A method for activating p21 gene expression

Abstract

The present invention provides a small activating RNA for activating or up-regulating human p21 gene expression, which is composed of a sense oligonucleotide chain and an antisense oligonucleotide chain, wherein the sense nucleic acid chain or the antisense nucleic acid chain has a homology of more than 75% with any continuous segment of 16-35 nucleotides in a human p21 gene promoter target gene sequence, wherein the target sequence of the human p21 gene is selected from the group consisting of sequences shown in SEQ ID NO: 5, 6, 7, 8, 9, 10, 11, and 12.

Description

Technical Field

[0001] The present invention relates to the field of molecular biology, and in particular to positively regulating gene expression by using double-stranded small RNA targeting gene promoter sequence. Background Art

[0002] Double-stranded small nucleic acid molecules, including chemically synthesized oligoribonucleotides such as small activating RNA (saRNA) and naturally occurring oligoribonucleotides such as microribonucleotides (miRNA), have been shown to be able to target regulatory sequences of protein-coding genes such as promoter sequences in a sequence-specific manner and positively regulate gene expression levels at the transcriptional and epigenetic levels. This phenomenon is called RNA activation (RNAa) (Li, Okino et al. (2006) Proc Natl Acad Sci USA 103:17337-17342; Janowski, Younger et al. (2007) Nat Chem Biol 3:166-173; Place, Li et al. (2008) Proc Natl Acad Sci US A105:1608-1613; Huang, Place et al. (2012) Nucleic Acids Res 40:1695-1707; Li (2017) Adv Exp Med Biol 983:1-20). Studies have shown that RNA activation is an endogenous molecular mechanism that is evolutionarily conserved from Caenorhabditis elegans to humans (Huang, Qin et al. (2010) PLoS One 5:e8848; Seth, Shirayama et al. (2013) Dev Cell 27:656-663; Turner, Jiao et al. (2014) Cell Cycle 13:772-781).

[0003] How to safely and selectively enhance the expression of endogenous genes or proteins in human cells remains a huge challenge in the field of gene therapy. Traditional viral-based gene therapy systems have inherent defects, including the possibility of altering the host genome and causing side effects such as immune responses. RNA activation has the advantage of being able to activate endogenous gene expression without changing the genome, representing a new strategy for activating endogenous gene expression, and has great application value in disease treatment.

[0004] p21 WAF1 / CIP1The (also known as CDKN1A, hereinafter referred to as p21) gene is a cyclin-dependent kinase (CDK) inhibitor and an important tumor suppressor gene (Harper, Adami et al. (1993) Cell 75: 805-816; Fang, Igarashi et al. (1999) Oncogene 18: 2789-2797). Studies have shown that overexpression of p21 by ectopic vectors or activation of endogenous p21 transcription can effectively inhibit the growth of tumor cells and in vivo tumors (Harper, Adami et al. (1993) Cell 75: 805-816; Eastham, Hall et al. (1995) Cancer Res 55: 5151-5155; Wu, Bellas et al. (1998) J Exp Med 187: 1671-1679; Harrington, Spitzweg et al. (2001) J Urol 166: 1220-1233). Therefore, the method of targeted activation of the p21 gene may have a wide range of application value in the treatment of many diseases such as cancer. Summary of the invention

[0005] The present invention provides a small activating RNA, characterized in that one chain of the small activating RNA has at least 75% homology or complementarity with any continuous fragment of 16-35 nucleotides in the sequence shown in SEQ ID NO: 5, 6, 7, 8, 9, 10, 11, 12, and the small activating RNA activates or upregulates the expression of the p21 gene by targeting the human p21 gene promoter sequence.

[0006] In an embodiment of the present invention, the target gene of the human p21 gene is selected from the group consisting of sequences shown in SEQ ID NOs: 5-12, wherein the human p21 gene promoter sequence is located at -893 to -801 bp (SEQ ID NO: 5), -717 to -632 bp (SEQ ID NO: 6), -585 to -551 bp (SEQ ID NO: 7), -554 to -504 bp (SEQ ID NO: 8), -514 to -485 bp (SEQ ID NO: 9), -442 to -405 bp (SEQ ID NO: 10), -352 to -313 bp (SEQ ID NO: 11), and -325 to -260 bp (SEQ ID NO: 12) upstream of the transcription start site (TSS).

[0007] In one embodiment, the small activating RNA comprises a sense nucleic acid chain and an antisense nucleic acid chain, wherein the sense nucleic acid chain and the antisense nucleic acid chain contain complementary regions, and the complementary regions can form a double-stranded nucleic acid structure, wherein the sense nucleic acid chain or the antisense nucleic acid chain has more than 75%, more than 80%, more than 90%, more than 95%, more than 99% or 100% homology with any continuous segment of 16-35 nucleotides in the human p21 gene promoter sequence.

[0008] In one embodiment, as described above, the sense nucleic acid chain and the antisense nucleic acid chain exist on two different nucleic acid chains; the sense nucleic acid chain and the antisense nucleic acid chain exist on the same nucleic acid chain, which is a hairpin-type single-stranded nucleic acid molecule, wherein the complementary regions of the sense nucleic acid chain and the antisense nucleic acid chain form a double-stranded nucleic acid structure.

[0009] In a specific embodiment, at least one strand of the small activating RNA has a 3' overhang with a length of 0 to 6 nucleotides; both strands of the small activating RNA have a 3' overhang with a length of 2-3 nucleotides.

[0010] In one embodiment, the sense nucleic acid strand or the antisense nucleic acid strand has a length of 16-35 nucleotides, for example, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleotides.

[0011] In a specific embodiment, the sense nucleic acid strand or the antisense nucleic acid strand is SEQ ID NO: 13-30, 35-46, 59-62, 67-74, 77-80, 85-96, 103-108, 111-118, 121-132, 139-140, 147-180, 185-186, 189-190, 195-198, 201-202, 209-212, 215-218, 225-240, 243-246, 249-258, 261-262, 265-270, 275-280, 283-3 The sequences shown in 00, 303-308, 317-320, 323-324, 329-348, 351-352, 357-358, 361-366, 371-392, 399-400, 405-412, 415-416, 419-424, 429-432, 439-442, 447-450, 453-458, 463-468 have at least 75%, such as 80%, 85%, 90%, 95%, 99%, 100% homology.

[0012] In a specific embodiment, the sense nucleic acid strand or antisense nucleic acid strand sequence is as shown in SEQ ID NO: 13-30, 35-46, 59-62, 67-74, 77-80, 85-96, 103-108, 111-118, 121-132, 139-140, 147-180, 185-186, 189-190, 195-198, 201-202, 209-212, 215-218, 225-240, 243-246, 249-258, 261-262, 265-2 70, 275-280, 283-300, 303-308, 317-320, 323-324, 329-348, 351-352, 357-358, 361-366, 371-392, 399-400, 405-412, 415-416, 419-424, 429-432, 439-442, 447-450, 453-458, 463-468 as shown in any one of the nucleotide sequences.

[0013] The present invention also provides a method for preparing the small activating RNA as described in any of the preceding items, characterized in that the method comprises the following steps: 1) using the target gene promoter sequence as a template, selecting a sequence containing 19 bases as a target site; 2) synthesizing an RNA sequence having more than 75% homology with the target site sequence obtained in step 1) to obtain a sense oligonucleotide chain; 3) the sequence is complementary to the sense oligonucleotide chain obtained in step 2); 4) mixing the sense oligonucleotide chain obtained in step 2) and the antisense oligonucleotide chain obtained in step 3) in an RNA annealing buffer at the same molar number, heating, and then naturally cooling to room temperature to obtain a double-stranded small activating RNA; wherein the human p21 gene promoter sequence is selected from the group consisting of the sequences shown in SEQ ID NO: 5, 6, 7, 8, 9, 10, 11, and 12.

[0014] In one embodiment, at least one nucleotide in the small activating RNA is a chemically modified nucleotide, and the chemical modification is at least one of the following modifications:

[0015] (1) modifying the phosphodiester bonds connecting nucleotides in the nucleotide sequence of the small activating RNA;

[0016] (2) modification of the 2'-OH of the ribose in the nucleotide sequence of the small activating RNA;

[0017] (3) modification of bases in the nucleotide sequence of the small activating RNA;

[0018] (4) At least one nucleotide in the nucleotide sequence of the small activating nucleic acid molecule is a locked nucleic acid.

[0019] In one embodiment, the activation or upregulation of the expression of the p21 gene is upregulation by at least 10%, such as 15%, 20%, 30%, 40%, 50%, 80%, 100%, 200% or more.

[0020] The present invention also provides the use of small activating RNA in preparing a preparation for activating or up-regulating the expression of p21 gene in cells. The small activating RNA is directly introduced into the cells, or is produced in the cells after the nucleotide sequence encoding the small activating RNA is introduced into the cells. The cells are mammalian cells, preferably human cells, more preferably human tumor cells. The human cells can be isolated human cell lines, or can exist in the human body.

[0021] In one embodiment, the human body is a patient suffering from a tumor caused by insufficient expression of p21 protein, and the small activating nucleic acid molecule is administered in an effective amount to achieve treatment of the tumor, and the tumor is preferably bladder cancer, prostate cancer, liver cancer or colorectal cancer.

[0022] In yet another aspect, the present invention also provides an isolated p21 gene small activating RNA target site, wherein the target site is any continuous 16-35 nucleotide sequence selected from any one of SEQ ID No 5-12.

[0023] In another aspect, the present invention discloses a method for activating or up-regulating the expression of human p21 gene in cells, wherein the method comprises administering the small activating RNA as described in any of the preceding items to a subject or a cell. The small activating RNA can be directly introduced into the cell, or it can be produced in the cell after the nucleotide sequence encoding the small activating RNA is introduced into the cell. The cell is a mammalian cell, preferably a human cell, more preferably a human tumor cell, and even more preferably a human bladder cancer, prostate cancer, liver cancer or colorectal cancer cell.

[0024] The present invention further discloses a composition comprising the aforementioned small activating RNA and a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier is a liposome, a high molecular polymer or a polypeptide.

[0025] The present invention also discloses the use of the aforementioned small activating RNA or the aforementioned composition in the preparation of a drug for activating or up-regulating the expression of the p21 gene, preferably in the preparation of a drug for anti-tumor or benign proliferative lesions, more preferably, the tumor is bladder cancer, prostate cancer, liver cancer or colorectal cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The p21 gene promoter sequence is shown, from -1000 bp upstream of the transcription start site (TSS) to 3 bp downstream of the TSS. The TSS is indicated by a curved arrow.

[0027] Figure 2 Screening of small activating RNA hotspots on the p21 gene promoter is shown. Figure 1 The p21 promoter sequence shown was designed and 439 double-stranded RNA molecules were chemically synthesized and transfected into PC3 human prostate cancer cells. After 72 hours, the p21 gene mRNA level was analyzed using the QuantiGene 2.0 method. (A) The fold change of p21 mRNA level caused by each of the 439 double-stranded RNA molecules (X-axis) relative to the control treatment (Y-axis). The double-stranded RNA molecules on the X-axis are sorted according to their positions relative to the TSS of the p21 gene (from RAG-898 at the most upstream to RAG-177 at the most downstream). Eight hotspot areas (light gray rectangular boxes) are marked in the figure. (B) The data of (A) are sorted from low to high according to the fold size of the change in p21 mRNA expression induced by each double-stranded RNA molecule. The dotted lines in (A) and (B) indicate 2-fold induction.

[0028] FIG. 3 shows the activation effect of double-stranded RNA molecules in hotspot regions 1 to 8 on the p21 gene.

[0029] Figure 4 The results of the experiment using QuantiGene 2.0 are shown by analyzing the mRNA level of the p21 gene using the RT-qPCR method. (A) 439 double-stranded RNA molecules were divided into four regions (groups) according to their activity in inducing the expression of p21 mRNA. Five double-stranded RNA molecules were randomly selected from each group and transfected into PC3 cells at a concentration of 10 nM. After 72 hours, total cell RNA was extracted and the mRNA level of the p21 gene was analyzed by the RT-qPCR method after reverse transcription. (B) The correlation between the relative mRNA levels of the p21 gene induced by double-stranded RNA molecules detected by QuantiGene 2.0 (X axis) and RT-qPCR (Y axis).

[0030] Figure 5 The effect of small activating RNA on inducing p21 mRNA expression and inhibiting KU-7 cell proliferation is shown. The three small activating RNAs shown were transfected into KU-7 cells at 10 nM for 72 hours. (A) RT-qPCR analysis of p21 gene mRNA expression levels. (B) CCK-8 method to assess cell viability. The viability of cells in the saRNA-treated group is expressed as a percentage of the viability of cells in the control-treated group (Mock). (C) Representative cell images at the end of transfection (100×).

[0031] Figure 6The effect of small activating RNA inducing p21 mRNA expression and inhibiting HCT116 cell proliferation is shown. The three small activating RNAs shown were transfected into HCT116 cells at 10 nM for 72 hours. (A) RT-qPCR analysis of p21 gene mRNA expression levels. (B) CCK-8 method was used to evaluate cell viability, and the viability of cells in the saRNA-treated group was expressed as a percentage of cell viability relative to the control-treated group (Mock). (C) Representative cell images at the end of transfection (100×).

[0032] Figure 7 The effect of small activating RNA on inducing p21 mRNA expression and inhibiting HepG2 cell proliferation is shown. The three small activating RNAs shown were transfected into HepG2 cells at 10 nM for 72 hours. (A) RT-qPCR analysis of p21 gene mRNA expression levels. (B) CCK-8 method was used to assess cell viability, and the viability of cells in the saRNA-treated group was expressed as a percentage of cell viability relative to that of the control-treated group (Mock). (C) Representative cell images at the end of transfection (100×). DETAILED DESCRIPTION

[0033] The present invention will be further illustrated by detailed description below.

[0034] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0035] As used herein, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

[0036] definition

[0037] As used herein, the term "complementarity" refers to the ability of two oligonucleotide chains to form base pairs with each other. Base pairs are usually formed by hydrogen bonds between nucleotide units in antiparallel oligonucleotide chains. Complementary oligonucleotide chains can be base paired in the Watson-Crick manner (e.g., AT, AU, CG), or in any other manner (e.g., Hoogsteen type or reverse Hoogsteen type base pairing) that allows the formation of a duplex. "100% pairing" or "complete complementarity" refers to having 100% complementarity, i.e., the nucleotide units of the two chains are all hydrogen bonded to each other.

[0038] Complete complementarity or 100% pairing refers to the situation that each nucleotide unit from the first oligonucleotide chain in the double-stranded region of the double-stranded oligonucleotide molecule can form hydrogen bonds with the second oligonucleotide chain without "mismatching". Incomplete complementarity refers to the situation that the nucleotide units of the two chains cannot all hydrogen bond with each other. For example, for two oligonucleotide chains with a double-stranded region of 20 nucleotides in length, if only two base pairs on each chain can hydrogen bond with each other, the oligonucleotide chains show 10% complementarity. In the same example, if 18 base pairs on each chain can hydrogen bond with each other, the oligonucleotide chains show 90% complementarity. Basic complementarity refers to about 79%, about 80%, about 85%, about 90%, about 95% or more complementarity.

[0039] The term "oligonucleotide" as used herein refers to a polymer of nucleotides, and includes, but is not limited to, single-stranded or double-stranded molecules of DNA, RNA, or DNA / RNA hybrids, oligonucleotide chains comprising regularly and irregularly alternating deoxyribose and ribose moieties, as well as modifications of these types of oligonucleotides and with naturally occurring or non-naturally occurring backbones.

[0040] The term "oligoribonucleotide" as used herein refers to an oligonucleotide comprising two or more modified or unmodified ribonucleotides and / or analogs thereof.

[0041] As used herein, the terms "oligonucleotide chain" and "oligonucleotide sequence" are interchangeable and refer to a general term for a short chain of nucleotides of less than 50 bases (the nucleotides may be deoxyribonucleic acid DNA or ribonucleic acid RNA). In the present invention, the length of the oligonucleotide chain may be any length of 17 to 30 nucleotides.

[0042] As used herein, the term "gene" refers to the entire nucleotide sequence required to encode a polypeptide chain or transcribe a functional RNA. "Gene" can be a gene that is endogenous or completely or partially recombinant to a host cell (for example, due to the introduction of exogenous oligonucleotides encoding promoters and coding sequences or the introduction of heterologous promoters adjacent to endogenous coding sequences into host cells). For example, the term "gene" includes nucleic acid sequences that can be composed of exons and introns. The sequence encoding a protein is, for example, a sequence within an exon in an open reading frame between a start codon and a stop codon, and as used herein, "gene" can refer to all other sequences including, for example, gene regulatory sequences such as promoters, enhancers, and transcription, expression, or activity of another gene known in the art, regardless of whether the other gene contains a coding sequence or a non-coding sequence. In one case, for example, "gene" can be used to describe a functional nucleic acid comprising a regulatory sequence such as a promoter or an enhancer. The expression of a recombinant gene can be controlled by one or more heterologous regulatory sequences.

[0043] As used herein, the term "target gene" can be a nucleic acid sequence naturally present in an organism, a transgenic, a viral or bacterial sequence, a chromosome or extrachromosomal and / or transiently or stably transfected or incorporated into a cell and / or its chromatin. The target gene can be a protein coding gene or a non-protein coding gene (e.g., a microRNA gene, a long non-coding RNA gene). The target gene usually contains a promoter sequence, and a small activating RNA designed to have identity (also known as homology) with the promoter sequence can achieve positive regulation of the target gene, which is manifested as upregulation of target gene expression. "Target gene promoter sequence" refers to the non-coding sequence of the target gene. In the present invention, the target gene promoter sequence in "complementary to the target gene promoter sequence" refers to the coding strand of the sequence, also known as the non-template strand, that is, a nucleic acid sequence with the same sequence as the gene coding sequence. "Target sequence" refers to a sequence fragment homologous or complementary to the sense oligonucleotide strand or antisense oligonucleotide of the small activating RNA in the target gene promoter sequence.

[0044] As used herein, the terms "sense strand" and "sense oligonucleotide strand" are interchangeable, and the sense oligonucleotide strand of the small activating RNA refers to the first RNA strand in the small activating RNA duplex that contains the coding strand that is identical to the promoter sequence of the target gene.

[0045] As used herein, the terms "antisense strand" and "antisense oligonucleotide strand" are interchangeable, and the antisense oligonucleotide strand of the small activating RNA refers to the second ribonucleic acid strand complementary to the sense oligonucleotide strand in the small activating RNA duplex.

[0046] As used herein, the term "coding strand" refers to the DNA strand in the target gene that cannot be transcribed, and the nucleotide sequence of this strand is consistent with the sequence of the RNA generated by transcription (in RNA, U replaces T in DNA). The coding strand of the double-stranded DNA sequence of the target gene promoter described in the present invention refers to the promoter sequence on the same DNA strand as the coding strand of the target gene DNA.

[0047] As used herein, the term "template strand" refers to the other strand of the double-stranded DNA of the target gene that is complementary to the coding strand, and can be used as a template to transcribe into RNA, which is complementary to the transcribed RNA base (AU, GC). During transcription, RNA polymerase binds to the template strand and moves along the 3'→5' direction of the template strand, catalyzing the synthesis of RNA in the 5'→3' direction. The template strand of the double-stranded DNA sequence of the target gene promoter described in the present invention refers to the promoter sequence on the same DNA strand as the target gene DNA template strand.

[0048] As used herein, the term "promoter" refers to a nucleic acid sequence that does not encode a protein and regulates the transcription of a protein-coding or RNA-coding nucleic acid sequence by being positionally associated with the nucleic acid sequence. Typically, a eukaryotic promoter contains 100-5,000 base pairs, although this length range is not meant to limit the term "promoter" as used herein. Although promoter sequences are generally located at the 5' end of a protein-coding or RNA-coding sequence, in some cases promoter sequences are also present in exon and intron sequences.

[0049] As used herein, the term "transcription start site" refers to the nucleotide that marks the start of transcription on the template strand of a gene. The transcription start site may appear on the template strand of the promoter region. A gene may have more than one transcription start site.

[0050] As used herein, the term "identity" or "homology" means that one oligonucleotide strand (sense strand or antisense strand) of the small activating RNA has at least 75% similarity with the coding strand or template strand of a region of the promoter sequence of the target gene.

[0051] As used herein, the terms "overhang", "overhang" and "dangling" are interchangeable and refer to the non-base paired nucleotides at the end (5' or 3') of the oligonucleotide chain, which are generated by extending beyond one of the chains in the double-stranded oligonucleotide. The single-stranded region extending beyond the 3' and / or 5' end of the duplex is called an overhang.

[0052] As used herein, the terms "gene activation" or "activated gene" are interchangeable and refer to measuring the transcription, translation or expression or active increase of a certain nucleic acid by measuring gene transcription level, mRNA level, protein level, enzyme activity, methylation state, chromatin state or configuration, translation level or its activity or state in a cell or biological system. These activities or states can be measured directly or indirectly. In addition, "gene activation", "activated gene" refers to the increase in activity associated with a nucleic acid sequence, regardless of the mechanism by which this activation occurs, such as it plays a regulatory role as a regulatory sequence, is transcribed into RNA, is translated into protein and increases protein expression.

[0053] As used herein, the terms "small activating RNA", "saRNA", "small activating nucleic acid molecule" are interchangeable and refer to ribonucleic acid molecules that can promote gene expression, and can be composed of a first ribonucleic acid chain (antisense chain, also called antisense oligonucleotide chain) comprising a ribonucleotide sequence having sequence identity with a non-coding nucleic acid sequence of a target gene (e.g., a promoter, an enhancer, etc.) and a second ribonucleic acid chain (sense chain, also called sense chain or sense oligonucleotide chain) comprising a nucleotide sequence complementary to the first chain, wherein the first chain and the second chain form a duplex. Small activating RNA can also be composed of a single-stranded RNA molecule that can form a double-stranded region hairpin structure that is synthesized or expressed by a vector, wherein the first region comprises a nucleic acid sequence having sequence identity with a promoter target sequence of a gene, and the second region comprises a nucleic acid sequence that is complementary to the first region. The duplex region length of the small activating RNA molecule is generally about 10 to about 50 base pairs, about 12 to about 48 base pairs, about 14 to about 46 base pairs, about 16 to about 44 base pairs, about 18 to about 42 base pairs, about 20 and about 40 base pairs, about 22 and about 38 base pairs, about 24 and about 36 base pairs, about 26 and about 34 base pairs, about 28 and about 32 base pairs, typically about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50 base pairs. In addition, the terms "saRNA" and "small activating RNA", "small activating nucleic acid molecule" also contain nucleic acids other than ribonucleotide portions, including but not limited to modified nucleotides or analogs.

[0054] As used herein, the term "hotspot" refers to a gene promoter region with a length of at least 30 bp, in which functional small activating nucleic acid molecule targets are clustered, that is, at least 60% of the small activating nucleic acid molecules targeting these hotspot regions can induce target gene mRNA expression by 1.5 times or more.

[0055] As used herein, the term "p21" refers to p21 WAF1 / CIP1 The gene, also known as CDKN1A, is a cyclin-dependent kinase (CDK) inhibitor and an important tumor suppressor gene, sometimes referred to as a "target gene." Overexpression of p21 or activation of endogenous p21 transcription can effectively inhibit the growth of tumor cells and in vivo tumors.

[0056] As used herein, the term "synthesis" refers to the synthesis method of oligonucleotides, including any method capable of synthesizing RNA, such as chemical synthesis, in vitro transcription, vector expression, etc. The preparation method of the small activating nucleic acid molecule provided by the present invention includes sequence design and sequence synthesis. The synthesis of the small activating nucleic acid molecule sequence can be carried out by chemical synthesis, or it can be commissioned to a biotechnology company specializing in nucleic acid synthesis. Generally speaking, the chemical synthesis method includes the following four processes: (1) synthesis of oligoribonucleotides; (2) deprotection; (3) purification and separation; (4) desalting and annealing. For example, the specific steps of the chemical synthesis of double-stranded RNA molecules such as saRNA described in the present invention are as follows:

[0057] (1) Synthesis of oligoribonucleotides

[0058] The automatic DNA / RNA synthesizer (e.g., Applied Biosystems EXPEDITE8909) is set to synthesize 1 micromole of RNA, and the coupling time of each cycle is set to 10-15 minutes. The starting material is a solid-phase-linked 5'-O-p-dimethoxytrityl-thymidine support. In the first cycle, one base is linked to the solid support. Then, in the nth (19≥n≥2) cycle, one base is linked to the base linked in the n-1th cycle. This cycle is repeated until the synthesis of the entire nucleic acid sequence is completed.

[0059] (2) Deprotection

[0060] The solid support connected with saRNA is added to the test tube, and 1 ml of ethanol / ammonia solution (volume ratio of 1:3) is added to the test tube, then sealed and placed in a 25-70°C incubator for 2-30 hours, the solution of the solid support containing saRNA is filtered and the filtrate is collected, the solid support is rinsed with double distilled water twice (1 ml each time) and the filtrate is collected, the eluate is combined and collected, and dried under vacuum conditions for 1-12 hours. Then, 1 ml of tetrabutylammonium fluoride tetrahydrofuran solution (1M) is added, and the mixture is placed at room temperature for 4-12 hours, and then 2 ml of n-butanol is added, and the precipitate is collected by high-speed centrifugation to obtain a crude product of saRNA single strand.

[0061] (3) Purification and separation

[0062] The obtained crude saRNA product was dissolved in 2 ml of 1 mol / ml ammonium acetate aqueous solution, and then separated by high pressure liquid chromatography reverse phase C18 column to obtain a purified saRNA single-stranded product.

[0063] (4) Desalting and annealing

[0064] The salt was removed by size exclusion gel filtration, and the single-stranded oligoribonucleic acid of the sense chain and the antisense chain were mixed in 1-2 ml of buffer (10 mM Tris, pH = 7.5-8.0, 50 mM NaCl) at the same molar ratio. The solution was heated to 95°C and then slowly cooled to room temperature to obtain a solution containing saRNA.

[0065] Materials and methods

[0066] Cell culture and transfection

[0067] Cell lines RT4, KU-7, T24, J82, TCCSUP and HT-1197 were cultured in modified McCoy's 5A medium (Gibco); cell lines 5637, PC3 and Bel-7402 were cultured in RPMI1640 medium (Gibco); UM-UC-3 cell line was cultured in basal medium (Gibco). All media contained 10% fetal calf serum (Sigma-Aldrich) and 1% penicillin / streptomycin (Gibco). Cells were cultured at 5% CO2 and 37°C. Double-stranded RNA molecules designed in the experiment were transfected at a concentration of 10 nM (unless otherwise specified) using RNAiMax (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions.

[0068] RNA isolation and reverse transcription polymerase chain reaction

[0069] The cells were 2-3×10 5 Cells / well were seeded in 6-well plates and reverse transfected with oligonucleotide duplexes. Total cellular RNA was extracted using the RNeasy Plus Mini kit (Qiagen) according to its instructions. RNA (1 μg) was reverse transcribed into cDNA using the PrimeScript RT kit containing gDNA Eraser (Takara, Shlga, Japan). qPCR was performed using ABI7500Fast Real-time PCR System (Applied Biosystems) and SYBR Premix Ex Taq II (Takara, Shlga, Japan) reagents, and the reaction conditions were: 95°C for 3 seconds, 60°C for 30 seconds, and amplification for 40 cycles. GAPDH was used as an internal reference. All primer sequences are listed in Table 1.

[0070] Table 1. Primer sequences for qRT-PCR analysis

[0071]

[0072] Cell proliferation assay

[0073] The cells were cultured at 2-4 × 10 3 Cells / well were plated in 96-well plates, cultured overnight, and transfected with oligonucleotide duplexes. Three days after transfection, cell proliferation was detected using CCK8 (Dojindo) according to its instructions. The experimental steps are briefly described as follows: 10 μL CCK8 solution was added to each well, incubated at 37°C for 1 hour, and then the absorbance at 450nm was measured using an ELISA reader.

[0074] QuantiGene 2.0 analysis

[0075] Cells were plated in 96-well plates and transfected with oligonucleotide duplexes. After 72 hours of transfection, the target gene mRNA level was quantitatively detected using the QuantiGene2.0 kit (AffyMetrix). The QuantiGene2.0 kit is a hybridization-based method that uses gene-specific probes to directly quantify mRNA levels. The experimental steps are briefly described as follows: Lysis buffer was added to lyse the transfected cells, and the cell lysate was loaded into a capture well plate coated with CDKN1A (p21) and HPRT1 (housekeeping gene) probes and hybridized overnight at 55°C. To enhance the hybridization signal, 2.0PreAMP, 2.0AMP and 2.0Lable Probe were hybridized in sequence in 100 μL of the corresponding buffer (provided by the Quantigene 2.0 kit). All hybridizations were shaken at 50-55°C for 1 hour. After the last wash step, 2.0Substrate was added and incubated at room temperature for 5 minutes. The light signal was then detected using an Infinite 200PRO plate reader (Tecan, Switzerland).

[0076] Statistical analysis

[0077] The results are expressed as mean ± standard deviation. One-way ANOVA followed by Tukey's t test was used for statistical analysis using GraphPad Prism software (GraphPad Software). The criteria for statistical significance were set at *p<0.05, **p<0.01 and ***p<0.001.

[0078] Example

[0079] The present invention is further illustrated by the following examples. The examples are provided for illustrative purposes only and should not be construed as limiting the scope or content of the present invention in any way.

[0080] Example 1: Screening for functional small activating RNA (saRNA) targeting the promoter region of the p21 gene

[0081] In order to screen for functional small activating RNA that can activate p21 gene expression, the 1 kb promoter sequence of p21 gene was retrieved from the UCSC Genome database ( Figure 1 ). A target of 19 bp in size was selected from -1 kb upstream of the transcription start site (TSS), and a total of 982 target sequences were obtained by moving 1 bp each time to the TSS site. The target sequences were filtered to exclude target sequences with a GC content higher than 65% or lower than 35%, and containing 5 or more consecutive identical nucleotides. After filtering, the remaining 439 target sequences entered the screening process as candidates. Based on these candidate sequences, the corresponding double-stranded double-stranded RNA molecules were chemically synthesized. Among them, the length of the sense and antisense strands of the double-stranded RNA molecules used in this experiment were both 21 nucleotides, and the 19 nucleotides in the 5' region of the first ribonucleic acid chain (sense chain) of the double-stranded RNA molecule, such as the double-stranded saRNA, had 100% identity with the promoter target sequence, and its 3' end contained dTdT protrusion; the 19 nucleotides in the 5' region of the second ribonucleic acid chain were completely complementary to the 19 nucleotides in the 3' region sequence of the first ribonucleic acid chain, and its 3' end contained dTdT protrusion.

[0082] The aforementioned double-stranded RNA molecules were transfected into PC3 prostate cancer cells at a final concentration of 10 nM. After 72 hours, the p21 gene mRNA level was detected using the QuantiGene 2.0 kit. The fold change of p21 mRNA level of each double-stranded RNA molecule relative to the blank control was calculated and plotted on Figure 2 The fold changes of p21 gene mRNA caused by all double-stranded RNA molecules in this study ranged from 0.66 (inhibition) to 8.12 (induction) ( Figure 2 B). 361 (82.2%) double-stranded RNA molecules induced p21 expression by 1.01 to 8.12 folds; 74 (16.9%) double-stranded RNA molecules showed an inhibitory effect (0.99 to 0.66 folds); and 4 double-stranded RNA molecules (0.9%) had no effect on the p21 gene mRNA level (1.0 fold).

[0083] Among the 439 double-stranded RNA molecules screened, 132 double-stranded RNA molecules (30.1%) were able to induce p21 mRNA at least 2 times; 229 (52.4%) double-stranded RNA molecules induced p21 mRNA at least 1.5 times, and these double-stranded RNA molecules that induced p21 mRNA expression to increase by more than 10% were saRNA, that is, functional saRNA. These functional saRNAs are scattered throughout the p21 promoter region. However, in 8 free areas, there are clusters of functional small activating RNAs, which are called "hotspots." Hotspot areas are defined as areas containing at least 10 corresponding small activating RNAs, of which at least 60% of the small activating RNAs can induce p21 mRNA expression by 1.5 times or more ( Figure 2 A and Figure 3). The target sequences of hotspots 1 to 8 and the corresponding small activating RNA sequences are listed in Tables 2 and 3, respectively.

[0084] Table 2 Target sequences of p21 promoter hotspot regions

[0085]

[0086] Table 3 Designed double-stranded RNA molecules located in the hotspot region of p21 promoter

[0087]

[0088]

[0089]

[0090]

[0091]

[0092]

[0093]

[0094]

[0095]

[0096]

[0097] These hot spots include hot spot region 1, the corresponding target sequence is -893 bp to -801 bp of the p21 promoter sequence, and the sequence is shown in SEQ ID NO: 93. 44 functional saRNAs were found in this region ( Table 3 , Figure 3A ), namely RAG-834, RAG-845, RAG-892, RAG-846, RAG-821, RAG-884, RAG-864, RAG-843, RAG-854, RAG-844, RAG-887, RAG-838, RAG-858, RAG-835, RAG-876, RAG-870, RAG-853, RAG-881, RAG-828, and RAG-864. 872, RAG-841, RAG-831, RAG-829, RAG-820, RAG-822, RAG-868, RAG-849, RAG-862, RAG-865, RAG-893, RAG-848, RAG-82 4. RAG-866, RAG-840, RAG-875,, RAG-880, RAG-871, RAG-888, RAG-885, RAG-894, RAG-833, RAG-825, RAG-889, RAG-823;

[0098] The corresponding target sequence of hotspot region 2 (Table 3, Figure 3B) is -717 to -632 bp of the p21 promoter sequence, as shown in SEQ ID As shown in NO:94, 31 functional saRNAs were found in this region, namely RAG-693, RAG-692, RAG-688, RAG-696, RAG-694, RAG-687, RAG-691, RAG-690, RAG-689, RAG-682, RAG-686, RAG-662, RAG-695, RAG-654, RAG-658, RAG-685, RAG-704, RAG-714, RAG-705, RAG-661, RAG-656, RAG-698, RAG-697, RAG-657, RAG-715, RAG-652, RAG-651, RAG-650, RAG-716, RAG-717, RAG-711;

[0099] Hotspot 3 ( Table 3 , Figure 3C ), the corresponding target sequence is −585 bp to −551 bp of the p21 promoter sequence, and the sequence is shown in SEQ ID NO: 95. In this region, it was found that it contained 9 functional saRNAs, namely RAG-580, RAG-577, RAG-569, RAG-576, RAG-570, RAG-574, RAG-585, RAG-579, and RAG-584;

[0100] Hotspot 4 (Table 3, Figure 3D), the corresponding target sequence is -554 bp to -505 bp of the p21 promoter sequence, and the sequence is shown in SEQ ID NO: 96. In this region, 17 functional saRNAs were found, namely RAG-524, RAG-553, RAG-537, RAG-526, RAG-554, RAG-523, RAG-534, RAG-543, RAG-525, RAG-535, RAG-546, RAG-545, RAG-542, RAG-531, RAG-522, RAG-529, and RAG-552;

[0101] Hotspot 5 (Table 3, Figure 3E), the corresponding target sequence is -514 bp to -485 bp of the p21 promoter sequence, and the sequence is shown in SEQ ID NO: 97. Nine functional saRNAs were found in this region, namely RAG-503, RAG-504, RAG-505, RAG-506, RAG-507, RAG-508, RAG-509, RAG-510, RAG-511, RAG-512, RAG-513, and RAG-514;

[0102] Hotspot 6 ( Table 3 , Figure 3F ), ​​the corresponding target sequence is −442 bp to −405 bp of the p21 promoter sequence, and the sequence is shown in SEQ ID NO: 98. In this region, 12 functional saRNAs were found, namely RAG-427, RAG-430, RAG-431, RAG-423, RAG-425, RAG-433, RAG-435, RAG-434, RAG-439, RAG-426, RAG-428, and RAG-442;

[0103] Hotspot 7 ( Table 3 , Figure 3G ), the corresponding target sequence is −352 bp to −313 bp of the p21 promoter sequence, and the sequence is shown in SEQ ID NO: 99. In this region, 13 functional saRNAs were found, namely RAG-335, RAG-351, RAG-352, RAG-331, RAG-344, RAG-342, RAG-341, RAG-333, RAG-345, RAG-346, RAG-336, RAG-332, and RAG-343;

[0104] Hotspot 8 (Table 3, Figure 3H), the corresponding target sequence is -325bp to -260bp of the p21 promoter sequence, and the sequence is shown in SEQ ID NO: 100. In this region, 18 functional saRNAs were found, namely RAG-294, RAG-285, RAG-286, RAG-292, RAG-291, RAG-284, RAG-279, RAG-280, RAG-325, RAG-293, RAG-322, RAG-321, RAG-281, RAG-289, RAG-278, RAG-283, RAG-282, and RAG-295.

[0105] To verify the results of QuantiGene 2.0, 439 double-stranded RNA molecules were divided into four groups (bins) according to the activity of activating p21mRNA expression. Five double-stranded RNA molecules were randomly selected from each group and transfected into PC3 cells at a concentration of 10nM. After 72 hours, total cell RNA was extracted and reverse transcribed, and the p21 gene mRNA level was analyzed by RT-qPCR. The p21 gene mRNA expression levels revealed by the two methods showed a significant correlation (R 2 =0.82)( Figure 4 ). All functional saRNAs obtained by the QuantiGene2.0 method can be verified as true functional small activating RNAs by RT-qPCR method, and some of them show stronger induction of p21 mRNA expression in RT-qPCR analysis (Table 4).

[0106] Table 4. Validation of the QuantiGene 2.0 method

[0107]

[0108]

[0109] In summary, the above data indicate that many specific sites in the p21 promoter region can be used as saRNA target sequences to induce p21 expression, some of which are more sensitive and their corresponding saRNAs are more likely to activate p21 expression.

[0110] Example 2: saRNA induces p21 gene mRNA expression and inhibits cancer cell proliferation

[0111] In order to further evaluate the effect of p21 saRNA in inducing p21 gene mRNA expression and inhibiting cancer cell proliferation, saRNA (RAG1-431, RAG1-553, RAG1-688) screened by QuantiGene 2.0 was transfected into cancer cell lines KU-7 (bladder cancer), HCT116 (colon cancer) and HepG2 (hepatocellular carcinoma). The results showed that in all the above cell lines, saRNA could induce at least twice the expression level of p21 gene mRNA and inhibit cell proliferation, revealing the efficacy of saRNA-mediated p21 induction. Specifically, RAG1-431, RAG1-553, and RAG1-688 were transfected into KU7 cells, respectively, inducing p21 mRNA expression by 14.0, 36.9, and 31.9 times, respectively, and producing survival rates of 71.7%, 60.7%, and 67.4% ( Figure 5 ). RAG1-431, RAG1-553, and RAG1-688 were transfected into HCT116 cells, respectively, and induced p21 mRNA expression by 2.3, 3.5, and 2.4 times, respectively, and produced survival rates of 45.3%, 22.5%, and 38.5% relative to the blank treatment ( Figure 6 ). RAG1-431, RAG1-553, and RAG1-688 were transfected into HepG2 cells, respectively, and induced p21 mRNA expression by 2.2, 3.3, and 2.0 times, respectively, and produced survival rates of 76.7%, 64.9%, and 79.9% relative to blank treatment ( Figure 7 ).

[0112] Incorporated by Reference

[0113] The entire disclosure of each patent and scientific article cited herein is incorporated by reference for all purposes.

[0114] Equivalence

[0115] The present invention may be implemented in other specific forms without departing from its essential features. Therefore, the foregoing embodiments are considered to be illustrative rather than limiting of the present invention described herein. The scope of the present invention is indicated by the appended claims rather than by the foregoing description, and it is intended that all changes within the meaning and scope of the equivalents of the claims be included therein. Sequence Listing <110> Sino-US Ruikang Nucleic Acid Technology (Nantong) Research Institute Co., Ltd. <120> A method for activating p21 gene expression <130> AJ2349PCT1901CN-DIV <150> CN201810316672.1 <151> 2018-04-10 <160> 468 <170> SIPOSequenceListing 1.0 <210> 1 <211> twenty one <212> DNA <213> Artificial Sequence <400> 1 atcaccatct tccaggagcg a 21 <210> 2 <211> twenty one <212> DNA <213> Artificial Sequence <400> 2 ttctccatgg tggtgaagac g 21 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <400> 3 ggaagaccat gtggacctgt 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <400> 4 ggattagggc ttcctcttgg 20 <210> 5 <211> 92 <212> DNA <213> Homo sapiens <400> 5 ggctatgtgg ggagtattca ggagacagac aactcactcg tcaaatcctc cccttcctgg 60 ccaacaaagc tgctgcaacc acagggattt ct 92 <210> 6 <211> 86 <212> DNA <213> Homo sapiens <400> 6 ggtagtctct ccaattccct ccttcccgga agcatgtgac aatcaacaac tttgtatact 60 taagttcagt ggacctcaat ttcctc 86 <210> 7 <211> 35 <212> DNA <213> Homo sapiens <400> 7 ctttgttggg gtgtctaggt gctccaggtg cttct 35 <210> 8 <211> 51 <212> DNA <213> Homo sapiens <400> 8 ttctgggaga ggtgacctag tgagggatca gtgggaatag aggtgatatt g 51 <210> 9 <211> 30 <212> DNA <213> Homo sapiens <400> 9 aggtgatatt gtggggcttt tctggaaatt 30 <210> 10 <211> 38 <212> DNA <213> Homo sapiens <400> 10 attaatgtca tcctcctgat cttttcagct gcattggg 38 <210> 11 <211> 40 <212> DNA <213> Homo sapiens <400> 11 tctaacagtg ctgtgtcctc ctggagagtg ccaactcatt 40 <210> 12 <211> 66 <212> DNA <213> Homo sapiens <400> 12 gtgccaactc attctccaag taaaaaaagc cagatttgtg gctcacttcg tggggaaatg 60 tgtcca 66 <210> 13 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 13 ucguggggaa auguguccat t 21 <210> 14 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 14 uggacacauu uccccacgat t 21 <210> 15 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 15 uucgugggga aaugugucct t 21 <210> 16 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 16 ggacacauuu ccccacgaat t 21 <210> 17 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 17 cuucgugggg aaauguguct t 21 <210> 18 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 18 gacacauuuc cccacgaagt t 21 <210> 19 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 19 acuucguggg gaaaugugut t 21 <210> 20 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 20 acacauuucc ccacgaagut t 21 <210> twenty one <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> twenty one cacuucgugg ggaaaugugt t 21 <210> twenty two <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> twenty two cacauuuccc cacgaagugt t 21 <210> twenty three <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> twenty three ucacuucguggggaaaugut t 21 <210> twenty four <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> twenty four acauuucccc acgaagugat t 21 <210> twenty five <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> twenty five cucacuucgu ggggaaaugt t 21 <210> 26 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 26 cauuucccca cgaagugagt t 21 <210> 27 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 27 gcucacuucg uggggaaaut t 21 <210> 28 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 28 auuuccccac gaagugagct t 21 <210> 29 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 29 ggcucacuuc guggggaaat t 21 <210> 30 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 30 uuuccccacg aagugagcct t 21 <210> 31 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 31 uggcucacuu cguggggaat t 21 <210> 32 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 32 uuccccacga agugagccat t 21 <210> 33 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 33 guggcucacu ucguggggat t 21 <210> 34 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 34 uccccacgaa gugagccact t 21 <210> 35 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 35 uguggcucac uucguggggt t 21 <210> 36 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 36 ccccacgaag ugagccacat t 21 <210> 37 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 37 uuuguggcuc acuucguggt t 21 <210> 38 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 38 ccacgaagug agccacaaat t 21 <210> 39 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 39 auuuguggcu cacuucgugt t 21 <210> 40 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 40 cacgaaguga gccacaaaut t 21 <210> 41 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 41 gauuuguggc ucacuucgut t 21 <210> 42 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 42 acgaagugag ccacaaauct t 21 <210> 43 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 43 agauuugugg cucacuucgt t 21 <210> 44 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 44 cgaagugagc cacaaaucut t 21 <210> 45 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 45 cagauuugug gcucacuuct t 21 <210> 46 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 46 gaagugagcc acaaaucugt t 21 <210> 47 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 47 ccagauuugu ggcucacuut t 21 <210> 48 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 48 aagugagcca caaaucuggt t 21 <210> 49 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 49 gccagauuug uggcucacut t 21 <210> 50 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 50 agugagccac aaaucuggct t 21 <210> 51 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 51 agccagauuu guggcucact t 21 <210> 52 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 52 gugagccaca aaucuggcut t 21 <210> 53 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 53 aagccagauu uguggcucat t 21 <210> 54 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 54 ugagccacaa aucuggcuut t 21 <210> 55 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 55 aaagccagau uuguggcuct t 21 <210> 56 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 56 gagccacaaa ucuggcuuut t 21 <210> 57 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 57 aaaagccaga uuuguggcut t 21 <210> 58 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 58 agccacaaau cuggcuuuut t 21 <210> 59 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 59 caacucauuc uccaaguaat t 21 <210> 60 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 60 uuacuuggag aaugaguugt t 21 <210> 61 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 61 ccaacucauu cuccaaguat t 21 <210> 62 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 62 uacuuggaga augaguuggt t 21 <210> 63 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 63 gccaacucau ucuccaagut t 21 <210> 64 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 64 acuuggagaa ugaguuggct t 21 <210> 65 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 65 ugccaacuca uucuccaagt t 21 <210> 66 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 66 cuuggagaau gaguuggcat t 21 <210> 67 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 67 gugccaacuc auucuccaat t 21 <210> 68 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 68 uuggagaaug aguuggcact t 21 <210> 69 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 69 uggagagugc caacucauut t 21 <210> 70 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 70 aaugaguugg cacucuccat t 21 <210> 71 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 71 cuggagagug ccaacucaut t 21 <210> 72 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 72 augaguuggc acucuccagt t 21 <210> 73 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 73 ccuggagagu gccaacucat t 21 <210> 74 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 74 ugaguuggca cucuccaggt t 21 <210> 75 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 75 uccuggagag ugccaacuct t 21 <210> 76 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 76 gaguuggcac ucuccaggat t 21 <210> 77 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 77 cuccuggaga gugccaacut t 21 <210> 78 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 78 aguuggcacu cuccaggagt t 21 <210> 79 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 79 ccuccuggag agugccaact t 21 <210> 80 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 80 guuggcacuc uccaggaggt t 21 <210> 81 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 81 uccuccugga gagugccaat t 21 <210> 82 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 82 uuggcacucu ccaggaggat t 21 <210> 83 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 83 guccuccugg agagugccat t 21 <210> 84 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 84 uggcacucuc caggaggact t 21 <210> 85 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 85 uguguccucc uggagagugt t 21 <210> 86 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 86 cacucuccag gaggacacat t 21 <210> 87 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 87 cuguguccuc cuggagagut t 21 <210> 88 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 88 acucuccagg aggacacagt t 21 <210> 89 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 89 gcuguguccu ccuggagagt t 21 <210> 90 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 90 cucuccagga ggacacagct t 21 <210> 91 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 91 ugcugugucc uccuggagat t 21 <210> 92 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 92 ucuccaggag gacacagcat t 21 <210> 93 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 93 gugcuguguc cuccuggagt t 21 <210> 94 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 94 cuccaggagg acacagcact t 21 <210> 95 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 95 agugcugugu ccuccuggat t 21 <210> 96 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 96 uccaggagga cacagcacut t 21 <210> 97 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 97 acagugcugu guccuccugt t 21 <210> 98 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 98 caggaggaca cagcacugut t 21 <210> 99 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 99 aacagugcug uguccuccut t 21 <210> 100 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 100 aggaggacac agcacuguut t 21 <210> 101 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 101 uaacagugcu guguccucct t 21 <210> 102 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 102 ggaggacaca gcacuguuat t 21 <210> 103 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 103 cuaacagugc uguguccuct t 21 <210> 104 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 104 gaggacacag cacuguuagt t 21 <210> 105 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 105 ucuaacagug cuguguccut t 21 <210> 106 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 106 aggacacagc acuguuagat t 21 <210> 107 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 107 ucuuuucagc ugcauugggt t 21 <210> 108 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 108 cccaaugcag cugaaaagat t 21 <210> 109 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 109 aucuuuucag cugcauuggt t 21 <210> 110 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 110 ccaaugcagc ugaaaagaut t 21 <210> 111 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 111 gaucuuuuca gcugcauugt t 21 <210> 112 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 112 caaugcagcu gaaaagauct t 21 <210> 113 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 113 ugaucuuuuc agcugcauut t 21 <210> 114 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 114 aaugcagcug aaaagaucat t 21 <210> 115 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 115 cugaucuuuu cagcugcaut t 21 <210> 116 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 116 augcagcuga aaagaucagt t 21 <210> 117 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 117 ccugaucuuu ucagcugcat t 21 <210> 118 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 118 ugcagcugaa aagaucaggt t 21 <210> 119 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 119 uccugaucuu uucagcugct t 21 <210> 120 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 120 gcagcugaaa agaucaggat t 21 <210> 121 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 121 cuccugaucu uuucagcugt t 21 <210> 122 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 122 cagcugaaaa gaucaggagt t 21 <210> 123 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 123 ccuccugauc uuuucagcut t 21 <210> 124 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 124 agcugaaaag aucaggaggt t 21 <210> 125 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 125 uccuccugau cuuuucagct t 21 <210> 126 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 126 gcugaaaaga ucaggaggat t 21 <210> 127 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 127 auccuccuga ucuuuucagt t 21 <210> 128 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 128 cugaaaagau caggaggaut t 21 <210> 129 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 129 cauccuccug aucuuuucat t 21 <210> 130 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 130 ugaaaagauc aggaggaugt t 21 <210> 131 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 131 ucauccuccu gaucuuuuct t 21 <210> 132 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 132 gaaaagauca ggaggaugat t 21 <210> 133 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 133 gucauccucc ugaucuuuut t 21 <210> 134 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 134 aaaagaucag gaggaugact t 21 <210> 135 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 135 ugucauccuc cugaucuuut t 21 <210> 136 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 136 aaagaucagg aggaugacat t 21 <210> 137 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 137 augucauccu ccugaucuut t 21 <210> 138 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 138 aagaucagga ggaugacaut t 21 <210> 139 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 139 aaugucaucc uccugaucut t 21 <210> 140 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 140 agaucaggag gaugacauut t 21 <210> 141 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 141 uaaugucauc cuccugauct t 21 <210> 142 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 142 gaucaggagg augacauuat t 21 <210> 143 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 143 uuaaugucau ccuccugaut t 21 <210> 144 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 144 aucaggagga ugacauuaat t 21 <210> 145 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 145 auuaauguca uccuccugat t 21 <210> 146 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 146 ucaggaggau gacauuaaut t 21 <210> 147 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 147 uggggcuuuu cuggaaauut t 21 <210> 148 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 148 aauuuccaga aaagccccat t 21 <210> 149 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 149 guggggcuuu ucuggaaaut t 21 <210> 150 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 150 auuuccagaa aagccccact t 21 <210> 151 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 151 uguggggcuu uucuggaaat t 21 <210> 152 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 152 uuuccagaaa agccccacat t 21 <210> 153 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 153 uuguggggcu uuucuggaat t 21 <210> 154 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 154 uuccagaaaa gccccacaat t 21 <210> 155 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 155 auuguggggc uuuucuggat t 21 <210> 156 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 156 uccagaaaag ccccacaaut t 21 <210> 157 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 157 uauugugggg cuuuucuggt t 21 <210> 158 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 158 ccagaaaagc cccacaauat t 21 <210> 159 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 159 auauugugggcuuuucugt t 21 <210> 160 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 160 cagaaaagcc ccacaauaut t 21 <210> 161 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 161 gauauugugg ggcuuuucut t 21 <210> 162 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 162 agaaaagccc cacaauauct t 21 <210> 164 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 164 ugauauugug gggcuuuuct t 21 <210> 165 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 165 gaaaagcccc acaauaucat t 21 <210> 166 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 166 gugauauugu ggggcuuuut t 21 <210> 167 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 167 aaaagcccca caauaucact t 21 <210> 168 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 168 ggugauauug uggggcuuut t 21 <210> 169 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 169 aaagccccac aauaucacct t 21 <210> 170 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 170 aggugauauu guggggcuut t 21 <210> 171 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 171 aagccccaca auaucaccut t 21 <210> 171 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 171 gggaauagag gugauauugt t 21 <210> 172 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 172 caauaucacc ucuauuccct t 21 <210> 173 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 173 ugggaauaga ggugauauut t 21 <210> 174 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 174 aauaucaccu cuauucccat t 21 <210> 175 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 175 gugggaauag aggugauaut t 21 <210> 176 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 176 auaucaccuc uauucccact t 21 <210> 177 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 177 agugggaaua gaggugauat t 21 <210> 178 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 178 uaucaccucu auucccacut t 21 <210> 179 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 179 cagugggaau agaggugaut t 21 <210> 180 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 180 aucaccucua uucccacugt t 21 <210> 181 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 181 ucagugggaa uagaggugat t 21 <210> 182 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 182 ucaccucuau ucccacugat t 21 <210> 183 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 183 aucaguggga auagaggugt t 21 <210> 184 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 184 caccucuauu cccacugaut t 21 <210> 185 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 185 gaucaguggg aauagaggut t 21 <210> 186 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 186 accucuauuc ccacugauct t 21 <210> 187 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 187 ggaucagugg gaauagaggt t 21 <210> 188 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 188 ccucuauucc cacugaucct t 21 <210> 189 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 189 gggaucagug ggaauagagt t 21 <210> 190 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 190 cucuauuccc acugauccct t 21 <210> 191 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 191 agggaucagu gggaauagat t 21 <210> 192 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 192 ucuauuccca cugaucccut t 21 <210> 193 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 193 gagggaucag ugggaauagt t 21 <210> 194 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 194 cuauucccac ugaucccuct t 21 <210> 195 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 195 ugagggauca gugggaauat t 21 <210> 196 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 196 uauucccacu gaucccucat t 21 <210> 197 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 197 gugagggauc agugggaaut t 21 <210> 198 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 198 auucccacug aucccucact t 21 <210> 199 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 199 agugagggau cagugggaat t 21 <210> 200 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 200 uucccacuga ucccucacut t 21 <210> 201 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 201 uagugaggga ucagugggat t 21 <210> 202 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 202 ucccacugau cccucacuat t 21 <210> 203 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 203 cuagugaggg aucagugggt t 21 <210> 204 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 204 cccacugauc ccucacuagt t 21 <210> 205 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 205 accuagugag ggaucagugt t 21 <210> 206 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 206 cacugauccc ucacuaggut t 21 <210> 207 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 207 gaccuaguga gggaucagut t 21 <210> 208 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 208 acugaucccu cacuagguct t 21 <210> 209 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 209 ugaccuagug agggaucagt t 21 <210> 210 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 210 cugaucccuc acuaggucat t 21 <210> 211 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 211 gugaccuagu gagggaucat t 21 <210> 212 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 212 ugaucccuca cuaggucact t 21 <210> 213 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 213 ggugaccuag ugagggauct t 21 <210> 214 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 214 gaucccucac uaggucacct t 21 <210> 215 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 215 aggugaccua gugagggaut t 21 <210> 216 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 216 aucccucacu aggucaccut t 21 <210> 217 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 217 gaggugaccu agugagggat t 21 <210> 218 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 218 ucccucacua ggucaccuct t 21 <210> 219 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 219 ggagagguga ccuagugagt t 21 <210> 220 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 220 cucacuaggu caccucucct t 21 <210> 221 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 221 gggagaggug accuagugat t 21 <210> 222 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 222 ucacuagguc accucuccct t 21 <210> 223 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 223 ugggagaggu gaccuagugt t 21 <210> 224 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 224 cacuagguca ccucucccat t 21 <210> 225 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 225 cugggagagg ugaccuagut t 21 <210> 226 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 226 acuaggucac cucucccagt t 21 <210> 227 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 227 ucugggagag gugaccuagt t 21 <210> 228 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 228 cuaggucacc ucucccagat t 21 <210> 229 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 229 uucugggaga ggugaccuat t 21 <210> 230 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 230 uaggucaccu cucccagaat t 21 <210> 231 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 231 aggugcucca ggugcuucut t 21 <210> 232 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 232 agaagcaccu ggagcaccut t 21 <210> 233 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 233 uaggugcucc aggugcuuct t 21 <210> 234 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 234 gaagcaccug gagcaccuat t 21 <210> 235 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 235 ugucuaggug cuccaggugt t 21 <210> 236 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 236 caccuggagc accuagacat t 21 <210> 237 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 237 ggugucuagg ugcuccaggt t 21 <210> 238 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 238 ccuggagcac cuagacacct t 21 <210> 239 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 239 gggugucuag gugcuccagt t 21 <210> 240 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 240 cuggagcacc uagacaccct t 21 <210> 241 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 241 ggggugucua ggugcuccat t 21 <210> 242 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 242 uggagcaccu agacacccct t 21 <210> 243 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 243 uggggugucu aggugcucct t 21 <210> 244 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 244 ggagcaccua gacaccccat t 21 <210> 245 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 245 uugggguguc uaggugcuct t 21 <210> 246 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 246 gagcaccuag acaccccaat t 21 <210> 247 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 247 uuguuggggu gucuaggugt t 21 <210> 248 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 248 caccuagaca ccccaacaat t 21 <210> 249 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 249 uuuguugggg ugucuaggut t 21 <210> 250 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 250 accuagacac cccaacaaat t 21 <210> 251 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 251 cuuuguuggg gugucuaggt t 21 <210> 252 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 252 ccuagacacc ccaacaaagt t 21 <210> 253 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 253 aguggaccuc aauuuccuct t 21 <210> 254 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 254 gaggaaauug agguccacut t 21 <210> 255 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 255 caguggaccu caauuuccut t 21 <210> 256 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 256 aggaaauuga gguccacugt t 21 <210> 257 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 257 ucaguggacc ucaauuucct t 21 <210> 258 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 258 ggaaauugag guccacugat t 21 <210> 259 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 259 uucaguggac cucaauuuct t 21 <210> 260 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 260 gaaauugagg uccacugaat t 21 <210> 261 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 261 guucagugga ccucaauuut t 21 <210> 262 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 262 aaauugaggu ccacugaact t 21 <210> 263 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 263 aguucagugg accucaauut t 21 <210> 264 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 264 aauugagguc cacugaacut t 21 <210> 265 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 265 aaguucagug gaccucaaut t 21 <210> 266 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 266 auugaggucc acugaacuut t 21 <210> 267 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 267 uaaguucagu ggaccucaat t 21 <210> 268 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 268 uugaggucca cugaacuuat t 21 <210> 269 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 269 uuaaguucag uggaccucat t 21 <210> 270 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 270 ugagguccac ugaacuuaat t 21 <210> 271 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 271 cuuaaguuca guggaccuct t 21 <210> 272 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 272 gagguccacu gaacuuaagt t 21 <210> 273 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 273 acuuaaguuc aguggaccut t 21 <210> 274 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 274 agguccacug aacuuaagut t 21 <210> 275 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 275 uacuuaaguu caguggacct t 21 <210> 276 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 276 gguccacuga acuuaaguat t 21 <210> 277 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 277 auacuuaagu ucaguggact t 21 <210> 278 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 278 guccacugaa cuuaaguaut t 21 <210> 279 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 279 gugacaauca acaacuuugt t 21 <210> 280 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 280 caaaguuguu gauugucact t 21 <210> 281 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 281 caugugacaa ucaacaacut t 21 <210> 282 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 282 aguuguugau ugucacaugt t 21 <210> 283 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 283 gcaugugaca aucaacaact t 21 <210> 284 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 284 guuguugauu gucacaugct t 21 <210> 285 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 285 agcaugugac aaucaacaat t 21 <210> 286 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 286 uuguugauug ucacaugcut t 21 <210> 287 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 287 aagcauguga caaucaacat t 21 <210> 288 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 288 uguugauugu cacaugcuut t 21 <210> 289 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 289 gaagcaugug acaaucaact t 21 <210> 290 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 290 guugauuguc acaugcuuct t 21 <210> 291 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 291 ggaagcaugu gacaaucaat t 21 <210> 292 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 292 uugauuguca caugcuucct t 21 <210> 293 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 293 cggaagcaug ugacaaucat t 21 <210> 294 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 294 ugauugucac augcuuccgt t 21 <210> 295 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 295 ccggaagcau gugacaauct t 21 <210> 296 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 296 gauugucaca ugcuuccggt t 21 <210> 297 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 297 cccggaagca ugugacaaut t 21 <210> 298 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 298 auugucacau gcuuccgggt t 21 <210> 299 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 299 ucccggaagc augugacaat t 21 <210> 300 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 300 uugucacaug cuuccgggat t 21 <210> 301 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 301 uucccggaag caugugacat t 21 <210> 302 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 302 ugucacaugc uuccgggaat t 21 <210> 303 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 303 cuucccggaa gcaugugact t 21 <210> 304 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 304 gucacaugcu uccgggaagt t 21 <210> 305 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 305 ccuucccgga agcaugugat t 21 <210> 306 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 306 ucacaugcuu ccgggaaggt t 21 <210> 307 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 307 uccuucccgg aagcaugugt t 21 <210> 308 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 308 cacaugcuuc cgggaaggat t 21 <210> 309 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 309 cuccuucccg gaagcaugut t 21 <210> 310 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 310 acaugcuuccgggaaggagt t 21 <210> 311 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 311 ccuccuuccc ggaagcaugt t 21 <210> 312 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 312 caugcuuccg ggaaggaggt t 21 <210> 313 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 313 cccuccuucc cggaagcaut t 21 <210> 314 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 314 augcuuccgg gaaggagggt t 21 <210> 315 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 315 ucccuccuuc ccggaagcat t 21 <210> 316 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 316 ugcuuccggg aaggagggat t 21 <210> 317 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 317 auucccuccu ucccggaagt t 21 <210> 318 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 318 cuuccgggaa ggagggaaut t 21 <210> 319 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 319 aauucccucc uucccggaat t 21 <210> 320 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 320 uuccgggaag gagggaauut t 21 <210> 321 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 321 ucuccaauuc ccuccuucct t 21 <210> 322 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 322 ggaaggaggg aauuggagat t 21 <210> 323 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 323 cucuccaauu cccuccuuct t 21 <210> 324 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 324 gaaggaggga auuggagagt t 21 <210> 325 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 325 ucucuccaau ucccuccuut t 21 <210> 326 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 326 aaggagggaa uuggagagat t 21 <210> 327 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 327 gucucuccaa uucccuccut t 21 <210> 328 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 328 aggagggaau uggagagact t 21 <210> 329 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 329 aguccucca auucccucct t 21 <210> 330 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 330 ggagggaauu ggagagacut t 21 <210> 331 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 331 uagucucucc aauucccuct t 21 <210> 332 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 332 gagggaauug gagagacuat t 21 <210> 333 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 333 guagucucuc caauucccut t 21 <210> 334 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 334 agggaauugg agagacuact t 21 <210> 335 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 335 gguagucucu ccaauuccct t 21 <210> 336 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 336 gggaauugga gagacuacct t 21 <210> 337 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 337 ugcaaccaca gggauuucut t 21 <210> 338 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 338 agaaaucccu gugguugcat t 21 <210> 339 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 339 cugcaaccac agggauuuct t 21 <210> 340 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 340 gaaaucccug ugguugcagt t 21 <210> 341 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 341 gcugcaacca cagggauuut t 21 <210> 342 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 342 aaaucccugu gguugcagct t 21 <210> 343 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 343 ugcugcaacc acagggauut t 21 <210> 344 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 344 aaucccugug guugcagcat t 21 <210> 345 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 345 cugcugcaac cacagggaut t 21 <210> 346 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 346 aucccugugg uugcagcagt t 21 <210> 347 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 347 gcugcugcaa ccacagggat t 21 <210> 348 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 348 ucccuguggu ugcagcagct t 21 <210> 349 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 349 agcugcugca accacagggt t 21 <210> 350 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 350 cccugugguu gcagcagcut t 21 <210> 351 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 351 aaagcugcug caaccacagt t 21 <210> 352 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 352 cugugguugc agcagcuuut t 21 <210> 353 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 353 caaagcugcu gcaaccacat t 21 <210> 354 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 354 ugugguugca gcagcuuugt t 21 <210> 355 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 355 acaaagcugc ugcaaccact t 21 <210> 356 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 356 gugguugcag cagcuuugut t 21 <210> 357 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 357 aacaaagcug cugcaaccat t 21 <210> 358 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 358 ugguugcagc agcuuuguut t 21 <210> 359 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 359 caacaaagcu gcugcaacct t 21 <210> 360 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 360 gguugcagca gcuuuguugt t 21 <210> 361 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 361 ccaacaaagc ugcugcaact t 21 <210> 362 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 362 guugcagcag cuuuguuggt t 21 <210> 363 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 363 gccaacaaag cugcugcaat t 21 <210> 364 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 364 uugcagcagc uuuguuggct t 21 <210> 365 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 365 ggccaacaaa gcugcugcat t 21 <210> 366 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 366 ugcagcagcu uuguuggcct t 21 <210> 367 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 367 uggccaacaa agcugcugct t 21 <210> 368 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 368 gcagcagcuu uguuggccat t 21 <210> 369 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 369 cuggccaaca aagcugcugt t 21 <210> 370 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 370 cagcagcuuu guuggccagt t 21 <210> 371 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 371 ccuggccaac aaagcugcut t 21 <210> 372 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 372 agcagcuuug uuggccaggt t 21 <210> 373 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 373 uuccuggcca acaaagcugt t 21 <210> 374 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 374 cagcuuuguu ggccaggaat t 21 <210> 375 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 375 cuuccuggcc aacaaagcut t 21 <210> 376 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 376 agcuuuguug gccaggaagt t 21 <210> 377 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 377 cccuuccugg ccaacaaagt t 21 <210> 378 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 378 cuuuguuggc caggaagggt t 21 <210> 379 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 379 ccccuuccug gccaacaaat t 21 <210> 380 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 380 uuuguuggcc aggaaggggt t 21 <210> 381 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 381 uccccuuccu ggccaacaat t 21 <210> 382 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 382 uuguuggcca ggaaggggat t 21 <210> 383 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 383 cuccccuucc uggccaacat t 21 <210> 384 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 384 uguuggccag gaaggggagt t 21 <210> 385 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 385 uccuccccuu ccuggccaat t 21 <210> 386 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 386 uuggccagga aggggaggat t 21 <210> 387 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 387 auccuccccu uccuggccat t 21 <210> 388 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 388 uggccaggaa ggggaggaut t 21 <210> 389 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 389 ucaaauccuc cccuuccugt t 21 <210> 390 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 390 caggaagggg aggauuugat t 21 <210> 391 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 391 gucaaauccu ccccuuccut t 21 <210> 392 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 392 aggaagggga ggauuugact t 21 <210> 393 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 393 cgucaaaucc uccccuucct t 21 <210> 394 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 394 ggaaggggag gauuugacgt t 21 <210> 395 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 395 ucgucaaauc cuccccuuct t 21 <210> 396 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 396 gaaggggagg auuugacgat t 21 <210> 397 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 397 cucgucaaau ccuccccuut t 21 <210> 398 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 398 aaggggagga uuugacgagt t 21 <210> 399 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 399 acucgucaaa uccuccccut t 21 <210> 400 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 400 aggggaggau uugacgagut t 21 <210> 401 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 401 ucacucguca aauccuccct t 21 <210> 402 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 402 gggaggauuu gacgagugat t 21 <210> 403 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 403 cucacucguc aaauccucct t 21 <210> 404 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 404 ggaggauuug acgagugagt t 21 <210> 405 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 405 acucacucgu caaauccuct t 21 <210> 406 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 406 gaggauuuga cgagugagut t 21 <210> 407 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 407 caacucacuc gucaaaucct t 21 <210> 408 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 408 ggauuugacg agugaguugt t 21 <210> 409 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 409 acaacucacu cgucaaauct t 21 <210> 410 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 410 gauuugacga gugaguugut t 21 <210> 411 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 411 gacaacucac ucgucaaaut t 21 <210> 412 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 412 auuugacgag ugaguuguct t 21 <210> 413 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 413 agacaacuca cucgucaaat t 21 <210> 414 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 414 uuugacgagu gaguugucut t 21 <210> 415 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 415 cagacaacuc acucgucaat t 21 <210> 416 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 416 uugacgagug aguugucugt t 21 <210> 417 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 417 acagacaacu cacucgucat t 21 <210> 418 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 418 ugacgaguga guugucugut t 21 <210> 419 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 419 gacagacaac ucacucguct t 21 <210> 420 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 420 gacgagugag uugucuguct t 21 <210> 421 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 421 agacagacaa cucacucgut t 21 <210> 422 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 422 acgagugagu ugucugucut t 21 <210> 423 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 423 gagacagaca acucacucgt t 21 <210> 424 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 424 cgagugaguu gucugucuct t 21 <210> 425 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 425 ggagacagac aacucacuct t 21 <210> 426 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 426 gagugaguug ucugucucct t 21 <210> 427 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 427 aggagacaga caacucacut t 21 <210> 428 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 428 agugaguugu cugucuccut t 21 <210> 429 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 429 caggagacag acaacucact t 21 <210> 430 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 430 gugaguuguc ugucuccugt t 21 <210> 431 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 431 ucaggagaca gacaacucat t 21 <210> 432 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 432 ugaguugucu gucuccugat t 21 <210> 433 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 433 uucaggagac agacaacuct t 21 <210> 434 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 434 gaguugucug ucuccugaat t 21 <210> 435 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 435 auucaggaga cagacaacut t 21 <210> 436 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 436 aguugucugu cuccugaut t 21 <210> 437 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 437 uauucaggag acagacaact t 21 <210> 438 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 438 guugucuguc uccugaauat t 21 <210> 439 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 439 21 <210> 440 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 440 uugucugucu ccugaauact t 21 <210> 441 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 441 aguauucagg agacagacat t 21 <210> 442 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 442 ugucugucuc cugaauacut t 21 <210> 443 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 443 gaguauucag gagacagact t 21 <210> 444 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 444 gucugucucc ugaauacuct t 21 <210> 445 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 445 ggaguauuca ggagacagat t 21 <210> 446 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 446 ucugucuccu gaauacucct t 21 <210> 447 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 447 gggaguauuc aggagacagt t 21 <210> 448 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 448 cugucuccug aauacuccct t 21 <210> 449 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 449 ggggaguauu caggagacat t 21 <210> 450 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 450 ugucuccuga auacucccct t 21 <210> 451 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 451 uggggaguau ucaggagact t 21 <210> 452 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 452 gucuccugaa uacuccccat t 21 <210> 453 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 453 guggggagua uucaggagat t 21 <210> 454 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 454 ucuccugaau acuccccact t 21 <210> 455 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 455 uguggggagu auucaggagt t 21 <210> 456 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 456 cuccugaaua cuccccacat t 21 <210> 457 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 457 auguggggag uauucaggat t 21 <210> 458 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 458 uccugaauac uccccacaut t 21 <210> 459 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 459 uaugugggga guauucaggt t 21 <210> 460 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 460 ccugaauacu ccccacauat t 21 <210> 461 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 461 cuaugugggg aguauucagt t 21 <210> 462 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 462 cugaauacuc cccacauagt t 21 <210> 463 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 463 gcuauguggg gaguauucat t 21 <210> 464 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 464 ugaauacucc ccacauagct t 21 <210> 465 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 465 ggcuaugugg ggaguauuct t 21 <210> 466 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 466 gaauacuccc cacauagcct t 21 <210> 467 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 467 gggcuaugug gggaguauut t 21 <210> 468 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 468 aauacucccc acauagccct t 21

Claims

1. An isolated p21 gene small activating RNA target site, wherein the target site is a continuous 19 nucleotide sequence from position 60 to position 78 of SEQ ID NO:

5.

2. A small activating RNA, wherein the small activating RNA comprises a sense nucleic acid chain and an antisense nucleic acid chain, wherein the sense nucleic acid chain and the antisense nucleic acid chain contain complementary regions, and the complementary regions can form a double-stranded nucleic acid structure, wherein the nucleotide sequences of the sense nucleic acid chain and the antisense nucleic acid chain are shown in SEQ ID NO: 363 and SEQ ID NO: 364, respectively.

3. The small activating RNA of claim 2, wherein at least one nucleotide in the small activating RNA is a chemically modified nucleotide, and the chemical modification is at least one of the following modifications: (1) Modification of the phosphodiester bonds connecting nucleotides in the nucleotide sequence of the small activating RNA; (2) modification of the 2'-OH of the ribose in the nucleotide sequence of the small activating RNA; (3) modification of bases in the nucleotide sequence of the small activating RNA; (4) At least one nucleotide in the nucleotide sequence of the small activating RNA is a locked nucleic acid.

4. Use of the small activating RNA as claimed in claim 2 or 3 in the preparation of a preparation for activating or up-regulating the expression of the p21 gene in cells.

5. The use according to claim 4, wherein the small activating RNA is directly introduced into the cell.

6. The use according to claim 5, wherein the cells are mammalian cells.

7. The use according to claim 5, wherein the cells are human cells.

8. A method for activating or upregulating the expression of p21 gene in cells for non-diagnostic and non-therapeutic purposes, wherein the method comprises administering the small activating RNA according to claim 2 or 3 to the cells.

9. The method of claim 8, wherein the small activating RNA is introduced directly into the cell.

10. The method of claim 8 or 9, wherein the cell is a mammalian cell.

11. The method of claim 10, wherein the cells are human cells.

12. A composition comprising the small activating RNA according to claim 2 or 3 and a pharmaceutically acceptable carrier.

13. The composition according to claim 12, characterized in that The pharmaceutically acceptable carrier is a liposome, a high molecular weight polymer or a polypeptide.

14. Use of the small activating RNA according to claim 2 or 3 or the composition according to claim 12 or 13 in the preparation of a preparation for activating or up-regulating p21 gene expression.

Images