KR102193869B1 - RNA interference inducing nucleic acids suppressing noncanonical targets of microRNA and use thereof - Google Patents

Abstract

The present invention relates to an RNA interference-inducing nucleic acid that suppresses a noncanonical target gene of a microRNA by modifying some sequence of a specific microRNA. When using the RNA interference-inducing nucleic acid according to the present invention, the microRNA is non-normal It has the effect of efficiently increasing the biological function expressed by suppressing the target gene, or selectively expressing only the biological function expressed by suppressing some of the functions exhibited by the existing microRNA, that is, the non-normal target gene. Through this, cell cycle, differentiation, dedifferentiation, morphology, migration, division, proliferation or death can be controlled, so it is expected to be used in various fields such as drugs and cosmetics.

Description

RNA interference inducing nucleic acids suppressing noncanonical targets of microRNA and use thereof

The present invention relates to an RNA interference-inducing nucleic acid that inhibits gene expression and its use, and to an interference-inducing nucleic acid having a useful effect exerted by selectively inhibiting an irregular target of microRNA and its use.

RNA interference refers to a phenomenon that suppresses the expression of a gene in the post-transcriptional stage. The naturally occurring RNA interference phenomenon is caused by micro RNA (miRNA). MicroRNAs are composed of 18-25 bases, most of which are small RNAs composed of about 21 bases, and are complementary to the messenger RNAs (mRNAs) of the target genes through the Argonaute protein. Arrange it. In the case of animal microRNA, it binds to the argonet protein and performs partial nucleotide sequence with the target messenger RNA.At this time, the seed region defined by the 1st to 8th nucleic acids from the 5'end of the microRNA ), if at least 6 consecutive nucleotides are sequenced, it is recognized as a target, and most importantly, it is sufficient when conjugated with the target messenger RNA in a sequence of 6 nucleotides from the 2nd to the 7th based on the 5'end. It degrades the target messenger RNA or inhibits its expression by inhibiting translation (Lewis BP, et.al, 2003, Cell, 115 (7), 787-98). Because microRNAs thus recognize the messenger RNA of a target gene through partial nucleotide sequence, a single microRNA can usually affect the expression of hundreds to thousands of genes.

The inhibition of target gene expression of microRNA is one of the important mechanisms of gene expression. Under normal conditions, it is involved in the differentiation and growth of cells, and when there is a function abnormality, it causes cancer, degenerative diseases, and diabetes, which is the key to life. Is attracting attention. Therefore, in order to artificially induce the function of inhibiting gene expression of microRNAs, an RNA interference material (siRNA or shRNA) containing an initiating region of the microRNA is designed and introduced into the cell to artificially differentiate or change the function, In some cases, it is also used as a treatment for diseases. Therefore, the complementary nucleotide sequence in the microRNA initiation region, which is mediated by an agonist and recognizes the target messenger RNA, is important for the function of the RNA interference material including the microRNA. In particular, in order to utilize such an RNA interference material, it is required to understand the function of each microRNA, and at this time, the function of the microRNA is determined by which target gene is suppressed, so the total target gene of the microRNA (target Sieve) was needed.

At the transcript volume level, studies on microRNA targets were first performed by the present inventors through the Ago HITS-CLIP (or called CLIP-Seq) experiment. In the Ago HITS CLIP test method, a cell or tissue sample is irradiated with UV light to form a complex through covalent bonds between RNA and Argonaute (Ago) in the cell, and this RNA-agonal complex is specific to Argonut. This is a method of analyzing the isolated RNA by next-generation sequencing after separation by immunoprecipitation using an appropriately recognized antibody. Through this, it is possible to accurately analyze the microRNA bound to the agonut, the target messenger RNA group with complementary base sequence, and the location thereof.

As a result, the present inventors have found that when the microRNA binds to the target messenger RNA, it may bind even if it is not exactly complementary to the microRNA seed region. More specifically, the microRNA seed region defined by the 1st to 8th nucleotide sequences based on the 5'end of the microRNA binds to the messenger RNA in an arrangement of at least 6 consecutive and complete nucleotide sequences, especially Among them, the most important of which is binding in the 2nd to 7th nucleotide sequence based on the 5'end is called canonical target recognition, deviating from the above rules, and is continuous with the microRNA seed region. Recognizing and binding as a target of microRNA, even if it is not an exact complementary sequence relationship, is called non-canonical target recognition. According to the results of Ago HITS-CLIP analysis, it can be seen that the frequency of recognizing targets irregularly through the initiating region of microRNAs is about 50% of the frequency of regular recognition.

Therefore, it can be seen that the RNA interference material (eg, siRNA or shRNA) designed to include the sequence of the initiating region of the existing microRNA exhibits biological functions by suppressing not only regular target genes but also various irregular target genes.

Chi S et al, Nature, 2009, 460 (7254): 479-86

Accordingly, the problem to be solved by the present invention is to solve the limitations of the RNA interference material designed by including the sequence of the originating region of the existing microRNA as it is and to increase the efficiency.

More specifically, the RNA interference material designed by including the sequence of the originating region of the existing microRNA as it is, suppresses both the regular target gene and the irregular target gene, but strongly suppresses the regular target gene, whereas the non-normal target gene is very weak. Suppress. Therefore, the problem to be solved by the present invention is to efficiently increase the biological function exhibited by the existing microRNA by suppressing the irregular target gene, or by suppressing some of the functions represented by the existing microRNA, that is, the irregular target gene. It is to provide an RNA interference-inducing nucleic acid having an effect that selectively exhibits only a biological function.

In order to solve the above problems, the present invention provides an RNA interference-inducing nucleic acid that suppresses a noncanonical target gene of a microRNA by modifying some sequences of a specific microRNA.

More specifically,

In the present invention, in one or more single strands of the double strands of nucleic acids that induce RNA interference, some sequences of specific microRNAs are modified to induce RNA interference that suppresses noncanonical target genes of microRNAs. As a nucleic acid,

The RNA interference-inducing nucleic acid is based on the 2nd to 7th nucleotide sequence based on the 5'end, which is the site that plays the most important role in binding to the target messenger RNA in a specific microRNA, from the 2nd to the 5th from the 5'end. Contains 4 base sequences of, the 6th and 7th bases are the same, and the 6th and 7th bases have a base sequence complementary to a base that can be aligned with the 6th base of a specific microRNA, and the specific microRNA The bases that can be aligned with the 6th base of are all complementary bases including the G:A and G:U wobble sequences, so that the bulge in the target gene between the 5th and 6th of the microRNA. It is characterized in that the ridge disappears from the non-normal target nucleotide sequence that binds while the) is formed and binds to a continuous nucleotide sequence, or

The RNA interference-inducing nucleic acid is a nucleotide sequence between the 5'end and the ninth base of a specific microRNA, preferably the guanine base is uracil in the 2nd to 7th sequence based on the 5'end or By having a modified base sequence substituted with at least one or more adenine, characterized in that the G:A or G:U wobble arrangement of the site becomes a regular base sequence of U:A or A:U, RNA interference inducing nucleic acids are provided.

Preferably, the specific microRNA is selected from the group consisting of miR-124, miR-155, miR-122, miR-1, let-7, miR-133, miR-302 and miR-372 and has the same initiating sequence. It provides an RNA interference-inducing nucleic acid, characterized in that it is composed of 18-24 bases.

Preferably, the RNA interference-inducing nucleic acid provides an RNA interference-inducing nucleic acid, characterized in that the 2nd to 7th nucleotide sequence at the 5'end is at least one of the following:

RNA interference-inducing nucleic acid (miR-124-BS) showing the nucleotide sequence of 5'-AA GGC C-3' as an RNA interference-inducing nucleic acid that suppresses an irregular target gene of miR-124 (SEQ ID NO: 1);

RNA interference-inducing nucleic acid (miR-122-BS) (SEQ ID NO: 2) showing the nucleotide sequence of 5'-GG AGU U-3' as an RNA interference-inducing nucleic acid that suppresses an irregular target gene of miR-122;

RNA interference-inducing nucleic acid (miR-155-BS) showing the nucleotide sequence of 5'-UA AUG G-3' as an RNA interference-inducing nucleic acid that suppresses an irregular target gene of miR-155 (SEQ ID NO: 3); or

RNA interference-inducing nucleic acid (miR-1-BS) showing the nucleotide sequence of 5'-GG AAU U-3' as an RNA interference-inducing nucleic acid that suppresses an irregular target gene of miR-1 (SEQ ID NO: 4).

Preferably, it provides an RNA interference-inducing nucleic acid, characterized in that the base sequence of the RNA interference-inducing nucleic acid is represented by one or more of the following:

an RNA interference-inducing nucleic acid (miR-124-BS) showing the nucleotide sequence of 5'-UAA GGC CAC GCG GUG AAU GCC-3' as an RNA interference-inducing nucleic acid that suppresses an irregular target gene of miR-124 (SEQ ID NO: 5);

RNA interference-inducing nucleic acid (miR-122-BS) showing the nucleotide sequence of 5'-UGG AGU UGU GAC AAU GGU GUU-3' as an RNA interference-inducing nucleic acid that suppresses an irregular target gene of miR-122 (SEQ ID NO: 6);

RNA interference-inducing nucleic acid (miR-155-BS) showing the nucleotide sequence of 5'-UUA AUG GCU AAU CGU GAU AGG-3' as an RNA interference-inducing nucleic acid that suppresses an irregular target gene of miR-155 (SEQ ID NO: 7); or

RNA interference-inducing nucleic acid (miR-1-BS) showing the nucleotide sequence of 5'-UGG AAU UGU AAA GAA GUA UGU-3' as an RNA interference inducing nucleic acid that suppresses an irregular target gene of miR-1 (SEQ ID NO: 8).

Preferably, the RNA interference-inducing nucleic acid provides an RNA interference-inducing nucleic acid, characterized in that the nucleotide sequence between the 5'end and the ninth base is at least one of the following:

As an RNA interference-inducing nucleic acid that suppresses an irregular target gene of miR-124, preferably, a modified nucleotide sequence in which at least one Guanine base is substituted with Uracil in the 2nd to 7th sequence based on the 5'end is used. With 5'-UAA UGC ACG-3' (miR-124-G4U) (SEQ ID NO: 9), 5'-UAA GUC ACG-3' (miR-124-G5U) (SEQ ID NO: 10) or 5'- RNA interference inducing nucleic acid comprising the nucleotide sequence of UAA UUC ACG-3' (miR-124-G4,5U) (SEQ ID NO: 11);

As an RNA interference-inducing nucleic acid that suppresses an irregular target gene of miR-1, preferably a modified nucleotide sequence in which at least one Guanine base is substituted with Uracil from the 2nd to 7th sequence based on the 5'end is used. To have, 5'-UUG AAU GUA-3' (miR-1-G2U) (SEQ ID NO: 12), 5'-UGU AAU GUA-3' (miR-1-G3U) (SEQ ID NO: 13), 5'- UGG AAU UUA-3' (miR-1-G7U) (SEQ ID NO: 14), 5'-UUU AAU GUA-3' (miR-1-G2,3U) (SEQ ID NO: 15), 5'-UGU AAU UUA- 3'(miR-1-G3,7U) (SEQ ID NO: 16), 5'-UUG AAU UUA-3' (miR-1-G2,7U) (SEQ ID NO: 17) or 5'-UUU AAU UUA-3' RNA interference-inducing nucleic acid comprising the nucleotide sequence of (miR-1-G2,3,7U) (SEQ ID NO: 18);

As an RNA interference-inducing nucleic acid that suppresses the irregular target gene of miR-122

5'-UUG AGU GUG-3' (miR-122-G2U) (SEQ ID NO: 19), 5'-UGU AGU GUG-3' (miR-122-G3U) (SEQ ID NO: 20), 5'-UGG AUU GUG -3' (miR-122-G5U) (SEQ ID NO: 21), 5'-UGG AGU UUG-3' (miR-122-G7U) (SEQ ID NO: 22), 5'-UGG AGU GUU-3' (miR- 122-G9U) (SEQ ID NO: 23), 5'-UUU AGU GUG-3' (miR-122-G2,3U) (SEQ ID NO: 24), 5'-UUG AUU GUG-3' (miR-122-G2, 5U) (SEQ ID NO: 25), 5'-UUG AGU UUG-3' (miR-122-G2,7U) (SEQ ID NO: 26), 5'-UUG AGU GUU-3' (miR-122-G2,9U) (SEQ ID NO: 27), 5'-UGU AUU GUG-3' (miR-122-G3,5U) (SEQ ID NO: 28), 5'-UGU AGU UUG-3' (miR-122-G3,7U) (SEQ ID NO: Number 29), 5'-UGU AGU GUU-3' (miR-122-G3,9U) (SEQ ID NO: 30), 5'-UGG AUU UUG-3' (miR-122-G5,7U) (SEQ ID NO: 31 ), 5'-UGG AUU GUU-3' (miR-122-G5,9U) (SEQ ID NO: 32), or 5'-UGG AGU UUU-3 (miR-122-G7,9U) (SEQ ID NO: 33) RNA interference-inducing nucleic acid comprising a nucleotide sequence, preferably in the second to seventh sequence based on the 5'end, by having a modified nucleotide sequence in which at least one guanine base is substituted with uracil, 5 '-UUG AGU GUG-3' (miR-122-G2U) (SEQ ID NO: 19), 5'-UGU AGU GUG-3' (miR-122-G3U) (SEQ ID NO: 20), 5'-UGG AUU GUG- 3'(miR-122-G5U) (SEQ ID NO: 21), 5'-UGG AGU UUG-3' (miR-122-G7U) (SEQ ID NO: 22), 5'-UUU AGU GUG-3' (miR-122 -G2,3U) (sequence Number 24), 5'-UUG AUU GUG-3' (miR-122-G2,5U) (SEQ ID NO: 25), 5'-UUG AGU UUG-3' (miR-122-G2,7U) (SEQ ID NO: 26 ), 5'-UGU AUU GUG-3' (miR-122-G3,5U) (SEQ ID NO: 28), 5'-UGU AGU UUG-3' (miR-122-G3,7U) (SEQ ID NO: 29), RNA interference inducing nucleic acid comprising the nucleotide sequence of 5'-UGG AUU UUG-3' (miR-122-G5,7U) (SEQ ID NO: 31);

As an RNA interference-inducing nucleic acid that suppresses an irregular target gene of miR-133, preferably a modified nucleotide sequence in which at least one Guanine base is substituted with Uracil in the 2nd to 7th sequence based on the 5'end is used. With 5'-UUU UGU CCC-3' (miR-133-G4U) (SEQ ID NO: 34), 5'-UUU GUU CCC-3' (miR-133-G5U) (SEQ ID NO: 35) or 5'- RNA interference inducing nucleic acid comprising the nucleotide sequence of UUU UUU CCC-3' (miR-124-G4,5U) (SEQ ID NO: 36);

As an RNA interference-inducing nucleic acid that suppresses the irregular target gene of let-7

5'-UUA GGU AGU-3' (let-7-G2U) (SEQ ID NO: 37), 5'-UGA UGU AGU-3' (let-7-G4U) (SEQ ID NO: 38), 5'-UGA GUU AGU -3' (let-7-G5U) (SEQ ID NO: 39), 5'-UGA GGU AUU-3' (let-7-G8U) (SEQ ID NO: 40), 5'-UUA UGU AGU-3' (let- 7-G2,4U) (SEQ ID NO: 41), 5'-UUA GUU AGU-3' (let-7-G2,5U) (SEQ ID NO: 42), 5'-UUA GGU AUU-3' (let-7- G2,8U) (SEQ ID NO: 43), 5'-UGA UUU AGU-3' (let-7-G4,5U) (SEQ ID NO: 44), 5'-UGA UGU AUU-3' (let-7-G4, 8U) (SEQ ID NO: 45), or an RNA interference inducing nucleic acid comprising the nucleotide sequence of 5'-UGA GUU AUU-3' (let-7-G5,8U) (SEQ ID NO: 46), preferably at the 5'end In the reference 2nd to 7th sequence, the guanine base has a modified nucleotide sequence in which at least one base is substituted with Uracil, and 5'-UUA GGU AGU-3' (let-7-G2U) (sequence 37), 5'-UGA UGU AGU-3' (let-7-G4U) (SEQ ID NO: 38), 5'-UGA GUU AGU-3' (let-7-G5U) (SEQ ID NO: 39), 5' -UUA UGU AGU-3' (let-7-G2,4U) (SEQ ID NO: 41), 5'-UUA GUU AGU-3' (let-7-G2,5U) (SEQ ID NO: 42), 5'-UGA RNA interference inducing nucleic acid comprising the base sequence of UUU AGU-3' (let-7-G4,5U) (SEQ ID NO: 44);

As an RNA interference-inducing nucleic acid that suppresses an irregular target gene of miR-302a, preferably a modified base sequence in which at least one Guanine base is substituted with Uracil in the 2nd to 7th sequence based on the 5'end is used. With 5'-UAA UUG CUU-3' (miR-302a-G4U) (SEQ ID NO: 47), 5'-UAA GUU CUU-3' (miR-302a-G6U) (SEQ ID NO: 48), or 5' RNA interference inducing nucleic acid comprising the nucleotide sequence of -UAA UUU CUU-3' (miR-302a-G4,6U) (SEQ ID NO: 49); or

As an RNA interference-inducing nucleic acid that suppresses an irregular target gene of miR-372, preferably a modified nucleotide sequence in which at least one Guanine base is substituted with Uracil from the 2nd to 7th sequence based on the 5'end is used. To have, 5'-AAA UUG CUG-3' (miR-372-G4U) (SEQ ID NO: 50), 5'-AAA GUU CUG-3' (miR-372-G6U) (SEQ ID NO: 51), 5'- AAA GUG CUU-3' (miR-372-G9U) (SEQ ID NO: 52), 5'-AAA UUU CUG-3' (miR-372-G4,6U) (SEQ ID NO: 53), 5'-AAA UUG CUU- RNA interference induction comprising the nucleotide sequence of 3'(miR-372-G4,9U) (SEQ ID NO: 54), or 5'-AAA GUU CUU-3' (miR-372-G6,9U) (SEQ ID NO: 55) Nucleic acid.

Preferably, it provides an RNA interference-inducing nucleic acid, characterized in that the base sequence of the RNA interference-inducing nucleic acid is represented by one or more of the following:

As an RNA interference-inducing nucleic acid that suppresses the irregular target gene of miR-124

5'-UAA UGC ACG CGG UGA AUG CCA A-3' (miR-124-G4U) (SEQ ID NO: 56), 5'-UAA GUC ACG CGG UGA AUG CCA A-3' (miR-124-G5U) (SEQ ID NO: RNA interference inducing nucleic acid showing the nucleotide sequence of 5'-UAA UUC ACG CGG UGA AUG CCA A-3' (miR-124-G4,5U) (SEQ ID NO: 58);

As an RNA interference-inducing nucleic acid that suppresses the irregular target gene of miR-1

5'-UUG AAU GUA AAG AAG UAU GUA U-3' (miR-1-G2U) (SEQ ID NO: 59), 5'-UGU AAU GUA AAG AAG UAU GUA U-3' (miR-1-G3U) (SEQ ID NO: Number 60), 5'-UGG AAU UUA AAG AAG UAU GUA U-3' (miR-1-G7U) (SEQ ID NO: 61), 5'-UUU AAU GUA AAG AAG UAU GUA U-3' (miR-1- G2,3U) (SEQ ID NO: 62), 5'-UGU AAU UUA AAG AAG UAU GUA U-3' (miR-1-G3,7U) (SEQ ID NO: 63), 5'-UUG AAU UUA AAG AAG UAU GUA U Base of -3' (miR-1-G2,7U) (SEQ ID NO: 64) or 5'-UUU AAU UUA AAG AAG UAU GUA U-3' (miR-1-G2,3,7U) (SEQ ID NO: 65) RNA interference inducing nucleic acid showing sequence;

5'-UUG AGU GUG ACA AUG GUG UUU G-3' (miR-122-G2U) (SEQ ID NO: 66), 5'-UGU AGU GUG ACA AUG as an RNA interference-inducing nucleic acid that suppresses an irregular target gene of miR-122 GUG UUU G-3 (miR-122-G3U) (SEQ ID NO: 67), 5'-UGG AUU GUG ACA AUG GUG UUU G-3' (miR-122-G5U) (SEQ ID NO: 68), 5'-UGG AGU UUG ACA AUG GUG UUU G-3' (miR-122-G7U) (SEQ ID NO: 69), 5'-UGG AGU GUU ACA AUG GUG UUU G-3' (miR-122-G9U) (SEQ ID NO: 70), 5 '-UUU AGU GUG ACA AUG GUG UUU G-3' (miR-122-G2,3U) (SEQ ID NO: 71), 5'-UUG AUU GUG ACA AUG GUG UUU G-3' (miR-122-G2,5U ) (SEQ ID NO: 72), 5'-UUG AGU UUG ACA AUG GUG UUU G-3' (miR-122-G2,7U) (SEQ ID NO: 73), 5'-UUG AGU GUU ACA AUG GUG UUU G-3' (miR-122-G2,9U) (SEQ ID NO: 74), 5'-UGU AUU GUG ACA AUG GUG UUU G-3 (miR-122-G3,5U) (SEQ ID NO: 75), 5'-UGU AGU UUG ACA AUG GUG UUU G-3 (miR-122-G3,7U) (SEQ ID NO: 76), 5'-UGU AGU GUU ACA AUG GUG UUU G-3 (miR-122-G3,9U) (SEQ ID NO: 77), 5 '-UGG AUU UUG ACA AUG GUG UUU G-3' (miR-122-G5,7U) (SEQ ID NO: 78), 5'-UGG AUU GUU ACA AUG GUG UUU G-3' (miR-122-G5,9U ) (SEQ ID NO: 79) or 5'-UGG AGU UUU ACA AUG GUG UUU G-3' (miR-122-G7,9U) (SEQ ID NO: 80) RNA interference inducing nucleic acid showing the base sequence;

5'-UUU UGU CCC CUU CAA CCA GCU G -3' (miR-133-G4U) (SEQ ID NO: 81), 5'-UUU GUU CCC CUU CAA as an RNA interference-inducing nucleic acid that suppresses an irregular target gene of miR-133 Base sequence of CCA GCU G-3' (miR-133-G5U) (SEQ ID NO: 82) or 5'-UUU UUU CCC CUU CAA CCA GCU G-3' (miR-124-G4,5U) (SEQ ID NO: 83) RNA interference inducing nucleic acid comprising a;

5'-UUA GGU AGU AGG UUG UAU AGU U-3' (let-7-G2U) (SEQ ID NO: 84), 5'-UGA UGU AGU AGG UUG as an RNA interference-inducing nucleic acid that suppresses an irregular target gene of let-7 UAU AGU U-3' (let-7-G4U) (SEQ ID NO: 85), 5'-UGA GUU AGU AGG UUG UAU AGU U-3' (let-7-G5U) (SEQ ID NO: 86), 5'-UGA GGU AUU AGG UUG UAU AGU U-3' (let-7-G8U) (SEQ ID NO: 87), 5'-UUA UGU AGU AGG UUG UAU AGU U-3' (let-7-G2,4U) (SEQ ID NO: 88 ), 5'-UUA GUU AGU AGG UUG UAU AGU U-3' (let-7-G2,5U) (SEQ ID NO: 89), 5'-UUA GGU AUU AGG UUG UAU AGU U-3' (let-7- G2,8U) (SEQ ID NO: 90), 5'-UGA UUU AGU AGG UUG UAU AGU U-3' (let-7-G4,5U) (SEQ ID NO: 91), 5'-UGA UGU AUU AGG UUG UAU AGU U -3' (let-7-G4,8U) (SEQ ID NO: 92) or 5'-UGA GUU AUU AGG UUG UAU AGU U-3' (let-7-G5,8U) (SEQ ID NO: 93) RNA interference inducing nucleic acids shown;

5'-UAA UUG CUU CCA UGU UUU GGU GA-3' (miR-302a-G4U) (SEQ ID NO: 94), 5'-UAA GUU CUU CCA UGU as an RNA interference inducing nucleic acid that inhibits an irregular target gene of miR-302a Base of UUU GGU GA-3' (miR-302a-G6U) (SEQ ID NO: 95), or 5'-UAA UUU CUU CCA UGU UUU GGU GA-3' (miR-302a-G4,6U) (SEQ ID NO: 96) RNA interference inducing nucleic acid showing sequence; or

5'-AAA UUG CUG CGA CAU UUG AGC GU -3' (miR-372-G4U) (SEQ ID NO: 97), 5'-AAA GUU CUG CGA CAU as an RNA interference-inducing nucleic acid that suppresses an irregular target gene of miR-372 UUG AGC GU -3' (miR-372-G6U) (SEQ ID NO: 98), 5'-AAA GUG CUU CGA CAU UUG AGC GU -3' (miR-372-G9U) (SEQ ID NO: 99), 5'-AAA UUU CUG CGA CAU UUG AGC GU -3' (miR-372-G4,6U) (SEQ ID NO: 100), 5'-AAA UUG CUU CGA CAU UUG AGC GU -3' (miR-372-G4,9U) (SEQ ID NO: No. 101), or an RNA interference-inducing nucleic acid showing the nucleotide sequence of 5′-AAA GUU CUU CGA CAU UUG AGC GU -3′ (miR-372-G6,9U) (SEQ ID NO: 102).

It provides a composition for inhibiting the expression of a non-normal target gene of microRNA comprising the RNA interference-inducing nucleic acid of the present invention.

It provides a composition for controlling cell cycle, differentiation, dedifferentiation, morphology, migration, dedifferentiation, division, proliferation or death, comprising the RNA interference-inducing nucleic acid of the present invention.

Preferably, the composition provides a composition, characterized in that it is any one or more of the following:

a composition for inducing cancer cell death comprising an RNA interference-inducing nucleic acid that suppresses an irregular target gene of miR-124;

a composition for inducing neurite branch differentiation comprising an RNA interference-inducing nucleic acid that suppresses an irregular target gene of miR-124;

a composition for inducing cell cycle arrest in liver cancer cells comprising an RNA interference-inducing nucleic acid that suppresses an irregular target gene of miR-122;

a composition for promoting myocyte differentiation or promoting muscle fibrosis, comprising an RNA interference-inducing nucleic acid that suppresses an irregular target gene of miR-1;

a composition for inducing myocyte death comprising an RNA interference-inducing nucleic acid that suppresses an irregular target gene of miR-155;

a composition for promoting apoptosis of neuroblastoma comprising an RNA interference-inducing nucleic acid that suppresses an irregular target gene of miR-124;

a composition for promoting cell division and proliferation of neuroblastoma comprising an RNA interference-inducing nucleic acid that suppresses an irregular target gene of miR-124;

a composition for inducing myocardial hypertrophy comprising an RNA interference-inducing nucleic acid that suppresses an irregular target gene of miR-1;

a composition for inducing myocardial hypertrophy comprising an RNA interference-inducing nucleic acid that suppresses an irregular target gene of miR-133;

a composition for inducing cell cycle arrest of cancer cells, comprising an RNA interference-inducing nucleic acid that suppresses an irregular target gene of let-7;

a composition for inducing cell cycle progression activity of hepatocytes comprising an RNA interference-inducing nucleic acid that suppresses an irregular target gene of let-7;

a composition for promoting dedifferentiation comprising an RNA interference-inducing nucleic acid that suppresses an irregular target gene of miR-302a;

a composition for promoting dedifferentiation comprising an RNA interference-inducing nucleic acid that suppresses an irregular target gene of miR-372; or

A composition for inhibiting cell migration of liver cancer cells comprising an RNA interference-inducing nucleic acid that suppresses an irregular target gene of miR-122.

In the present invention, in one or more single strands of the double strands of nucleic acids that induce RNA interference, including the following steps, some sequences of specific microRNAs are modified to form noncanonical target genes of microRNAs. ) It provides a method for producing an RNA interference-inducing nucleic acid that inhibits the expression of:

It contains the 2nd to 5th 4 nucleotide sequences from the 5'end of the specific microRNA, the 6th and 7th bases are the same, and the 6th and 7th bases can be aligned with the 6th base of a specific microRNA. It has a base sequence complementary to a base, and all complementary bases including G:A, G:U wobble arrangement are included in the base that can be aligned with the 6th base of the specific microRNA. Constructing an RNA interference-inducing nucleic acid so that a bulge occurs in the target gene between the 5th and 6th and the corresponding bulge disappears from the binding irregular target nucleotide sequence and binds to a continuous nucleotide sequence, or

Of the nucleotide sequence between the 5'end and the ninth nucleotide of a specific microRNA, the guanine base has a modified nucleotide sequence in which at least one base is substituted with uracil, and G:A or G:U of the corresponding site Constructing an RNA interference-inducing nucleic acid such that the wobble sequence becomes a normal nucleotide sequence of U:A or A:U.

In addition, the present invention provides a method for screening a test substance for regulating cell cycle, differentiation, dedifferentiation, morphology, migration, division, proliferation, dedifferentiation or death, including the following steps:

Transfecting a target cell with an RNA interference-inducing nucleic acid;

Treating the test substance to the cell of interest; And

Checking the expression level or whether the non-normal target gene of the microRNA inhibited by the RNA interference-inducing nucleic acid in the target cell.

In addition, the present invention provides the following 1 to 3 for RNA interference inducing nucleic acids:

1. About 2'OMe modified RNA interference-inducing nucleic acid

In the present invention, in one or more single strands of the double strands of nucleic acids that induce RNA interference, some sequences of specific microRNAs are modified to induce RNA interference that suppresses noncanonical target genes of microRNAs. As a nucleic acid,

The RNA interference-inducing nucleic acid contains the 2nd to 5th 4 base sequences from the 5'end of the specific microRNA, the 6th and 7th bases are the same, and the 6th and 7th bases are the 6th of the specific microRNA. Has a base sequence that is complementary to a base that can be aligned with a base, and the base that can be aligned with the 6th base of the specific microRNA includes all complementary bases including G:A and G:U wobble arrangements. In this way, a bulge occurs in the target gene between the 5th and 6th microRNA, and the bulge disappears from the non-normal target nucleotide sequence to which it binds and binds to a continuous nucleotide sequence,

The specific microRNA provides an RNA interference-inducing nucleic acid, characterized in that a methyl group (2'OMe) is added to the 2'position of the ribosyl ring of the 6th nucleotide from the 5'end.

Preferably, the RNA interference-inducing nucleic acid provides an RNA-interference-inducing nucleic acid, characterized in that it suppresses only the expression of a target gene for the normal initiation of the corresponding RNA interference-inducing nucleic acid.

Preferably, the RNA interference-inducing nucleic acid provides an RNA interference-inducing nucleic acid, characterized in that it specifically suppresses only the irregular nucleus site of a specific microRNA and removes the irregular nucleus site that may occur newly.

In addition, the present invention provides a composition for suppressing the expression of non-normal target genes of microRNAs, including RNA interference-inducing nucleic acids.

In addition, in the present invention, in one or more single strands of the double strands of nucleic acids inducing RNA interference, including the following steps, some sequences of specific microRNAs are modified to be noncanonical target genes of microRNAs (noncanonical As a method for producing RNA interference-inducing nucleic acids that inhibit the expression of target gene),

It contains the 2nd to 5th 4 nucleotide sequences from the 5'end of the specific microRNA, the 6th and 7th bases are the same, and the 6th and 7th bases can be aligned with the 6th base of a specific microRNA. It has a base sequence complementary to a base, and all complementary bases including G:A, G:U wobble arrangement are included in the base that can be aligned with the 6th base of the specific microRNA. Constructing an RNA interference-inducing nucleic acid so that a bulge occurs in the target gene between the 5th and 6th and the corresponding bulge disappears from the non-normal target nucleotide sequence and binds to a continuous nucleotide sequence; And

It provides a method for producing an RNA interference-inducing nucleic acid, comprising the step of adding a methyl group (2'OMe) to the 2'position of the ribosyl ring of the 6th nucleotide from the 5'end of the specific microRNA.

2. About RNA interference-inducing nucleic acids (SEQ ID NOs: 103 to 528) that inhibit microRNA atypical nucleation target sites

In the present invention, in one or more single strands of the double strands of nucleic acids that induce RNA interference, some sequences of specific microRNAs are modified to induce RNA interference that suppresses noncanonical target genes of microRNAs. As a nucleic acid,

The RNA interference-inducing nucleic acid contains the 2nd to 5th 4 base sequences from the 5'end of the specific microRNA, the 6th and 7th bases are the same, and the 6th and 7th bases are the 6th of the specific microRNA. Has a base sequence that is complementary to a base that can be aligned with a base, and the base that can be aligned with the 6th base of the specific microRNA includes all complementary bases including G:A and G:U wobble arrangements. Induction of RNA interference, characterized in that the bulge is formed in the target gene between the 5th and 6th of the microRNA, and the bulge disappears from the non-normal target nucleotide sequence and binds to a continuous nucleotide sequence. Nucleic acids are provided.

Preferably, the RNA interference-inducing nucleic acid provides an RNA interference-inducing nucleic acid, characterized in that it selectively inhibits only the irregular nucleus target site and not the normal target gene of the microRNA.

Preferably, the specific microRNA is let-7/98/4458/4500, miR-125a-5p/125b-5p/351/670/4319, miR-124/124ab/506, miR-9/9ab, miR- 29abcd, miR-103a/107/107ab, miR-221/222/222ab/1928, miR-26ab/1297/4465, miR-15abc/16/16abc/195/322/424/497/1907, miR-126- 3p, miR-30abcdef/30abe-5p/384-5p, miR-33ab/33-5p, miR-34ac/34bc-5p/449abc/449c-5p, miR-19ab, miR-99ab/100, miR-17/ 17-5p/20ab/20b-5p/93/106ab/427/518a-3p/519d, miR-27abc/27a-3p, miR-218/218a, miR-22/22-3p, miR-185/882/ 3473/4306/4644, miR-181abcd/4262, miR-338/338-3p, miR-127/127-3p, miR-101/101ab, miR-149, miR-324-5p, miR-24/24ab/ 24-3p, miR-33a-3p/365/365-3p, miR-139-5p, miR-138/138ab, miR-143/1721/4770, miR-25/32/92abc/363/363-3p/ 367, miR-574-5p, miR-7/7ab, miR-145, miR-135ab/135a-5p, miR-148ab-3p/152, miR-28-5p/708/1407/1653/3139, miR- 130ac/301ab/301b/301b-3p/454/721/4295/3666, miR-3132, miR-155, miR-485-3p, miR-132/212/212-3p, hsa-miR-9-3p, miR-374ab, miR-129-3p/129ab-3p/129-1-3p/129-2-3p, hsa-miR-126-5p, miR-425/425-5p/489, miR-423-3p, miR-21/590-5p, miR-3 1, hsa-miR-20b-3p, hsa-let-7d-3p, miR-191, miR-18ab/4735-3p, miR-369-3p, hsa-miR-5187-5p, miR-382, miR- 485-5p/1698/1703/1962, hsa-miR-136-3p, miR-576-3p, miR-204/204b/211, miR-769-5p, miR-342-5p/4664-5p, miR- 361-5p, miR-199ab-3p/3129-5p, miR-142-3p, miR-299-5p/3563-5p, miR-193/193b/193a-3p, hsa-miR-1277-5p, miR- 140/140-5p/876-3p/1244, hsa-miR-30a/d/e-3p, hsa-let-7i-3p, miR-409-5p/409a, miR-379/1193-5p/3529, miR-136, miR-154/872, miR-4684-3p, miR-361-3p, miR-335/335-5p, miR-423a/423-5p/3184/3573-5p, miR-371/373/ 371b-5p, miR-1185/3679-5p, miR-3613-3p, miR-93/93a/105/106a/291a-3p/294/295/302abcde/372/373/428/519a/520be/520acd- 3p/1378/1420ac, miR-876-5p/3167, miR-329/329ab/362-3p, miR-582-5p, miR-146ac/146b-5p, miR-380/380-3p, miR-499- 3p/499a-3p, miR-551a, miR-142-5p, hsa-miR-17-3p, miR-199ab-5p, miR-542-3p, miR-1277, hsa-miR-29c-5p, miR- 3145-3p, hsa-miR-106b-3p, hsa-miR-22-5p, miR-744/1716, hsa-miR-132-5p, miR-488, miR-501-3p/502-3p/500/ 502a, miR-486-5p/3107, miR-450a/451a, hsa-miR-30c-3p , miR-499-5p, miR-421, miR-197, miR-296-5p, miR-326/330/330-5p, miR-214/761/3619-5p, miR-612/1285/3187-5p , miR-409-3p, miR-378/422a/378bcdefhi, miR-342-3p, miR-338-5p, miR-625, miR-200bc/429/548a, hsa-miR-376a-5p, miR-584 , miR-411, miR-573/3533/3616-5p/3647-5p, miR-885-5p, hsa-miR-99-3p, miR-876-3p, miR-654-3p, hsa-miR-340 -3p, miR-3614-5p, hsa-miR-124-5p, miR-491-5p, miR-96/507/1271, miR-548a-3p/548ef/2285a, hsa-miR-32-3p, miR -3942-5p/4703-5p, miR-34b/449c/1360/2682, hsa-miR-23a/b-5p, miR-362-5p/500b, miR-677/4420, miR-577, miR-3613 -5p, miR-369-5p, miR-150/5127, miR-544/544ab/544-3p, hsa-miR-29a-5p, miR-873, miR-3614-3p, miR-186, miR-483 -3p, hsa-miR-374a-3p, miR-196abc, hsa-miR-145-3p, hsa-miR-29b-2-5p, hsa-miR-221-5p, miR-323b-3p, miR-616 , miR-330-3p, hsa-miR-7-3p, miR-187, hsa-miR-26a-3p, miR-452/4676-3p, miR-129-5p/129ab-5p, miR-223, miR -4755-3p, miR-1247, miR-3129-3p, hsa-miR-335-3p, miR-542-5p, hsa-miR-181a-3p, hsa-miR-186-3p, hsa-miR-27b -5p, miR-491-3p, miR-4687-3p, hsa-miR-101-5 p, miR-4772-5p, miR-337-3p, hsa-miR-223-5p, hsa-miR-16/195-3p, miR-3677-3p, hsa-miR-766-5p, miR-299/ 299-3p/3563-3p, miR-3140-3p, miR-532-5p/511, hsa-miR-24-5p, miR-4778-5p, miR-642b, miR-483-5p, miR-767- 5p, hsa-miR-31-3p, miR-574-3p, miR-3173-3p, miR-2127/4728-5p, hsa-miR-103a-2-5p, miR-3591-3p, hsa-miR- 625-3p, hsa-miR-15b-3p, miR-522/518e/1422p, miR-548d-3p/548acbz, hsa-miR-452-3p, miR-192/215, miR-1551/4524, hsa- miR-425-3p, miR-3126-3p, hsa-miR-125b-2-3p, miR-324-3p/1913, hsa-miR-141-5p, hsa-miR-365a/b-5p, hsa- miR-29b-1-5p, miR-563/380-5p, miR-1304, miR-216c/1461/4684-5p, hsa-miR-2681-5p, miR-194, miR-296-3p, hsa- miR-205-3p, miR-888, miR-4802-3p, hsa-let-7a/g-3p, miR-762/4492/4498, hsa-miR-744-3p, hsa-miR-148b-5p, miR-514/514b-3p, miR-28-3p, miR-550a, hsa-miR-125b-1-3p, hsa-miR-506-5p, hsa-miR-1306-5p, miR-3189-3p, miR-675-5p/4466, hsa-miR-34a-3p, hsa-miR-454-5p, miR-509-5p/509-3-5p/4418, hsa-miR-19a/b-5p, miR- 4755-5p, hsa-miR-93-3p, miR-3130-5p/4482, hsa-miR-488-5p, hsa-miR-378a-5p, miR-575/4676-5p, miR-1307, miR-3942-3p, miR-4677-5p, miR-339-3p, miR-548b-3p, hsa-miR-642b-5p, miR-188-5p, hsa-miR-652-5p, miR-2114, miR-3688-5p, hsa-miR-15a-3p, hsa-miR-181c-3p, miR-122/122a/1352, miR-556-3p, hsa- miR-218-2-3p, miR-643, miR-140-3p, miR-1245, hsa-miR-2115-3p, miR-518bcf/518a-3p/518d-3p, miR-3200-3p, miR- 545/3065/3065-5p, miR-1903/4778-3p, hsa-miR-302a-5p, hsa-miR-183-3p, miR-3144-5p, miR-582-3p, miR-4662a-3p, miR-3140-5p, hsa-miR-106a-3p, hsa-miR-135a-3p, miR-345/345-5p, miR-125a-3p/1554, miR-3145-5p, miR-676, miR- 3173-5p, hsa-miR-5586-3p, miR-615-3p, miR-3688-3p, miR-4662a-5p, miR-4659ab-5p, hsa-miR-5586-5p, hsa-miR-514a- 5p, miR-10abc/10a-5p, hsa-miR-888-3p, miR-3127-5p, miR-508-3p, hsa-miR-185-3p, hsa-miR-200c-5p, hsa-miR- 550a-3p, miR-513c/514b-5p, miR-490-3p, hsa-miR-5187-3p, miR-3664-3p, miR-3189-5p, miR-4670-3p, miR-105/105ab, hsa-miR-135b-3p, hsa-miR-5010-3p, miR-493/493b, miR-3605-3p, miR-188-3p, hsa-miR-449c-3p, miR-4761-5p, miR- 224, miR-4796-5p, hsa-miR-551b-5p , miR-556-5p, hsa-miR-122-3p, miR-4677-3p, miR-877, miR-576-5p, miR-490-5p, hsa-miR-589-3p, miR-4786-3p , hsa-miR-374b-3p, hsa-miR-26b-3p, miR-3158-3p, miR-4423-3p, miR-518d-5p/519bc-5p520c-5p/523b/526a, miR-4707-3p , hsa-miR-10a-3p, miR-526b, hsa-miR-676-5p, hsa-miR-660-3p, hsa-miR-5004-3p, miR-193a-5p, hsa-miR-222-5p , miR-4661-3p, hsa-miR-25-5p, miR-4670-5p, miR-659, miR-1745/3194-3p, hsa-miR-182-3p, miR-298/2347/2467-3p , hsa-miR-130b-5p, miR-4746-3p, miR-1893/2277-5p, miR-3619-3p, hsa-miR-138-1-3p, miR-4728-3p, miR-3127-3p , miR-671-3p, hsa-miR-211-3p, hsa-miR-2114-3p, hsa-miR-877-3p, miR-3157-5p, miR-502-5p, miR-500a, miR-548g , miR-523, hsa-miR-584-3p, miR-205/205ab, miR-4793-5p, hsa-miR-363-5p, hsa-miR-214-5p, miR-3180-5p, miR-1404 /2110, miR-3157-3p, hsa-miR-191-3p, miR-1346/3940-5p/4507, miR-4746-5p, miR-3939, hsa-miR-181a-2-3p, hsa-miR -500a-3p, hsa-miR-196b-3p, hsa-miR-675-3p, hsa-miR-548aj/g/x-5p, miR-4659ab-3p, hsa-miR-5001-3p, hsa-miR -1247-3p, miR-2890/4707-5p, hsa-miR-150-3p, h sa-miR-629-3p, miR-2277-3p, miR-3547/3663-3p, miR-34bc-3p, miR-518ef, miR-3187-3p, miR-1306/1306-3p, miR-3177- 3p, miR-1ab/206/613, miR-128/128ab, miR-1296, miR-598/598-3p, miR-887, miR-1-5p, miR-376c/741-5p, miR-374c/ 655, miR-494, miR-651, miR-1301/5047, miR-381-5p, miR-216a, miR-300/381/539-3p, miR-1249, miR-579, miR-656, miR- 433, miR-1180, miR-597/1970, miR-190a-3p, miR-1537, miR-874-5p, miR-410/344de/344b-1-3p, miR-370, miR-219-2- 3p/219-3p, miR-3620, miR-504/4725-5p, miR-2964/2964a-5p, miR-450a-2-3p, miR-511, miR-6505-3p, miR-433-5p, miR-6741-3p, miR-370-5p, miR-579-5p, miR-376c-5p,miR-376b-5p, miR-552/3097-5p, miR-1910, miR-758, miR-6735- It is selected from the group consisting of 3p, miR-376a-2-5p, miR-585, miR-451, and miR-137/137ab, and contains 4 nucleotide sequences from the 2nd to the 5th from the 5'end from the same starting sequence And, the 6th and 7th bases are the same, and the 6th and 7th bases are composed of 6-24 bases while having a base sequence complementary to a base that can be aligned with the 6th base of a specific microRNA. , RNA interference-inducing nucleic acids are provided.

Preferably, the RNA interference-inducing nucleic acid provides an RNA interference-inducing nucleic acid, characterized in that it comprises a nucleotide sequence represented by any one or more of SEQ ID NOs: 103 to 528 (see Table 3).

In addition, the present invention provides a composition for suppressing the expression of non-normal target genes of microRNAs, including RNA interference-inducing nucleic acids.

In addition, in the present invention, in one or more single strands of the double strands of nucleic acids inducing RNA interference, including the following steps, some sequences of specific microRNAs are modified to be noncanonical target genes of microRNAs (noncanonical As a method for producing RNA interference-inducing nucleic acids that inhibit the expression of target gene),

It contains the 2nd to 5th 4 nucleotide sequences from the 5'end of the specific microRNA, the 6th and 7th bases are the same, and the 6th and 7th bases can be aligned with the 6th base of a specific microRNA. It has a base sequence complementary to a base, and all complementary bases including G:A, G:U wobble arrangement are included in the base that can be aligned with the 6th base of the specific microRNA. It characterized in that it comprises the step of constructing an RNA interference-inducing nucleic acid so that a bulge occurs in the target gene between the 5th and 6th and the corresponding bulge disappears from the non-normal target nucleotide sequence and binds to a continuous nucleotide sequence. , It provides a method of preparing a nucleic acid inducing RNA interference.

3. About RNA interference-inducing nucleic acids (SEQ ID NOs: 529 to 863) that inhibit microRNA irregular G:A wobble target sites

In the present invention, in one or more single strands of the double strands of nucleic acids that induce RNA interference, some sequences of specific microRNAs are modified to induce RNA interference that suppresses noncanonical target genes of microRNAs. As a nucleic acid,

The RNA interference-inducing nucleic acid has a modified nucleotide sequence in which at least one guanine base is substituted with a uracil base among the nucleotide sequences between the 2nd to 9th bases from the 5'end of a specific microRNA, and the corresponding It provides an RNA interference-inducing nucleic acid, characterized in that the G:A wobble of the region becomes the normal nucleotide sequence of U:A.

Preferably, the RNA interference-inducing nucleic acid includes 6 to 8 consecutive nucleotide sequences starting at the second base from the 5'end of the specific microRNA,

The nucleotide sequence provides an RNA interference-inducing nucleic acid, characterized in that at least one or more guanine bases are substituted with uracil bases.

Preferably, the RNA interference-inducing nucleic acid is characterized in that it selectively inhibits only the non-normal target gene of the microRNA bound to the G:A wobble arrangement and does not inhibit the normal target gene of the microRNA. Provides.

Preferably, the specific microRNA is hsa-miR-1-3p, hsa-miR-194-5p, hsa-miR-193a-5p, hsa-miR-15b-3p, hsa-miR-200c-5p, hsa- miR-214-5p, hsa-miR-134-5p, hsa-miR-145-3p, hsa-miR-22-5p, hsa-miR-423-3p, hsa-miR-873-3p, hsa-miR- 122-5p, hsa-miR-143-3p, hsa-miR-485-5p, hsa-miR-409-5p, hsa-miR-24-3p, hsa-miR-223-3p, hsa-miR-144- 5p, hsa-miR-379-5p, hsa-miR-146b-5p/hsa-miR-146a-5p, hsa-miR-539-5p, hsa-miR-296-5p, hsa-miR-767-5p, hsa-miR-34a-5p/hsa-miR-34c-5p, hsa-let-7f-5p/hsa-let-7d-5p/hsa-let-7b-5p/hsa-let-7a-5p/hsa- let-7e-5p/hsa-miR-202-3p/hsa-let-7i-5p/hsa-miR-98-5p/hsa-let-7c-5p/hsa-let-7g-5p, hsa-miR- 1271-3p, hsa-miR-138-5p, hsa-miR-19b-3p/hsa-miR-19a-3p, hsa-miR-27a-5p, hsa-miR-146b-3p, hsa-miR-7- 5p, hsa-miR-423-5p, hsa-miR-324-5p, hsa-miR-629-5p, hsa-miR-139-3p, hsa-miR-30d-5p/hsa-miR-30e-5p/ hsa-miR-30a-5p/hsa-miR-30c-5p/hsa-miR-30b-5p, hsa-miR-221-3p/hsa-miR-222-3p, hsa-miR-509-3p, hsa- miR-769-5p, hsa-miR-142-3p, hsa-miR-185-5p, hsa-miR-508-3p/hsa-miR-219a-5p, hsa-miR-31-5p, hsa-miR- 103a-3p/hsa-miR-107, hsa-miR-542-3p, hsa-miR-219a-2-3p, hsa-miR-29c-3p/hsa-miR-29a-3p/hsa-miR-29b-3p, hsa-miR-125b-1- 3p, hsa-miR-411-5p, hsa-miR-196a-5p/hsa-miR-196b-5p, hsa-miR-3622a-5p, hsa-miR-127-5p, hsa-miR-22-3p, hsa-miR-153-3p, hsa-miR-15b-5p/hsa-miR-16-5p/hsa-miR-424-5p, hsa-let-7g-3p/hsa-miR-493-5p/hsa- let-7c-3p, hsa-let-7i-3p, hsa-miR-218-5p, hsa-miR-1307-5p, hsa-miR-127-3p, hsa-miR-210-3p, hsa-miR- 187-3p, hsa-miR-192-3p, hsa-miR-192-5p, hsa-miR-21-5p, hsa-miR-500a-3p, hsa-miR-203a-3p, hsa-miR-30c- 2-3p, hsa-miR-488-3p, hsa-miR-301a-3p/hsa-miR-301b-3p, hsa-miR-126-3p, hsa-miR-26b-3p, hsa-miR-324- 3p, hsa-miR-3065-3p, hsa-miR-124-5p, hsa-miR-345-5p, hsa-miR-615-3p, hsa-miR-889-5p/hsa-miR-135a-5p/ hsa-miR-135b-5p, hsa-miR-18a-5p, hsa-miR-708-5p/hsa-miR-28-5p, hsa-miR-224-5p, hsa-miR-100-3p, hsa- miR-873-5p, hsa-miR-4662a-5p, hsa-miR-99b-3p/hsa-miR-99a-3p, hsa-miR-433-5p, hsa-miR-3605-5p, hsa-miR- 744-5p, hsa-miR-1296-5p, hsa-miR-133a-3p, hsa-miR-382-5p, hsa-miR-425-5p, hsa-miR-377-5p, hsa-miR-3180- 3p, h sa-miR-758-3p, hsa-miR-93-3p, hsa-miR-154-5p, hsa-miR-124-3p, hsa-miR-194-3p, hsa-miR-375, hsa-miR- 148a-5p, hsa-miR-2277-5p, hsa-miR-17-3p, hsa-miR-4772-3p, hsa-miR-329-5p, hsa-miR-182-5p/hsa-miR-96- 5p, hsa-miR-2467-5p/hsa-miR-485-5p, hsa-miR-149-5p, hsa-miR-29b-2-5p, hsa-miR-122-3p, hsa-miR-302a- 3p/hsa-miR-520a-3p/hsa-miR-519b-3p/hsa-miR-520b/hsa-miR-519c-3p/hsa-miR-520c-3p/hsa-miR-519a-3p, hsa- miR-532-5p, hsa-miR-132-5p, hsa-miR-541-5p, hsa-miR-671-3p, hsa-miR-518e-3p, hsa-miR-487a-5p, hsa-miR- 589-5p/hsa-miR-146b-5p/hsa-miR-146a-5p, hsa-miR-196b-5p/hsa-miR-196a-5p, hsa-miR-486-3p, hsa-miR-378a- 3p, hsa-miR-27b-5p, hsa-miR-6720-3p, hsa-miR-574-3p, hsa-miR-29a-5p, hsa-miR-30c-2-3p/hsa-miR-30c- 1-3p, hsa-miR-199b-3p, hsa-miR-574-5p, hsa-miR-4677-3p, hsa-miR-654-3p, hsa-miR-652-3p, hsa-miR-19a- 3p/hsa-miR-19b-3p, hsa-let-7c-5p/hsa-miR-98-5p/hsa-let-7g-5p/hsa-let-7f-5p/hsa-miR-202-3p/ hsa-let-7b-5p/hsa-let-7e-5p/hsa-let-7a-5p/hsa-let-7d-5p/hsa-let-7i-5p, hsa-miR-3663-3p, hsa- miR-152-3p/hsa-mi R-148b-3p/hsa-miR-148a-3p, hsa-miR-193b-5p, hsa-miR-502-3p/hsa-miR-501-3p, hsa-miR-299-3p, hsa-miR- 140-5p, hsa-miR-96-5p/hsa-miR-182-5p, hsa-miR-193b-3p, hsa-miR-365a-3p, hsa-miR-486-5p, hsa-miR-493- 3p, hsa-miR-548am-5p, hsa-miR-20b-5p/hsa-miR-20a-5p/hsa-miR-93-5p/hsa-miR-17-5p/hsa-miR-106b-5p, hsa-miR-541-3p, hsa-miR-452-5p, hsa-miR-221-5p, hsa-miR-518f-3p, hsa-miR-370-3p, hsa-miR-107/hsa-miR- 103a-3p, hsa-miR-338-3p, hsa-miR-409-3p, hsa-let-7d-5p/hsa-let-7g-5p/hsa-let-7i-5p/hsa-let-7f- 5p/hsa-let-7e-5p/hsa-let-7a-5p/hsa-let-7b-5p/hsa-let-7c-5p, hsa-miR-130b-3p/hsa-miR-301a-3p/ hsa-miR-130a-3p/hsa-miR-301b-3p, hsa-miR-512-3p, hsa-miR-191-5p, hsa-miR-509-3-5p, hsa-miR-92a-3p/ hsa-miR-92b-3p/hsa-miR-363-3p/hsa-miR-25-3p/hsa-miR-32-5p, hsa-miR-183-5p, hsa-miR-1307-3p, hsa- miR-499a-5p/hsa-miR-208a-3p, hsa-miR-186-5p, hsa-miR-450b-5p, hsa-miR-450a-5p, hsa-miR-101-3p/hsa-miR- 144-3p, hsa-miR-320a, hsa-miR-199b-5p/hsa-miR-199a-5p, hsa-miR-135a-5p, hsa-miR-145-5p, hsa-miR-26a-5p, hsa-miR-34c-5p, h sa-miR-125b-5p, hsa-miR-526b-5p, hsa-miR-16-5p/hsa-miR-15b-5p/hsa-miR-424-5p/hsa-miR-15a-5p, hsa- miR-9-3p, hsa-miR-363-5p, hsa-miR-1298-3p, hsa-miR-148a-3p, hsa-miR-302a-3p, hsa-miR-9-5p, hsa-miR- 28-3p, hsa-miR-508-3p, hsa-miR-137, hsa-miR-5010-5p, hsa-miR-523-5p, hsa-miR-128-3p, hsa-miR-199a-5p/ hsa-miR-199b-5p, hsa-miR-181a-2-3p, hsa-miR-27a-3p/hsa-miR-27b-3p, hsa-let-7d-3p, hsa-miR-129-5p, hsa-miR-424-3p, hsa-miR-181a-3p, hsa-miR-10a-5p, hsa-miR-196b-5p, hsa-miR-92a-1-5p, hsa-miR-483-5p, hsa-miR-1537-3p, hsa-miR-106b-5p/hsa-miR-20a-5p/hsa-miR-17-5p/hsa-miR-93-5p, hsa-miR-30a-3p/hsa- miR-30e-3p, hsa-miR-374a-3p, hsa-miR-675-5p, hsa-miR-503-5p, hsa-miR-340-5p, hsa-miR-208a-3p, hsa-miR- 200b-3p/hsa-miR-200c-3p, hsa-miR-518f-5p/hsa-miR-523-5p, hsa-miR-625-3p, hsa-miR-194-5p, hsa-let-7g- 3p, hsa-miR-514a-5p, hsa-miR-381-3p, hsa-miR-513c-5p/hsa-miR-514b-5p, hsa-miR-520a-5p, hsa-miR-125b-5p/ hsa-miR-125a-5p, hsa-miR-141-3p, hsa-miR-874-3p, hsa-miR-202-5p, hsa-miR-140-3p, hsa-miR-361-3p, hsa- miR -513b-5p, hsa-miR-33a-5p, hsa-let-7a-5p/hsa-let-7c-5p/hsa-let-7b-5p/hsa-let-7d-5p/hsa-let-7f -5p/hsa-let-7e-5p/hsa-let-7i-5p/hsa-let-7g-5p, hsa-miR-136-3p, hsa-miR-508-5p, hsa-miR-204-5p /hsa-miR-211-5p, hsa-miR-146a-5p/hsa-miR-146b-5p, hsa-miR-23a-3p, hsa-miR-21-3p, hsa-miR-877-5p, hsa -miR-302a-5p, hsa-miR-139-5p, hsa-miR-99a-5p/hsa-miR-100-5p/hsa-miR-99b-5p, hsa-miR-216a-5p, and hsa- It is selected from the group consisting of miR-3157-3p, preferably from the 2nd to 7th sequence or the 2nd to 9th sequence based on the 5'end, in which at least one guanine base is substituted with Uracil. It provides an RNA interference-inducing nucleic acid, characterized in that it is composed of 6-24 bases while having a modified nucleotide sequence so that the G:A wobble arrangement of the corresponding region becomes a regular nucleotide arrangement of U:A.

Preferably, the RNA interference-inducing nucleic acid has 6 to 8 consecutive nucleotide sequences starting at the 2nd base from the 5'end and up to the 7th or starting at the 2nd base to the 9th, SEQ ID NOs: 529 to 863 (Table 4), it provides an RNA interference-inducing nucleic acid, characterized in that represented by any one or more of.

In addition, the present invention provides a composition for suppressing the expression of non-normal target genes of microRNAs comprising the RNA interference-inducing nucleic acid.

In addition, in the present invention, in one or more single strands of the double strands of nucleic acids inducing RNA interference, including the following steps, some sequences of specific microRNAs are modified to be noncanonical target genes of microRNAs (noncanonical As a method for producing RNA interference-inducing nucleic acids that inhibit the expression of target gene),

RNA interference to have a modified nucleotide sequence in which at least one guanine base is substituted with a uracil base among 6 to 8 consecutive base sequences starting at the second base from the 5'end of a specific microRNA It provides a method for producing an RNA interference-inducing nucleic acid, comprising the step of constructing an inducible nucleic acid.

Hereinafter, the present invention will be described.

When the microRNA binds to the target messenger RNA, it is recognized as a target of the microRNA and binds to the messenger RNA even if it is not exactly complementary to the microRNA seed region.This is a non-canonical target. recognition). The present inventors have completed the present invention by developing an RNA interference-inducing nucleic acid that selectively inhibits irregular target genes of microRNAs by modifying some sequences of microRNAs, focusing on the recognition of such an irregular target of microRNA, and revealing its efficacy. .

The present invention is an RNA that inhibits only the expression of a noncanonical target gene of a microRNA by modifying a part of the sequence of a specific microRNA in one or more single strands of a nucleic acid that induces RNA interference. As an interference inducing nucleic acid,

The RNA interference-inducing nucleic acid contains the 2nd to 5th 4 base sequences from the 5'end of the specific microRNA, the 6th and 7th bases are the same, and the 6th and 7th bases are the 6th of the specific microRNA. Has a base sequence that is complementary to a base that can be aligned with a base, and the base that can be aligned with the 6th base of the specific microRNA includes all complementary bases including G:A and G:U wobble arrangements. So that a bulge occurs in the target gene between the 5th and 6th of the microRNA, and the bulge disappears from the non-normal target nucleotide sequence to which it binds and binds to a continuous nucleotide sequence, or

The RNA interference-inducing nucleic acid is a nucleotide sequence between the 5'end and the ninth base of a specific microRNA, preferably the guanine base is uracil in the 2nd to 7th sequence based on the 5'end or By having a modified base sequence substituted with at least one or more adenine, characterized in that the G:A or G:U wobble arrangement of the site becomes a regular base sequence of U:A or A:U, RNA interference inducing nucleic acids are provided.

The RNA interference-inducing nucleic acid according to the present invention is one or more single strands of the double-stranded nucleic acid that induces RNA interference, preferably microRNA, small hairpin RNA (shRNA), and small interfering RNA (small). interfering RNA, siRNA), DsiRNA, lsiRNA, ss-siRNA, asiRNA, piRNA, or endo-siRNA.

The RNA interference-inducing nucleic acid according to the present invention is based on two non-canonical target recognition of microRNAs.

More specifically, microRNAs recognize not only canonical target genes but also irregular target genes. The non-canonical target gene recognized by the microRNA does not have an exact complementary relationship with the microRNA.

One aspect of the non-normal target gene recognized by the microRNA and the RNA interference-inducing nucleic acid that suppresses the non-normal target gene is as follows.

One recognition type of non-canonical target genes to which the microRNA binds has a continuous complementary relationship with all of the bases from the 5'end to the 5th of the microRNA. However, the gene recognized by the 6th base at the 5'end of the microRNA (aligned with the 6th base at the 5'end of the microRNA) is a base having a complementary relationship with the 5th base at the 5'end of the microRNA. Rather than a base that is immediately contiguous with and, the immediately contiguous base is pushed out in a raised shape, and then the base that has a complementary relationship with the microRNA becomes the base recognized by the 6th base from the 5'end of the microRNA.

The RNA interference-inducing nucleic acid that suppresses the non-normal target gene recognized by the microRNA of the above aspect includes 4 base sequences from the 2nd to the 5th from the 5'end of the specific microRNA. In addition, two bases consecutive to the four base sequences are identical to each other, and the two base sequences have a base sequence that is complementary to a base that can be aligned with the 6th base of a specific microRNA. Bases that can be aligned with the 6th base include all complementary bases including the G:A and G:U wobble configurations.

RNA interference-inducing nucleic acids that suppress irregular target genes recognized by these specific microRNAs share at least 4 nucleotide sequences from the 5'end to the 5'end of the specific microRNA compared to the nucleotide sequence of the microRNA. Include as.

In addition, the 6th base sequence and the 7th base sequence from the 5'end of the RNA interference-inducing nucleic acid may be identical, and these two base sequences are base sequences complementary to bases that can be aligned with the 6th base of a specific microRNA. And all complementary bases including G:A and G:U wobble arrangements are included in the bases that can be aligned with the 6th base of the specific microRNA. For example, the 6th base of a specific microRNA: a base that can be aligned with the 6th base: a base that is complementary to a base that can be aligned with the 6th base is (A: U,G: A,C), (G : A, U, C: U, A, G), (U: G, A: C, U) or (C: G: C). For example, when the 6th base of a specific microRNA is A, the 6th base sequence and 7th base sequence from the 5'end of the RNA interference inducing nucleic acid may be AA or CA; When the 6th base of a specific microRNA is G, the 6th base sequence and 7th base sequence from the 5'end of the RNA interference inducing nucleic acid may be UG, AG, or GG; When the 6th base of a specific microRNA is U, the 6th base sequence and 7th base sequence from the 5'end of the RNA interference-inducing nucleic acid may be CU or UU; Alternatively, when the 6th base of a specific microRNA is C, the 6th base sequence and the 7th base sequence from the 5'end of the RNA interference-inducing nucleic acid may be CC.

Preferably, the 6th base sequence from the 5'end of the RNA interference-inducing nucleic acid and the 6th base sequence from the 5'end of the specific microRNA may be the same, preferably the 7th base sequence from the 5'end of the RNA interference-inducing nucleic acid The 6th nucleotide sequence from the 5'end of a particular microRNA may be identical.

In addition, preferably, the RNA interference-inducing nucleic acid may include a 7th base sequence from the 5'end of a specific microRNA as an 8th base sequence from the 5'end.

In another aspect, an aspect of the non-normal target gene recognized by the microRNA and the RNA interference-inducing nucleic acid that suppresses the non-normal target gene is as follows.

For non-normal target genes recognized by microRNA, the bases of the microRNA and the target gene recognized by the microRNA are A:U, G:C, C:G, U:A, along with a complementary relationship arrangement of G:A. Contains an array of or an array of G:U wobble relationships.

When the microRNA and the target gene recognized by the microRNA are in a wobble relationship of G:A, the base of the specific microRNA: the base of the target gene recognized by the specific microRNA: the target gene recognized by the specific microRNA is suppressed The nucleotide sequence of the RNA interference-inducing nucleic acid is G:A:U. In addition, when the microRNA and the target gene recognized by the microRNA are in a wobble relationship of G:U, the base of the specific microRNA: the base of the target gene recognized by the specific microRNA: the target gene recognized by the specific microRNA The arrangement of the bases of the RNA interference-inducing nucleic acid that inhibits G:U:A.

The RNA interference-inducing nucleic acid according to the present invention has a modified nucleotide sequence in which at least one guanine (G) base is substituted with uracil (U) among the nucleotide sequences between the 5'end and the ninth base of a specific microRNA. Alternatively, in the RNA interference-inducing nucleic acid according to the present invention, one or more guanine (G) bases from the nucleotide sequence from the 5'end to the ninth base of the specific microRNA, preferably from the 2nd to the 7th sequence based on the 5'end, are adenine ( It has a modified base sequence substituted with A). When one or more guanine (G) bases are included in the base sequence between the 5'end of a specific microRNA and the ninth base, all included guanine bases may be replaced with uracil (U) or adenine (A), At least one or more guanine bases are substituted with uracil (U) or adenine (A). If at least one guanine (G) base is included in the nucleotide sequence between the 5'end and the ninth base of a specific microRNA, preferably in the 2nd to 7th sequence based on the 5'end, it is substituted with a uracil or adenine base. In addition to the base to be used, the remaining bases may be the same as those of a specific microRNA.

The RNA interference-inducing nucleic acid according to the present invention selectively inhibits non-normal target genes of microRNA and does not inhibit normal target genes of microRNA.

According to a specific example 2 of the present invention (see Fig. 2), the function of regulating the expression of messenger RNA against the target gene of the RNA interference-inducing nucleic acid according to the present invention is specific for non-normal target genes, and suppressed for normal target genes. Did not show any effect.

Therefore, when the RNA interference-inducing nucleic acid according to the present invention is used, only the expression of an irregular target gene can be selectively suppressed, and therefore, only an expected effect can be selectively induced through the inhibition of RNA interference-induced nucleic acid expression.

In the present invention, the specific microRNA may be at least one selected from the group consisting of miR-124, miR-155, miR-122, miR-1, miR-133, let-7, mR-302a, and miR-372.

In the present invention, the nucleotide sequences of human miR-124, miR-155, miR-122, miR-1, miR-133, let-7, miR-302a and miR-372, respectively, are miRBase, a sequence database of microRNAs. As described in MIMAT0000422, MIMAT0000646, MIMAT0000421, MIMAT0000416, MIMAT0000427, MIMAT0000062, MIMAT0000684, and MIMAT0000724 at (http://www.mirbase.org/).

However, the specific microRNA according to the present invention is not limited to human microRNAs, and includes animal microRNAs, including mammals.

As an RNA interference-inducing nucleic acid that suppresses an irregular target gene recognized by a specific microRNA according to the present invention, it contains the 2nd to 5th 4 base sequences from the 5'end of the specific microRNA, and the 6th and 7th bases May be the same, and the 6th and 7th bases have a base sequence that is complementary to a base that can be aligned with the 6th base of a specific microRNA, and the base that can be aligned with the 6th base of the specific microRNA includes G: RNA interference-inducing nucleic acids, characterized in that all complementary bases including A, G:U wobble sequences, are included, include 6 or more nucleotide sequences, but are not limited thereto, but are usually about 21 or 24 It may have the following nucleotide sequence.

The RNA interference-inducing nucleic acid according to the present invention is an RNA interference-inducing nucleic acid that suppresses an irregular target gene of miR-124 when a specific microRNA is miR-124, and is from the 2nd to 5th end of the 5'end of miR-124. It contains 4 base sequences, the 6th and 7th bases may be the same, and the 6th and 7th bases have a base sequence complementary to a base that can be aligned with the 6th base of miR-124, and the miR- It is an RNA interference-inducing nucleic acid that includes all complementary bases including G:A and G:U wobble sequences in the bases that can be aligned with the 6th base of 124.

For example, the RNA interference-inducing nucleic acid may be an RNA interference-inducing nucleic acid (miR-124BS) in which the 2nd to 7th base sequence at the 5'end represents the base sequence of 5'-AA GGC C-3'. More preferably, the RNA interference-inducing nucleic acid may be an RNA interference-inducing nucleic acid (miR-124BS) showing the nucleotide sequence of 5′-UAA GGC CAC GCG GUG AAU GCC-3′.

The RNA interference-inducing nucleic acid according to the present invention is an RNA interference-inducing nucleic acid that suppresses an irregular target gene of miR-122 when a specific microRNA is miR-122, and is the 2nd to 5th from the 5'end of miR-122. It contains 4 base sequences, the 6th and 7th bases are the same, and the 6th and 7th bases have a base sequence complementary to a base that can be aligned with the 6th base of miR-122. It is an RNA interference-inducing nucleic acid that includes all complementary bases including G:A and G:U wobble arrangements as bases that can be aligned with the 6th base.

For example, the RNA interference-inducing nucleic acid may be an RNA interference-inducing nucleic acid (miR-122BS) in which the 2nd to 7th base sequence of the 5'end represents the base sequence of 5'-GG AGU U-3'. More preferably, the RNA interference inducing nucleic acid may be an RNA interference inducing nucleic acid (miR-122BS) showing the nucleotide sequence of 5′-UGG AGU UGU GAC AAU GGU GUU-3′.

The RNA interference-inducing nucleic acid according to the present invention is an RNA interference-inducing nucleic acid that suppresses an irregular target gene of miR-155 when a specific microRNA is miR-155, from the 2nd to 5th end of the 5'end of miR-155. It contains 4 base sequences, the 6th and 7th bases are the same, the 6th and 7th bases have a base sequence complementary to a base that can be aligned with the 6th base of miR-155, It is an RNA interference-inducing nucleic acid that includes all complementary bases including G:A and G:U wobble arrangements as bases that can be aligned with the 6th base.

For example, the RNA interference-inducing nucleic acid may be an RNA interference-inducing nucleic acid (miR-155BS) in which the 2nd to 7th base sequence of the 5'end represents the base sequence of 5'-UA AUG G-3'. More preferably, the RNA interference inducing nucleic acid may be an RNA interference inducing nucleic acid (miR-155BS) showing the nucleotide sequence of 5′-UUA AUG GC UAA U CGU GAU AGG-3′.

The RNA interference-inducing nucleic acid according to the present invention is an RNA interference-inducing nucleic acid that suppresses an irregular target gene of miR-1 when a specific microRNA is miR-1, and the 2nd to 5th end from the 5'end of miR-1 It includes 4 base sequences, the 6th and 7th bases are the same, and the 6th and 7th bases have a base sequence complementary to a base that can be aligned with the 6th base of miR-1. It is an RNA interference-inducing nucleic acid that includes all complementary bases including G:A and G:U wobble arrangements as bases that can be aligned with the 6th base.

For example, the RNA interference-inducing nucleic acid may be an RNA interference-inducing nucleic acid (miR-1BS) in which the 2nd to 7th base sequence of the 5'end represents the base sequence of 5'-GG AAU U-3''. More preferably, the RNA interference inducing nucleic acid may be an RNA interference inducing nucleic acid (miR-1BS) showing the nucleotide sequence of 5′-UGG AAU UGU AAA GAA GUA UGU-3′.

As an RNA interference-inducing nucleic acid that suppresses an irregular target gene recognized by a specific microRNA according to the present invention, of the nucleotide sequence between the 5'end and the ninth base of the specific microRNA, preferably from the 2nd to the 7th based on the 5'end. The length of the RNA interference-inducing nucleic acid, characterized in that the guanine (G) base has at least one modified base sequence substituted with uracil (U) or adenine (A), has a length of 6 or more base sequences. Although not limited, it may have a sequence of 24 or less, usually around 21.

The RNA interference-inducing nucleic acid according to the present invention is an RNA interference-inducing nucleic acid that suppresses an irregular target gene of miR-124 when a specific microRNA is miR-124, and the base between the 5'end and the ninth base of miR-124 Among the sequences, preferably, it is an RNA interference-inducing nucleic acid having a modified nucleotide sequence in which at least one guanine (G) base is substituted with uracil (U) or adenine (A) base from the 2nd to 7th sequence based on the 5'end. .

For example, the RNA interference-inducing nucleic acid has a nucleotide sequence of 5'-UAA UGC AC-3' (miR-124-G4U), 5'-UAA GUC AC-3' (miR-124) between the 5'end and the ninth base. -G5U) or 5'-UAA UUC AC-3' (miR-124-G4,5U). More preferably, the RNA interference inducing nucleic acid is 5'-UAA UGC ACG CGG UGA AUG CCA A-3' (miR-124-G4U), 5'-UAA GUC ACG CGG UGA AUG CCA A-3' (miR-124 -G5U) or 5'-UAA UUC ACG CGG UGA AUG CCA A-3' (miR-124-G4,5U).

The RNA interference-inducing nucleic acid according to the present invention is an RNA interference-inducing nucleic acid that suppresses an irregular target gene of miR-1 when a specific microRNA is miR-1, and a base between the 5'end and the 9th base of miR-1 It is an RNA interference-inducing nucleic acid having a modified nucleotide sequence in which at least one guanine (G) base in the sequence is substituted with uracil (U) or adenine (A) base.

For example, the RNA interference inducing nucleic acid has a base sequence between the 5'end and the ninth base 5'-UUG AAU GUA-3' (miR-1-G2U), 5'-UGU AAU GUA-3' (miR-1 -G3U), 5'-UGG AAU UUA-3' (miR-1-G7U), 5'-UUU AAU GUA-3' (miR-1-G2,3U), 5'-UGU AAU UUA-3' (miR-1-G3,7U), 5'-UUG It may be an RNA interference inducing nucleic acid representing AAU UUA-3' (miR-1-G2,7U) or 5'-UUU AAU UUA-3' (miR-1-G2,3,7U). More preferably, the RNA interference inducing nucleic acid is 5'-UUG AAU GUA AAG AAG UAU GUA U-3' (miR-1-G2U), 5'-UGU AAU GUA AAG AAG UAU GUA U-3' (miR-1 -G3U), 5'-UGG AAU UUA AAG AAG UAU GUA U-3' (miR-1-G7U), 5'-UUU AAU GUA AAG AAG UAU GUA U-3' (miR-1-G2,3U), 5'-UGU AAU UUA AAG AAG UAU GUA U-3' (miR-1-G3,7U), 5'-UUG AAU UUA AAG AAG UAU GUA U-3' (miR-1-G2,7U) or 5' -UUU AAU UUA AAG AAG UAU GUA U-3' (miR-1-G2,3,7U) may be an RNA interference inducing nucleic acid showing the base sequence.

The RNA interference-inducing nucleic acid according to the present invention is an RNA interference-inducing nucleic acid that suppresses an irregular target gene of miR-122 when a specific microRNA is miR-122, and the base between the 5'end and the ninth base of miR-122 In the sequence, preferably, in the 2nd to 7th sequence based on the 5'end, the guanine (G) base is an RNA interference-inducing nucleic acid having a modified base sequence in which at least one base is substituted with uracil (U) or adenine (A) base. .

For example, the RNA interference-inducing nucleic acid has a nucleotide sequence between the 5'end and the ninth base 5'-UUG AGU GUG-3' (miR-122-G2U), 5'-UGU AGU GUG-3' (miR-122 -G3U), 5'-UGG AUU GUG-3' (miR-122-G5U), 5'-UGG AGU UUG-3' (miR-122-G7U), 5'-UGG AGU GUU-3' (miR- 122-G9U), 5'-UUU AGU GUG-3' (miR-122-G2,3U), 5'-UUG AUU GUG-3' (miR-122-G2,5U), 5'-UUG AGU UUG- 3'(miR-122-G2,7U), 5'-UUG AGU GUU-3' (miR-122-G2,9U), 5'-UGU AUU GUG (miR-122-G3,5U), 5'- UGU AGU UUG-3' (miR-122-G3,7U), 5'-UGU AGU GUU-3' (miR-122-G3,9U), 5'-UGG AUU UUG-3' (miR-122-G5 ,7U), 5′-UGG AUU GUU-3′ (miR-122-G5,9U), or 5′-UGG AGU UUU-3 (miR-122-G7,9U). . More preferably, the RNA interference-inducing nucleic acid is 5'-UUG AGU GUG ACA AUG GUG UUU G-3' (miR-122-G2U), 5'-UGU AGU GUG ACA AUG GUG UUU G-3 (miR-122- G3U), 5'-UGG AUU GUG ACA AUG GUG UUU G-3' (miR-122-G5U), 5'-UGG AGU UUG ACA AUG GUG UUU G-3' (miR-122-G7U), 5'- UGG AGU GUU ACA AUG GUG UUU G-3' (miR-122-G9U), 5'-UUU AGU GUG ACA AUG GUG UUU G-3' (miR-122-G2,3U), 5'-UUG AUU GUG ACA AUG GUG UUU G-3' (miR-122-G2,5U), 5'-UUG AGU UUG ACA AUG GUG UUU G-3' (miR-122-G2,7U), 5'-UUG AGU GUU ACA AUG GUG UUU G-3' (miR-122-G2,9U), 5'-UGU AUU GUG ACA AUG GUG UUU G-3 (miR-122-G3,5U), 5'-UGU AGU UUG ACA AUG GUG UUU G- 3 (miR-122-G3,7U), 5'-UGU AGU GUU ACA AUG GUG UUU G-3 (miR-122-G3,9U), 5'-UGG AUU UUG ACA AUG GUG UUU G-3' (miR -122-G5,7U), 5'-UGG AUU GUU ACA AUG GUG UUU G-3' (miR-122-G5,9U) or 5'-UGG AGU UUU ACA AUG GUG UUU G-3' (miR-122 -G7,9U) may be an RNA interference inducing nucleic acid showing the nucleotide sequence.

The RNA interference-inducing nucleic acid according to the present invention is an RNA interference-inducing nucleic acid that suppresses an irregular target gene of miR-133 when a specific microRNA is miR-133, and the base between the 5'end and the ninth base of miR-133 In the sequence, preferably, in the 2nd to 7th sequence based on the 5'end, the guanine (G) base is an RNA interference-inducing nucleic acid having a modified base sequence in which at least one base is substituted with uracil (U) or adenine (A) base. .

For example, the RNA interference-inducing nucleic acid has a base sequence between the 5'end and the ninth base 5'-UUU UGU CCC-3' (miR-133-G4U), 5'-UUU GUU CCC-3' (miR-133 -G5U) or 5'-UUU UUU CCC-3' (miR-124-G4,5U). More preferably, the RNA interference-inducing nucleic acid is 5'-UUU UGU CCC CUU CAA CCA GCU G-3' (miR-133-G4U), 5'-UUU GUU CCC CUU CAA CCA GCU G-3' (miR-133 -G5U) or 5'-UUU UUU CCC CUU CAA CCA GCU G-3' (miR-124-G4,5U) may be an RNA interference inducing nucleic acid showing the base sequence.

The RNA interference-inducing nucleic acid according to the present invention is an RNA interference-inducing nucleic acid that suppresses an irregular target gene of let-7 when a specific microRNA is let-7, and the base between the 5'end and the ninth base of let-7 Among the sequences, preferably, it is an RNA interference-inducing nucleic acid having a modified nucleotide sequence in which at least one guanine (G) base is substituted with uracil (U) or adenine (A) base from the 2nd to 7th sequence based on the 5'end. .

For example, the RNA interference-inducing nucleic acid has a base sequence between the 5'end and the ninth base 5'-UUA GGU AGU-3' (let-7-G2U), 5'-UGA UGU AGU-3' (let-7 -G4U), 5'-UGA GUU AGU-3' (let-7-G5U), 5'-UGA GGU AUU-3' (let-7-G8U), 5'-UUA UGU AGU-3' (let- 7-G2,4U), 5'-UUA GUU AGU-3' (let-7-G2,5U), 5'-UUA GGU AUU-3' (let-7-G2,8U), 5'-UGA UUU AGU-3' (let-7-G4,5U), 5'-UGA UGU AUU-3' (let-7-G4,8U), or 5'-UGA GUU AUU-3' (let-7-G5, 8U) may be an RNA interference inducing nucleic acid. More preferably, the RNA interference inducing nucleic acid is 5'-UUA GGU AGU AGG UUG UAU AGU U-3' (let-7-G2U), 5'-UGA UGU AGU AGG UUG UAU AGU U-3' (let-7 -G4U), 5'-UGA GUU AGU AGG UUG UAU AGU U-3' (let-7-G5U), 5'-UGA GGU AUU AGG UUG UAU AGU U-3' (let-7-G8U), 5' -UUA UGU AGU AGG UUG UAU AGU U-3' (let-7-G2,4U), 5'-UUA GUU AGU AGG UUG UAU AGU U-3' (let-7-G2,5U), 5'-UUA GGU AUU AGG UUG UAU AGU U-3' (let-7-G2,8U), 5'-UGA UUU AGU AGG UUG UAU AGU U-3' (let-7-G4,5U), 5'-UGA UGU AUU RNA interference inducing nucleic acid showing the base sequence of AGG UUG UAU AGU U-3' (let-7-G4,8U) or 5'-UGA GUU AUU AGG UUG UAU AGU U-3' (let-7-G5,8U) Can be

The RNA interference-inducing nucleic acid according to the present invention is an RNA interference-inducing nucleic acid that inhibits an irregular target gene of miR-302a when a specific microRNA is miR-302a, and the base between the 5'end and the ninth base of miR-302a In the sequence, preferably, in the 2nd to 7th sequence based on the 5'end, the guanine (G) base is an RNA interference-inducing nucleic acid having a modified base sequence in which at least one base is substituted with uracil (U) or adenine (A) base. .

For example, the RNA interference-inducing nucleic acid has a base sequence between the 5'end and the ninth base 5'-UAA UUG CUU-3' (miR-302a-G4U), 5'-UAA GUU CUU-3' (miR-302a -G6U), or an RNA interference inducing nucleic acid representing 5'-UAA UUU CUU-3' (miR-302a-G4,6U). More preferably, the RNA interference-inducing nucleic acid is 5'-UAA UUG CUU CCA UGU UUU GGU GA-3' (miR-302a-G4U), 5'-UAA GUU CUU CCA UGU UUU GGU GA-3' (miR-302a -G6U), or may be an RNA interference inducing nucleic acid showing the nucleotide sequence of 5'-UAA UUU CUU CCA UGU UUU GGU GA-3' (miR-302a-G4,6U).

The RNA interference-inducing nucleic acid according to the present invention is an RNA interference-inducing nucleic acid that suppresses an irregular target gene of miR-372 when a specific microRNA is miR-372, and the base between the 5'end and the ninth base of miR-372 In the sequence, preferably, in the 2nd to 7th sequence based on the 5'end, the guanine (G) base is an RNA interference-inducing nucleic acid having a modified base sequence in which at least one base is substituted with uracil (U) or adenine (A) base. .

For example, the RNA interference-inducing nucleic acid has a nucleotide sequence between the 5'end and the ninth base 5'-AAA UUG CUG-3' (miR-372-G4U), 5'-AAA GUU CUG-3' (miR-372 -G6U), 5'-AAA GUG CUU-3' (miR-372-G9U), 5'-AAA UUU CUG-3' (miR-372-G4,6U), 5'-AAA UUG CUU-3' ( miR-372-G4,9U), or an RNA interference inducing nucleic acid representing 5'-AAA GUU CUU-3' (miR-372-G6,9U). More preferably, the RNA interference-inducing nucleic acid is an RNA interference-inducing nucleic acid that suppresses an irregular target gene of miR-372, and is 5'-AAA UUG CUG CGA CAU UUG AGC GU -3' (miR-372-G4U), 5'- AAA GUU CUG CGA CAU UUG AGC GU -3' (miR-372-G6U), 5'-AAA GUG CUU CGA CAU UUG AGC GU -3' (miR-372-G9U), 5'-AAA UUU CUG CGA CAU UUG AGC GU -3' (miR-372-G4,6U), 5'-AAA UUG CUU CGA CAU UUG AGC GU -3' (miR-372-G4,9U), or 5'-AAA GUU CUU CGA CAU UUG AGC It may be an RNA interference-inducing nucleic acid showing the nucleotide sequence of an RNA interference-inducing nucleic acid showing the nucleotide sequence of GU-3′ (miR-372-G6,9U).

In addition, the present invention is an RNA that suppresses noncanonical target genes of microRNAs by modifying some sequences of specific microRNAs in one or more single strands of a nucleic acid that induces RNA interference. As an interference inducing nucleic acid,

The RNA interference-inducing nucleic acid contains the 2nd to 5th 4 base sequences from the 5'end of the specific microRNA, the 6th and 7th bases are the same, and the 6th and 7th bases are the 6th of the specific microRNA. Has a base sequence that is complementary to a base that can be aligned with a base, and the base that can be aligned with the 6th base of the specific microRNA includes all complementary bases including G:A and G:U wobble arrangements. In this way, a bulge occurs in the target gene between the 5th and 6th microRNA, and the bulge disappears from the non-normal target nucleotide sequence to which it binds and binds to a continuous nucleotide sequence,

The specific microRNA provides an RNA interference-inducing nucleic acid, characterized in that a methyl group (2'OMe) is added to the 2'position of the ribosyl ring of the 6th nucleotide from the 5'end.

The RNA interference-inducing nucleic acid according to the present invention includes the 2nd to 5th 4 nucleotide sequences from the 5'end of the specific microRNA, the 6th and 7th bases are the same, and the 6th and 7th bases are specific microRNAs. It has a base sequence that is complementary to a base that can be aligned with the 6th base of the specific microRNA, and is a base that can be aligned with the 6th base of the specific microRNA, and is all complementary including a G:A, G:U wobble arrangement. If a base is included and a methyl group (2'OMe) is added at the 2'position of the ribosyl ring of the 6th nucleotide from the 5'end of the microRNA, the rest of the nucleotide sequence other than this is not limited, and any base Any sequence may be included, and in this case, the RNA interference-inducing nucleic acid may contain 6 to 24 nucleotide sequences, preferably 6 to 15, more preferably 6 to 8 nucleotide sequences. However, there is no particular limitation on the length of the nucleic acid.

The RNA interference-inducing nucleic acid according to the present invention includes a nucleotide sequence chemically modified by adding a methyl group (2'OMe) to the 2'position of the ribosyl ring of the 6th nucleotide from the 5'end of the microRNA, and the 2 'Ome-modified RNA interference-inducing nucleic acids selectively inhibit only the expression of its normal initiating target gene and may not inhibit the expression of non-normal initiation target genes. Thus, the 2'OMe modified RNA interference-inducing nucleic acid maintains the object of the present invention to specifically inhibit only the irregular nucleus site of a specific microRNA, while the newly occurring irregular nucleus site can be completely removed. have.

In the RNA interference-inducing nucleic acid according to the present invention, there is no restriction on the kind of microRNA to which the methyl group (2'OMe) can be added, and the 2'position of the ribosyl ring of the 6th nucleotide from the 5'end of the microRNA If so, 2'OMe may be added to any microRNA, but according to a specific embodiment of the present invention, the microRNA to which the 2'OMe may be added may be miR-124 or miR-1.

In addition, the present invention is an RNA that suppresses noncanonical target genes of microRNAs by modifying some sequences of specific microRNAs in one or more single strands of a nucleic acid that induces RNA interference. As an interference inducing nucleic acid,

The RNA interference-inducing nucleic acid contains the 2nd to 5th 4 base sequences from the 5'end of the specific microRNA, the 6th and 7th bases are the same, and the 6th and 7th bases are the 6th of the specific microRNA. Has a base sequence that is complementary to a base that can be aligned with a base, and the base that can be aligned with the 6th base of the specific microRNA includes all complementary bases including G:A and G:U wobble arrangements. Induction of RNA interference, characterized in that the bulge is formed in the target gene between the 5th and 6th of the microRNA, and the bulge disappears from the non-normal target nucleotide sequence and binds to a continuous nucleotide sequence. Nucleic acids are provided.

The RNA interference-inducing nucleic acid according to the present invention includes the 2nd to 5th 4 nucleotide sequences from the 5'end of the specific microRNA, the 6th and 7th bases are the same, and the 6th and 7th bases are specific microRNAs. It has a base sequence that is complementary to a base that can be aligned with the 6th base of the specific microRNA, and is a base that can be aligned with the 6th base of the specific microRNA, and is all complementary including a G:A, G:U wobble arrangement. If a base is included, there is no restriction on the remaining base sequences except for this, and any base sequence may be included, and in this case, the RNA interference-inducing nucleic acid may include 6 to 24 base sequences, preferably May include 6 to 15, more preferably 6 to 8 nucleotide sequences, but there is no particular limitation on the length of the nucleic acid.

The RNA interference-inducing nucleic acid according to the present invention includes the 2nd to 5th 4 nucleotide sequences from the 5'end of the specific microRNA, the 6th and 7th bases are the same, and the 6th and 7th bases are specific microRNAs. It has a base sequence that is complementary to a base that can be aligned with the 6th base of RNA, and the bases that can be aligned with the 6th base of the specific microRNA are all complementary including G:A and G:U wobble arrangements. The target site for irregular nucleus fusion by including an enemy base so that a bulge occurs in the target gene between the 5th and 6th of the microRNA, and the bulge disappears from the binding irregular target nucleotide sequence and binds to a continuous nucleotide sequence. Only selectively inhibits the microRNA, but not the canonical target gene of the microRNA.

The RNA interference-inducing nucleic acid according to the present invention may include a nucleotide sequence represented by any one or more of SEQ ID NOs: 103 to 528 (see Table 3).

In addition, the present invention inhibits only the expression of noncanonical target genes of microRNAs by modifying some sequences of specific microRNAs in one or more single strands of nucleic acid that induces RNA interference. As an RNA interference inducing nucleic acid,

The RNA interference-inducing nucleic acid is a nucleotide sequence between the 2nd to 9th bases from the 5'end of the specific microRNA, or the Guanine base in the 2nd to 7th sequence based on the 5'end as a Uracil base. It provides an RNA interference-inducing nucleic acid, characterized in that it has at least one or more substituted modified nucleotide sequences, and the G:A wobble of the corresponding region becomes a regular nucleotide sequence of U:A.

The RNA interference-inducing nucleic acid according to the present invention includes a sequence between the 2nd base and the 9th base from the 5'end of a specific microRNA, or of the 2nd to 7th sequence based on the 5'end, the Guanine base is Uracil ) At least one base may be substituted, and the RNA interference-inducing nucleic acid has at least one guanine base between the 2nd base and the 9th base from the 5'end of a specific microRNA as a uracil base. In the case of including a modified nucleotide sequence that is more than substituted, there is no restriction on the remaining nucleotide sequences except for this, and any nucleotide sequence may be included. At this time, the base sequence between the 2nd base and the 9th base from the 5'end of the specific microRNA, or the base substituted with the uracil (U) base among the 2nd to 7th sequences based on the 5'end, the remaining bases are specified. It may be the same as the base of the microRNA.

As an RNA interference-inducing nucleic acid that suppresses an irregular target gene recognized by a specific microRNA according to the present invention, a nucleotide sequence between the 2nd to 9th bases from the 5'end of the specific microRNA, or the 2nd to 7th based on the 5'end The RNA interference-inducing nucleic acid, characterized in that it has a modified nucleotide sequence in which at least one guanine base is substituted with a uracil base, may include 6 to 24 nucleotide sequences, and is preferably May include 6 to 15, more preferably 6 to 8 nucleotide sequences, but guanine among 6 to 8 consecutive nucleotide sequences starting at the second base from the 5'end of the specific microRNA If the (Guanine) base is an RNA interference-inducing nucleic acid containing at least one modified base sequence substituted with at least one uracil base, there is no particular limitation on the length of the nucleic acid. At this time, in the RNA interference-inducing nucleic acid, a Guanine base among 6 to 8 consecutive base sequences starting from the second base from the 5'end of the specific microRNA is at least one uracil base. In the case of including a modified nucleotide sequence that is more than substituted, there is no restriction on the type of nucleotide sequence other than this, and any nucleotide sequence may be included.

The RNA interference-inducing nucleic acid according to the present invention is a modified nucleotide sequence in which at least one guanine (G) base is substituted with uracil (U) base among 6 to 8 consecutive nucleotide sequences starting from the second base from the 5'end. By making the G:A wobble sequence at the site become a regular nucleotide sequence of U:A, it selectively inhibits only the non-normal target genes of microRNAs that bind to the G:A wobble sequence, and is a regular target of microRNAs. It does not suppress the gene.

The RNA interference-inducing nucleic acid according to the present invention may include a nucleotide sequence represented by any one or more of SEQ ID NOs: 529 to 863 (see Table 4).

When the above-described RNA interference-inducing nucleic acid according to the present invention is used, it is possible to specifically suppress an irregular target gene of microRNA.

Accordingly, the present invention provides a composition for inhibiting the expression of an irregular target gene of a microRNA comprising an RNA interference-inducing nucleic acid or a kit for suppressing the expression of an irregular target gene of a microRNA comprising an RNA interference-inducing nucleic acid.

In addition, by using the above-described RNA interference-inducing nucleic acid according to the present invention, it is possible to selectively control the action that occurs due to suppression of the expression of the non-normal target gene of the microRNA by specifically inhibiting the non-normal target gene of the microRNA.

More specifically, the use of the RNA interference-inducing nucleic acid according to the present invention specifically suppresses the non-normal target gene of the microRNA. Therefore, using the above-described RNA interference-inducing nucleic acid according to the present invention, expression of the non-normal target gene of the microRNA. Actions according to (eg, cell cycle, differentiation, dedifferentiation, morphology, migration, division, proliferation, or death) can be specifically regulated.

Accordingly, the present invention provides a composition or kit for regulating cell cycle, differentiation, dedifferentiation, morphology, migration, division, proliferation, or death, including RNA interference-inducing nucleic acids.

In the present invention, the'cell cycle' is a continuous process in which cells grow, divide, and grow again, and the cells are in M phase (cell division), G1 phase (first division preparation), S phase (DNA synthesizer), It means repeating the 4 phases of the G2 phase (second fission preparation phase). Stage G1 is the preparation stage for DNA synthesis from immediately after cell division to the start of DNA synthesis, and stage G2 is the preparation stage for cell division from the end of DNA synthesis to the start of cell division.

In the present invention, the'cell cycle regulation' includes inducing a cell to fluctuate between the four phases, or promoting or arresting the fluctuation, for example, G2/M cells in G1 Inducing fluctuations in the /G2 phase may be included as an example of cell cycle regulation.

In the present invention, the'cell differentiation' is a process in which a cell acquires morphological and functional specificity, and a cell changes its size, shape, membrane potential, metabolic activity, and response to signals through differentiation. .

In the present invention, the'regulation of cell differentiation' includes promoting or delaying the rate at which cells differentiate, inducing differentiation to start, or inhibiting differentiation so that differentiation does not start, such as inducing neurons to differentiate Inducing the acquisition of morphological specificity (eg neurite differentiation) can be included as an example of cell differentiation regulation. Or, for example, inducing muscle cells to differentiate and inducing them to have morphological specificity (eg, muscle fibrosis of muscle cells) may be included as an example of cell differentiation regulation.

In the present invention, the term'reverse differentiation' refers to a phenomenon in which the progression of differentiation is reversed or the pluripotency of the differentiated cells is acquired at the time of undifferentiation before differentiation, and further changes like mesenteric cells.

In the present invention, the'regulation of dedifferentiation' includes inducing, promoting, or inhibiting or delaying the progress of dedifferentiation so that cells are dedifferentiated, and the result of dedifferentiation control is, for example, a cell growth pattern (eg, cluster formation, etc.) , It can be determined by checking the activity of dedifferentiation factors (Oct3/4, Sox2, c-Myc, Klf4).

In the present invention, the'cell type' refers to a phenotypic characteristic including the shape, structure, and size of a cell, and the cell type is different depending on the type of tissue or organ even in the same organism. Varies.

In the present invention, the'cell morphology control' is to induce a change in phenotypic characteristics including the shape, structure, size of the cell, etc., promoting, delaying, inducing or inhibiting the change in the natural morphology of the cell. It includes promoting or inducing non-naturally changing morphology. For example, inducing myocardial hypertrophy of cardiomyocytes may be included as an example.

In the present invention, the'cell migration' refers to the movement of cells, and is an important process for the growth and physiological activity of animals, whereas cells mainly respond quickly to a given stimulus and usually move in a non-collective shape, Other cell types can only migrate during specific growth periods or under specific circumstances. Cell migration occurs mainly during the initiation of neural and vasculature or wound healing of epithelial tissues, and cell population migration contributes to the metastasis of a variety of tumors.

In the present invention, the'regulation of cell migration' means to delay, promote, induce or inhibit (inhibit) the movement of cells.

In the present invention,'cell division and proliferation' refers to a process in which a parent cell is divided into two daughter cells, and means a process of increasing the number of cells.

In the present invention,'cell division and proliferation control' includes delaying, promoting, inhibiting or inducing cell division and proliferation. Cell division and proliferation can be induced when the phase of the cell is induced in the M phase (cell division) during the cell cycle. For example, when the number of cells distributed in the G0/G1 phase is decreased and the number of cells distributed in the G2/M phase is increased, it may be included as an example of cell division and proliferation regulation.

In the present invention,'apoptosis' refers to a step leading to death by genetic properties while maintaining a functional role of a cell, and includes both early and late cell death. According to apoptosis, the entire cell is atrophy, and a new protein is synthesized and death is induced by the cell's suicide gene.

In the present invention,'regulating cell death' includes inducing, delaying, promoting or inhibiting cell death.

More specifically, the present invention is a composition for inducing cancer cell death comprising an RNA interference-inducing nucleic acid (preferably, miR-124BS) that suppresses an irregular target gene of miR-124;

a composition for inducing neurite branch differentiation comprising an RNA interference-inducing nucleic acid (preferably, miR-124BS) that suppresses an irregular target gene of miR-124;

a composition for inducing cell cycle arrest of liver cancer cells, comprising an RNA interference-inducing nucleic acid (preferably, miR-122BS) that suppresses an irregular target gene of miR-122;

a composition for promoting myocyte differentiation or promoting muscle fibrosis, comprising an RNA interference-inducing nucleic acid (preferably, miR-1BS) that suppresses an irregular target gene of miR-1;

a composition for inducing myocyte death comprising an RNA interference-inducing nucleic acid (preferably, miR-155BS) that suppresses an irregular target gene of miR-155;

a composition for promoting apoptosis of neuroblastoma, comprising an RNA interference-inducing nucleic acid (preferably, miR-124-G5U) that suppresses an irregular target gene of miR-124;

a composition for promoting cell division and proliferation of neuroblastoma comprising an RNA interference-inducing nucleic acid (preferably, miR-124-G4U) that suppresses an irregular target gene of miR-124;

a composition for inducing myocardial hypertrophy comprising an RNA interference-inducing nucleic acid (preferably, miR-1-G2U, miR-1-G3U, miR-1-G7U) that suppresses an irregular target gene of miR-1;

a composition for inducing myocardial hypertrophy comprising an RNA interference-inducing nucleic acid (preferably, miR-133-G4U) that suppresses an irregular target gene of miR-133;

a composition for inducing cell cycle arrest of cancer cells, comprising an RNA interference-inducing nucleic acid (preferably, let-7-G2U, let-7-G2, 4U) that suppresses an irregular target gene of let-7;

a composition for inducing cell cycle progression activity of hepatocytes comprising an RNA interference-inducing nucleic acid (preferably, let-7-G4U) that suppresses an irregular target gene of let-7;

a composition for promoting dedifferentiation comprising an RNA interference-inducing nucleic acid (preferably, miR-302a-G4U) that suppresses an irregular target gene of miR-302a;

a composition for promoting dedifferentiation comprising an RNA interference-inducing nucleic acid (preferably, miR-372-G4U) that suppresses an irregular target gene of miR-372; or

A composition for inhibiting cell migration of liver cancer cells comprising an RNA interference-inducing nucleic acid (preferably, miR-122-G2U, or miR-122-G2,3U) that inhibits an irregular target gene of miR-122.

In addition, by using the RNA interference-inducing nucleic acid according to the present invention, it is possible to selectively regulate the action of the non-normal target gene of the microRNA by specifically inhibiting the non-normal target gene of the microRNA, and various fields (eg, pharmaceutical, Cosmetics, cytology, etc.).

In addition, the RNA interference-inducing nucleic acid according to the present invention can be used in a method of screening a test substance for regulating cell cycle, differentiation, dedifferentiation, morphology, migration, division, proliferation or death.

More specifically, introducing the RNA interference-inducing nucleic acid according to the present invention into a target cell;

Treating the test substance to the cell of interest; And

Comprising the step of confirming the expression amount or whether the non-normal target gene of the microRNA inhibited by the RNA interference-inducing nucleic acid in the target cell,

It provides a method for screening a test substance for regulating cell cycle, differentiation, dedifferentiation, morphology, migration, division, proliferation or death.

In the present invention, the target cell is included without limitation as long as it is derived from mammals including humans, and the tissue or type from which the target cell is extracted is not limited, and for example, nerve cells, cardiomyocytes, cancer cells, muscle cells, etc. I can.

In the present invention, the introduction of the RNA interference-inducing nucleic acid into the target cell may be appropriately performed according to various methods known in the art, for example, the introduction is transfection using calcium phosphate (Graham, FL, etc., Virology, 52:456. (1973)), transfection with DEAE dextran, transfection by microinjection (Capecchi, MR, Cell, 22:479(1980)), transfection with cationic lipids (Wong, TK et al., Gene, 10 :87 (1980)), electroporation (Neumann E et al., EMBO J, 1:841 (1982)), transduction or transfection as described in Basic methods in molecular biology, Davis et al., 1986 and Molecular cloning: A laboratory manual, Davis et al., 1986), and may be performed according to methods well known to those of ordinary skill in the art to which the present invention belongs. Preferably, it can be by transfection. Transfection efficiency can vary greatly depending on the cell type as well as the cell culture conditions and transfection reagents.

In the present invention, the test substance is an unknown substance used in screening to examine whether it affects the cell cycle, differentiation, dedifferentiation, morphology, migration, division, proliferation or death by controlling the expression of the non-normal target gene of the microRNA. Means, and includes, but is not limited to, compounds, extracts, proteins or nucleic acids.

In the present invention, the expression amount or whether the microRNA of the non-normal target gene can be confirmed by various expression analysis methods known in the art, such as RT-PCR, ELISA (see Sambrook, J et al, Molecular Cloning A Laboratory Manual, 3rd ed Cold Spring Harbor Press (2001)), western blot, FACS analysis, etc. may be used, but are not limited thereto.

In the present invention, the amount or expression of the non-normal target gene of the microRNA is checked, and as a result, compared with the case of treating only the RNA interference-inducing nucleic acid without a test substance in the expression amount or expression of the non-normal target gene of the microRNA. If there is a difference, it can be determined that the test substance has an effect on the expression of the non-canonical target gene of the microRNA. Furthermore, the test substance may be determined to have a function of regulating cell cycle, differentiation, dedifferentiation, morphology, migration, division, proliferation or death.

In addition, the present invention provides a method for preparing the above-described RNA interference-inducing nucleic acid.

More specifically, in one or more single strands of the double-stranded nucleic acid that induces RNA interference, including the following steps, some sequence of a specific microRNA is modified to be a noncanonical target gene of the microRNA. gene) to inhibit RNA interference-inducing nucleic acids:

It contains the 2nd to 5th 4 nucleotide sequences from the 5'end of the specific microRNA, the 6th and 7th bases are the same, and the 6th and 7th bases can be aligned with the 6th base of a specific microRNA. RNA interference-inducing nucleic acid is used to include all complementary bases including G:A and G:U wobble arrangements in bases that have a base sequence complementary to a base and can be aligned with the 6th base of the specific microRNA. Constructing, or

Constructing an RNA interference-inducing nucleic acid to have a modified base sequence in which at least one guanine base is substituted with uracil or adenine among the base sequence between the 5'end and the ninth base of a specific microRNA.

In addition, in the present invention, in one or more single strands of the double strands of nucleic acids inducing RNA interference, including the following steps, some sequences of specific microRNAs are modified to be noncanonical target genes of microRNAs (noncanonical As a method for producing RNA interference-inducing nucleic acids that inhibit the expression of target gene),

It contains the 2nd to 5th 4 nucleotide sequences from the 5'end of the specific microRNA, the 6th and 7th bases are the same, and the 6th and 7th bases can be aligned with the 6th base of a specific microRNA. It has a base sequence complementary to a base, and all complementary bases including G:A, G:U wobble arrangement are included in the base that can be aligned with the 6th base of the specific microRNA. Constructing an RNA interference-inducing nucleic acid so that a bulge occurs in the target gene between the 5th and 6th and the corresponding bulge disappears from the non-normal target nucleotide sequence and binds to a continuous nucleotide sequence; And

It provides a method for producing an RNA interference-inducing nucleic acid, comprising the step of adding a methyl group (2'OMe) to the 2'position of the ribosyl ring of the 6th nucleotide from the 5'end of the specific microRNA.

In addition, in the present invention, in one or more single strands of the double strands of nucleic acids inducing RNA interference, including the following steps, some sequences of specific microRNAs are modified to be noncanonical target genes of microRNAs (noncanonical As a method for producing RNA interference-inducing nucleic acids that inhibit the expression of target gene),

It contains the 2nd to 5th 4 nucleotide sequences from the 5'end of the specific microRNA, the 6th and 7th bases are the same, and the 6th and 7th bases can be aligned with the 6th base of a specific microRNA. It has a base sequence complementary to a base, and all complementary bases including G:A, G:U wobble arrangement are included in the base that can be aligned with the 6th base of the specific microRNA. It characterized in that it comprises the step of constructing an RNA interference-inducing nucleic acid so that a bulge occurs in the target gene between the 5th and 6th and the corresponding bulge disappears from the non-normal target nucleotide sequence and binds to a continuous nucleotide sequence. , It provides a method of preparing a nucleic acid inducing RNA interference.

In addition, in the present invention, in one or more single strands of the double strands of nucleic acids inducing RNA interference, including the following steps, some sequences of specific microRNAs are modified to be noncanonical target genes of microRNAs (noncanonical As a method for producing RNA interference-inducing nucleic acids that inhibit the expression of target gene),

RNA interference to have a modified nucleotide sequence in which at least one guanine base is substituted with a uracil base among 6 to 8 consecutive base sequences starting at the second base from the 5'end of a specific microRNA It provides a method for producing an RNA interference-inducing nucleic acid, comprising the step of constructing an inducible nucleic acid.

Since the RNA interference-inducing nucleic acid has already been described, description is omitted in order to avoid overlapping description.

RNA interference-inducing nucleic acid construction follows a variety of methods known in the art, and is not limited to a specific method.

In addition, the present invention provides an interference-inducing nucleic acid modified by inserting a methyl group (2'OME) at the 2'position of the ribosyl ring of the 6th nucleotide from the 5'end of a specific microRNA.

The modified interference-inducing nucleic acid maintains the object of the present invention to specifically inhibit only the irregular nucleus site of the corresponding miRNA, and has the effect of completely eliminating the newly appearing irregular nucleus linkage.

When the interference-inducing nucleic acid according to the present invention is used, the biological function exhibited by the conventional microRNA suppressing the non-normal target gene can be efficiently increased, or some of the functions exhibited by the existing microRNA, that is, the biological exhibited by suppressing the irregular target gene. It can have the effect of selectively displaying only functions. In addition, since such interference-inducing nucleic acids can regulate cell cycle, differentiation, dedifferentiation, morphology, migration, division, proliferation or death, it is expected to be used in various fields such as drugs and cosmetics.

1 schematically shows the mechanism of action of an interference-inducing nucleic acid that suppresses an irregular target of a microRNA.
FIG. 2 is a non-normal target specific expression of miR-124-BS by measuring target gene suppression of a normal target (starter: Seed) and a non-regular nucleation target (nucleation bulge) of miR-124 with the enzyme of a luciferase reporter. They confirmed the inhibitory effect.
3 shows apoptosis induced by a normal target (start: Seed) and an irregular nucleation target (nucleation bulge) of miR-124 in cervical cancer cells (HeLa) in miR-124-BS. Cell sorting: FACS) shows the results confirmed through analysis.
Figure 4 is a cell morphology and cell morphology through expression of miR-124-BS in human neuroblastoma (Sh-Sy-5y) of neurite differentiation induced by an irregular nucleation target (nucleation bulge) of miR-124. It shows the results observed by the expression pattern of the marker.
5 shows miR-124-BS(4753) and miR-124, which are the natural forms of miR-124-BS that regulates miR-124's nucleation target (nucleation bulge) with a regular target (starter: Seed). This is the result of observing the differentiation of neurite branches induced by miR-124-(3714) having the same starting sequence as in cell morphology and molecular biological characteristics.
Figure 6 shows that in mouse neuroblastoma (N2a), neurite branch differentiation induced by an irregular nucleation target (nucleation bulge) of miR-124 is a mechanism through regulation of the expression of the MAPRE1 gene. It shows the observed result.
7 shows the results of confirming the suppression of the expression of an irregular nucleation target (nucleation bulge) gene induced by miR-124-BS in mouse neuroblastoma (N2a) cells through RNA-Seq analysis at the transcript level. .
FIG. 8 shows the results of observing cell cycle arrest induced by an irregular nucleation target (nucleation bulge) of miR-122 in human liver cancer cell line (HepG2) through flow cytometry with miR-122-BS.
9 shows the effect of thickening of muscle cells induced by an irregular nucleation target (nucleation bulge) of miR-1 in a myocyte line and myocyte differentiation induced by a miR-155 irregular nucleation target (nucleation bulge). Inhibition and induction of apoptosis were observed by miR-1-BS and miR-155-BS, respectively, in terms of cell morphology, molecular biological characteristics, and flow cytometric analysis.
10 is a result of bioinformatically analyzing the results of Ago HITS-CLIP, which is microRNA and target RNA data, which bind to the agonut protein in human brain tissue, as well as the normal initiation target (initiation: seed), as well as non-normal GA This is the result of confirming that the G:A wobble target naturally exists.
FIG. 11 shows that miR-124 in mouse neuroblastoma (N2a) cells shows a completely different function from miR-124 in brain cell differentiation of miR-124 with the 4th irregular GA sequence initiating target and the 5th non-normal GA sequence initiating target. It shows the results of observation of cell morphology and cell death by flow cytometry through G4U and miR-124-G5U.
Figure 12 shows the biological functions of the inhibition of miR-124 of the 4th irregular GA sequence initiation target and the 5th non-normal GA sequence initiation target in human neuroblastoma (SH-sy-5y) cells, miR-124-G4U, miR-124- It shows the results of observation by flow cytometry (Fluorescence-activated cell sorting: FACS) for cell proliferation and cell cycle analysis through G5U.
FIG. 13 shows the results of confirming that the target of the 7th irregular GA sequence of miR-1 has a gene suppression function in the myocardial cell line (h9c2) through flow cytometry (Fluorescence-activated cell sorting: FACS) in a fluorescent protein reporter. .
Figure 14 shows the function of the 2nd, 3rd, 7th irregular GA sequence initiating target of miR-1 in the neonatal cardiomyocytes of rats miR-1-G2U, miR-1-G3U, miR After expressing -1-G7U, the results confirmed through observation of cell morphology and molecular biological characteristics for the effect of inducing myocardial hypertrophy are shown.
Figure 15 shows the result of observing the cell morphological characteristics for the myocardial hypertrophy induction effect after expressing miR-133-G4U in cardiomyocytes (h9c2) for the function of the 4th irregular GA sequence initiating target of miR-133.
Figure 16 shows that miR-122 inhibits gene expression through the 2nd and 3rd irregular GA sequence initiating targets, a human liver cancer cell line (HepG2) The results observed through the measurement of the enzyme activity of the luciferase reporter are shown.
Figure 17 shows the function of inhibiting the expression of the 2nd irregular GA sequence initiation target and the 2nd and 3rd irregular GA sequence initiation target by miR-122 inhibiting the migration of human liver cancer cell line (HepG2) miR-122-G2U, miR- It shows the results confirmed by observation of the cell morphology through the expression of 122-G2,3,U.
Figure 18 shows that the suppression of the expression of the 2nd and 3rd irregular GA sequence initiating target by miR-122 induces cell cycle arrest of the human liver cancer cell line (HepG2), showing the expression of miR-122-G2,3U. It shows the results confirmed by flow cytometry.
FIG. 19 shows that inhibition of expression of the 2nd and 3rd irregular GA sequence initiation target and the 2nd and 7th irregular GA sequence initiation target by miR-122 induces cell cycle arrest of human liver cancer cell line (HepG2). It shows the results confirmed by flow cytometry through the expression of miR-122-G2,3U and miR-122-G2,7U.
Figure 20 shows that inhibition of the expression of the 2nd or 2nd, 4th irregular GA sequence initiating target by let-7 induces cell cycle arrest of the human liver cancer cell line (HepG2) and 4 non-normal GA sequence initiation targets The results confirmed by flow cytometry through the expression of let-7a-G2U, let-7a-G2,4U, and let-7-G4U that inhibition of expression promotes the cell cycle.
Figure 21 shows miR-372-G4U or miR-302a-G4U, which induces inhibition of the expression of the 4 irregular GA sequence triggering target by miR-372 or miR-302a, promotes dedifferentiation together with miR-372 or miR-302a It shows the result of confirming that it is done with an Oct4 gene expression reporter.
22 shows the result of confirming the phenomenon that the 2'OMe modification at the 6th of the 5'end cannot inhibit the irregular nucleus target of the corresponding RNA interference derivative.
Figure 23 shows the results confirming that the 2'OMe modification at the 6th of the 5'end cannot suppress the entire non-normal nucleus target mRNA in the transcript.
FIG. 24 shows the results of analyzing microRNAs that bind to irregular nucleus sites through the Ago HITS CLIP experiment.
FIG. 25 shows the results of identifying a G>U mutation that recognizes an irregular GA sequence initiating target as a normal target by analyzing a sequence mutation in a cancer patient microRNA sequencing database (TCGA) in a tumor microRNA.

Hereinafter, the present invention will be described in more detail through examples. However, the following examples are only examples of the present invention, and the present invention is not limited to the following examples.

[ Example One] MicroRNA Mechanism of action of interference-inducing nucleic acids that inhibit irregular targets

Through the Ago HITS CLIP experiment, the present inventors determined that in addition to the normal initiation target in which the microRNA is nucleotide sequenced with the target in the agonut protein, the microRNA initiation and the complete complementary arrangement are in addition to the microRNA initiation and a complete complementary arrangement. It was confirmed through analysis of the RNA complex sequence result (Ago HITS-CLIP) bound to the Argonut protein that it binds to the target messenger RNA without doing so. In addition, as a result of analyzing the nucleotide sequence of the RNA with these characteristics, base 6 of the microRNA induces a nucleation bulge in the target and the target body (microRNA irregular nucleus target site) induces nucleotide sequence, It was confirmed that a target (microRNA irregular GA sequence initiating target site) that allows GA sequence (wobble) naturally exists in the microRNA initiation region (FIG. 1).

In the schematic example shown in FIG. 1, miR-124 is at least 6 for the base sequence 5'-GUGCCUUA-3' of the target through the starting nucleotide sequence (5'-UAAGGCAC-3') via the agonut protein. It has been reported as a canonical seed site for normal initiation of microRNAs, and targets having such a sequence of complementary nucleotide sequences (Nature, 2009, 460 (7254): 479-86).

In addition, among the RNA complexes bound to Argonut, miR-124 starting nucleotide sequence (5'-UAAGGCAC-3') and microRNA irregular nucleus target site (5'- GUGGCCUU -3') based on the 5'end of miR-124. Between the 5th and 6th, a target with a complementary nucleotide sequence was identified so that the G of the target messenger RNA was arranged in a bulge, and based on this, the nucleotide sequence was complementary to the microRNA irregular nucleus target site. The starting nucleotide sequence of miR-124 has a nucleotide sequence (5-UAAGGCCA-3) in which base 6 is repeated once more between 6 and 7 of the miR-124 initiation, and a continuous and perfect complementary sequence to this is miR It was used as miR-124BS (BS: Bulge site) siRNA that suppresses the irregular nucleus target of -124.

When analyzing the sequence of the target messenger RNA bound to the microRNA in the RNA complex bound to the agonut, in addition to the existing complementary nucleotide sequence, the G:A wobble nucleotide sequence was found in the binding of the microRNA initiating sequence and the target messenger RNA. Confirmed to happen. Therefore, in the case of miR-124, the 4th G base based on the 5'end in the starting base sequence (5'-UAAGGCAC-3') performs a G:A wobble arrangement, and the microRNA irregular GA sequence initiating target site (5'- UGGCAUU- 3′), and based on this, miR-124-GU was invented by designing siRNA with a sequence that is continuous and completely complementary to the target site for starting the irregular GA sequence of miR-124. It was used as an siRNA that inhibits the expression of the GA sequence initiating target.

[ Example 2] Irregular Nuclear proliferation target Site and Contiguous and completely complementary nucleotide sequence in the starting region MicroRNA -BS Inhibitory ability Confirm

Among the naturally occurring regular starting target sites of miR-124, the 5'-GUGCCUU-3' base sequence complementary to the 2nd to 7th bases based on the 5'end of miR-124. The regular initiation target site is a fully complementary nucleotide sequence with the initiation of miR-124 (5'-UAAGGCAC-3') and at least 6 consecutive bases (Fig. 2a).

The nucleation bulge site (Nuc site; nucleation bulge site) of miR-124 discovered through Ago HITS CLIP experiment is 5'- GUGGCCUU -3', and base 6 of miR-124 forms a nucleus with the target. As a result, the G of the target RNA arranged between the 5th and 6th of the 5'end of miR-124 is formed into a bulge. Based on this, a siRNA was designed using a nucleotide sequence (5'-UAAGGCCA-3') that is continuously and completely complementary to a non-normal nucleation bulge site and named as miR-124-BS siRNA. (Fig. 2a). miR-124-BS recognizes the nucleus target site, which is an irregular target of miR-124, as a regular initiating target, and it is thought that it will specifically and more strongly inhibit the gene expression of the irregular target having it. This fact was confirmed through the luciferase reporter experiment (Fig. 2b-c).

First of all, in order to check how much miR-124 can suppress the irregular target nuclear blasting target site compared to the existing regular target seed site, the corresponding site for miR-124 in the luciferase reporter is described. In addition, after introducing miR-124 at various concentrations (0, 0.01, 0.1, 0.5, 2.5, 15 nM) into the cells, the IC ₅₀ (inhibitory concentration 50) was measured by the activity of luciferase. It was confirmed that the site was about 0.5 nM, and the irregular nucleus site was about 0.2 nM (Fig. 2b). However, when looking at the rate of suppression at the highest level, it was found that the inhibition through the non-regular nuclear fusion site was somewhat weak, with about 50% of the regular initiation sites and about 80% of the irregular nuclear fusion sites. In particular, when looking at the initiation of gene expression inhibition, it was confirmed through luciferase activity measurement that gene suppression begins at concentrations of about 0.01 nM or more for the normal initiating target and 0.1 nM for the irregular nucleus target ( Fig. 2b). Therefore, it was found that miR-124 regulates the expression of regular targets and non-regular nucleus targets, and has high efficiency in regulating expression of regular targets.

The luciferase reporter experiment conducted at this time was performed by cloning the corresponding miR-124 target site sequence into the 3'UTR (3'untranslated region) portion of the Renilla luciferase gene of the psi-check2 vector (Promega) in a second sequence. . As for the irregular ridge site sequence, a non-normal ridge target site naturally present in the 3'UTR portion of MINK1 was used. miR-124 is a human-derived sequence, according to the miRBase database, the corresponding guide strand and the carrier strand are chemically synthesized by Bioneer, separated by HPLC, and the method provided by the company. Was prepared as a guide strand and a transport strand duplex. The miR-124 and luciferase reporter vector were co-transfected into approximately 10,000 cervical cancer cells (HeLa: ATCC CCL-2) using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's protocol. HeLa cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% FBS (fetal bovine serum), 100 U/ml penicillin, and 100 μg/ml steptomycin, and complete medium without antibiotics when performing transfection. The cells were cultured in. After 24 hours of transfection, luciferase activity was measured using Promega's Dual-luciferase reporter assay system according to the manufacturer's protocol, and Renilla luciferase activity was measured at least 3 by using promega's Glomax Luminometer. The experiment was repeated more than once, and the calculation was normalized by the activity of firefly luciferase.

In order to confirm the function of miR-124-BS, which was invented to specifically suppress only the non-canonical nucleus target of miR-124, the same experiment was performed using a luciferase reporter. As a result, miR-124-BS did not inhibit the luciferase reporter enzyme activity at any concentration (0-10 nM) with respect to the miR-124 normal initiating target, but at a concentration of 0.1 nM or more for the miR-124 irregular nucleus target reporter. It was confirmed the effect of inhibiting the luciferase reporter enzyme activity in (Fig. 2c). When the gene suppression efficiency at this time was measured by IC ₅₀ , the suppression against the non-regular nucleus target was about 0.5 nM. In view of this, it can be interpreted that the function of regulating the expression of the miR-124 irregular nucleus target of miR-124-BS is specific to the irregular nucleus target site (Fig. 2c). At this time, the luciferase reporter experiment was produced by synthesizing miR-124-BS at Bioneer in the same manner as before, and transfection (co-) into cervical cancer cells (HeLa: ATCC CCL-2) with the corresponding luciferase reporter vector. 24 hours after transfection), luciferase enzyme activity was measured and performed.

In the above examples, the inventors invented miRNA-BS containing an initiating region sequence that is continuous and completely complementary to the site sequence for the purpose of suppressing only irregular organic targets of microRNA, and to verify the effect. For this reason, when applied to miR-124 and confirmed through a luciferase reporter experiment, it was possible to verify the characteristics that miR-124-BS could not inhibit the expression of the normal initiating target and effectively suppress the expression of the irregular raised target. .

[ Example 3] miR -124 irregular Nuclear proliferation target Observation of apoptosis of cervical cancer cells induced through inhibition of expression

After confirming in the previous examples that miR-124-BS inhibits only irregular raised targets among the various targets inhibited by miR-124, it was attempted to confirm whether it can only exhibit specific functions of miR-124. In the case of miR-124, since it is generally expressed specifically only in neurons, it is known to play a role mainly related to neurons. However, it has been observed that the expression of miR-124 in tumor-generated nocturnal gliomas is reduced, and correlated with this, if the expression of miR-124 is artificially induced into tumor cells, it can cause the death of tumor cells. Therefore, in order to confirm whether the various and different functions of miR-124 differ depending on the type of recognizing the target, miR-124-BS was introduced into cervical cancer cells (HeLa) and the cell death was observed by flow cytometry ( Fig. 3a).

In this experiment, 75nM miR-124 or miR-124-BS duplex was transfected into cervical cancer cells (HeLa: ATCC CCL-2) using RNAiMAX reagent (Invitrogen) according to the manufacturer's protocol. , After 72 hours, the cells were removed from the culture dish by trypsin treatment, and the cells were treated with BD Pharmingen's Annexin V: FITC Apoptosis Detection Kit II according to the method provided by the manufacturer, and then Annexin V with BD Aria I (BD Biosciences). Apoptosis was measured with a furnace and necrosis with a PI (Propidium Iodide). At this time, as a control, it was synthesized and used in the same manner as the sequence of cel-miR-67, a microRNA of C.elegans.

As a result of dividing these experiments into control, miR-124 expression, and miR-124-BS expression, respectively, when miR-124 was expressed, apoptosis (the number of cells stained with Annexin V) in cervical cancer cells (HeLa) compared to the control group. ), it was observed that the rate at which the death proceeds increased from about 30% to about 78%, and similarly, it was confirmed that miR-124-BS increased more than twice to about 73% (FIG. 3A). In view of the results of the previous Example 2, in which miR-124-BS did not inhibit the normal initiating site of miR-124 but only suppressed the irregular raised site, cervical cancer cell death occurred due to suppression of the expression of the irregular nucleus target. Able to know.

miR-124 is a brain tissue-specific microRNA, and it has been reported that it does not have a brain tissue-specific function in completely other cells without tissue cell-specific transcript expression (Farh K et al, 2005, Science, 310 (5755). ) 1817-21). Therefore, in order to observe the function that miR-124 causes by inhibiting the irregular elevation target in nerve cells, after introducing miR-124-BS into the cerebellar neural stem cell line C17.2, cell differentiation was observed cellular morphology ( Figure 3b, above). As a result, when miR-124 was introduced, the differentiation of the neurite branches of C17.2 cells was made longer and promoted. In the case of miR-124-BS, the differentiation was observed, but the branches were differently observed. Therefore, it can be seen that the normal neurite branch differentiation caused by miR-124 occurs by suppressing the normal initiating site target. At this time, the introduction of miRNA was carried out with the RNAiMax reagent by synthesizing 50nM miRNA in the same manner as in Example 2.

In addition, since miR-124-BS induces apoptosis in previous HeLa cells, it was observed by flow cytometry in C17.2 cells in the same manner as in Example 2, whether this function appeared in neurons. As a result, in the case of miR-124 expression, the rate at which apoptosis occurs is 22.4%, which is almost the same as that of 21% of the control group, whereas miR-124-BS slightly increases to 33.4% (Fig. 3b, middle drawing. ). It was found that apoptosis in cervical cancer cells is the same in that apoptosis occurs through the inhibition of the irregular raised target of miR-124, but the increase rate is very small than that of cervical cancer, which is about twice as high. miR-124 plays a role mainly related to differentiation in neural stem cells, and in this case, regulation of the cell cycle can play an important role. Therefore, after the introduction of miR-124 and miR-124-BS into C17.4 cells, the cell cycle was observed by applying flow cytometry through PI (Propidium Iodide) staining (FIG. 3B, the figure below). At this time, miR-124 was found to induce cell cycle arrest at G0/G1 to 83%, slightly increased compared to 69% of the control group. In contrast, it could be observed that the expression of miR-124-BS did not significantly arrest the cell cycle. In this experiment, the microRNA was transfected and cultured for 48 hours, as in the previous example, and then the cells were removed and fixed with ethanol, and 1 mg/ml of propidium iodide (PI; Sigma-Aldrich) was 0.2 mg/ml. After reacting with ml of RnaseA at 37° C. for 30 minutes, flow cytometry was performed with BD FACSCalibur (BD Biosciences).

Summarizing the results in the above examples, just when miR-124 irregular nucleus target expression is inhibited by miR-124-BS, it induces death of cervical cancer cells (Hela) in the same manner as miR-124, From this, it can be seen that the inhibitory function of miR-124's irregular elevation target is the death of cancer cells. In addition, in the case of neural stem cells, the differentiation of neurites occurs due to the expression of miR-124, resulting in slight apoptosis and cell cycle arrest. In miR-124-BS, which inhibits only the irregular raised target of miR-124, this function It appears in a somewhat different form. Therefore, it can be seen that the neuronal differentiation function of miR-124 is mainly caused by the suppression of the known regular initiating target gene, and in other forms, it could be thought that it can occur through non-regular raised targets.

[ Example 4] miR -124-BS miR -124 Irregular Nuclear proliferation target By inhibiting expression, different types of branched neurons with short processes neurite outgrowth) inducing function

After confirming in the previous example that miR-124-BS causes neural dendritic differentiation in a different form from miR-124, in order to examine this in more detail, the sh-sy-5y cell line (CRL-2266), which is a human neuroblastoma. ) Was used to experiment. At this time, in addition to miR-124-BS, miR-124 has the same initiating sequence that can recognize and suppress the irregular nucleus site, but other sequences have the same microRNA sequence as miR-124. An experiment was conducted by synthesizing one miR-124-BS (4753) by the method described in the previous example. The sequence of miR-124-BS (4753) used at this time is 5'-CAAGGCCAAAGGAAGAGAACAG-3', and as a control, cel-miR-67 (NT; non-), a microRNA of C.elegans, is used as a control. targeting) was synthesized and used in the same manner as the sequence. Corresponding miRNA was transfected in the same manner as described in the previous example, and the cells were cultured for 60 hours, and then cell morphological changes were observed with an optical microscope (FIG. 4A).

As a result, the expression of miR-124 could be observed to change the morphology of long differentiation of the neurite out of human neuroblastoma (Sh-sy-5y), whereas it was possible to suppress only the irregular elevation target of miR-124. In the case of miR-124-BS (4753) having the same sequence as miR-124-BS, a short but branched neurite outgrowth was observed in which a large number of neurite branches were formed in one cell (Fig. 4a). ). Based on this, the same experiment was conducted in additional catecholamine-based neurons (CAD, CRL-11179). At this time, the microRNA was transfected, and the cells were cultured for 52 hours, and then observed (Fig. 4b). As a result, miR-124 has a small number but forms a long process, similar to the results shown in human neuroblastoma (Sh-sy-5y, CRL-2266) cells, and miR-124-BS (4753) is short but many neurons. It was possible to observe the eruption of the protruding branches. (Fig. 4b). Additionally, when miR-124 and miR-124-BS (4753) were delivered intracellularly at the same ratio together, cells with long neurites and cells with many neurite branches were simultaneously observed. This shows the possibility that when expressed together with the invented miR-124-BS and the existing miR-124, it can artificially promote various types of neural differentiation similar to brain cells seen in more complex neural networks.

Additionally, the neuronization of CAD cell lines promoted by these microRNAs was confirmed through immunostaining for Tuj1, a neuron-specific marker (FIG. 4B, below). In this experiment, 52 hours after transfection of the corresponding microRNA, CAD cells were fixed with 4% paraformaldehyde, and the primary antibody, Tuj1 (MRB-435P, Covance Antibody Products) was 1:1000. At the ratio of, Alexa 594 fluorescent substance-conjugated secondary antibody (Abcam: ab150076) was subjected to antibody staining (immune-staining) at a ratio of 1:1000, followed by staining the nucleus with DAPI.

From the results of the above examples, it can be seen that miR-124 has a function of forming short but many neurite branches, which are different from the functions of the existing miR-124 by inhibiting only the irregular nuclear ridge target. In particular, this fact was observed using miR-124-BS (4753), which is different from miR-124-BS, but it has the same sequence of the starting region, which can only suppress irregular nucleus targets. It turns out that the RNA interference derivatives containing a starting sequence capable of recognizing miR-124-BS have the same effect as miR-124-BS. In addition, it indicates that the complex neurite branches produced by miR-124-BS can be used as a method that can promote various types of neural differentiation similar to human brain cells showing complex neural networks.

[ Example 5] miR -124-BS siRNA and shRNA After expression as a vector, the morphology and molecular biological characteristics of branched neurite outgrowth induced thereby confirmed.

To further confirm the specific neurite differentiation induced by the expression of miR-124-BS in mouse neuroblastoma N2a (CCL-131), miR-124, miR-124-BS (4753) as in Example 4 Was introduced into the cells and cultured for 72 hours to observe the change in the morphology of the cells (FIG. 5A). In addition, miR-124 (3714), which has the same starting sequence of miR-124 at the same time, but has a different sequence for other parts, is synthesized with the 5'-GAAGGCAGCAGUGCUCCCCUGU-3' sequence to form a duplex in the form of siRNA. Introduced and experimented. At this time, as a control, it was synthesized and used in the same manner as the sequence of cel-miR-67 (NT; non-targeting), a microRNA of C. elegans.

As a result, in the experimental group to which miR-124 was delivered, neurons having long neurites similar to those seen in CAD, a catecholamine-based neuron, were observed, and in the cells transfected with miR-124-BS(4753), various directions were observed. It was possible to confirm the features of the cell morphology with short neurite outstretched by branches (Fig. 5a, above). In addition, in the experimental group transfected with miR-124 (3714), it was possible to observe a large number of cells showing relatively long cell protrusions, similar to miR-124 (Fig. 5a, above). Such changes in cell morphology include antibodies against the protein expressed in differentiated neurons, Tuj1 (Covance Antibody Products, MRB-435P), vGLUT1 (synaptic systes, 135-303), and MAP2 (millipore, MAB3418). It was confirmed that the amount of expression was increased compared to the expression of the four control proteins (ACT-B, TUB-A, GAPDH, H3B) through immunoblotting (Fig. 5a, below). Through this, it was confirmed that the neuron differentiation by miR-124-BS (4753), which is similar to miR-124 but shows a different morphology, is slightly different in morphology, but induces differentiation in the same way as a gene expression marker. As expected, miR-124(3714), which has the same initiating sequence and is likely to suppress the regular initiating target in the same way, shows the characteristic of long differentiation of neuronal branches like miR-124 as expected. In addition, when both miR-124-BS (4753) and miR-124 (3714) were expressed, the amount of protein expressed in differentiated neurons increased. All of them have molecular characteristics of differentiated neurons. It could be confirmed that there is.

The expression of microRNAs in the examples so far was carried out by introducing the synthesized duplex into cells, but the expression of microRNAs is also possible by introducing a vector expressing the shRNA form, which is a hairpin-like RNA capable of generating it. . Therefore, a vector expressing miR-124, miR-124-BS (4753), and miR-124 (3714) in the form of shRNA was constructed using the pCAG-miR30 plasmid (addgene #14758), a vector expressing pre-miRNA. . The pCAG-miR30 vector used at this time uses a CAG promoter to strongly express microRNA, and is designed to express the backbone of microRNA-30, so it has the advantage of maximizing microRNA expression (Paddison P et al, Nature methods, 2004, 1(2): 163-7).

The pCAG vector made to express miR-124, miR-124-BS (4753), and miR-124 (3714), respectively, was introduced into N2a cells using a pCAG vector expressing nothing as a control. Was observed (Fig. 5b). The experimental group of the hairpin structure introduced into the neuroblastoma N2a showed a change similar to that of the cell morphology seen by miR-124, miR-124-BS (4753), and miR-124 (3714) duplexes. When cultured for 72 hours after transfection, neuroblastomas with long neurites were observed in the experimental group expressing miR-124 and miR-124 (3714). On the contrary, in the experimental group in which miR-124-BS(4753) was expressed, neurocytomas with neurites extending in short and various directions could be observed (FIG. 5B, above). This change in cell morphology is the expression of Tuj1 (O. von Bohlen et al. 　 2007 Cell and Tissue Research, 329, 3, 409-20), a protein expressed in differentiated neurons, but increased expression of Synaptophysin (SYP). It was confirmed (Fig. 5b, below), and thus miR-124, miR-124-BS (4753), and miR-124 (3714) hairpin vector-introduced cells have the characteristics of differentiated neurons compared to the control group. Could be confirmed. The increase of TUJ1 was observed by immunoblot experiment in the same manner as in the previous example, and Synaptophysin was primer described in the previous paper (SEKwon et al. 2011　 Neuron 70, 847-54) through qPCR (Quantitative Polymerase Chain Reaction) experiment. Proceed using the sequence. In this experiment, the vector was transfected using the Lipofectamine 3000 reagent (Invitrogen) in N2a cells according to the company's protocol, and after 24 hours, the total RNA was extracted with the RNeasy kit (Qiagen), and the protocol According to the method, reverse transcription was performed with Superscript III RT (Invitrogen), and qPCR was performed with SYBR® Green PCR Master Mix (Applied Biosystems) to measure the amount of messenger RNA of SYP with the corresponding primer, and normalized to the GAPDH messenger RNA value.

From the results of the above examples, even if a vector that allows the introduction of microRNA to be expressed in the form of shRNA is used, if only the sequence at the beginning of miR-124 is the same (miR-124(3714)), miR As with -124, it can be seen that normal long neurite differentiation is caused by suppressing the expression of the target gene. In addition, if the irregular raised target of miR-124 is also expressed in the form of shRNA in the same way as miR-124-BS (miR-124-BS (4753)), branches can cause more neurite differentiation. In this way, both normal target suppression and irregular elevation target suppression by miR-124 showed molecular gene marker expression in the form of neurite differentiation of neurons, and through this, it was confirmed that both were differentiated into neurons.

[ Example 6] miR -124-BS MAPRE1 Confirmation of induction of neurite differentiation of mouse neuroblastoma (N2a) through expression control of

Additionally, the induction of neurite outgrowth in neuroblastoma (N2a) of miR-124, miR-124-BS (4753), and miR-124 (3714) was observed in terms of cell morphology, and this was quantitatively analyzed (FIG. 6a ). In quantitative analysis, pUltra (addgene #24129) expressing a green fluorescent protein (GFP) 　 plasmid and the corresponding microRNA expression vector were used in the previous examples in order to distinguish neural branches derived from each neuron. In the same way as, the number of neurite branches was measured while observing the neurite with a fluorescence microscope (Leica) while performing the cell culture for 72 hours. The length was analyzed through the Image J program.

As a result, for 100 cells, the number of neurite branches expressed per cell was about 3 to 5 in miR-124, miR-124-BS (4753), and miR-124 (3714) compared to the control group. It was confirmed that the fold increased (Fig. 6A, lower left). However, when miR-124-BS (4753) was expressed, significantly more branches were observed than when miR-124 or miR-124 (3714) was expressed. In addition, if it could be confirmed that the length of neurites generated per cell increased 4-6 times in the miR-124-BS (4753) and miR-124 (3714) experimental groups compared to the control group, in more detail miR-124 or miR-124 When (3714) was expressed, it could be observed that longer neurite branches were formed than when miR-124-BS(4753) was expressed (Fig. 6a, lower right). This is the same result as observed in the cell morphology in the previous examples (Fig. 4, Fig. 5, Fig. 6).

In order to find out whether the specific neuronal differentiation caused by miR-124-BS is caused by the irregular elevation site of a certain gene, search for the irregular elevation target sequence of miR-124 in the sequence at the 3'UTR part of the gene related to neuronal differentiation. saw. As a result, MAPRE1 (Microtubule-associated protein RP/EB family member 1: Elena 　 　 　 et al. 2013 EMBO 　 　 　 32, 　 1293-1306) gene was found that was reported to regulate neuronal differentiation. MAPRE1 is a protein attached to the end of a microtuble that constitutes a neurite when it is generated, and can determine the shape of a neurite through regulation of the formation of the corresponding microtubule. Therefore, in order to confirm whether this MAPRE1 gene is recognized as an irregular raised target of miR-124 and its expression is inhibited, the method of carrying out the amount of messenger RNA of MAPRE1 in Example 5b after introducing miR-124-BS into N2a cells According to the qPCR experiment was performed and measured (Fig. 6b). When qPCR was performed with two different primers, both results showed that the miR-124-BS-introduced experimental group decreased MAPRE1 expression to a level of 70-40% compared to the control group. Through this, it was confirmed that the expression of MAPRE1 was inhibited as an irregular elevation site of miR-124.

If MAPRE1 is an irregular uplift target of miR-124 and is a very important target, the neurological differentiation pattern that appears due to the suppression of the irregular uplift target of miR-124-BS should be further promoted due to decreased expression of MAPRE-1. Therefore, in order to confirm this point, two siRNAs capable of inhibiting MAPRE1 were produced, and miR-124 was transfected into N2a cells together with this, and an experiment was performed to confirm whether a change in cell morphology was shown (FIG. 6c ). As a result, compared to the control group expressing only miR-124, it was observed that the number of neurite branches increased significantly in the experimental group expressing the siRNA for miR-124 and MAPRE1 together, which was achieved by using two different sequences of MAPRE1 siRNA. The same was observed in the experiment (Fig. 6c). In this experiment, if RNA was introduced into the cells in the same manner as in the previous examples, the morphology was observed while culturing the cells for 48 hours thereafter.

Through this, it was confirmed that miR-124-BS-induced short and branched neurite differentiation is a biological function that can be manifested by at least partially inhibiting the expression of the MAPRE1 gene through an irregular elevation target site of miR-124.

[ Example 7] Transcript At the level miR Upon introduction of -124-BS into cells miR Analysis of the inhibition tendency of the non-normal elevation target gene of -124

In mouse neuroblastoma (N2a), it was confirmed that the gene suppressed through miR-124-BS ultimately induces neuronal differentiation, which produces a large number of neuroblastoma neurites (FIG. 5). This phenomenon is thought to appear by a mechanism in which the initiating sequence of miR-124-BS recognizes and suppresses the irregular raised target of miR-124, and such a possibility was confirmed by MAPRE1 found as a target gene (FIG. 6). However, in reality, it may be caused by the inhibition of hundreds of miR-124 irregularly raised target genes.

The miR-124-BS invented by the present inventors introduced miR-124 and miR-124-BS into N2a cells in order to identify only the irregularly raised target genes of miR-124 at the level of the transcript where all genes are expressed. Later, RNA-Seq analysis was performed. As a control at this time, if the same nucleotide sequence as miR-124 but a duplex (miR-124-6pi) whose 5'end is a non-base (pi) was used, this modification is the 6th based on the 5'end of the miRNA. It has been reported that it does not align with the target messenger RNA at all because there is no base in ((Lee HS et. Al. 2015, Nat Commun. 6:10154).

In the RNA-Seq experiment, N2a cells were converted from the sequence of cel-miR-67, a microRNA of C.elegans, to a non-base 6 of the 5'end as an experimental control (NT-6pi). The miR-124, miR-124-BS, and miR-124-6pi duplex were delivered at a concentration of 75 nM, respectively, and after 24 hours incubation, the total RNA was extracted with an RNAeasy (Qiagen) kit, and a library was prepared in Otogenetics and the next generation Next-generation sequencing was performed. Thereafter, the FASTAQ file, which is the sequence data obtained in the above experiment, was mapped to the mouse genome sequence (mm9) with the TopHat2 program, and the expression value (FPKM) was obtained with the Cufflink and Cuffdiff programs, and a mouse neuroblastoma in which the control NT-6pi was introduced ( N2a) was normalized to the results in cells and expressed as a log ratio (fold change, log2 ratio) for analysis.

In order to analyze from the RNA-Seq results whether the amount of messenger RNA of the gene carrying the microRNA target site is inhibited by the expression of the corresponding microRNA, the normal starting site of miR-124 (from the 2nd to the 8th based on the 5'end) And 7mer) in the 3'UTR, and in order to select that the sequence of the site is conserved in several species, a gene with a phastCons score of 0.9 or higher was selected, and this profile result was inhibited dependently on the expression of the corresponding microRNA. It was compared and analyzed by cumulative fraction in the order of In addition, by the same method, irregular raised sites (nuc, 7mer) of miR-124 were also identified, and the rate of inhibition of the corresponding gene was analyzed by the same cumulative rate. At this time, since the gene with the non-normal bulging site of miR-124 has a regular starting site at the same time, it may be difficult to judge the inhibitory effect.Therefore, when the entire messenger RNA does not have the normal starting sequence of miR-124 (nuc only) and On the contrary, only regular starting sites and no irregular raised sites (seed only) were also analyzed.

In the RNA-Seq sequencing analysis of the miR-124-expressing experimental group, when the fold change according to the miR-124 expression was analyzed according to the cumulative fraction, the conserved regular starting site of miR-124 was 3' It was confirmed that the gene (miR-124 seed) in the UTR or the gene (miR-124 seed only) having only the site was inhibited very much compared to the distribution in the total gene (Total mRNA) (Fig. 7a, above). In contrast, it could be observed that the gene having the irregular raised site of miR-124 was significantly inhibited but very weakly inhibited (Fig. 7a, below). However, this inhibition was not observed in the cells introduced with the control miR-124-6pi (Fig. 7b).

In the case of expressing the miR-124-BS developed by the present inventor, when the fold change according to the expression of miR-124-BS was analyzed according to the cumulative fraction in RNA-Seq sequencing, miR- The gene (miR-124 seed) having the conserved normal initiation site of 124 in 3'UTR showed a slight inhibitory effect, but there was no irregular elevation site of miR-124, and the gene having only the normal initiation site (miR-124 seed only), it was confirmed that there was no change at all (Fig. 7c, above). However, in the case of a gene with an irregular elevation target of miR-124 (miR-124 nuc) or a gene with only the target (miR-124 nuc only), it can be seen that gene suppression occurs strongly and effectively. Was (Figure 7c, below). In Figure 7c, when miR-124-BS was expressed, the gene having only the normal initiating site of miR-124 (miR-124 seed only) did not inhibit the normal initiating site of miR-124 at all. Although it can be concluded that miR-124-BS somewhat inhibited the initiating site target of miR-124, perhaps, but in the presence of the regular site of miR-124, the irregular elevation site of miR-124 may be present together. It is presumed to exhibit any inhibitory effect.

From the results of the above examples, it was found that miRNA at the transcript level very strongly and efficiently inhibits the suppression of the existing normal initiating target gene, but very weakly inhibits the irregular raised target. It was confirmed that it suppressed the expression of the irregular raised target of miRNA at the cadaver level, but not the existing normal initiating sequence.

[ Example 8] miR -122-BS is human Liver cancer cell line ( HepG2 ) Of cell cycle arrest induced in Flow cytometry Confirmation through analysis

Based on the results of observing the induction of neuronal differentiation of miR-124 by the irregular nucleus target of miR-124, an experiment was conducted on the biological function of the irregular nucleus target of other microRNAs. miR-122 has 5'-UGG AGU GU-3' as the starting nucleotide sequence, and miR-122-BS, which is the nucleotide sequence of siRNA that nucleotides sequence with its irregular nucleus target site, repeats U once again In this case, the 5'-UGG AGU U GUG ACA AUG GUG -3' sequence is at the 5'end and consists of 19 bases, and the 20th and 21st bases are dt (Deoxy Thymine Nucleotide). It is done. In particular, in the duplex structure, the dt portion was composed of a duplex of a guide and a carrier strand so that two nucleotide overhangs were formed at the 3'end. At this time, the carrier strand performs a complete complementary base bond with the guide strand, and both the first and the second based on the 5'end were modified to 2'OMe, and two dt were included at the end of the base sequence (FIG. 8A). The guide strand and the carrier strand of miR-122-BS were chemically synthesized by Bioneer and separated by HPLC, and prepared as a duplex of the guide strand and the carrier strand according to the method provided by the company.

The prepared miR-122-BS (FIG. 8A) was introduced into a human liver cancer cell line (HepG2), and then the change in cell cycle was observed through flow cytometry. In this experiment, the duplex of the control group NT-6pi, miR-122, miR-122-BS was transferred to HepG2 at a concentration of 50 nM using RNAiMAX (Invitrogen) reagent, transferred to cells (transfection), and cultured for 24 hours. Nocodazole was treated at 100ng/ml for 16 hours to synchronize the cells to G2/M (dividing preparation/dividing stage), and then using Muse ^TM Cell Cycle Kit (Catalog No. MCH100106, Milipore). Thus, the cell cycle was analyzed with Muse Cell Analyzer (Milipore) according to the experimental method provided by the company.

As a result, compared to the cell cycle analysis of the control group to which NT-6pi was delivered, the miR-122-BS experimental group showed that the G0/G1 phase cells increased about 2 times from 10.7% to 24.3%. It was confirmed that the cell cycle arrest of HepG2 was increased (Fig. 8b-c). This G0/G1 phase cell increase was not observed in the cells to which miR-122 was delivered. Subsequently, the G2/M phase cell distribution of the human liver cancer cell line (HepG2) to which miR-122-BS was delivered decreased from 83.4% to 67.3% compared to the control, and these results were found in the cells to which miR-122 was delivered. Not observed (Fig. 8b,c).

Through this, miR-122-BS shows a biological function of inducing cell cycle arrest in human liver cancer cell lines through the regulation of the expression of the irregular nucleus target of miR-122, which is completely different from the biological function of miR-122. It could be observed that it is a function.

[ Example 9] miR- 1-BS is skeletal muscle cells ( C2C12 ) Induced muscle fibrosis function and miR- 155-BS confirmed apoptosis function

Since miRNA-BS has observed a new biological function different from the function that appears by inhibiting the expression of the normal initiating target of miRNA, miR-1 plays an important function while expressed in muscle cells to observe how these functions appear in muscle cells. , miRNA-BS was applied to miR-155.

First of all, miR-1 is a muscle tissue-specific microRNA and is reported to function in myocyte differentiation (Chen J et.al, Nature genetics, 2006, 38(2): 228-233), miR-1- BS is a skeletal muscle cell line After expression in C2C12, cell morphological changes (FIG. 9A) and the presence or absence of expression of gene markers expressed during differentiation (FIG. 9B) were investigated. At this time, miR-1-BS was synthesized by the same method used in the previous example and transfected into cells, and when cells were cultured for 48 hours, 10% FBS (fetal bovine serum), 100 U/ml penicillin, and 100 μg/ Using Dulbecco's modified Eagle's medium (Invitrogen) supplemented with ml steptomycin, it was in a growth condition (GM) state (Fig. 9A, above) and a deprivation media condition (DM) in which FBS was removed after cultivation. ) Was observed by dividing into a state that was further cultured for 48 hours (Fig. 9a, bottom). Afterwards, the cells were treated with 4% para-form aldehyde (PFA) to fix the cells and reacted with the myosin 2 heavy chain protein expressed in differentiated muscle cells with a primary antibody (MF20: Developmental studies hybridoma bank). Then, by staining with a mouse secondary antibody (ab150105) with Alexa 488 fluorescent substance, cell morphology and differentiation were observed (FIG. 9A).

As a result, mouse skeletal muscle cells (C2C12), when miR-1-BS was expressed under proliferation culture conditions (GM), a greater number of differentiated myocytes compared to the cells to which NT or miR-1 duplexes were transferred were transferred to MF20. It was confirmed by the staining result. In the deprivation media condition (DM), which reduces serum during culture, all of the differentiation into muscle fibers was observed, but miR-1-BS was more than that in skeletal muscle cells expressing NT, the control group. It was possible to observe the thick form (Fig. 9a). This may be the result of miR-1-BS inducing myocyte differentiation more quickly under proliferation culture conditions. In the differentiation of these muscle cells, the amount of expression of the gene that increases in expression during differentiation can be molecularly identified as a marker. Accordingly, the amount of sk-actin and Myogenin expression is determined by a primer specific for the messenger RNA. -PCR was performed to detect the amount of the messenger RNA of b-actin, which did not differ in the expression level even during differentiation (Fig. 9b). As a result, it was confirmed that the amount of transcription of sk-actin and Myogenin increased when both miR-1(1) and miR-1-BS(1BS) were expressed. In the case of Myogenin, the amount of miR-1-BS It could be observed that it increased more than miR-1

miR-155 is a microRNA that inhibits myocyte differentiation (Seok H et. Al, JBC, 286(41):35339-46　2011), and the effect of miR-155-BS was demonstrated in the same way as for miR-1 above. It was observed through differentiation of skeletal muscle cells (C2C12) (Fig. 9c). As a result, after expressing the control group NT, miR-155, miR-155-BS, no significant morphological difference was observed in the growth culture conditions (GM), but the deficient culture conditions that induce the differentiation of muscle cells ( In DM), the control group is differentiated, and when miR-155 is expressed, it is suppressed, but when miR-155-BS is introduced, the differentiation is eliminated by an immuno-staining experiment using myosin 2 heavy chain protein. It was confirmed through (Fig. 9c). In particular, when looking closely at C2C12 cells expressing miR-155-BS, it was observed that miR-155-BS induced cell death. In contrast, no cell death was observed in the myocytes to which miR-155 was delivered (FIG. 9C, bottom). In order to investigate this in more detail, C2C12 cells expressing the corresponding microRNA were grown in proliferation culture conditions for 48 hours, and then cell death was examined by applying the same flow cytometric analysis as in Example 3 (FIG. 9D). As a result, it was confirmed that the experimental group to which miR-155-BS was delivered increased apoptosis by about 1.5 to 2.5 times or more compared to the control group (FIG. 9d, e).

Through the above examples, miR-1-BS promotes the differentiation of skeletal muscle cells to induce the differentiation of thick muscle fibers, and miR-155-BS induces the death of skeletal muscle cells. It was confirmed that the function of miR-1 to contract cells and miR-155 to inhibit muscle cell differentiation were different.

[ Example 10] In Ago HITS CLIP analysis Wobble (wobble) base sequence MicroRNA Irregular target allowed by the initiation value Site discovery

As a method for analyzing the target of microRNA at the transcript level, an experimental method called Ago HITS CLIP has been developed and is widely used (Nature, 2009, 460 (7254): 479-86). In the Ago HITS CLIP experiment, a cell or tissue sample is irradiated with UV light to induce a covalent bond between the RNA and the argonet protein in the cell, and the resulting RNA-agonal complex uses an antibody that specifically recognizes the agonut. This is a method of sequencing the separated RNA by immunoprecipitation and then analyzing the separated RNA by next-generation sequencing. The obtained nucleotide sequence data can not only identify the target mRNA of the microRNA through bioinformatics analysis, but also accurately analyze its binding position and sequence (Nature, 2009, 460 (7254): 479-86). . Ago HITS-CLIP was first applied to the cerebral cortical tissue of mice to map the targets of microRNAs in the brain tissue, and the method has been applied to various tissues until now. Through these various Ago HITS-CLIP analyzes, the microRNA binding sites in various tissues were revealed. In particular, although these target sequences are similar to the initiating binding of microRNAs, other forms of non-canonical binding targets in the target mRNA sequence It was found that many exist (Nat Struct Mol Biol. 2012 Feb 12;19(3):321-7).

It was observed that some of the revealed irregular binding laws exist as wobbles through the G:U base sequence in addition to the known base sequence. Unlike conventional nucleotide sequences, wobble nucleotide sequences are found in specific RNAs, and it has been suggested that well-known G:U wobbles can be arranged in irregular targets of microRNAs. However, compared to this, the binding was weak and was not frequently observed. In particular, G:A wobbles can form a nucleotide sequence in a specific RNA, but other than that, it has not been investigated at all. However, in the case of the starting sequence of microRNA, since the free energy of the nucleotide sequence is structurally stabilized by the argonet protein, in general, the weak G:A nucleotide sequence may be relatively important. Paying attention to, the following analysis was conducted.

First, in order to confirm whether the wobble nucleotide sequence including G:A is present in the microRNA target in human brain tissue, data obtained by performing Ago HITS-CLIP on gray matter of brain tissue after death (Boudreau RL et al, Neuron, Vol. 81 (2), 2014, 294-305) was analyzed (Fig. 10A). The analysis at this time was carried out according to the method of developing Ago HITS-CLIP (Nature, 2009, 460 (7254): 479-86), and the RNA sequence that was bound together with Argonaut-microRNA was used in the sequenced FASTQ file with the Bowtie2 program. It was carried out by arranging the human genome sequence, and at the same time, the sequencing result was arranged on the human microRNA sequence, and 20 kinds of microRNAs (top20 miRNA: FIG. 10b) frequently bound to the agonut protein in the brain tissue were also analyzed. Finally, for the above-identified top20 miRNA, a canonical target site having a sequence complementary to the 2nd to 8th from the 5'end, which is the seed sequence, was examined (Fig. 10b). At this time, it was observed that a fairly large number of normal initiating target sites were found at the microRNA binding site (Fig. 3b, median value 1292.5 sites, error range +/- 706.62). After that, it was examined in the Ago HITS-CLIP data whether there is an irregular target site capable of binding to the G:U or G:A wobble nucleotide sequence in the starting nucleotide sequence of this microRNA. At this time, as a control for the analysis, a target sequence in which complementary alignment of bases was impossible due to the same base sequence as the microRNA seed base sequence was used for analysis.

As a result of the analysis, both G:A wobble-allowed targets (median 1119.5 sites, error range+/- 526.48) and G:U wobble-allowed targets (median 995 sites, error range+/- 523.22) sites were both control (median 891) Site, error range +/- 410.71) showed a higher distribution, and in particular, microRNA target sites with G:A wobbles showed that there were about 1.1 times more than G:U wobbles (Fig. 10b). MiR-124, which is specifically expressed in brain tissue, has two Gs at the 4th and 5th from the 5'end at the beginning, so it can perform G:U, G:A wobble nucleotide sequence (Fig. 10c). Therefore, as a result of distinguishing and analyzing the G:A, G:U wobble nucleotide sequence by G at this specific position (FIG. 10D), the most common target sites for the normal initiation of miR-124 (seed) were found (1,992). However, it was found that the fourth made up G:U wobbles (1,542) to a degree comparable to this, and then observed that it made up G:A wobbles (1,542). In addition, there were many target sites consisting of G:U wobbles (1,800) and G:A wobbles (1,198) in G of No. 5??, and all sites examined in this way exist more than the number of control groups (647). It could be confirmed that (Fig. 10d). Therefore, when microRNA recognizes the target mRNA through the nucleotide sequence of the starting part, as well as the regular nucleotide sequence through the starting part, the microRNA allows irregular G:U or G:A wobble nucleotide sequence, especially G:A It was found that the wobble sequence can bind to the target mRNA through this binding. The target recognition of the microRNA through the G:A wobble base is a new result that has not been reported, and thus it was confirmed that it was recognized as an irregular target in several microRNAs as observed in the above examples.

Through the above examples, there are cases in which the G:A wobble arrangement is allowed in the starting sequence for the recognition of irregular targets of microRNAs, through which many microRNAs bind to the messenger RNA of the target gene and suppress the expression of the gene. It is believed to do.

[ Example 11] MicroRNA Irregular target G:A Starting arrangement Site Recognized mIRNA -GU development and miR When -124 is applied miR -124- G5U Confirmation of the effect of increasing neuronal cell death

In Example 10, it was found that there are cases in which the G:A wobble arrangement is allowed in the starting sequence for the recognition of an irregular target of microRNA, and this is called an irregular G:A starting sequence site, and can be arranged complementarily. The sequence of a new RNA interference inducing substance, miRNA-GU, was invented as follows. When this sequencing technique is applied to miR-124, miR-124 has an initiating sequence of 5'-AAGGCAC-3', and the target for initiating an irregular GA sequence is G:A base 4 and base G 5, respectively. It is a nucleotide sequence capable of wobble nucleotide sequence, and therefore, if this G:A wobble sequence is replaced with the sequence of miRNA-GU, which is a format that converts the existing complementary nucleotide sequence, miR-124-G4U (5'-AAUGCAC-3'), It can be changed to miR-124-G5U (5'-AAGUCAC-3'). The modified miR-124-G4U and miR-124-G5U can bind to the irregular target site that the existing miR-124 weakly binds in the G:A wobble configuration and can bind to the recognized irregular target site in a complementary configuration. The function of the non-canonical G:A initiating arrangement target can be indicated.

Therefore, in order to investigate the biological function mediated by the miRNA irregular GA sequence initiating target, an experiment was performed by applying the non-normal GA sequence initiating target to miR-124. To this end, NT (described as N2a in Fig. 11B), miR-124, miR-124-G4U, and miR-124-G5U were introduced into the neuroblastoma N2a as controls according to the same method as in the previous example, and the shape of the cell was It was observed while incubating for 72 hours (FIG. 11B). As a result, when miR-124 was expressed, a phenomenon of differentiation occurs in which long nerve branch processes are formed, but in the case of the experimental group to which miR-124-G4U and miR-124-G5U were delivered, no differentiation occurred (Fig. 11b). In this way, the existing function of miR-124 was not observed when the function of miRNA-GU was changed to miRNA-GU, so a change in the degree of apoptosis was observed in the human neuroblastoma Sh-sy-5y cell line. Similar to the method used in Example 9, the flow cytometry at this time was performed using the Muse Annexin V and Dead Cell Assay Kit (millipore), and performed through the Muse Cell Analyzer (Milipore). As a result, it was confirmed that the expression of miR-124-G-5U increased apoptosis by more than 2 times compared to the control group (Fig. 11c), and this was subdivided into early apoptosis or late apoptosis. In the case of analysis, compared to the experimental group to which miR-124 was delivered, the experimental group to which miR-124-G-5U was delivered significantly increased in both cases of early apoptosis and late apoptosis. Became (Fig. 11D). In the case of the experimental group to which miR-124-G-4U was delivered, late apoptosis was significantly suppressed compared to the experimental group to which miR-124 was delivered (Fig. 11 c and d). Could be (Fig. 11 b).

Based on this, it was confirmed that inhibition of miR-124 irregular GA sequence initiation target by miR-124-GU disappears the ability of neuron differentiation induced by miR-124, and instead represents another biological function, apoptosis regulation function. .

[ Example 12] miR -124- G4U Promotes cell division of neuroblasts induced by

In the previous example, in the case of miR-124-G4U, nerve cell differentiation ability was lost, and no other function appeared even in apoptosis, so cell division was examined. That is, after introducing control NT, miR-124, miR-124-G4U, and miR-124-G5U into neuroblastoma cells N2a in the same manner as in the previous example, flow cytometric analysis for cell division and proliferation Was carried out (Fig. 12A). In this experiment, cells were treated according to the experimental method provided by the manufacturer using the Muse Ki67 Proliferation Kit (millipore 　 company) 　, and then the cell division was analyzed with Muse 　 Cell Analyzer (Milipore). This method quantitatively analyzes the number of cells with increased cell division proliferation by measuring the number of cells stained with Ki67. When miR-124 is expressed in N2a cells, cells are differentiated compared to the control, NT, and the number of cells with reduced Ki67 staining intensity increases. However, in the experimental group delivered with miR-124-G4U, the number of Ki67-stained cells, which are proliferated cells, slightly increased from 61% to 65% compared to the control group (NT). In contrast, it could be observed that there was no difference in miR-14-G5U compared to the control group.

In addition, in order to investigate the effect of miR-124-G4U on the cell cycle, flow cytometry was performed using the human neuroblastoma sh-sy-5y cell line (FIG. 12B). The analysis at this time was analyzed by Muse Cell Analyzer (Milipore) using Muse ^TM Cell Cycle Kit (Catalog No. MCH100106, Milipore) to find out the functions of miR-124-G4U and miR-124-G5U by cell cycle. . As a result, in the case of miR-124, cell cycle arrest was observed with an increase in the number of cell cycles in G0/G1, but miR-124-G4U and miR-124-G5U were cells induced by miR-124. It was observed that the cessation of division disappeared and the cell cycle returned as in the control group.

Through the above examples, it was found that miR-124-G4U among the RNA interference-inducing modified sequences that inhibit the miR-124 irregular GA sequence initiation target has a function of increasing cell division and proliferation of neuroblasts.

[ Example 13] Myocardial cell line in miR -1 starts an irregular GA arrangement target That we can suppress the expression of genes Fluorescent protein Confirmation with Reporter

In the above example, after discovering that the microRNA can irregularly allow G:A wobble arrangement in the initiating sequence to bind to the target mRNA, and this may have a new function, this binding actually A reporter experiment was conducted to confirm at the cellular level whether it could cause inhibition. At this time, the confirmation experiment was conducted on miR-1, which is known to be highly expressed in muscle cells, such as h9c2, a cardiomyocyte.In particular, paying attention to the G present at the 7th from the 5'end of miR-1, through which G: A wobble nucleotide sequence was performed as a target (FIG. 13). In order to measure gene suppression by microRNA more precisely at the level of individual cells, a fluorescent protein expression reporter system was constructed and used (FIG. 13). The fluorescent protein expression reporter system was constructed so that two-colored fluorescent proteins were expressed in one vector. At this time, a green fluorescent protein (GFP) was expressed in the SV40 promoter, and the 3'UTR (3'untranslated region). Several target sites of microRNA were sequentially arranged in the part, so that the effect of gene suppression by microRNA could be measured through the intensity of the green fluorescent protein, and at the same time, the red fluorescent protein (RFP) is the TK promoter. The green fluorescent signal was normalized to the level of the intensity by allowing it to be expressed by (Fig. 13A, above).

The fluorescent protein expression reporter vector constructed in this way is complementary to the bases from the 2nd to the 8th from the 5'end of miR-1, and G is G for the 7th base; A non-normal target site (7G :A-site) was inserted to prepare a reporter (GFP-7G:A site), and it was confirmed through an experiment whether this was inhibited by miR-1. At this time, in the experiment, 500ng GFP-7G:A site reporter vector and 25uM of miR-1 were simultaneously introduced (co-transfected) into the cardiomyocyte line H9c2 using a lipofectamine 2000 (Invitrogen) reagent according to the protocol provided by the company. After 24 hours, each fluorescence signal was measured by flow cytometry through Life Technologu's Attune NxT. As a result, the 7G:A site of miR-1 was very efficiently suppressed (Fig. 13A, center) by the expression of miR-1 compared with the cells introduced with a control nucleic acid (cont; NT) (Fig. 13A, left). Confirmed. That is, as a result of analysis in four parts (Q5-1, Q5-2, Q5-3, Q5-4) according to the expression level of the two fluorescent proteins in the cardiomyocyte line h9c2, in the case of cells into which miR-1 was introduced, the control nucleic acid the decrease in the case where the introduction of more than ³ of the 10 century, red fluorescent protein and 10 ^3rd century the cellular distribution of the visible green fluorescent protein 59.51% (control:: Q5-3) 41.80% (Q5-3 miR-1) from Observed.

It was observed in the control group that the GFP-7G:A site reporter vector was somewhat inhibited in h9c2 cells even without the introduction of miR-1. This can be expected to be inhibited by miR-1 already expressed in h9c2 cells through G;A wobble nucleotide sequence with reporter target mRNA. To confirm this, a miRIDIAN microRNA Hairpin inhibitor (miR-1 inhibitor, Fig. 13a, right), which can inhibit the expression of miR-1 in h9c2 cells, was purchased from Dharmacon and transfected into cells at a concentration of 50 uM. I did. These results, the distribution of the visible (Fig. 13a, left side of the figure) miR-1 inhibitor one case (Fig. 13a, right figure), ten ^3rd century red fluorescent protein and green fluorescent protein of 10 ³ intensity of introducing than in control cells 81.56% (miR-1 inhibitor: Q5-3), it was confirmed that miR-1 present in the cell can suppress gene expression through the 7G:A site of miR-1 (Fig. 13a) .

In addition, a control (GFP-no site) in which the microRNA target site was not added to the fluorescent protein expression reporter and the GFP-7G:A site, a reporter containing the 7G:A site of miR-1, were introduced into H9c2 cells to compare their activity. It was confirmed, and as a positive control, it was confirmed by quantitatively comparing with the case of introducing miR-1 together (Fig. 13b). In this analysis, the red fluorescent protein (RFP) value measured by the flow cytometer was divided by section, and the green fluorescent protein (GFP) value at this time was averaged and converted into a log ratio. At this time, the green fluorescence of the control GFP-no site The protein level was set to 1 (relative log GFP) and was calculated relatively. At this time, for mR-1 and 7 G bases, when there is a site that is complementarily nucleotide sequenced through G:A wobble (GFP-7G:A site), in particular, a red fluorescent protein with a value of 1.5-3 In the section where the expression of (log RFP) is visible, it was confirmed that the expression of green fluorescent protein (relative log GFP) through G:A wobble was lowered by about 10% than 1. In the positive control, it was observed that the expression of the green fluorescent protein (relative log GFP) was inhibited by 40%-10% in the expression of the red fluorescent protein (log RFP) in the 1.5-4 section, especially in the positive control. .

In order to confirm that the results observed in the above examples are not affected by the difference in the intensity of the different promoters used in the corresponding fluorescent protein reporter and the fluorescence activity of the two different fluorescent proteins, the two fluorescent proteins were interchanged. A fluorescent protein reporter p.UTA.3.0 (Lemus-Diaz N et al, Scientific Reports, (7), 2017) was purchased and used from Addgene (plasmid # 82447). At this time, in the reporter experiment, miR-1-7G; A sequence was repeatedly inserted into the 3'UTR part of the red fluorescent protein (RFP) regulated by the SV40 promoter in the p.UTA.3.0 vector, and then the reporter A vector (RFP-7G:A site) was prepared and proceeded, in which case the expression level of the red fluorescent protein was used as a standardization reflecting the degree of introduction into the cell by using the value of the green fluorescent protein (GFP) expressed by the Tk promoter. It was modified and used (Fig. 13c, above). As a result, as in the previous example, 7G of miR-1; 7G of miR-1 and a control group of a reporter (RFP-no site) without a miRNA target site showed that the A site was inhibited by miR-1 expressed in h9c2; The results of the reporter containing the A site (RFP-7G:A site) were compared and confirmed, and at the same time, the same phenomenon as in the previous example was observed in co-transfection with miR-1 as a positive control group (Fig. 4c-d).

From the above, it was found that the non-normal target of the microRNA discovered through the Ago HITS-CLIP analysis could not only bind through the G:A wobble arrangement in the starting sequence of the microRNA, but also suppress the expression of the target gene. Confirmed. Therefore, from this point of view, it is possible to sufficiently bind to microRNA using a target site through the G;A wobble array, and if this point can be vigorously induced through miRNA-GU, it is possible to suppress irregular targets. It is thought that only the biological function of microRNA can be utilized.

[ Example 14] miR - 1 of 2, 3, Number 7 Initiation of irregular GA sequence for G base target Through gene regulation Cardiomyocyte Confirmation of hypertrophy induction function

In order to confirm the possibility through miRNA-GU that the G:A wobble nucleotide sequence may act as an atypical target of microRNAs and regulate biological functions, such an irregular gene suppression phenomenon, miR-1 of cardiomyocytes as described in the previous example. The experiment was conducted centering on.

In order to investigate whether the non-canonical G:A wobble target has a specific biological function, miR-1 must not recognize the regular target, but only recognize the irregular G:A wobble sequence target site. MiRNA-GU was used in the experiment by substituting U for G so that G in the starting region nucleotide sequenced with A instead of C. In this experiment, primary rat neonatal cardiomyocyte cultures were cultured from cardiac tissues of rats in order to perform more physiologically similar to the heart. At this time, in the culture of cardiomyocytes, cardiac myocardial cells were isolated from the heart tissues of rats on day 1 after birth through an enzymatic reaction (Cellutron's neomyt kit), and myocardial cells were 10% FBS (fetal bovine serum), 5% HS. (horse serum), 100 U/ml penicillin, and Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 100 µg/ml steptomycin and M199 (WellGene) medium were mixed at 4:1, and brdU was added to the prepared medium. It was cultured to differentiate it from other cells having a cell cycle present in the tissue.

It was first checked whether the myocardial hypertrophy is induced in the primary cultured cardiomyocytes thus obtained (Fig. 14a). Cardiomyocytes have the characteristics of stopping the cell cycle and increasing the size of myocardial cells, which is called myocardial hypertrophy. Myocardial hypertrophy has played a role in compensating for the deterioration of cardiomyocyte function, is also known as an intermediate pathology of heart disease, and is a heart disease model system well known for cell morphological observation and gene expression profile changes. In a cell model of myocardial hyperplasia, treatment with an adrenal receptor agonist such as phenylephrine (PE) on myocardial cells can induce myocardial hypertrophy (Molkentin, JD et al, 1998, Cell 93(2), 215-228). Therefore, the cultured cardiomyocytes were treated with 100 uM of phenylephrine to induce myocardial hypertrophy, and accordingly, it was confirmed morphologically that the size of the myocardial cells increased compared to the control group (Fig. 6a, rCMC) that was not treated with anything. (Fig. 14A, +PE). In addition, when miR-1 was introduced into the primary cardiomyocytes, it was observed that the size of the cardiomyocytes decreased (FIG. 14A, upper row, right). This is consistent with the well-known myocardial hypertrophy and miR-1 function in cardiomyocytes (Molkentin, JD et al, 1998, Cell 93(2), 215-228, Ikeda S et al, Mol cell boil, 2009, vol 29 , 2193-2204).

miR-1 contains three Gs in the starting region, and thus miR-1 substituted with U in order to recognize only the irregular G:A wobble sequence target site without recognizing the regular target. Was designed, and according to the position of the G, the 2nd position (miR-1-G2U), 3rd position (miR-1-G3U), 7th?? It was synthesized by applying to position (miR-1-G7U). At this time, the substitution nucleotide sequence of miR-1 used in FIG. 14 is as follows (miR-1-G2U: 5'p-UUGAAUGUAAAGAAGUAUGUAU-3'; miR-1-G3U: 5'p-UGUAAUGUAAAGAAGUAUGUAU-3'; miR -1-G7U: 5'p-UGGAAUUUAAAGAAGUAUGUAU-3') This was synthesized as a guide strand, and the carrier strand was designed for the existing miR-1, chemically synthesized by Bioneer, separated by HPLC, and provided by the company. According to one method, a guide strand and a transport strand were prepared and used as a duplex. The transfection of the microRNA into the primary cardiomyocyte line was performed at a concentration of 50uM using RNAimax (Invitrogen) reagent.

As a result, when miR-1-G2U or miR-1-G3U or miR-1-G7U was introduced into the primary cardiomyocyte culture, the shape of the cardiomyocyte was similar to that of the cells inducing myocardial hypertrophy (Fig. 14A, +PE). It could be observed that it became larger (Fig. 14a). At this time, in order to quantitatively analyze the size of each cell, more than 100 cells were quantified through the Image J program (NIH), and accordingly, the miR-1 substituent synthesized to recognize only the G:A wobble sequence site of miR-1 ( Both miR-1-G2U, miR-1-G3U, and miR-1-G7U) were analyzed to have increased in size by about 1.5 to 1.8 times compared to when the control (NT) nucleic acid was introduced. This is similar to the size of the cardiomyocytes in the model of cardiomyocyte hypertrophy induced by the phenylephrine (PE) drug, and miR-1 itself has an effect of reducing the cell size in the form of cardiomyocytes, so its regular target inhibitory effect is achieved. It was observed that it exhibits a different function in cell morphology than target inhibition through G;A wobble (FIG. 14B). In addition, in order to further confirm the myocardial hypertrophy observed in this example at a molecular level, the expression of the ANP (atrial naturiuretic peptide) gene, which is known to increase its expression as a marker, was evaluated by qPCR (Quantitative Polymerase Chain Reaction) experiment. Compared to and measured as a relative value (Fig. 14c). As a result, when the synthesized miR-1 substitutions (miR-1-G2U, miR-1-G3U, miR-1-G7U) were introduced into cardiomyocytes to recognize only the G:A wobble sequence site of miR-1 It was confirmed through four repeated experiments that the ANP expression level was significantly increased by 1.2 to 1.5 times compared to the control group (NT). This increase in ANP expression is less than that of the cardiomyocyte test group treated with the phenylephrine (PE) drug, which increased in size by about two times compared to the control group, but when the original miR-1 was introduced, ANP expression was significantly decreased. Contrary to this phenomenon, it can be seen that miR-1 exhibits completely different biological functions through the G:A wobble arrangement site.

From the above, it was finally seen that the non-canonical target inhibited through the G:A wobble arrangement in the microRNA initiation region may exhibit a biologically different function from the conventional normal target recognition. These findings indicate that microRNA functions through regular targets and non-canonical targets are different from each other, and from this point, if only the G:A wobble target of microRNA can be suppressed, the G:A wobble target Only the biological functions caused by it could be selectively controlled.

[ Example 15] myocardial cell line ( H9c2 )in miR -133- G4U Through expression Hypertrophic Judo observation

For the irregular target phenomenon recognized by the G:A wobble nucleotide sequence acting on the starting part of the microRNA, in order to additionally confirm its biological function in cardiomyocytes, it functions in cardiomyocytes other than the miR-1 discussed in the previous example. MiRNA-GU was applied to known miR-133. miR-133 is known to regulate myocyte development and disease pathology, and to suppress myocardial hypertrophy (Nat Med. 2007, 13(5): 613-618; Ikeda S et al, Mol cell boil, 2009, vol29, 2193-2204). Therefore, the present inventors aimed at miR-133-G4U (5'-UUUUGUCCCCUUCAACCAGCUG-3', an interference-inducing nucleic acid that specifically inhibits target recognition through the 4th G based on the 5'end among the target sites for the non-normal GA sequence of miR-133). ) Was synthesized and produced by Bioneer as in the above example, and transferred to the myocardial cell line h9c2 using an RNAiMAX reagent (Invitrogen) as a duplex having a concentration of 50 nM, and the change in cell morphology was observed (FIG. 15A). As a result, the expression of miR-133-G4U increased the size of H9c2 cells compared to the control (NT) (FIG. 15A), and the size of 100 or more cells was quantitatively analyzed through the Image J (NIH) program. , It was confirmed that the increase was significantly increased by about 2 times (FIG. 15B).

Therefore, it is inferred that miR-133's deterioration in the expression of the non-regular GA sequence initiation target caused by miR-133-G4U induces cardiac hypertrophy, which is a new function different from the normal initiation target inhibition function of miR-133, which inhibits cardiac hypertrophy. can do.

[ Example 16] liver cancer Intracellular miR Initiation of irregular GA arrangement by -122 Site The effect of inhibiting gene expression Luciferase Confirmed through reporter

In the discovery that miRNA in the previous Example 10 irregularly binds to the G:A wobble sequence initiating site, miRNA-GU that specifically recognizes only such irregular target binding was invented, and miR-124, miR- 1, When applied to miR-133, it was observed in Examples 11, 12, 14, and 14 that an effect different from the existing function appeared. In addition, in order to confirm whether microRNA can actually inhibit the expression of a target gene having an irregular G:A wobble sequence initiating site, its function was confirmed in a myocardial tissue cell line targeting miR-1 in Example 13. I did. In addition, it was attempted to confirm using the luciferase reporter system whether it can inhibit the expression of the non-normal G:A wobble sequence initiating target gene targeting miR-122, which is specifically expressed in liver tissues and liver cancer cells (FIG. 16).

MiR-122, which functions by expressing and functioning specifically in liver cancer or liver tissue, is 2nd and 3rd based on the 5'end in its initiating sequence (base 1-8 of the 5'start: 5'- UGG AGU GU-3'). It has a G that can do a G:A wobble arrangement at the 1st, 5th and 7th. Therefore, first of all, in order to measure the inhibitory efficiency for the target site where the second G is irregularly recognized through the G:A wobble arrangement, IC ₅₀ (Inhibitory concentration 50) was measured and compared with the regular starting site (Fig. 16a).

The luciferase reporter vector for conducting the experiment was to detect the suppression of the expression of the non-normal GA sequence initiating target capable of GA base sequence for the 2 G base of miR-122. GA sequence initiating site (2G:A) sequence (base 1-9 of 5'start: 5'-CAC ACU CAA-3') is 3'of Renilla luciferase gene in psi-check2 (Promega) vector. It was fabricated by introducing it to be sequentially arranged 5 times in the UTR (3'untranslated region) part. At the same time, a luciferase reporter vector for the regular initiation site (base 1-9 based on 5'start: 5'-CAC ACU CCA-3') was also constructed in the same way.

With the luciferase reporter vector constructed in this way, the target gene having a normal starting site (seed site) and an irregular GA sequence starting site (2G:A site) through the second G based on the 5'end at various concentrations of miR-122 The expression inhibition effect was measured by the change in the activity of the corresponding luciferase reporter, and as a result of examining the IC ₅₀ (inhibitory concentration 50) value, miR-122 was about 0.5 nM for the normal initiation target site, and the non-normal GA sequence initiation site (2G: A site) was measured to be about 7 nM, and it was confirmed that miR-122 inhibits the expression of a gene having an irregular GA sequence initiating site (2G:A site), and its efficiency was low compared to the normal initiating site. There is (Fig. 16a). In addition, when the concentration at which gene expression inhibition begins was examined by the luciferase reporter in the non-regular GA sequence initiating target, it was confirmed that the luciferase enzyme activity was inhibited under the concentration condition of 1.5 nM or more (FIG. 16A). This showed the difference between miR-122 inhibiting the expression of the normal initiating target site and about twice the inhibition efficiency.

In this experiment, the duplex of miR-122 was synthesized by the same method used in the above example, and it was Lipofectamine 2000 reagent with the corresponding psi-check2 vector (50 ng) at various concentrations (0, 1, 5, 10, 25 nM). (Invitrogen) was delivered to approximately 10,000 liver cancer cell lines (Huh7, KCLB: 60104) 　 according to the manufacturer's protocol, and cultured in 96 wells, and lucifer was performed in the same manner as in Example 2. It was carried out by measuring the enzyme activity.

After confirming that miR-122 inhibits the expression of the irregular GA sequence initiating site gene, to confirm whether it is equally regulated by miR-122 present in the cells, the Huh7 cell line, a liver cancer cell in which miR-122 exists. Using, a luciferase reporter experiment was performed for an irregular GA sequence initiating site (2G:A) caused by the 2nd G and the 3rd G based on the 5'end (FIG. 16B). In order to proceed with this experiment, additionally, a luciferase reporter vector (luc-3G:A site) for an irregular GA sequence initiating site (3G:A) caused by the 3rd G at the 5'end, the 2nd and 3rd at the 5'end. The luciferase reporter vector for the non-canonical GA sequence initiating site (luc-2G:A, 3G:A site) caused by the G-th, a luciferase reporter vector for measuring a sequence completely complementary to the sequence of the entire miR-122 (luc -PM site) was prepared in the same manner as described above, and a luciferase reporter vector (luc-2G:A site) for the irregular GA sequence initiating site (2G:A) caused by the 2nd G based on the 5'end Together, the experiment was performed by measuring the activity of luciferase after introduction into a liver cancer cell line (Fig. 16b).

As a result, it was observed that when the luc-PM site vector was introduced into Huh7, which is known to exist with miR-122, its activity decreased by about 50% compared to the control in which only the luciferase vector itself was introduced. It was confirmed that 122 was present, and at the same time, miR-122 was the 3rd irregular GA sequence triggering target reporter (Luc-3G:A), the 2nd and 3rd irregular GA sequence triggering target reporter (Luc-2G:A, 3G:A). It was confirmed that inhibiting the activity of. In addition, in order to know whether this inhibitory effect was induced by miR-122, an expression inhibitor of miR-122 (hsa-miR-122-5p inhibitor, IH-300591-06-0010) was purchased from Damacon and treated with 50 nM. In this case, it was confirmed that all these inhibition phenomena disappeared.

Through the above examples, it was confirmed that miR-122, although weaker than the regular initiating site, can inhibit the expression of the 2 non-normal GA sequence initiating target based on the 5'end. It was found that the present miR-122 inhibited the expression of the target gene for initiating the 3rd irregular GA sequence and the target gene for the 2nd and 3rd non-normal GA sequence based on the 5'end. In particular, it was confirmed that the inhibitory effect made in Huh7, a liver cancer cell, disappeared by treatment with an expression inhibitor of miR-122, which was regulated by miR-122 (FIG. 16B). Therefore, it can be seen that miR-122 has a regulatory function to suppress the expression of the target gene for initiating the irregular GA sequence.

[ Example 17] miR -122 specific irregular GA array Through suppression of the expression of the initiating target gene Liver cancer cell line Confirmation of cell migration inhibition

In order to find out the function of the non-normal GA sequence initiating target of miR-122 in liver cancer cells, miRNA-GU, which complementarily recognizes and inhibits the non-normal GA sequence initiating target site, was applied and introduced into hepG2, a liver cancer cell, Among the properties of liver cancer cells, a wound healing assay was performed to investigate how the cell migration ability, which is important for cancer metastasis, changes.

In the analysis of wound healing in hepG2, a liver cancer cell line, first, based on the 5'end of miR-122, the 2nd irregular GA sequence initiation target, the 3rd non-normal GA sequence initiation target, and the 2nd and 3rd irregular GA sequence initiation targets were recognized complementarily. After synthesizing the miRNA-GU with the corresponding sequence, it was introduced into hepG2 cells in the same manner as in the above example, and after culturing for 24 hours, a scratch was made on the cell layer using a 1000ul tip. And, the cells were cultured for up to 48 hours, and the migration of the cells of each experimental group was observed and compared with the introduction of the control group NT-6pi. The sequence of the interference-inducing nucleic acid used in the experiment is as follows (miR-122: 5'-UG GAG UGU GAC AAU GGU GUU UG-3'; miR-122-G2U: 5'-UU GAG UGU GAC AAU GGU GUU UG-3'; miR-122-G3U: 5'-UG UAG UGU GAC AAU GGU GUU UG-3'; miR-122-G2,3U: 5'-UU UAG UGU GAC AAU GGU GUU UG-3').

As a result, when miR-122 was introduced as in the case of NT-6pi as a control group, cell migration was observed that almost completely filled the defects made when the cells were cultured for 48 hours. However, in the experimental group to which miR-122-G2U, miR-122-G2, 3U siRNA was delivered, this cell migration was inhibited, and this inhibition was most strongly in the experimental group to which miR-122-G2,3U siRNA was delivered. (Fig. 17a). As a result of quantitatively measuring this, it was confirmed that the miR-122-G2U, miR-122-G2, 3U siRNA inhibited cell migration 2-3 times compared to the control group (FIG. 17 b ). For the quantitative analysis, the cell migration of miR-122 was observed in the form of a cell, and then the observation result was analyzed and measured using the Image J program (NIH). In addition, as a result of conducting an experiment on the cell migration induced by miR-122-G3U siRNA, it was not possible to observe the function of inhibiting cell migration by miR-122-G3U (Fig. 17c-d).

Based on this, inhibition of miR-122's non-regular GA sequence initiating target inhibits the migration of liver cancer cells, which preferentially acts on the GA sequence of base 2, followed by the addition of the GA sequence of base 3, cells It can be seen that the enhancement of the migration inhibitory function is induced.

[ Example 18] miR Irregular by -122-G2,3U GA array Confirmation of increased cell cycle arrest due to initiating target regulation

Cell migration is closely related to cell division, and is particularly observed in cancer cells (Cancer research, 2004, 64(22):8420-8427). Therefore, flow cytometry experiments were performed in the same manner as in Example 12 to confirm whether miR-122-G2,3U-induced inhibition of liver cancer cell migration is related to cell cycle regulation. At this time, NT-6pi, miR-122, miR-122-G2, miR-122-3U, miR-122-G2, 3U were delivered to HepG2, a liver cancer cell line, and the cell cycle of each cell was measured. As a result, miR-122- In the G2,3U-delivered experimental group, it was confirmed that the proportion of cells showing a G0/G1 distribution increased from 52% on average to 68% of the control (NP-6pi), and it was confirmed that the cell distribution in the G2/M phase was remarkably low. (Fig. 18). However, no significant differences were observed for miR-122, miR-122-G2U, and miR-122-G3U compared to the control group. At this time, in the cell cycle analysis experiment, the synthesized siRNA duplex was delivered to HepG at a concentration of 50 nM, cultured for 24 hours, and then, using the Muse Cell Cycle Kit (Catalog No. MCH100106, Milipore), according to the protocol provided by the company. The experiment was carried out and measured with a Muse Cell Analyzer (Milipore).

Therefore, it was confirmed that the cancer cell function regulated by miR-122-G2,3U lowers cell division (G2/M) and induces cell cycle arrest (G0/G1), according to this example. The results can be interpreted that miR-122 represents the function of preventing the progression of the liver cancer cell cycle by simultaneously recognizing both the 2nd G and the 3rd G based on the 5'end of the non-normal GA sequence initiating site by G:A wobble arrangement.

[ Example 19] miR -122-G2,3U, miR Irregular by -122-G2,7U GA array Observation of induction of cell cycle arrest due to initiating target control

In the previous example 17, miR-122-G2,3U was found to have a function of inhibiting the migration of hepatocytes as a function of the target for initiating the irregular GA sequence of miR-122, in particular the expression of miR-122-G2,3U Inhibition of cell migration induced by, was observed that the function was maximized when the modulatory effect of G base 3 was added to the biological function of target regulation at the initiation of the irregular GA sequence of base 2 G (Fig. 17). Therefore, in order to investigate whether the biological function of target inhibition at the initiation of the irregular GA sequence is changed by an additional combination of G bases 2, 3, and 7 of miR-122, miR-122-G7U, miR-122-G3,7U, miR-122 An experiment was conducted to additionally confirm the cell cycle regulation function of -G2,7U siRNA.

In this experiment, hepG2, a liver cancer cell line, was used in the same manner, and the miRNA-GU duplex was prepared in the same manner as in Example 18 and transfected into cells at a concentration of 50 nM. However, in this cell cycle observation, after 24 hours incubation, nocodazole was treated at 100 ng/ml for 16 hours to synchronize HepG2 cells to G2/M (dividing preparation/dividing stage), and then how many Whether or not the cells were stopped at G2/M, the amount of cells in the cell cycle arrested state at G0/G1 was measured with a Muse Cell Analyzer (Milipore) flow cytometer (FIG. 19).

As a result, miR-122-G2U did not have a difference in the number of cells in G0/G1 in cell cycle arrest compared to NT-6pi, the control group, but miR-122-G2,3U siRNA, miR-122-G2,7U In addition to the second G, when the non-normal GA sequence initiating target site was regulated, it was confirmed that the rate of cell cycle arrest (G0/G1) increased (FIG. 19A). However, miR-122-G3, 7U did not show any significant increase (Fig. 19a). Quantitative analysis showed that in the case of the experimental group to which miR-122-G2, 3U siRNA was delivered, the rate of cell cycle arrest (G0/G1) increased by about twice or more compared to the control group, followed by miR-122-G2, In the 7U experimental group, it was observed that the ratio of cell cycle arrest (G0/G1) increased by about 1.5 times (FIG. 19B).

Based on this, it was confirmed that the cell cycle arrest status can be increased through the regulation of the non-normal GA sequence initiating target by miR-122-G2,3U siRNA and miR-122-G2,7U siRNA.

[ Example 20] let-7a- G2U , let-7a-G2,4U denormal GA array Observation of cell cycle arrest induction induced by initiating target regulation

A representative microRNA that functions as an anticancer control (Tumor suppressor) is the let-7 family. It was reported that the function that regulates the development process of the pretty little nematode was reported (Nature, 2000, 403(6772): 901-906), and was then suppressed in various cancer cells (Cancer Res., 2004, 64(11): 3753- 3756) and anti-cancer function (Cell, 2005, 120(5): 635-647 ;Genes Dev. 2007, 21(9):1025-1030) through regulation of tumor formation process (Tumorigenesis) have been reported. It has been studied as an important gene with potential for cancer diagnosis and development of anticancer drugs. Accordingly, the present inventors conducted an experiment to find out the biological function induced by the inhibition of the target for initiating an irregular GA sequence of let-7.

The starting sequence from the 1st to the 9th of the 5'end of let-7a is 5'-UGA GGU AGU-3', and the G:A wobble base sequence is the 2nd, 4th, 5th, 8th Exists in The present inventors paid attention to the second and fourth G among them, synthesized by transforming them into miRNA-GU, and introduced them into HepG2 cells, a liver cancer cell line, and then how they affected the cell cycle in the previous Example 19. The experiment was carried out in the same manner as. The nucleotide sequence for let-7 synthesized and used at this time is as follows (let-7a: 5'- U GAG GUA GUA GGU UGU AUA G UU-3';let-7a-G2U: 5'-U UAG GUA GUA GGU UGU AUA G UU-3'; let-7a-G4U: 5'-U GAU GUA GUA GGU UGU AUA G UU-3'; let-7a-G2,4U: 5'- U UAU GUA GUA GGU UGU AUA G UU -3').

As a result, in the case of let-7a, there was no significant difference compared to the control NT-6pi, but let-7a-G2U and let-7a-G2,4U increase cell cycle arrest in G0/G1, It could be observed that let-7-G4U, on the contrary, increases the amount of cells in G2/M to rapidly cycle the cell cycle (FIG. 20A). Quantifying this by 4 repeated experiments. In the case of the let-7a-G2U experimental group, the distribution of G1/G0 cells increased by about 1.5 times compared to the control (Nt-6pi), and the distribution of G2/M cells decreased. Could be observed (Fig. 20b). Through this, it was confirmed that the inhibition of the expression of the 2nd irregular GA sequence initiating target of let-7a induces cell cycle arrest in HepG2 cells. Then, in the case of G base 4 present at the beginning of let-7a, the single GA sequence (let-7a-G4U) reduces the induction of the cell cycle arrest state of HepG2, but rather rapidly proceeds to the G2/M state and is nocoda. The result was that a large number of cells were retained by treatment with the sol drug. In addition, when the 2nd and 4th GAs were arranged at the same time (let-7a-G2,4U), the induction of the cell cycle arrest state of HepG2 could be observed. Such cell cycle arrest was not observed in HepG2 cells to which let-7a was delivered (FIG. 20 b ). Thus, inhibition of the expression of the 2nd and 2nd, 4th irregular GA sequence initiating targets of let-7a induces a cell cycle arrest state of HepG2 cells, which is a function that let-7a is seen in the regulation of HepG2 cells performed by the present inventors. It was confirmed that is a completely different function.

Through the above examples, let-7 plays a role of promoting cancer cell cycle arrest by inhibiting the genes of the irregular GA starting sequence site through G at the 5'end, more preferably 2 times at the 5'end. It can be seen that the effect is greatest when G and No. 4 G work together in a G;A wobble arrangement. In addition, it is believed that the non-regular GA initiating sequence site through the 5'terminal standard G of let-7 promotes the cell cycle progression of cancer cells and induces the proliferation of cancer cells.

[ Example 21] miR -302- 4GU , miR -372- To 4GU By irregular GA array Inducing target inhibition Retrodifferentiation Promote OCT4 Observation with promoter reporter

Differentiated cells can acquire differentiation capacity again by artificial expression of several transcription factors, and cells that have finally acquired the same omnipotent differentiation capacity as embryonic cells are called induced pluripotent stem cells. This technique introduces four factors (Oct3/4, Sox2, c-Myc, Klf4) into mouse embryonic fibroblasts (MEM), and induces pluripotent stem cells (iPS) with differentiation diversity like stem cells. cell: inducible pluripotent stem cell) has been reported (Cell, 2006,126 (4): 663-676). Similarly, it has been reported that induced pluripotent stem cells can be produced through the delivery of four factors (OCT4, SOX2, NANOG, and LIN28) to human somatic cells (Science, 2007, 318(5858): 1917-1920). This is a field with high application potential in that it can create regeneration potential through any cell including human cells through the process of dedifferentiation. After the initial discovery, the generation of induced pluripotent stem cells Efforts have been made to increase efficiency. MicroRNA-induced pluripotent stem cells (mirPS) reported as part of this are also one of the methods for efficiently inducing pluripotency.MicroRNAs such as miR-302a and miR-372 are used alone or with four factors (Oct3/ 4, Sox2, c-Myc, Klf4) has been reported to reverse differentiation of cells (RNA, 2008 14(10): 2115-2124; Nat Biotechnol. 2011, 29 (5): 443-448). Therefore, the present inventors conducted an experiment to find out whether inhibition of the expression of targets initiating the irregular GA sequence of miR-302a and miR-372 can promote the process of dedifferentiating cells.

First, in order to monitor pluripotency induction, a plasmid containing the Oct4 promoter, which is reported to be activated in stem cells, and a green fluorescent protein expressed depending on it, was purchased (Addgene #21319) (FIG. 21A). When the Oct4 expression reporter vector (pOct4:GFP) was introduced into the differentiated cervical cancer cells HeLa, green fluorescent protein was not expressed, but when the cells were dedifferentiated to obtain differentiation capacity, the green fluorescent protein was expressed in the Oct4 expression reporter. It will be. The experiment was performed on the microRNAs miR-302a and miR-372, which are known to cause dedifferentiation together with the Oct4 expression reporter vector. Of the starting sequences of miR-302a and miR-372, the 2nd to 8th sequences based on the 5'end are "AA GUG CU", so the non-normal GA sequence starting sequence is possible through the 4th G and 6th G based on the 5'end. Do. However, the present inventors performed an experiment by synthesizing miR-302-G4U and miR-372-G4U by paying attention to the fourth G.

Firstly, in the experiment, an Oct4 expression reporter vector (pOct4:GFP) was delivered to HeLa cells according to the manufacturer's protocol using a reagent Lipofectamine 2000 (Invitrogen), followed by a guide strand according to the sequence of human miR-302 and miR-372, The carrier strand was synthesized in Bioneer as in the above example, prepared as a duplex, and sequentially delivered at a concentration of 50 nM using an RNAimax (Invitrogen) reagent. In addition, the preparation of target-specific siRNA for the initiation of the irregular GA sequence of miR-302 and miR-372 was prepared in the same manner as in the above example by substituting U for the G base in the initiation, and delivered to the cells at a concentration of 50 nM. The nucleotide sequence of the nucleic acid used here is as follows (miR-302: 5'-UAAGUGCUUCCAUGUUUUGGUGA-3'; miR-302-4GU: 5'-UAAUUGCUU CCAUGUUUUGGUGA-3'; miR-302 carrier strand: 5'- ACUUAAACGUGGAUGUACUUGCU- 3'; miR-372: 5'- AAAGUGCUGCG ACAUUUGAGCGU-3'; miR-372-G4U: 5'- AAAUUGC UGCGACA UUUGAGCGU-3', miR-372 carrier strand: 5'-CCUCAAAUGUGGAGCACUAUUCU-3'). HeLa cells into which the reporter vector and RNA were introduced were cultured in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% FBS (fetal bovine serum), 100 U/ml penicillin, and 100 μg/ml steptomycin for 14 days. , When performing transfection, cells were cultured in complete medium without antibiotics.

As a result, the experimental group in which miR-302 or miR-372 was delivered alone showed colony-like cell growth compared to the control group, but expression of green fluorescent protein indicating the activity of the Oct4 promoter was not observed until the 14-day culture period. Did. However, in the experimental group into which the miR-302 and miR-372 irregular GA sequence initiating target suppression siRNA was introduced, not only the cell growth in the form of a cluster, but also the expression of green fluorescent protein indicating the activity of the Oct4 promoter was observed (Fig. 21 bc, red arrow). , When miR-302 and miR-372 and each of the non-normal GA sequence initiating target inhibitory siRNAs were simultaneously delivered to the cells, a stronger and larger green fluorescent protein-expressing cell population could be observed (FIG. 21 bc). Based on this, it can be seen that inhibition of miR-302 and miR-372 non-normal GA sequence initiating targets can promote cell dedifferentiation and acquire differentiation capacity through Oct4 expression.

Through the above examples, miR-372-G4U and miR-302a-G4U can promote cell dedifferentiation more efficiently than existing miR-372 and miR-302 by expressing only miR-372 and miR-302 alone. It can be observed that miR-372-G4U and miR-302a-G4U can be used as materials to promote cell dedifferentiation.

[ Example 22] 5 '2 at the end of the 6th OMe Modification is irregular of the corresponding RNA interference derivative Nuclear proliferation Identify the phenomenon of not being able to suppress the target

In the above embodiment, the non-normal fusion target shows a new biological function completely different from the normal initiation target, and therefore, miRNA-BS, an RNA interference-inducing nucleic acid that regulates only the inhibition of the expression of the non-normal fusion target, was invented, and its function was confirmed. I did. However, in the case of miRNA-BS, the starting sequence was changed to complementarily align the existing miRNA's irregular nucleus site, but it was confirmed that a new nucleus site was created again through the changed starting sequence. Therefore, it was found that RNA interference-inducing nucleic acids such as miRNA-BS need a technique that recognizes only the canonical initiation sequence and not the irregular nucleus site. Accordingly, the present inventors believe that the base at position 6 based on the 5'end in the starting sequence is important in order to perform irregular nucleus bonding, and the degree of occurrence of irregular nucleus bonding varies depending on the alignment strength of the base sequence at this 6 position. In order to reduce the nucleotide sequence intensity at position 6, the following experiment was conducted. In other words, a methyl group (2' OMe) was added to the 2'position of the ribosyl ring of nucleotide 6 of miR-124 and modified, and the modified interference-inducing nucleic acid (miR-124-6me) was a non-normal nucleus target of miR-124. Whether there was an inhibitory function was observed by the luciferase reporter experiment. 2'OMe-modified miR-124 was prepared through synthesis (Bionia Corporation), and a luciferase reporter experiment was conducted in the same manner as in the previous Example 2, and gene suppression efficiency was measured by IC ₅₀ (FIG. 22A ). The luciferase reporter vector used at this time was used for the normal initiation site (Luc-seed) of miR-124 and the non-normal nucleation site (Luc-nucleation bulge).

As a result, the inhibitory effect of miR-124 on the target gene for irregular elevation as seen in Example 2 disappeared, whereas the suppression of gene expression through the normal initiation site had an IC ₅₀ value of 0.3 nM, and 2'OMe was the 6th nucleotide. Even when added to was observed to still exhibit an excellent inhibitory effect (Fig. 22a). Additionally, in the same way, 2'OMe was applied to the 6th nucleotide of miR-1, a microRNA other than miR-124 (miR-1-6me), and this was the normal starting site of miR-1 (Luc-seed). When examining the effect of inhibiting gene expression on the target with a luciferase vector including a Luc-nucleation bulge (FIG. 22b), irregular bulge by miR-1 as the result of previous miR-124 The target gene inhibitory effect disappeared, whereas the gene expression suppression through the normal initiation site was confirmed to have an excellent IC ₅₀ value of 0.7 nM. Through the above example, when the 2'OMe modification is applied to the 6th 5'end of the nucleic acid that induces RNA interference, the effect of inhibiting the expression of the target gene for normal initiation induced by the corresponding RNA interference-inducing nucleic acid is maintained, It was confirmed that the suppression of the expression of the target gene for abnormal nuclear fusion disappears.

[ Example 23] 5 ‘2 at the end of the 6th’ OMe Transformation In the transcript All irregular nuclear fusion targets mRNA RNA- Seq Confirmed by analysis

In Example 22, when a 2'OM modification was applied to the 6th nucleotide of miR-1 (miR-1-6me), the effect of inhibiting the expression of the target gene for normal initiation was maintained, while the suppression of the expression of the target gene for the irregular nucleus was It was confirmed that it decreased. Therefore, it was confirmed that miR-1-6me invented by the present inventors actually reduced the inhibitory function of the non-normal elevation target gene of miR-1 in the transcript of h9c2, a cardiomyocyte cell line in which all genes related to miR-1 function were expressed. To do this, RNA-Seq analysis was performed after introducing miR-1 and miR-1-6me into h9c2 cells. The RNA-Seq experiment was performed using a non-base 6 of the 5'end in the sequence of cel-miR-67, a microRNA of C.elegans, as an experimental control (NT-6pi). The experiment was carried out in the same manner as in 7. At this time, the RNA-Seq library was prepared using Lexogen's SENSE Total RNA-Seq Library Kit, and then sequenced using Illumina's MiniSeq.

Thereafter, the FASTAQ file, which is the sequence data obtained by the above experiment, was mapped to the mouse genome sequence (rn6) with the TopHat2 program, and the expression value (FPKM) was obtained with the Cufflink and Cuffdiff programs, and the control NT-6pi was introduced in h9c2 cells. The results were standardized and expressed as a log ratio (Fold change, log2 ratio) for analysis. At this time, in order to analyze from the RNA-Seq results whether the amount of messenger RNA of the gene carrying the microRNA target site is inhibited by the expression of the corresponding microRNA, the normal starting site of miR-1 (from the 2nd to 8th based on the 5'end) A gene having a 3'UTR) was selected and the result of this profile was compared and analyzed as a cumulative fraction in the order of inhibition dependent on the expression of the corresponding microRNA (Fig. 23a). . As a result, it was confirmed that both miR-1 and miR-1-6me can suppress the gene (seed) having the normal starting site of miR-1 in 3'UTR compared to the distribution of the total gene (Total mRNA). . After that, by the same method, the gene that has an irregular ridge of miR-1 (nuc, 7mer) in the 3'UTR was also analyzed at the cumulative ratio, and the target gene of the miracle of miR-1 by miR-1-6me The amount of the corresponding target mRNA in the cells to which miR-1 was introduced and the cells to which miR-1-6me was introduced was compared to see if the inhibition of expression of was decreased (Fig. 23b). As a result, it was observed that in miR-1, when compared to the expression in miR-1-6me, the non-normal nucleus target gene was still significantly inhibited compared to the total mRNA, through which miR-1- In 6me, it was confirmed that the inhibition of the expression of the non-normal nucleus target gene was reduced.

Judging from the results of the above examples, at the transcript level, the existing microRNA efficiently suppresses the non-normal elevation target gene together with the existing normal initiation target gene, but a 2'OMe modification was added to the 6th nucleotide. It was found that microRNA (miRNA-6me) still suppressed the normal initiating target gene, but not the irregular raised target gene. Therefore, the 2'OMe modification applied to the 6th of the 5'end is applied to miRNA-BS, maintaining the original intention to specifically suppress only the irregular nucleus site of the corresponding miRNA, while the newly appearing irregular nucleus binding is not By completely removing it, you can minimize its side effects.

[ Example 24] Ago Irregular through HITS CLIP experiment analysis Nuclear proliferation On site Combined MicroRNA Sympathy

In order to identify microRNAs that can be applied to the invention that allows microRNAs to recognize and suppress only irregular nucleus sites, we analyzed the results of Ago HITS CLIP, a method that can analyze the target of microRNA at the transcript level. , MicroRNA that binds to the irregular nucleus site was analyzed. First of all, Ago HITS-CLIP data performed in the cerebral cortex of a mouse (p13) 13 days after birth was sequenced in the same manner as in Example 10. First, the top 20 microRNAs of expression that bind together with Agonut were It was confirmed that it binds to the target mRNA and the irregular nucleus site (FIG. 24A). Therefore, when recognizing the target mRNA through the nucleotide sequence of the starting part, the corresponding microRNA (FIG. 24B) can bind to the target mRNA through the nucleus site irregularly as well as the regular nucleotide sequence through the starting part. Could know.

Expanding on the mouse Ago HITS-CLIP data analysis of the above example, other Ago HITS-CLIP results performed on human brain and heart tissues (boudreau RL et al, 2014, Neuron, 81(2) 294-305, Spengler RM et al, 2016, Nucleic Acids Res, 44(15) 7120-7131) was analyzed in the same manner as in the above example, and the frequency of the normal initiation site and the irregular nucleus site to which Ago and the corresponding microRNA are bound was calculated ( Human brain microRNA, Table 1; Human heart microRNA, Table 2).

As a result, it was confirmed that the microRNAs (Tables 1 to 2) described in the corresponding result table were bound to the irregular nucleus site in the tissue. Therefore, when applying the present invention to modify the microRNA discovered by synthesizing all the data analyzed for Ago HITS-CLIP in the above example to recognize an irregular nucleus site as a regular initiation site, it is shown in Table 3 below. As shown, a total of 426 sequences (BS sequences) are composed of nucleotides from the 2nd to the 7th based on the 5'end of the RNA interfering nucleic acid, and the modified RNA interfering nucleic acid is only the irregular raised target of the corresponding microRNA. Will combine and show only those functions.

Through the Ago HITS-CLIP experimental sequencing analysis of the above examples, microRNAs that bind to irregular raised targets in various tissue cells were identified, and accordingly, when applying the present invention to modify the irregular nucleus site to be recognized as a normal starting site. A total of 426 sequences (BS sequences) were created, and accordingly, a technique was completed that shows only the function of inhibiting non-normal targets of microRNA.

[ Example 25] Cancer patients MicroRNA Sequencing database ( TCGA ) To analyze the sequence variation, and through this G:A Wobble Starting arrangement On site Combined MicroRNA Sympathy

Through the above examples, it was confirmed that the microRNA inhibited the expression of the target gene by irregularly binding to the G:A wobble site, and through this, the sequence to recognize the irregular G:A wobble site as a regular initiating site in the present invention. A microRNA sequence conversion method (miRNA-BS) was developed to modify. In this way, if binding to A of target mRNA through wobble arrangement through G in the existing microRNA initiating sequence changes the function of microRNA, this mechanism can be seen through sequence mutation of microRNA in diseases such as tumors. The results of sequencing microRNAs in the tissues of cancer patients (miRNA-Seq) were analyzed. At this time, 8105 cancer-related microRNA sequence experiments were obtained from the TCGA database, and 468 cancer-related microRNA sequence results were additionally obtained from the Gene Expression Omnibus (GEO) database, and a total of 8,573 tumor microRNA sequence results were obtained in Example 10. In the same way as in the method used in, the mapping through the bowtie2 program and the place where the mutation occurred was analyzed. The microRNA sequence data used in the analysis for each cancer type at this time is as shown in FIG. 25A.

In the result, the frequency (fraction) of mutations was classified by the position of the 5'end of the microRNA, and the distribution of the most frequent mutations (top 10000) and the hypothesized mutations (bottom 10000) was examined. (FIG. 25B), it could be observed that in the case of 10,000 mutations that occur most often, they mainly occur at the beginning of the microRNA (between the 2nd and 8th). In addition, when looking at the most frequently occurring mutations 100 and the least occurring mutations 100 on the heatmap (FIG. 25C), it was found that the mutations were concentrated in the initiating region in the same manner as in the Example. The sequence mutations of tumor microRNAs that show a tendency to appear at the beginning are reverse-transcribed and sequenced with DNA.When looking at the results of sequencing as A, T, C, and G, only G is formed in the starting sequence. Showed a tendency to become (FIG. 25D). This tendency was confirmed again that the mutation mainly occurs in G not only for the top 100 mutation rates, but also in all cases where mutation occurs beyond this (FIG. 25E).

This may be a phenomenon in which the G of the microRNA that recognizes the irregular G:A wobble that the present inventors noted is mutated to U in order to better bind the A of the target mRNA on the other side. As a result of analyzing which base was changed in (FIG. 25F), it was observed that most of them were reverse-transcribed and sequenced with DNA, indicating that G was changed from G to T. These results show that in cancer tissues, in fact, in order to better recognize the non-regular G:A wobble initiating target as a normal initiation, the G present in the initiation part of the microRNA changes its G to U and nucleotide sequence with the target A. It means, and therefore, this variant of the method tells us how to apply the sequencing method to recognize the irregular G:A wobble initiating sequence developed in the present invention as a canonical initiating sequence to which position G of which microRNA. Therefore, by combining all of these results, microRNAs with a normalized mutation frequency of 2 or more per cancer patient were selected (see Table 4). As shown in Table 4, a total of 335 sequences (sequence (G>U)) consist of nucleotides from the 2nd reference to the 5'end of the RNA interference nucleic acid, and the modified RNA interference nucleic acid Only irregular G:A wobble elevation targets will be bound and will exhibit only that function.

Through the microRNA sequencing mutation analysis of the above example, a mutant microRNA that recognizes an irregular G:A wobble initiating target as a regular target in several cancer patients was identified, and thus to recognize an irregular G:A wobble initiating site as a regular initiation site. When applying the present invention (miRNA-GU) to modify, a total of 335 sequences (sequence (G>U)) were created (Table 4), and accordingly, a technique was completed showing only the function of inhibiting non-normal targets of microRNA.

<110> Korea University <120> RNA interference inducing nucleic acids suppressing noncanonical targets of microRNA and use thereof <130> MP19-147 <160> 863 <170> KoPatentIn 3.0 <210> 1 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> 5'2~7 of miR-124-BS <400> 1 aaggcc 6 <210> 2 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> 5'2~7 of miR-122-BS <400> 2 aaggcc 6 <210> 3 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> 5'2~7 of miR-155-BS <400> 3 uaaugg 6 <210> 4 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> 5'2~7 of miR-1-BS <400> 4 ggaauu 6 <210> 5 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> miR-124-BS <400> 5 uaaggccacg cggugaaugc c 21 <210> 6 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> miR-122-BS <400> 6 uggaguugug acaauggugu u 21 <210> 7 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miR-155-BS <400> 7 uuaaugggcu aaucgugaua gg 22 <210> 8 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> miR-1-BS <400> 8 uggaauugua aagaaguaug u 21 <210> 9 <211> 9 <212> RNA <213> Artificial Sequence <220> <223> 5'1~9 of miR-124-G4U <400> 9 uaaugcacg 9 <210> 10 <211> 9 <212> RNA <213> Artificial Sequence <220> <223> 5'1~9 of miR-124-G5U <400> 10 uaagucacg 9 <210> 11 <211> 9 <212> RNA <213> Artificial Sequence <220> <223> 5'1~9 of miR-124-G4,5U <400> 11 uaauucacg 9 <210> 12 <211> 9 <212> RNA <213> Artificial Sequence <220> <223> 5'1~9 of miR-1-G2U <400> 12 uugaaugua 9 <210> 13 <211> 9 <212> RNA <213> Artificial Sequence <220> <223> 5'1~9 of miR-1-G3U <400> 13 uguaaugua 9 <210> 14 <211> 9 <212> RNA <213> Artificial Sequence <220> <223> 5'1~9 of miR-1-G7U <400> 14 uggaauuua 9 <210> 15 <211> 9 <212> RNA <213> Artificial Sequence <220> <223> 5'1~9 of miR-1-G2,3U <400> 15 uuuaaugua 9 <210> 16 <211> 9 <212> RNA <213> Artificial Sequence <220> <223> 5'1~9 of miR-1-G3,7U <400> 16 uguaauuua 9 <210> 17 <211> 9 <212> RNA <213> Artificial Sequence <220> <223> 5'1~9 of miR-1-G2,7U <400> 17 uugaauuua 9 <210> 18 <211> 9 <212> RNA <213> Artificial Sequence <220> <223> 5'1~9 of miR-1-G2,3,7U <400> 18 uuuaauuua 9 <210> 19 <211> 9 <212> RNA <213> Artificial Sequence <220> <223> 5'1~9 of miR-122-G2U <400> 19 uugagugug 9 <210> 20 <211> 9 <212> RNA <213> Artificial Sequence <220> <223> 5'1~9 of miR-122-G3U <400> 20 uguagugug 9 <210> 21 <211> 9 <212> RNA <213> Artificial Sequence <220> <223> 5'1~9 of miR-122-G5U <400> 21 uggauugug 9 <210> 22 <211> 9 <212> RNA <213> Artificial Sequence <220> <223> 5'1~9 of miR-122-G7U <400> 22 uggaguuug 9 <210> 23 <211> 9 <212> RNA <213> Artificial Sequence <220> <223> 5'1~9 of miR-122-G9U <400> 23 uggaguguu 9 <210> 24 <211> 9 <212> RNA <213> Artificial Sequence <220> <223> 5'1~9 of miR-122-G2,3U <400> 24 uuuagugug 9 <210> 25 <211> 9 <212> RNA <213> Artificial Sequence <220> <223> 5'1~9 of miR-122-G2,5U <400> 25 uugauugug 9 <210> 26 <211> 9 <212> RNA <213> Artificial Sequence <220> <223> 5'1~9 of miR-122-G2,7U <400> 26 uugaguuug 9 <210> 27 <211> 9 <212> RNA <213> Artificial Sequence <220> <223> 5'1~9 of miR-122-G2,9U <400> 27 uugaguguu 9 <210> 28 <211> 9 <212> RNA <213> Artificial Sequence <220> <223> 5'1~9 of miR-122-G3,5U <400> 28 uguauugug 9 <210> 29 <211> 9 <212> RNA <213> Artificial Sequence <220> <223> 5'1~9 of miR-122-G3,7U <400> 29 uguaguuug 9 <210> 30 <211> 9 <212> RNA <213> Artificial Sequence <220> <223> 5'1~9 of miR-122-G3,9U <400> 30 uguaguguu 9 <210> 31 <211> 9 <212> RNA <213> Artificial Sequence <220> <223> 5'1~9 of miR-122-G5,7U <400> 31 uggauuuug 9 <210> 32 <211> 9 <212> RNA <213> Artificial Sequence <220> <223> 5'1~9 of miR-122-G5,9U <400> 32 uggauuguu 9 <210> 33 <211> 9 <212> RNA <213> Artificial Sequence <220> <223> 5'1~9 of miR-122-G7,9U <400> 33 uggaguuuu 9 <210> 34 <211> 9 <212> RNA <213> Artificial Sequence <220> <223> 5'1~9 of miR-133-G4U <400> 34 uuuuguccc 9 <210> 35 <211> 9 <212> RNA <213> Artificial Sequence <220> <223> 5'1~9 of miR-133-G5U <400> 35 uuuguuccc 9 <210> 36 <211> 9 <212> RNA <213> Artificial Sequence <220> <223> 5'1~9 of miR-124-G4,5U <400> 36 uuuuuuccc 9 <210> 37 <211> 9 <212> RNA <213> Artificial Sequence <220> <223> 5'1~9 of let-7-G2U <400> 37 uuagguagu 9 <210> 38 <211> 9 <212> RNA <213> Artificial Sequence <220> <223> 5'1~9 of let-7-G4U <400> 38 ugauguagu 9 <210> 39 <211> 9 <212> RNA <213> Artificial Sequence <220> <223> 5'1~9 of let-7-G5U <400> 39 ugaguuagu 9 <210> 40 <211> 9 <212> RNA <213> Artificial Sequence <220> <223> 5'1~9 of let-7-G8U <400> 40 ugagguauu 9 <210> 41 <211> 9 <212> RNA <213> Artificial Sequence <220> <223> 5'1~9 of let-7-G2,4U <400> 41 uuauguagu 9 <210> 42 <211> 9 <212> RNA <213> Artificial Sequence <220> <223> 5'1~9 of let-7-G2,5U <400> 42 uuaguuagu 9 <210> 43 <211> 9 <212> RNA <213> Artificial Sequence <220> <223> 5'1~9 of let-7-G2,8U <400> 43 uuagguauu 9 <210> 44 <211> 9 <212> RNA <213> Artificial Sequence <220> <223> 5'1~9 of let-7-G4,5U <400> 44 ugauuuagu 9 <210> 45 <211> 9 <212> RNA <213> Artificial Sequence <220> <223> 5'1~9 of let-7-G4,8U <400> 45 ugauguauu 9 <210> 46 <211> 9 <212> RNA <213> Artificial Sequence <220> <223> 5'1~9 of let-7-G5,8U <400> 46 ugaguuauu 9 <210> 47 <211> 9 <212> RNA <213> Artificial Sequence <220> <223> 5'1~9 of miR-302a-G4U <400> 47 uaauugcuu 9 <210> 48 <211> 9 <212> RNA <213> Artificial Sequence <220> <223> 5'1~9 of miR-302a-G6U <400> 48 uaaguucuu 9 <210> 49 <211> 9 <212> RNA <213> Artificial Sequence <220> <223> 5'1~9 of miR-302a-G4,6U <400> 49 uaauuucuu 9 <210> 50 <211> 9 <212> RNA <213> Artificial Sequence <220> <223> 5'1~9 of miR-372-G4U <400> 50 aaauugcug 9 <210> 51 <211> 9 <212> RNA <213> Artificial Sequence <220> <223> 5'1~9 of miR-372-G6U <400> 51 aaaguucug 9 <210> 52 <211> 9 <212> RNA <213> Artificial Sequence <220> <223> 5'1~9 of miR-372-G9U <400> 52 aaagugcuu 9 <210> 53 <211> 9 <212> RNA <213> Artificial Sequence <220> <223> 5'1~9 of miR-372-G4,6U <400> 53 aaauuucug 9 <210> 54 <211> 9 <212> RNA <213> Artificial Sequence <220> <223> 5'1~9 of miR-372-G4,9U <400> 54 aaauugcuu 9 <210> 55 <211> 9 <212> RNA <213> Artificial Sequence <220> <223> 5'1~9 of miR-372-G6,9U <400> 55 aaaguucuu 9 <210> 56 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miR-124-G4U <400> 56 uaaugcacgc ggugaaugcc aa 22 <210> 57 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> miR-124-G5U <400> 57 uaagucacgc ggugaaugcc a 21 <210> 58 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miR-124-G4,5U <400> 58 uaauucacgc ggugaaugcc aa 22 <210> 59 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miR-1-G2U <400> 59 uugaauguaa agaaguaugu au 22 <210> 60 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miR-1-G3U <400> 60 uguaauguaa agaaguaugu au 22 <210> 61 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miR-1-G7U <400> 61 uggaauuuaa agaaguaugu au 22 <210> 62 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miR-1-G2,3U <400> 62 uuuaauguaa agaaguaugu au 22 <210> 63 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miR-1-G3,7U <400> 63 uguaauuuaa agaaguaugu au 22 <210> 64 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miR-1-G2,7U <400> 64 uugaauuuaa agaaguaugu au 22 <210> 65 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miR-1-G2,3,7U <400> 65 uuuaauuuaa agaaguaugu au 22 <210> 66 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miR-122-G2U <400> 66 uugaguguga caaugguguu ug 22 <210> 67 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miR-122-G3U <400> 67 uguaguguga caaugguguu ug 22 <210> 68 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miR-122-G5U <400> 68 uggauuguga caaugguguu ug 22 <210> 69 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miR-122-G7U <400> 69 uggaguuuga caaugguguu ug 22 <210> 70 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miR-122-G9U <400> 70 uggaguguua caaugguguu ug 22 <210> 71 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miR-122-G2,3U <400> 71 uuuaguguga caaugguguu ug 22 <210> 72 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miR-122-G2,5U <400> 72 uugauuguga caaugguguu ug 22 <210> 73 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miR-122-G2,7U <400> 73 uugaguuuga caaugguguu ug 22 <210> 74 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miR-122-G2,9U <400> 74 uugaguguua caaugguguu ug 22 <210> 75 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miR-122-G3,5U <400> 75 uguauuguga caaugguguu ug 22 <210> 76 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miR-122-G3,7U <400> 76 uguaguuuga caaugguguu ug 22 <210> 77 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miR-122-G3,9U <400> 77 uguaguguua caaugguguu ug 22 <210> 78 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miR-122-G5,7U <400> 78 uggauuuuga caaugguguu ug 22 <210> 79 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miR-122-G5,9U <400> 79 uggauuguua caaugguguu ug 22 <210> 80 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miR-122-G7,9U <400> 80 uggaguuuua caaugguguu ug 22 <210> 81 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miR-133-G4U <400> 81 uuuugucccc uucaaccagc ug 22 <210> 82 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miR-133-G5U <400> 82 uuuguucccc uucaaccagc ug 22 <210> 83 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miR-124-G4,5U <400> 83 uuuuuucccc uucaaccagc ug 22 <210> 84 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> let-7-G2U <400> 84 uuagguagua gguuguauag uu 22 <210> 85 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> let-7-G4U <400> 85 ugauguagua gguuguauag uu 22 <210> 86 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> let-7-G5U <400> 86 ugaguuagua gguuguauag uu 22 <210> 87 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> let-7-G8U <400> 87 ugagguauua gguuguauag uu 22 <210> 88 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> let-7-G2,4U <400> 88 uuauguagua gguuguauag uu 22 <210> 89 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> let-7-G2,5U <400> 89 uuaguuagua gguuguauag uu 22 <210> 90 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> let-7-G2,8U <400> 90 uuagguauua gguuguauag uu 22 <210> 91 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> let-7-G4,5U <400> 91 ugauuuagua gguuguauag uu 22 <210> 92 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> let-7-G4,8U <400> 92 ugauguauua gguuguauag uu 22 <210> 93 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> let-7-G5,8U <400> 93 ugaguuauua gguuguauag uu 22 <210> 94 <211> 23 <212> RNA <213> Artificial Sequence <220> <223> miR-302a-G4U <400> 94 uaauugcuuc cauguuuugg uga 23 <210> 95 <211> 23 <212> RNA <213> Artificial Sequence <220> <223> miR-302a-G6U <400> 95 uaaguucuuc cauguuuugg uga 23 <210> 96 <211> 23 <212> RNA <213> Artificial Sequence <220> <223> miR-302a-G4,6U <400> 96 uaauuucuuc cauguuuugg uga 23 <210> 97 <211> 23 <212> RNA <213> Artificial Sequence <220> <223> miR-372-G4U <400> 97 aaauugcugc gacauuugag cgu 23 <210> 98 <211> 23 <212> RNA <213> Artificial Sequence <220> <223> miR-372-G6U <400> 98 aaaguucugc gacauuugag cgu 23 <210> 99 <211> 23 <212> RNA <213> Artificial Sequence <220> <223> miR-372-G9U <400> 99 aaagugcuuc gacauuugag cgu 23 <210> 100 <211> 23 <212> RNA <213> Artificial Sequence <220> <223> miR-372-G4,6U <400> 100 aaauuucugc gacauuugag cgu 23 <210> 101 <211> 23 <212> RNA <213> Artificial Sequence <220> <223> miR-372-G4,9U <400> 101 aaauugcuuc gacauuugag cgu 23 <210> 102 <211> 23 <212> RNA <213> Artificial Sequence <220> <223> miR-372-G6,9U <400> 102 aaaguucuuc gacauuugag cgu 23 <210> 103 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> let-7/98/4458/4500 BS seq <400> 103 gagguu 6 <210> 104 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-125a-5p/125b-5p/351/670/4319 BS seq <400> 104 cccugg 6 <210> 105 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-124/124ab/506 BS seq <400> 105 aaggcc 6 <210> 106 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-9/9ab BS seq <400> 106 cuuugg 6 <210> 107 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-29abcd BS seq <400> 107 agcacc 6 <210> 108 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-103a/107/107ab BS seq <400> 108 gcagcc 6 <210> 109 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-221/222/222ab/1928 BS seq <400> 109 gcuacc 6 <210> 110 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-26ab/1297/4465 BS seq <400> 110 ucaagg 6 <210> 111 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-15abc/16/16abc/195/322/424/497/1907 BS seq <400> 111 agcagg 6 <210> 112 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-126-3p BS seq <400> 112 cguacc 6 <210> 113 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-30abcdef/30abe-5p/384-5p BS seq <400> 113 guaaaa 6 <210> 114 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-33ab/33-5p BS seq <400> 114 ugcauu 6 <210> 115 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-34ac/34bc-5p/449abc/449c-5p BS seq <400> 115 ggcagg 6 <210> 116 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-19ab BS seq <400> 116 gugcaa 6 <210> 117 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-99ab/100 BS seq <400> 117 acccgg 6 <210> 118 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-17/17-5p/20ab/20b-5p/93/106ab/427/518a-3p/519d BS seq <400> 118 aaaguu 6 <210> 119 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-27abc/27a-3p BS seq <400> 119 ucacaa 6 <210> 120 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-218/218a BS seq <400> 120 ugugcc 6 <210> 121 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-22/22-3p BS seq <400> 121 agcugg 6 <210> 122 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-185/882/3473/4306/4644 BS seq <400> 122 ggagaa 6 <210> 123 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-181abcd/4262 BS seq <400> 123 acauuu 6 <210> 124 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-338/338-3p BS seq <400> 124 ccagcc 6 <210> 125 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-127/127-3p BS seq <400> 125 cggauu 6 <210> 126 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-101/101ab BS seq <400> 126 acaguu 6 <210> 127 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-149 BS seq <400> 127 cuggcc 6 <210> 128 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-324-5p BS seq <400> 128 gcaucc 6 <210> 129 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-24/24ab/24-3p BS seq <400> 129 ggcucc 6 <210> 130 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-33a-3p/365/365-3p BS seq <400> 130 aaugcc 6 <210> 131 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-139-5p BS seq <400> 131 cuacaa 6 <210> 132 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-138/138ab BS seq <400> 132 gcuggg 6 <210> 133 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-143/1721/4770 BS seq <400> 133 gagauu 6 <210> 134 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-25/32/92abc/363/363-3p/367 BS seq <400> 134 auugcc 6 <210> 135 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-574-5p BS seq <400> 135 gagugg 6 <210> 136 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-7/7ab BS seq <400> 136 ggaagg 6 <210> 137 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-145 BS seq <400> 137 uccagg 6 <210> 138 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-135ab/135a-5p BS seq <400> 138 auggcc 6 <210> 139 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-148ab-3p/152 BS seq <400> 139 cagugg 6 <210> 140 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-28-5p/708/1407/1653/3139 BS seq <400> 140 aggagg 6 <210> 141 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-130ac/301ab/301b/301b-3p/454/721/4295/3666 BS seq <400> 141 agugcc 6 <210> 142 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-3132 BS seq <400> 142 ggguaa 6 <210> 143 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-155 BS seq <400> 143 uaaugg 6 <210> 144 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-485-3p BS seq <400> 144 ucauaa 6 <210> 145 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-132/212/212-3p BS seq <400> 145 aacagg 6 <210> 146 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-9-3p BS seq <400> 146 uaaagg 6 <210> 147 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-374ab BS seq <400> 147 uauaaa 6 <210> 148 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-129-3p/129ab-3p/129-1-3p/129-2-3p BS seq <400> 148 agcccc 6 <210> 149 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-126-5p BS seq <400> 149 auuauu 6 <210> 150 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-425/425-5p/489 BS seq <400> 150 augacc 6 <210> 151 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-423-3p BS seq <400> 151 gcucgg 6 <210> 152 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-21/590-5p BS seq <400> 152 agcuuu 6 <210> 153 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-31 BS seq <400> 153 ggcaaa 6 <210> 154 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-20b-3p BS seq <400> 154 cuguaa 6 <210> 155 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-let-7d-3p BS seq <400> 155 uauacc 6 <210> 156 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-191 BS seq <400> 156 aacggg 6 <210> 157 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-18ab/4735-3p BS seq <400> 157 aagguu 6 <210> 158 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-369-3p BS seq <400> 158 auaauu 6 <210> 159 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-5187-5p BS seq <400> 159 gggauu 6 <210> 160 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-382 BS seq <400> 160 aaguuu 6 <210> 161 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-485-5p/1698/1703/1962 BS seq <400> 161 gaggcc 6 <210> 162 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-136-3p BS seq <400> 162 aucauu 6 <210> 163 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-576-3p BS seq <400> 163 agaugg 6 <210> 164 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-204/204b/211 BS seq <400> 164 ucccuu 6 <210> 165 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-769-5p BS seq <400> 165 gagacc 6 <210> 166 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-342-5p/4664-5p BS seq <400> 166 gggguu 6 <210> 167 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-361-5p BS seq <400> 167 uaucaa 6 <210> 168 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-199ab-3p/3129-5p BS seq <400> 168 caguaa 6 <210> 169 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-142-3p BS seq <400> 169 guaguu 6 <210> 170 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-299-5p/3563-5p BS seq <400> 170 gguuuu 6 <210> 171 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-193/193b/193a-3p BS seq <400> 171 acuggg 6 <210> 172 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-1277-5p BS seq <400> 172 aauauu 6 <210> 173 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-140/140-5p/876-3p/1244 BS seq <400> 173 aguggg 6 <210> 174 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-30a/d/e-3p BS seq <400> 174 uuucaa 6 <210> 175 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-let-7i-3p BS seq <400> 175 ugcgcc 6 <210> 176 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-409-5p/409a BS seq <400> 176 gguuaa 6 <210> 177 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-379/1193-5p/3529 BS seq <400> 177 gguagg 6 <210> 178 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-136 BS seq <400> 178 cuccaa 6 <210> 179 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-154/872 BS seq <400> 179 agguuu 6 <210> 180 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-4684-3p BS seq <400> 180 guugcc 6 <210> 181 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-361-3p BS seq <400> 181 cccccc 6 <210> 182 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-335/335-5p BS seq <400> 182 caagaa 6 <210> 183 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-423a/423-5p/3184/3573-5p BS seq <400> 183 gagggg 6 <210> 184 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-371/373/371b-5p BS seq <400> 184 cucaaa 6 <210> 185 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-1185/3679-5p BS seq <400> 185 gaggaa 6 <210> 186 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-3613-3p BS seq <400> 186 caaaaa 6 <210> 187 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-93/93a/105/106a/291a-3p/294/295/302abcde/372/373/428/519a/520 be/520acd-3p/1378/1420ac BS seq <400> 187 aagugg 6 <210> 188 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-876-5p/3167 BS seq <400> 188 ggauuu 6 <210> 189 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-329/329ab/362-3p BS seq <400> 189 acacaa 6 <210> 190 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-582-5p BS seq <400> 190 uacagg 6 <210> 191 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-146ac/146b-5p BS seq <400> 191 gagaaa 6 <210> 192 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-380/380-3p BS seq <400> 192 auguaa 6 <210> 193 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-499-3p/499a-3p BS seq <400> 193 acaucc 6 <210> 194 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-551a BS seq <400> 194 cgaccc 6 <210> 195 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-142-5p BS seq <400> 195 auaaaa 6 <210> 196 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-17-3p BS seq <400> 196 cugcaa 6 <210> 197 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-199ab-5p BS seq <400> 197 ccaguu 6 <210> 198 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-542-3p BS seq <400> 198 gugacc 6 <210> 199 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-1277 BS seq <400> 199 acguaa 6 <210> 200 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-29c-5p BS seq <400> 200 gaccgg 6 <210> 201 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-3145-3p BS seq <400> 201 gauauu 6 <210> 202 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-106b-3p BS seq <400> 202 cgcacc 6 <210> 203 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-22-5p BS seq <400> 203 guucuu 6 <210> 204 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-744/1716 BS seq <400> 204 gcgggg 6 <210> 205 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-132-5p BS seq <400> 205 ccgugg 6 <210> 206 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-488 BS seq <400> 206 ugaaaa 6 <210> 207 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-501-3p/502-3p/500/502a BS seq <400> 207 augcaa 6 <210> 208 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-486-5p/3107 BS seq <400> 208 ccuguu 6 <210> 209 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-450a/451a BS seq <400> 209 uuugcc 6 <210> 210 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-30c-3p BS seq <400> 210 ugggaa 6 <210> 211 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-499-5p BS seq <400> 211 uaagaa 6 <210> 212 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-421 BS seq <400> 212 ucaacc 6 <210> 213 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-197 BS seq <400> 213 ucaccc 6 <210> 214 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-296-5p BS seq <400> 214 gggccc 6 <210> 215 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-326/330/330-5p BS seq <400> 215 cucugg 6 <210> 216 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-214/761/3619-5p BS seq <400> 216 cagcaa 6 <210> 217 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-612/1285/3187-5p BS seq <400> 217 cugggg 6 <210> 218 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-409-3p BS seq <400> 218 aauguu 6 <210> 219 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-378/422a/378bcdefhi BS seq <400> 219 cuggaa 6 <210> 220 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-342-3p BS seq <400> 220 cucacc 6 <210> 221 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-338-5p BS seq <400> 221 acaauu 6 <210> 222 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-625 BS seq <400> 222 gggggg 6 <210> 223 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-200bc/429/548a BS seq <400> 223 aauacc 6 <210> 224 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-376a-5p BS seq <400> 224 uagauu 6 <210> 225 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-584 BS seq <400> 225 uauggg 6 <210> 226 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-411 BS seq <400> 226 aguagg 6 <210> 227 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-573/3533/3616-5p/3647-5p BS seq <400> 227 ugaagg 6 <210> 228 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-885-5p BS seq <400> 228 ccauuu 6 <210> 229 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-99-3p BS seq <400> 229 aagcuu 6 <210> 230 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-876-3p BS seq <400> 230 gguggg 6 <210> 231 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-654-3p BS seq <400> 231 augucc 6 <210> 232 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-340-3p BS seq <400> 232 ccgucc 6 <210> 233 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-3614-5p BS seq <400> 233 cacuuu 6 <210> 234 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-124-5p BS seq <400> 234 guguuu 6 <210> 235 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-491-5p BS seq <400> 235 gugggg 6 <210> 236 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-96/507/1271 BS seq <400> 236 uuggcc 6 <210> 237 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-548a-3p/548ef/2285a BS seq <400> 237 aaaacc 6 <210> 238 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-32-3p BS seq <400> 238 aauuuu 6 <210> 239 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-3942-5p/4703-5p BS seq <400> 239 agcaaa 6 <210> 240 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-34b/449c/1360/2682 BS seq <400> 240 aggcaa 6 <210> 241 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-23a/b-5p BS seq <400> 241 ggguuu 6 <210> 242 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-362-5p/500b BS seq <400> 242 auccuu 6 <210> 243 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-677/4420 BS seq <400> 243 ucacuu 6 <210> 244 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-577 BS seq <400> 244 agauaa 6 <210> 245 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-3613-5p BS seq <400> 245 guuguu 6 <210> 246 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-369-5p BS seq <400> 246 gaucgg 6 <210> 247 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-150/5127 BS seq <400> 247 cucccc 6 <210> 248 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-544/544ab/544-3p BS seq <400> 248 uucugg 6 <210> 249 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-29a-5p BS seq <400> 249 cugauu 6 <210> 250 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-873 BS seq <400> 250 caggaa 6 <210> 251 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-3614-3p BS seq <400> 251 agccuu 6 <210> 252 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-186 BS seq <400> 252 aaagaa 6 <210> 253 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-483-3p BS seq <400> 253 cacucc 6 <210> 254 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-374a-3p BS seq <400> 254 uuaucc 6 <210> 255 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-196abc BS seq <400> 255 agguaa 6 <210> 256 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-29b-2-5p BS seq <400> 256 gauucc 6 <210> 257 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-29b-2-5p BS seq <400> 257 ugguuu 6 <210> 258 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-221-5p BS seq <400> 258 ccuggg 6 <210> 259 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-323b-3p BS seq <400> 259 ccaauu 6 <210> 260 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-616 BS seq <400> 260 gucauu 6 <210> 261 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-330-3p BS seq <400> 261 caaagg 6 <210> 262 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-7-3p BS seq <400> 262 aacaaa 6 <210> 263 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-187 BS seq <400> 263 cguguu 6 <210> 264 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-26a-3p BS seq <400> 264 cuauuu 6 <210> 265 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-452/4676-3p BS seq <400> 265 acuguu 6 <210> 266 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-129-5p/129ab-5p BS seq <400> 266 uuuuuu 6 <210> 267 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-223 BS seq <400> 267 gucagg 6 <210> 268 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-4755-3p BS seq <400> 268 gccagg 6 <210> 269 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-1247 BS seq <400> 269 cccguu 6 <210> 270 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-3129-3p BS seq <400> 270 aacuaa 6 <210> 271 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-335-3p BS seq <400> 271 uuuucc 6 <210> 272 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-542-5p BS seq <400> 272 cggggg 6 <210> 273 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-181a-3p BS seq <400> 273 ccaucc 6 <210> 274 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-186-3p BS seq <400> 274 cccaaa 6 <210> 275 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-27b-5p BS seq <400> 275 gagcuu 6 <210> 276 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-491-3p BS seq <400> 276 uuaugg 6 <210> 277 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-4687-3p BS seq <400> 277 ggcugg 6 <210> 278 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-101-5p BS seq <400> 278 aguuaa 6 <210> 279 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-4772-5p BS seq <400> 279 gaucaa 6 <210> 280 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-337-3p BS seq <400> 280 uccuaa 6 <210> 281 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-223-5p BS seq <400> 281 guguaa 6 <210> 282 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-16/195-3p BS seq <400> 282 caauaa 6 <210> 283 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-3677-3p BS seq <400> 283 ucgugg 6 <210> 284 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-766-5p BS seq <400> 284 ggaggg 6 <210> 285 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-299/299-3p/3563-3p BS seq <400> 285 augugg 6 <210> 286 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-3140-3p BS seq <400> 286 gcuuuu 6 <210> 287 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-532-5p/511 BS seq <400> 287 augccc 6 <210> 288 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-24-5p BS seq <400> 288 gccuaa 6 <210> 289 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-4778-5p BS seq <400> 289 auucuu 6 <210> 290 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-642b BS seq <400> 290 gacacc 6 <210> 291 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-483-5p BS seq <400> 291 agacgg 6 <210> 292 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-767-5p BS seq <400> 292 gcaccc 6 <210> 293 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-31-3p BS seq <400> 293 gcuauu 6 <210> 294 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-574-3p BS seq <400> 294 acgcuu 6 <210> 295 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-3173-3p BS seq <400> 295 aaggaa 6 <210> 296 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-2127/4728-5p BS seq <400> 296 gggagg 6 <210> 297 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-103a-2-5p BS seq <400> 297 gcuucc 6 <210> 298 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-3591-3p BS seq <400> 298 aacacc 6 <210> 299 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-625-3p BS seq <400> 299 acuauu 6 <210> 300 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-15b-3p BS seq <400> 300 gaaucc 6 <210> 301 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-522/518e/1422p BS seq <400> 301 aaaugg 6 <210> 302 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-548d-3p/548acbz BS seq <400> 302 aaaaaa 6 <210> 303 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-452-3p BS seq <400> 303 ucaucc 6 <210> 304 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-192/215 BS seq <400> 304 ugaccc 6 <210> 305 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-1551/4524 BS seq <400> 305 uagcaa 6 <210> 306 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-425-3p BS seq <400> 306 ucgggg 6 <210> 307 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-3126-3p BS seq <400> 307 aucugg 6 <210> 308 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-125b-2-3p BS seq <400> 308 cacaaa 6 <210> 309 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-324-3p/1913 BS seq <400> 309 cugccc 6 <210> 310 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-141-5p BS seq <400> 310 aucuuu 6 <210> 311 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-365a/b-5p BS seq <400> 311 gggacc 6 <210> 312 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-29b-1-5p BS seq <400> 312 cugguu 6 <210> 313 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-563/380-5p BS seq <400> 313 gguugg 6 <210> 314 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-1304 BS seq <400> 314 uugagg 6 <210> 315 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-216c/1461/4684-5p BS seq <400> 315 ucucuu 6 <210> 316 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-2681-5p BS seq <400> 316 uuuuaa 6 <210> 317 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-194 BS seq <400> 317 guaacc 6 <210> 318 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-296-3p BS seq <400> 318 aggguu 6 <210> 319 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-205-3p BS seq <400> 319 auuucc 6 <210> 320 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-888 BS seq <400> 320 acucaa 6 <210> 321 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-4802-3p BS seq <400> 321 acaugg 6 <210> 322 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-let-7a/g-3p BS seq <400> 322 uguacc 6 <210> 323 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-762/4492/4498 BS seq <400> 323 gggcuu 6 <210> 324 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-744-3p BS seq <400> 324 uguugg 6 <210> 325 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-148b-5p BS seq <400> 325 aguucc 6 <210> 326 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-514/514b-3p BS seq <400> 326 uugacc 6 <210> 327 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-28-3p BS seq <400> 327 acuagg 6 <210> 328 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-550a BS seq <400> 328 gugccc 6 <210> 329 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-125b-1-3p BS seq <400> 329 cggguu 6 <210> 330 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-506-5p BS seq <400> 330 auucaa 6 <210> 331 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-1306-5p BS seq <400> 331 caccuu 6 <210> 332 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-3189-3p BS seq <400> 332 ccuugg 6 <210> 333 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-675-5p/4466 BS seq <400> 333 ggugcc 6 <210> 334 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-34a-3p BS seq <400> 334 aaucaa 6 <210> 335 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-454-5p BS seq <400> 335 cccuaa 6 <210> 336 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-509-5p/509-3-5p/4418 BS seq <400> 336 acugcc 6 <210> 337 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-19a/b-5p BS seq <400> 337 guuuuu 6 <210> 338 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-4755-5p BS seq <400> 338 uucccc 6 <210> 339 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-93-3p BS seq <400> 339 cugcuu 6 <210> 340 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-3130-5p/4482 BS seq <400> 340 acccaa 6 <210> 341 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-488-5p BS seq <400> 341 ccagaa 6 <210> 342 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-378a-5p BS seq <400> 342 uccugg 6 <210> 343 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-575/4676-5p BS seq <400> 343 agccaa 6 <210> 344 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-1307 BS seq <400> 344 cucggg 6 <210> 345 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-3942-3p BS seq <400> 345 uucagg 6 <210> 346 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-4677-5p BS seq <400> 346 uguucc 6 <210> 347 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-339-3p BS seq <400> 347 gagcgg 6 <210> 348 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-548b-3p BS seq <400> 348 aagaaa 6 <210> 349 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-642b-5p BS seq <400> 349 guuccc 6 <210> 350 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-188-5p BS seq <400> 350 aucccc 6 <210> 351 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-652-5p BS seq <400> 351 aacccc 6 <210> 352 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-2114 BS seq <400> 352 aguccc 6 <210> 353 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-3688-5p BS seq <400> 353 guggcc 6 <210> 354 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-15a-3p BS seq <400> 354 aggccc 6 <210> 355 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-181c-3p BS seq <400> 355 accauu 6 <210> 356 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-122/122a/1352 BS seq <400> 356 ggaguu 6 <210> 357 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-556-3p BS seq <400> 357 uauuaa 6 <210> 358 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-218-2-3p BS seq <400> 358 augguu 6 <210> 359 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-643 BS seq <400> 359 cuuguu 6 <210> 360 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-140-3p BS seq <400> 360 accacc 6 <210> 361 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-1245 BS seq <400> 361 agugaa 6 <210> 362 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-2115-3p BS seq <400> 362 aucagg 6 <210> 363 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-518bcf/518a-3p/518d-3p BS seq <400> 363 aaagcc 6 <210> 364 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-3200-3p BS seq <400> 364 accuuu 6 <210> 365 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-545/3065/3065-5p BS seq <400> 365 caacaa 6 <210> 366 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-1903/4778-3p BS seq <400> 366 cuucuu 6 <210> 367 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-302a-5p BS seq <400> 367 cuuaaa 6 <210> 368 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-183-3p BS seq <400> 368 ugaauu 6 <210> 369 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-3144-5p BS seq <400> 369 ggggaa 6 <210> 370 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-582-3p BS seq <400> 370 aacugg 6 <210> 371 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-4662a-3p BS seq <400> 371 aagauu 6 <210> 372 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-3140-5p BS seq <400> 372 ccugaa 6 <210> 373 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-106a-3p BS seq <400> 373 ugcaaa 6 <210> 374 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-135a-3p BS seq <400> 374 auaggg 6 <210> 375 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-345/345-5p BS seq <400> 375 cugacc 6 <210> 376 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-125a-3p/1554 BS seq <400> 376 cagguu 6 <210> 377 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-3145-5p BS seq <400> 377 acuccc 6 <210> 378 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-676 BS seq <400> 378 uguccc 6 <210> 379 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-3173-5p BS seq <400> 379 gcccuu 6 <210> 380 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-5586-3p BS seq <400> 380 agaguu 6 <210> 381 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-615-3p BS seq <400> 381 ccgagg 6 <210> 382 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-3688-3p BS seq <400> 382 auggaa 6 <210> 383 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-4662a-5p BS seq <400> 383 uagccc 6 <210> 384 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-4659ab-5p BS seq <400> 384 ugccaa 6 <210> 385 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-5586-5p BS seq <400> 385 auccaa 6 <210> 386 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-514a-5p BS seq <400> 386 acucuu 6 <210> 387 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-10abc/10a-5p BS seq <400> 387 acccuu 6 <210> 388 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-888-3p BS seq <400> 388 acugaa 6 <210> 389 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-3127-5p BS seq <400> 389 ucaggg 6 <210> 390 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-508-3p BS seq <400> 390 gauugg 6 <210> 391 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-185-3p BS seq <400> 391 ggggcc 6 <210> 392 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-200c-5p,hsa-miR-550a-3p BS seq <400> 392 gucuuu 6 <210> 393 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-513c/514b-5p BS seq <400> 393 ucucaa 6 <210> 394 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-490-3p BS seq <400> 394 aaccuu 6 <210> 395 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-5187-3p BS seq <400> 395 cugaaa 6 <210> 396 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-3664-3p BS seq <400> 396 cucagg 6 <210> 397 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-3189-5p BS seq <400> 397 gccccc 6 <210> 398 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-4670-3p BS seq <400> 398 gaaguu 6 <210> 399 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-105/105ab BS seq <400> 399 caaauu 6 <210> 400 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-135b-3p BS seq <400> 400 uguagg 6 <210> 401 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-5010-3p BS seq <400> 401 uuuguu 6 <210> 402 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-493/493b BS seq <400> 402 gaaggg 6 <210> 403 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-3605-3p BS seq <400> 403 cuccgg 6 <210> 404 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-188-3p BS seq <400> 404 ucccaa 6 <210> 405 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-449c-3p BS seq <400> 405 ugcuaa 6 <210> 406 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-4761-5p BS seq <400> 406 caaggg 6 <210> 407 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-224 BS seq <400> 407 aagucc 6 <210> 408 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-4796-5p BS seq <400> 408 gucuaa 6 <210> 409 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-551b-5p BS seq <400> 409 aaaucc 6 <210> 410 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-556-5p BS seq <400> 410 augagg 6 <210> 411 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-122-3p BS seq <400> 411 acgccc 6 <210> 412 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-4677-3p BS seq <400> 412 cugugg 6 <210> 413 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-877 BS seq <400> 413 uagagg 6 <210> 414 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-576-5p BS seq <400> 414 uucuaa 6 <210> 415 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-490-5p BS seq <400> 415 cauggg 6 <210> 416 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-589-3p BS seq <400> 416 cagaaa 6 <210> 417 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-4786-3p BS seq <400> 417 gaagcc 6 <210> 418 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-374b-3p BS seq <400> 418 uuagcc 6 <210> 419 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-26b-3p BS seq <400> 419 cuguuu 6 <210> 420 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-3158-3p BS seq <400> 420 agggcc 6 <210> 421 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-4423-3p BS seq <400> 421 uaggcc 6 <210> 422 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-518d-5p/519bc-5p520c-5p/523b/526a BS seq <400> 422 ucuagg 6 <210> 423 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-4707-3p BS seq <400> 423 gcccgg 6 <210> 424 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-10a-3p BS seq <400> 424 aaauuu 6 <210> 425 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-526b BS seq <400> 425 ucuugg 6 <210> 426 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-676-5p BS seq <400> 426 cuucaa 6 <210> 427 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-660-3p BS seq <400> 427 ccuccc 6 <210> 428 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-5004-3p BS seq <400> 428 uuggaa 6 <210> 429 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-193a-5p BS seq <400> 429 gggucc 6 <210> 430 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-222-5p BS seq <400> 430 ucaguu 6 <210> 431 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-4661-3p BS seq <400> 431 aggauu 6 <210> 432 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-25-5p BS seq <400> 432 ggcggg 6 <210> 433 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-4670-5p BS seq <400> 433 agcgaa 6 <210> 434 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-659 BS seq <400> 434 uugguu 6 <210> 435 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-1745/3194-3p BS seq <400> 435 gcucuu 6 <210> 436 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-182-3p BS seq <400> 436 gguucc 6 <210> 437 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-298/2347/2467-3p BS seq <400> 437 gcagaa 6 <210> 438 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-130b-5p BS seq <400> 438 cucuuu 6 <210> 439 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-4746-3p BS seq <400> 439 gcgguu 6 <210> 440 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-1893/2277-5p BS seq <400> 440 gcgcgg 6 <210> 441 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-3619-3p BS seq <400> 441 ggaccc 6 <210> 442 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-138-1-3p BS seq <400> 442 cuacuu 6 <210> 443 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-4728-3p BS seq <400> 443 augcuu 6 <210> 444 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-3127-3p BS seq <400> 444 ccccuu 6 <210> 445 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-671-3p BS seq <400> 445 ccgguu 6 <210> 446 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-211-3p BS seq <400> 446 cagggg 6 <210> 447 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-2114-3p BS seq <400> 447 gagccc 6 <210> 448 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-877-3p BS seq <400> 448 ccucuu 6 <210> 449 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-3157-5p BS seq <400> 449 ucagcc 6 <210> 450 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-502-5p BS seq <400> 450 uccuuu 6 <210> 451 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-500a BS seq <400> 451 aauccc 6 <210> 452 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-548g BS seq <400> 452 aaacuu 6 <210> 453 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-523 BS seq <400> 453 aacgcc 6 <210> 454 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-584-3p BS seq <400> 454 caguuu 6 <210> 455 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-205/205ab BS seq <400> 455 ccuucc 6 <210> 456 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-4793-5p BS seq <400> 456 cauccc 6 <210> 457 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-363-5p BS seq <400> 457 gggugg 6 <210> 458 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-214-5p BS seq <400> 458 gccugg 6 <210> 459 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-3180-5p BS seq <400> 459 uuccaa 6 <210> 460 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-1404/2110 BS seq <400> 460 uggggg 6 <210> 461 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-3157-3p BS seq <400> 461 ugcccc 6 <210> 462 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-191-3p BS seq <400> 462 cugcgg 6 <210> 463 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-1346/3940-5p/4507 BS seq <400> 463 uggguu 6 <210> 464 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-4746-5p BS seq <400> 464 cggucc 6 <210> 465 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-3939 BS seq <400> 465 acgcgg 6 <210> 466 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-181a-2-3p BS seq <400> 466 ccacuu 6 <210> 467 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-500a-3p BS seq <400> 467 ugcacc 6 <210> 468 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-196b-3p BS seq <400> 468 cgacaa 6 <210> 469 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-675-3p BS seq <400> 469 uguauu 6 <210> 470 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-548aj/g/x-5p BS seq <400> 470 gcaaaa 6 <210> 471 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-4659ab-3p BS seq <400> 471 uucuuu 6 <210> 472 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-5001-3p BS seq <400> 472 ucugcc 6 <210> 473 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-1247-3p BS seq <400> 473 cccggg 6 <210> 474 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-2890/4707-5p BS seq <400> 474 ccccgg 6 <210> 475 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-150-3p BS seq <400> 475 ugguaa 6 <210> 476 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-629-3p BS seq <400> 476 uucucc 6 <210> 477 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-2277-3p BS seq <400> 477 gacagg 6 <210> 478 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-3547/3663-3p BS seq <400> 478 gagcaa 6 <210> 479 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-34bc-3p BS seq <400> 479 aucacc 6 <210> 480 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-518ef BS seq <400> 480 aagcgg 6 <210> 481 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-3187-3p BS seq <400> 481 uggccc 6 <210> 482 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-1306/1306-3p BS seq <400> 482 cguugg 6 <210> 483 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-3177-3p BS seq <400> 483 gcacgg 6 <210> 484 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-1ab/206/613 BS seq <400> 484 ggaauu 6 <210> 485 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-128/128ab BS seq <400> 485 cacagg 6 <210> 486 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-1296 BS seq <400> 486 uagggg 6 <210> 487 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-598/598-3p BS seq <400> 487 acgucc 6 <210> 488 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-887 BS seq <400> 488 ugaacc 6 <210> 489 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-1-5p BS seq <400> 489 cauacc 6 <210> 490 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-376c/741-5p BS seq <400> 490 acauaa 6 <210> 491 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-374c/655 BS seq <400> 491 uaauaa 6 <210> 492 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-494 BS seq <400> 492 gaaacc 6 <210> 493 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-651 BS seq <400> 493 uuaggg 6 <210> 494 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-1301/5047 BS seq <400> 494 ugcagg 6 <210> 495 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-381-5p BS seq <400> 495 gcgagg 6 <210> 496 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-216a BS seq <400> 496 aaucuu 6 <210> 497 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-300/381/539-3p BS seq <400> 497 auacaa 6 <210> 498 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-1249 BS seq <400> 498 cgcccc 6 <210> 499 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-579 BS seq <400> 499 ucauuu 6 <210> 500 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-656 BS seq <400> 500 auauuu 6 <210> 501 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-433 BS seq <400> 501 ucaugg 6 <210> 502 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-1180 BS seq <400> 502 uuccgg 6 <210> 503 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-597/1970 BS seq <400> 503 gugucc 6 <210> 504 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-190a-3p BS seq <400> 504 uauauu 6 <210> 505 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-1537 BS seq <400> 505 aaaccc 6 <210> 506 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-874-5p BS seq <400> 506 ggcccc 6 <210> 507 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-410/344de/344b-1-3p BS seq <400> 507 auauaa 6 <210> 508 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-370 BS seq <400> 508 ccugcc 6 <210> 509 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-219-2-3p/219-3p BS seq <400> 509 gaauuu 6 <210> 510 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-3620 BS seq <400> 510 cacccc 6 <210> 511 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-504/4725-5p BS seq <400> 511 gacccc 6 <210> 512 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-2964/2964a-5p BS seq <400> 512 gauguu 6 <210> 513 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-450a-2-3p BS seq <400> 513 uugggg 6 <210> 514 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-511 BS seq <400> 514 ugucuu 6 <210> 515 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-6505-3p BS seq <400> 515 gacuuu 6 <210> 516 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-433-5p BS seq <400> 516 acgguu 6 <210> 517 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-6741-3p BS seq <400> 517 cggcuu 6 <210> 518 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-370-5p BS seq <400> 518 aggucc 6 <210> 519 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-579-5p BS seq <400> 519 cgcggg 6 <210> 520 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-376c-5p,miR-376b-5p BS seq <400> 520 guggaa 6 <210> 521 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-552/3097-5p BS seq <400> 521 acaggg 6 <210> 522 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-1910 BS seq <400> 522 cagucc 6 <210> 523 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-758 BS seq <400> 523 uugugg 6 <210> 524 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-6735-3p BS seq <400> 524 ggccuu 6 <210> 525 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-376a-2-5p BS seq <400> 525 guagaa 6 <210> 526 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-585 BS seq <400> 526 gggcgg 6 <210> 527 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-451 BS seq <400> 527 aaccgg 6 <210> 528 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> miR-137/137ab BS seq <400> 528 uauugg 6 <210> 529 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-1-3p G2U <400> 529 ugaaug 6 <210> 530 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-194-5p G2U <400> 530 uuaaca 6 <210> 531 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-193a-5p G2U <400> 531 uggucu 6 <210> 532 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-15b-3p G2U <400> 532 uaauca 6 <210> 533 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-200c-5p G2U <400> 533 uucuua 6 <210> 534 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-214-5p G2U <400> 534 uccugu 6 <210> 535 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-134-5p G2U <400> 535 uugacu 6 <210> 536 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-145-3p G2U <400> 536 uauucc 6 <210> 537 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-22-5p G2U <400> 537 uuucuu 6 <210> 538 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-423-3p G2U <400> 538 ucucgg 6 <210> 539 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-873-3p G2U <400> 539 uagacu 6 <210> 540 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-122-5p G2U <400> 540 ugagug 6 <210> 541 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-143-3p G2U <400> 541 uagaug 6 <210> 542 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-485-5p G2U <400> 542 uaggcu 6 <210> 543 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-409-5p G2U <400> 543 uguuac 6 <210> 544 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-24-3p G2U <400> 544 ugcuca 6 <210> 545 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-223-3p G2U <400> 545 uucagu 6 <210> 546 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-144-5p G2U <400> 546 uauauc 6 <210> 547 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-379-5p G2U <400> 547 uguaga 6 <210> 548 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-146b-5p/hsa-miR-146a-5p G2U <400> 548 uagaac 6 <210> 549 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-539-5p G2U <400> 549 uagaaa 6 <210> 550 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-296-5p G2U <400> 550 uggccc 6 <210> 551 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-767-5p G2U <400> 551 ucacca 6 <210> 552 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-34a-5p/hsa-miR-34c-5p G2U <400> 552 ugcagu 6 <210> 553 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-let-7f-5p/hsa-let-7d-5p/hsa-let-7b-5p/hsa-let-7a-5p/hsa-let-7 e-5p/hsa-miR-202-3p/hsa-let-7i-5p/hsa-miR-98-5p/hsa-let-7c-5p/hsa -let-7g-5p G2U <400> 553 uaggua 6 <210> 554 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-1271-3p G2U <400> 554 uugccu 6 <210> 555 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-138-5p G2U <400> 555 ucuggu 6 <210> 556 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-19b-3p/hsa-miR-19a-3p G2U <400> 556 uugcaa 6 <210> 557 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-27a-5p G2U <400> 557 uggcuu 6 <210> 558 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-146b-3p G2U <400> 558 ucccug 6 <210> 559 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-7-5p G2U <400> 559 ugaaga 6 <210> 560 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-423-5p G2U <400> 560 uagggg 6 <210> 561 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-324-5p G2U <400> 561 ucaucc 6 <210> 562 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-629-5p G2U <400> 562 ugguuu 6 <210> 563 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-139-3p G2U <400> 563 ugagac 6 <210> 564 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-30d-5p/hsa-miR-30e-5p/hsa-miR-30a-5p/hsa-miR-30c-5p/hsa-m iR-30b-5p G2U <400> 564 uuaaac 6 <210> 565 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-221-3p/hsa-miR-222-3p G2U <400> 565 ucuaca 6 <210> 566 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-509-3p G2U <400> 566 uauugg 6 <210> 567 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-769-5p G2U <400> 567 uagacc 6 <210> 568 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-142-3p G2U <400> 568 uuagug 6 <210> 569 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-185-5p G2U <400> 569 ugagag 6 <210> 570 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-508-3p/hsa-miR-219a-5p G2U <400> 570 uauugu 6 <210> 571 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-31-5p G2U <400> 571 ugcaag 6 <210> 572 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-103a-3p/hsa-miR-107 G2U <400> 572 ucagca 6 <210> 573 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-542-3p G2U <400> 573 uugaca 6 <210> 574 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-219a-2-3p G2U <400> 574 uaauug 6 <210> 575 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-29c-3p/hsa-miR-29a-3p/hsa-miR-29b-3p G3U <400> 575 aucacc 6 <210> 576 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-125b-1-3p G3U <400> 576 cugguu 6 <210> 577 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-7-5p G3U <400> 577 guaaga 6 <210> 578 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-411-5p G3U <400> 578 auuaga 6 <210> 579 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-196a-5p/hsa-miR-196b-5p G3U <400> 579 auguag 6 <210> 580 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-3622a-5p G3U <400> 580 augcac 6 <210> 581 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-127-5p G3U <400> 581 uuaagc 6 <210> 582 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-22-3p G3U <400> 582 aucugc 6 <210> 583 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-153-3p G3U <400> 583 uucaua 6 <210> 584 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-193a-5p G3U <400> 584 gugucu 6 <210> 585 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-185-5p G3U <400> 585 guagag 6 <210> 586 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-1-3p G3U <400> 586 guaaug 6 <210> 587 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-15b-5p/hsa-miR-16-5p/hsa-miR-424-5p G3U <400> 587 aucagc 6 <210> 588 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-let-7g-3p/hsa-miR-493-5p/hsa-let-7c-3p G3U <400> 588 uuuaca 6 <210> 589 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-let-7i-3p G3U <400> 589 uucgca 6 <210> 590 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-218-5p G3U <400> 590 uuugcu 6 <210> 591 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-1307-5p G3U <400> 591 cuaccg 6 <210> 592 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-127-3p G3U <400> 592 cugauc 6 <210> 593 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-210-3p G3U <400> 593 uuugcg 6 <210> 594 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-187-3p G3U <400> 594 cuuguc 6 <210> 595 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-192-3p G3U <400> 595 uuccaa 6 <210> 596 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-192-5p G3U <400> 596 uuaccu 6 <210> 597 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-34a-5p/hsa-miR-34c-5p G3U <400> 597 gucagu 6 <210> 598 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-21-5p G3U <400> 598 aucuua 6 <210> 599 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-500a-3p G3U <400> 599 uucacc 6 <210> 600 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-629-5p G3U <400> 600 guguuu 6 <210> 601 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-379-5p G3U <400> 601 guuaga 6 <210> 602 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-203a-3p G3U <400> 602 uuaaau 6 <210> 603 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-24-3p G3U <400> 603 gucuca 6 <210> 604 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-30c-2-3p G3U <400> 604 uuggag 6 <210> 605 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-488-3p G3U <400> 605 uuaaag 6 <210> 606 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-301a-3p/hsa-miR-301b-3p G3U <400> 606 auugca 6 <210> 607 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-126-3p G3U <400> 607 cuuacc 6 <210> 608 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-122-5p G3U <400> 608 guagug 6 <210> 609 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-27a-5p G3U <400> 609 gugcuu 6 <210> 610 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-26b-3p G4U <400> 610 cuuuuc 6 <210> 611 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-324-3p G4U <400> 611 cuuccc 6 <210> 612 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-3065-3p G4U <400> 612 caucac 6 <210> 613 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-423-5p G4U <400> 613 gauggg 6 <210> 614 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-124-5p G4U <400> 614 guuuuc 6 <210> 615 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-345-5p G4U <400> 615 cuuacu 6 <210> 616 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-615-3p G4U <400> 616 ccuagc 6 <210> 617 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-889-5p/hsa-miR-135a-5p/hsa-miR-135b-5p G4U <400> 617 auugcu 6 <210> 618 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-193a-5p G4U <400> 618 gguucu 6 <210> 619 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-18a-5p G4U <400> 619 aaugug 6 <210> 620 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-125b-1-3p G4U <400> 620 cguguu 6 <210> 621 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-708-5p/hsa-miR-28-5p G4U <400> 621 aguagc 6 <210> 622 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-224-5p G4U <400> 622 aauuca 6 <210> 623 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-100-3p G4U <400> 623 aaucuu 6 <210> 624 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-873-5p G4U <400> 624 caugaa 6 <210> 625 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-4662a-5p G4U <400> 625 uaucca 6 <210> 626 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-99b-3p/hsa-miR-99a-3p G4U <400> 626 aaucuc 6 <210> 627 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-433-5p G4U <400> 627 acugug 6 <210> 628 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-542-3p G4U <400> 628 guuaca 6 <210> 629 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-3605-5p G4U <400> 629 gaugau 6 <210> 630 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-744-5p G4U <400> 630 gcuggg 6 <210> 631 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-1296-5p G4U <400> 631 uauggc 6 <210> 632 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-133a-3p G4U <400> 632 uuuguc 6 <210> 633 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-382-5p G4U <400> 633 aauuug 6 <210> 634 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-425-5p G4U <400> 634 auuaca 6 <210> 635 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-377-5p G4U <400> 635 gauguu 6 <210> 636 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-127-3p G4U <400> 636 cguauc 6 <210> 637 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-3180-3p G4U <400> 637 ggugcg 6 <210> 638 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-143-3p G4U <400> 638 gauaug 6 <210> 639 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-758-3p G4U <400> 639 uuuuga 6 <210> 640 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-93-3p G4U <400> 640 cuucug 6 <210> 641 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-128-2-5p G4U <400> 641 ggugcc 6 <210> 642 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-134-5p G4U <400> 642 guuacu 6 <210> 643 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-154-5p G4U <400> 643 aguuua 6 <210> 644 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-3622a-5p G4U <400> 644 agucac 6 <210> 645 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-124-3p G4U <400> 645 aaugca 6 <210> 646 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-27a-5p G4U <400> 646 ggucuu 6 <210> 647 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-194-3p G4U <400> 647 cauugg 6 <210> 648 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-375 G4U <400> 648 uuuuuc 6 <210> 649 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-148a-5p G4U <400> 649 aauuuc 6 <210> 650 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-2277-5p G4U <400> 650 gcucgg 6 <210> 651 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-769-5p G4U <400> 651 gauacc 6 <210> 652 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-17-3p G4U <400> 652 cuucag 6 <210> 653 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-873-3p G4U <400> 653 gauacu 6 <210> 654 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-4772-3p G4U <400> 654 cuucaa 6 <210> 655 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-329-5p G4U <400> 655 aguuuu 6 <210> 656 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-182-5p/hsa-miR-96-5p G4U <400> 656 uuugca 6 <210> 657 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-2467-5p/hsa-miR-485-5p G4U <400> 657 gaugcu 6 <210> 658 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-149-5p G4U <400> 658 cuugcu 6 <210> 659 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-29b-2-5p G4U <400> 659 uguuuu 6 <210> 660 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-122-3p G4U <400> 660 acucca 6 <210> 661 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-302a-3p/hsa-miR-520a-3p/hsa-miR-519b-3p/hsa-miR-520b/hsa- miR-519c-3p/hsa-miR-520c-3p/hsa-miR-519a-3p G4U <400> 661 aauugc 6 <210> 662 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-532-5p G4U <400> 662 auuccu 6 <210> 663 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-132-5p G4U <400> 663 ccuugg 6 <210> 664 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-541-5p G4U <400> 664 aaugau 6 <210> 665 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-671-3p G4U <400> 665 ccuguu 6 <210> 666 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-296-5p G4U <400> 666 gguccc 6 <210> 667 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-518e-3p G4U <400> 667 aaucgc 6 <210> 668 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-487a-5p G4U <400> 668 uguuua 6 <210> 669 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-589-5p/hsa-miR-146b-5p/hsa-miR-146a-5p G4U <400> 669 gauaac 6 <210> 670 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-196b-5p/hsa-miR-196a-5p G4U <400> 670 aguuag 6 <210> 671 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-486-3p G4U <400> 671 ggugca 6 <210> 672 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-629-5p G4U <400> 672 gguuuu 6 <210> 673 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-378a-3p G4U <400> 673 cuugac 6 <210> 674 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-27b-5p G4U <400> 674 gaucuu 6 <210> 675 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-6720-3p G4U <400> 675 gcuccu 6 <210> 676 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-574-3p G4U <400> 676 acucuc 6 <210> 677 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-29a-5p G4U <400> 677 cuuauu 6 <210> 678 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-30c-2-3p/hsa-miR-30c-1-3p G4U <400> 678 ugugag 6 <210> 679 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-199b-3p G4U <400> 679 cauuag 6 <210> 680 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-574-5p G4U <400> 680 gauugu 6 <210> 681 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-539-5p G4U <400> 681 gauaaa 6 <210> 682 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-4677-3p G4U <400> 682 cuuuga 6 <210> 683 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-654-3p G4U <400> 683 auuucu 6 <210> 684 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-652-3p G4U <400> 684 auugcg 6 <210> 685 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-19a-3p/hsa-miR-19b-3p G4U <400> 685 guucaa 6 <210> 686 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-let-7c-5p/hsa-miR-98-5p/hsa-let-7g-5p/hsa-let-7f-5p/hsa-miR-2 02-3p/hsa-let-7b-5p/hsa-let-7e-5p/hsa-let-7a-5p/hsa-let-7d-5p/hsa -let-7i-5p G4U <400> 686 gaugua 6 <210> 687 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-3663-3p G4U <400> 687 gaucac 6 <210> 688 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-152-3p/hsa-miR-148b-3p/hsa-miR-148a-3p G4U <400> 688 cauugc 6 <210> 689 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-193b-5p G4U <400> 689 gguguu 6 <210> 690 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-502-3p/hsa-miR-501-3p G4U <400> 690 auucac 6 <210> 691 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-299-3p G4U <400> 691 auuugg 6 <210> 692 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-140-5p G5U <400> 692 aguugu 6 <210> 693 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-96-5p/hsa-miR-182-5p G5U <400> 693 uuguca 6 <210> 694 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-193b-3p G5U <400> 694 acuugc 6 <210> 695 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-365a-3p G5U <400> 695 aauucc 6 <210> 696 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-486-5p G5U <400> 696 ccuuua 6 <210> 697 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-125b-1-3p G5U <400> 697 cgguuu 6 <210> 698 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-210-3p G5U <400> 698 uguucg 6 <210> 699 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-493-3p G5U <400> 699 gaaugu 6 <210> 700 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-548am-5p G5U <400> 700 aaauua 6 <210> 701 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-218-5p G5U <400> 701 uguucu 6 <210> 702 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-20b-5p/hsa-miR-20a-5p/hsa-miR-93-5p/hsa-miR-17-5p/hsa-miR -106b-5p G5U <400> 702 aaauug 6 <210> 703 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-541-3p G5U <400> 703 gguugg 6 <210> 704 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-452-5p G5U <400> 704 acuuuu 6 <210> 705 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-221-5p G5U <400> 705 ccuugc 6 <210> 706 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-518f-3p G5U <400> 706 aaaucg 6 <210> 707 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-370-3p G5U <400> 707 ccuucu 6 <210> 708 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-107/hsa-miR-103a-3p G5U <400> 708 gcauca 6 <210> 709 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-122-5p G5U <400> 709 ggauug 6 <210> 710 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-338-3p G5U <400> 710 ccauca 6 <210> 711 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-409-3p G5U <400> 711 aauuuu 6 <210> 712 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-124-3p G5U <400> 712 aaguca 6 <210> 713 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-let-7d-5p/hsa-let-7g-5p/hsa-let-7i-5p/hsa-let-7f-5p/hsa-let-7 e-5p/hsa-let-7a-5p/hsa-let-7b-5p/hsa-let-7c-5p G5U <400> 713 gaguua 6 <210> 714 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-130b-3p/hsa-miR-301a-3p/hsa-miR-130a-3p/hsa-miR-301b-3p G5U <400> 714 aguuca 6 <210> 715 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-512-3p G5U <400> 715 aguucu 6 <210> 716 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-191-5p G5U <400> 716 aacuga 6 <210> 717 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-509-3-5p G5U <400> 717 acuuca 6 <210> 718 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-92a-3p/hsa-miR-92b-3p/hsa-miR-363-3p/hsa-miR-25-3p/hsa-mi R-32-5p G5U <400> 718 auuuca 6 <210> 719 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-18a-5p G5U <400> 719 aaguug 6 <210> 720 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-183-5p G5U <400> 720 auguca 6 <210> 721 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-138-5p G5U <400> 721 gcuugu 6 <210> 722 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-1307-3p G5U <400> 722 cucugc 6 <210> 723 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-423-5p G5U <400> 723 gagugg 6 <210> 724 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-499a-5p/hsa-miR-208a-3p G5U <400> 724 uaauac 6 <210> 725 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-378a-3p G5U <400> 725 cuguac 6 <210> 726 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-186-5p G5U <400> 726 aaauaa 6 <210> 727 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-450b-5p G5U <400> 727 uuuuca 6 <210> 728 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-450a-5p G5U <400> 728 uuuucg 6 <210> 729 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-142-3p G5U <400> 729 guauug 6 <210> 730 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-101-3p/hsa-miR-144-3p G5U <400> 730 acauua 6 <210> 731 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-320a G5U <400> 731 aaaucu 6 <210> 732 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-199b-5p/hsa-miR-199a-5p G5U <400> 732 ccauug 6 <210> 733 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-1296-5p G5U <400> 733 uagugc 6 <210> 734 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-185-5p G5U <400> 734 ggauag 6 <210> 735 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-135a-5p G5U <400> 735 augucu 6 <210> 736 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-411-5p G6U <400> 736 aguaua 6 <210> 737 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-145-5p G6U <400> 737 uccauu 6 <210> 738 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-26a-5p G6U <400> 738 ucaauu 6 <210> 739 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-193b-3p G6U <400> 739 acuguc 6 <210> 740 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-34c-5p G6U <400> 740 ggcauu 6 <210> 741 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-379-5p G6U <400> 741 gguaua 6 <210> 742 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-125b-5p G6U <400> 742 cccuua 6 <210> 743 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-221-5p G6U <400> 743 ccuguc 6 <210> 744 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-526b-5p G6U <400> 744 ucuuua 6 <210> 745 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-7-5p G6U <400> 745 ggaaua 6 <210> 746 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-16-5p/hsa-miR-15b-5p/hsa-miR-424-5p/hsa-miR-15a-5p G6U <400> 746 agcauc 6 <210> 747 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-9-3p G6U <400> 747 uaaauc 6 <210> 748 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-363-5p G6U <400> 748 ggguug 6 <210> 749 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-1298-3p G6U <400> 749 aucuug 6 <210> 750 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-509-3p G6U <400> 750 gauuug 6 <210> 751 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-744-5p G6U <400> 751 gcggug 6 <210> 752 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-148a-3p G6U <400> 752 caguuc 6 <210> 753 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-302a-3p G6U <400> 753 aaguuc 6 <210> 754 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-1296-5p G6U <400> 754 uagguc 6 <210> 755 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-423-5p G6U <400> 755 gaggug 6 <210> 756 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-9-5p G6U <400> 756 cuuuug 6 <210> 757 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-138-5p G6U <400> 757 gcuguu 6 <210> 758 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-22-3p G6U <400> 758 agcuuc 6 <210> 759 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-28-3p G6U <400> 759 acuaua 6 <210> 760 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-508-3p G6U <400> 760 gauuuu 6 <210> 761 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-137 G6U <400> 761 uauuuc 6 <210> 762 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-5010-5p G6U <400> 762 ggggua 6 <210> 763 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-523-5p G6U <400> 763 ucuaua 6 <210> 764 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-191-5p G6U <400> 764 aacgua 6 <210> 765 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-128-3p G6U <400> 765 cacauu 6 <210> 766 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-199a-5p/hsa-miR-199b-5p G7U <400> 766 ccaguu 6 <210> 767 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-181a-2-3p G7U <400> 767 ccacuu 6 <210> 768 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-27a-3p/hsa-miR-27b-3p G7U <400> 768 ucacau 6 <210> 769 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-let-7d-3p G7U <400> 769 uauacu 6 <210> 770 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-129-5p G7U <400> 770 uuuuuu 6 <210> 771 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-210-3p G7U <400> 771 ugugcu 6 <210> 772 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-219a-2-3p G7U <400> 772 gaauuu 6 <210> 773 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-424-3p G7U <400> 773 aaaacu 6 <210> 774 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-18a-5p G7U <400> 774 aagguu 6 <210> 775 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-488-3p G7U <400> 775 ugaaau 6 <210> 776 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-1-3p G7U <400> 776 ggaauu 6 <210> 777 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-181a-3p G7U <400> 777 ccaucu 6 <210> 778 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-199b-3p G7U <400> 778 caguau 6 <210> 779 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-10a-5p G7U <400> 779 acccuu 6 <210> 780 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-196b-5p G7U <400> 780 agguau 6 <210> 781 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-92a-1-5p G7U <400> 781 gguugu 6 <210> 782 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-483-5p G7U <400> 782 agacgu 6 <210> 783 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-17-3p G7U <400> 783 cugcau 6 <210> 784 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-363-5p G7U <400> 784 gggugu 6 <210> 785 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-9-5p G7U <400> 785 cuuugu 6 <210> 786 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-1537-3p G7U <400> 786 aaaccu 6 <210> 787 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-106b-5p/hsa-miR-20a-5p/hsa-miR-17-5p/hsa-miR-93-5p G7U <400> 787 aaaguu 6 <210> 788 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-143-3p G7U <400> 788 gagauu 6 <210> 789 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-30a-3p/hsa-miR-30e-3p G7U <400> 789 uuucau 6 <210> 790 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-423-3p G7U <400> 790 gcucgu 6 <210> 791 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-1298-3p G7U <400> 791 aucugu 6 <210> 792 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-1307-5p G7U <400> 792 cgaccu 6 <210> 793 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-423-5p G7U <400> 793 gagggu 6 <210> 794 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-122-5p G7U <400> 794 ggaguu 6 <210> 795 <211> 7 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-374a-3p G8U <400> 795 uuaucau 7 <210> 796 <211> 7 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-675-5p G8U <400> 796 ggugcgu 7 <210> 797 <211> 7 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-92a-1-5p G8U <400> 797 gguuggu 7 <210> 798 <211> 7 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-1307-5p G8U <400> 798 cgaccgu 7 <210> 799 <211> 7 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-503-5p G8U <400> 799 agcagcu 7 <210> 800 <211> 7 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-24-3p G8U <400> 800 ggcucau 7 <210> 801 <211> 7 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-340-5p G8U <400> 801 uauaaau 7 <210> 802 <211> 7 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-509-3-5p G8U <400> 802 acugcau 7 <210> 803 <211> 7 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-34a-5p/hsa-miR-34c-5p G8U <400> 803 ggcaguu 7 <210> 804 <211> 7 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-526b-5p G8U <400> 804 ucuugau 7 <210> 805 <211> 7 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-203a-3p G8U <400> 805 ugaaauu 7 <210> 806 <211> 7 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-153-3p G8U <400> 806 ugcauau 7 <210> 807 <211> 7 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-208a-3p G8U <400> 807 uaagacu 7 <210> 808 <211> 7 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-200b-3p/hsa-miR-200c-3p G8U <400> 808 aauacuu 7 <210> 809 <211> 7 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-518f-5p/hsa-miR-523-5p G8U <400> 809 ucuagau 7 <210> 810 <211> 7 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-625-3p G8U <400> 810 acuauau 7 <210> 811 <211> 7 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-194-5p G8U <400> 811 guaacau 7 <210> 812 <211> 7 <212> RNA <213> Artificial Sequence <220> <223> hsa-let-7g-3p G8U <400> 812 uguacau 7 <210> 813 <211> 7 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-1298-3p G8U <400> 813 aucuggu 7 <210> 814 <211> 7 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-514a-5p G8U <400> 814 acucugu 7 <210> 815 <211> 7 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-483-5p G8U <400> 815 agacggu 7 <210> 816 <211> 7 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-126-3p G8U <400> 816 cguaccu 7 <210> 817 <211> 7 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-128-3p G8U <400> 817 cacaguu 7 <210> 818 <211> 7 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-381-3p G8U <400> 818 auacaau 7 <210> 819 <211> 7 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-320a G8U <400> 819 aaagcuu 7 <210> 820 <211> 7 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-513c-5p/hsa-miR-514b-5p G8U <400> 820 ucucaau 7 <210> 821 <211> 7 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-138-5p G8U <400> 821 gcugguu 7 <210> 822 <211> 7 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-520a-5p G8U <400> 822 uccagau 7 <210> 823 <211> 7 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-125b-5p/hsa-miR-125a-5p G8U <400> 823 cccugau 7 <210> 824 <211> 7 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-141-3p G8U <400> 824 aacacuu 7 <210> 825 <211> 7 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-874-3p G8U <400> 825 ugcccuu 7 <210> 826 <211> 7 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-202-5p G8U <400> 826 uccuauu 7 <210> 827 <211> 7 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-140-3p G8U <400> 827 accacau 7 <210> 828 <211> 7 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-361-3p G8U <400> 828 cccccau 7 <210> 829 <211> 7 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-513b-5p G8U <400> 829 ucacaau 7 <210> 830 <211> 7 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-134-5p G8U <400> 830 gugacuu 7 <210> 831 <211> 7 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-33a-5p G8U <400> 831 ugcauuu 7 <210> 832 <211> 7 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-512-3p G8U <400> 832 agugcuu 7 <210> 833 <211> 7 <212> RNA <213> Artificial Sequence <220> <223> hsa-let-7a-5p/hsa-let-7c-5p/hsa-let-7b-5p/hsa-let-7d-5p/hsa-let-7 f-5p/hsa-let-7e-5p/hsa-let-7i-5p/hsa-let-7g-5p G8U <400> 833 gagguau 7 <210> 834 <211> 7 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-375 G8U <400> 834 uuguucu 7 <210> 835 <211> 7 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-136-3p G8U <400> 835 aucaucu 7 <210> 836 <211> 7 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-508-5p G8U <400> 836 acuccau 7 <210> 837 <211> 8 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-124-3p G9U <400> 837 aaggcacu 8 <210> 838 <211> 8 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-204-5p/hsa-miR-211-5p G9U <400> 838 ucccuuuu 8 <210> 839 <211> 8 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-210-3p G9U <400> 839 ugugcguu 8 <210> 840 <211> 8 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-146a-5p/hsa-miR-146b-5p G9U <400> 840 gagaacuu 8 <210> 841 <211> 8 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-508-3p G9U <400> 841 gauuguau 8 <210> 842 <211> 8 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-23a-3p G9U <400> 842 ucacauuu 8 <210> 843 <211> 8 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-219a-2-3p G9U <400> 843 gaauuguu 8 <210> 844 <211> 8 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-21-3p G9U <400> 844 aacaccau 8 <210> 845 <211> 8 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-122-5p G9U <400> 845 ggaguguu 8 <210> 846 <211> 8 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-877-5p G9U <400> 846 uagaggau 8 <210> 847 <211> 8 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-129-5p G9U <400> 847 uuuuugcu 8 <210> 848 <211> 8 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-526b-5p G9U <400> 848 ucuugagu 8 <210> 849 <211> 8 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-302a-5p G9U <400> 849 cuuaaacu 8 <210> 850 <211> 8 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-532-5p G9U <400> 850 augccuuu 8 <210> 851 <211> 8 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-520a-5p G9U <400> 851 uccagagu 8 <210> 852 <211> 8 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-139-5p G9U <400> 852 cuacaguu 8 <210> 853 <211> 8 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-99a-5p/hsa-miR-100-5p/hsa-miR-99b-5p G9U <400> 853 acccguau 8 <210> 854 <211> 8 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-320a G9U <400> 854 aaagcugu 8 <210> 855 <211> 8 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-216a-5p G9U <400> 855 aaucucau 8 <210> 856 <211> 8 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-127-3p G9U <400> 856 cggauccu 8 <210> 857 <211> 8 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-125b-1-3p G9U <400> 857 cggguuau 8 <210> 858 <211> 8 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-381-3p G9U <400> 858 auacaagu 8 <210> 859 <211> 8 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-27a-3p/hsa-miR-27b-3p G9U <400> 859 ucacaguu 8 <210> 860 <211> 8 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-5010-5p G9U <400> 860 gggggauu 8 <210> 861 <211> 8 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-3157-3p G9U <400> 861 ugcccuau 8 <210> 862 <211> 8 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-17-3p G9U <400> 862 cugcaguu 8 <210> 863 <211> 8 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-523-5p G9U <400> 863 ucuagagu 8

Claims (5)

As an RNA interference-inducing nucleic acid that suppresses a noncanonical target gene of microRNA by modifying a partial sequence of a specific microRNA in one or more single strands of a nucleic acid that induces RNA interference,
The RNA interference inducing nucleic acid is
RNA interference-inducing nucleic acid, characterized in that the 2nd to 7th nucleotide sequence of the 5'end is at least one of SEQ ID NOs: 103 to 528 shown in the following table:

The method of claim 1,
The RNA interference-inducing nucleic acid selectively inhibits only the non-normal nucleus target site and does not inhibit the normal target gene of the microRNA.
delete A composition for inhibiting expression of a non-normal target gene of microRNA comprising the RNA interference-inducing nucleic acid of claim 1 or 2.
In one or more single strands of a nucleic acid inducing RNA interference, including the following steps, a partial sequence of a specific microRNA is modified to express a noncanonical target gene of a microRNA As a method for producing an RNA interference-inducing nucleic acid that inhibits,
A method for producing an RNA interference-inducing nucleic acid comprising the step of constructing an RNA interference-inducing nucleic acid, characterized in that the second to seventh nucleotide sequence of the 5'end is at least one of SEQ ID NOs: 103 to 528 shown in the following table:

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