KR20090014676A - Small interfering rna (sirna) having a shortened nucleotide sequence - Google Patents

Abstract

The present invention relates to short-length interfering RNAs (siRNA) and uses thereof, and more particularly, interference-inducing RNAs consisting of less than 19 base sequences shorter than the conventional 19 base sequences and The present invention relates to a therapeutic agent for a variety of gene-control related diseases. Since the interference-inducing RNA of the present invention is composed of a small number of nucleotide sequences, the production cost is low, it shows higher gene control efficiency per unit weight, and the clinical application is easy in the preparation of the multi-target interference-inducing RNA. It can be widely used in the treatment of various cancers or viral diseases to replace drugs based on interference-induced RNA.

Description

Short Interference Induction RNA {Small interfering RNA (siRNA) having a shortened nucleotide sequence}

The present invention relates to short length interfering RNA (hereinafter referred to as "siRNA") and its use, more specifically, less than 19 base sequences shorter than the conventional 19 base sequences It relates to interference inducing RNAs consisting of and a therapeutic agent for various gene-control-related diseases using the same.

Small interferencing RNA (siRNA) is a short RNA molecule with a double helix structure of 19-21 bp in size, which is introduced into a cell to cleave intracellular messenger RNA (mRNA) having complementary base sequences and Functions to inhibit expression. This interference inducing RNA (siRNA), along with short hairpin RNA (shRNA), is highly efficient and selectively cleaves the target mRNA via the RNA interference (RNAi) pathway in vivo. [1]. Genetic control technology using such RNA interference induction is much higher in success rate and efficiency compared to various control methods using existing genes. Therefore, it is in the spotlight as a research tool of functional genomics and also as a candidate therapeutic substance such as anticancer agent or antiviral agent against various cancers and viral diseases [2].

The Tuschel group first introduced interference-induced RNA of 19 + 2 structure in 2001, which contains two 3'-two-nucleotide overhangs at the 3'-site of a 19 bp duplex. Produced. In addition, it was confirmed that the interference-inducing RNA can induce specific gene suppression while excluding non-specific gene repression in mammalian cells [3]. Therefore, from now on, most of the experiments on interference-inducing RNA have been carried out using siRNA having the 19 + 2 structure.

Recently, si RNAs having a structure modified from these 19 + 2 siRNAs have been developed. Specifically, a long siRNA or a 29 bp shRNA, such as a 27 bp duplex, has been developed. In addition, these interference-induced RNAs were confirmed to be able to efficiently suppress the expression of target mRNA which was not previously inhibited by gene expression by 19 bp double stranded siRNA [4, 5].

Therefore, various antiviral therapeutics are currently being researched and developed using such interference-induced RNAi. However, siRNAs or shRNAs that target only a portion of the viral genome can induce mutations in the target sequences of viruses with high mutation rates, resulting in viral mutants that are ultimately resistant. Indeed, there have been reported cases of viral mutations [6]. Therefore, there is an urgent need to develop interference-inducing RNA that can lower the drug resistance caused by these mutations.

Recently, in order to overcome the disadvantage of the interference-induced RNA, a multi-target interference-induced RNA (multi-target RNAi) technology that can suppress the expression of a plurality of target genes at the same time has been attempted to develop [7]. Such multi-target RNAi can be usefully used not only for antiviral therapeutics but also for the development of therapeutics for various diseases such as cancer. Indeed, multi-target RNAi can control the growth of cancer cells synergistically by controlling multiple cancer-related pathways in the cell. As such, the basic concept in the control of expression of a plurality of genes in a cell through RNAi is called "combinatorial RNAi", and a lot of researches on this are in progress [7].

To date, most multi-target RNAi have been developed based primarily on short lengths of hairpin RNA (shRNA). Indeed, it has been reported that such shRNAs have been implemented with multi-target RNAi using recombinant expression vectors comprising one or more hairpin structures [8]. However, unlike shRNAs that are introduced into cells to function during gene expression, siRNAs must be manufactured according to chemical synthesis and then introduced into the living body. Therefore, siRNAs are required to be developed into multi-target siRNAs, in particular, through a process of structural modification.

Recently, the Guo group has proposed a method of introducing a plurality of siRNAs into cells using a bacteriophage-based RNA nanostructure called Phi29 RNA [9]. However, this method has a limitation in that it can be developed as a real therapeutic agent because the total size of the RNA structure is too large to facilitate chemical synthesis. In addition, the Tussell Group has developed some complementary siRNAs (sense complement) and sisense (antisense strand) of siRNA, each of which can control two target genes simultaneously by inhibiting different target genes. [10]. However, this method also has a problem that the control efficiency of the target gene is low and the siRNA double strands are structurally unstable.

The present inventors have also reported various experiments on the inhibitory activity of interference-induced RNAi according to the structural change by conducting a study of chemically synthesizing various interference-inducing RNAs and varying their structures [11].

Accordingly, the present inventors have continued to develop an effective anticancer agent or anti-barrier therapeutic agent using various interference inducing RNAs. As a result, the present inventors have shorter than 15 to 19 shorter lengths than siRNAs consisting of 19 base sequences. By designing and synthesizing interference-inducing RNAs consisting of sequencing and confirming that they can effectively replace the expression of target genes by conventional interference-inducing RNA by controlling the expression of target genes in cells alone or in the form of multi-target nanostructures. The present invention has been successfully completed.

An object of the present invention is to provide a variety of gene-control-related therapeutics, including interference-inducing RNAs shorter than the conventional 19 base sequences and an anticancer or antiviral therapeutic agent using the same.

In order to achieve the above object, the present invention provides a short interfering RNA (siRNA) of short length consisting of less than 19 base sequences.

In the present invention, the short-length interference inducing RNA is preferably composed of 15 to less than 19 base sequences, preferably at least 9 or more base sequences, and also includes some single stranded base sequences. desirable.

The present invention provides the use of the short length interference inducing RNA to prepare multi-target interference inducing RNA to control the expression of one or more target genes.

In addition, the present invention provides a therapeutic agent for gene-control-related diseases, including an anticancer agent and / or an antiviral agent using the short-length interference induction RNA (siRNA) as an active ingredient.

As described above, the present invention relates to interference-inducing RNAs consisting of less than 19 base sequences shorter than the conventional 19 base sequences, and a therapeutic agent for gene-control-related diseases using the same.

The interference-inducing RNA of the present invention is composed of fewer nucleotide sequences than the conventional one, and thus, the production cost is low, the gene control efficiency per unit weight is higher, and the clinical application such as the introduction into the cell during the preparation of the multi-target interference-inducing RNA. Since it is easy, it can be widely used to develop a therapeutic agent for various cancers or viral diseases, etc., replacing the drugs based on conventional interference-inducing RNA.

Hereinafter, the present invention will be described in detail.

The present invention provides small interfering RNAs (siRNAs) consisting of short length sequences that control the expression of target genes.

Specifically, the present invention provides a short length interference inducing RNA consisting of less than 19 base sequences, unlike conventional interference inducing RNA consisting of 19 or more base sequences.

The short length interference inducing RNA is preferably composed of 15 to less than 19 base sequences, preferably at least 9 or more base sequences, and also preferably includes some single stranded base sequences.

In addition, siRNAs for controlling the target genes, which constitute the short-length interference inducing RNA of the present invention, are SEQ ID NO: 7 having Lamin as a target gene and siLamin and TIG3 of SEQ ID NO: 8 as target genes. It is preferable to select from siTIG3 of 9 and SEQ ID NO: 10, and SEQ ID NO: 11 having DBP as the target gene, siDBP of SEQ ID NO: 12, and siOASIS of SEQ ID NO: 13 with SEQ ID NO: 14 and OASIS as the target gene. In addition to the target genes, any gene associated with anticancer or antiviral action may be selected and used as the target gene, and further, all genes required for the preparation of interference-inducing RNA may be selected and used as the target gene.

In addition, the short-length interference inducing RNA of the present invention is preferably used to prepare multiple interference-inducing RNAs combined to control a plurality of target genes.

Provided are short length interference inducing RNAs that control one target gene of the invention.

Specifically, the present invention provides a short length interference inducing RNA, siLamin (17) of SEQ ID NO: 1 and SEQ ID NO: 2 consisting of 17 nucleotide sequences for controlling the Lamin gene.

In another aspect, the present invention provides a short length interference induced RNA, siTIG3 (17) of SEQ ID NO: 3 and SEQ ID NO: 4 consisting of 17 nucleotide sequences for controlling the TIG3 gene.

In another aspect, the present invention provides a short length interference induced RNA, siTIG3 (15) of SEQ ID NO: 5 and SEQ ID NO: 6 consisting of 15 nucleotide sequences for controlling the TIG3 gene.

The present invention provides a short length of multiple interference inducing RNA combining (binding) siRNAs to control two target genes.

At least one of the siRNAs controlling the target genes is preferably composed of less than 19 nucleotide sequences, more preferably 15 to less than 19 nucleotide sequences, more preferably at least 9 or more nucleotide sequences It is preferable to make, and it is also preferable to include some single stranded nucleotide sequence.

In addition, siRNAs that control the target genes, which constitute the short-length interference-inducing RNA, are SEQ ID No. 7 and SEQ ID NO. 10, which use lamin as the target gene, and siLamin and TIG3 as SEQ ID. Preferably, siTIG3 and DBP are selected from SEQ ID NO: 11, siDBP of SEQ ID NO: 12, and siOASIS of SEQ ID NO: 13 and OASIS 14 of SEQ ID NO: 14 as target genes. In addition to the target genes, all genes associated with anticancer or antiviral action may be selected and used as the target genes, and further, all genes required for the production of interference-inducing RNA may be used as the target genes.

Specifically, the present invention provides a short length interference-inducing RNA, dsiLamin (3'17) -TIG3 (5'17), comprising the nucleotide sequences of SEQ ID NO: 23 and SEQ ID NO: 24 controlling the Lamin gene and the TIG3 gene. do.

In another aspect, the present invention provides a short length interference induced RNA, dsiLamin (5'17) -TIG3 (5'15) comprising the nucleotide sequence of SEQ ID NO: 25 and SEQ ID NO: 26 to control the Lamin gene and TIG3 gene. .

The present invention also provides a short length interference inducing RNA, dsiLamin (3'17) -TIG3 (5'15), comprising the nucleotide sequences of SEQ ID NO: 27 and SEQ ID NO: 28 controlling the Lamin gene and the TIG3 gene. .

In another aspect, the present invention provides a short length interference induced RNA, dsiLamin (5'15) -TIG3 (5'15), comprising the nucleotide sequences of SEQ ID NO: 29 and SEQ ID NO: 30 controlling the Lamin gene and the TIG3 gene. .

In another aspect, the present invention provides a short length interference induced RNA, dsiLamin (3'15) -TIG3 (5'15), comprising the nucleotide sequences of SEQ ID NO: 31 and SEQ ID NO: 32 controlling the Lamin gene and the TIG3 gene. .

In another aspect, the present invention provides a short length interference induced RNA, dsiLamin (M15) -TIG3 (5′15), comprising the nucleotide sequences of SEQ ID NO: 33 and SEQ ID NO: 34 controlling the Lamin gene and the TIG3 gene.

The present invention provides short length multiple interference induced RNAs that combine siRNAs to control three target genes.

At least one of the siRNAs controlling the target genes is preferably composed of less than 19 nucleotide sequences, more preferably 15 to less than 19 nucleotide sequences, more preferably at least 9 nucleotide sequences It is preferable to include some single-stranded base sequence.

In addition, siRNAs that control the target genes, which constitute the short-length interference-inducing RNA, are SEQ ID No. 7 and SEQ ID NO. 10, which use lamin as the target gene, and siLamin and TIG3 as SEQ ID. It is preferable to select from siTIG3 and DBP of SEQ ID NO: 11, siDBP of SEQ ID NO: 12 and siOASIS of SEQ ID NO: 13 and OASIS of SEQ ID NO: 14 as target genes. In addition to the target genes, all genes associated with anticancer or antiviral action may be selected and used as the target genes, and further, all genes required for the production of interference-inducing RNA may be used as the target genes.

Specifically, the present invention is a short length multiple interference induced RNA, tsiLamin (19) -DBP (19) -TIG3 (17) to simultaneously control the Lamin gene, DBP gene and TIG3 gene as shown in Figure 19 (a) To provide.

In addition, the present invention provides a short length multiple interference induced RNA, tsiLamin (19) -DBP (19) -TIG3 (15), as shown in Figure 19 (b).

In addition, the present invention is a short length multiple interference induced RNA, tsiLamin (17) -DBP (19) -TIG3 (17) to simultaneously control the Lamin gene, DBP gene and TIG3 gene as shown in Figure 20 (a) to provide.

In addition, the present invention provides a short length multiple interference induced RNA, tsiLamin (17) -DBP (19) -TIG3 (15), as shown in Figure 20 (b).

In addition, the present invention is a short length multiple interference induced RNA, tsiLamin (19) -DBP (19) which simultaneously controls the Lamin gene, DBP gene and TIG3 gene and includes some single-stranded sequences as shown in FIG. -0.5 TIG3 (17).

In addition, the present invention, as shown in Figure 22, simultaneously controlling the Lamin gene, DBP gene and TIG3 gene and a short length of multiple interference inducing RNA, including a single stranded nucleotide sequence, tsi0.5Lamin (17) -DBP ( 19) -TIG3 19 is provided.

In addition, the present invention, as shown in Figure 23 (a) and simultaneously control the Lamin gene, DBP gene and TIG3 gene and a short length multiple interference induced RNA, tsi0.5Lamin (17) comprising some single-stranded base sequence -Provide DBP (19) -TIG3 (17).

In addition, the present invention provides a short length multiple interference induced RNA, tsi0.5Lamin (17) -DBP (19) -TIG3 (15), including some single stranded nucleotide sequences as shown in FIG. 23 (b). .

The present invention provides short length multiple interference induced RNAs that combine siRNAs to control four target genes.

At least one of the siRNAs controlling the target genes is preferably composed of less than 19 nucleotide sequences, more preferably 15 to less than 19 nucleotide sequences, more preferably at least 9 nucleotide sequences It is preferable to include some single-stranded base sequence.

In addition, siRNAs that control the target genes, which constitute the short-length interference inducing RNA, are SEQ ID No. 9 and SEQ ID No. 10, which use lamin as the target gene, and siLamin and TIG3 as SEQ ID. It is preferable to select from siTIG3 and DBP of SEQ ID NO: 11, siDBP of SEQ ID NO: 12 and siOASIS of SEQ ID NO: 13 and OASIS of SEQ ID NO: 14 as target genes. In addition to the target genes, all genes associated with anticancer or antiviral action may be selected and used as the target genes, and further, all genes required for the production of interference-inducing RNA may be used as the target genes.

Specifically, the present invention is a short length multiple interference induction RNA, qsiDBP (19) -OASIS (19)-to simultaneously control the Lamin gene, DBP gene, TIG3 gene and OASIS gene as shown in Figure 25 (a) TIG3 (17) -Lamin (19) is provided.

In addition, the present invention provides a short length multiple interference induced RNA, qsiDBP (19) -OASIS (19) -TIG3 (15) -Lamin (19), as shown in FIG. 25 (b).

In addition, the present invention is a short length multiple interference induced RNA, qsiLamine (17) -TIG3 (15) -OASIS (19)-to simultaneously control the Lamin gene, DBP gene, TIG3 gene and OASIS gene as shown in FIG. DBP 19 is provided.

In addition, the present invention, as shown in Figure 28 (a) is a short length multiple interference induced RNA, qsiDBP (19) including some single-stranded base sequence to remove the Lamin gene, DBP gene, TIG3 gene and OASIS gene at the same time (19) ) -OASIS (19) -0.5TIG3 (17) -Lamin (19).

In addition, the present invention, as shown in Figure 28 (b) is a short length multi-interference induced RNA, including some single-stranded nucleotide sequence, qsi0.5Lamin (17) -TIG3 (19) -OASIS (19) -DBP ( 19).

In addition, the present invention, as shown in FIG. 29 (a), simultaneously controlling the Lamin gene, DBP gene, TIG3 gene and OASIS gene, and short length multiple interference induced RNA, qsiDBP (19) ) -OASIS (19) -TIG3 (15) -0.5Lamin (17).

In addition, the present invention is a short length multiple interference induced RNA, qsiLamin (17) -0.5TIG3 (15) -OASIS (19) -DBP (19) as shown in Figure 29 (b) ).

The present invention provides the use of such short length interference inducing RNA (siRNA) as a therapeutic agent for various gene-control related diseases, including anticancer and / or antiviral therapies.

In addition to being used as an active ingredient such as an anticancer agent or an antiviral agent, the multi-silencing siRNA combining the short-length siRNAs of the present invention is more effective in controlling all the expressions of one or more target genes simultaneously. It can be widely used to prevent and treat them.

Hereinafter, the present invention will be described in more detail with reference to Examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 siRNA Preparation and Gene Expression Inhibition with Short Base Sequences

The inventors have identified the possibility of efficiently controlling the target gene even in siRNA having fewer than 19 nucleotide sequences through previous studies.

The siTIG3 15 bp of SEQ ID NO: 5 and SEQ ID NO: 6 was prepared, and transformed into HeLa cells using lipofectamine 2000 (lipofectamine 2000) at a concentration of 100 nM or 10 nM, respectively. In addition, mRNA level of the target gene TIG3 was quantified by real-time reverse transcriptase polymerase chain reaction (real-time RT-PCR). Furthermore, the mRNA amount was converted and the efficiency of siTIG3 expression inhibition of target genes was calculated and compared.

As a result, as shown in Figure 2, it was confirmed that siTIG3 15 bp can control the target gene with an efficiency similar to siTIG3 having a length of 19 bp.

Example 2 Construction of Double-Target Interference Inducing RNAs

The inventors have already conducted previous studies [11] to express gene expression of siRNA even when a duplex having a different sequence is attached to the 3'-side of the antisense strand of the interference-inducing siRNA. It was confirmed that the inhibition efficiency was maintained. Based on this fact, the inventors predicted that when two 19 bp siRNAs were attached to each other, the gene control efficiency before attachment of the new siRNA construct formed could be maintained.

As shown in FIG. 3, an interference-inducing RNA dsiLamin comprising the nucleotide sequences of SEQ ID NO: 15 and SEQ ID NO: 16, which forms a double structure by combining siLamin, which is a lamin as a target gene, and siTIG3, which is a TIG3 as a target gene, is formed. (19) -TIG3 (19) [dual silencing siRNA Lamin (19) -TIG3 (19)].

Example 3 Investigation of Gene Expression Inhibition of Double-Target Interference Inducing RNAs I

In the present invention, siLamin of SEQ ID NO: 7 and SEQ ID NO: 8 including only Lamin gene, siTIG3 of SEQ ID NO: 9 and SEQ ID NO: 10 including only TIG3 gene, The mixture of siLamin and siTIG3, and dsiLamin (19) -TIG3 (19) of SEQ ID NO: 15 and SEQ ID NO: 16, which are the dual target interference inducing RNAs of the present invention, were concentrations of 100 in HeLa cells in the same manner as in Example 1, respectively. Transfection was performed using lipofectamine 2000 at a concentration of nM or 10 nM. In addition, mRNA levels of the target genes Lamin and TIG3 were quantified by real-time reverse transcriptase polymerase chain reaction (real-time RT-PCR). Furthermore, the efficiency of inhibiting the expression of target genes was calculated and compared by converting the mRNA amount. As a result, as shown in Figure 4, the dual-target interference-induced RNA dsiLamin (19) -TIG3 (19) of the present invention compared to the control group that simply mixed a single target gene siLamin and siTIG3 of the gene expression after cell transformation The inhibition was confirmed to be equivalent.

Example 4 Investigation of Gene Expression Inhibition of Double-Target Interference Inducing RNAs II

In the present invention, as described in Example 3, whether the double-target siRNA structure has a universal expression inhibitory activity even in the general target gene was investigated as follows. Specifically, interference-inducing RNA dsiDBP (19) -TIG3 (19) of SEQ ID NO: 17 and SEQ ID NO: 18, dsiLamin (19) -DBP (19) of SEQ ID NO: 19 and SEQ ID NO: 20, and dsiOASIS of SEQ ID NO: 21 and SEQ ID NO: 22 A combination of various interference inducing RNAs including different target genes was prepared, including (19) -TIG3 (19) (see FIGS. 5, 7 and 9).

As a result, as shown in Figures 6, 8 and 10, respectively, the dual-targeting (dual silencing) siRNAs were able to effectively inhibit the expression of both target genes with a slight difference. Therefore, the dual-target siRNAs linking two siRNAs having a size of 19 bp were found to have the same level of inhibition of gene expression after cell transformation compared to the control mixed with two single-target 19 bp siRNAs.

Example 5 Construction of Short-Length Double-Target Interference Inducing RNAs

In the present invention, since the dual-target interference-inducing siRNAs described in Example 3 are composed of 38 bp sequences, even when the expression of target genes is smoothly inhibited, compared to the conventional siRNAs consisting of 19 bp or 27 bp sequences. It was determined to have a relatively long problem. Therefore, it was investigated whether the dual-target siRNA shorter than 38 bp could effectively inhibit gene expression as follows.

As shown in FIG. 11, the short-length double-strengthening process was performed by reducing the length of each siRNA from the dual-target dsiLamin-TIG3 siRNA construct to 17 bp + 17 bp, 17 bp + 15 bp and 15 bp + 15 bp, respectively. Target siRNA (dual silencing siRNA) was synthesized. Specifically, Lamin (3'17) of SEQ ID NO: 23 and SEQ ID NO: 24 is a duplex having a size of 17 bp from the 3 'side of the antisense sequence, Lamin (5'17 of SEQ ID NO: 25 and SEQ ID NO: 26 ) Is a duplex of 17 bp from the 5 'side of the 19 mer antisense sequence of siLamin of 19 bp. In addition, Lamin (5'15) of SEQ ID NO: 27 and SEQ ID NO: 28 is a duplex of 15 bp from the 5 'side of the antisense sequence of siLamin of 19 bp, the above SEQ ID NO: 31 and SEQ ID NO: 32 Lamin (3'15) is a duplex of 15 bp from the 3 'side of the antisense sequence, Lamin (M15) of SEQ ID NO: 33 and SEQ ID NO: 34 is the 15 bp middle portion of the antisense sequence of siLamin 19 bp The duplex of size was used. In addition, siRNAs that inhibit the TIG3 gene were reduced to only 17 bp and 15 bp in length while maintaining the 5 'side antisense end. TIG3 (5'17) uses duplexes of 17 bp size from the 5 'side of the antisense sequence of siTIG3, and TIG3 (5'15) uses duplexes of 15 bp size from the 5' side of the antisense sequence of siTIG3. Inhibition of expression of target genes in cells by the short length dual silencing siRNA constructs prepared above was measured and shown in FIG. 12.

Example 6 Investigation of Gene Expression Inhibition of Short-Length Double-Target Interference Inducing RNAs

In the present invention, gene expression inhibition of interference-inducing RNAs including a short-length dual-target gene was measured by the same procedure as described in Example 1 above. As a result, among the dual-target siRNAs, the TIG3 gene was found to sufficiently reduce the mRNA level of the TIG3 gene with only 15 bp or 17 bp duplex from the 5 'side of the antisense strand, which was shorter than the 19 bp duplex. Could. In addition, when the Lamin gene uses a 17 bp duplex, the 17 bp duplex from the 3 'side of the antisense strand shows excellent activity in reducing the mRNA level of the Lamin gene, while the 17 bp duplex from the 5' side. It was found that the sieve showed little inhibitory activity (see FIG. 12). Thus, the dual-target siRNA constructs of the present invention incorporating two siRNA units have been shown to be able to sufficiently inhibit specific gene expression even with sequence-dependent siRNA combinations shorter than the conventional 19 bp. It was.

As such, short-length dual-target siRNA constructs target genes even though they reduce the number of bases to a lower base number, i.e. up to 64 mer for the 17 bp + 15 bp duplex, compared to a construct with two 19 bp siRNA combinations. It has the advantage of being able to effectively suppress their expression. Therefore, in the future, when developing a new drug using the siRNA of the present invention as an active ingredient, the production cost can be reduced efficiently.

Example 7 Construction of Some Single Strand Double-Target Interference Inducing RNAs

In the present invention, interference-inducing RNAs including a dual-target gene consisting of some single strands were prepared as follows. For reference, through conventional prior studies, siRNAs are known to be capable of inhibiting expression of target genes by inducing RNAi in the form of a single strand rather than a double strand [12]. In the present invention, as shown in Figure 13, by removing the sense strand (sense strand) of one siRNA unit from the double-target siRNA structure (denoted '0.5' in Figure 13) to make a single strand including the double stranded part together A dual-target dsiRNA was prepared in mixed form. Specifically, as shown in FIG. 13, among some single-stranded dual-target siRNAs, dsi0.5Lamin (19) -TIG3 (19) of SEQ ID NO: 35 and SEQ ID NO: 36 is a single 19 bp sized molecule behind the TIG3 siRNA duplex. The siLamin antisense strand was bound, and dsiLamin (19) -0.5TIG3 (19) of SEQ ID NO: 37 and SEQ ID NO: 39 bound a single siTIG3 antisense strand of 19 bp to the back of the Lamin siRNA duplex. In addition, dsi0.5Lamin (3'17) -TIG3 (5'15) of SEQ ID NO: 39 and SEQ ID NO: 40 was formed with a 17 mer single strand in the antisense 3 'direction of the Lamin gene behind the 15 bp siTIG3 double strand. Combined.

Example 8. Investigation of Gene Expression Inhibition of Some Single Strand Double-Target Interference Inducing RNAs

In the present invention, the gene expression inhibition of interference-inducing RNAs including a double-target gene consisting of some single strands was measured by the same procedure as described in Example 3.

As a result, as shown in Figure 14, the lamin gene and TIG3 in the case of dsiLamin (19) -0.5TIG3 (19) of SEQ ID NO: 37 and SEQ ID NO: 38, which binds a 19 mer single siTIG3 antisense strand to the back of the Lamin siRNA double strand It was confirmed that the mRNA expression of all genes can be effectively suppressed. However, in the case of dsi0.5Lamin (19) -TIG3 (19) of SEQ ID NO: 35 and SEQ ID NO: 36, which binds a 19 mer single siLamin antisense strand behind the TIG3 siRNA double strand, it was confirmed that only TIG3 gene expression could be suppressed. . However, in the case of dsi0.5Lamin (3'17) -TIG3 (5'15) of SEQ ID NO: 39 and 17 SEQ ID NO: 40 which bound 17 mer single strands from the antisense 3 'direction of the Lamin gene behind the 15 bp siTIG3 double strand It was confirmed that the expression of both the TIG3 gene and the Lamin gene was efficiently suppressed. Thus, some single stranded dual-target interference RNAs of the present invention can simultaneously inhibit two target genes with much fewer bases, ie less than 47 mer, compared to two 19 bp siRNAs (total 76 mer). When manufacturing a new drug, it has the advantage of significantly lowering the production cost.

Example 9 Construction of Triple-Target Interference Inducing RNAs and Investigation of Gene Expression Inhibition

In the present invention, interference-inducing RNAs containing triple-target genes were prepared as follows to investigate gene expression inhibition. Specifically, as a process of combining three 19 bp siRNAs as shown in FIG. 17, an interference inducing RNA (triple silencing siRNA; tsiRNA) including a triple-target gene was prepared by chemical synthesis. It was also confirmed that this triple-target siRNA effectively inhibited the expression of three target genes similarly to the case of simply mixing and transfecting three siRNAs of 19 bp in size (see FIG. 18). . Therefore, it was found that one RNA construct can effectively inhibit the expression of three or more target genes simultaneously. In addition, the triple-target tsiRNA of the present invention was also able to reduce the length of the interference-inducing RNA double strands or to modify some of them into single-stranded form in the same way as the double-target dsiRNA, and even in this case effectively inhibited expression of the target genes. .

Example 10 Construction of Multi-Target Interference Inducing RNAs Including Aptamer Sequences and Investigation of Gene Expression Inhibition

In the present invention, multi-target interference inducing RNAs including an aptamer sequence were prepared to investigate expression inhibition of target genes as follows. Specifically, a small RNA nanostructure capable of replacing a duplex portion of one of the triple-target tsiRNAs with a cell-specific aptamer sequence and selectively introducing two or more siRNAs into a specific cell Was made (see FIG. 30). In addition, the same procedure as described in Example 1 confirmed that the multi-target interference induced RNA effectively inhibits the expression of target genes.

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Figure 1 shows various sequence structures of short length interference inducing RNA (siRNA) consisting of less than 19 nucleotide sequences controlling one target gene of the present invention.

Figure 2 shows the expression inhibition of the target gene of siTIG3 (15) of the present invention.

FIG. 3 shows the sequence structure of a dual-target siRNA, dsiLamin (19) -TIG3 (19), which combines two interference inducing RNAs (siRNA) of the present invention.

Figure 4 shows the expression inhibition of the target genes of the double-target siRNA of the present invention, dsiLamin (19) -TIG3 (19).

5 shows the sequence structure of the dual-target siRNA, dsiDBP (19) -TIG3 (19) of the present invention.

Figure 6 shows the expression inhibition of the target genes of the dual-target siRNA, dsiDBP (19) -TIG3 (19) of the present invention.

Figure 7 shows the sequence structure of the dual-target siRNA of the present invention, dsiLamin (19) -DBP (19).

Figure 8 shows the expression inhibition of the target genes of the double-target siRNA of the present invention, dsiLamin (19) -DBP (19).

9 shows the sequence structure of the dual-target siRNA, dsiOASIS (19) -TIG3 (19) of the present invention.

Figure 10 shows the expression inhibition of the target genes of the dual-target siRNA, dsiOASIS (19) -TIG3 (19) of the present invention.

FIG. 11 shows various sequence structures of dual-target siRNAs combining short length siRNAs consisting of less than 19 nucleotide sequences of the present invention.

Figure 12 shows the expression inhibition of the target genes of the dual-target siRNAs in combination with the short length siRNA of the present invention.

FIG. 13 shows various sequence structures of dual-target siRNAs (Lamin-TIG3) combining some single stranded siRNAs of the present invention.

14 shows the expression inhibition of these target genes of dual-target siRNAs combining some single stranded siRNAs of the present invention.

FIG. 15 shows various sequence structures of dual-target siRNAs (DBP-TIG3) combining some single stranded siRNAs of the present invention. FIG.

Figure 16 shows the expression inhibition of these target genes of some single stranded dual-target siRNAs of the present invention.

Figure 17 shows the sequence structure of triple silencing siRNA (triple silencing siRNA) combining three interference inducing RNA (siRNA) of the present invention, (a) is the sequence structure of tsiLamin-DBP-TIG3, (b) Is the sequence structure of tsiTIG3-DBP-Lamin.

Figure 18 shows the expression inhibition of the target genes of the triple-target siRNAs, tsiLamin-DBP-TIG3 and tsiTIG3-DBP-Lamin of the present invention.

Figure 19 shows the sequence structure of the triple-target siRNA combining one short length siRNA of the present invention, (a) is the sequence structure of tsiLamin-DBP-TIG3 (17), (b) is tsiLamin-DBP -Sequence structure of TIG3 (15).

Figure 20 shows the sequence structure of triple-target siRNA combining one or more short length siRNA of the present invention, (a) is the sequence structure of tsiLamin (17) -DBP-TIG3 (17), (b) is The sequence structure of tsiLamin (17) -DBP-TIG3 (15).

Figure 21 shows the sequence structure of a triple-target siRNA combining one or more short length siRNAs of the present invention with some or all short lengths of single stranded siRNAs, (a) is the sequence structure of tsiLamin-DBP-0.5TIG3. And (b) is the sequence structure of tsiLamin-DBP-0.5TIG3 (17).

FIG. 22 shows the sequence structure of the triple-target siRNA, tsi0.5Lamin (17) -DBP-TIG3, combining one or more short length siRNAs of the present invention with some or all short lengths of single stranded siRNAs.

FIG. 23 shows the sequence structure of triple-target siRNA combining one or more short length siRNAs of the present invention with some or all short lengths of single stranded siRNA, wherein (a) is tsiLamin (17) -DBP-TIG3 ( 17), and (b) is the sequence structure of tsiLamin (17) -DBP-TIG (15).

Figure 24 shows the sequence structure of quadruple silencing siRNA (quadruple silencing siRNA) combining four interference inducing RNA (siRNA) of the present invention, (a) is the sequence structure of qsiDBP-OASIS-TIG3-Lamin, ( b) is the sequence structure of qsiLamin-TIG3-OASIS-DBP.

Figure 25 shows the sequence structure of the quadruple-target siRNA combining one short length siRNA of the present invention, (a) is the sequence structure of qsiDBP-OASIS-TIG3 (17) -Lamin, (b) is qsiDBP The sequence structure of -OASIS-TIG3 (15) -Lamin.

FIG. 26 shows the sequence structure of a quad-target siRNA, qsiLamin (17) -TIG3 (15) -OASIS-DBP, combining one or more short length siRNAs of the present invention.

Figure 27 shows the sequence structure of the quadruple-target siRNA, qsiDBP-OASIS-0.5TIG3-Lamin, which combines some single stranded siRNAs of the present invention.

Figure 28 shows the sequence structure of a quadruple-target siRNA combining some short-length single stranded siRNA of the present invention, (a) is the sequence structure of qsiDBP-OASIS-0.5TIG3 (17) -Lamin, (b ) Shows the sequence structure of qsi0.5Lamin (17) -TIG3-OASIS-DBP.

Figure 29 shows the sequence structure of a quadruple-target siRNA combining one short-length siRNA and some or all short-length siRNA of the present invention, (a) is qsiDBP-OASIS-TIG3 (15)- The sequence structure of 0.5 Lamin (17), (b) is the sequence structure of qsi Lamin (17) -0.5TIG3 (15) -OASIS-DBP.

FIG. 30 schematically shows the structure of an aptamer-multiple target interference inducing RNA (siRNA) combining a cell specific aptamer with interference inducing RNAs (siRNAs) of the present invention. Aptamer-tsiRNA structure combining a dual-target siRNA and aptamer, (b) is aptamer-qsiRNA structure combining a triple-target siRNA and aptamer.

<110> Postech Academy-Industry Foundation <120> Small Interfering RNA (siRNA) having a shortened nucleotide          sequence <130> P07-E304 <160> 44 <170> KopatentIn 1.71 <210> 1 <211> 17 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 1 uucuucugga aguccag 17 <210> 2 <211> 17 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 2 aagaagaccu ucagguc 17 <210> 3 <211> 17 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 3 uagagaacgc cugagac 17 <210> 4 <211> 17 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 4 aucucuugcg gacucug 17 <210> 5 <211> 15 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 5 uagagaacgc cugag 15 <210> 6 <211> 15 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 6 aucucuugcg gacuc 15 <210> 7 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 7 uguucuucug gaaguccag 19 <210> 8 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 8 acaagaagac cuucagguc 19 <210> 9 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 9 uagagaacgc cugagacag 19 <210> 10 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 10 aucucuugcg gacucuguc 19 <210> 11 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 11 ugagaagcga ugucuucga 19 <210> 12 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 12 acucuucgcu acagaagcu 19 <210> 13 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 13 ucguagaaua ggaggcuuc 19 <210> 14 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 14 agcaucuuau ccuccgaag 19 <210> 15 <211> 38 <212> RNA <213> Artificial Sequence <220> <223> dsiRNA <400> 15 uguucuucug gaaguccagc ugucucaggc guucucua 38 <210> 16 <211> 38 <212> RNA <213> Artificial Sequence <220> <223> dsiRNA <400> 16 acaagaagac cuucaggucg acagaguccg caagagau 38 <210> 17 <211> 38 <212> RNA <213> Artificial Sequence <220> <223> dsiRNA <400> 17 ugagaagcga ugucuucgac ugucucaggc guucucua 38 <210> 18 <211> 38 <212> RNA <213> Artificial Sequence <220> <223> dsiRNA <400> 18 acucuucgcu acagaagcug acagaguccg caagagau 38 <210> 19 <211> 38 <212> RNA <213> Artificial Sequence <220> <223> dsiRNA <400> 19 uguucuucug gaaguccagu cgaagacauc gcuucuca 38 <210> 20 <211> 38 <212> RNA <213> Artificial Sequence <220> <223> dsiRNA <400> 20 acaagaagac cuucagguca gcuucuguag cgaagagu 38 <210> 21 <211> 38 <212> RNA <213> Artificial Sequence <220> <223> dsiRNA <400> 21 ucguagaaua ggaggcuucc ugucucaggc guucucua 38 <210> 22 <211> 38 <212> RNA <213> Artificial Sequence <220> <223> dsiRNA <400> 22 agcaucuuau ccuccgaagg acagaguccg caagagau 38 <210> 23 <211> 34 <212> RNA <213> Artificial Sequence <220> <223> dsiRNA <400> 23 uucuucugga aguccagguc ucaggcguuc ucua 34 <210> 24 <211> 34 <212> RNA <213> Artificial Sequence <220> <223> dsiRNA <400> 24 aagaagaccu ucagguccag aguccgcaag agau 34 <210> 25 <211> 32 <212> RNA <213> Artificial Sequence <220> <223> dsiRNA <400> 25 uguucuucug gaagucccuc aggcguucuc ua 32 <210> 26 <211> 32 <212> RNA <213> Artificial Sequence <220> <223> dsiRNA <400> 26 acaagaagac cuucagggag uccgcaagag au 32 <210> 27 <211> 32 <212> RNA <213> Artificial Sequence <220> <223> dsiRNA <400> 27 uucuucugga aguccagcuc aggcguucuc ua 32 <210> 28 <211> 32 <212> RNA <213> Artificial Sequence <220> <223> dsiRNA <400> 28 aagaagaccu ucaggucgag uccgcaagag au 32 <210> 29 <211> 30 <212> RNA <213> Artificial Sequence <220> <223> dsiRNA <400> 29 uguucuucug gaagucucag gcguucucua 30 <210> 30 <211> 30 <212> RNA <213> Artificial Sequence <220> <223> dsiRNA <400> 30 acaagaagac cuucagaguc cgcaagagau 30 <210> 31 <211> 30 <212> RNA <213> Artificial Sequence <220> <223> dsiRNA <400> 31 cuucuggaag uccagcucag gcguucucua 30 <210> 32 <211> 30 <212> RNA <213> Artificial Sequence <220> <223> dsiRNA <400> 32 gaagaccuuc aggucgaguc cgcaagagau 30 <210> 33 <211> 30 <212> RNA <213> Artificial Sequence <220> <223> dsiRNA <400> 33 uucuucugga agucccucag gcguucucua 30 <210> 34 <211> 30 <212> RNA <213> Artificial Sequence <220> <223> dsiRNA <400> 34 aagaagaccu ucagggaguc cgcaagagau 30 <210> 35 <211> 38 <212> RNA <213> Artificial Sequence <220> <223> dsiRNA <400> 35 uguucuucug gaaguccagc ugucucaggc guucucua 38 <210> 36 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> dsiRNA <400> 36 gacagagucc gcaagagau 19 <210> 37 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> dsiRNA <400> 37 uguucuucug gaaguccag 19 <210> 38 <211> 38 <212> RNA <213> Artificial Sequence <220> <223> dsiRNA <400> 38 acaagaagac cuucaggucg acagaguccg caagagau 38 <210> 39 <211> 32 <212> RNA <213> Artificial Sequence <220> <223> dsiRNA <400> 39 uucuucugga aguccagcuc aggcguucuc ua 32 <210> 40 <211> 15 <212> RNA <213> Artificial Sequence <220> <223> dsiRNA <400> 40 gaguccgcaa gagau 15 <210> 41 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> dsiRNA <400> 41 ugagaagcga ugucuucga 19 <210> 42 <211> 38 <212> RNA <213> Artificial Sequence <220> <223> dsiRNA <400> 42 acucuucgcu acagaagcug acagaguccg caagagau 38 <210> 43 <211> 38 <212> RNA <213> Artificial Sequence <220> <223> dsiRNA <400> 43 ugagaagcga ugucuucgac ugucucaggc guucucua 38 <210> 44 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> dsiRNA <400> 44 gacagagucc gcaagagau 19  

Claims (7)

Small interfering RNA (siRNA) of short length consisting of less than 19 sequences. The method of claim 1, The less than 19 base sequences are short-length interference induction RNA (siRNA), characterized in that 15 to less than 19 base sequences. The method of claim 1, The less than 19 nucleotide sequences are at least 9 or more nucleotide sequences of the short, interference-induced RNA (siRNA) characterized in that. The method of claim 1, The less than 19 nucleotide sequences of the short length interference-induced RNA (siRNA), characterized in that it comprises some single stranded nucleotide sequence. The method of claim 1, Short length interference induced RNA (siRNA), characterized in that it is used to prepare multiple interference induced RNAs that control the expression of two or more target genes. A therapeutic agent for gene-control-related diseases comprising the short-length interference-inducing RNA (siRNA) of claim 1 as an active ingredient. The method of claim 6, The gene-controlling related disease comprises a cancer or a viral disease.

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