JP4915809B2 - Double-stranded RNA with high gene knockdown ability - Google Patents

Description

  The development of a medicine that efficiently treats intractable diseases such as cancer and AIDS is a major issue in the life science field. In recent years, an RNAi (RNA interference) method using a long double-stranded RNA has attracted attention as one of the promising methods that can overcome this problem. (Refer nonpatent literature 1). However, this RNAi method was confirmed to induce an interferon reaction in mammalian cells, and it was considered difficult to apply the RNAi method in mammalian cell systems. Therefore, Tuschl et al. Avoided the interferon response by chemically synthesizing a short double-stranded RNA with a base length of 21 bases with a dangling end at the 3 'end, and introduced it directly into mammalian cells. It has been reported that it has the ability to suppress gene expression (see Non-Patent Document 2). In Non-Patent Document 2, a 21-base siRNA (small interference RNA) having a 2-base dangling end at the 3 ′ ends of both the sense strand and the antisense strand was observed to have a very high RNA interference effect. However, it has been reported that no significant RNA interference effect was observed in other short double-stranded RNAs. According to this report, RNA interferometry using a 21-base long double-stranded RNA is now common. In recent years, it has become clear that double-stranded RNA consisting of 27 base pairs exhibits an RNA interference effect that is about 100 times higher than siRNA consisting of 21 bases in length. (See Non-Patent Document 3).

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

However, there are reports that even in siRNA methods using short RNAs, interferon responses are confirmed when high concentrations of RNA molecules are used and in cell types. In addition, in order to clinically apply RNA molecules, it is necessary that the RNA molecules have resistance in vivo, that is, have nuclease resistance. Against the background of such conventional technology, development of a new type of RNA interference molecule having high gene knockdown ability (RNA interference effect) and excellent nuclease resistance even at low concentrations is expected.
Fire et.al, Nature, 391, 806-811 (1998) Tuschl et.al., EMBO Journal, 20, 6877-6888 (2001) J. Rossi et.al. Nature Biotech., 23, 222-226 (2005)

  Accordingly, an object of the present invention is to solve the above-described problems of the conventional art. Specifically, an object of the present invention is to provide a double-stranded RNA that has high nuclease resistance 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, a sense strand containing a base sequence complementary to a target sequence in a target gene, and an antisense containing a base sequence complementary to the sense strand In a double-stranded RNA having a strand and capable of suppressing the expression of the target gene, by satisfying the following constitutions (i) to (vi), the nuclease resistance is high, and the RNA interference effect is excellent. It was found that double-stranded RNA can be obtained.
(i) A dangling end is provided only at either the 3 ′ end of the sense strand or the 3 ′ end of the antisense strand.
(ii) On the side not having the dangling end, the sense strand and the antisense strand are perfectly complementarily paired to form a blunt end.
(iii) In the sense strand or the antisense strand, the first to sixth nucleotides from the blunt end side may be composed of deoxyribonucleotides.
(iv) When a dangling end is formed at the 3 ′ end of the sense strand, the antisense strand is composed of 23 bases, and a dangling end is formed at the 3 ′ end of the antisense strand. Is formed, the antisense strand is composed of 23 to 26 bases.

The present invention has been completed by making further improvements based on the above findings. That is, the present invention provides the following double-stranded RNA.
Item 1. A double-stranded RNA having a sense strand containing a base sequence complementary to a target sequence in a target gene, and an antisense strand containing a base sequence complementary to the sense strand, and capable of suppressing the expression of the target gene There,
Of both ends of the double-stranded RNA, one has a dangling end, the other forms a blunt end,
The dangling end is formed at one of the 3 ′ end of the sense strand or the 3 ′ end of the antisense strand,
On the blunt end side, the sense strand and the antisense strand are paired in a completely complementary manner,
In the sense strand or the antisense strand, when the first to sixth nucleotides from the blunt end side may be composed of deoxyribonucleotides, and a dangling end is formed at the 3 ′ end of the sense strand The antisense strand is composed of 23 bases, and when the dangling end is formed at the 3 ′ end of the antisense strand, the antisense strand is composed of 23 to 26 bases. A double-stranded RNA, characterized in that
Item 2. Item 2. The double-stranded RNA according to Item 1, wherein the dangling end is composed of 2 to 6 nucleotides.
Item 3. Item 3. The double-stranded RNA according to Item 1 or 2, wherein a dangling end is formed at the 3 ′ end of the antisense strand.
Item 4. The dangling end is formed at the 3 ′ end of the antisense strand, and the first and second nucleotides from the 3 ′ end side of the sense strand are composed of deoxyribonucleotides. Double-stranded RNA according to 1.
Item 5. The sense strand is composed of 21 nucleotides, the antisense strand is composed of 23 nucleotides, and the antisense strand is composed of 2 nucleotides at the 3 ′ end. Item 5. The double-stranded RNA according to Item 3 or 4, wherein a ring end is formed.
Item 6. Item 3. The double-stranded RNA according to Item 1 or 2, wherein a dangling end is formed only at the 3 ′ end of the sense strand.
Item 7. The sense strand is composed of 25 nucleotides, the antisense strand is composed of 23 nucleotides, and dangling composed of 2 nucleotides at the 3 ′ end of the sense strand Item 7. The double-stranded RNA according to item 6, wherein an end is formed.

  According to the double-stranded RNA of the present invention, the nuclease resistance is high, an excellent RNA interference effect is exhibited, and the sustainability of the effect is also excellent. Furthermore, since the double-stranded RNA of the present invention does not require the use of artificially modified RNA, no special treatment is required in terms of synthesis, and the manufacturing cost is different from the conventional method. There is no point. In addition, since large-scale synthesis is possible, it can be said that its application range such as drug development and academic use is wide and its industrial utility value is extremely high.

The double-stranded RNA of the present invention has a sense strand containing a base sequence complementary to the target sequence in the target gene, and an antisense strand containing a base sequence complementary to the sense strand, and the target gene A double-stranded RNA capable of suppressing expression, comprising the following constitutions (1) to (5).
(1) Of both ends of the double-stranded RNA, one has a dangling end and the other forms a blunt end,
(2) The dangling end is formed at one of the 3 ′ end of the sense strand or the 3 ′ end of the antisense strand,
(3) On the blunt end side, the sense strand and the antisense strand are paired in a completely complementary manner,
(4) In the sense strand or the antisense strand, the first to sixth nucleotides from the blunt end side may be composed of deoxyribonucleotides, and
(5) When a dangling end is present at the 3 ′ end of the sense strand, the antisense strand is composed of 23 bases, and a dangling end is present at the 3 ′ end of the antisense strand. In some cases, the antisense strand is composed of 23 to 26 bases.

  Hereinafter, the double-stranded RNA of the present invention will be described in detail.

  The double-stranded RNA of the present invention includes a sense strand containing 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 double-stranded RNA of the present invention, the target gene is not particularly limited and can be appropriately selected based on the use of the double-stranded 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 is an A / U pair, (ii) the 5 ′ end of the sense strand is a G / C pair, and (iii) the 5 ′ end side of the antisense strand. (Vi) Double-stranded RNA with high RNA interference effect by designing double-stranded RNA with no more than 9 G / C pairs in the double strand Can be designed (Ui-Tei et. Al, Nucleic Acids Res., 32, 936-948 (2004)).

  In the double-stranded RNA of the present invention, when the dangling end is present at the 3 ′ end of the sense strand, the sense strand constitutes the dangling end at the 3 ′ end of the base sequence complementary to the target sequence. It consists of a base sequence in which base sequences are linked. When the dangling end is bonded to the 3 ′ end of the antisense strand, the sense strand is composed of a base sequence complementary to the target sequence.

  In the double-stranded RNA of the present invention, the number of nucleotides constituting the sense strand differs depending on whether the dangling end described later is present in the sense strand or the antisense strand, but for example 19 to 30, preferably Is 21 to 29, more preferably 21 to 27. More specifically, if the dangling end is present at the 3 ′ end of the sense strand, the number of nucleotides constituting the sense strand is usually 24-29, preferably 24-27, more preferably In addition, when the dangling end is present at the 3 ′ end of the antisense strand, the number of nucleotides constituting the sense strand is usually 21 to 24, preferably 21 to 23. More preferably, 21 to 22, and particularly preferably 21 are mentioned. Here, when a dangling end is present at the 3 ′ end of the sense strand, the number of nucleotides constituting the sense strand is complementary to the number of nucleotides constituting the dangling end and the target sequence. This means the total number of nucleotides constituting a simple base sequence. When a dangling end is present at the 3 ′ end of the antisense strand, the number of nucleotides constituting the sense strand means the number of nucleotides constituting a base sequence complementary to the target sequence.

  Moreover, the double-stranded RNA of the present invention has an antisense strand containing a base sequence complementary to the sense strand.

  In the double-stranded RNA of the present invention, when the dangling end is present at the 3 ′ end of the sense strand, the antisense strand is complementary to the “base sequence complementary to the target sequence” portion of the sense strand. It consists of a target base sequence. On the other hand, when the dangling end is present at the 3 ′ end of the antisense strand, the antisense strand has a nucleotide sequence complementary to the “base sequence complementary to the target sequence” portion of the sense strand. It consists of a base sequence in which the base sequence constituting the dangling end is linked to the 3 ′ end.

  When a dangling end is present at the 3 'end of the sense strand, the number of nucleotides constituting the antisense strand is 23. When the dangling end is present at the 3 ′ end of the antisense strand, the number of nucleotides constituting the antisense strand is 23 to 26, preferably 23 to 25, and more preferably 23 to 24. And particularly preferably 23.

  In the present invention, the sense strand and the antisense strand are RNA strands, and the nucleotides constituting them are basically ribonucleotides, but for the purpose of improving the resistance to degrading enzymes, 2′-O-methyl modification is performed. 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.

  As described above, the double-stranded RNA of the present invention has a dangling end only at one of the 3 'end of the sense strand or the 3' end of the antisense strand. In the present specification, the term “dangling end” means a portion of a base sequence in which a single strand is present alone without being able to form a double strand because there is no pairing base.

  In the double-stranded RNA of the present invention, the number of nucleotides constituting the dangling end may be appropriately set according to the presence position of the dangling end, etc., but usually 2 to 6, preferably 2 to 4, More preferably, two are exemplified.

  Also, the nucleotide sequence constituting the dangling end is not particularly limited. For example, the sequence may be set as appropriate by combining ribonucleotides, deoxynucleotides, or a combination thereof. In addition, the dangling end sequence contains various chemically modified nucleotides such as 2'-O-methyl modified type, 2'-F modified type, LNA (Locked Nucleic Acid), etc. for the purpose of improving resistance to degradation enzyme. May be.

  The double-stranded RNA of the present invention is a hybrid of the sense strand and the antisense strand, and has a dangling end at one of the 3 ′ end of the sense strand or the 3 ′ end of the antisense strand, On the side where there is no dangling end, the sense strand and the antisense strand are paired in a completely complementary manner to form a blunt end. Here, “completely complementary pairing” means that the sense strand and the antisense strand are hybridized without the presence of a dangling end.

  In addition, in the double-stranded RNA of the present invention, from the viewpoint of more effectively exerting an RNA interference effect, the sense strand or the anti-antigen is provided on the side having no dangling end (that is, the blunt end side). The 1st to 6th, preferably 1st to 4th, and more preferably 1st and 2nd nucleotides from the blunt end side of the sense strand may be composed of deoxyribonucleotides. In particular, when the 3 ′ end side of the sense strand forms a blunt end, it is 1-4th, preferably 1-3, more preferably from the blunt end side (ie, 3 ′ end side) of the sense strand. Since the first and second nucleotides are composed of deoxyribonucleotides, a more effective RNA interference effect can be achieved.

Furthermore, as the double-stranded RNA of the present invention, the double-stranded RNAs of the following embodiments (A) to (D) are preferably exemplified from the viewpoint of expressing a particularly excellent RNA interference effect.
(A) The sense strand is composed of 21 nucleotides, the antisense strand is composed of 23 nucleotides, and there is a dangling end composed of 2 nucleotides at the 3 ′ end of the antisense strand. Double-stranded RNA.
(B) The sense strand is composed of 21 nucleotides, the antisense strand is composed of 23 nucleotides, and the dangling end composed of 2 nucleotides is present at the 3 ′ end of the antisense strand. And a double-stranded RNA in which the first and second nucleotides from the 3 ′ end of the sense strand are composed of deoxyribonucleotides.
(C) The sense strand is composed of 23 nucleotides, the antisense strand is composed of 25 nucleotides, and there is a dangling end composed of 2 nucleotides at the 3 ′ end of the antisense strand. And a double-stranded RNA in which the first and second nucleotides from the 3 ′ end of the sense strand are composed of deoxyribonucleotides.
(D) The sense strand is composed of 25 nucleotides, the antisense strand is composed of 23 nucleotides, and there is a dangling end composed of 2 nucleotides at the 3 ′ end of the sense strand. Double-stranded RNA.

  The double-stranded RNA of the present invention is used by being introduced into a cell for the purpose of suppressing the expression of a target gene. Specifically, the double-stranded 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. Design and synthesis of various types of double-stranded RNA Single-stranded RNA (21-27 bases in length) that has a homologous sequence with the mRNA of the synthetic Renilla luciferase gene and can suppress Renilla luciferase gene expression A sense strand) and a 21-27 base RNA (sense strand) complementary to the single strand were designed and synthesized. The double-stranded RNA can form various forms of double strands by combining the antisense strand and the sense strand. In this double-stranded RNA, DS (double strand) RNA is a complete double-stranded RNA that does not have a dangling end (single-stranded region), and two strands have a dangling end (overhang) at both ends of the double-stranded RNA. 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 for each type of double-stranded RNA is A (A1 or A2) for the sense strand and B (for B1 only) for the antisense strand, and describes the number of bases in the single-stranded RNA that becomes the sense strand and antisense strand It is distinguished by. Some RNAs have DNA at the 2 ′ base at the 3 ′ end of RNA, and are described as dd next to the number of bases in single-stranded RNA. Since two types of sense strands are designed, A1 and A2 are used for distinction. For example, a double-stranded double-stranded RNA consisting of 27 bases in length is a DS 27A1 / 27B1 RNA; it has a dangling end of 2 bases on the right side and consists of a 27-base long sense strand and a 25-base long antisense strand. RO 27A1 / 25B1 RNA with a dangling end of 2 bases on the left side, consisting of a 21-base sense strand and a 23-base antisense strand, and 2 at the 3 'end of the sense strand A double-stranded RNA having a base DNA is referred to as LO 21ddA2 / 23B1 RNA. Fig. 1 shows the designed and synthesized double-stranded RNA. The RNA sequences used are as follows.
<Sense strand>
27nt 27A1: 5'-CUGGCCUUUCACUACUCCUACGAGCAC-3 '
27A2: 5'-GGCCUUUCACUACUCCUACGAGCACCA-3 '
25nt 25A1: 5'-CUGGCCUUUCACUACUCCUACGAGC-3 '
25A2: 5'-GGCCUUUCACUACUCCUACGAGCAC-3 '
23nt 23A1: 5'-CUGGCCUUUCACUACUCCUACGA-3 '
23A2: 5'-GGCCUUUCACUACUCCUACGAGCGC-3 '
23A2: 5'-GGCCUUUCACUACUCCUACGA dGdC -3 '
21nt 21A1: 5'-CUGGCCUUUCACUACUCCUAC-3 '
21A2: 5'-GGCCUUUCACUACUCCUACGA-3 '
21A2: 5'-GGCCUUUCACUACUCCUAC dGdA -3 '
<Antisense strand>
27nt 27B1: 5'-GUGCUCGUAGGAGUAGUGAAAGGCCAG-3 '
25nt 27B1: 5'-GCUCGUAGGAGUAGUGAAAGGCCAG -3 '
23nt 27B1: 5'-UCGUAGGAGUAGUGAAAGGCCAG-3 '
21nt 27B1: 5'-GUAGGAGUAGUGAAAGGCCAG-3 '
(Underlined sites are deoxyribonucleotides).

  All RNAs were purchased from Hayashi Kasei Co., Ltd. (HPLC purification, MALDI-TOF MS analysis completed), and the concentration was calculated by measuring the absorbance at 260 nm using a UV spectrum detector. In addition, in the universal buffer (manufactured by Hayashi Kasei Co., Ltd.), the same-strand sense strand and antisense strand single-stranded RNA are mixed, heated at 92 ° C for 2 minutes, and then gradually lowered to 4 ° C. did. 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.

2. Degradative enzyme resistance of various double-stranded RNAs
2-1. Degradation enzyme resistance of DS RNA (reference test)
The nuclease resistance of DS RNA (DS 27A1 / 27B1 RNA, DS 25A1 / 25B1 RNA, DS 23A1 / 23B1 RNA, DS 21A1 / 21B1 RNA: see FIG. 1), which is a complete double-stranded RNA, was examined. In the experiment, various DS RNAs adjusted to a final concentration of 2 μM were incubated at 37 ° C. in RPMI-1640 medium (Invitrogen) containing 10% FBS (Sanko Junyaku Co., Ltd.) (final volume 110 μl). 0 h, 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, 24 h, and 48 h, 10 μl was taken 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 (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 comparison, Si 21A2 / 21B1 RNA consisting of 21-base RNA generally used in the siRNA method and having a 2-base-long dangling end at the 3 ′ end was also examined for resistance to degrading enzymes by the same method.

  Figure 2 shows the results of DS RNA degradation enzyme resistance. From this result, it was suggested that the longer strand length of double-stranded RNA is superior in resistance to degrading enzyme.

2-2. Degradation enzyme resistance of Si RNA (reference test)
Si RNAs with 2 nt dangling ends on both ends of double-stranded RNA (Si 27A2 / 27B1 RNA, Si 25A2 / 25B1 RNA, Si 23A2 / 23B1 RNA, Si 21A2 / 21B1 RNA: see Fig. 1) ) Nuclease resistance was examined. The experiment was performed under the same conditions as in 2-1.

  FIG. 3 shows the results of resistance to degradation enzymes of Si RNA. As a result, the 27-base-length Si RNA (each Si 27A2 / 27B1 RNA) was relatively stable even in a medium containing 10% serum. On the other hand, Si RNA (Si 25A2 / 25B1 RNA, Si 23A2 / 23B1 RNA, Si 21A2 / 21B1 RNA) consisting of RNAs of 25, 23, and 21 bases has a degradation rate as the RNA chain length decreases. In particular, Si 21A2 / 21B1 RNA was completely degraded in about 10 hours.

2-3. RO RNA (RO 27A1 / 25B1 RNA, RO 27A1 / 23B1 RNA) is a double-stranded RNA with a dangling end (overhang) on the right when the 5 'end of the degrading enzyme-resistant sense strand is shown on the left. , RO 27A1 / 21B1 RNA, RO 25A1 / 23B1 RNA (double-stranded RNA of the present invention), RO 25A1 / 21B1 RNA, RO 23A1 / 21B1 RNA, RO 23A1 / 27B1 RNA, RO 23A1 / 25B1 RNA, RO 21A1 / 25B1 RNA , RO 21A1 / 23B1 RNA: see Fig. 1) was examined for nuclease resistance. The experiment was performed under the same conditions as in 2-1.

  4 and 5 show the results of RO RNA degradation enzyme resistance. As a result, RO RNA having a dangling end at the 3 'end, like DS RNA, decreased its resistance to degradation enzyme as the RNA chain length was shortened. Moreover, the shorter the length of the single-stranded RNA region serving as the dangling end, the higher the resistance to degrading enzymes. RO 25A1 / 23B1 RNA (the double-stranded RNA of the present invention) is slightly inferior to that of the most excellent RO 27A1 / 25B1 RNA, but shows superior resistance to degradation enzymes, compared to Si 21A2 / 21B1 RNA Even so, it was revealed that the resistance was extremely high (FIG. 4).

  On the other hand, RO RNA having a dangling end at the 5 ′ end was rapidly degraded in all double-stranded RNAs, and the degradation rate was higher than that of Si 21A2 / 21B1 RNA (FIG. 5).

  Each half-life is about 82 hours for RO 27A1 / 25B1 RNA, about 32 hours for RO 27A1 / 23B1 RNA, about 2.5 hours for RO 27A1 / 21B1 RNA, about 26 hours for RO 25A1 / 23B1 RNA, and RO 25A1. / 21B1 RNA about 2 hours, RO 23A1 / 21B1 RNA about 6 hours, RO 23A1 / 27B1 0.5 hours or less, RO 23A1 / 25B1 0.5 hours or less, RO 21A1 / 25B1 0.5 hours or less, and RO 21A1 / 23B1 It was 0.5 hours or less.

2-4. LO RNA-degrading enzyme resistant LO RNA (LO 25A2 / 27B1 RNA, LO 23A2 / 25B1 RNA) that has a dangling end (overhang) on the left when the 5 'end of the sense strand is shown on the left , LO 21A2 / 23B1 RNA (double-stranded RNA of the present invention: see FIG. 1) was examined for nuclease resistance. The experiment was performed under the same conditions as in 2-1.

  FIG. 6 shows the results of LO RNA degradation enzyme resistance. As a result, LO RNA with a longer chain length tended to show higher degrading enzyme resistance than LO RNA with a shorter chain length, but LO 21A2 / 23B1 RNA (the double-stranded RNA of the present invention) showed superior degradation. It was confirmed to have enzyme resistance. Also, when comparing RO RNA and LO RNA that have similar apparent structures (eg, RO 27A1 / 25B1 RNA and LO 25A2 / 27B1 RNA, RO 23A1 / 21A1 RNA and LO 21A2 / 23B1 RNA), LO RNA It has been revealed that is resistant to degradative enzymes better than RO RNA.

  Each LO RNA has a half-life of 100 hours or longer for LO 25A2 / 27B1 RNA, 100 hours or longer for LO 23A2 / 25B1 RNA, and approximately 53 hours for LO 21A2 / 23B1 RNA (the double-stranded RNA of the present invention). there were.

3. Dicer processing of various double-stranded RNAs
3-1. DS RNA processing by Dicer (reference test)
Processing of each DS RNA (see FIG. 1) by Dicer was examined. Dicer cleavage experiment was performed by adjusting the final concentration to 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 RNA 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). Further, as a control, siRNA (Si 21A2 / 21B1 RNA) consisting of double-stranded RNA of 21 base length not treated with Dicer was used.

  The results are shown in FIG. As a result, a band of DS 27A1 / 27B1 RNA was confirmed at the same position as Si 21A2 / 21B1 RNA in the presence of the recombinant Dicer, and a 21-base siRNA containing a 2-base dangling end was generated by Dicer cleavage. It was strongly suggested that DS 25A1 / 25B1 RNA was also processed by the recombinant Dicer. DS 23A1 / 23B1 RNA was partially processed in double stranded RNA, but not in most double stranded RNA. DS 21A1 / 21B1 RNA was not processed at all by Dicer. As a result, it was suggested that double-stranded RNA having a longer RNA chain length is more susceptible to processing by Dicer (FIG. 7).

3-2. Si RNA processing by Dicer (reference test)
Processing of Si RNA (see FIG. 1) by Dicer was examined. Dicer cutting experiments were performed under the same conditions as in 3-1. The results are shown in FIG. As a result, it was revealed that the Si 27A2 / 27B1 RNA was processed to a double-stranded RNA having a length of 21 bases by a recombinant Dicer. Similarly, it was revealed that Si 25A2 / 25B1 RNA was also processed to a double-stranded RNA having a length of 21 bases by a recombinant Dicer. Si 23A2 / 23B1 RNA was mostly processed by recombinant Dicer to produce 21-base double-stranded RNA, but a portion of the RNA produced products other than 21-base RNA. It was. From these results, it was found that the longer RNA chain in Si RNA is more susceptible to processing by Dicer. It was also revealed that Si RNA is more susceptible to processing by Dicer than DS RNA (FIG. 8).

3-3. Processing of RO RNA with Dicer
RO RNA (RO 27A1 / 25B1 RNA, RO 27A1 / 23B1 RNA, RO 27A1 / 21B1 RNA, RO 25A1 / 23B1 RNA (double-stranded RNA of the present invention), RO 25A1 / 21B1 RNA, RO 23A1 / 21B1 RNA, RO 23A1 / 27B1 RNA, RO 23A1 / 25B1 RNA, RO 21A1 / 25B1 RNA, RO 21A1 / 23B1 RNA (see Fig. 1) were examined for processing by Dicer. Dicer cutting experiments were performed under the same conditions as in 3-1. The obtained results are shown in FIG. As a result, RO RNA (RO 27A1 / 25B1 RNA, RO 27A1 / 23B1 RNA, RO 27A1 / 21B1 RNA) with a 27-base long single-stranded RNA in the sense strand is processed by recombinant Dicer in all types, A band was confirmed at the same position as the 21-base long double-stranded RNA. For RO 25A1 / 23B1 RNA (double-stranded RNA of the present invention) and RO 25A1 / 21B1 RNA with a 25-base long single-stranded RNA in the sense strand and a dangling end at the 3 'end, after treatment with recombinant Dicer Although the band was confirmed at the same position as the double-stranded RNA of 21 bases in RO 25A1 / 21B1 RNA, double-stranded RNA of 21 bases in RO 25A1 / 23B1 RNA (the double-stranded RNA of the present invention) No band was confirmed at the position of, suggesting the possibility of different Dicing products. In addition, both double-stranded RNAs had a broadened RNA band after Dicer treatment. The same applies to RO 23A1 / 21B1 RNA having a single-stranded RNA of 23 bases in the sense strand and a dangling end at the 3 ′ end, and RO 23A1 / 21B1p RNA phosphorylated at the 5 ′ end of the antisense strand. Evaluated. A part of RO 23A1 / 21B1 RNA was confirmed to have the same band as the 21-base long double-stranded RNA in the processing reaction by Dicer. It was revealed that all molecules of RO 23A1 / 21B1p RNA were processed by Dicer. This result suggests that the phosphorylated one is more easily recognized by Dicer. On the other hand, RO RNAs with dangling ends at the 5 'end (RO 23A1 / 25B1 RNA, RO 21A1 / 25B1 RNA, RO 21A1 / 23B1 RNA) are almost in the same position after treatment with recombinant Dicer. A band was confirmed, revealing that it was not processed by Dicer (FIG. 9).

3-4. LO RNA processing by Dicer LO RNA (LO 25A2 / 27B1 RNA, LO 23A2 / 25B1 RNA) is a double-stranded RNA with a dangling end (overhang) on the left when the 5 'end of the sense strand is shown on the left. , LO 21A2 / 23B1 RNA (double-stranded RNA of the present invention: see FIG. 1) was examined for processing by Dicer. In addition, LO RNA having a 2-base DNA at the 3 ′ end of the LO RNA sense strand (LO 23ddA2 / 25B1 RNA (double-stranded RNA of the present invention) and LO 21ddA2 / 23B RNA (double-stranded RNA of the present invention): Similarly, the processing by Dicer was also examined for FIG. Dicer cutting experiments were performed under the same conditions as in 3-1.

  The results are shown in FIGS. This result strongly suggests that the LO 25A2 / 27B1 RNA was treated with the recombinant Dicer, and a band was confirmed at almost the same position as the 21-base-length double-stranded RNA, and it was processed into a 21-base-length siRNA. It was done. Similarly, LO 23A2 / 25B1 RNA was also processed by Dicer, and a band was confirmed at almost the same position as the 21-base long double-stranded RNA. In addition, even though LO 21A2 / 23B1 RNA (the double-stranded RNA of the present invention) has an extremely short RNA chain length, most of the double-stranded RNA molecules are processed by Dicer to produce a 21-base long RNA. It had been. In addition, LO 21A2 / 23B1 RNA that was not processed was in the same position as RNA that was not treated with Dicer (FIG. 10). For LO 23ddA2 / 25B1 RNA (double-stranded RNA of the present invention) and LO 21ddA2 / 23B RNA (double-stranded RNA of the present invention), LO 23ddA2 / 25B1 RNA is processed by Dicer and double-stranded RNA of 21 base length. A band was confirmed at almost the same position, but it was found that LO 21ddA2 / 23B RNA had a band at the same position in the presence and absence of Dicer and was not easily processed (FIG. 11).

4). RNA interference effects of various types of double-stranded RNA
4-1. RNA interference effect of DS RNA (reference test)
The RNA interference effect of each DS RNA (see FIG. 1) 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 on the wells, each 80 [mu] l added to fresh medium without containing an antibiotic to the well, a vector expressing firefly and Renilla luciferase (psiCHECK ^TM -2 Vector: manufactured by Promega) and Lipofectamine ^TM 2000 ( 10 μl of a complex solution (trade name, manufactured by Invitrogen) 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 . After that, double-stranded RNA containing an antisense sequence homologous to the genetic sequence of Renilla luciferase was added to Lipofectamine ^TM 2000 (Invitrogen) so that the final concentration would be 0nM, 0.2nM, 0.5nM, 1nM, 2nM, 5nM, 10nM. And 10 μl of the complex solution was added to the HeLa cells into which the expression vector was 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 double-stranded RNA, incubate for 48 hours, and measure the expression level of firefly and Renilla luciferase using Dula-Glo ^™ Luciferase Assay System (Promega) with a luminometer (MicroLumat LB96p: manufactured by BERTHOLD) The expression level (%) of Renilla luciferase was calculated using the expression level of firefly luciferase as a control.

  FIG. 12 shows the results of RNA interference effects of various DS RNAs at a concentration of 0.5 nM. As a result, the expression level of each luciferase at 0.5 nM was 59.32 (± 3.19)% for DS 25A1 / 25B1 RNA and 69.37 (± 2.37)% for DS 21A1 / 21B1 RNA.

4-2. RNA interference effect of Si RNA
The RNA interference effect of Si RNA (see FIG. 1) was evaluated using Renilla luciferase as a target. The experiment was performed under the same conditions as in 4-1.

  FIG. 13 shows the results of the RNA interference effect of Si RNA at a concentration of 0.5 nM. As a result, the expression level of luciferase was 55.21 (± 1.60)% for Si 25A2 / 25B1 RNA.

4-3. RNA interference effect of RO RNA The RNA interference effect of each RO RNA (see FIG. 1) was evaluated using Renilla luciferase as a target. The experiment was performed under the same conditions as in 4-1 above.

  FIG. 14 shows the results of the RNA interference effect of various RO RNAs at a concentration of 0.5 nM. As a result, high gene expression suppression is achieved in RNAs with a 23-base long RNA in the antisense strand, such as RO 27A1 / 23B1 RNA (double-stranded RNA of the present invention) and RO 25A1 / 23B1 RNA (double-stranded RNA of the present invention). And an excellent RNA interference effect was observed. In particular, RO 25A1 / 23B1 RNA (the double-stranded RNA of the present invention) shows a very excellent RNA interference effect, and even compared with DS RNA and Si RNA, RO 25A1 / 23B1 RNA (the double-stranded RNA of the present invention). Remarkably high RNA interference effect was observed. The RNA interference effect of RO 27A1 / 23B1 RNA was comparable to that of DS 27A1 / 27B1 RNA and Si 21A2 / 21B1 RNA. On the other hand, other RO RNA (RO 27A1 / 25B1 RNA, RO 27A1 / 21B1 RNA, RO 27A1 / 21B1 RNA, RO 23A1 / 21B1 RNA, RO 23A1 / 21B1p RNA, RO 23A1 / 25B1 RNA, RO 23A1 / 25B1 RNA, RO 21A1 / 25B1 RNA and RO 21A1 / 23B1 RNA) were not observed to have a particularly high RNA interference effect. The luciferase expression levels at 0.5 nM were 58.15 (± 3.14)% for RO 27A1 / 25B1 RNA, 30.09 (± 2.17)% for RO 27A1 / 23B1 RNA (double-stranded RNA of the present invention), and RO 27A1 / 21B1. 61.74 (± 0.71)% for RNA, 13.25 (± 0.70)% for RO 25A1 / 23B1 RNA (double-stranded RNA of the present invention), 73.14 (± 7.79)% for RO 25A1 / 21B1 RNA, 80.06 for RO 23A1 / 21B1 RNA (± 1.62)%, RO 23A1 / 21B1p RNA 57.79 (± 1.97)%, RO 23A1 / 25B1 RNA 38.19 (± 1.99)%, RO 21A1 / 25B1 RNA 65.32 (± 4.31)%, and RO 21A1 / 23B1 The RNA was 52.81 (± 2.84)%.

  From these results, when RO RNA is used, those with a dangling end at the 3 ′ end of the sense strand and an antisense length of 23 bases are suitable, and RO 25A / 23B RNA is particularly excellent. The knowledge that possessed the ability to suppress gene expression was obtained.

4-4. RNA interference effect of LO RNA The RNA interference effect of each LO RNA (see FIG. 1) was evaluated using Renilla luciferase as a target. The experiment was performed under the same conditions as in 4-1 above.

  FIG. 15 shows the results of RNA interference effects of various LO RNAs (LO 23A2 / 25B1 RNA, LO 21A2 / 23B1 RNA) at a concentration of 0.5 nM, and FIG. 16 has DNA at two bases at the 3 ′ end of the sense strand. The result of the RNA interference effect of LO RNA (LO 23ddA2 / 25B1 RNA, LO 21ddA2 / 23B1 RNA) is shown.

  As a result, high gene expression suppression ability was observed in the LO RNA used this time (LO 23A2 / 25B1 RNA, LO 21A2 / 23B1 RNA, LO 23ddA2 / 25B1 RNA, LO 21ddA2 / 23B1 RNA; all of the double-stranded RNA of the present invention). It was. In particular, LO 21A2 / 23B1 RNA, LO 23ddA2 / 25B1 RNA, and LO 21ddA2 / 23B1 RNA exhibit particularly excellent RNA interference effects, and are at least several tens to several times higher than DS RNA or RO RNA. Met. The expression level of each luciferase at 0.5 nM was 25.18 (± 1.97)% for LO 23A2 / 25B1 RNA, 9.57 (± 0.39)% for LO 21A2 / 23B1 RNA, 8.57 (± 0.79)% for LO 23ddA2 / 25B1 RNA, LO The 21ddA2 / 23B1 RNA was 8.65 (± 2.19)%.

  From these results, it was revealed that LO RNA having a dangling end at the 3 'end of the antisense strand exhibits a very high RNA interference effect. The reason for this is that RNA with the LO RNA structure is likely to be recognized by a group of proteins required for RNA interference reactions such as Dicer and RISC in the cell. Among LO RNAs, LO 21A / 23B RNA and LO 23ddA2 / 25B1 RNA and LO 21ddA2 / 23B1 RNA having 2 base DNA at the 3 ′ end of the sense strand showed particularly excellent RNA interference effects. Among the various double-stranded RNAs used, the RNA interference effect was remarkably excellent.

5. Persistence of RNA interference effects of various double-stranded RNAs Next, the persistence of RNA interference effects of various double-stranded RNAs was examined. In order to evaluate the persistence of the RNA interference effect, double-stranded RNA adjusted to 50 nM was incubated with HeLa cells (human cervical cancer cells, Institute of Aging Medicine, Tohoku University) for 7 days, followed by RNA interference The effect was tracked. The target used in the gene expression suppression experiment was Renilla luciferase, and a vector (psiCHECK ^TM -2 Vector: Promega) with firefly and Renilla luciferase genes 48 hours before measurement was Lipofectamine ^TM 2000 (Invitrogen) Was introduced into cells. In the gene expression suppression analysis, the expression level of firefly and Renilla luciferase was measured with a luminometer using Dula-Glo ^™ Luciferase Assay System (Promega), and the expression level of Renilla luciferase was controlled using the expression level of firefly luciferase as a control ( %) Was calculated. 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. 17, FIG. 18, and FIG. As a result, RO 27A1 / 23B1 RNA (invented double-stranded RNA) and RO 25A1 / 23B1 RNA (invented double-stranded RNA), which had high RNA interference effects in RO RNA, were observed to show high gene expression suppression on day 7. It was revealed that the RNA interference effect is excellent in sustainability. In addition, high gene expression suppression was observed in LO 23A2 / 25B1 RNA and LO 21A2 / 25B1 RNA on the 7th day, and especially the long-lasting RNA interference effect in LO 21A2 / 23B1 RNA (the double-stranded RNA of the present invention). Was observed.

6). Cytotoxicity of various double-stranded RNAs to HeLa cells Cytotoxicity of double-stranded RNAs (DS RNA, Si RNA, RO RNA, LO RNA) to HeLa cells was evaluated. 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, the old medium on the wells was removed and 90 μl of fresh medium without antibiotics was added to each well. Cytotoxicity of various RNAs was evaluated by positively introducing double-stranded RNA into cells with Lipofectamine ^™ 2000 (Invitrogen) in order to evaluate the intracellular toxicity of RNA. Complexes were formed with Lipofectamine ^TM 2000 (Invitrogen) so that the final concentration of double-stranded RNA was 0 nM, 0.2 nM, 0.5 nM, 1 nM, 2 nM, 5 nM, 10 nM, and 10 μl of the above complex was mixed with 90 μl of the above. The final volume per well was 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. HeLa cells into which double-stranded RNA had been introduced were incubated at 37 ° C. for 48 hours in the presence of 5% CO ₂ . Then, add 50μl of CellTiter-Glo Reagent in CellTiter-Glo Luminescent Cell Viability Assay (Promega) to each well, and after stirring for about 60 min, measure the amount of Luminescence in each well with a luminometer (MicroLumat LB96p: manufactured by BERTHOLD) did. Since the measured amount of Luminescence depends on ATP in living cells, the amount of Luminescence of control cells (when RNA is 0 nM, Lipofectamine ^TM 2000 is 0 μl. This is defined as 100% survival rate) and various double strands are introduced. The survival rate in each well was calculated by calculating the relative value of the Luminescence amount of the obtained cells. (See Promega CellTiter-Glo Luminescent Cell Viability Assay manual.)
FIG. 20 shows the results of cell viability when the maximum concentration of double-stranded RNA used this time is 10 nM. In FIG. 20, “A” is the result of cell viability when using DS RNA, “B” is Si RNA, “C” is RO RNA, and “D” is LO RNA. As a result, no significant cytotoxicity was observed in all double-stranded RNAs. Among the results obtained this time, those with a relatively low survival rate (survival rate of about 80%) showed a higher survival rate than when Lipofectamine ^™ 2000 alone was added in the same amount (0.2 μl). (Survival rate about 60%). That is, it is considered that even those with a relatively low survival rate are not sufficiently toxic by RNA, but are sufficiently high in the effect of toxicity by Lipofectamine ^™ 2000.

FIG. 3 is a diagram showing the structures of various double-stranded RNAs synthesized. It is a figure which shows the nuclease resistance result of DS RNA. It is a figure which shows the nuclease resistance result of Si RNA. It is a figure which shows the nuclease resistance result of RO RNA (RO 27A1 / 25B1 RNA, RO 27A1 / 23B1 RNA, 27A1 / 21B1 RNA, RO 25A1 / 23B1 RNA, RO 25A1 / 21B1 RNA, RO 23A1 / 21B1 RNA). It is a figure which shows the nuclease resistance result of RO RNA (RO 23A1 / 27B1 RNA, RO 23A1 / 25B1 RNA, RO 21A1 / 25B1 RNA, RO 21A1 / 23B1 RNA). It is a figure which shows the nuclease resistance result of LO RNA. It is a figure which shows the result of having examined processing by DSer of DS RNA. It is a figure which shows the result of having examined processing of Si RNA by Dicer. It is a figure which shows the result of having investigated the processing by ROcer of Dicer. It is a figure which shows the result of having examined processing by LOcer of Dicer. It is a figure which shows the result of having investigated processing by LOcer which has DNA of 2 bases at 3 'terminal of a sense strand by Dicer. It is a figure which shows the result of the RNA interference effect of DS RNA in the case of 0.5 nM density | concentration. It is a figure which shows the result of the RNA interference effect of Si RNA in the case of 0.5 nM density | concentration. It is a figure which shows the result of the RNA interference effect of RO RNA in the case of 0.5 nM density | concentration. It is a figure which shows the result of the RNA interference effect of LO RNA in 0.5 nM density | concentration. It is a figure which shows the result of the RNA interference effect of LO RNA LO RNA which has DNA of 2 bases at 3 'terminal of a sense strand at the concentration of 0.5 nM. It is a figure which shows the persistence of the RNA interference effect of double stranded RNA (DS25A1 / 25B1 RNA, DS21A1 / 21B1 RNA, Si25A2 / 25B1 RNA, Si23A2 / 23B1 RNA, Si21A2 / 21B1 RNA). It is a figure which shows the persistence of the RNA interference effect of double stranded RNA (RO27A1 / 25B1 RNA, RO27A1 / 23B1 RNA, RO25A1 / 23B1 RNA, RO25A1 / 21B1 RNA, RO23A1 / 21B1 RNA). It is a figure which shows the persistence of the RNA interference effect of double stranded RNA (LO 23A2 / 25B1 RNA, LO 21A2 / 23B1 RNA). It is a figure which shows cell viability when various double stranded RNA of 10 nM density | concentration is added to HeLa cell.

Claims (7)

A double-stranded RNA having a sense strand containing a base sequence complementary to a target sequence in a target gene, and an antisense strand containing a base sequence complementary to the sense strand, and capable of suppressing the expression of the target gene There,
Of both ends of the double-stranded RNA, one has a dangling end, the other forms a blunt end,
The dangling end is formed at one of the 3 ′ end of the sense strand or the 3 ′ end of the antisense strand,
On the blunt end side, the sense strand and the antisense strand are paired in a completely complementary manner,
In the sense strand or the antisense strand, when the first to sixth nucleotides from the blunt end side may be composed of deoxyribonucleotides, and a dangling end is formed at the 3 ′ end of the sense strand The antisense strand is composed of 23 bases, and when the dangling end is formed at the 3 ′ end of the antisense strand, the antisense strand is composed of 23 to 24 bases. A double-stranded RNA characterized by The double-stranded RNA according to claim 1, wherein the dangling end is composed of 2 to 6 nucleotides. The double-stranded RNA according to claim 1 or 2, wherein a dangling end is formed at the 3 'end of the antisense strand. 4. A dangling end is formed at the 3 ′ end of the antisense strand, and the first and second nucleotides from the 3 ′ end side of the sense strand are composed of deoxyribonucleotides. A double-stranded RNA according to claim 1. The sense strand is composed of 21 nucleotides, the antisense strand is composed of 23 nucleotides, and the antisense strand is composed of 2 nucleotides at the 3 ′ end. The double-stranded RNA according to claim 3 or 4, wherein a ring end is formed. The double-stranded RNA according to claim 1 or 2, wherein a dangling end is formed only at the 3 'end of the sense strand. The sense strand is composed of 25 nucleotides, the antisense strand is composed of 23 nucleotides, and dangling composed of 2 nucleotides at the 3 ′ end of the sense strand The double-stranded RNA according to claim 6, wherein an end is formed.

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