 15 to about 49 base pairs.

[0127] In some embodiments, the double-stranded region of the siRNA can comprise 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, or 49 base pairs.

[0128] In certain embodiments, the RNAi molecule can be active in the RISC complex and have a length of the double-stranded region that is active against RISC.

[0129] In a further embodiment, the RNAi molecule can be active as a Dicer substrate that is converted into an RNAi molecule that is active in the RISC complex.

[0130] In some aspects, the RNAi molecule can have a guide and a passenger sequence portion that are complementary to opposing ends of a long molecule, such that the molecule can form a double-stranded region in the complementary sequence portion, and each strand is joined at one end of the double-stranded region by either a nucleotide or a non-nucleotide linker. For example, a hairpin sequence stem and loop sequence. The interaction between each strand and the linker can be a covalent or non-covalent interaction.

[0131] The RNAi molecules of the present disclosure may include a nucleotide, non-nucleotide, or nucleotide / non-nucleotide mixed linker that binds the sense region of the nucleic acid to the antisense region of the nucleic acid. The nucleotide linker can be a linker having a length of 2 nucleotides or more, for example, about 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. The nucleotide linker may also be a nucleic acid aptamer. As used herein, "aptamer" or "nucleic acid aptamer" means a nucleic acid molecule that specifically binds to a target molecule, where the nucleic acid molecule has a sequence that includes a sequence recognized by the target molecule in its natural context. Alternatively, the aptamer may be a nucleic acid molecule that binds to a target molecule, where the target molecule does not have to bind to the nucleic acid naturally. For example, an aptamer can bind to the ligand-binding domain of a protein and can be used to prevent the interaction between a naturally occurring ligand and the protein. See, for example, Gold et al., Annu Rev Biochem, 1995, Vol. 64, pp. 763-797; Brody et al., J. Biotechnol., 2000, Vol. 74, pp. 5-13; Hermann et al., Science, 2000, Vol. 287, pp. 820-825.

[0132] Examples of non-nucleotide linkers include abasic nucleotides, polyethers, polyamines, polyamides, peptides, carbohydrates, lipids, polyhydrocarbons or other polymeric compounds, such as polymeric compounds having 2 to 100 ethylene glycol units, such as polyethylene glycol. Some examples are described in Seela et al., Nucleic Acids Research, 1987, Vol. 15, pp. 3113-3129; Cload et al., J. Am. Chem. Soc., 1991, Vol. 113, pp. 6324-6326; Jaeschke et al., Tetrahedron Lett., 1993, Vol. 34, p. 301; Arnold et al., WO1989 / 002439; Usman et al., WO1995 / 006731; Dudycz et al., WO1995 / 011910; and Ferentz et al., J. Am. Chem. Soc., 1991, Vol. 113, pp. 4000-4002.

[0133] The RNAi molecule can have one or more overhangs from the double-stranded region. An overhang is a single-stranded region that is not base-paired and may be 1 to 8 nucleotides or more in length. The overhang may be a 3'-end overhang having a single-stranded region of 1 to 8 nucleotides at the 3'-end of the strand. The overhang may be a 5'-end overhang having a single-stranded region of 1 to 8 nucleotides at the 5'-end of the strand.

[0134] The overhangs of the RNAi molecule may have the same length or different lengths.

[0135] The RNAi molecule can have one or more blunt ends where the double-stranded region ends without an overhang and the strands base pair to the end of the double-stranded region.

[0136] The RNAi molecules of the present disclosure can have one or more blunt ends, or can have one or more overhangs, or can have a combination of blunt ends and overhangs.

[0137] The 5' end of the strand of the RNAi molecule may be a blunt end or may have an overhang. The 3' end of the strand of the RNAi molecule may be a blunt end or may have an overhang.

[0138] The 5' end of the strand of the RNAi molecule may be a blunt end and the 3' end may have an overhang. The 3' end of the strand of the RNAi molecule may be a blunt end and the 5' end may have an overhang.

[0139] In some embodiments, both ends of the RNAi molecule are blunt ends.

[0140] In further embodiments, both ends of the RNAi molecule have overhangs.

[0141] The 5'- and 3'-terminal overhangs may be of different lengths.

[0142] In certain embodiments, the RNAi molecule may have blunt ends where the 5' end of the antisense strand and the 3' end of the sense strand do not have overhang nucleotides.

[0143] In further embodiments, the RNAi molecule may have blunt ends where the 3' end of the antisense strand and the 5' end of the sense strand do not have overhang nucleotides.

[0144] The RNAi molecule may have mismatches in base pairing in the double-stranded region.

[0145] Any nucleotide in the overhang of the RNAi molecule can be a deoxyribonucleotide or a ribonucleotide.

[0146] One or more deoxyribonucleotides may be present at the 5' end, where the 3' end of the other strand of the RNAi molecule may not have an overhang and may not have a deoxyribonucleotide overhang.

[0147] One or more deoxyribonucleotides may be present at the 3'-end, where the 5'-end of the other strand of the RNAi molecule may not have an overhang and may not have a deoxyribonucleotide overhang.

[0148] In some embodiments, one or all of the overhang nucleotides of the RNAi molecule may be 2'-deoxyribonucleotides.

[0149] Dicer substrate RNAi molecules In some aspects, the RNAi molecule can be of a length suitable as a Dicer substrate that is processed to produce a RISC-active RNAi molecule. See, e.g., Rossi et al., US2005 / 0244858.

[0150] The double-stranded RNA (dsRNA) that is a Dicer substrate can be of a length sufficient to be processed by Dicer to produce an active RNAi molecule and may further include one or more of the following characteristics: (i) the Dicer substrate dsRNA can be asymmetric, e.g., having a 3'-overhang on the antisense strand, and (ii) the Dicer substrate dsRNA may have a modified 3'-end on the sense strand that directs the orientation of Dicer binding and processes the dsRNA into an active RNAi molecule.

[0151] In certain embodiments, the longest strand in the Dicer substrate dsRNA can be 24 to 30 nucleotides in length.

[0152] The Dicer substrate dsRNA can be symmetric or asymmetric.

[0153] In some embodiments, the Dicer substrate dsRNA can have a sense strand of 22 to 28 nucleotides and an antisense strand of 24 to 30 nucleotides.

[0154] In certain embodiments, the dicer substrate dsRNA may have an overhang at the 3' end of the antisense strand.

[0155] In further embodiments, the dicer substrate dsRNA can have a sense strand that is 25 nucleotides in length and an antisense strand that is 27 nucleotides in length including a 2-base 3'-overhang. The overhang can be 1, 2, or 3 nucleotides in length. The sense strand may also have a 5' phosphate.

[0156] The asymmetric dicer substrate dsRNA can have two deoxyribonucleotides at the 3' end of the sense strand instead of two ribonucleotides.

[0157] The sense strand of the dicer substrate dsRNA can be about 22 to about 30, or about 22 to about 28, or about 24 to about 30, or about 25 to about 30, or about 26 to about 30, or about 26 and 29, or about 27 to about 28 nucleotides in length.

[0158] The sense strand of the dicer substrate dsRNA can be 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.

[0159] In certain embodiments, the dicer substrate dsRNA may have sense and antisense strands that are at least about 25 nucleotides in length and do not exceed about 30 nucleotides in length.

[0160] In certain embodiments, the dicer substrate dsRNA may have sense and antisense strands that are 26 to 29 nucleotides in length.

[0161] In certain embodiments, the dicer substrate dsRNA may have a sense strand and an antisense strand that are 27 nucleotides in length.

[0162] The sense and antisense strands of the dicer substrate dsRNA can have blunt ends and be of the same length, or can be of different lengths with overhangs, or can have blunt ends and protrusions.

[0163] The dicer substrate dsRNA may have a double-stranded region 19, 20, 21, 22, 23, 24, 25, 26, or 27 nucleotides in length.

[0164] The antisense strand of the dicer substrate dsRNA can have any sequence that anneals to at least a part of the sequence of the sense strand under biological conditions such as in the cytoplasm of eukaryotic cells.

[0165] The dicer substrate having a sense strand and an antisense strand can be linked by a third structure such as a linker group or a linker oligonucleotide. The linker can, for example, link the two strands of the dsRNA and form a hairpin upon annealing.

[0166] The sense and antisense strands of the dicer substrate are generally complementary, but may have mismatches in base pairing.

[0167] In some embodiments, the dicer substrate dsRNA may be asymmetric such that the sense strand has 22-28 nucleotides and the antisense strand has 24-30 nucleotides.

[0168] One of the strands of the dicer substrate dsRNA, particularly the region of the antisense strand, may have a sequence length of at least 19 nucleotides, and these nucleotides are within a 21-base region adjacent to the 3' end of the antisense strand and are sufficiently complementary to the nucleotide sequence of the RNA produced from the target gene.

[0169] The antisense strand of the dicer substrate dsRNA can have 1 to 9 ribonucleotides at the 5' end so that its length is 22 to 28 nucleotides. When the length of the antisense strand is 21 nucleotides, 1 to 7 ribonucleotides, or 2 to 5 ribonucleotides, or 4 ribonucleotides can be added to the 3' end. The added ribonucleotides can have any sequence.

[0170] The sense strand of the dicer substrate dsRNA may have 24 to 30 nucleotides. The sense strand may be substantially complementary to the antisense strand and anneal to the antisense strand under biological conditions.

[0171] Methods of using RNAi molecules The nucleic acid molecules and RNAi molecules of the present invention can be delivered to cells or tissues by direct application of the molecules or by using molecules combined with a carrier or diluent.

[0172] The nucleic acid molecules and RNAi molecules of the present invention can be delivered or administered to cells, tissues, organs or subjects by direct application of the molecules using a carrier or diluent or other delivery vehicle that functions to assist or facilitate entry into cells, such as viral sequences, viral substances or lipid or liposome formulations.

[0173] The nucleic acid molecules and RNAi molecules of the present invention can form a complex with a cationic lipid, be packaged within a liposome, or be delivered to a target cell or tissue. The nucleic acid or nucleic acid complex can be locally administered to relevant tissues in vitro or in vivo by direct skin application, transdermal application or injection.

[0174] Delivery systems may include, for example, aqueous and non-aqueous gels, creams, emulsions, microemulsions, liposomes, ointments, aqueous and non-aqueous solutions, lotions, aerosols, hydrocarbon bases and powders, and may include additives such as solubilizing agents and penetration enhancers.

[0175] The compositions and methods of the present disclosure can include an expression vector comprising a nucleic acid sequence encoding at least one RNAi molecule of the present invention so as to enable the expression of nucleic acid molecules.

[0176] The nucleic acid molecules and RNAi molecules of the present invention can be expressed from transcription units inserted into DNA or RNA vectors. The recombinant vector can be a DNA plasmid or a viral vector. Viral vectors that provide transient expression of nucleic acid molecules can be used.

[0177] For example, the vector may contain sequences encoding both strands of a double-stranded RNAi molecule, or a single nucleic acid molecule that is self-complementary and forms an RNAi molecule. The expression vector may contain nucleic acid sequences encoding two or more nucleic acid molecules.

[0178] The nucleic acid molecules can be expressed intracellularly from eukaryotic promoters. Those skilled in the art understand that any nucleic acid can be expressed in eukaryotic cells from an appropriate DNA / RNA vector.

[0179] In some embodiments, viral constructs can be used to introduce the expression construct into cells, which is for the transcription of the dsRNA construct encoded by the expression construct, and the dsRNA is active in RNA interference.

[0180] Lipid formulations can be administered to animals by intravenous, intramuscular or intraperitoneal injection, or orally or by inhalation, or by other methods known in the art.

[0181] Pharmaceutically acceptable formulations for administering oligonucleotides are known and can be used.

[0182] Exemplary protocol for in vitro knockdown One day before transfection, 100 μl of DMEM (HyClone catalog number SH30243.01) containing 10% FBS was seeded into a 96-well plate at 2×10 3 cells / well and cultured in an incubator at 37 °C with a humidified atmosphere in 5% CO2 air. Before transfection, the medium was changed to 90 μl of Opti-MEM I reduced serum medium (Life Technologies catalog number 31985-070) containing 2% FBS. Then, 0.2 μl of Lipofectamine RNAiMax (Life Technologies catalog number 13778-100) was mixed with 4.8 μl of Opti-MEM I at room temperature for 5 minutes. Next, 1 μl of siRNA was mixed with 4 μl of Opti-MEM I, mixed with the LF2000 solution, and gently mixed without vortexing. After 5 minutes at room temperature, the mixture was incubated at room temperature for an additional 10 minutes to form the RNA-RNAiMax complex. Furthermore, 10 μl of the RNA-RNAiMax complex was added to the well and the plate was gently shaken by hand. The cells were incubated in an incubator at 37 °C with a humidified atmosphere in 5% CO2 air for 2 hours. The medium was replaced with fresh Opti-MEM I reduced serum medium containing 2% FBS. Twenty-four hours after transfection, the cells were washed once with ice-cold PBS. The cells were lysed with 50 μl of Cell-to-Ct lysis buffer (Life Technologies catalog number 4391851C) for 5 - 30 minutes at room temperature. 5 μl of stop solution was added and incubated for 2 minutes at room temperature. The mRNA level was immediately measured by RT-qPCR using TAQMAN. The samples could be frozen at -80 °C and assayed later.

[0183] Exemplary protocol for serum stability 0.2 mg / ml of siRNA was incubated with 10% human serum at 37°C. At specific time points (0, 5, 15, and 30 minutes), 200 μl of the sample was aliquoted and extracted with 200 μl of an extraction solvent (chloroform:phenol:isoamyl alcohol = 24:25:1). The sample was vortexed and centrifuged at 13,000 rpm for 10 minutes at room temperature, and then the upper layer solution was transferred and filtered through a 0.45 μm filter. The filtrate was transferred to a 300 μl HPLC injection vial. For LCMS, the mobile phases were MPA: 100 mM HFIP + 7 mM TEA in H2O, and MPB: 50% methanol + 50% acetonitrile. Column: Waters Acquity OST 2.1×50 mm, 1.7 μm was used.

Example

[0184] Example 1: The siRNA of the present invention targeting GST-π was found to be active in gene silencing in vitro. The dose-dependent activity of GST-π siRNA for gene knockdown was found to show an IC50 of less than about 250 picomoles (pM) and a low IC50 of about 1 pM.

[0185] In vitro transfection was performed in the A549 cell line to determine the siRNA knockdown efficiency. As shown in Table 7, dose-dependent knockdown of GST-π mRNA was observed with the siRNA in Table 1.

[0186]

Table 7

[0187] As shown in Table 7, the activity of the GST-π siRNA in Table 3 was in the range of 17 - 235 pM and was suitable for many applications including drugs used in vivo.

[0188] Example 2: The structure of the GST-π siRNA of the present invention having a deoxynucleotide located in the seed region of the antisense strand of siRNA was shown to unexpectedly and advantageously increase the gene knockdown activity in vitro.

[0189] In vitro transfection was performed in the A549 cell line, and the knockdown efficiency of GST-π siRNA based on structure BU2' (SEQ ID NOs: 131 and 157) was determined. As shown in Table 8, a dose-dependent knockdown of GST-π mRNA was observed using GST-π siRNA based on structure BU2'.

[0190] [Table 8]

[0191] As shown in Table 8, the activity of GST-π siRNA based on structure BU2' having three deoxynucleotides in the seed region of the antisense strand was unexpectedly increased up to 6-fold compared to GST-π siRNA having no deoxynucleotide in the double-stranded region.

[0192] These data indicate that GST-π siRNA having a structure with three deoxynucleotides at positions 3, 5, and 7, or positions 4, 6, and 8 in the seed region of the antisense strand has surprisingly high gene knockdown activity compared to GST-π siRNA having no deoxynucleotide in the double-stranded region.

[0193] The activity shown in Table 8 for GST-π siRNA having three deoxynucleotides in the seed region of the antisense strand was in the range of 5 - 8 pM and was very suitable for many applications including drugs used in vivo.

[0194] Example 3: The structure of the GST-π siRNA of the present invention having a deoxynucleotide located in the seed region of the antisense strand of siRNA was shown to unexpectedly and advantageously increase the gene knockdown activity in vitro.

[0195] In vitro transfection was performed in the A549 cell line to determine the knockdown efficiency of GST-π siRNA based on structure A9’ (SEQ ID NOs: 183 and 195). As shown in Table 9, dose-dependent knockdown of GST-π mRNA was observed with GST-π siRNA based on structure A9’.

[0196]

Table 9

[0197] As shown in Table 9, the activity of GST-π siRNA based on structure A9’ having 3 to 6 deoxynucleotides in the seed region of the antisense strand was surprisingly increased up to 24-fold compared to GST-π siRNA having no deoxynucleotides in the double-stranded region.

[0198] These data indicate that GST-π siRNA having a structure with 3 to 6 deoxynucleotides at positions 4, 6, and 8, or positions 1, 3, 5, and 7, or positions 3 to 8, or positions 5 to 8, or positions 3, 5, and 7 in the seed region of the antisense strand has unexpectedly high gene knockdown activity compared to GST-π siRNA having no deoxynucleotides in the double-stranded region.

[0199] The activity shown in Table 9 for GST-π siRNA having 3 to 6 deoxynucleotides in the seed region of the antisense strand was in the range of 1 to 15 pM, which was very suitable for many applications including drug agents used in vivo.

[0200] Example 4: The structure of GST-π siRNA having deoxynucleotides located in the seed region of the antisense strand of siRNA was shown to unexpectedly and advantageously increase gene knockdown activity in vitro.

[0201] In vitro transfection was performed in the A549 cell line to determine the knockdown efficiency of GST-π siRNA based on structure B13’ (SEQ ID NOs: 207 and 222). As shown in Table 10, a dose-dependent knockdown of GST-π mRNA was observed with GST-π siRNA based on structure B13’.

[0202]

Table 10

[0203] As shown in Table 10, the activity of GST-π siRNA based on structure B13’, which has three deoxynucleotides in the seed region of the antisense strand, unexpectedly increased compared to GST-π siRNA that does not have deoxynucleotides in the double-stranded region.

[0204] These data indicate that GST-π siRNA having a structure with three deoxynucleotides at positions 4, 6, and 8 in the seed region of the antisense strand has unexpectedly high gene knockdown activity compared to GST-π siRNA that does not contain deoxynucleotides in the double-stranded region.

[0205] The activity shown in Table 10 for GST-π siRNA having three deoxynucleotides in the seed region of the antisense strand was in the picomolar range of 11 pM and was very suitable for many applications including drug agents used in vivo.

[0206] Example 5: The structure of GST-π siRNA having deoxynucleotides located in the seed region of the antisense strand of siRNA unexpectedly and advantageously increased gene knockdown activity in vitro.

[0207] In vitro transfection was performed in the A549 cell line to determine the knockdown efficiency of GST-π siRNA based on structure B4’ (SEQ ID NOs: 261 and 273). As shown in Table 11, a dose-dependent knockdown of GST-π mRNA was observed with GST-π siRNA based on structure B4’.

[0208]

Table 11

[0209] As shown in Table 11, the activity of GST-π siRNA based on Structure B4’ having 6 deoxynucleotides in the seed region of the antisense strand was unexpectedly increased by more than 2-fold compared to GST-π siRNA having no deoxynucleotides in the double-stranded region.

[0210] These data indicate that GST-π siRNA having a structure with 6 deoxynucleotides located at positions 3 - 8 within the seed region of the antisense strand has surprisingly high gene knockdown activity compared to GST-π siRNA having no deoxynucleotides in the double-stranded region.

[0211] The activity shown in Table 11 of GST-π siRNA having 6 deoxynucleotides in the seed region of the antisense strand was in the picomolar range of 113 pM and was very suitable for many applications including drug agents used in vivo.

[0212] Example 6: The structure of GST-π siRNA having deoxynucleotides located in the seed region of the antisense strand of siRNA unexpectedly and advantageously increased the gene knockdown activity in vitro.

[0213] In vitro transfection was performed in the A549 cell line to determine the knockdown efficiency of GST-π siRNA based on Structure B2’ (SEQ ID NOs: 237 and 249). As shown in Table 12, dose-dependent knockdown of GST-π mRNA was observed using GST-π siRNA based on Structure B2’.

[0214]

Table 12

[0215] As shown in Table 12, the activity of GST-π siRNA based on Structure B2’ having 3 to 4 deoxynucleotides within the seed region of the antisense strand was surprisingly increased up to 4-fold compared to GST-π siRNA having no deoxynucleotides in the double-stranded region.

[0216] These data indicate that GST-π siRNA having a structure with 3 to 4 deoxynucleotides located at positions 5 to 8, or positions 1, 3, 5 and 7, or positions 3, 5 and 7 in the seed region of the antisense strand has unexpectedly high gene knockdown activity compared to GST-π siRNA not containing deoxynucleotides in the double-stranded region.

[0217] The activity shown in Table 12 for GST-π siRNA having 3 to 4 deoxynucleotides in the seed region of the antisense strand is in the range of 30 to 100 pM, which is very suitable for many applications including drug agents used in vivo.

[0218] Example 7: The structure of GST-π siRNA containing one or more 2'-deoxy-2'-fluoro-substituted nucleotides showed unexpectedly increased gene knockdown activity in vitro.

[0219] In vitro transfection was performed in the A549 cell line to determine the knockdown efficiency of GST-π siRNA based on Structure BU2’ (SEQ ID NOs: 131 and 157). As shown in Table 13, dose-dependent knockdown of GST-π mRNA was observed using GST-π siRNA based on Structure BU2’.

[0220]

Table 13

[0221] As shown in Table 13, the activity of GST-π siRNA based on Structure BU2’ having one or more 2'-F deoxynucleotides was surprisingly increased up to 10-fold compared to GST-π siRNA having no 2'-F deoxynucleotides.

[0222] These data indicate that GST-π siRNA having a structure with one or more 2'-F deoxynucleotides has unexpectedly high gene knockdown activity compared to GST-π siRNA having no 2'-F deoxynucleotides.

[0223] The activity shown in Table 13 for GST-π siRNA having one or more 2'-F deoxynucleotides was in the range of 3 - 13 pM and was very suitable for many applications including drug agents used in vivo.

[0224] Example 8: The structure of GST-π siRNA containing one or more 2'-deoxy-2'-fluoro-substituted nucleotides unexpectedly showed increased gene knockdown activity in vitro.

[0225] In vitro transfection was performed in the A549 cell line to determine the knockdown efficiency of GST-π siRNA based on Structure B13’ (SEQ ID NOs: 207 and 222). As shown in Table 14, dose-dependent knockdown of GST-π mRNA was observed with GST-π siRNA based on Structure B13'.

[0226]

Table 14

[0227] As shown in Table 14, the activity of GST-π siRNA based on Structure B13’ having three 2'-F deoxynucleotides at non-overhang positions was surprisingly increased by about 3-fold compared to GST-π siRNA having no 2'-F deoxynucleotides.

[0228] These data indicate that GST-π siRNA having a structure with one or more 2'-F deoxynucleotides has unexpectedly high gene knockdown activity compared to GST-π siRNA having no 2'-F deoxynucleotides.

[0229] The activities shown in Table 14 for GST-π siRNA having one or more 2'-F deoxynucleotides were in the picomolar range of 6 pM and were very suitable for many applications including drug agents used in vivo.

[0230] Example 9: Orthotopic A549 lung cancer mouse model. The GST-π siRNA of the present invention can show a significant reduction of orthotopic lung tumors in vivo. In this example, GST-π siRNA brought about gene knockdown efficacy in vivo when administered in a liposomal formulation to orthotopic lung tumors of athymic nude mice.

[0231] Generally, orthotopic tumor models can show direct clinical relevance with respect to drug efficacy and potency, as well as improved predictive ability. In an orthotopic tumor model, tumor cells are directly transplanted into the same organ from which the cells are derived.

[0232] The antitumor efficacy of the siRNA formulation against human lung cancer A549 was evaluated by comparing the final primary tumor weights measured at autopsy in the treatment group and the vehicle control group.

[0233] Figure 1 shows the in vivo orthotopic lung tumor inhibition of GST-π siRNA based on structure BU2 (SEQ ID NOs: 61 and 126). An orthotopic A549 lung cancer mouse model was used at a relatively low dose of 2 mg / kg of siRNA targeting GST-π.

[0234] GST-π siRNA showed significant and unexpectedly favorable lung tumor inhibition efficacy in this 6-week study. As shown in Figure 1, after 43 days, GST-π siRNA showed significantly favorable tumor inhibition efficacy, and the final tumor average weight was significantly reduced by 2.8-fold compared to the control.

[0235] For this study, male NCr nu / nu mice at 5 - 6 weeks of age were used. During the experimental period, the experimental animals were maintained in a HEPA - filtered environment. The siRNA formulation was stored at 4°C before use and warmed to room temperature 10 minutes before injection into the mice.

[0236] On the day of surgical orthotopic implantation (SOI), for this A549 human lung cancer orthotopic model, the stock tumor was harvested from the subcutaneous site of animals with A549 tumor xenografts and placed in RPMI - 1640 medium. Necrotic tissue was removed, and the viable tissue was cut into pieces of 1.5 - 2 mm 3 in length. The animals were anesthetized by isoflurane inhalation, and the surgical area was sterilized with iodine and alcohol. A transverse incision of approximately 1.5 cm was made on the left chest wall of the mice using a pair of surgical scissors. An intercostal incision was made between the third and fourth ribs to expose the left lung. One A549 tumor fragment was transplanted onto the surface of the lung with 8 - 0 surgical sutures (nylon). The chest wall was closed with 6 - 0 surgical sutures (silk). The lung was re - inflated by intra - thoracic puncture using a 3 cc syringe with a 25G×1 / 2 needle to draw out the remaining air in the thoracic cavity. The chest wall was closed with 6 - 0 surgical silk sutures. All procedures of the above operations were performed using a 7 - fold magnifying microscope under a HEPA - filtered laminar flow hood.

[0237] Three days after tumor transplantation, the tumor - bearing model mice were randomly divided into groups of 10 mice per group. For the target group, the treatment of 10 mice was started three days after tumor transplantation.

[0238] For the target group, the formulation was a liposomal composition of (ionizable lipid:cholesterol:DOPE:DOPC:DPPE - PEG - 2K:DSPE - PEG - 2K). The liposomes encapsulated GST - π siRNA.

[0239] Regarding the test endpoint, the experimental mice were sacrificed 42 days after the start of treatment. The primary tumors were excised and weighed on an electronic balance for subsequent analysis.

[0240] Regarding the evaluation of the toxicity of the compound, the average body weights of the mice in the treatment group and the control group were maintained within the normal range throughout the experimental period. No other symptoms of toxicity were observed in the mice.

[0241] Example 10: The GST-π siRNA of the present invention showed a significant reduction in cancer xenograft tumors in vivo. The GST-π siRNA brought about a gene knockdown effect in vivo when administered to cancer xenograft tumors in a liposomal formulation.

[0242] Figure 2 shows the tumor inhibitory efficacy of GST-π siRNA (SEQ ID NOs: 156 and 182). A cancer xenograft model was used at a relatively low dose of 0.75 mg / kg of siRNA targeting GST-π.

[0243] The GST-π siRNA showed a significant and unexpectedly favorable tumor inhibitory efficacy within several days after administration. After 36 days, the GST-π siRNA showed a significantly favorable tumor inhibitory efficacy, and the tumor volume decreased by 2-fold compared to the control.

[0244] As shown in Figure 3, the GST-π siRNA showed a significant and unexpectedly favorable tumor inhibitory efficacy on the last day. In particular, the tumor weight decreased by more than 2-fold.

[0245] The GST-π siRNA was administered as a liposomal formulation having a composition (ionizable lipid:cholesterol:DOPE:DOPC:DPPE-PEG-2K) (25:30:20:20:5) by two injections (on day 1 and day 15).

[0246] For the cancer xenograft model, the A549 cell line was obtained from ATCC. The cells were maintained in a culture medium supplemented with 10% fetal bovine serum, 100 U / ml penicillin, and 100 μg / ml streptomycin. The cells were split 48 hours before inoculation to be in the logarithmic growth phase at the time of harvest. The cells were gently trypsinized with trypsin-EDTA and recovered from tissue culture. The number of viable cells was counted and measured with a hemocytometer in the presence of trypan blue (only viable cells were counted). The cells were resuspended in serum-free medium at 5×107 It was resuspended at a concentration of / ml. Subsequently, the cell suspension was thoroughly mixed with ice-thawed BD Matrigel at a ratio of 1:1 and used for injection.

[0247] The mice were Charles River Laboratory's thymic nude mice (nu / nu) female mice, immunodeficient, 6 - 8 weeks old, with 7 - 8 mice per group.

[0248] For tumor model preparation, using a 25G needle and syringe per mouse for one inoculation dose, 0.1 ml of an inoculum of 2.5×10 6 A549 cells were subcutaneously inoculated into the right flank. The mice were not anesthetized for inoculation.

[0249] For tumor volume measurement and randomization, the tumor size was measured to the nearest 0.1 mm. The tumor volume was calculated using the formula: tumor volume = length × width 2 / 2. When the established tumors reached approximately 120 - 175 mm 3 the average tumor volume was approximately 150 mm 3 and the mice were assigned to various vehicle control and treatment groups so that the average tumor volume of the treatment group was within 10% of the average tumor volume of the vehicle control group. Ideally, the CV% of the tumor volume was less than 25%. On the same day, the test article and control vehicle were administered according to the dosing regimen. The tumor volume was monitored 3 times in the first week and 2 times in the remaining weeks including the end of the study.

[0250] For drug administration, on the administration day, the test article was taken out from the -80°C freezer and thawed on ice. Before applying to the syringe, the bottle containing the formulation was hand - inverted several times. All test articles were administered IV at 0.75 mg / kg, q2wX2, at 10 ml / kg.

[0251] Regarding body weight, the mice were weighed to the nearest 0.1 g. The body weight was monitored and recorded daily within 7 days of the first administration. In the remaining weeks including the end of the study, the body weight was monitored for several weeks and recorded twice a week.

[0252] To collect tumors 28 days after the initial dosing, tumor volumes were measured, tumors were dissected for weighing, and stored for PD biomarker studies. Tumor weights were recorded.

[0253] Example 11: The GST-π siRNA of the present invention showed an increase in cancer cell death by apoptosis of cancer cells in vitro. GST-π siRNA brought about GST-π knockdown, resulting in upregulation of PUMA, a biomarker of apoptosis, along with a decrease in cell viability.

[0254] The GST-π siRNA SEQ ID NOs: 156 and 182, which contain a combination of deoxynucleotides, 2'-F-substituted deoxynucleotides, and 2'-OMe-substituted ribonucleotides in the seed region, unexpectedly increased apoptosis of cancer cells.

[0255] The expression levels of PUMA for GST-π siRNA SEQ ID NOs: 156 and 182 are shown in Figure 4. As shown in Figure 4, the expression of PUMA increased significantly 2 - 4 days after transfection with GST-π siRNA.

[0256] These data indicate that the structure of GST-π siRNA containing a combination of deoxynucleotides, 2'-F-substituted deoxynucleotides, and 2'-OMe-substituted ribonucleotides in the seed region unexpectedly increases apoptosis of cancer cells.

[0257] The protocol for the PUMA biomarker was as follows. One day before transfection, 100 μl of DMEM (HyClone catalog number SH30243.01) containing 10% FBS was added to a 96-well plate at 2×10 3Cells were seeded in wells and cultured in a 37°C incubator with a humidified atmosphere in 5% CO2 air. The next day, prior to transfection, the medium was exchanged with 90 μl of Opti-MEM I reduced serum medium (Life Technologies catalog number 31985-070) containing 2% FBS. Then, 0.2 μl of Lipofectamine RNAiMAX (Life Technologies catalog number 13778-100) was mixed with 4.8 μl of Opti-MEM I at room temperature for 5 minutes. 1 μl of siRNA (stock concentration 1 μM) was mixed with 4 μl of Opti-MEM I, mixed with the RNAiMAX solution, and then gently mixed. The mixture was incubated at room temperature for 10 minutes to form an RNA-RNAiMAX complex. 10 μl of the RNA-RNAiMAX complex was added per well to give a final siRNA concentration of 10 nM. The cells were incubated for 2 hours and the medium was exchanged with fresh Opti-MEM I reduced serum medium containing 2% FBS. At 1, 2, 3, 4 and 6 days after transfection, the cells were washed once with ice-cold PBS and then lysed at room temperature for approximately 5 - 30 minutes using 50 μl of Cell-to-Ct lysis buffer (Life Technologies catalog number 4391851C). 5 μl of stop solution was added and incubated at room temperature for 2 minutes. The mRNA level of PUMA (BBC3, Cat#Hs00248075, Life Technologies) was measured by qPCR using TAQMAN.

[0258] Example 12: The GST-π siRNA of the present invention was able to show a significant reduction in cancer xenograft tumors in vivo. The GST-π siRNA can provide gene knockdown efficacy in vivo when administered to cancer xenograft tumors in a liposomal formulation.

[0259] Figure 5 shows the tumor inhibitory efficacy of GST-π siRNA (SEQ ID NOs: 61 and 126). Dose-dependent knockdown of GST-π mRNA was observed in vivo with siRNA targeting GST-π. A cancer xenograft model was used with a relatively low dose of 0.75 mg / kg of siRNA targeting GST-π.

[0260] GST-π siRNA showed significant and unexpectedly favorable tumor inhibitory efficacy within several days after administration. As shown in Figure 5, treatment with GST-π siRNA significantly decreased GST-π mRNA expression 4 days after injection into the lipid formulation. At a high dose of 4 mg / kg, a significant decrease of about 40% was detected 24 hours after injection.

[0261] GST-π siRNA was administered as a single injection as a 10 mL / kg liposomal formulation with a composition (ionizable lipid:cholesterol:DOPE:DOPC:DPPE-PEG-2K) (25:30:20:20:5).

[0262] For the cancer xenograft model, the A549 cell line was obtained from ATCC. The cells were maintained in RPMI-1640 supplemented with 10% fetal bovine serum, 100 U / ml penicillin, and 100 μg / ml streptomycin. The cells were split 48 hours before inoculation to be in the logarithmic growth phase at the time of harvest. The cells were gently trypsinized with trypsin-EDTA and harvested from tissue culture. The number of viable cells was counted and measured with a hemocytometer in the presence of trypan blue (counting only viable cells). The cells were resuspended in serum-free RPMI medium at a concentration of 4×10 7 / ml. The cell suspension was then thoroughly mixed with ice-thawed BD Matrigel at a 1:1 ratio and used for injection.

[0263] The mice were Charles River Laboratory thymic nude (nu / nu) female mice, immunodeficient, 6 - 8 weeks old, with 3 mice per group.

[0264] For tumor model preparation, 0.1 ml of an inoculum of 2×10 6 A549 cells was subcutaneously inoculated into the right flank of each mouse using a 25G needle and syringe per inoculum dose. The mice were not anesthetized for inoculation.

[0265] For tumor volume measurement and randomization, tumor size was measured to the nearest 0.1 mm. Tumor volume was calculated using the formula: Tumor volume = length × width 2 / 2. Tumor volume was monitored twice a week. When the established tumor reached approximately 350 - 600 mm 3 the mice were assigned to groups at various time points. On the same day, the test article was administered according to the dosing regimen.

[0266] For drug administration, on the day when the established tumor reached approximately 350 - 600 mm 3 the test article was removed from the refrigerator at 4°C. Before applying to the syringe, the bottle containing the formulation was inverted by hand several times to make a homogeneous solution.

[0267] For body weight, the mice were weighed to the nearest 0.1 g. Body weight was monitored for several weeks including the end day of the study and recorded twice a week.

[0268] For tumor collection, the animals were sacrificed with excess CO2 and the tumors were dissected at 0, 24, 48, 72, and 96 (optional) and 168 hours after administration. The tumors were first moistened and divided into three parts for KD, distribution, and biomarker analysis. The samples were snap-frozen in liquid nitrogen and stored at -80°C until ready to be processed.

[0269] Example 13: The GST-π siRNA of the present invention inhibited pancreatic cancer xenograft tumors in vivo. The GST-π siRNA produced gene knockdown efficacy in vivo when administered to pancreatic cancer xenograft tumors in a liposomal formulation.

[0270] In this xenograft model, each mouse was subcutaneously inoculated in the right flank with 0.1 ml of a 2.5×10 6 PANC-1 cell inoculum. Thymus-deficient nude female mice (6 - 8 weeks old, Charles River) were used. Tumor size was measured to the nearest 0.1 mm. When the established tumor reached approximately 150 - 250 mm 3 (average tumor volume approximately 200 mm 3) When it reached, the mice were assigned to various vehicle controls and treatment groups such that the average tumor volume in the treatment group was within 10% of the average tumor volume in the vehicle control group. On the same day, the test article and the control vehicle were administered according to the dosing regimen. Tumor volume was monitored 3 times in the first week and 2 times in the remaining weeks including the end of the study.

[0271] Figure 6 shows the tumor inhibitory efficacy of GST-π siRNA (SEQ ID NOs: 63 and 128). As shown in Figure 6, the dose response was obtained at doses in the range of 0.375 mg / kg to 3 mg / kg of siRNA targeting GST-π. GST-π siRNA showed significant and unexpectedly favorable tumor inhibitory efficacy within several days after administration. Therefore, GST-π siRNA showed significant and unexpectedly favorable tumor inhibitory efficacy at the endpoint.

[0272] GST-π siRNA was administered in a liposomal formulation having a composition (ionizable lipid:cholesterol:DOPE:DOPC:DPPE-PEG-2K) (25:30:20:20:5).

[0273] Example 14: The GST-π siRNA of the present invention showed increased serum stability.

[0274] Figure 7 shows the incubation in human serum and the detection of the remaining siRNA at various time points by HPLS / LCMS. As shown in Figure 7, the half-life (t 1 / 2 ) in serum of both the sense strand (Figure 7, upper panel) and the antisense strand (Figure 7, lower panel) of GST-π siRNA (SEQ ID NOs: 61 and 126) was approximately 100 minutes.

[0275] Example 15: The GST-π siRNA of the present invention showed improved stability in the formulation in plasma.

[0276] Figure 8 shows the incubation of the formulation in plasma and the detection of the remaining siRNA at various time points. As shown in Figure 8, the plasma half-life (t 1 / 2) was significantly longer than 100 hours.

[0277] GST-π siRNA was prepared in a liposomal formulation having the composition (ionizable lipid:cholesterol:DOPE:DOPC:DPPE-PEG-2K) (25:30:20:20:5). The z-average size of the liposomal nanoparticles was 40.0 nm and the siRNA was 91% encapsulated.

[0278] The formulation was incubated in 50% human serum in PBS for 40 minutes, 1.5 hours, 3 hours, 24 hours, and 96 hours. The amount of GST-π siRNA was determined by an ELISA-based assay.

[0279] Example 16: The GST-π siRNA of the present invention showed a reduced off-target effect by the passenger strand.

[0280] For GST-π siRNA (SEQ ID NOs: 156 and 182), Figure 9 shows that the in vitro knockdown for the guide strand was almost exponential compared to a control having a scrambled sequence that showed no effect. The IC50 of this siRNA was measured at 5 pM. Figure 10 shows the in vitro knockdown of the passenger strand of the same GST-π siRNA. As shown in Figure 10, the off-target knockdown of the passenger strand for GST-π siRNA was reduced by more than 100-fold.

[0281] For GST-π siRNA (SEQ ID NOs: 187 and 199), (SEQ ID NOs: 189 and 201), and (SEQ ID NOs: 190 and 202), Figure 11 shows that the in vitro knockdown of the guide strand was approximately exponential. The IC50 of these siRNAs was measured at 6, 7, and 5 pM, respectively. As shown in Figure 12, the in vitro knockdown of the passenger strand of these GST-π siRNAs was significantly reduced by at least 10-fold. All of these GST-π siRNAs had deoxynucleotides in the seed region of the duplex region and no other modifications in the duplex region.

[0282] For GST-π siRNA (SEQ ID NOs: 217 and 232), FIG. 13 shows that the in vitro knockdown of the guide strand of this highly active GST-π siRNA was almost exponential. The IC50 of this siRNA was measured at 11 pM. As shown in FIG. 14, the in vitro knockdown of the passenger strand of this GST-π siRNA decreased significantly by more than 100-fold. This GST-π siRNA had deoxynucleotides in the seed region of the double-stranded region, and there were no other modifications in the double-stranded region.

[0283] The off-target effect was determined using the expression reporter plasmid psiCHECK-2 encoding the Renilla luciferase gene (Dual-Luciferase Reporter Assay System, Promega, Catalog No.: E1960). The siRNA concentration was typically 50 pM. Protocol: On day 1, HeLa cells were seeded at 5 - 7.5×10 3 / 100 μl / well. On day 2, co-transfection was performed at approximately 80% cell confluence. On day 3, the cells were harvested and luciferase activity was measured. Luciferase activity was measured using the Promega luciferase assay system (E4550) according to the manufacturer's protocol.

[0284] The psiCHECK-2 vector can monitor changes in the expression of the target gene fused to the reporter gene of Renilla luciferase. The siRNA construct was cloned into the multiple cloning region, and the vector was co-transfected into HeLa cells with the siRNA. When a specific siRNA binds to the target mRNA and initiates the RNAi process, the fusion Renilla luciferase:construct mRNA is cleaved and subsequently degraded, reducing the Renilla luciferase signal.

[0285] For example, the plasmid insert for siRNA having the BU2' structure was as follows: PsiCHECK-2 (F) plasmid insert: Accession number 285 ctcgag gggcaacTGAAGCCTTTTGAGACCCTGcTgTcccag gcggccgc PsiCHECK-2 (R) plasmid insert: Accession number 286 ctcgag cTgggacagCAGGGTCTCAAAAGGCTTCagTTgccc gcggccgc

[0286] Example 17: The GST-π siRNA of the present invention was shown to have a significantly reduced miRNA-like off-target effect, which is seed-dependent unintended off-target gene silencing.

[0287] For GST-π siRNAs (SEQ ID NOs: 156 and 182), (SEQ ID NOs: 187 and 199), (SEQ ID NOs: 189 and 201), (SEQ ID NOs: 190 and 202), and (SEQ ID NOs: 217 and 232), the off-target activity mimicking miRNA was found to be essentially negligible. The seed-dependent unintended off-target gene silencing of these GST-π siRNAs was at least 10- to 100-fold less than the target activity of the guide strand.

[0288] To test the miRNA-related off-target effect, seed-complementary target sequences with 1 to 4 repeats that are complementary to the entire seed-containing region at positions 1 to 8 at the 5'-end of the antisense strand but not to the remaining non-seed region (positions 9 to 21) were introduced into the region corresponding to the 3'UTR of luciferase mRNA to determine the efficiency of the seed-dependent unintended off-target effect. Using plasmid inserts, miRNAs with perfect matching in the seed region and mismatches (bulges) in the non-seed region were mimicked.

[0289] For example, the plasmid insert for the siRNA having the BU2' structure was as follows: PsiCHECK-2 (Fmi1) plasmid insert: Accession number 287 ctcgag gggcaacTCTACGCAAAACAGACCCTGcTgTcccag gcggccgc PsiCHECK-2 (Fmi2) plasmid insert: SEQ ID NO: 288 ctcgag gggcaacTCTACGCAAAACAGACCCTGcT CTACGCAAAACAGACCCTGcT gTcccag gcggccgc PsiCHECK-2 (Fmi3) plasmid insert: SEQ ID NO: 289 ctcgag gggcaacTCTACGCAAAACAGACCCTGcT CTACGCAAAACAGACCCTGcT CTACGCAAAACAGACCCTGcT gTcccag gcggccgc PsiCHECK-2 (Fmi4) plasmid insert: SEQ ID NO: 290 ctcgag gggcaacTCTACGCAAAACAGACCCTGcT CTACGCAAAACAGACCCTGcT CTACGCAAAACAGACCCTGcT CTACGCAAAACAGACCCTGcT gTcccag gcggccgc

[0290] The embodiments described herein are not limiting, and one of ordinary skill in the art can readily understand that specific combinations of the modifications described herein can be tested without undue experimentation to identify nucleic acid molecules having improved RNAi activity.

[0291] All publications, patents, and documents specifically mentioned herein are hereby incorporated by reference in their entirety for all purposes.

[0292] The present invention is not limited to the specific methodologies, protocols, materials, and reagents described, and it is understood that these can vary. It should also be understood that the terminology used herein is for the purpose of describing only specific embodiments and is not intended to limit the scope of the present invention. It will be readily apparent to those skilled in the art that various substitutions and modifications can be made to the descriptions disclosed herein without departing from the scope and spirit of the description, and that these embodiments are within the scope of this description and the appended claims.

[0293] Note that the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise. Similarly, the terms "a" (or "an"), "one or more", and "at least one" can be used interchangeably herein. The terms "comprises", "comprising", "containing", "including", and "has" can be used interchangeably and should be read broadly and without limitation.

[0294] The description of ranges of values herein is intended to serve as a shorthand method of referring individually to each separate value within the range, unless otherwise indicated herein, and each separate value is incorporated as if it were individually recited. Here, with respect to Markush groups, those skilled in the art will recognize that this description includes the individual members as well as sub-groups of members of the Markush group.

[0295] Without further elaboration, it is believed that one skilled in the art can, based on the above description, utilize the present invention to its fullest extent. Accordingly, the following specific embodiments are to be construed as merely illustrative and in no way limit the remainder of the disclosure.

[0296] All features disclosed herein can be combined in any combination. Each feature disclosed herein can be replaced by an alternative feature that serves the same, equivalent, or similar purpose.

[0297] SEQUENCE LISTING <110> NITTO DENKO CORPORATION <120> RNA INTERFERENCE AGENTS FOR GST-PI GENE MODULATION <130> P21-0878 <150> JP 2014-266198 <151> 2014-12-26 <150> US 62 / 184,239 <151> 2015-06-24 <150> US 62 / 266,664 <151> 2015-12-13 <160> 290 <170> PatentIn version 3.5 <210> 1 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 1 ucccagaacc agggaggcat t 21 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 2 cuuuugagac ccugcuguct t 21 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 3 cugucccaga accagggagt t 21 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 4 ugucccagaa ccagggaggt t 21 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 5 aagccuuuug agacccugct t 21 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 6 uugagacccu gcugucccat t 21 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 7 uuuugagacc cugcugucct t 21 <210> 8 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 8 gagacccugc ugucccagat t 21 <210> 9 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 9 gcuggaagga ggagguggut t 21 <210> 10 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 10 cuggaaggag gagguggugt t 21 <210> 11 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 11 ucagggccag agcuggaagt t 21 <210> 12 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 12 ugagacccug cugucccagt t 21 <210> 13 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 13 agggccagag cuggaaggat t 21 <210> 14 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 14 agcuggaagg aggagguggt t 21 <210> 15 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 15 agacccugcu gucccagaat t 21 <210> 16 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 16 gagcuggaag gaggaggugt t 21 <210> 17 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 17 ugcuguccca gaaccagggt t 21 <210> 18 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 18 cccagaacca gggaggcaat t 21 <210> 19 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 19 ccagaaccag ggaggcaagt t 21 <210> 20 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 20 uuugagaccc ugcuguccct t 21 <210> 21 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 21 gacccugcug ucccagaact t 21 <210> 22 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 22 gaucagggcc agagcuggat t 21 <210> 23 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 23 agccuuuuga gacccugcut t 21 <210> 24 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 24 gccuuuugag acccugcugt t 21 <210> 25 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 25 ccuuuugaga cccugcugut t 21 <210> 26 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 26 cgccuuuuga gacccugcat t 21 <210> 27 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 27 ccuacaccgu ggucuauuut t 21 <210> 28 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 28 ugugggagac cagaucucct t 21 <210> 29 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 29 gcgggaggca gaguuugcct t 21 <210> 30 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 30 ccuuucucca ggaccaauat t 21 <210> 31 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 31 acccugcugu cccagaacct t 21 <210> 32 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 32 ggucuauuuc ccaguucgat t 21 <210> 33 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 33 cccuggugga cauggugaat t 21 <210> 34 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 34 acaucucccu caucuacact t 21 <210> 35 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 35 gcaaggauga cuaugugaat t 21 <210> 36 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 36 ccuucgcuga cuacaaccut t 21 <210> 37 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 37 cuggcagauc agggccagat t 21 <210> 38 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 38 gacggagacc ucacccugut t 21 <210> 39 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 39 cgggcaagga ugacuaugut t 21 <210> 40 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 40 cuuuugagac ccugcuguat t 21 <210> 41 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 41 gagcuggaag gaggagguat t 21 <210> 42 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 42 acccugcugu cccagaacat t 21 <210> 43 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 43 ugcuguccca gaaccaggat t 21 <210> 44 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 44 agccuuuuga gacccugcat t 21 <210> 45 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 45 ccuuuugaga cccugcugat t 21 <210> 46 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 46 ugaagccuuu ugagacccut t 21 <210> 47 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 47 acugaagccu uuugagacct t 21 <210> 48 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 48 aggaugacua ugugaaggct t 21 <210> 49 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 49 ggaugacuau gugaaggcat t 21 <210> 50 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 50 gaugacuaug ugaaggcact t 21 <210> 51 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 51 cucccucauc uacaccaact t 21 <210> 52 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 52 gaagccuuuu gagacccugt t 21 <210> 53 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 53 ucucccucau cuacaccaat t 21 <210> 54 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 54 ccucaucuac accaacuaut t 21 <210> 55 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 55 cccucaucua caccaacuat t 21 <210> 56 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 56 caacugaagc cuuuugagat t 21 <210> 57 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 57 aacugaagcc uuuugagact t 21 <210> 58 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 58 cugaagccuu uugagaccct t 21 <210> 59 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 59 ucccucaucu acaccaacut t 21 <210> 60 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 60 gcucccucau cuacaccaat t 21 <210> 61 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 61 gaagccuuuu gagacccuat t 21 <210> 62 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 62 acugaagccu uuugagacat t 21 <210> 63 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 63 cucccucauc uacaccaaat t 21 <210> 64 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 64 ccucaucuac accaacuaat t 21 <210> 65 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 65 accaauaaaa uuucuaagat t 21 <210> 66 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 66 ugccucccug guucugggac a 21 <210> 67 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 67 gacagcaggg ucucaaaagg c 21 <210> 68 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 68 cucccugguu cugggacagc a 21 <210> 69 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 69 ccucccuggu ucugggacag c 21 <210> 70 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 70 gcagggucuc aaaaggcuuc a 21 <210> 71 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 71 ugggacagca gggucucaaa a 21 <210> 72 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 72 ggacagcagg gucucaaaag g 21 <210> 73 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 73 ucugggacag cagggucuca a 21 <210> 74 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 74 accaccuccu ccuuccagct c 21 <210> 75 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 75 caccaccucc uccuuccagc t 21 <210> 76 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 76 cuuccagcuc uggcccugat c 21 <210> 77 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 77 cugggacagc agggucucaa a 21 <210> 78 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 78 uccuuccagc ucuggcccug a 21 <210> 79 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 79 ccaccuccuc cuuccagcuc t 21 <210> 80 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 80 uucugggaca gcagggucuc a 21 <210> 81 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 81 caccuccucc uuccagcuct g 21 <210> 82 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 82 cccugguucu gggacagcag g 21 <210> 83 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 83 uugccucccu gguucuggga c 21 <210> 84 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 84 cuugccuccc ugguucuggg a 21 <210> 85 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 85 gggacagcag ggucucaaaa g 21 <210> 86 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 86 guucugggac agcaggguct c 21 <210> 87 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Artificial Sequence Description: Synthetic oligonucleotide <220> <223> Combined DNA / RNA Molecule Description: Synthetic oligonucleotide <400> 87 uccagcucug gcccugauct g 21 <210> 88 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Artificial Sequence Description: Synthetic oligonucleotide <220> <223> Combined DNA / RNA Molecule Description: Synthetic oligonucleotide <400> 88 agcagggucu caaaaggcut c 21 <210> 89 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Artificial Sequence Description: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 89 cagcaggguc ucaaaaggct t 21 <210> 90 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 90 acagcagggu cucaaaaggc t 21 <210> 91 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 91 ugcagggucu caaaaggcgt c 21 <210> 92 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 92 aaauagacca cgguguaggg c 21 <210> 93 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 93 ggagaucugg ucucccacaa t 21 <210> 94 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 94 ggcaaacucu gccucccgct c 21 <210> 95 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 95 uauugguccu ggagaaagga a 21 <210> 96 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 96 gguucuggga cagcaggguc t 21 <210> 97 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 97 ucgaacuggg aaauagacca c 21 <210> 98 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 98 uucaccaugu ccaccagggc t 21 <210> 99 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 99 guguagauga gggagaugua t 21 <210> 100 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 100 uucacauagu cauccuugcc c 21 <210> 101 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 101 agguuguagu cagcgaagga g 21 <210> 102 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 102 ucuggcccug aucugccagc a 21 <210> 103 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 103 acagggugag gucuccgucc t 21 <210> 104 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 104 acauagucau ccuugcccgc c 21 <210> 105 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 105 uacagcaggg ucucaaaagg c 21 <210> 106 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 106 uaccuccucc uuccagcuct g 21 <210> 107 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 107 uguucuggga cagcaggguc t 21 <210> 108 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 108 uccugguucu gggacagcag g 21 <210> 109 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 109 ugcagggucu caaaaggcut c 21 <210> 110 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 110 ucagcagggu cucaaaaggc t 21 <210> 111 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 111 agggucucaa aaggcuucag t 21 <210> 112 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 112 ggucucaaaa ggcuucagut g 21 <210> 113 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 113 gccuucacau agucauccut g 21 <210> 114 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 114 ugccuucaca uagucaucct t 21 <210> 115 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 115 gugccuucac auagucaucc t 21 <210> 116 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 116 guugguguag augagggaga t 21 <210> 117 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 117 cagggucuca aaaggcuuca g 21 <210> 118 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 118 uugguguaga ugagggagat g 21 <210> 119 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 119 auaguuggug uagaugaggg a 21 <210> 120 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 120 uaguuggugu agaugaggga g 21 <210> 121 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined D NA / RNA Molecule: Synthetic oligonucleotide <400> 121 ucucaaaagg cuucaguugc c 21 <210> 122 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 122 gucucaaaag gcuucaguug c 21 <210> 123 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 123 gggucucaaa aggcuucagt t 21 <210> 124 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 124 aguuggugua gaugagggag a 21 <210> 125 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 125 uugguguaga ugagggagct g 21 <210> 126 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 126 uagggucuca aaaggcuuca g 21 <210> 127 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 127 ugucucaaaa ggcuucagut g 21 <210> 128 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 128 uuugguguag augagggaga t 21 <210> 129 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 129 uuaguuggug uagaugaggg a 21 <210> 130 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 130 ucuuagaaau uuuauugguc c 21 <210> 131 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <220> <221> modified_base <222> (20)..(21) <223> a, c, t, g, u, unknown or other <400> 131 gaagccuuuu gagacccuan n 21 <210> 132 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 132 gaagccuuuu gagacccuau u 21 <210> 133 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 133 gaagccuuuu gagacccuau u 21 <210> 134 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 134 gaagccuuuu gagacccuau u 21 <210> 135 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 135 gaagccuuuu gagacccuau u 21 <210> 136 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 136 gaagccuuuu gagacccuau u 21 <210> 137 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 137 gaagccuuuu gagacccuau u 21 <210> 138 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 138 gaagccuuuu gagacccuau u 21 <210> 139 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 139 gaagccuuuu gagacccuau u 21 <210> 140 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 140 gaagccuuuu gagacccuau u 21 <210> 141 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 141 gaagccuuuu gagacccuau u 21 <210> 142 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 142 gaagccuuuu gagacccuat t 21 <210> 143 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Artificial Sequence Description: Synthetic oligonucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 143 gaagccuuuu gagacccuau u 21 <210> 144 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Artificial Sequence Description: Synthetic oligonucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 144 gaagccuuuu gagacccuau u 21 <210> 145 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Artificial Sequence Description: Synthetic oligonucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 145 gaagccuuuu gagacccuau u 21 <210> 146 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 146 gaagccuuuu gagacccuau u 21 <210> 147 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (19)..(19) <223> 2'-deoxy-2'-fluoro-nucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 147 gaagccuuuu gagacccuau u 21 <210> 148 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (14)..(14) <223> 2'-deoxy-2'-fluoro-nucleotide <220> <221> modified_base <222> (19)..(19) <223> 2'-deoxy-2'-fluoro-nucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 148 gaagccuuuu gagacccuau u 21 <210> 149 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (19)..(21) <223> 2'-OMe-nucleotide <400> 149 gaagccuuuu gagacccuau u 21 <210> 150 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (19)..(21) <223> 2'-OMe-nucleotide <400> 150 gaagccuuuu gagacccuau u 21 <210> 151 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (1)..(3) <223> 2'-OMe-nucleotide <220> <221> modified_base <222> ( 20)..(21) <223> 2'-OMe-nucleotide <400> 151 gaagccuuuu gagacccuau u 21 <210> 152 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (1)..(3) <223> 2'-OMe-nucleotide <220> <221> modified_base <222> (8)..(8) <223> 2'-OMe-nucleotide <220> <221> modified_base <222> (10)..(10) <223> 2'-OMe-nucleotide <220> <221> modified_base <222> (18)..(18) <223> 2'-OMe-nucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 152 gaagccuuuu gagacccuau u 21 <210> 153 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (1)..(3) <223> 2'-OMe-nucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 153 gaagccuuuu gagacccuau u 21 <210> 154 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (1)..(3) <223> 2'-OMe-nucleotide <220> <221> modified_base <222> (8)..(8) <223> 2'-OMe-nucleotide <220> <221> modified_base <222> (10)..(10) <223> 2'-OMe-nucleotide <220> <221> modified_base <222> (18)..(18) <223> 2'-OMe-nucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 154 gaagccuuuu gagacccuau u 21 <210> 155 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (1)..(3) <223> 2'-OMe-nucleotide <220> <221> modified_base <222> (8)..(8) <223> 2'-OMe-nucleotide <220> <221> modified_base <222> (10)..(10) <223> 2'-OMe-nucleotide <220> <221> modified_base <222> (18)..(21) <223> 2'-OMe-nucleotide <400> 155 gaagccuuuu gagacccuau u 21 <210> 156 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (1)..(3) <223> 2'-OMe-nucleotide <220> <221> modified_base <222> (8)..(8) <223> 2'-OMe-nucleotide <220> <221> modified_base <222> (10)..(10) <223> 2'-OMe-nucleotide <220> <221> modified_base <222> (18)..(21) <223> 2'-OMe-nucleotide <400> 156 gaagccuuuu gagacccuau u 21 <210> 157 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <220> <221> modified_base <222> (20)..(21) <223> a, c, t, g, u, unknown or other <400> 157 uagggucuca aaaggcuucn n 21 <210> 158 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 158 uagggucuca aaaggcuucu u 21 <210> 159 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <220> <221> modified_base <222> (1)..(1) <223> 2'-deoxy-nucleotide <220> <221> modified_base <222> (6)..(6) <223> 2'-deoxy-nucleotide <220> <221> modified_base <222> (8)..(8) <223> 2'-deoxy-nucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 159 uagggucuca aaaggcuucu u 21 <210> 160 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <220> <221> modified_base <222> (6)..(6) <223> 2'-deoxy-nucleotide <220> <221> modified_base <222> (8)..(8) <223> 2'-deoxy-nucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 160 uagggucuca aaaggcuucu u 21 <210> 161 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <220> <221> modified_base <222> (6)..(6) <223> 2'-deoxy-nucleotide <220> <221> modified_base <222> (8)..(8) <223> 2'-deoxy-nucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 161 uagggucuca aaaggcuucu u 21 <210> 162 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <220> <221> modified_base <222> (6)..(6) <223> 2'-deoxy-nucleotide <220> <221> modified_base <222> (8)..(8) <223> 2'-deoxy-nucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 162 uagggucuca aaaggcuucu u 21 <210> 163 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <220> <221> modified_base <222> (6)..(6) <223> 2'-deoxy-nucleotide <220> <221> modified_base <222> (8)..(8) <223> 2'-deoxy-nucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 163 uagggucuca aaaggcuucu u 21 <210> 164 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <220> <221> modified_base <222> (1)..(1) <223> 2'-deoxy-nucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 164 uagggucuca aaaggcuucu u 21 <210> 165 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 165 uagggucuca aaaggcuucu u 21 <210> 166 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <220> <221> modified_base <222> (6)..(6) <223> 2'-deoxy-nucleotide <220> <221> modified_base <222> (8)..(8) <223> 2'-deoxy-nucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 166 uagggucuca aaaggcuucu u 21 <210> 167 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <220> <221> modified_base <222> (6)..(6) <223> 2'-deoxy-nucleotide <220> <221> modified_base <222> (8)..(8) <223> 2'-deoxy-nucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 167 uagggucuca aaaggcuucu u 21 <210> 168 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <220> <221> modified_base <222> (6)..(6) <223> 2'-deoxy-nucleotide <220> <221> modified_base <222> (8)..(8) <223> 2'-deoxy-nucleotide <220> <221> modified_base <222> (14)..(14) <223> 2'-OMe-nucleotide <220> <221> modified_base <222> (16)..(16) <223> 2'-OMe-nucleotide <220> <221> modified_base <222> (18)..(18) <223> 2'-OMe-nucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 168 uagggucuca aaaggcuucu u 21 <210> 169 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (1)..(1) <223> 2'-OMe-nucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 169 uagggucuca aaaggcuucu u 21 <210> 170 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (1)..(1) <223> 2'-deoxy-2'-fluoro-nucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 170 uagggucuca aaaggcuucu u 21 <210> 171 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <220> <221> modified_base <222> (1)..(1) <223> 2'-deoxy-nucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 171 uagggucuca aaaggcuucu u 21 <210> 172 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (1)..(2) <223> Phosphorothioate linkage <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 172 uagggucuca aaaggcuucu u 21 <210> 173 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (1)..(1) <223> 2'-deoxy-2'-fluoro-nucleotide <220> <221> modified_base <222> (9)..(9) <223> 2'-deoxy-2'-fluoro-nucleotide <220> <221> modified_base <222> (17)..(17) <223> 2'-deoxy-2'-fluoro-nucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 173 uagggucuca aaaggcuucu u 21 <210> 174 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (1)..(1) <223> 2'-deoxy-2'-fluoro-nucleotide <220> <221> modified_base <222> (9)..(9) <223> 2'-deoxy-2'-fluoro-nucleotide <220> <221> modified_base <222> (11)..(12) <223> 2'-deoxy-2'-fluoro-nucleotide <220> <221> modified_base <222> (17)..(17) <223> 2'-deoxy-2'-fluoro-nucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 174 uagggucuca aaaggcuucu u 21 <210> 175 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (1)..(1) <223> 2'-OMe-nucleotide <220> <221> modified_base <222> (9)..(9) <223> 2'-OMe-nucleotide <220> <221> modified_base <222> (17)..(17) <223> 2'-OMe-nucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 175 uagggucuca aaaggcuucu u 21 <210> 176 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (1)..(1) <223> 2'-deoxy-2'-fluoro-nucleotide <220> <221> modified_base <222> (9)..(9) <223> 2'-OMe-nucleotide <220> <221> modified_base <222> (17)..(17) <223> 2'-OMe-nucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 176 uagggucuca aaaggcuucu u 21 <210> 177 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 177 uagggucuca aaaggcuucu u 21 <210> 178 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <220> <221> modified_base <222> (6)..(6) <223> 2'-deoxy-nucleotide <220> <221> modified_base <222> (8)..(8) <223> 2'-deoxy-nucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 178 uagggucuca aaaggcuucu u 21 <210> 179 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <220> <221> modified_base <222> (1)..(1) <223> 2'-OMe-nucleotide <220> <221> modified_base <222> (6)..(6) <223> 2'-deoxy-nucleotide <220> <221> modified_base <222> (8)..(8) <223> 2'-deoxy-nucleotide <220> <221> modified_base <222> (9)..(9) <223> 2'-OMe-nucleotide <220> <221> modified_base <222> (11)..(12) <223> 2'-OMe-nucleotide <220> <221> modified_base <222> (18)..(18) <223> 2'-OMe-nucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 179 uagggucuca aaaggcuucu u 21 <210> 180 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <220> <221> modified_base <222> (1)..(1) <223> 2'-OMe-nucleotide <220> <221> modified_base <222> (6)..(6) <223> 2'-deoxy-nucleotide <220> <221> modified_base <222> (8)..(8) <223> 2'-deoxy-nucleotide <220> <221> modified_base <222> (9)..(9) <223> 2'-OMe-nucleotide <220> <221> modified_base <222> (11)..(12) <223> 2'-OMe-nucleotide <220> <221> modified_base <222> (18)..(18) <223> 2'-OMe-nucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 180 uagggucuca aaaggcuucu u 21 <210> 181 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <220> <221> modified_base <222> (1)..(1) <223> 2'-OMe-nucleotide <220> <221> modified_base <222> (6)..(6) <223> 2'-deoxy-nucleotide <220> <221> modified_base <222> (8)..(8) <223> 2'-deoxy-nucleotide <220> <221> modified_base <222> (9)..(9) <223> 2'-OMe-nucleotide <220> <221> modified_base <222> (11)..(12) <223> 2'-OMe-nucleotide <220> <221> modified_base <222> (17)..(18) <223> 2'-OMe-nucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 181 uagggucuca aaaggcuucu u 21 <210> 182 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <220> <221> modified_base <222> (1)..(1) <223> 2'-deoxy-2'-fluoro-nucleotide <220> <221> modified_base <222> (6)..(6) <223> 2'-deoxy-nucleotide <220> <221> modified_base <222> (8)..(8) <223> 2'-deoxy-nucleotide <220> <221> modified_base <222> (9)..(9) <223> 2'-OMe-nucleotide <220> <221> modified_base <222> (11)..(12) <223> 2'-OMe-nucleotide <220> <221> modified_base <222> (17)..(18) <223> 2'-OMe-nucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 182 uagggucuca aaaggcuucu u 21 <210> 183 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <220> <221> modified_base <222> (20)..(21) <223> a, c, t, g, u, unknown or other <400> 183 ccuuuugaga cccugcugun n 21 <210> 184 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 184 ccucaucuac accaacuauu u 21 <210> 185 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modifi ed_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 185 ccucaucuac accaacuauu u 21 <210> 186 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 186 ccucaucuac accaacuauu u 21 <210> 187 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 187 ccucaucuac accaacuauu u 21 <210> 188 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 188 ccucaucuac accaacuauu u 21 <210> 189 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 189 ccucaucuac accaacuauu u 21 <210> 190 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 190 ccucaucuac accaacuauu u 21 <210> 191 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 191 ccucaucuac accaacuauu u 21 <210> 192 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 192 ccucaucuac accaacuauu u 21 <210> 193 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 193 ccucaucuac accaacuauu u 21 <210> 194 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (1)..(1) <223> 2'-OMe-nucleotide <220> <221> modified_base <222> (3)..(3) <223> 2'-OMe-nucleotide <220> <221> modified_base <222> (5)..(5) <223> 2'-OMe-nucleotide <220> <221> modified_base <222> (7)..(7) <223> 2'-OMe-nucleotide <220> <221> modified_base <222> (9)..(9) <223> 2'-OMe-nucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 194 ccucaucuac accaacuauu u 21 <210> 195 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <220> <221> modified_base <222> (20)..(21) <223> a, c, t, g, u, unknown or other <400> 195 acagcagggu cucaaaaggn n 21 <210> 196 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 196 auaguuggug uagaugaggu u 21 <210> 197 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <220> <221> modified_base <222> (2)..(2) <223> 2'-deoxy-nucleotide <220> <221> modified_base <222> (5)..(6) <223> 2'-deoxy-nucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 197 auaguuggug uagaugaggu u 21 <210> 198 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <220> <221> modified_base <222> (2)..(2) <223> 2'-deoxy-nucleotide <220> <221> modified_base <222> (5)..(6) <223> 2'-deoxy-nucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 198 auaguuggug uagaugaggu u 21 <210> 199 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <220> <221> modified_base <222> (5)..(6) <223> 2'-deoxy-nucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 199 auaguuggug uagaugaggu u 21 <210> 200 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <220> <221> modified_base <222> (5)..(6) <223> 2'-deoxy-nucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 200 auaguuggug uagaugaggu u 21 <210> 201 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <220> <221> modified_base <222> (5)..(6) <223> 2'-deoxy-nucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 201 auaguuggug uagaugaggu u 21 <210> 202 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <220> <221> modified_base <222> (5)..(5) <223> 2'-deoxy-nucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 202 auaguuggug uagaugaggu u 21 <210> 203 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <220> <221> modified_base <222> (5)..(5) <223> 2'-deoxy-nucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 203 auaguuggug uagaugaggu u 21 <210> 204 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <220> <221> modified_base <222> (2)..(2) <223> 2'-deoxy-nucleotide <220> <221> modified_base <222> (6)..(6) <223> 2'-deoxy-nucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 204 auaguuggug uagaugaggu u 21 <210> 205 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <220> <221> modified_base <222> (6)..(6) <223> 2'-deoxy-nucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 205 auaguuggug uagaugaggu u 21 <210> 206 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DN A / RNA Molecule: Synthetic oligonucleotide <220> <221> modified_base <222> (2)..(2) <223> 2'-deoxy-nucleotide <220> <221> modified_base <222> (5)..(6) <223> 2'-deoxy-nucleotide <220> <221> modified_base <222> (14)..(14) <223> 2'-OMe-nucleotide <220> <221> modified_base <222> (16)..(16) <223> 2'-OMe-nucleotide <220> <221> modified_base <222> (18)..(18) <223> 2'-OMe-nucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 206 auaguuggug uagaugaggu u 21 <210> 207 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <220> <221> modified_base <222> (20)..(21) <223> a, c, t, g, u, unknown or other <400> 207 gaugacuaug ugaaggcacn n 21 <210> 208 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 208 ggaugacuau gugaaggcau u 21 <210> 209 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 209 ggaugacuau gugaaggcau u 21 <210> 210 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 210 ggaugacuau gugaaggcau u 21 <210> 211 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 211 ggaugacuau gugaaggcau u 21 <210> 212 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 212 ggaugacuau gugaaggcau u 21 <210> 213 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 213 ggaugacuau gugaaggcau u 21 <210> 214 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 214 ggaugacuau gugaaggcau u 21 <210> 215 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 215 ggaugacuau gugaaggcau u 21 <210> 216 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 216 ggaugacuau gugaaggcau u 21 <210> 217 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 217 ggaugacuau gugaaggcau u 21 <210> 218 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (1)..(1) <223> 2'-OMe-nucleotide <220> <221> modified_base <222> (3)..(3) <223> 2'-OMe-nucleotide <220> <221> modified_base <222> (5)..(5) <223> 2'-OMe-nucleotide <220> <221> modified_base <222> (7)..(7) <223> 2'-OMe-nucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 218 ggaugacuau gugaaggcau u 21 <210> 219 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (11)..(11) <223> 2'-deoxy-2'-fluoro-nucleotide <220> <221> modified_base <222> (13)..(13) <223> 2'-deoxy-2'-fluoro-nucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 219 ggaugacuau gugaaggcau u 21 <210> 220 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (1)..(2) <223> 2'-OMe-nucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 220 ggaugacuau gugaaggcau u 21 <210> 221 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (1)..(2) <223> 2'-OMe-nucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 221 ggaugacuau gugaaggcau u 21 <210> 222 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <220> <221> modified_base <222> (20)..(21) <223> a, c, t, g, u, unknown or other <400> 222 gugccuucac auagucaucn n 21 <210> 223 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 223 ugccuucaca uagucauccu u 21 <210> 224 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <220> <221> modified_base <222> (1)..(1) <223> 2'-deoxy-nucleotide <220> <221> modified_base <222> (5)..(6) <223> 2'-deoxy-nucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 224 ugccuucaca uagucauccu u 21 <210> 225 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <220> <221> modified_base <222> (5)..(6) <223> 2'-deoxy-nucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 225 ugccuucaca uagucauccu u 21 <210> 226 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <220> <221> modified_base <222> (5)..(6) <223> 2'-deoxy-nucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 226 ugccuucaca uagucauccu u 21 <210> 227 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <220> <221> modified_base <222> (5)..(6) <223> 2'-deoxy-nucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 227 ugccuucaca uagucauccu u 21 <210> 228 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <220> <221> modified_base <222> (5)..(6) <223> 2'-deoxy-nucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 228 ugccuucaca uagucauccu u 21 <210> 229 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <220> <221> modified_base <222> (1)..(1) <223> 2'-deoxy-nucleotide <220> <221> modified_base <222> (5)..(5) <223> 2'-deoxy-nucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 229 ugccuucaca uagucauccu u 21 <210> 230 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <220> <221> modified_base <222> (5)..(5) <223> 2'-deoxy-nucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 230 ugccuucaca uagucauccu u 21 <210> 231 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <220> <221> modified_base <222> (6)..(6) <223> 2'-deoxy-nucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 231 ugccuucaca uagucauccu u 21 <210> 232 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <220> <221> modified_base <222> (6)..(6) <223> 2'-deoxy-nucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 232 ugccuucaca uagucauccu u 21 <210> 233 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <220> <221> modified_base <222> (5)..(6) <223> 2'-deoxy-nucleotide <220> <221> modified_base <222> (14)..(14) <223> 2'-OMe-nucleotide <220> <221> modified_base <222> (16)..(16) <223> 2'-OMe-nucleotide <220> <221> modified_base <222> (18)..(18) <223> 2'-OMe-nucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 233 ugccuucaca uagucauccu u 21 <210> 234 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Artificial Sequence Description: Synthetic oligonucleotide <220> <221> modified_base <222> (4)..(4) <223> 2'-deoxy-2'-fluoro-nucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 234 ugccuucaca uagucauccu u 21 <210> 235 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Artificial Sequence Description: Synthetic oligonucleotide <220> <221> modified_base <222> (9)..(9) <223> 2'-OMe-nucleotide <220> <221> modified_base <222> (11)..(11) <223> 2'-OMe-nucleotide <220> <221> modified_base <222> (13)..(13) <223> 2'-OMe-nucleotide <220> <221> modified_base <222> (15)..(15) <223> 2'-OMe-nucleotide <220> <221> modified_base <222> (17)..(17) <223> 2'-OMe-nucleotide <220> <221> modified_base <222> (19)..(21) <223> 2'-OMe-nucleotide <400> 235 ugccuucaca uagucauccu u 21 <210> 236 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (11)..(11) <223> 2'-OMe-nucleotide <220> <221> modified_base <222> (13)..(13) <223> 2'-OMe-nucleotide <220> <221> modified_base <222> (15)..(15) <223> 2'-OMe-nucleotide <220> <221> modified_base <222> (17)..(17) <223> 2'-OMe-nucleotide <220> <221> modified_base <222> (19)..(21) <223> 2'-OMe-nucleotide <400> 236 ugccuucaca uagucauccu u 21 <210> 237 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <220> <221> modified_base <222> (20)..(21) <223> a, c, t, g, u, unknown or other <400> 237 gaagccuuuu gagacccugn n 21 <210> 238 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 238 gaagccuuuu gagacccugu u 21 <210> 239 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 239 gaagccuuuu gagacccugu u 21 <210> 240 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 240 gaagccuuuu gagacccugu u 21 <210> 241 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 241 gaagccuuuu gagacccugu u 21 <210> 242 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 242 gaagccuuuu gagacccugu u 21 <210> 243 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 243 gaagccuuuu gagacccugu u 21 <210> 244 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 244 gaagccuuuu gagacccugu u 21 <210> 245 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 245 gaagccuuuu gagacccugu u 21 <210> 246 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 246 gaagccuuuu gagacccugu u 21 <210> 247 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 247 gaagccuuuu gagacccugu u 21 <210> 248 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (1)..(1) <223> 2'-OMe-nucleotide <220> <221> modified_base <222> (3)..(3) <223> 2'-OMe-nucleotide <220> <221> modified_base <222> (5)..(5) <223> 2'-OMe-nucleotide <220> <221> modified_base <222> (7)..(7) <223> 2'-OMe-nucleotide <220> <221> modified_base <222> (9)..(9) <223> 2'-OMe-nucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 248 gaagccuuuu gagacccugu u 21 <210> 249 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <220> <221> modified_base <222> (20)..(21) <223> a, c, t, g, u, unknown or other <400> 249 cagggucuca aaaggcuucn n 21 <210> 250 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 250 cagggucuca aaaggcuucu u 21 <210> 251 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <220> <221> modified_base <222> (6)..(6) <223> 2'-deoxy-nucleotide <220> <221> modified_base <222> (8)..(8) <223> 2'-deoxy-nucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 251 cagggucuca aaaggcuucu u 21 <210> 252 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <220> <221> modified_base <222> (6)..(6) <223> 2'-deoxy-nucleotide <220> <221> modified_base <222> (8)..(8) <223> 2'-deoxy-nucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 252 cagggucuca aaaggcuucu u 21 <210> 253 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <220> <221> modified_base <222> (6)..(6) <223> 2'-deoxy-nucleotide <220> <221> modified_base <222> (8)..(8) <223> 2'-deoxy-nucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 253 cagggucuca aaaggcuucu u 21 <210> 254 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <220> <221> modified_base <222> (6)..(6) <223> 2'-deoxy-nucleotide <220> <221> modified_base <222> (8)..(8) <223> 2'-deoxy-nucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 254 cagggucuca aaaggcuucu u 21 <210> 255 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <220> <221> modified_base <222> (6)..(6) <223> 2'-deoxy-nucleotide <220> <221> modified_base <222> (8)..(8) <223> 2'-deoxy-nucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 255 cagggucuca aaaggcuucu u 21 <210> 256 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 256 cagggucuca aaaggcuucu u 21 <210> 257 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 257 cagggucuca aaaggcuucu u 21 <210> 258 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <220> <221> modified_base <222> (6)..(6) <223> 2'-deoxy-nucleotide <220> <221> modified_base <222> (8)..(8) <223> 2'-deoxy-nucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 258 cagggucuca aaaggcuucu u 21 <210> 259 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <220> <221> modified_base <222> (6)..(6) <223> 2'-deoxy-nucleotide <220> <221> modified_base <222> (8)..(8) <223> 2'-deoxy-nucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 259 cagggucuca aaaggcuucu u 21 <210> 260 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <220> <221> modified_base <222> (6)..(6) <223> 2'-deoxy-nucleotide <220> <221> modified_base <222> (8)..(8) <223> 2'-deoxy-nucleotide <220> <221> modified_base <222> (14)..(14) <223> 2'-OMe-nucleotide <220> <221> modified_base <222> (16)..(16) <223> 2'-OMe-nucleotide <220> <221> modified_base <222> (18)..(18) <223> 2'-OMe-nucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 260 cagggucuca aaaggcuucu u 21 <210> 261 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <220> <221> modified_base <222> (20)..(21) <223> a, c, t, g, u, unknown or other <400> 261 ccucaucuac accaacuaun n 21 <210> 262 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 262 ccucaucuac accaacuauu u 21 <210> 263 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 263 ccucaucuac accaacuauu u 21 <210> 264 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 264 ccucaucuac accaacuauu u 21 <210> 265 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 265 ccucaucuac accaacuauu u 21 <210> 266 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 266 ccucaucuac accaacuauu u 21 <210> 267 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 267 ccucaucuac accaacuauu u 21 <210> 268 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 268 ccucaucuac accaacuauu u 21 <210> 269 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 269 ccucaucuac accaacuauu u 21 <210> 270 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 270 ccucaucuac accaacuauu u 21 <210> 271 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 271 ccucaucuac accaacuauu u 21 <210> 272 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (1)..(1) <223> 2'-OMe-nucleotide <220> <221> modified_base <222> (3)..(3) <223> 2'-OMe-nucleotide <220> <221> modified_base <222> (5)..(5) <223> 2'-OMe-nucleotide <220> <221> modified_base <222> (7)..(7) <223> 2'-OMe-nucleotide <220> <221> modified_base <222> (9)..(9) <223> 2'-OMe-nucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 272 ccucaucuac accaacuauu u 21 <210> 273 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <220> <221> modified_base <222> (20)..(21) <223> a, c, t, g, u, unknown or other <400> 273 auaguuggug uagaugaggn n 21 <210> 274 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 274 auaguuggug uagaugaggu u 21 <210> 275 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <220> <221> modified_base <222> (2)..(2) <223> 2'-deoxy-nucleotide <220> <221> modified_base <222> (5)..(6) <223> 2'-deoxy-nucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 275 auaguuggug uagaugaggu u 21 <210> 276 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <220> <221> modified_base <222> (2)..(2) <223> 2'-deoxy-nucleotide <220> <221> modified_base <222> (5)..(6) <223> 2'-deoxy-nucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 276 auaguuggug uagaugaggu u 21 <210> 277 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <220> <221> modified_base <222> (5)..(6) <223> 2'-deoxy-nucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 277 auaguuggug uagaugaggu u 21 <210> 278 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <220> <221> modified_base <222> (5)..(6) <223> 2'-deoxy-nucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 278 auaguuggug uagaugaggu u 21 <210> 279 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <220> <221> modified_base <222> (5)..(6) <223> 2'-deoxy-nucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 279 auaguuggug uagaugaggu u 21 <210> 280 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <220> <221> modified_base <222> (5)..(5) <223> 2'-deoxy-nucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 280 auaguuggug uagaugaggu u 21 <210> 281 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <220> <221> modified_base <222> (5)..(5) <223> 2'-deoxy-nucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 281 auaguuggug uagaugaggu u 21 <210> 282 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <220> <221> modified_base <222> (2)..(2) <223> 2'-deoxy-nucleotide <220> <221> modified_base <222> (6)..(6) <223> 2'-deoxy-nucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 282 auaguuggug uagaugaggu u 21 <210> 283 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <220> <221> modified_base <222> (6)..(6) <223> 2'-deoxy-nucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 283 auaguuggug uagaugaggu u 21 <210> 284 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Artificial Sequence Description: Synthetic oligonucleotide <220> <223> Combined DNA / RNA Molecule Description: Synthetic oligonucleotide <220> <221> modified_base <222> (2)..(2) <223> 2'-deoxy-nucleotide <220> <221> modified_base <222> (5)..(6) <223> 2'-deoxy-nucleotide <220> <221> modified_base <222> (14)..(14) <223> 2'-OMe-nucleotide <220> <221> modified_base <222> (16)..(16) <223> 2'-OMe-nucleotide <220> <221> modified_base <222> (18)..(18) <223> 2'-OMe-nucleotide <220> <221> modified_base <222> (20)..(21) <223> 2'-OMe-nucleotide <400> 284 auaguuggug uagaugaggu u 21 <210> 285 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 285 ctcgaggggc aactgaagcc ttttgagacc ctgctgtccc aggcggccgc 50 <210> 286 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 286 ctcgagctgg gacagcaggg tctcaaaagg cttcagttgc ccgcggccgc 50 <210> 287 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 287 ctcgaggggc aactctacgc aaaacagacc ctgctgtccc aggcggccgc 50 <210> 288 <211> 71 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 288 ctcgaggggc aactctacgc aaaacagacc ctgctctacg caaaacagac cctgctgtcc 60 caggcggccg c 71 <210> 289 <211> 92 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 289 ctcgaggggc aactctacgc aaaacagacc ctgctctacg caaaacagac cctgctctac 60 gcaaaacaga ccctgctgtc ccaggcggcc gc 92 <210> 290 <211> 113 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic oligonucleotide <400> 290 ctcgaggggc aactctacgc aaaacagacc ctgctctacg caaaacagac cctgctctac 60 gcaaaacaga ccctgctcta cgcaaaacag accctgctgt cccaggcggc cgc 113

Claims

1. An RNAi molecule capable of mediating RNA interference against GST-π gene expression, wherein the RNAi molecule comprises a polynucleotide sense strand and a polynucleotide antisense strand forming a double-stranded region with a length of 15 to 30 nucleotides, the sense strand and the antisense strand have a length of 15 to 30 nucleotides, a continuous region of 15 to 30 nucleotides located within the double-stranded region of the antisense strand is complementary to the sequence of mRNA encoding GST-π, at least a part of the sense strand is complementary to at least a part of the antisense strand, the antisense strand has deoxynucleotides at a plurality of positions, and the plurality of positions are as follows: each of positions 4, 6, and 8 from the 5'-end of the antisense strand; each of positions 3, 5, and 7 from the 5'-end of the antisense strand; each of positions 1, 3, 5, and 7 from the 5'-end of the antisense strand; each of positions 3 to 8 from the 5'-end of the antisense strand; or each of positions 5 to 8 from the 5'-end of the antisense strand; is one of them, the RNAi molecule.

2. The RNAi molecule according to claim 1, wherein one or more nucleotides within the double-stranded region are chemically modified.

3. Whether the chemically modified nucleotide is a 2'-deoxynucleotide, and / or the chemically modified nucleotide comprises a 2'-O-alkyl-substituted nucleotide, a 2'-deoxy-2'-fluoro-substituted nucleotide, a phosphorothioate nucleotide, a locked nucleotide, or any combination thereof, the RNAi molecule according to claim 2.

4. The RNAi molecule according to claim 1, wherein the antisense strand has one or more 2'-deoxy-2'-fluoro-substituted nucleotides within the double-stranded region.

5. A pharmaceutical composition comprising the RNAi molecule according to claim 1 and a pharmaceutically acceptable carrier.

6. The pharmaceutical composition according to claim 5, wherein the pharmaceutically acceptable carrier comprises a lipid molecule, a nanoparticle, or a liposome.

7. The pharmaceutical composition according to claim 5, for treating a disease associated with GST-π expression.

8. The pharmaceutical composition according to claim 7, wherein the disease associated with GST-π expression is a malignant tumor, cancer, cancer caused by cells expressing mutant KRAS, sarcoma, or carcinoma.

9. A method for preventing, treating, or ameliorating a disease associated with GST-π expression in a subject in need thereof by gene silencing, the method comprising administering the pharmaceutical composition according to claim 5 to the subject.

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