JP4900943B2 - Modified double-stranded RNA with excellent nuclease resistance and RNA interference effect - Google Patents

Description

  The present invention relates to a modified RNA that can effectively suppress the expression of a target gene. More specifically, the present invention relates to a sense strand RNA consisting of 23 bases (a sequence complementary to the target sequence in the target gene) and an antisense strand RNA consisting of 21 or 23 bases complementary to the sense strand RNA. The present invention relates to a modified RNA which has a high nuclease resistance 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) and 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. In mammalian cell systems, The RNAi method was considered difficult to apply. Therefore, Tuschl et al. Chemically synthesized a 21-base long double-stranded RNA with a dangling end at the 3 ′ end, and introduced it directly into mammalian cells, thereby avoiding the interferon response and having a high sequence-specific high gene. It was reported to show expression suppression ability (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 the 3 'end showed a very high RNA interference effect, but with all other types of short double-stranded RNA, significant RNA interference was observed. No effect was observed. According to this report, an RNA interference method using a double-stranded RNA that is 21 bases long and has a dangling end of 2 bases at the 3 'end is now common. Here, the method of inhibiting target gene expression using a short double-stranded RNA having a length of 21 bases is referred to as 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 media. 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, various chemically modified siRNAs have been reported in order to obtain a long-term and highly active RNA interference effect in synthetic siRNAs with excellent cell transduction properties. 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, it has already been reported that the RNA interference effect is significantly reduced in most cases of 21-base siRNA modified at the ends.

  Moreover, in order to give cell introduction property and tissue selectivity, cholesterol, long-chain alkyl, sugar chain, peptide and the like modified at the end of siRNA have been reported. SiRNA modified with cholesterol and long alkyl chain has not only improved cell transferability but also has been observed to accumulate in the liver in vivo. In addition, an improvement in cell introduction property has been observed in siRNA modified with sugar chains and peptides. However, in any case, compared with siRNA not modified, the same or lower RNA interference effect was observed, and nuclease resistance was not acquired.

  In addition, double-stranded RNA that exerts an RNA interference effect generally employs a structure having a dangling end (overhang) at the end, but does not have a dangling end at the end (that is, smooth) The RNA interference effect is also being studied for the structure having a terminal. However, in the structure where the 5 ′ end of the sense strand RNA is a blunt end, the RNA interference effect is almost the same as when the dangling end is on the 5 ′ end side of the sense strand RNA, or the RNA interference effect Is suggested to be reduced (see Non-Patent Document 3).

  Furthermore, by combining functional molecules (protein, peptide, cholesterol, etc.) with double-stranded RNA that exhibits RNA interference effects, in addition to RNA interference effects, useful effects based on the functional molecules are also exhibited. It is expected that However, it has been found that simply binding a functional molecule to a double-stranded RNA that exerts an RNA interference effect leads to a significant attenuation of the RNA interference effect. The present situation is that functional molecule-modified RNA having useful effects based on molecules has not been constructed.

As described above, in the prior art, what base length RNA and what structure RNA should be adopted in order to provide excellent nuclease resistance and RNA interference effect for double-stranded RNA that exhibits RNA interference effect? The current situation has not been elucidated.
Fire et.al, Nature, 391, 806-811 (1998) Tuschl et.al., EMBO Journal, 20, 6877-6888 (2001) JT Marques et.al., Nature Biotech., 24, 559-565 (2005)

  An object of the present invention is to provide a novel double-stranded RNA that has high nuclease resistance and can exhibit an excellent RNA interference effect. Furthermore, an object of the present invention is to provide a functional molecule-modified double-stranded RNA having both an excellent RNA interference effect and a useful effect based on a functional molecule.

The inventors of the present invention have made extensive studies to solve the above-mentioned problems. As a result, the present inventors include a sense strand RNA containing a base sequence complementary to the target sequence in the target gene, and a base sequence complementary to the sense strand RNA. In double-stranded RNA that has antisense strand RNA and can suppress the expression of the target gene, by providing all the features of (i) to (iii) below, it has high nuclease resistance and further excellent RNA interference effect It was found that the RNA to be played can be acquired.
(i) The sense strand RNA consists of 23 nucleotides, and the antisense strand RNA consists of 21 or 23 nucleotides.
(ii) The 5 ′ end of the sense strand RNA is blunt ended.
(iii) A substituent is bonded only to at least one of the first to sixth nucleotides from the 5 ′ end side of the sense strand RNA.

The present invention has been completed by making further improvements based on such findings. That is, the present invention provides the following modified RNAs.
Item 1. Two strands having a sense strand RNA consisting of a base sequence complementary to the target sequence in the target gene, and an antisense strand RNA having a base sequence complementary to the sense strand RNA, and capable of suppressing the expression of the target gene Strand RNA,
The sense strand RNA consists of 23 nucleotides, and the antisense strand RNA consists of 21 or 23 nucleotides;
The 5 ′ end side of the sense strand RNA is a blunt end,
The modified RNA is characterized in that a substituent is bonded only to at least one of the first to sixth nucleotides from the 5 ′ end of the sense strand RNA.
Item 2. Item 2. The modified RNA according to item 1, wherein at least one substituent is bonded to the first to fourth nucleotides from the 5 ′ end side of the sense strand RNA.
Item 3. Item 2. The modified RNA according to Item 1, wherein one substituent is bound only to the first nucleotide from the 5 ′ end of the sense strand RNA.
Item 4. Item 4. The modified RNA according to any one of Items 1 to 3, wherein the substituent is an aminoalkyl group.
Item 5. Item 4. The modified RNA according to any one of Items 1 to 3, wherein the substituent is an aminoalkyl group having 1 to 40 carbon atoms.

  The modified RNA of the present invention comprises (i) a sense strand RNA consisting of 23 nucleotides, and an antisense strand RNA consisting of 21 or 23 nucleotides. (Ii) a smooth 5 ′ end side of the sense strand RNA By satisfying all the three features of the terminator and (iii) binding a substituent only to at least one of the first to sixth nucleotides from the 5 ′ end of the sense strand RNA In particular, the RNA interference effect is improved.

  The modified RNA of the present invention comprises a sense strand RNA that is composed of 23 nucleotides and is a base sequence complementary to the target sequence in the target gene.

  Here, the target gene is a gene whose gene expression is to be suppressed by the RNA interference effect. In the modified RNA of the present invention, the target gene is not particularly limited, and can be appropriately selected based on the use of the modified RNA.

  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)).

  The sense strand RNA in the modified RNA of the present invention is a base sequence composed of 23 nucleotides complementary to the target sequence in the target gene, that is, a complementary strand having a length of 23 bases to the target sequence. Here, the number of nucleotides of the sense strand RNA means the number of nucleotides constituting a base sequence complementary to the target sequence in the target gene, and the sense strand RNA has a nucleic acid such as DNA or PNA as a substituent. In the case of having it, the number of nucleotides constituting these substituents is not included.

  In addition, a substituent is bonded to at least one of the first to sixth bases from the 5 'end of the sense strand RNA of the modified RNA of the present invention. On the other hand, in the modified RNA of the present invention, no substituent is bonded to a site other than the 5 'end 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 described later are not substituted with a substituent, and are composed only of nucleotides or deoxyribonucleotides. As described above, since the substituent is bonded only to the 5 'end side of the sense strand RNA, it is possible to express a significantly superior RNA interference effect.

  Here, the substituent is not particularly limited, and examples thereof include an amino group; a mercapto group; a nitro group; an alkyl group having 1 to 40 carbon atoms (preferably 2 to 20, more preferably 4 to 12); carbon An aminoalkyl group having 1 to 40 (preferably 2 to 20, more preferably 4 to 12); a thioalkyl group having 1 to 40 (preferably 2 to 20, more preferably 4 to 12) carbon atoms; 40 (preferably 2-20, more preferably 4-12) alkoxyl group; 1-40 carbon atoms (preferably 2-20, more preferably 4-12) aminoalkoxyl groups; 1-40 carbon atoms (preferably Is a thioalkoxyl group having 2 to 20 and more preferably 4 to 12); a mono- or dialkylamino group having 1 to 40 carbon atoms (preferably 2 to 20, more preferably 4 to 12); An alkylthio group having 1 to 40 (preferably 2 to 20 and more preferably 4 to 12); a polyethylene oxide group having 2 to 40 (preferably 2 to 20 and more preferably 4 to 12) carbon atoms; 39 (preferably 3 to 21, more preferably 3 to 12) polypropylene oxide groups. By binding these substituents, the RNA interference effect can be remarkably enhanced.

  Further, the substituent may be a group having a functional molecule other than those described above. Thus, when the substituent is a group having a functional molecule, it is possible to combine an excellent RNA interference effect and a useful effect based on the functional molecule.

  Here, examples of functional molecules include sugars, proteins, peptides, amino acids, DNA, RNA (including tRNA), aptamers, modified nucleotides, low molecular organic / inorganic materials, cholesterol, dendrimers, lipids, polymer materials, etc. Is done.

  Examples of the sugar include monosaccharides such as glucose, galactose, glucosamine, and galactosamine, and oligosaccharides or polysaccharides in which these are arbitrarily combined.

  As said protein, the protein which exists in the living body, the protein which has a pharmacological action, the protein which has a molecule recognition effect etc. can be used, Importin b protein, avidin, an antibody etc. can be mentioned as an example of this protein.

  Specific examples of the DNA include DNA having a base length of 5 to 50, preferably DNA having a base length of 5 to 25.

  Examples of the peptide include R8, nuclear localization signal peptide sequences (such as HIV-1 Tat and SV40T antigen), nuclear export signal peptides (such as HIV-1 Rev and MAPKK), and cell membrane fusion peptides. .

  Examples of the modified nucleotide include those obtained by modifying a phosphate skeleton such as phosphorothioate-type or boranophosphate-type DNA / RNA; 2′-modified nucleotides such as 2′-0Me-modified RNA and 2′-F-modified RNA; LNA (Locked Nucleic Acid) and ENA (2'-O, 4'-C-Ethylene-bridged nucleic acids), etc., modified nucleotides that are cross-linked sugar molecules; PNA (peptide nucleic acid), morpholino nucleotides, etc. Modified nucleotides and the like differing

  Examples of the low-molecular organic / inorganic material include fluorescent substances such as Cy3 and Cy5; biotin; quantum dots; gold fine particles.

  As said dendrimer, a polyamide amine dendrimer etc. are mentioned, for example.

  Examples of the lipid include linoleic acid and DOPE (1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine).

  Examples of the polymer material include polyethylene glycol and polyethyleneimine.

  Here, the group having a functional molecule may be a residue of the functional molecule itself, or a group represented by the following general formula (I), that is, a functional molecule residue of a bifunctional linker. It may be a group to which one functional group is bonded. That is, in the former case, the functional molecule is directly bonded to a predetermined site of the sense strand RNA, and in the latter case, the functional molecule is attached to a predetermined site of the sense strand RNA via a bifunctional linker. Are connected. The group having a functional molecule is preferably the latter group represented by the following general formula (I).

  Here, the bifunctional linker is not particularly limited as long as it includes two functional groups. 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) -iminodiacetic acid etc. can be used.

  In addition to the above, those having the following structure can also be used as the bifunctional linker.

  Here, in the general formulas (L-4) to (L-21), n1 represents an integer of 1 to 40, preferably an integer of 2 to 20, and more preferably an integer of 2 to 12.

  In the general formulas (L-22) and (L-23), 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-4) to (L-23), a functional molecule may be bonded to either the right side or the left side. Preferably, a functional molecule is bound on the left side, and a predetermined site of the sense strand RNA is bound on the right side.

  The bifunctional linker may be appropriately selected and used according to the type of functional molecule to be bound. For example, when cholesterol is used as the functional molecule, a linker of general formula (L-22) or (L-23), particularly a linker of general formula (L-22) is preferably used.

  When the functional molecule is DNA, it is desirable to directly bind the DNA to the 5 ′ end side of the sense strand RNA without using a linker.

  Among the groups containing the functional molecule, a group containing DNA or cholesterol is preferable. When DNA is employed as the substituent, it becomes possible to form a complex by further hybridizing DNA or RNA having a base sequence complementary to the DNA, and to provide a further useful function based on the DNA of the present invention. The modified RNA can be prepared. Further, when a group containing cholesterol is employed as the substituent, it is possible to obtain the advantage that the modified RNA of the present invention can be introduced into cells without using a gene introduction agent.

  Preferable examples of the substituent include aminoalkyl groups, thioalkyl groups, and groups having cholesterol, and particularly preferable examples include aminoalkyl groups. For example, when an aminoalkyl group and a thioalkyl group are employed as the substituent, the RNA interference effect can be dramatically improved, and further, a membrane-permeable peptide, sugar, protein, or not via a linker. The functional molecule such as can be covalently bound, and the advantage that various functions can be imparted to the RNA molecule can be obtained. Furthermore, for example, when a group containing cholesterol is employed as the substituent, the modified RNA of the present invention can be introduced into cells without using a gene introduction agent in addition to exhibiting a desired RNA interference effect. The advantage of becoming

  In the sense strand RNA, the nucleotide to which the substituent is bound is not particularly limited as long as it is the 1st to 6th nucleotide from the 5 ′ end side of the sense strand RNA, but preferably 1 to 5 from the 5 ′ end side. The fourth nucleotide, more preferably the first and / or second nucleotide from the 5 ′ end side, particularly preferably the 5 ′ end (first from the 5 ′ end side) nucleotide.

  Further, the binding site of the substituent is not particularly limited, but the substituent is bonded to the hydrogen atom constituting the hydroxyl group of the phosphate portion of the predetermined nucleotide of the sense strand RNA. It is preferable.

  The number of the substituents bound to the modified RNA of the present invention is not particularly limited, and examples thereof include 1 to 3, preferably 1 or 2, and more preferably 1.

  The binding of the substituent to the nucleotide on the 5 'end side of the sense strand RNA is performed according to a known chemical synthesis method according to the type of the substituent used.

  In addition, the modified RNA of the present invention is obtained by hybridizing antisense strand RNA to the above sense strand RNA so that the 5 ′ end of the sense strand RNA is a blunt end (brand end). This chain is formed. Here, the structure in which the 5 ′ end of the sense strand RNA is a blunt end means a structure in which not only the 5 ′ end of the sense strand RNA but also the 3 ′ end of the antisense strand RNA has no dangling end. To do.

  The antisense strand RNA is composed of 21 or 23 nucleotides, and the sense strand RNA is in a state where the 5 ′ end side of the sense strand RNA (that is, the 3 ′ end side of the antisense strand RNA) is a blunt end. RNA that can hybridize with and form a double strand. That is, when the antisense strand RNA is composed of 21 nucleotides, the antisense strand RNA is a base composed of 21 nucleotides that are completely complementary to the 1st to 21st nucleotides from the 5 ′ end of the sense strand. The modified RNA of the present invention is composed of a sequence, and has a structure in which the 5 ′ end of the sense strand is a blunt end and a dangling end consisting of 2 nucleotides at the 3 ′ end of the sense strand RNA. When the antisense strand RNA is composed of 23 nucleotides, the antisense strand RNA is composed of a base sequence composed of 23 nucleotides that is completely complementary to the sense strand, and the modified RNA of the present invention is a sense RNA. It has a blunt end structure on both the 3 'and 5' ends of the strand.

  The modified RNA of the present invention synthesizes a sense strand RNA having a substituent having the above structure and an antisense strand RNA having the above structure, and hybridizes these sense strand RNA and antisense strand RNA according to a known method. It is prepared by.

  The modified RNA of the present invention is used by being introduced into cells. The modified RNA of the present invention is introduced into a target cell and used in the same manner as RNA conventionally used as siRNA.

  Examples are given below to illustrate the present invention in more detail. These examples are illustrative only and do not limit the invention.

Example 1 5′-terminal amino-modified double-stranded RNA
Designed a double-stranded RNA of 23-19 bases long sense strand RNA and 23-19 bases long antisense strand RNA that has a homologous sequence with Renilla luciferase and can suppress the gene expression of Renilla luciferase. A double-stranded RNA with an amino group modified at the 5 ′ end and a non-modified double-stranded RNA were compared. The double-stranded RNA can form various forms of double strands by combining the antisense strand and the sense strand. The double-stranded RNA does not have a dangling end (single-stranded region). The complete double-stranded RNA is DS (double strand) RNA, and the double-stranded RNA has two dangling ends (overhangs) at both ends. Double-stranded RNA with a dangling end (overhang) only on the right side when the 5 'end of the sense strand is shown on the left when the strand RNA is Si RNA, RO (Right Overhang) RNA, and the 5' end of the sense strand When shown on the left side, a double-stranded RNA having a dangling end (overhang) only on the left side was named LO (Left Overhang) RNA. In addition, the nomenclature of various double-stranded RNAs is distinguished by describing the number of bases of the single-stranded RNA to be the sense strand and antisense strand, with the sense strand being A (A1 or A2), the antisense strand being B, Yes. In addition, since two types of sense strands are designed, A1 and A2 are used for distinction. In addition, a modification of the 5 ′ end of the sense strand with an amino group is described as AxN (x = 1, 2). The sequence of RNA used is as follows.
<Sense strand>
23nt 23A1: 5'-CUGGCCUUUCACUACUCCUACGA-3 '
23A2: 5'-GGCCUUUCACUACUCCUACGAGCGC-3 '
23A1N: 5'NH _2- (CH ₂ ) ₆ -PO ₃ -CUGGCCUUUCACUACUCCUACGA-3 '
23A2N: 5'NH _2- (CH ₂ ) ₆ -PO ₃ -GGCCUUUCACUACUCCUACGAGC-3 '
21nt 21A1: 5'-CUGGCCUUUCACUACUCCUAC-3 '
21A2: 5'-GGCCUUUCACUACUCCUACGA-3 '
21A1: 5'NH _2- (CH ₂ ) ₆ -PO ₃ -CUGGCCUUUCACUACUCCUAC -3 '
21A2: 5'NH _2- (CH ₂ ) ₆ -PO ₃ -GGCCUUUCACUACUCCUACGA-3 '
19nt 19A1: 5 '-GGCCUUUCACUACUCCUAC-3'
<Antisense strand>
27nt 27B: 5'-GUGCUCGUAGGAGUAGUGAAAGGCCAG-3 '
25nt 25B: 5'-GCUCGUAGGAGUAGUGAAAGGCCAG -3 '
23nt 23B: 5'-UCGUAGGAGUAGUGAAAGGCCAG-3 '
21nt 21B: 5'-GUAGGAGUAGUGAAAGGCCAG-3 '
19nt 19B: 5'-GUAGGAGUAGUGAAAGGCC-3 '

1. Structure of terminal amino-modified double-stranded RNA
A terminal amino-modified double-stranded RNA in which the 5 ′ end of the sense strand and the antisense strand of the double-stranded RNA was aminated was synthesized. A specific synthesis method is shown below. First, the terminal amino-modified RNA is a single-stranded RNA (purchased from Hayashi Kasei Co., Ltd .; HPLC purified and MALDI-TOF MS analyzed), and 5′-terminal amination is 5′-Amino-Modifier C6 ( Glen Research). In the synthesized 5 ′ terminal amino-modified double-stranded RNA, — (CH ₂ ) ₆ —NH ₂ is bound to the terminal (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. In addition, in the universal buffer (Hayashi Kasei Co., Ltd.), the same strand of sense and antisense strands were mixed, heated at 92 ° C for 2 minutes, and then gradually lowered to 4 ° C. . Each synthesized double-stranded RNA is electrophoresed for 60 minutes under a 250V condition using a 20% polyacrylamide gel, and then stained with a silver staining kit (GE Healthcare Bioscience). confirmed. FIG. 1 shows an unmodified double-stranded RNA whose end is not modified and a modified double-stranded RNA in which the 5 ′ end is modified with an amino group.

2. Was studied terminal amino-modified two resistance to enzymatic degradation unmodified double-stranded RNA and the 5 'end amino-modified double-stranded RNA nuclease resistance of the chain. In the experiment, 5′-terminal amino-modified double-stranded RNA adjusted to a final concentration of 2 μM was added to RPMI-1640 medium (Invitrogen) containing 10% FBS (Sanko Junyaku Co., Ltd.) (final amount 110 μl), 37 After incubation at 0 ° C., 10 μl was taken after 0 h, 0.5 h, 1 h, 2 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. 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 are shown in FIG. From these results, Ds 23A1 / 23B RNA, Ds 23A1N / 23B RNA, RO 23A1 / 21B RNA, RO 23A1N /, which are double-stranded RNAs having blunt ends at the 5 ′ end and / or 3 ′ end of the sense strand 21B RNA, LO 21A2 / 23B RNA and LO 21A2N / 23B RNA are 21 bases long and commonly used in RNA interference reactions, and have higher resistance to degradation than 21 siRNAs containing a 2 base dangling end at the 3 'end. Became clear. In addition, it was suggested that the longer strand length of the double-stranded RNA is superior in the resistance to decomposing enzyme, and that the amino group bound to the 5 ′ end is excellent in the decomposing enzyme resistance. Furthermore, it is clear that RO RNA having a blunt end at the 5 ′ end of the sense strand and a dangling end at the 5 ′ end of the antisense strand is rapidly degraded in a medium containing 10% FBS. became. From this result, (a) DS RNA having a sense strand consisting of 23 bases and both ends being blunt ends, (b) the sense strand comprising 23 bases and the 5 'end of the sense strand being blunt RO RNA having a dangling end at the 3 ′ end of the sense strand, or (c) the sense strand has a length of 23 bases, the 3 ′ end of the sense strand is a blunt end, and the antisense strand A new finding was obtained that LO RNA having a dangling end at the 3 ′ end possesses higher degradation enzyme resistance than 21 siRNA.

3. Processing of terminal amino-modified double-stranded RNA with Dicer Next, processing of each 5′-terminal amino-modified double-stranded RNA with Dicer was examined. Dicer cleavage experiments were performed at 5 'end adjusted to a final concentration of 2 μM with 0.5 U recombinant Dicer (Gene Therapy Systems) in 20 mM Tris-HCl (pH 8.0), 15 mM NaCl, 2.5 mM Mg ₂ Cl solution. 10 μl of amino-modified double-stranded RNA was prepared in a sample tube and incubated for 12 hours in an incubator set at 37 ° C. Thereafter, in order to stop the cleavage reaction by Dicer, 2 μl of Dicer Stop Solution (Gene Therapy Systems) was added to the reaction solution, and further 2 μl of loading dye was added. 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). As a control, siRNA (Si 21A2 / 21B1 RNA) consisting of double-stranded RNA of 21 base length not treated with Dicer was also measured at the same time.

  The results are shown in FIG. In FIG. 3, “A” is the result of processing of DS RNA, “B” is Si RNA, “C” is RO RNA, and “D” is the result of recombinant Dicer processing.

As a result, DS 23A1 / 23B1 RNA and DS 23A1N / 23B1 RNA were confirmed to be processed into double-stranded RNA of 21 base length in one part, but most of them were not processed by Dicer. In addition, DS 21A1 / 21B1 RNA and DS 21A1N / 21B1 RNA have not been confirmed to change in the presence of Dicer.
As for Si RNA, although part of Si 23A2 / 23B RNA and Si 23A2N / 25B RNA was not processed by Dicer, most RNA molecules were processed into 21-base-length SiRNA. No change was observed in Si 21A2N / 21B RNA in which the 5 ′ end of the sense strand of 21 SiRNA was aminated even in the presence of Dicer.

  In addition, in RO RNA, RO RNA with 23-base-long RNA in the sense strand was confirmed to be processed into double-stranded RNA of 21-base length in one part, but most of it was processed by Dicer. There wasn't. In addition, the phenomenon appeared remarkably in the case where the 5 'end of the sense strand was aminated. In addition, RO RNA having a 21-base long RNA molecule in the sense strand was not confirmed in the presence of Dicer, regardless of the presence of the modifying group at the 5 'end of the sense strand.

  On the other hand, in LO RNA, LO RNA having a 23-base-long RNA molecule in the sense strand is 21-base siRNA or RNA close to it when processed by Dicer, regardless of the presence of a modifying group at the 5 'end of the sense strand. The product has been confirmed by gel electrophoresis analysis. Similarly, LO RNA having a 21-base-long RNA molecule in the sense strand can also produce a 21-base-long siRNA or a similar RNA product by processing with Dicer, regardless of the presence of a modifying group at the 5 'end of the sense strand. The result was suggested to be obtained.

4). RNA interference effect of terminal amino-modified double-stranded RNA Next , the RNA interference effect of each terminal amino-modified double-stranded RNA was evaluated using Renilla luciferase as a target. 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 wells, add 80 μl each of fresh medium without antibiotics to the wells, and add firefly and Renilla luciferase-expressing vectors (psiCHECK ^TM -2 Vector: Promega) and Lipofectamine ^TM 2000 (trade name) , Invitrogen) was added to each well containing 10 μl of 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 . After that, terminal amino-modified double-stranded RNA containing an antisense sequence homologous to the genetic sequence of Renilla luciferase was added to Lipofectamine ^TM 2000 (final concentration to 0nM, 0.2nM, 0.5nM, 1nM, 2nM, 5nM, 10nM). Complexed with Invitrogen) and 10 μl of the complex solution was added to HeLa cells into which the expression vector had been introduced. Here, the final volume per well is 100 μl. A complex solution of RNA 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 at room temperature for 30 minutes. After introducing the RNA, 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 express the level of firefly luciferase As a control, the expression suppression effect of Renilla luciferase was calculated.

  FIG. 4 shows the results of the RNA interference effect of the terminal amino-modified double-stranded RNA at a concentration of 0.5 nM.

  As a result, by modifying the amino group with a blunt end at the 5 'end of the sense strand, such as DS RNA or RO RNA, a dramatic RNA interference effect compared to RNA molecules with similar RNA structure but not modified An improvement was observed. In addition, the effect is the same regardless of the length of the dangling end on the reverse side of the blunt end side in RO RNA, and when the dangling end is present at the 5 ′ end of the antisense strand, A high RNA interference effect was obtained by amino-modifying the 5 ′ end. On the other hand, in the case of Si RNA or LO RNA that has a dangling end on the 3 'end of the antisense strand, the RNA molecule that has the same structure without modification even if the 5' end of the sense strand is modified with an amino group The RNA interference effect was almost the same as that of. From this result, it is said that the RNA interference effect is dramatically improved by modifying only the 5 ′ end of the sense strand of the RNA interference molecule in which the 5 ′ end of the sense strand such as DS RNA or RO RNA is a blunt end. New findings were obtained.

5. Persistence of RNA interference effect of terminal amino-modified double-stranded RNA Next, the persistence of RNA interference effect of terminal amino-modified double-stranded RNA was examined. In order to evaluate the persistence of the RNA interference effect, the terminal amino-modified double-stranded RNA adjusted to 50 nM was applied to HeLa cells (human cervical cancer cells, Tohoku University Institute for Aging Medicine, respectively) for 2 days, 4 days, and 7 days. ) Followed by subsequent RNA interference effects. The target used in the gene expression suppression experiment was Renilla luciferase, and a vector (psiCHECK ^TM -2 Vector: Promega) containing the firefly and Renilla luciferase genes was introduced into the cells 48 hours before measurement using Lipofectamine ^TM 2000 . In addition, terminal amino-modified 27nt dsRNA was introduced into cells using Lipofectamine ^™ 2000, and the medium was changed every two days. For gene expression suppression analysis, firefly and Renilla luciferase expression levels were measured with a luminometer using the Dula-Glo ^™ Luciferase Assay System (Promega), and the Renilla luciferase expression suppression effect was calculated using the firefly luciferase expression level as a control did. The expression vector and RNA used here were complexed with Lipofectamine ^™ 2000 in the same manner as described above, and 10 μl of each sample was added to the cells. The final volume of the cell solution was adjusted to 100 μl.

  The obtained results are shown in FIG. As a result, the above-mentioned end-modified RNA interference molecules in which the 5 'end of the sense strand of DS RNA or RO RNA, which had a high RNA interference effect, was modified with an amino group also showed a high persistence of the RNA interference effect, and the cell Even after 7 days from the addition, the one in which the 5 ′ end of the sense strand was modified with an amino group showed a higher RNA interference effect than the unmodified RNA.

  From the above results, the sense strand RNA is composed of 23 nucleotides, the antisense strand RNA is composed of 21 or 23 nucleotides, and the sense strand RNA has a blunt end on the 5 ′ end side of the sense strand RNA. It has been clarified that it is possible to provide excellent characteristics in terms of both nuclease resistance and RNA interference effect by attaching a modifying group only to the 5 ′ end side of the strand RNA.

FIG. 3 is a diagram showing the structure of a terminal amino-modified double-stranded RNA synthesized in Example 1. In Example 1, it is a figure which shows the nuclease resistance result of terminal amino modification double stranded RNA. In Example 1, it is a figure which shows the result of having examined the processing by Dicer of terminal amino modification double stranded RNA. In Example 1, it is a figure which shows the result of the RNA interference effect of the terminal amino modification double strand RNA in the case of 0.5 nM density | concentration. In Example 1, it is a figure which shows the result of having evaluated the durability of the RNA interference effect of terminal amino modification double stranded RNA.

Claims (2)

Two strands having a sense strand RNA consisting of a base sequence complementary to the target sequence in the target gene, and an antisense strand RNA having a base sequence complementary to the sense strand RNA, and capable of suppressing the expression of the target gene Strand RNA,
The sense strand RNA consists of 23 nucleotides, and the antisense strand RNA consists of 21 or 23 nucleotides;
The 5 ′ end side of the sense strand RNA is a blunt end,
And wherein the sense strand one aminoalkyl group only from 5 'end to the first nucleotide of the RNA is bound, modified RNA. The aminoalkyl group is an aminoalkyl group having 1 to 40 carbon atoms, modified RNA according to claim 1.

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