JP5252618B2 - Aromatic ring-modified siRNA with high RNA interference effect - Google Patents

Description

  The present invention relates to an aromatic ring-modified siRNA that can efficiently suppress the expression of a target gene. More specifically, the present invention relates to an aromatic ring-modified siRNA that has a high nuclease resistance and a high intracellular introduction rate and can exhibit an excellent RNA interference effect.

  The development of a medicine that efficiently treats intractable diseases such as cancer and AIDS is a major issue in the life science field. One of the promising methods that can overcome this problem is a gene drug that acts only on a specific gene. Among these gene medicines, the RNA interference (RNAi) method using a short double-stranded RNA (siRNA) of 21 bases has recently attracted attention. This RNAi method was first reported by Fire et al. In 1998 (see Non-Patent Document 1). According to the report of Fire et al., By introducing a double-stranded RNA of about 100 base pairs homologous to a specific region of the gene whose function is to be inhibited into the cell, 2 to about 20-25 base pairs by the action of Dicer in the cytoplasm. It is decomposed into single-stranded RNA, and then forms multiple RNA / protein complexes with this protein (this complex is called RICS: RNA-induced silencing complex), which binds to the homologous site of mRNA produced from the target gene. It strongly suppresses gene expression. However, in mammalian cells, when a long double-stranded RNA of about 30 base pairs or more is introduced, an interferon reaction, which is a viral response reaction, is induced, resulting in cell death. The RNAi method was considered difficult to apply. Therefore, Tuschl et al. Avoided the interferon response by chemically synthesizing a 21-base long double-stranded RNA with dangling ends at both 3 'ends of the double-stranded RNA and introducing it directly into mammalian cells. It has been reported that it shows a high gene expression suppression ability in a sequence-specific manner (see Non-Patent Document 2). They also synthesize short double-stranded RNAs with double-stranded regions of 19 base pairs and various dangling end strands at the 3 'end or 5' end to examine the RNA interference effect. As a result, 21-base siRNA with 2 base dangling ends at both 3 'ends showed a very high RNA interference effect, but it was notable for all other types of short double-stranded RNA. No interference effect was observed. According to this report, RNA interference using a double-stranded RNA that is 21 bases long and has 2 bases of dangling ends at both 3 'ends is now common. Here, the method of inhibiting target gene expression using a short double-stranded RNA having a length of 21 bases is called the siRNA method in distinction from the RNAi method.

  Since this siRNA method uses synthetic RNA, sample preparation is relatively easy, the handling operation is simple, and it has a very powerful effect, so it attracts great attention not only in the life science field but also in the biobusiness field. ing.

However, this excellent siRNA method also has a problem that must be solved. As described above, siRNA is composed of RNA molecules, and is rapidly degraded by the action of nucleases contained in cells and in the medium . The double-stranded RNA region shows relatively high nuclease resistance compared to single-stranded RNA, but double-stranded RNA consisting of 19 base pairs hardly shows the conventional RNA interference effect. Therefore, synthetic siRNA shows a high gene expression suppression effect for about 2 days to 4 days after introduction into cells with the target gene sequence, but after that, the RNA interference effect declines sharply, and after about 7 days the RNA interference effect Has been reported to almost disappear.

Recently, in synthetic siRNA, attempts have been made to variously modify siRNA in order to increase nuclease resistance and acquire a highly active RNA interference effect. For example, in order to acquire degradation resistance from exonuclease, siRNA in which the end of siRNA is modified with an amino group, a thiol group, or basic is synthesized. However, in most cases of 21-base siRNA modified at the end, RNA interference effects have already been reported to be significantly reduced even if nuclease resistance and cell introduction rate can be improved. Currently, siRNA is chemically modified so that it has high nuclease resistance and cell introduction rate, and has not been able to enhance the RNA interference effect.
Fire et.al, Nature, 391, 806-811 (1998) Tuschl et.al., EMBO Journal, 20, 6877-6888 (2001)

  An object of the present invention is to provide a novel siRNA that has a high nuclease resistance and a high introduction rate into cells and can exhibit an excellent RNA interference effect.

  The inventors of the present invention have made extensive studies in order to solve the above-mentioned problems. As a result, in siRNA, at least one of the first to sixth nucleotides from the 5 ′ end of the sense strand RNA is directly or via a linker. A modified siRNA that can achieve high nuclease resistance and high cell introduction rate, and has excellent RNA interference effect by binding a family compound (particularly, an aromatic compound that can be intercalated or stacked with the siRNA) I found out that it can be built. The present invention has been completed by making further improvements based on such findings.

That is, the present invention provides the aromatic ring-modified siRNA listed below.
Item 1. Antisense strand RNA comprising a nucleotide sequence complementary to a target sequence in a target gene, and has a ruse Nsu stranded RNA having a nucleotide sequence complementary to the antisense strand RNA, and the expression of the target gene An aromatic ring modification characterized in that it is an siRNA that can be suppressed, and an aromatic compound is bonded to at least one of the first to sixth nucleotides from the 5 ′ end of the sense strand RNA directly or via a linker siRNA.
Item 2. Item 2. The aromatic ring-modified double-stranded RNA according to Item 1, wherein the aromatic compound is a compound capable of intercalating or stacking with siRNA.
Item 3. Item 2. The aromatic ring-modified double-stranded RNA according to Item 1, wherein the aromatic compound is a compound containing a phenyl group, a phenol group, a naphthyl group, or a pyrene group.

  In the aromatic ring-modified siRNA of the present invention, the 5 ′ end side of the sense strand RNA is modified with an aromatic compound, thereby greatly improving the RNA interference effect. Although not intended to be limited to interpretation, the aromatic ring-modified siRNA of the present invention is structurally more distinct from the 5 ′ end of the sense strand RNA due to the specific aromatic compound present on the 5 ′ end side of the sense strand RNA. It is also presumed that a protein complex that induces an RNA interference effect is likely to interact with the 5 ′ end of the antisense strand RNA. Therefore, the protein complex can positively interact with the antisense strand that exhibits the RNA interference effect rather than the sense strand that does not exhibit the RNA interference effect, and thereby can exhibit the RNA interference effect more efficiently. Conceivable.

  Furthermore, since the aromatic ring-modified siRNA of the present invention exhibits excellent intracellular translocation ability, it can be introduced into cells with a reduced amount of conventional gene transfer reagents. Therefore, the aromatic ring-modified siRNA of the present invention can reduce the expression of cytotoxicity that is a concern due to the use of conventional gene introduction reagents, and can ensure high safety in clinical applications.

  The aromatic ring-modified siRNA of the present invention is a modified siRNA in which an aromatic compound is bound to a specific site of an siRNA directly or via a linker.

In siRNA, a 21-base long sense strand RNA and a 21-base long antisense strand RNA are hybridized so as to have a 2-base dangling end at both 3 ′ ends to form a double strand. That is, in siRNA, the 1st to 19th nucleotides from the 5 ′ end of the sense strand RNA and the 3rd to 21st nucleotides from the 3 ′ end of the antisense strand RNA are hybridized, and 1 from the 3 ′ end of the sense strand RNA. It has a structure in which the second nucleotide and the first and second nucleotides from the 3 ′ end of the antisense strand RNA each form a dangling end.

antisense strand RNA used for siRNA is at the 3 'end of the sequence consisting of the complementary 19 bases to a target sequence in a target gene, the nucleotide sequence sequence ing from 2 bases constituting dangling end linked It is. Here, the target gene is a gene whose gene expression is to be suppressed by the RNA interference effect. In the aromatic ring-modified siRNA of the present invention, the target gene is not particularly limited, and can be appropriately selected based on the use of the aromatic ring-modified siRNA.

  The target sequence in the target gene is not particularly limited as long as it is a sequence capable of suppressing gene expression due to RNA interference effect, and is appropriately determined by a known method, specifically using NCBI BLAST search or the like. Can do. For example, a region consisting of 19 to 30 bases following the base “AA” in the exon portion 50 to 100 bases downstream from the start codon of the target gene coding region (ORF) and having a GC content of around 50% May be the target sequence. Employing such a complementary strand to the target sequence has empirically revealed in the art that an excellent RNA interference effect can be obtained. Further, for example, the target sequence can be set according to the manual (Dicer Substrate RNAi Design) of IDT (Integrated DNA Technologies, INC). Recently, (i) the 5 ′ end of the antisense strand RNA is an A / U pair, (ii) the 5 ′ end of the sense strand RNA is a G / C pair, and (iii) the antisense strand RNA 5 'It has a high RNA interference effect by designing double-stranded RNA that has about 5 A / U pairs on the end side and (vi) no more than 9 G / C pairs in the double strand 2 It has been reported that single-stranded RNA can be designed (Ui-Tei et. Al, Nucleic Acids Res., 32, 936-948 (2004)).

  Further, the two bases constituting the dangling end are not particularly limited, and may be composed of bases complementary to the target sequence in the target gene, but bases complementary to the target sequence in the target gene. Not necessarily.

siRNA to be used Rousset Nsu strand RNA is the 3 'end of the sequence consisting of the complementary 19 bases relative to 19 th base sequence from the 5' end of the antisense strand, the dangling end This is a base sequence in which the two base sequences are linked . Cell for the types of bases constituting the dangling end of Nsu strand RNA, not particularly limited. In the aromatic ring-modified siRNA of the present invention, the nucleotides constituting the sense strand RNA and the antisense strand RNA are basically ribonucleotides, but for the purpose of improving degradation enzyme resistance, 2′-O-methyl modification The RNA sequence may contain a type, 2′-F modified type, various chemically modified nucleotides such as LNA (Locked Nucleic Acid), or deoxyribonucleotide. Specific examples of such chemically modified nucleotides include phosphorothioate-type and boranophosphate-type DNA / RNA-modified phosphate skeletons; 2′-0Me-modified RNA, 2′-F-modified RNA, etc. Modified nucleotides such as LNA (Locked Nucleic Acid) and ENA (2'-O, 4'-C-Ethylene-bridged nucleic acids); Examples include modified nucleotides having different basic skeletons such as forinonucleotides; base-modified nucleotides such as 5-fluorouridine and 5-propyluridine. In addition, the constituent nucleotides of the dangling ends in the sense strand RNA and the antisense strand RNA in the aromatic ring-modified siRNA of the present invention may be deoxyribonucleotides.

  In the aromatic ring-modified siRNA of the present invention, an aromatic compound is bound to at least one of the first to sixth nucleotides from the 5 ′ end side of the sense strand RNA. In the aromatic ring-modified siRNA of the present invention, no other substituent is bonded to a site other than the 5 ′ end side of the sense strand RNA. That is, the portion other than the 5 ′ end side of the sense strand RNA and the antisense strand RNA portion are not substituted with other substituents, and are composed only of nucleotides. As described above, since the aromatic compound is bonded only to the 5 ′ end side of the sense strand RNA, a significantly superior RNA interference effect can be expressed.

  In the aromatic ring-modified siRNA of the present invention, the aromatic compound bound to the sense strand RNA is not particularly limited as long as it has a benzene ring. Aromatic compounds that can be calated or stacked are preferred.

  Here, the aromatic compound capable of intercalating siRNA is an aromatic compound that can reversibly enter between two strands formed from a sense strand and an antisense strand. Specific examples of such aromatic compounds include compounds having a skeleton such as naphthalene, anthracene, pyrene, indole, quinolizidine, phenanthridine, acridine, β-carboline, and ellipticine.

  In addition, the aromatic compound that can be stacked on the siRNA is a space between the benzene ring of the aromatic compound and the purine nucleus and / or the pyrimidine nucleus of the nucleotide constituting the dangling of the 3 ′ end of the antisense strand of the siRNA. It is an aromatic compound that can take such an arrangement that the benzene ring is stacked on the nucleotide base constituting the dangling of the 3 ′ end of the antisense strand by working π-π interaction. Specific examples of such aromatic compounds include compounds having a skeleton such as benzene, naphthalene, anthracene, pyrene, indole, quinolizidine, phenanthridine, acridine, β-carboline, and ellipticine.

  The aromatic compound used in the aromatic ring-modified siRNA of the present invention preferably includes a compound having a benzene skeleton, a naphthalene skeleton, or a pyrene skeleton; and more preferably includes a phenyl group, a phenol group, a naphthyl group, or a pyrene group. Compound; particularly preferably a compound containing a phenyl group, a phenol group or a naphthyl group; more preferably a compound containing a phenyl group or a phenol group. By binding such an aromatic compound, the intracellular introduction rate and the RNA interference effect can be further improved. In these aromatic compounds, the phenyl group, the phenol group, the naphthyl group, or the pyrene group is substituted with a halogen atom and / or an alkyl group having 1 to 5 carbon atoms as long as the effects of the present invention are not hindered. It may be.

  In the aromatic ring-modified siRNA of the present invention, the binding mode of the aromatic compound and the sense strand is not particularly limited, and these may be directly bonded or may be bonded via a linker. . Here, the linker is not particularly limited as long as the aromatic compound and the sense strand can be linked, and specific examples of the linker include those having the following structures.

  In the general formula (L-2), n0 represents an integer of 0 to 10, preferably an integer of 1 to 5, and more preferably an integer of 2 to 5. In the general formulas (L-1), (L-2) and (L-6) to (L-23), n1 is an integer of 1 to 40, preferably an integer of 2 to 20, more preferably An integer of 2 to 12 is shown.

  In the general formulas (L-24) and (L-25), n2 represents an integer of 1 to 20, preferably an integer of 1 to 10, and more preferably an integer of 1 to 6.

In the linkers represented by the general formulas (L-1) to (L-25), a sense strand RNA may be bound to either the right side or the left side. Preferably, a predetermined site of the sense strand RNA is bonded to the right side of the linker represented by the general formulas (L-1) to (L-25), and the aromatic compound is bonded to the left side of the linker. It has a shall.

  Further, the binding site of the linker in the aromatic compound may be appropriately set according to the type of aromatic compound and the type of linker. As a preferable bonding mode between the linker and the aromatic compound, there may be mentioned one in which the linker is bonded to a hydrogen atom bonded to the benzene ring of the aromatic compound.

The linker is appropriately selected according to the type of aromatic compound to be linked. For example, when using a compound containing a phenyl group or a naphthyl group as the aromatic compound, the above-mentioned linker, the linker is suitable for the general formula (L-1). Further, for example, in the case of using a compound containing a phenol group or a pyrene group as the aromatic compound, the above-mentioned linker, the linker is suitable for the general formula (L-2).

In addition to the linker, depending on the type of aromatic compound to be bonded, for example, N-succinimidyl = 3- (2-pyridyldithio) propinate, N-4-maleimidobutyric acid, S- (2-pyridyldithio) Cysteamine, iodoacetoxysuccinimide, N- (4-maleimidobutyryloxy) succinimide, N- [5- (3′-maleimidopropylamide) -1-carboxypentyl] iminodiacetic acid, N- (5-aminopentyl) It is also possible to use a bifunctional linker (linker containing two functional groups) such as) -iminodiactec acid.

The number of aromatic compounds bound to the aromatic ring-modified siRNA of the present invention is not particularly limited, and examples thereof include 1 to 3, preferably 1 or 2, and more preferably 1.

  The aromatic ring-modified siRNA of the present invention synthesizes the sense strand RNA and the antisense strand RNA to which an aromatic compound is linked, and hybridizes the sense strand RNA and the antisense strand RNA according to a known method. It is manufactured. The sense strand RNA to which an aromatic compound is linked can also be produced using a known synthesis technique.

  Since the aromatic ring-modified siRNA of the present invention can suppress the expression of a target gene when introduced into a cell, it can be used as a medicament for the purpose of suppressing the expression of a target gene. The amount and method of introducing the aromatic ring-modified siRNA of the present invention into cells are the same as in the conventional siRNA. In addition, since the aromatic ring-modified siRNA of the present invention alone exhibits excellent intracellular translocation ability, the conventional gene introduction reagent can be used without using the conventional gene introduction reagent used for introduction of siRNA into cells. It is also possible to reduce the amount of reagent used and introduce it into the cell.

Hereinafter, the present invention will be described in detail based on examples, but the present invention is not limited thereto.
Example 1
1. Synthesis of aromatic ring-modified 21nt siRNA 21nt siRNA capable of suppressing the expression of luciferase gene or VEGF (vascular endothelial growth factor) gene was synthesized. The sequence of the synthesized RNA is shown below.
Target: Luciferase Sense strand: 5'-GGCCUUUCACUACUCCUACGA-3 '
Antisense strand: 3'-GACCGGAAAGUGAUGAGGAUG-5
Target: VEGF
Sense strand: 5'-UCCUACAGCACAACAAAUGUG-3 '
Antisense strand: 3'-GAAGGAUGUCGUGUUGUUUAC-5.

  Conjugate RNA (21 base length) in which an aromatic ring group was bonded to the 5 'end of this sense strand RNA was synthesized. In the conjugated RNA, the aromatic compound is covalently bonded via a modified aminoalkyl group (Amino Modifier C6; Glen Research) to the 5 'end of the sense strand RNA. Aromatic ring-modified RNA was synthesized by reacting an aromatic ring compound having an active ester with a sense strand RNA aminated at the 5 'end in a liquid phase.

A specific synthesis method is shown below. In order to aminate the 5 ′ end of the sense strand RNA, a normal method (phosphoramidite synthesis method) may be performed using 5′-Amino-Modifier C6 (Glen Research) on RNA solid phase synthesis, As a result, 5 ′ terminal aminoalkyl group-modified sense strand RNA (21 bases in length) can be synthesized. The 5′-terminal aminoalkyl group-modified sense strand RNA was purchased from Hayashi Kasei Co., Ltd. after HPLC purification and MALDI-TOF MS analysis. In the synthesized 5 ′ terminal aminoalkyl group-modified sense strand RNA, — (CH ₂ ) ₆ —NH ₂ is bonded to the terminal (the phosphate residue of the first nucleotide from the 5 ′ terminal side). The concentration of the synthesized single-stranded RNA was calculated by measuring the absorbance at 260 nm using a UV spectrum detector.

Subsequently, this aminoalkyl group-modified single-stranded RNA was adjusted with water (RNase Free water) to 200 μM and used for synthesis of aromatic ring-modified RNA. In order to bind the aminoalkyl group-modified single-stranded RNA and the aromatic ring compound, 20 μl of RNA solution (200 μM) and 8 μl of active esterified aromatic compound (100 mM / DMF) are mixed together, and 100 mM TEAA buffer ( 8 μl of pH 8.0) was added, and finally the final volume was 40 μl with water (RNase Free water). An appropriate amount of DIEA (diisopropylethylamine) was added to the reaction system for the purpose of promoting the reaction. The sample was then allowed to react for approximately 12 hours at room temperature. The active esterified aromatic ring compounds used were Phenoxy acetic acid N-hydroxy succinimide ester (Sigma-Aldrich), 3- (4-Hydroxy phenyl) propionic acid N-hydroxy succinimide ester (Kanto Chemical), (2-Naphthoxy) acetic acid N-hydroxy succinimide ester (Kanto Chemical) and 1-Pyrene butyric acid N-hydroxy succinimide ester (Sigma-Aldrich). After the reaction, the sample was purified by HPLC in order to remove unnecessary reagents in the RNA sample to which the aromatic compound was bound. For HPLC purification, A: 100% 20 mM TEAA (pH 7.0), B: 70% CH ₃ CN / 20 mM TEAA (pH 7.0) were used as buffers, and 10% B buffer to 100% B buffer were used for 50 minutes. Purification was performed with a linear gradient of. Further, CAP CELL (4.6 × 150 mm, 5 μm; SHISEIDO) was used as a purification column. The results of HPLC analysis of unmodified 21mer RNA and aromatic ring-modified 21mer RNA are shown in FIG. In FIG. 1, “A” is unmodified RNA and Phenly-RNA conjugate, “B” is unmodified RNA and Phenol-RNA conjugate, “C” is unmodified RNA and Naphtyl-RNA conjugate, and “D” is unmodified. It is a HPLC analysis result of modified RNA and Pyrene-RNA conjugate. The aromatic ring-modified RNA purified by HPLC was lyophilized, dissolved again in 20 μl of purified water, and then the concentration and synthesis yield were calculated by UV spectrum analysis.

  The structural model and yield of aromatic ring-modified RNA are shown below. The yield of aromatic ring-modified RNA varies depending on the type of target gene and the type of aromatic compound to be bound (Luc; sense strand RNA targeting the luciferase gene, VEGF; sense strand RNA targeting the vascular endothelial growth factor gene. ).

  Hereinafter, the Phenyl-RNA may be referred to as “Phenyl-siRNA” or “Phe-siRNA.” The Phenol-RNA may also be referred to as “Phenol-siRNA”. The Naphtyl-RNA may be referred to as “Naphtyl-siRNA” or “Nap-siRNA”. The Pyrene-RNA may be referred to as “Pyrene-siRNA”.

  The synthesized 21-base long aromatic ring-modified RNA (sense strand) was allowed to form a double strand with a 21-base long complementary strand RNA (antisense strand). At this time, the 3 ′ end of the sense strand and the antisense strand of the double-stranded RNA is designed to have a dangling end (overhang) of 2 bases. For comparison, unmodified RNA whose aromatic ring was not modified and complementary strand RNA were also formed into 21-base double-stranded RNA containing a 2-base dangling end at the 3 ′ end. Double strands are formed in a universal buffer (Hayashi Kasei Co., Ltd.) by mixing equimolar amounts of an aromatic ring-modified or unmodified sense strand RNA and antisense strand RNA, heating at 92 ° C for 2 minutes, Created by gradually lowering the temperature.

2. The ability of the aromatic ring-modified siRNA to form a double strand and the processing by Dicer were confirmed. To confirm the ability to form double strands, a 2 μM concentration RNA sample (10 μl) was electrophoresed for 70 minutes at 250 V using a 20% polyacrylamide gel, and the product was stained with a silver staining kit (GE Healthcare Bioscience) ( Staining conditions were confirmed by referring to the product manual). As a result, it was confirmed that the aromatic ring-modified siRNA formed a double strand in the same manner as the unmodified siRNA.

In addition, processing of aromatic ring-modified siRNA by Dicer was confirmed. Dicer cleavage experiment was performed using 0.5 U recombinant Dicer (Gene Therapy Systems) and final concentration of 2 μM in 20 mM Tris-HCl (pH 8.0), 15 mM NaCl, 2.5 mM Mg ₂ Cl solution. 10 μl of the circle-modified siRNA was prepared in a sample tube and incubated for 12 hours in an incubator set at 37 ° C. Thereafter, 2 μl of Dicer Stop Solution (Gene Therapy Systems) was added to the reaction solution, and 2 μl of loading dye was added to stop the cleavage reaction by Dicer. The obtained product was subjected to electrophoresis using a 20% polyacrylamide gel at 250 V for 70 minutes. Thereafter, the product was stained with a silver staining kit (manufactured by GE Healthcare Bioscience) (see the product manual for staining conditions), and gel analysis was performed with ChemiImager 4000 (manufactured by Alpha Innotech corporation). As a control, the same experiment was performed using unmodified siRNA not treated with Dicer. The results are shown in FIG. As a result, in all aromatic ring-modified siRNAs, changes in the presence and absence of Dicer were not confirmed, and it was revealed that they were not degraded by Dicer.

3. Degradation enzyme resistance of aromatic ring modified siRNA The nuclease resistance of aromatic ring modified siRNA was investigated. First, unmodified and aromatic ring-modified siRNA adjusted to a final concentration of 2 μM in RPMI-1640 medium (Invitrogen) containing 10% FBS (Sanko Junyaku Co., Ltd.) (final amount 110 μl) at 37 ° C. After incubation, 10 μl was taken after 0 h, 1 h, 2 h, 3 h, 4 h, 6 h, 8 h, 12 h, 24 h, and 48 h, and added to a sample tube containing 2 μl of loading die. Subsequently, in order to stop the decomposition reaction, the sample was frozen in liquid nitrogen immediately after sampling and stored at -20 ° C. The obtained product was subjected to electrophoresis using a 20% polyacrylamide gel at 250 V for 70 minutes. Thereafter, the product was stained with a silver staining kit (GE Healthcare Bioscience) (see the product manual for staining conditions), and gel analysis was performed with ChemiImager 4000 (Alpha Innotech Corporation). The results of nuclease resistance of the aromatic ring-modified siRNA are shown in FIG. For comparison, the results of nuclease resistance of unmodified siRNA are also shown. As a result, it was clarified that the aromatic ring-modified siRNA covalently bound to the aromatic ring compound has higher resistance to degradation from nuclease than the unmodified siRNA. In particular, it has been clarified that aromatic ring-modified siRNA having a phenyl group or a Naphtyl group bonded has high nuclease resistance.

4). RNA interference effect of aromatic ring-modified siRNA (suppression of luciferase gene expression)
The RNA interference effect of the synthesized aromatic ring-modified siRNA and unmodified siRNA was evaluated using Renilla luciferase as a target. The experiment was performed according to the following procedure. Before the experiment, HeLa cells (human cervical cancer cells, Tohoku University Institute for Aging Medicine) adjusted to 1 × 10 ⁵ cells / ml were plated on a 96-well plate, and incubated at 37 ° C. overnight. The next day, remove the old medium in the well, add 80 μl of fresh medium without antibiotics (MEM medium; GIBCO) to each well, and add a vector that expresses firefly and Renilla luciferase (psiCHECK ^TM -2 Vector: Promega). 10 μl of Lipofectamine ^™ 2000 (trade name, Invitrogen) complex solution was added to each well containing HeLa cells. Here, the expression vector was set to 0.02 μg per well and Lipofectamine ^™ 2000 was set to 0.2 μl per well, and the required amount was adjusted with OptiMem (Invitrogen). In order to form a complex, the expression vector and Lipofectamine ^™ 2000 were mixed using OptiMem and then incubated at room temperature for 30 minutes. After adding the complex solution, the cells were incubated for 4 hours at 37 ° C. in the presence of 5% CO ₂ . Thereafter, Lipofectamine as unmodified siRNA comprising a gene sequence and a phase complementary antisense sequences of the Renilla luciferase, the final concentration of the aromatic ring-modified siRNA becomes 0 nM, 0.2 nM, 0.5 nM, 1 nM, 2 nM, 5 nM, in 10nM ^A complex was formed with ^TM 2000 (Invitrogen), and 10 μl of the complex solution was added to HeLa cells into which the expression vector was introduced. Here, the final volume per well is 100 μl. A complex solution of siRNA and Lipofectamine ^™ 2000 was prepared by mixing 5 µl of RNA aqueous solution and 5 µl of Lipofectamine ^™ 2000 (0.2 µl) OptiMem solution per well and incubating for 30 minutes at room temperature. After introducing siRNA, incubate for 48 hours, measure the expression level of firefly and Renilla luciferase using Dula-Glo ^™ Luciferase Assay System (Promega) with a luminometer (MicroLumat LB96p: BERTHOLD), and the expression level of firefly luciferase As a control, the expression suppression effect of Renilla luciferase was calculated.

  The results when the concentration of unmodified siRNA and aromatic ring-modified siRNA is 0.5 nM are shown in FIG. As a result, the aromatic ring-modified siRNA was confirmed to have a higher RNA interference effect than the unmodified siRNA that is generally widely used. In particular, in the aromatic ring-modified siRNA to which a phenyl group was bound, a significantly improved RNA interference effect was confirmed as compared with the unmodified siRNA. This result revealed a new finding that the RNA interference effect of siRNA is significantly improved by binding an aromatic ring compound to the 5 'end of the sense strand of siRNA.

5. RNA interference effect of aromatic ring-modified siRNA (suppression of VEGF gene expression)
The RNA interference effect of the synthesized aromatic ring-modified siRNA and unmodified siRNA was evaluated using HeLa cells (human cervical cancer cells, Institute of Aging Medicine, Tohoku University) targeting the VEGF gene.

The experiment was performed as follows. Prior to the experiment, 500 μl of HeLa cells adjusted to 1 × 10 ⁵ cells / ml were spread on a 24-well plate and incubated overnight at 37 ° C. The next day, the old medium in the wells was removed and 450 μl each of fresh medium without antibiotics was added to the wells. The medium was MEM medium (GIBCO). The final concentration of double-stranded RNA that contains a gene sequence and a phase complementary antisense sequences of VEGF to form Lipofectamine ^TM 2000 (Invitrogen) and the complex so as to be 200 nM, the siRNA solution 50μl into the cells 450μl added. Here, the final volume per well is 500 μl. A complex solution of siRNA and Lipofectamine ^™ 2000 was prepared by mixing 25 μl of RNA aqueous solution and 25 μl of Lipofectamine ^™ 2000 (2 μl) OptiMem solution per well and incubating for 30 minutes at room temperature. After introducing the siRNA, it was incubated at 37 ° C. for 48 hours in the presence of 5% CO ₂ . After incubation, the cells were washed three times with PBS (−), and total RNA in the cells was extracted with RNeasy Plus Mini Kit (Qiagen). Then, RT-PCR reaction was performed in order to measure the amount of mRNA of VEGF. Qiagen OneStep RT-PCR Kit (Qiagen) was used for RT-PCR reaction, and 5'-CCC TGA TGA GAT CGA GTA CAT CTT-3 'and 5'-ACC GCC TCG GCT TGT CAC- 3 'was used. As a control, the GAPDH gene was measured by the same method. As GAPDH primers, 5′-GGA AAG CTG TGG CGT GAT G-3 ′ and 5′-CTG TTG CTG TAG CCG TAT TC-3 ′ were used. RT-PCR reaction is RT (Reverse Transcripratase) reaction at 50 ° C for 30 minutes, PCR reaction is 92 ° C for 30 seconds double strand dissociation, 55 ° C for 30 seconds annealing reaction, 68 ° C for 45 seconds extension reaction This was repeated 25 to 28 times (depending on the cells used), and finally incubated at 68 ° C. for 10 minutes, and the temperature was lowered to 4 ° C. to complete the reaction. Reagents, total-RNA, primers and the like used for RT-PCR were prepared according to the reaction conditions of Qiagen OneStep RT-PCR Kit (Qiagen). After RT-PCR reaction, 2 μl of loading dye was added, and RT-PCR products from VEGF and GAPDH mRNA were confirmed on a 2% agarose gel. The evaluation of the gene expression suppression effect is to measure the VEGF expression level of the cells into which unmodified or aromatic ring-modified siRNA is introduced when the VEGF gene expression level of control cells (cells without siRNA introduction) is 100. It went by. Moreover, the error in the expression level between cells was corrected by the gene expression level of the control gene (GAPDH).

  FIG. 5 shows the results of the RNA interference effect of aromatic ring-modified siRNA and unmodified siRNA when targeting VEGF. As a result, it was confirmed that the aromatic ring-modified siRNA in which an aromatic ring compound was bound to the 5 ′ end of the sense strand RNA of the siRNA had a significantly higher RNA interference effect than the unmodified siRNA. From this result, it was confirmed that the aromatic ring-modified siRNA has a high gene expression suppression ability without being restricted by the type of the target gene.

6). Examination of the introduction of aromatic ring-modified 21nt siRNA into cells Before the experiment, 1 ml each of HeLa cells (human cervical cancer cells, Tohoku University Institute for Aging Medicine) adjusted to 1x10 ⁵ cells / ml was spread on a 24-well plate. % Fetal bovine serum (FBS: manufactured by Sanko Junyaku Co., Ltd.) and antibiotics (Kanamycin Sulfate: manufactured by Invitrogen) were cultured at 37 ° C. in the presence of 5% CO ₂ in a medium (MEM: manufactured by Invitrogen). Prior to introduction of the fluorescently labeled oligonucleotide, the medium was changed to a medium (450 μl) containing no antibiotics. As the fluorescence-labeled oligonucleotide, an antisense strand RNA whose F ′ label was used at the 5 ′ end was used, and a double strand was formed with an unmodified sense strand RNA and a sense strand RNA with an aromatic ring modified. Unmodified siRNA and aromatic ring-modified siRNA were introduced into cells using Lipofectamine ^™ 2000. The siRNA was adjusted to a final concentration of 200 nM, and a complex with Lipofectamine ^™ 2000 was prepared by the same method as described above. The prepared 50 μl of siRNA / Lipofectamine ^™ 2000 complex was added to cells containing 450 μl of medium and incubated for 4 hours at 37 ° C. in the presence of 5% CO ₂ . Thereafter, the cells were washed three times with a medium or PBS (−), and the cell transduction properties of unmodified siRNA and aromatic ring-modified siRNA were observed using a confocal fluorescence laser microscope and flow cytometry. In addition, cell introduction into A562 cells (human lung cancer cells, Tohoku University Institute of Aging Medicine) and SH10-TC cells (human gastric cancer cells, Institute of Aging Medicine, Tohoku University) was also evaluated by the same method.

In the evaluation with the confocal fluorescence laser microscope, the Radiance 2000 system (Bio Rad) was used, and fluorescence was observed using an argon laser. For flow cytometry, a coulter EPICS XL cytometer (Beckman coulter) was used to measure the cell transferability per 10,000 counts of cells. For flow cytometry analysis, XL EXPO32 ^™ software (Beckman coulter) was used.

  As a result, it was confirmed that the aromatic ring-modified siRNA was higher in cell introduction than the unmodified siRNA. In particular, a significant difference in transmissibility was observed for A-549 cells and SH10-TC cells compared to unmodified siRNA. In addition, among the aromatic ring-modified siRNAs, it was also revealed that siRNA having a phenyl group bound exhibits a high cell introduction property.

In Example 1, it is a figure which shows the HPLC analysis result of aromatic ring modification single strand RNA. In Example 1, it is a figure which shows the result of having examined processing by Dicer of each aromatic ring modification siRNA. In Example 1, it is a figure which shows the nuclease tolerance result of aromatic ring modification siRNA. In Example 1, it is a figure which shows the evaluation result of the RNA interference effect (luciferase gene expression suppression effect) of aromatic ring modification siRNA in the case of 0.5 nM density | concentration. In Example 1, it is a figure which shows the evaluation result of the RNA interference effect (VEGF gene expression suppression) of aromatic ring modification siRNA.

Claims (3)

SiRNA having an antisense strand RNA consisting of a base sequence complementary to a target sequence in a target gene, and a sense strand RNA having a base sequence complementary to the antisense strand RNA, and capable of suppressing the expression of the target gene a is, via a linker to a phosphate group of the nucleotides of the 5 'end of the sense strand RNA, and wherein the phenyl group, is one selected from the group consisting of phenol group, or a naphthyl group is bonded Aromatic ring-modified siRNA. The aromatic ring-modified siRNA according to claim 1, wherein the linker is represented by the following formula (L-1) or (L-2);
_{-O-CH 2 -CO-NH- (} CH 2) n1 - (L-1)
_{- (CH 2) n0 -CO-} NH- (CH 2) n1 - (L-2)
(In formula (L-1) and formula (L-2), n1 represents an integer of 1 to 10, and in formula (L-2), n0 represents an integer of 0 to 10). The following chemical formulas (1) to (3) are added to the phosphate group of the nucleotide at the 5 ′ end of the sense strand RNA.

The aromatic ring-modified siRNA according to claim 1 or 2, to which a group represented by any of the above is bonded.

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